Microorganisms and Cyanide · MICROORGANISMS ANDCYANIDE 653 ofhigherplants of70to 80families, including agriculturally important species such as cas- sava,flax, sorghum,alfalfa, peaches,

BAcrzuiowIcAL Rzvimws, Sept. 1976, p. 652-680 Vol. 40, No.3Copyright 0 1976 American Society for Microbiology Printed in U.S.A.

Microorganisms and CyanideCHRISTOPHER J. KNOWLES

Biological Laboratories, University ofKent, Canterbury CT2 7NJ, Kent, Great Britain

INTRODUCTION............................................................. 652PLANTCYANOGENESIS,....................................................... 652CYANIDE PRODUCTION BY FUNGI .......... ............................... 653Cyanide-Linked Diseases .................... ................................ 653Snow mold disease ........................................................ 653Fairy ring disease ......................................................... 655Copperspot disease......................................................... 655

Cyanide Production in Culture ................ ............................... 656Formation of Cyanogens . .................................................... 656

CYANIDE PRODUCTION BY BACTERIA .......... ........................... 657Species Producing Cyanide .................... .............................. 657Conditions of Cyanide Production ............. ............................... 657Metabolic Precursors of Cyanide .............. ............................... 658

SPECULATIONS ON THE METABOLIC PATHWAY OF CYANIDE PRODUCTIONBY MICROORGANISMS ................. ............................... 658

CYANIDE ASSIMILATION BY CYANOGENIC MICROORGANISMS ..... ...... 660Alanine Formation .......................................................... 660Glutamic Acid Formation.................................................... 661a-Aminobutyric Acid Formation .............. ............................... 661j3-Cyanoalanine Formation ................... ............................... 661'y-Cyano-a-aminobutyric Acid Formation..... 665

GROWTH OF MICROORGANISMS ONCYANIDE. 665UTILIZATION OF CYANIDE BY MICROBIAL ECOSYSTEMS ..... ........... 666

Soil Ecosystems.............................................................. 666Disposal of Cyanide Wastes by Sewage Systems ............................... 667

MECHANISMS OF CYANIDE RESISTANCE AND DETOXICATION ..... ...... 669Cyanide-Resistant Respiration .............. ................................. 669Achromobacter ............................................................ 669Bacilus cereus ........................................................... 670Chromobacterium violaceum ................. .............................. 670Ewcherichia colU .......................................................... 671Tetrahymenapyriformni.n................................................... 671

Cyanide Detoxication ....................................................... 672CONCLUDING REMARKS..................................................... 673LITERATURECITED. ............... 674

INTRODUCTIONThe susceptibility of cytochrome oxidases to

cyanide means that, although it is a valuableinhibitor for respiratory studies, cyanide is ex-

ceedingly toxic to living cells and, therefore,cyanide pollution causes great damage to mi-crobial and other ecosystems.

Unfortunately, many industrial processes

produce cyanide as a by-product and largequantities of it must be disposed of. Chemicalmethods of cyanide degradation are expensive;there have been many cases reported ofcyanidepollution and the dangerous practice of'dump-ing" cyanide wastes. Moreover, industrial ef-fluents are not the only sources of cyanidefound in the environment. A remarkable num-

ber of plants, including many of agriculturalimportance, are cyanogenic, releasing much cy-

anide into the soil.

Thus, in addition to being of intrinsic scien-tific interest, an understanding of microbialmechanisms of cyanide resistance and utiliza-tion is of wider environmental, agricultural,and economic importance. It is the aim of thisreview to discuss cyanide production, utiliza-tion, degradation, and resistance by microorga-nisms.

PLANT CYANOGENESISTo comprehend microbial cyanide produc-

tion, it is necessary to have a knowledge ofplant cyanogenesis. Luckily, there have beenseveral recent reviews of plant cyanogenesis(35-37, 45, 151), and therefore it will be neces-sary only to briefly discuss this topic here.Cyanophoric plants synthesize compounds

(cyanogens) that, on hydrolysis, liberate hydro-gen cyanide (cyanogenesis). At least 800 species

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MICROORGANISMS AND CYANIDE 653

of higher plants of 70 to 80 families, includingagriculturally important species such as cas-sava, flax, sorghum, alfalfa, peaches, almonds,and beans, are known to be cyanogenic (45).West African cassava flour contains sufficientquantities of cyanogens such that the humandaily consumption of cyanide, when cassava isthe staple part of the diet, is equivalent toabout one-half the lethal dose, which is proba-bly the reason for the widespread and chronicneurological disorders found in this area (36,117).Most of the known plant cyanogens are glu-

cosidic derivatives of a-hydroxynitriles (cy-anohydrins), although some plants form cyano-lipids and other cyanogens (151). The glucosidiccyanogens are formed from several aminoacids, e.g., lotaustralin from isoleucine, lina-marin from valine, amygdalin and prunasinfrom phenylalanine, and dhurrin from tyro-sine.

Experiments, involving feeding radiolabeledamino acids to cyanogenic plants, have shownthat the a-carbon atom and the amino nitrogenof the amino acid are used to form the cyanidegroup of glucosidic cyanogens (Fig. 1), appar-ently without cleavage of the covalent CNlinkage. The carboxyl group of the amino acidis lost, and the P-caron atom is the center forglucosylation.

Although the pathway of glucosidic cyanogenformation is not yet clear, there is enough evi-dence to suggest that aldoximes are involved asintermediates (36, 101). The probable pathwayis given in Fig. 1.Catabolism of plant glucosidic cyanogens to

release hydrogen cyanide occurs in two steps. Aglucosidase (EC 3.2.1.21, a-D-glucose gluco-hydrolase) acts to remove glucose and release

the aglycone, and an oxynitrilase then causesrelease of cyanide (Fig. 2).Some plants can also metabolize cyanide (2,

15, 16, 48, 49, 69); this will be discussed inanother section (page 661).

CYANIDE PRODUCTION BY FUNGIFormation of hydrogen cyanide by a microor-

ganism was first demonstrated by von L6seckein 1871, when he observed it in the fungusMarasmius oreades (183).

Since then cyanide production has been ob-served in a wide range of fungi. Bach reviewedthe early work on fungal cyanogenesis andlisted 31 species ofmany genera that form cya-nide, including several strains of Clitocybe,Marasmius, Pholiota, Polyporus, and Tricho-loma (9). Locquin claimed that 300 species ofbasidiomycetes from 52 genera and several as-comycetes are cyanogenic (92). Singer (156) andHutchinson (76) have more recently briefly re-viewed fungal cyanogenesis. There has, to myknowledge, been only one report of cyanogene-sis in eukaryotic microorganisms other thanthe fungi: the alga Chlorella vulgaris is able toproduce low concentrations of cyanide (57).The antibiotic activity of several cyanogenic

fungi against other fungi and bacteria is re-lated to their production of cyanide (92, 103,140), and cyanide produced by Fomes scutella-tus inhibits germination of lettuce seeds andgrowth of seedlings (103).

Cyanide-Linked DiseasesSnow mold disease. Winter crown rot or

snow mold disease, so called because it developsunder snow cover, is a disease of alfalfa (Medi-cago sativa) and other forage plants. It is

R1 COOH)H-H(

R2 NH2

Amino acid

Rione or, XCH-CH=NOHseveralsteps

Aldoxime

R,-a )H-CAN

R2

Nitrile

lo] Rt /OHoxygenase

R2 C N

[UDP - glucose]glvcosvltransferase

a-Hydroxynitrile(the aglycone)

RI /O-glucoseRBCN

Glucosidiccyanogen

FIG. 1. Probable pathway offormation ofplant glucosidic cyanogens. The symbols show the fates ofa- and#-carbon atoms and the amino-nitrogen atom ofamino acids on conversion to cyanogens. UDP, Uridine 5-diphosphate.

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RI\ /O-glucose

R2 C NGlucosidiccyanogen

R1\ /OH,8-glucosidase / \ -NR2 CoNe

aglyconeIoxynitrilase

RI/C=O + HCN

R2

FIG. 2. Release of cyanide from plant glucosidic cyanogens.

caused by a psychrophilic, cyanide-producingbasidiomycete which is particularly prevalentin western Canada (87, 88). This fungus has a

wide range of hosts, damaging forage legumes,winter wheat, turf grass, and perennials (38).Under artificial conditions maximal infection

and death occurs when the soil, infected by thefungus, is kept at 2 to 4TC and insulated fromthe atmosphere by a layer of expanded mica orother insulant, mimicking the effect of snowcover (87, 88). The fungus must become associ-ated with the host in the fall or early winter ifthe disease is to develop, and, after infection,the winter-dormant plants become increasinglysusceptible (38). Damage is most severe whenthere is a slow thaw. The disease is able todevelop when the crown bud tissues are in-fected (38, 87, 88).From crown buds and shoots infected with

the fungus and kept at winter soil tempera-tures (2 to 40C), invasion of the tissues does notbegin for about 30 days (87, 88), at which timelethal doses of cyanide have been produced inthe crown tissues (88). Mass fungal invasion,both intracellular and intercellular, occursafter 45 to 60 days; the infected tissues thencontain massive doses of cyanide many-foldgreater than those found in the surroundingsoil-fungus mat (87).That cyanide production in the crown buds

probably precedes invasion by the fungus isindicated also by experiments in the field (86).Here, analyses of infected alfalfa showed thatin diseased plants damage to the host was pro-portional to its cyanide content. The crownbuds of plants we're infected by the basidiomy-cete in October, before winter dormancy com-menced, and cyanide was observed to be pres-ent in the crown tissues from January to April,being at its maximum at the end ofMarch anddisappearing by the end of April, when thespring thaw started. The basidiomycete couldbe seen attached to the outer portions of thecrown during the period up to the start of Feb-ruary but penetration did not occur untilMarch, when there was extensive mycelial in-

vasion and disintegration of the buds.Additional evidence for the role of cyanide in

pathogenesis of the disease is provided by acorrelation between the concentration of cya-nide formed in the plant tissues and the sever-ity of infection of highly sensitive and less sen-sitive plant species. Thus, cold-hardy grasses(Bromus inermis, Poa pratensis) and alfalfa(Medicago falcata) are more resistant to thepathogen than cold-sensitive alfalfa (M. sativa)and grass (Agrostis tennis), and in the sensi-tive species the concentration of cyanide ismuch greater than in resistant species (85).Although certain types of the basidiomycete

can themselves produce cyanide (see next sec-tion), it seems probable that all or at least themajor part of the cyanide found in the infectedalfalfa is derived from host plant glucosidiccyanogens. Pathogenic types of the basidiomy-cete have been shown to produce an extracellu-lar ,8-glucosidase that releases cyanide from theglucosidic cyanogen amygdalin, as well as fromhomogenates of crown bud tissues of the sensi-tive strain of alfalfa, M. sativa, but not therelatively resistant M. falcata (34). Cyanide isreleased by the reaction:

plant cyanogen fungal4-i+g/3-glucosidase glucose

non-enzymicor

oxynitrilase(plant or fungal?)

HCN + R*CHO

Three types of snow mold fungus have beenobtained (186); their properties are summarizedin Table 1. Type C do not produce cyanide andare not pathogenic. They are obtained in thespring from superficial mycelia on plants andare saprophytic. Type A cause production ofhigh levels of cyanide in crown tissues and are'very virulent; type B are less virulent and thereis less cyanide formation in the host plant.Pathogenicity is therefore approximately pro-

+ glucose

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TABLz 1. Characteristics of three types of isolates ofa psychrophilic basidiomyceteeIsolate

CharacteristicTypeA TypeB TypeC

Colony characteristics Relatively slowly grow- Fairly rapidly growing, Heterogenous group;ing, mycelium ap- abundant fluffy aerial very rapidly grow-pressed to substra- mycelium; stromata ing; few aerial myce-tum; abundant not usually seen liastroma-like bodies

Cyanide formation in cul- None Prolific Noneture

Cyanide formation on al- Prolific (600-655 jug/ml Moderate (175-240 j.g/ Nonefalfa (Medicago sativa) in crown buds) ml in crown buds)in the field

Pathogenicity toM. sativa (a) 3-18% survival (a) 85-95% survival (a) 100% survivalin the field (b) 0-3% survival (b) 47-60% survival (b) 100% survival

Pathogenicity toM. sativa 28-38% survival 68-81% survival 98% survivalin a controlled environ-ment

Pathogenicity to a grass 62-70% survival 3342% survival 100% survival(Dactylis glomerata) inthe field

Cyanide tolerance in cul- Low (up to 50 tLg ofHCN High (up to 250 ,g of Low (up to 50 j&g ofture per ml) HCN per ml) HCN per ml)a Data summarized from Ward et al. (186) through the courtesy of The National Research Council of

Canada. All three types of isolates grow at 0 to 25°C (optimally 12 to 17.5°C) and at pH values 4.0 to 9.0(optimally at pH 6.0 to 7.0).

portional to cyanide production in the host.Contrary to their effects on alfalfa, type B aremore virulent then type A in a grass (Dactylisglomerata); unfortunately, the relationship tocyanide concentration has not been reported.Fairy ring disease. Fairy ring disease is

caused by fungi of the order Agaricales, partic-ularly Marasmius oreades. M. oreades is de-structive of grasslands, including lawns, pas-tures, and parks (89). Pathogenesis is initiatedby a toxic excretion from M. oreades that dam-ages the root tops of the host plant, weakeningit and permitting mass invasion by the fungus(13, 46, 47).This toxic excretion could be a glucosidase

that acts to promote cyanide production in theplant, causing initial damage before fungal in-vasion. Thus, although considerable work isneeded to confirm it, it is possible that theetiology of fairy ring disease is related to snowmold disease and copperspot disease (see nextsection). Unlike the snow mold basidiomycete(38), M. oreades grows poorly below 15TC, indi-cating that it has little activity in the coolersections of the year (89).That cyanide can cause damage to plants

sensitive to M. oreades has been shown by sus-pending root systems of three different grassesabove cyanide-evolving cultures of the fungus;root discoloration and damage occurred (47).Copperspot disease. Stemphylium loti

causes copperspot disease of birdsfoot trefoil(Lotus corniculatus) (58). Stem cankers, red orbrown leaf spots, and defoliation of affectedleaflets are the principal symptoms of the dis-ease.Drake showed that S. loti grows intercellu-

larly on trefoil, apparently producing a sub-stance that damages the plant before mass in-tracellular invasion and collapse of the plantcells (43).

Millar and Higgins have shown that the toxicexcretion from S. loti is a (3-glucosidase thatcauses release of cyanide from the cyanogenicglucosides (linamarin and lotaustralin) of theplant; the released cyanide is lethal to the plant(107). Two types of trefoil were used; bothformed cyanogenic glucosides, but only onetype (HCN+) released cyanide. The other type(HCN-) was unable to form cyanide because itdid not contain a (3-glucosidase. S. loti waspathogenic to both HCN+ and HCN- plants,

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causing cyanide release from shoots of theplants and excised leaflets: the f3-glucosidasemust therefore have originated from the fun-gus. Release of cyanide was accompanied bydepletion of the plant cyanogens, showing thatthese, and not cyanogenic compounds excretedby S. loti, were the source of cyanide.Several other fungi, not pathogenic to trefoil,

were shown to cause cyanide release from tre-foil substrate when they had been grown invitro on autoclaved trefoil shoots (107). S. loti ispathogenic because it is able to adapt to growthin the presence of cyanide, forming a cyanide-tolerant respiratory system (51, 52) and synthe-sizing cyanide hydratase (53) for detoxicationof cyanide to formamide.

Cyanide Production in CultureIn the early papers (cf. Bach, reference 9) on

fungal cyanogenesis, there was considerableconfusion as to when cyanide formation occursduring the growth cycle. There was generalagreement that fruiting bodies form cyanidebut conflicting evidence for its formation bymycelia. Bach (9) showed that cyanide was pro-duced in young, fresh fruiting bodies of Pholi-ota aurea only when they had been damaged.Older fruiting bodies and old mycelia alsoformed cyanide. Cyanide production was re-lated to availability of oxygen.Lebeau and Dickson (87, 88) were the first to

study fungal cyanide production in liquid cul-ture. They showed that the snow mold basidi-omycete grew as mycelia with clamp connec-tions in stationary liquid culture in a basalsalts medium, in soybean meal soil, or groundalfalfa crown tissues. For both growth and cya-nide production the optimum temperature was120C. Cyanide production was found to occurduring active growth on all media, but espe-cially on the complex media.The isolation of three types of the snow mold

fungus (186) has already been mentioned. Onlytype B produce cyanide in culture, whereastype A are more virulent in alfalfa (M. sativa)and cause more cyanide production in the hostplant (Table 1). It is therefore possible that typeA and B isolates differ in their abilities to formthe enzymes for cyanide production and to pro-duce the extracellular 3-glucosidase thatcauses release of cyanide from the host plantcyanogens.

In shake flasks a B-type isolate of the snowmold fimgus, growing on complex and syntheticmedia, produced cyanide, but only during theautolytic phase, several days after growth hadreached a maximum (185). This result wouldseem to contradict the observations on cyanideproduction during active growth of mycelial

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surface colonies (87, 88). However, in mycelia,the growing outer edge is followed by an innerarea of autolyzing organisms; the latter frac-tion of the colony could be responsible for cya-nide production.

Inclusion of glycine in the growth mediumresulted in a stimulation of cyanide productionby a B-type isolate of the fungus growing inshake flasks on a glucose-basal salts medium(188). Unlike the response to it by bacteria (27,104, 122, 195), glycine here appeared to stimu-late cyanide production during growth ratherthan in the stationary phase. However, thiscould have been due to cyanogen formation (seenext section) in the logarithmic phase (184),since assay of cyanide was by steam distillationof the culture: the cyanide observed was thetotal of free cyanide plus heat-labile cyanogen.Cyanide is produced directly from glycine,

with the cyanide carbon derived from the gly-cine methylene group; addition of [1-14C]- or [2-14C]glycine to growing cultures of the snowmold fungus resulted in formation of H14CNonly when the label was in the [2-'4C]-positionof the added glycine (188). A lower level ofcyanide was produced from serine, presumablydue to the conversion of serine to glycine viaserine hydroxymethyl transferase (EC 2.1.2.1)or other enzymes.

Despite earlier reports to the contrary (110,but see reference 9) mycelia of M. oreades, aswell as fruiting bodies, produce cyanide (89).Cyanide is produced when the organism isgrowing under natural conditions as fairy rings(46, 47), as well as in shake flask cultures dur-ing the autolytic phase of the mycelial growthcycle (89).

Formation of CyanogensWard (184) has shown that a B-type isolate of

the snow mold basidiomycete produces an un-stable, probably not glucosidic, cyanogen inshake cultures. There was no free cyanide inthe cultures until the start of the stationaryphase, but an increasing rate of synthesis ofthecyanogen occurred during active growth; at alltimes the concentration of the cyanogen wasmuch greater than that of the free cyanide.Unfortunately, the reasons for release of cya-nide from the cyanogen during autolysis werenot studied. Perhaps induction of an oxynitri-lase, causing breakdown of the cyanogen, oc-curred during this phase. Alternatively, on au-tolysis, the enzyme may become accessible tothe cyanogen, or an increase in pH may occur,destabilizing the cyanogen.

Free cyanide was released slowly from thecyanogen by a non-enzymatic process, not af-fected by a 03-glucosidase formed by the fungus


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or by a commercial emulsin preparation. Thepartially purified cyanogen was unstable attemperatures above 400C and at pH valueshigher than 6.0 (184).Tapper and MacDonald (174) have shown

that the major cyanogen present in extracts ofthe snow mold fungus is glyoxylic acid cyano-hydrin. Lower concentrations of pyruvic acid cy-anohydrin are also formed. Glyoxylic acid cy-anohydrin has pH and temperature character-istics similar to those of the cyanogen isolatedby Ward (184) and is presumably the samecompound.

Stevens and Strobel (168) measured the effec-tiveness of various amino acids as substratesfor cyanogenesis by the snow mold and con-cluded that valine and isoleucine were the bestprecursors of hydrogen cyanide. Valine and iso-leucine are the precursors of linamarin andlotaustralin, respectively, in higher plants (23,24); Stevens and Strobel (168) claim to havedetected small quantities of these glycosidiccyanogens in the snow mold fungus. However,their suggestion that valine and isoleucine actas the preferred precursors of the cyanogen(s)in the fungus has been severely criticized byWard et al. (189), who also confirmed that gly-cine is the best precursor of cyanide. Further-more, Tapper and MacDonald (174) have beenunable to detect linamarin or lotaustralin inthe fungus.

Stevens and Strobel (168) also showed thatthe snow mold formed an oxynitrilase and two,6-glucosidases of differing substrate specifici-ties. They contend that formation of these en-

zymes supports their hypothesis that linamarinand lotaustralin are the precursors of cyanide.However, the f-glucosidases are probably re-sponsible for the extracellular glucosidic activ-ity noted by others (34, 184) to cause cyaniderelease from host plant cyanogenic glucosides.The oxynitrilase, since it is of broad specificityto aliphatic nitrites, could be used for release ofcyanide from the fungal glyoxylic acid cyanohy-drin (174) or breakdown ofthe aglycones ofhostplant cyanogens (34).

Plantlike cyanogenic glucosides have notbeen detected in cultures ofM. oreades (59, 110,183), but this fungus forms a cyanogen (187) ofseemingly similar properties to that ofthe snowmold basidiomycete (184, 189). This cyanogen istherefore probably the cyanohydrin of glyoxylicacid (174).CYANIDE PRODUCTION BY BACTERIA

Species Producing CyanideAlthough cyanide production is widespread

in fungi and cyanide is probably a normal fim-gal metabolite, certain pseudomonads and

Chromobacterium violaceum are the only bac-terial species known to produce cyanide (Table2). Conflicting reports on the types of pseudo-monads producing cyanide could be the resultof either the conditions of culture tested, sincesignificant cyanide production occurs only un-der specific growth conditions (see next section),or the assay procedures. In the earlier studiesinsensitive techniques were used (smell andpicric acid-Na2CO3 indicator papers), whereasin recent studies more sensitive methods havebeen used (calorimetric tests and gas chroma-tography-mass spectroscopy), enabling detec-tion of much lower levels of cyanide.

Conditions of Cyanide ProductionLorck was the first to show clearly that opti-

mal cyanide production by bacteria is depend-ent on the inclusion of glycine in the growthmedium (93). Growth ofPseudomonas aerugi-nosa on nutrient broth resulted in the forma-tion ofa high level ofcyanide, but in a glycerol-synthetic medium cyanide evolution only ap-proached that found for growth on nutrientbroth if glycine was included as a nitrogensource. Others have since confirmed the de-pendence of cyanide production by bacteria on

TABLE 2. Cyanide-producing bacteria

Organism Comments Reference

C. violaceum Mesophilic C. viola- 162-164ceum species werecyanide producing;C. lividum speciesdid not form cya-nide

C. violaceum 4 out of 9 strains 104tested were cyanideproducing; C. livi-dum was found notto form cyanide

C. violaceum 122P. chlorophis 2 strains formed cya-

nideP. aureofaciens 2 strains formed cya- 104

nidePseudomonas 22, 195, 196

sp.P. aeruginosa 74 out of 110 strains 27

formed cyanideP. fluorescens 1 out of 5 strains

formed cyanideP. polycolor (= 2 out of 4 strains

P. aerugi- formed cyanidenosa)

P. fluorescens 50B. pyocyaneus 9 strains formed cya- 126

(= P. aerugi- nidenosa)

B. pyocyaneus 33

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the inclusion of glycine in the growth medium(19, 20, 27, 104, 197).Michaels and Corpe showed (104) that with

C. violaceum, which grew well on peptone or ona minimal salts medium containing -gluta-mate, DL-alanine, L-histidine, or glucose plusammonia cyanide production was much greateron peptone than on the other substrates. How-ever, addition of guanine, alanine, glycine, ormethionine, but not any of45 other amino acidsor nitrogenous compounds, to cells grown to thestationary phase on glutamate caused a stimu-lation of cyanide production without furthergrowth. A mixture of glycine and methioninecaused a much greater stimulation of cyanideproduction, to a level similar to that found forgrowth on peptone. Glycine could be replacedby several related compounds, and glycinemethyl ester gave an even greater cyanideyield.

Cyanide was also formed when glycine andmethionine were added to harvested, washedcells suspended in basal salts, which had beenfirst grown to the stationary phase on gluta-mate-basal salts medium; addition of glycineand methionine to the growth medium 2 h be-fore harvesting substantially increased the cya-nide yield (104). Addition of succinate (or mal-ate or fumarate) to the washed cell suspensionin basal salts containing glycine and methio-nine doubled the yield of cyanide without fur-ther growth. The stimulatory effect ofsuccinatewas inhibited by both azide and dinitrophenol,suggesting that it acts as a respiratory energysource, although it could also be a precursor forextra glycine or another substrate.

Glycine is thus the direct substrate for cya-nide production. However, the role of methio-nine is less clear. It can be replaced by betaine,dimethyl glycine, and choline and could there-fore act as a methyl donor (104).Both cyanide yield and rate of synthesis of

cyanide in bacterial growth media are maximalat the start ofthe stationary phase (27, 104, 122,195). Castric noted three phases of cyanide for-mation during aerobic growth ofP. aeruginosa:no cyanide was formed during lag and logarith-mic growth phases, active cyanogenesis oc-curred during the transition from logarithmicto stationary phases, and limited cyanogenesisoccurred in the stationary phase (27). The dataof Michaels and Corpe (104) and Niven et al.(122) show that there is a similar situation in C.violaceum. All these workers used growth con-ditions of relatively low efficiency of oxygentransfer, and therefore the phase of decelerat-ing growth could be due to oxygen limitation,indicating that cyanide formation is maximalunder conditions of low aeration or anaerobio-

sis. However, growth of P. aeruginosa underconditions of very low aeration (stationary cul-ture) led to a drastic decrease in cyanide pro-duction (27). Anaerobic growth in the presenceofnitrate, which permits anaerobic respiration,also caused a decrease in cyanide production,suggesting a requirement for molecular oxygenrather than a respiratory-driven removal of re-ducing equivalents or a supply of energy. Wiss-ing, using a Pseudomonas species, elegantlydemonstrated (196) that cyanide production incells suspended in phosphate buffer plus gly-cine was low under anaerobic conditions andnot only oxygen, but also artificial electron ac-ceptors (phenazine methosulfate, methyleneblue, 2,6-dichlorophenol indophenol, and ferri-cyanide), could greatly stimulate cyanide for-mation. Some inhibitors of flavin-linked en-zymes, although not amytal or rotenone,caused partial inhibition of oxygen-driven cya-nide formation. Other respiratory inhibitors,uncoupling agents, or ionophores were nottested, and it is not possible to tell whether theflavin-associated step is respiratory linked, per-oxidative, an oxygenase, etc.Cyanide production does not itself inhibit

growth of the bacteria themselves or cause lossof viability, because addition of cyanide in thelag or logarithmic phases ofcultures ofC. viola-ceum has no effect on the final growth yield(104).Cyanide production in growing P. aerugi-

nosa has a narrower temperature range thangrowth; it also requires the presence ofiron andis inhibited by high phosphate concentrations(27). Addition of chloramphenicol to late-loga-rithmic-phase P. aeruginosa inhibits cyanideformation, showing a requirement for proteinsynthesis.There have been no reports of the formation

of any cyanogenic compounds by bacteria.Metabolic Precursors of Cyanide

Michaels et al. grew C. violaceum on a gluta-mate-basal salts medium and incorporated me-thionine and glycine into the medium 2 h beforeharvesting, to adapt the cells for cyanide pro-duction (105). Cells were harvested, washed,and resuspended in basal salts containing suc-cinate, glycine, and methionine. In differentexperiments, [1-'4C]- or [2-14C]glycine or [1 -methyl -"4C]methionine were labeled. Cyanideand CO2 formation in the non-proliferatingcells were followed for 6 h, and the distributionsofthe radioactivity was measured. About 7 and2% of the [2-14C]- and [1-14C]glycine, respec-tively, were taken up by the cells, mainly intothe protein fraction. About 10% of the [1-14Ciglycine (the carboxyl carbon) was converted

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to C02, and 11% of the [2-'4C]glycine (the meth-ylene carbon) was converted to cyanide. Thespecific activity of cyanide formed from [2-'4C]-glycine was 94% of the specific activity of thesubstrate, whereas when [1-14C]glycine or [1-methyl-'4ClCmethionine were the substrates thespecific activity ofcyanide was less than 0.1% ofthe substrate's specific activity.The carbon ofthe cyanide formed must there-

fore have originated from the methylene groupof the glycine, and the CO2 presumablystemmed from the carboxyl group:

NH2*CH2 *COOH - HCN + C02+ 4 [H]Brysk et ad (20) grew C. violaceum on gluta-

mate-basal salts and then supplemented themedium with methionine plus [2 -

'4C15N]glycine and continued growth for 18 h.Ofthe glycine consumed, 25% was incorporatedinto free cyanide and 16% into /-cyanoalanine.,3-Cyanoalanine is formed from cyanide andserine and is the first step of cyanide utilizationby non-proliferating cells of C. violaceum (cf.reference 19 and page 661). However, radioac-tivity in (3-cyanoalanine could have arisen dur-ing metabolism of glycine to serine or by otherpathways, rather than via cyanide formation.Analysis of the carbon distribution of radioac-tivity in (8-cyanoalanine showed that 44% oftheactivity was incorporated into the cyano carbon(i.e., 7% of the total glycine consumed). There-fore, at least 25 + 7 = 32% of the glycine uti-lized was converted to cyanide. This figure is anunderestimate because it does not take intoaccount the further metabolism of 3-cyanoalan-ine. Thus, synthesis ofcyanide is an important,possibly major, pathway of glycine metabolismunder these conditions. The '5N/14C ratios oftheadministered glycine, unconsumed glycine, andHCN and (3-cyanoalanine formed were essen-tially the same, indicating that the methylenecarbon of glycine was converted to cyanidewithout cleavage of the carbon-nitrogen bond.Reversible transamination and oxidative deam-ination of glycine therefore did not occur.Wissing (196) has measured the molar ratio

CH2-NH2C==o

OH

0

IIC-NH2

(02)

IOOH

of cyanide produced to total glycine consumedby a Pseudomonas species suspended in phos-phate buffer (in the absence of methionine orother methyl donors and without succinate orother energy sources). A ratio of 1.0:1.1 wasfound. Therefore, no further metabolism of


either cyanide or glycine (e.g., to ,B-cyanoalan-ine) occurred, and future studies of the meta-bolic pathway of cyanogenesis from glycineshould be facilitated by use of these conditions.Cyanide production by the Pseudomonas spe-cies was low unless oxygen or an artificial elec-tron acceptor was present, and the process wasinhibited by several flavin inhibitors, indicat-ing that it is probably linked to a flavoproteinoxidase, oxygenase, or respiratory dehydrogen-ase (196, 197).

Recently (196), glycine-driven cyanide pro-duction has been obtained in a cell-free extractof the Pseudomonas species (derived by sonica-tion), which will enable studies of the enzymol-ogy of the system. Optimal cyanide-producingactivity was obtained when the cell-free extractwas prepared and activity was assayed in thepresence of phenazine methosulfate and flavinadenine dinucleotide, which presumably act asan oxidizing agent or electron carrier and coen-zyme, respectively. Sucrose density gradientfractionation of cell-free extracts shows thatactivity is associated with particulate ratherthan soluble material. Interestingly, this orga-nism contains internal membrane structuresreminiscent of those found in type II methylo-trophic bacteria (133), although C. violaceumhas internal membranes of the mesosomal type(143).

SPECULATIONS ON THE METABOLICPATHWAY OF CYANIDE PRODUCTION

BY MICROORGANISMSGlycine is the metabolic precursor ofcyanide,

with the carbon derived from the methylenecarbon of glycine, in both bacteria and fungi(20, 105, 188). It seems reasonable, therefore, tosuggest that the mechanism of cyanide produc-tion is similar in all the fungi and bacteria sofar studied.On the basis of their studies with C. viola-

ceum, Michaels et al. (105) proposed the follow-ing scheme for cyanide formation, althoughwithout evidence for formation of the interme-diates or assay of enzyme activities:

C N

CH20 O - HCN + CO2OH

Conn has suggested (36) that cyanide may beformed by microorganisms via a pathway anal-ogous to that for formation of plant cyanogenicglucosides (Fig. 1). A relatively simple reactionsequence from glycine would be involved, withformaldoxime as an intermediate:


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7COOHOH!

\NH,-- H2C=NOH -- HC=N

Wissing (196) has more recently proposed apathway of bacterial cyanide production fromglycine, which could also be applicable to fun-gal cyanogenesis. Two flavoprotein steps are

Cyanide is a highly reactive group and easilyreacts (non-enzymatically) with keto groups toform cyanohydrins (136). The cyanohydrins ofglyoxylic acid and pyruvic acid found in thesnow mold fungus (174) could therefore arisefrom direct chemical interaction of cyanidewith the acids. Glyoxylic acid is obtained fromglycine via glycine oxidase, and the cyanohy-drin would then be formed by:

(a)0+CHO COOH + NH,H2N-CH2--COOH -* (HN=CH-COOH) HO-CH-COOH

-HC + C02 C=N

involved:

H2N-CH2-COOH oxidation

flavoprotein

HN= CH- COOH oxid-ion , HCN + CO0flavoproteinThis is a simplification and, if this scheme is

operative, it is probable that there are two oxi-dative steps:H2N-CH2-COOH HN=CH-COOH

2(H) 2(H)

N-C-COOH (iii) HCN + CO2There are at least two enzymes known for

glycine oxidation: (a) an amino acid oxidase(glycine:oxygen oxidoreductase [deaminat-ing]), which is flavin linked and peroxidative(121, 137); and (b) glycine dehydrogenase (gly-cine:nicotinamide adenine dinucleotide[NAD+]oxidoreductase [deaminating], EC1.4.1.10). Since cyanogenic oxidation of glycineis flavin linked (196), it is possible that step (i)is peroxidative and flavin linked and that theenzyme is related to glycine oxidase. On theother hand, there are obvious energetic advan-tages to the organism ifthe enzymes catalyzingsteps (i) and (ii) are classical dehydrogenasesassociated with the respiratory system. Itshould also be noted that the unstable imine(HN = CH.COOH), proposed as an intermedi-ate of cyanide formation, is almost certainly anintermediate in glycine oxidase (62).Release of cyanide from the aglycones of

plant cyanogenic glucosides occurs by the ac-tion of an oxynitrilase. Plant oxynitrilaseseither have flavin adenine dinucleotide as aprosthetic group (14, 150) or are devoid offlavin(150, 17). The requirement for flavin in thescheme given above could therefore be for oxi-dation of the unstable nitrile via step (iii)rather than for the oxidative steps. It will beinteresting to see whether the oxynitrilasepresent in the snow mold fungus (168) requiresflavin for activity. The nitrile (N C-COOH)is likely to be highly unstable and step (iii)could also be non-enzymatic.

In the presence of significant intracellular lev-els of glyoxylic acid (or other keto compoundssuch as pyruvic acid or a-oxoglutaric acid),there is not likely to be much free cyanidepresent. Oxynitrilase would then act to releasehydrogen cyanide from the cyanohydrins andmight be expected to have a broad specificity.

I would like to emphasize the speculativenature of this hypothetical pathway: it is pro-posed in the hope that it will stimulate researchin this area. In particular, the properties andsubstrate specificity of the fungal oxynitrilaseneed to be further examined, and a searchneeds to be made for glyoxylic acid cyanohydrinand other cyanogens in bacteria, as well asevidence for or against the formation of theintermediates of the proposed pathway.CYANIDE ASSIMILATION BY CYANO-

GENIC MICROORGANISMSAs will be discussed below, metabolic path-

ways of cyanide assimilation have so far beenstudied mainly in species that are also able toproduce cyanide, but, as far as is known, areunable to grow on it as a carbon and/or nitrogensource. In these cases cyanide may be a normalintracellular metabolite, possibly present asglyoxylic acid cyanohydrin or other cyanogens.Therefore, formation of high concentrations ofcyanide could be due to excretion of an abnor-mal excess of it, or as the end product for re-moval of some other metabolite such as glycinethat has been overproduced. It is noteworthythat production ofappreciable concentrations ofcyanide only occurs on certain media, under spe-cific growth conditions, and in the stationaryphase of growth (27, 104, 185).

Alanine FormationStrobel first showed that the cyanide-produc-

ing snow mold basidiomycete could assimilatecyanide (169). He incubated mycelia withH'4CN for 20 h and then harvested, disrupted,and fractionated the cells. Alanine and, to alesser extent, glutamate were the only labeledamino acids. In shorter-term experiments, ala-nine was the only labeled amino acid. Control

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studies with '4CO2 showed that cyanide was notconverted to C02 before assimilation.Cyanide assimilation into several other fungi

has been tested (4). M. oreades, Pholiota adi-posa, Pholiota aurivella, Pholiota praecox, andRhizopus nigricans incorporated H14CN intoalanine and, to a lesser extent, into other aminoacids, whereas Fusarium nivale incorporatedcyanide into asparagine only. No radiolabeledamino acids were detected in Clitocybe illu-dens. Rhizoctonia solani, Aspergillus flavus,and Fusarium solani.

Strobel has shown that mycelia of the snowmold basidiomycete incubated with H14CNformed labeled a-aminopropionitrile and L-ala-nine (170). The label appeared in the a-amino-propionitrile during the first 12 h of incubationand then decreased, whereas the quantity ofradioactive alanine increased steadily for 24 h.When H'4CI5N was used as the substrate, car-bon labeling appeared exclusively in the C1position of both products and nitrogen labelingappeared in the cyano group of a-aminopro-pionitrile. These results are consistent with thefollowing pathway:

0 NH,IIHC OH,2C-0CH,-O-H 2H,.CH3--CN )I

NH,~~~N~H NH,

acetaldehyde a-aminopropionitrile

NH2

CH,-C-COOHH

L-alanineIn support of this, incubation ofa crude enzymeextract from the fungus with KCN,(NH4)2HPO4, and acetaldehyde resulted in for-mation of a-aminopropionitrile. The conversionpresumably occurs via the intermediary forma-tion ofa cyanohydrin, with subsequent replace-ment of the hydroxyl group by an amino group;one or both steps could be enzymatically driven.Intracellularly, acetaldehyde could arise fromoxidation of ethanol by alcohol dehydrogenase(EC 1.1.1.1., alcohol:NAD+ oxidoreductase) orother sources. The breakdown of the nitrile toalanine could occur either via the formation ofa-aminopropionamide (cf. reference 53) or inone stage by a nitrilase (EC 3.5.5.1, nitrileamiinohydrolase; 141, 176).

Glutamic Acid FormationThe earlier demonstration that labeled gluta-

mate is formed when the snow mold fungus is


fed H14CN (169) has been confirmed (172). In apathway analogous to that resulting in alanineformation, glutamate is derived from cyanideand succinic semialdehyde via 4-amino-4-cy-anobutyric acid:

* o

HC=O CN

uth *0

OH-NH2CIHeHCN,1CHO1H2 NH, UCH2

COOH OH2OOOH

OOOH

2H,2O OH-NH2

0NH,

COOH

In extracts, aminocyanobutyrate nitrilase ac-tivity was observed, but there was little amino-cyanobutyrate synthase activity (172). Gluta-mate decarboxylase, succinate semialdehydedehydrogenase, and 3-aminobutyrate-gluta-mate transaminase activities were also pres-ent. A cyclic conversion of cyanide to CO2 wasproposed (Fig. 3). Ifthis is the case, there wouldbe no assimilation of cyanide-carbon by theorganism. The pathway would serve to detoxifythe cyanide, possibly with assimilation of thecyanide-nitrogen.

a-Aminobutyric Acid FormationThe fungus Rhizoctonia solani, which has

been reported not to produce cyanide (4), has apathway of cyanide incorporation into propi-nonaldehyde to give a-aminobutyronitrile anda-aminobutyric acid (118):

CHOHCNCyH2 NH,

CH,

ON

H2N-Hfl

-NH2,H3CH,

COOH

HN-CH

CH2

CH,

The enzymology of this pathway is presumablysimilar to the acetaldehyde and succinate semi-aldehyde pathways mentioned above (170, 172).In cultures fed ammonia, cyanide, and propion-aldehyde, the formation of the nitrile precededformation of a-aminobutyrate. a-Aminobuty-ronitrilase activity was detected (118).

(3-Cyanoalanine FormationMany plants are able to assimilate cyanide

into asparagine (15) or the dipeptide y-gluta-myl-,8-cyanoalanine (49). The latter compoundis a normal constituent ofVicia species but onlyoccurs in other plants when they are fed cya-nide (139). In plants fed H14CN, the label entersthe amide carbon ofasparagine (15). Formationofboth the dipeptide and asparagine occurs via/3-cyanoalanine:


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,asparagineHCN -. B-cyanoalanine

Initially, it was thought that f3-cyanoalaninewas synthesized by plants from serine and cya-nide (15, 48). However, in extracts of Lotustennis (birdsfoot trefoil), the rate of formationof (3-cyanoalanine is 50-fold greater when cya-nide and cysteine are the substrates than whenHCN and serine are supplied (48). (3-Cyanoal-anine synthase (EC 4.4.1.9, i-cysteine-hydrogensulfide-lyase [adding HCN]) has been purifiedfrom mitochondria of Lipinus angustifolia(blue lupine). It catalyzes (8-cyanoalanine for-mation from cysteine and HCN (2, 16, 69):H2N-CH-COOH

+ HCN-.CH2SH

H2N-CH-COOH+ H2S

CH2CNSerine is not a substrate, but the enzyme is alsoable to utilize O-acetyl-L-serine (about 20 timesless effectively than cysteine):H2N-CH-COOH

+ HCNCH20-CO.CH3

H2N-CH-COOH+ CH3COOH

CH2CNBlue lupine also contains (2, 69) a soluble 0-acetyl-L-serine sulfhydrase (EC 4.2.99.8, O-ace-tyl-L-serine acetate lyase [adding H2S]). Thisenzyme catalyzes formation of cysteine fromsulfide and O-acetyl-L-serine but is also able touse O-acetyl-I-serine and cyanide, at a low rate,to form 3-cyanoalanine.When Chlorella pyrenoidosa is fed cyanide,

it forms ,3-cyanoalanine and y-glutamyl-,1-cy-anoalanine (49), and Escherichia coli is able toconvert cyanide to B3-cyanoalanine. Dunnilland Fowden (44) suggested that the E. coli 38-cyanoalanine synthase activity represents afunction of an indispensible but nonspecific en-zyme, possibly serine sulfhydrase (EC 4.2.1.22,L-serine hydro-lyase). The primary function is:

serine + H2S -- cysteine

The other possible reactions are:

serine + HCN -- (3-cyanoalaninecysteine + HCN -> /8-cyanoalanine + H2S

Catalysis of these reactions by E. coli extractsis stimulated fourfold by 10 mM adenosine 5'-triphosphate.

y-glutamyl - (8-cyanoalanineBrysk et al. have shown that cyanide pro-

duced by C. violaceum (strain 9) can be assimi-lated into 3-cyanoalanine and then into theamide carbon of asparagine (19):

H2N-CH-COOH HCNCH2OHserine

H2N-CH-COOH H20 H2N-CH-COOHCH2-CN CH2C0NH2

,3-cyanoalanine asparagine

When washed suspensions of C. violaceum,grown on glutamate-minimal salts medium,were incubated with glycine, methionine, andsuccinate, A-cyanoalanine accumulated in themedium. Maximal concentrations of 83-cyanoal-anine occurred with 5 h of incubation; longerperiods of incubation resulted in its disappear-ance.

Incubation of washed cells with serine,K14CN, and succinate resulted in incorporationof radioactivity into cells and formation of (8-cyanoalanine. Replacement of serine by gly-cine, and glycine plus NN-dimethyl glycine ormethionine, gave less, but still significant, for-mation of 83-cyanoalanine and incorporation ofradioactivity into the cells. Radioactive incor-poration from K14CN was into the C4 position of,8-cyanoalanine and asparagine. When [2-1"O]glycine was used (plus KCN and succinate),radioactivity was found mainly in the C2, C3,and C4 atoms of (8-cyanoalanine. With labeledsuccinate as substrate, there was extensive in-corporation of radioactivity into the cells butnot into (8-cyanoalanine: succinate was proba-bly acting as an energy donor for adenosine 5'-triphosphate formation. However, with[14C]formaldehyde, but not [14C]formate, assubstrate (plus KCN and succinate), the ,3-cy-anoalanine formed was radioactive.

Since the label from radioactive glycine wasincorporated into (3-cyanoalanine, it was pro-posed that glycine can act not only as a precur-sor of cyanide, but also to supply serine (19).Figure 4 shows a possible pathway of glycineand cyanide metabolism by C. violaceum strain9. In this scheme, utilization of three moleculesof glycine is required for formation of f8-cy-ano'hlanine. One molecule is converted to cya-nide and C02, one to a C-I unit (and presum-ably ammonia and carbon dioxide), and one

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COOH

OH2OH.2COOH

succinic acid

NADtW >Jsuccinic semialdehyde

Ndehydrogenase

NADH + ,

COOH H-C

CH-NH2 C

(

CH2

OOH succiniculsemialdehydeglutamic acid sie

transaminaseCOOH

0=0

OCH2 NH2-4

OH2

1-oxoglutaric acid

r ,Q:NH2-CH

CH2

COOH4-amino-4-cyanobutyric

acid

COOH

CH2 glutamic decarboxylase NH2-H

CIHi I HCH2 /uCH2

OHICOOk?j2 OOH2

3-aminobutyric acid glutamic acidFIG. 3. Possible pathway for detoxication ofcyanide by the snow mold basidiomycete, involving formation

of 4-amino-4-cyanobutyric acid. Redrawn from Strobel (170) by courtesy of the Jorunal of BiologicalChemistry.

links up with the C-I unit to form serine, whichthen combines with cyanide to form (8-cyanoal-anine. An alternative possibility, not discussedby the authors (19) but consistent with theirdata, is that two molecules of glycine are con-verted to cyanide. One of the cyanide moleculescould be converted to formamide and then for-mate (53), which acts as a supply of C-I units foraddition to the third molecule of glycine, con-verting it to serine.In view ofthe possibility that /3-cyanalanine

is synthesized in E. coli by a nonspecific fimc-tion of serine sulfhydrase (44) and that inplants cysteine. is the donor for its formation(69), it is a pity that the properties of the (3-cyanoalanine synthase enzyme ofC. violaceumhave not been studied. Does addition ofcysteineplus KCN to washed cells of C. violaceum alsocause stimulation of 3-cyanoalanine formation?

Castric and Strobel isolated a cyanide-resist-ant and -metabolizing strain ofBacillus mega-terium from Fargo clay that had been planted

with the cyanogenic plant, flax, for many years(28). The organism was grown on Trypticasesoy broth containing 1 mM cyanide: growthcoincided with cyanide disappearance from themedium. When K14CN was added to the me-dium, the label was incorporated into C02, theamide carbon of asparagine, and the C4 car-boxyl of aspartate. Washed cells fed serine con-verted it to ,B-cyanoalanine and asparagine,and cells fed (3-cyanoalanine converted it toasparagine, but (3-cyanoalanine was not de-tected during conversion of serine to asparagineby cell-free extracts: possibly nitrilase activityfor conversion to 83-cyanoalanine to asparagineis more active than 3-cyanoalanine synthaseactivity under the conditions used in the latterexperiment. Cysteine did not act as a substratefor (8-cyanoalanine or asparagine synthesis inextracts or in washed cell suspensions. It wasproposed that (3-cyanoalanine was synthesizedfrom serine and cyanide, which was convertedto asparagine and then aspartate.

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H2N-CH2-COOH -

NHe glycine glycine, oxidase

CHO.COOHglyoxylatecoilC1 unit

(methylene-tetrahydro-folate?)

t

(formaldehyde)

serine hydroxymethyl-transferase

H2N-OH-COOHI f31CH20H

--4[H]

-CO2

HCN

*cyanoalanine synthase(stimulated by ATP [?],

Serine H20 ret 73)-11

--- H2N-H-COOHnitrile hydratase IH-CN

H2N-CH-COOH ~ ~ ~ C2-CH2N H-I H j8-cyanoalanine

CH2-CONH2asparagine

nitrilase (

inase, ref

asparaginas H2N-C OOH\pIXH3 UH2 COOH

aspartate

(or asparag-L. 82).

FIG. 4. Incorporation ofcyanide and glycine into ,&cyanoalanine and asparagine by C. violaceum strain 9.Adapted from the data of Brysk et al. (19) by courtesy of the Journal of Bacteriology. For details of C1conversions involving tetrahydrofolate, see reference 39. ATP, Adenosine 5'-triphosphate.

Castric and Conn have more recently shownformation of (8-cyanoalanine in extracts of thecyanide-metabolizing strain of B. megaterium(29). Whereas serine (plus cyanide) could act asa substrate for /3-cyanoalanine synthesis, cys-teine and O-acetyl-serine were 17 to 18 timesmore effective as substrates. This suggests thatone of the enzymes of cysteine biosynthesis isable to catalyze (8-cyanoalanine formation. Itwas reasoned that if 8-cyanoalanine synthesisfrom cysteine and cyanide is of physiologicalsignificance, then this activity should be induc-ible by inclusion of cyanide in the medium: thisdid not occur. If, on the other hand, the enzymethat catalyzes B3-cyanoalanine synthesis is anenzyme involved in cysteine biosynthesis, andthe B3-cyanoalanine synthase activity is only asecondary function of it, then the enzymeshould be repressed by inclusion of cysteine inthe growth medium. Activities of 3-cyanoalan-ine (from cyanide and cysteine) and cysteine(from O-acetyl-serine and sulfide) synthaseswere equally repressed by growth with cysteineas the sulfur source rather than sulfate or djen-

kolic acid. Furthermore, O-acetyl-serinesylfhydrase was purified 30-fold and the activ-ity of f8-cyanoalanine synthesis exactly paral-leled the purification (but at a 4 x 10-4-foldlower activity).

This data (29) and the earlier report (28) thatserine stimulates (-cyanoalanine and aspara-gine synthesis inB. megaterium raise the ques-tion as to how the conversion from serine occursin this organism (and possibly also in C. viola-ceum; see reference 19 and above). Serine can(i) act as a direct substrate for 3-cyanoalanineformation by a serine-linked P3-cyanoalaninesynthase, (ii) be converted to cysteine by serinesulfhydrase, which is then converted to (8-cy-anoalanine by a cysteine-linked /3-cyanoalan-ine synthase or by a secondary function of 0-acetyl-serine sulfhydrase, or (iii) be convertedto O-acetyl-serine by serine acetyltransferase(EC 2.3.1.30., acetyl-coenzyme A:L-serine 0-acetyl-transferase) and then to 83-cyanoalanineby O-acetyl-serine sulfhydrase. In B. megate-rium, pathway (ii) probably occurs, and if ser-ine transferase is present, possibly by (iii),

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whereas the serine-linked (8-cyanoalanine syn-thase (i) does not appear to be formed.

y-Cyano-a-Aminobutyric Acid FormationBrysk and Ressler have reported that strain

D341 of C. violaceum is able to form 'y-cyano-a-i-aminobutyric acid (21). This compound wasnot detected during growth on a glutamate-minimal salts medium, when cyanide and (3-cyanoalanine were formed, but when washed,early logarithmic-phase cells were incubatedwith cyanide plus glutamate, serine, or threo-nine there was accumulation ofboth /3-cyanoal-anine and y-cyano-a-aminobutyric acid. Whencyanide and aspartate or asparagine were used,only y-cyano-a-aminobutyric acid accumu-lated. Aspartate could have been acting as anend product repressor of the (8-cyanoalaninepathway, hence preventing (3-cyanoalaninesynthesis (21). Aldehydes can act as metabolicprecursors of nitrile formation (118, 170, 172,and above), and it is possible that aspartatesemialdehyde acts as the precursor of y-cyano-a-amino-butyric acid in C. violaceum (21).However, incubation ofthe organism with cya-nide and aspartate semialdehyde did not resultin its formation, although this could have beendue to permeability problems for entry of thesemialdehyde into the cells. Labeling experi-ments showed that the cyano group of 'y-cyano-a-butyric acid stemmed from cyanide and the C1to C4 atoms from aspartate. Thus, either aspar-tate acted as a direct precursor of the nitrile ora substance derivable from it participated with-out degradation ofthe carbon chain. There wasno evidence in in vivo experiments for furthermetabolism of y-cyano-a-aminobutyric acid,and in sonic extracts no nitrilase activity wasdetected, suggesting that the nitrile is a detoxi-cation product of cyanide rather than the firstmetabolite ofa pathway ofcyanide assimilation(21).

Ressler and her co-workers have since puri-fied an enzyme from C. violaceum strain D341that catalyzes synthesis of'y-cyano-a-aminobu-tyric acid from the unusual amino acid homo-cystine and from cyanide (138). The first step isnon-enzymatic formation ofy-thiocyano-a-ami-nobutyric acid and homocysteine from cyanideand homocystine:

H2N-CH-COOH H2N-CH-COOH

CHI +HCNUH24 &}C2

H2N-CH-COOH

CH2CH2SCN

H2N-CH-COOH

+ CH2

CHISH


The enzyme y-thiocyano-a-aminobutyric acidthiocyano-lyase (adding CN; EC 4.4.1.) cata-lyzes the formation of y-cyano-a-aminobutyricacid and thiocyanate:HN-CH-COOH

+HCN

CH2SCN

H2N-CH-COOHC1H2

CHtIN

+ HSCN

The enzyme requires pyridoxal phosphate as acofactor. 63-Cyano-a-aminobutyric acid syn-thase activity does not appear to be a secondaryfunction ofthe enzyme. In particular, O-acetyl-homoserine is only a poor substrate.Washed cell suspensions of C. violaceum

therefore probably convert aspartate to homo-cystine before addition of cyanide and forma-tion of y-thiocyano-a-aminobutyric acid and y-cyano-a-aminobutyric acid. Ressler et al. havealso shown that incubation of C. violaceumwith y-thiocyano-a-aminobutyric acid and cya-nide results in a greater formation of y-cyano-a-butyric acid than incubation with aspartateand cyanide (138).

GROWTH OF MICROORGANISMS ONCYANIDE

In view of the large quantities of cyanideeffluents produced by industry and the forma-tion of cyanide by many plants, it is remarka-ble how few studies there have been of thegrowth of microorganisms on cyanide as a car-bon and/or nitrogen source. Furthermore, cya-nide is a one-carbon compound, and the utiliza-tion of other C1 compounds by microorganismshas been extensively studied because of theirscientific (133) and industrial (81) importance.

Pettet and Ware (128) and Ware and Painter(190) isolated a cyanide-utilizing bacterium. Asmall percolating filter was seeded with cya-nide-acclimatized sewage sludge and percolatedwith up to 100 ,ug of KCN per ml in tap waterfor several weeks. Cyanide-utilizing organismscould not be obtained by plating the cyanide-free effluent onto nutrient agar, but small colo-nies were isolated when silica gel plates con-taining cyanide in tap water were inoculatedwith some of the crust lining the coke particlesof the percolating filter. The colonies weremixed cultures of a gram-negative bacillus anda gram-positive filamentous organism. The for-mer was removed by plating in the presence ofdihydrostreptomycin sulfate.The gram-positive filamentous bacterium is


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probably an actinomycete (128). On silica gelplates it forms white colonies with segmentedhyphae. Aerial hyphae that bear conidia are

produced, but the organism is not resistant todrying. Growth occurs at pH 7.5 to 9.0, and theoptimum temperature is "below 30oC." It is astrict autotroph; growth is slow and sparse andinhibited by agar or peptone. The optimal cya-nide concentration for growth is about 1.5 mM,but up to 6 mM cyanide can be utilized. Cya-nide is converted to ammonia.Winter (194) utilized a sample ofthe cyanide-

acclimatized sludge of Pettet and Ware to seedcoke-filled percolating filters, and isolated fromthem two organisms capable ofgrowing on cya-nide as a carbon and nitrogen source. Theseorganisms are facultative autotrophs, able togrow rapidly and profusely on cyanide and avariety of organic nutrients. Both organismsgrow as filamentous, gram-positive rods withsparse aerial hyphae. Coccoid, spore-like cellsform, which germinate to form fresh mycelia.They are catalase positive and have an opti-mum temperature range of 25 to 30°C. They areboth actinomycetes, probably of the genus No-cardia.

Skowronski and Strobel (157) have isolated astrain of Bacillus pumilus from samples ofFargo clay that had been planted with flax. Theorganism was isolated in media containing 2.5M cyanide, and it degrades cyanide to carbondioxide and ammonia when grown in Trypti-case soy broth containing 0.1 M cyanide. Incyanide-containing media, the organism grewin a filamentous form, which did not revert tothe rod form when regrown in cyanide-free me-dia. In a basal salts medium containing cyanideas the sole carbon and nitrogen source, a 10-foldincrease in cell numbers was noted.Sakurai (144) (quoted by Mikami and Misono

[106]) has isolated a cyanide-decomposing bac-terium, and Howe (73) cites the isolation of acyanide-utilizing strain ofArthrobacter by Aas-lestad.There have also been several reports of mi-

croorganisms able to use cyanide as a nitrogensource. Rangaswami and Balasubramanian(134, 135) and Iwanoff and Zwetkoff (77) claimto have isolated strains of Aspergillus nigerthat utilize cyanide as the sole source of nitro-gen, and Furuki and co-workers (54) have stud-ied the conditions of optimum growth of a bac-terium that utilizes cyanide as a nitrogensource. The latter organism grows best at pH10.2 in the presence of 400 to 500 ,ug of cyanideper ml and with glucose, fructose, mannose, or

galactose as carbon sources.Trelawny et al. have isolated a soil bacte-

rium that can utilize cyanoacetate as the sole

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source ofnitrogen and carbon (178). It is unableto utilize cyanide as a carbon and nitrogensource, even though free cyanide is formed dur-ing metabolism of cyanoacetate, but in thepresence of succinate it utilizes cyanide, thiocy-anate, and ferri- or ferrocyanide as nitrogensources.There have been several reports of auto-

trophic bacteria able to grow on thiocyanate asa carbon and nitrogen or a nitrogen source (64,65, 75, 132, 167, 199). Putilina reported that athiocyanate utilizer (claimed to be Pseudomo-nas nonliquefaciens) was also able to oxidizecyanide as a carbon and nitrogen source (132).There have been no reports of the metabolic

pathways of cyanide assimilation by organismsable to utilize it as a carbon and/or nitrogensource. However, cyanide is a relatively simplemolecule, and it is possible to predict some ofthe pathways of incorporation.Formamide hydrolyase (cyanide hydratase),

purified from the fungus S. loti, degrades cya-nide to formamide (53). Many methylotrophicbacteria can grow on formamide as a carbonand nitrogen source (133), and it is thereforepossible that some methylotrophs or closely re-lated organisms are able to utilize cyanide.Cyanide could also be incorporated into mi-

croorganisms by the pathways of assimilationutilized by cyanogenic organisms (page 660).For example, in the case of the f3-cyanoalaninepathway, a variant of the methylotrophic serinepathway (133) can be envisaged:

HCN

C1 unit HCN-- - glycine _ . serine k-

13-cyanoalanine - asparagine -- -Howe has suggested several different path-

ways of cyanide incorporation, including path-ways involving thiocyanate and cyanate as in-termediates (73, 74).UTILIZATION OF CYANIDE BY MICRO-

BIAL ECOSYSTEMSSoil Ecosystems

Despite the large number of reports of plantcyanogenesis, there has been little research onthe effect of cyanide release into the soil on theneighboring microbial ecosystems.Rangaswami and Balasubramanian (134,

135) analyzed the cyanide content ofthe roots ofsix varieties of sorghum: in each case the cya-nide content was high in seedlings but reducedsomewhat with age. As with non-cyanogenicplants, there was a distinct rhizosphere effect;


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i.e., there were more microorganisms in theroot rhizosphere than in the surrounding soil,presumably due to exudation of nutrients bythe plants. No clear relationship was estab-lished between microbial population and cya-nide content of the roots, although there weremore gram-negative and fewer gram-positiveand sporeforming bacteria in the rhizospheresthan in the surrounding soil. No strains of theplant pathogensHelminthosporium andFusar-ium could be detected in the rhizospheres.When a volatile exudate of the roots of

sorghum plants, containing cyanide, waspassed through a suspension of the rhizospheremicroflora of sorghum, the bacterial populationincreased fivefold, the fungal population de-creased fivefold, and the population of the acti-nomycetes was unaffected (135). Germinationand growth of the pathogens Helminthospo-rium turcicum and Fusarium monoliformewere delayed by 1 to 2- days by the sorghumexudate but were not further inhibited, nor wasgrowth of the fungi when they were added tosoils containing growing sorghum plants.

Strobel has shown that cyanide can be me-tabolized by various soil ecosystems (171).Fargo clay that had been planted with flax formany years was especially able to utilize cya-nide. Double-labeled cyanide (H'4C15N) wasmetabolized by Fargo clay to 14CO2, 15NH3, andother products. Measurements of the ratio of14C-15N in the administered HCN to the ratio inthe evolved CO2-NH3 (other than NH3 itself andhydrolizable nitriles) suggested that more cya-nide nitrogen than carbon was retained by thesoil.Allen and Strobel have made the intriguing

proposal that a "cyanide microcycle" operatesin the soil (4). The occurrence of both cyanide-evolving and -utilizing plants, fungi, and bacte-ria means that in the microatmosphere of thesoil molecules of HCN could be directly trans-ferred from plants to microorganisms, and viceversa, as sources of nitrogen and/or carbonwithout prior conversion to CO2 and NH3.

Disposal of Cyanide Wastes bySewage Systems

The electroplating, steel, carbonization, andother important industries produce large quan-tities of cyanide wastes. To prevent damage tonatural ecosystems, the cyanide content ofthese effluents must be reduced to essentiallyzero before discharge into the environment.Even for discharge into a sewer there must be adrastic initial reduction to no more than 1 to 2gg of cyanide per ml (128). Conventional chem-ical methods of disposal of cyanide wastes arewasteful of resources, because other chemicals,


such as chlorine, are required for detoxicationof the cyanide (72, 161). In addition, chemicaldegradation is relatively expensive and oftenrequires further disposal ofthe products (73).

It was long thought, due to the toxicity ofcyanide and its affinity for cytochrome oxidase,that biological disposal of cyanide wastes wouldbe impractical. Furthermore, cyanide wastesusually contain other extremely toxic chemi-cals such as phenol, organic nitriles, cyanate,thiocyanate, and, in electroplating wastes,heavy metals (Cd, Cr, Cu, Fe, Ni, and Zn).Shock treatment of sewage systems does, infact, lead to loss of viability, but it becameapparent about 20 years ago that gradual accli-matization of sewage systems enables them todegrade moderate concentrations of cyanidewith a high degree of efficiency. More recently,several patents (e.g., U.S. patents 3,145,166and 3,165,166) have been taken out for biologi-cal methods of decomposition of cyanide wastesand some commercial sewage plants have beenconstructed. High initial capital costs are in-volved in the construction of suitable sewagesystems, but running costs are much lowerthan for chemical methods of cyanide disposal;biological treatment is therefore particularlysuitable for regular disposal of large quantitiesof wastes of moderate cyanide concentration(up to about 100 jig/ml).Howe has reviewed the literature on cyanide

decomposition by sewage systems (72-74).Some of the information cited is of greater in-terest to pollution control engineers than mi-crobiologists, and only the papers ofmore directmicrobial relevance will be discussed here; thereader should refer to Howe for other informa-tion and references (see also 106, 152-154).

Pettet and Mills investigated the effects offeeding settled domestic sewage containingKCN and the cyanide complexes of Zn, Cd, Cu,Ni, and Fe to small percolating filters filledwith metallurgical coke (127). Zn, Cd, and Niwere present as K2M(CN)4 (M = metal), Cuwas present as K3Cu(CN)4, and Fe was presentas K4Fe(CN)6: stabilities of the complexes in-crease in the order Cd, Zn, Cu, Ni, Fe.The filters were gradually acclimatized to

increasing concentrations of the metal cya-nides. Exposure to 1 to 4 ,ug of the cyanides perml caused initial disturbances in biological oxy-gen demand and nitrification, but recovery oc-curred within a few days and up to 150 ,ug ofcyanide per ml could be tolerated. Very littlecyanide was detected in the effluents from fil-ters fed 1 to 100 ,ug of K, Zn, and Cd cyanidesper ml, but on filters fed 100 ug of Cu, Ni, andFe cyanides per ml the cyanide contents of theeffluents were 2.7, 15, and 38 ug/ml, respec-


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tively. This result was probably due to the in-hibitory effects of the more stable Cu and Nicyanides and the inactivity of the sewage sys-tem towards ferrocyanide.

Filters acclimatized at 100 pug of the metalcyanides per ml in sewage were subsequentlytreated by the cyanides containing graduallydecreasing concentrations of sewage, untileventually the metal cyanides in tap waterwere used as the feedstuffs. The K, Zn, and Cdcyanides were totally degraded, and most ofthenitrogen was recovered as nitrate and nitrite.The Cu and Ni complexes were 75 to 80% de-graded and nitrogen was recovered as ammo-nia, nitrate, and nitrite, but ferrocyanide wasattacked only slightly. Thus, sewage microor-ganisms not only degrade cyanide, but some ofthem at least are also able to subsist on cya-nide.Winter seeded laboratory-scale trickling fil-

ters with two cyanide-utilizing actinomycetes(page 666) and fed the filters KCN and cyanideplating wastes (194). The filters degraded over90% of the cyanide when fed 25 ttg ofKCN perml for several weeks, but with an influent con-taining 45 to 137 ,ug of KCN per ml, the effl-ciency of degradation was only 63%. FeedingCu(CN)2 or a 4:1 mixture of Cu(CN)2-Zn(CN)2at up to 18 ,ug ofmetal per ml (plus KCN to give50 to 100 pmg of cyanide per ml) resulted in a 70to 90% degradation of cyanide. When "grabsamples" of cyanide plating wastes, containing0 to 50 ,ug of Ni, Zn, Cu, Fe, Cr, and Al per ml(7 to 35 ,ug of cyanide per ml, total), were fedto the filter, cyanide degradation declined toalmost zero but totally recovered when refedKCN. Maintaining the-grab samples at pH 6.5gave an efficiency of cyanide decomposition ofonly 40%, but when composite cyanide platingwastes, of greater uniformity ofmetal composi-tion, were used the efficiency was 95 to 100% (atpH 8 and 20 to 25 ,ug of cyanide per ml).Degradation of cyanide has also been studied

in activated sludge systems. Nesbitt et al. (121)gradually acclimatized settled, primary sludgeto cyanide and then fed 3-liter aerated, acti-vated sludge systems with 60 or 120 mg of cya-nide per day for several months. Cyanide, asthe sole source of carbon and nitrogen, wascompletely metabolized, and there was no lossof suspended solids. About 98% of the cyanide-carbon was converted to C02 and 75 to 90% ofthe cyanide-nitrogen was converted to ammo-nia, nitrate, and nitrite.Ludzack and Schaffer (94) studied degrada-

tion of cyanide, cyanate, and thiocyanate in alaboratory-scale, complete-mixing activatedsludge unit. Utilization ofcyanide was found to

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be improved by addition of dextrose to the in-fluent. Up to 60 mg of cyanide per liter ofinfluent was completely degraded, with no lossof suspended solids and with recovery of thecyanide-nitrogen as ammonia and nitrate plusnitrite. The periods of acclimatization to in-creasing concentrations of cyanide were foundto be crucial, although acclimatized sludgesreadily accepted variable influent levels of cya-nide. On the other hand, shock (slug) doses ofcyanide were found to be metabolized by unac-climatized sludges, but it took the system sev-eral days to recover normal operating capac-ity: cyanide was probably bacteriostatic ratherthan bacteriocidal.

Activated sludge systems were also found todegrade thiocyanate and cyanate, although cy-anate was more difficult to handle and cyanatesludges were unstable (94). It has been arguedthat cyanate and thiocyanate are intercon-verted (199) or converted to cyanide (72, 74)during their utilization. Therefore, sewage sys-tems acclimatized to cyanate, thiocyanate, orcyanide might exhibit cross-acclimatization tothe other chemicals. However, this was notfound to be the case (94), and degradation ofeach of these chemicals probably occurs viapathways not involving the formation of theothers as intermediates.

Aerated, activated sludge systems are able,like percolating filters, to degrade cyanide inthe presence of significant concentrations ofheavy metals. Cyanide-acclimatized activatedsludges were shown by Mikami and Misono(106) to totally degrade 100 to 150 ,ug of cyanideper ml, and 85 to 95% of the cyanide-nitrogenwas found in the effluent stream as ammonia,nitrate, and nitrite. Provided the sludge wasgradually acclimatized to Cr(VI) (up to 15 ,ug/ml) or Cu(II) (up to 10 ,ug/ml) and the level ofcyanide loading (at 150 ,ug of cyanide per ml)was suitably adjusted, total degradation of cya-nide and the nitrogen balance were main-tained. When Ni(MM) was fed to the systemmixed with the cyanide influent, it formed anondegradable complex; the complex did notform when separate cyanide and Ni(ll) (up to 3,g/ml) feeds were used and cyanide degrada-tion proceeded, but with enhanced nitrification.Because cytochrome oxidases are usually

highly cyanide sensitive, it might be expectedthat anaerobic digestion systems would have agreater capacity to degrade cyanide wastesthan aerobic systems, particularly when otherorganic foodstuffs are present. A two-stage an-aerobic digester has been developed (73) thatuses a carefully controlled primary stage andhas partical recycling of the sludge from the


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second stage, and that is notably efficient in itsability to degrade relatively high concentra-tions ofcyanide wastes at high loading rates.

MECHANISMS OF CYANIDE RESIST-ANCE AND DETOXICATION

It is a common myth that cyanide is a specificinhibitor of cytochrome oxidase. In fact, cya-nide inhibits a wide range ofenzymes, many ofwhich, though by no means all, are hemopro-teins or other metal-containing oxidases or oxy-genases. Dixon and Webb have listed some ofthe enzymes known to be inhibited by cyanide(41). Cyanide is a very reactive molecule, and itis not surprising that it is catholic in its inhibi-tory tastes. It forms stable complexes withmany metals, reacts with keto groups to formcyanohydrins, and reduces thiol groups (136).At concentrations of about 10-4 M or lower,cyanide is usually highly inhibitory to cyto-chrome oxidase but has little effect on otherenzymes, which require 1O-4 to 10-2 M cyanidefor significant inhibition; there are, of course,many exceptions to this generalization. There-fore, treatment of cells with "low" concentra-tions of cyanide often results in an apparentlyspecific inactivation of respiration.How, then, do some microorganisms manage

to adapt to growth in the presence of cyanide?They can either induce enzymes for degrada-tion and detoxication of cyanide or form cya-nide-resistant enzymes. Because cytochromeoxidase is sensitive to cyanide poisoning andrespiration is central to functioning of the cell,the development of cyanide-resistant respira-tory systems is of particular interest and, con-sequently, there have been several studies ofcyanide-resistant respiration, but there hasbeen, to my knowledge, no research on theadaptation of other cyanide-sensitive enzymesto cyanide resistance.

Differentiation between cyanide-sensitiveand cyanide-resistant organisms has been usedas a taxonomic test. Moller developed a cya-nide-containing medium (116), which has beenimproved by inclusion ofan indicator (56) and ahigher concentration of cyanide (119). It hasbeen shown that the Enterobacteriaceae can besubdivided into cyanide-sensitive (Escherichia,Shigella, Edwardsiella, Salmonella, Arizona)and cyanide-resistant (Citrobacter, Klebsiella,Enterobacter, Serratia, Proteus, Providencia,Aeromonas) species (79, 119).

Preliminary studies in my laboratory haveshown that the active component of complexcyanide differentiation media is probably cys-teine (N. Porter, D. E. F. Harrison, and C. J.Knowles, unpublished observations).

Cyanide-Resistant RespirationThere have been innumerable reports on the

effects of cyanide on respiration by intact mi-croorganisms, mitochondria, and cell-free ex-tracts (10, 55, 68, 91, 160). Cyanide-resistant"alternate oxidases" (60, 69, 70, 77, 82, 91, 146,148, 157), which are not cytochromes, have beenfound in various eukaryotic microorganisms;such oxidases can be induced by manipulatingthe growth conditions or by forming mutants(60, 70, 82, 124, 146). Seemingly, they are syn-thesized to counteract a depression in the levelofcytochrome oxidase or other respiratory com-ponents. Branched respiratory systems also oc-cur in bacteria, with one branch less cyanidesensitive than the other (1, 78, 192, 193).

It is not relevant to this review to discussfurther the mechanism of inhibition of respira-tion by cyanide or cyanide-resistant alternateoxidases. Only the respiratory systems of cya-nide-evolving microorganisms and adaptationof the respiratory systems of cyanide-resistantmicroorganisms to growth in the presence ofcyanide will be discussed.One method by which microorganisms could

exhibit cyanide-resistant respiration dependson the spatial organization of the respiratorysystem across the cytoplasmic or mitochondrialmembrane. For example, a bacterial cyto-chrome oxidase could be located on the cyto-plasmic (inner) side of the plasma membrane,with the membrane impermeable to exoge-nously added cyanide. However, at physiologi-cal pH values, hydrogen cyanide (pKa = 9.1) isessentially undissociated, and as it is a smallmolecule it probably permeates membranesrapidly (3, 31). Using the turbidity change tech-nique (3) with NH4CN and KCN as substrates(plus ionophores, see reference 63), Wissing andKnowles (unpublished observations) haveshown that a cyanide-evolving pseudomonad isreadily permeated by cyanide. It is thereforeimprobable that crypticity of cytochrome oxi-dase to cyanide is an explanation for cyanide-resistant respiration.Achromobacter. Arima and his colleagues

(5, 111-115, 123) have studied the respiratorysystem of a strain of Achromobacter isolatedfrom soil which grows rapidly in rich mediacontaining 1 mM cyanide; cyanide is not de-graded during growth (111).Cytochromes b and o were formed during

logarithmic growth under conditions of highaeration, but cytochromes d and a, were onlyinduced in the stationary phase (5, 123). When1 mM cyanide was included in the growth me-dium, the cytochrome b concentration of loga-rithmic-phase cells doubled, cytochrome o de-

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creased by 40%, the cytochrome d content was10-fold higher, and cytochrome al was induced.Under conditions of limited aeration the cyto-chrome composition was similar to that foundfor aerobic growth in the presence of cyanide(5, 114).A shift from aerobic growth in the presence of

cyanide to aerobic growth in its absence causedcytochromes a, and d to disappear and the cyto-chrome b content to halve but had little effecton the cytochrome o content (5). Conversely,addition of 1 mM cyanide to aerobic culturescaused a massive induction of cytochromes a,and d and small decreases in the concentrationsof cytochromes b and o.

Succinate oxidase activities of intact cellsand sonic extracts and succinate dehydrogenaseactivity of extracts of cyanide-sensitive cells(aerobic growth) were 50 to 200% greater thanthe corresponding activities of cyanide-resist-ant cells (aerobic growth; 1 mM cyanide),whereas the activities of glucose oxidase in in-tact cells and NADH oxidase in extracts wereboth 50% greater in the resistant cells (5).Furthermore, resistant cells required 200-foldmore cyanide for inhibition of respiration thansensitive cells (2 x 10-3 M and 10-5 M, respec-tively) (123). Adaptation of cyanide-resistantcells to cyanide sensitivity and vice versa re-sulted in the appropriate changes of cyanidesensitivity of the oxidases (5, 112).To determine which, of the three possible

cytochromes (al, d, or o), is the cyanide-resist-ant oxidase in Achromobacter, Arima and Oka(5, 123) adopted two approaches. The first ap-proach was to examine the effect of cyanide onthe level and rate of reduction of the cyto-chromes of resistant cells after addition of suc-cinate under aerobic conditions (5). The resultssuggested a cyanide-resistant pathway of elec-tron flow from cytochrome b to cytochrome d,and that cytochrome a, was sensitive to cyanideinhibition. The role of cytochrome o could notbe determined in these experiments, but as it isthe only oxidase found in sensitive cells and itsconcentration decreases in resistant cells, it ispresumably the cyanide-sensitive oxidase.The second approach used action spectra to

detect relief of CO inhibition of oxidase activi-ties in the presence and absence of cyanide(123); intense light of appropriate wavelengthcan specifically relieve inhibition of respirationvia a particular oxidase (25, 26). Oka and Ar-ima (123) used blue and red light for relief ofinhibition of cytochromes o and a, and cyto-chrome d, respectively. For cyanide-sensitiveAchromobacter (containing only cytochrome oas a possible oxidase), both red and blue lightrelieved inhibition by CO, but in the presence

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of 0.5 mM cyanide only red light restored activ-ity. The cyanide-resistant oxidase was thusconfirmed as cytochrome d.Bacillus cereus. McFeters et al. have iso-

lated a strain of B. cereus able to grow inTrypticase soy broth containing 1 mM cyanideand which adaptively develops respiratory re-sistance to cyanide inhibition (102). Suspen-sions of stationary-phase cells grown in mediawith or without cyanide showed biphasic curvesof respiratory inhibition by cyanide. The second,sharper inhibition phase required a 10-foldgreater cyanide concentration for cells grown inthe presence of cyanide. Reduced minus oxi-dized difference spectra showed that the bacte-rium normally formed type b and a (probably a+ a3) cytochromes but that when grown in thepresence of cyanide the contents of these cyto-chromes were 50 and 100% greater, respec-tively, than in cells grown in cyanide-free me-dia. Reduced plus CO minus reduced differencespectra and photodissociation spectra of CO-inhibited bacteria (CO action spectra) con-firmed that in cyanide-resistant organisms cy-tochrome a3 was the oxidase.

C. violaceum. Niven et al. (122) studied therespiratory system of C. violaceum grown un-der conditions of high and low cyanide evolu-tion; the maximal cyanide levels in the cultureswere 1.8 mM and 30 ,uM, respectively, by thestart of the stationary phase.A particulate fraction derived from station-

ary-phase, cyanide-evolving cells was unable tooxidize the high-potential electron donor, re-duced N,N,N',N'-tetramethyl-p-phenylenedi-amine (TMPD), whereas a comparable particu-late fraction from cells grown under conditionsof low cyanide evoluation rapidly oxidized re-duced TMPD, and the oxidase activity washighly sensitive to inhibition by cyanide orazide. Oxidation of reduced TMPD by othermicroorganisms is also very cyanide sensitive(70, 78, 82, 192).The particulate fraction from cyanide-evolv-

ing cells of C. violaceum oxidized NADH andsuccinate by oxidases that were very resistantto inhibition by cyanide and azide; the inhibi-tion curves were monophasic (123). The particu-late fraction from cells evolving little cyanidealso oxidized NADH and succinate, but the cya-nide and azide inhibition curves were biphasic.In each case there was an extensive plateauregion, at about 50% of the overall oxidase ac-tivity, separating the highly inhibitor-sensitiveand relatively inhibitor-resistant phases.

It seems likely that the particulate fractionfrom cyanide-evolving cells synthesizes a linearrespiratory system with cyanide- and azide-re-sistant oxidases, whereas the particulate frac-


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tion from cells evolving little cyanide forms anadditional respiratory branch with a cyanide-sensitive oxidase; reduced TMPD is oxidized bythe latter pathway.

C. violaceum, grown either under conditionsofhigh or low cyanide evolution, formed a com-plex complement of cytochromes (122). It hasnot so far been possible to determine whichcytochrome is the cyanide-resistant oxidase.Cyanide-evolving cells contained cytochromesal, d, o, and c as the CO-binding cytochromes,and hence possible oxidases, whereas cellsevolving little cyanide formed only cytochromeso and c as the CO-binding cytochromes. Cyto-chromes a,, d, and o are known bacterial oxi-dases (25, 26), but CO-binding, nondenatured c-type cytochromes are comparatively rare and ithas yet to be clearly established (192) that theyare oxidases, although in methylotrophic bacte-ria a CO-binding cytochrome c is involved inmethane oxygenase activity (177). The CO-binding cytochrome c of C. violaceum, unlikethe membrane-bound cytochromes a1, d, and o,is mainly soluble in cell extracts, although inthe intact cell it is probably periplasmic in loca-tion (122).E. colU. Ashcroft and Haddock have shown

thatE. coli strain C-1 can grow aerobically, ona medium containing n-xylose as the carbonsource, in the presence of up to 1 mM cyanidewith little effect on the growth rate, althoughthe lag period is extended and the growth yielddecreases (6). When succinate, which can pro-vide energy only by the respiratory system, isthe carbon source, 250 uM cyanide is the maxi-mal cyanide concentration that can be toler-ated. On succinate the mean generation timeincreases from 2.4 h in the absence of inhibitorto about 7 h in the presence of 150 ,uM cyanide.The respiratory system was examined in par-

ticulate fractions derived from early logarith-mic-phase cells ofE. coli that had been grownon succinate in the presence and absence of 150,uM cyanide (6). Growth in the presence of cya-nide had little effect on the ubiquinone andtotal b-type cytochrome contents, but the me-naquinone, cytochrome d, and cytochrome al,levels increased substantially and the cyto-chrome o level decreased somewhat. Withgrowth in the absence of cyanide, cytochromesb5w and b5a were the only spectrally distinctcytochromes, but in the presence of cyanidecytochrome b5m was also observed. NADH oxi-dase activity was unaffected by growth in thepresence of cyanide, but its sensitivity to inhi-bition by cyanide was dramatically lowered(from 75 to 500 ,uM cyanide for 50% inhibition).Thus, the overall effect of inclusion of cya-

nide in the growth medium is the formation ofa


respiratory system of similar composition tothat found for anaerobic growth ofE. coli or foroxygen-limited, stationary-phase aerobic cul-tures (see Haddock and Schairer [611 for refer-ences). Pudek and Bragg had previously shown(130, 131) that mid-logarithmic-phase cells ofE.coli strain NRC-482 formed equal quantities ofcytochromes d and o, whereas in the stationaryphase the content of cytochrome d increasedbut there was very little cytochrome o present.NADH oxidase of the particulate fraction fromstationary-phase cells was much more resistantto cyanide inhibition than particles from log-arithmic-phase cells. The increased resistanceof stationary-phase cells was partially due tothe greater overall cytochrome d content andpartially due to its increased content relative tothe cyanide-sensitive oxidase, cytochrome o.Cytochrome a1 is also formed in stationary-phase cells of E. coli, or in logarithmic-phasecells grown in the presence of cyanide, and, asit is also CO binding, it could also be an oxi-dase. Kinetic evidence, however, suggests thatit is not an oxidase inE. coli (reference 42, citedin reference 6).Tetralymena pyriformis. There have been

several reports of the adaptation of growth andrespiration of the protozoan. T. pyriformis, tocyanide, which have been reviewed by Dan-forth (40).McCashland and Pace showed that very low

concentrations of cyanide (e.g., 10-9 M) stimu-late growth and respiration of T. pyriformisstrain W but that higher concentrations inhibitboth of these processes (98). McCashland (95,96) grew strain W in media containing gradu-ally increasing concentrations of cyanide (from10-12 to 10-4 M). Cells adapted to 10-4M cyanidehave 82% of the volume of normal, nonadaptedcells, and their nucleus-cytoplasmic volume ra-tio is greater. Respiration of adapted cells ismore resistant to inhibition by 1 mM cyanidethan normal cells, and adapted cells lose theirresistance to cyanide when regrown on cyanide-free media. Glucose is used more rapidly byadapted than normal cells, but this does notreflect a switch to a fermentative type ofmetab-olism, because the lactate output is similar innormal and adapted cells (about 50% of theglucose is converted to lactate) (99). The grossmetabolism of fats is unchanged, but adaptedcells utilize amino acids more rapidly duringgrowth than do normal cells.The sensitivity of seven strains of T. pyrifor-

mis to cyanide has been examined (97). Milli-molar cyanide inhibits growth of all the strainsby 80% or more; strains E and W are particu-larly sensitive. Respiration of strains E and Wis 75 to 80% inhibited by 1 mM cyanide,


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whereas respiration of the other strains is only0 to 10% inhibited by 1 mM cyanide and 8 to50% inhibited by 5 mM cyanide.

Cyanide DetoxicationMany microorganisms are resistant, or can

induce resistance, to cyanide by degrading it tonontoxic products. Thus, cyanide-acclimatizedsewage systems (72, 94, 121, 127) and specificmicroorganisms (157, 190, 194) have been re-ported to convert cyanide to carbon dioxide andammonia, nitrite, or nitrate. In addition, it ispossible that cyanide may be detoxified by con-version to ,3-cyanoalanine or other products re-ported to be intermediates of cyanide assimila-tion (page 661). However, such pathways ofdetoxication would be wasteful of the cells' re-sources (e.g., loss of serine or cysteine in thecase of /8-cyanoalanine formation). It is possiblethat these pathways act to recycle intermedi-ates, with the ultimate conversion of cyanide toammonia and carbon dioxide rather than itsassimilation, although this would seem to be aneedlessly complex method of detoxication.The simplest method of detoxifying cyanide

is to convert it to formate and then to carbondioxide (by formate dehydrogenase). Conver-sion to formate could be direct (by a nitrilase) orvia formamide (by cyanide hydratase and for-mamidase):

wide range of reactions involving transforma-tions of sulfur-containing molecules. A sul-phane-containing anion donates sulfur to rho-danese, forming a rhodanese-sulfur complex,which then interacts with a thiophilic acceptoranion to regenerate free rhodanese:

RSO.S- + rhodanese = RSOx-+ rhodanese - S

rhodanese - S + X- rhodanese + SX-.The sulfur donors include thiosulfate, thiosulfo-nate, persulfide, polysulfide, ethanesulfonateand thiotaurine, and the sulfur acceptors in-clude cyanide, sulfite, organic sulfinates,thiols, dithiols, dithionite, and lipoate. The cy-anide-to-thiocyanate reaction is atypical amongthe rhodanese-catalyzed reactions in that it isirreversible.Long suggested that rhodanese, found in

mammalian tissues, acts to detoxify cyanide(83). The later discoveries that rhodanese islocated in the mitochondria ofmammalian cells(165) and that cyanide-inhibited cytochrome ox-idase is reactivated by addition of rhodaneseand thiosulfate (166) would appear to confirmthis hypothesis. However, more recently it hasbeen realized that rhodanese has an importantrole in the metabolism of various sulfur-con-taining compounds (cf. 142, 191).Rhodanese has been discovered in a wide

H20 H,2 NAD+ NADH + H+HCN -L-- H.CO.NH2 H.COOH

\

e C02

2HO NH'<

In support of this possible pathway, spores ofS. loti have been shown to induce a cyanidehydratase, forming formamide as the detoxica-tion product; the enzyme has been partiallypurified (53). In addition, Fusarium solani cat-alyzes release of ammonia from cyanide (thecarbon product is unknown), and the enzymeresponsible has been purified (153).

In 1933, Lang reported the discovery of rho-danese [EC 2.8.1.1, thiosulfate:cyanide sulfur-transferase) from a wide variety of animal tis-sues and E. coli (83). The distribution, proper-ties, and biological function of rhodanese havebeen reviewed recently (142, 191), and it willtherefore only be briefly discussed here.Lang showed (83) that rhodanese catalyzes

the formation of thiocyanate and sulfite fromthiosulfate and cyanide:

S2032- + CN- SO32- + SCN-

Rhodanese has since been shown to catalyze a

range of bacteria. The frequent observations ofrhodanese in Thiobacillus species (18, 31, 32,90, 100, 145, 173, 175), in photosynthetic bacte-ria (159, 198), and in Desulfphomaculatum (11)suggest that, in these organisms at least, ithas a role in sulfur metabolism rather than oneas a cyanide detoxifying agent. Rhodanese hasalso been found in several non-photosyntheticheterotrophic microorganisms (7, 8, 83, 149,180, 181). Some reports suggest that E. coliforms rhodanese, whereas others indicate thatit does not (83, 149, 180): Westley has pointedout that nonspecific reactions can be mistakenfor rhodanese activity (191).

In B. subtilis and B. stearothermophilus,rhodanese is constitutive (8, 181). Rhodaneseactivity in photosynthetic bacteria does not cor-relate with the ability to metabolize thiosulfateor the pathway of thiosulfate metabolism (198).On the other hand, in Thiobacillus denitrifi-cans rhodanese can be induced threefold by

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growth in media containing 40 or 400 uM cya-nide (18) and growth of Rhodopseudomonaspalustris in cyanide-containing media resultsin a doubling of rhodanese activity (198).In summary, it seems likely, but has yet to be

clearly established, that rhodanese has a role incyanide detoxication by some microorganismsin addition to its function in the metabolism ofsulfur-containing compounds.Mercaptopyruvate sulfurtransferase (EC

2.8.1.2, 3-mercaptopyruvate:cyanide sulfur-transferase) catalyzes the formation of pyru-vate and thiocyanate from mercaptopyruvateand cyanide (142, 191):

HS.CH2.CO.COOH + CNe -- CH3.CO.COOH+ SCN-

It is specific for mercaptopyruvate, but sulfitemay replace cyanide, and the enzyme canrelease colloidal sulfur from mercaptopyruvatealone. In animals it functions as a transsulfur-ase (142). It has also been found in E. coli (80),where its function is not yet clear, but presum-ably it has a role in mercaptopyruvate metabo-lism rather than in detoxication of cyanide.

CONCLUDING REMARKSIn experiments designed to simulate the con-

ditions of the prebiotic earth, significant con-centrations ofhydrogen cyanide have been rou-tinely found (108, 109). Moreover, under similarconditions cyanide is readily converted toamino acids, adenine, and other biologicallyimportant compounds (124, 125).

It therefore seems probable that among themost primitive organisms (which must havebeen anaerobes since the early earth did nothave an oxygen-containing atmosphere) weresome that could metabolize cyanide, perhaps inconjunction with other carbon and nitrogensources. At present there is some, though farfrom adequate, understanding of the pathwaysof cyanide assimilation by aerobic microorga-nisms, but virtually nothing is known aboutcyanide assimilation by anaerobic microorga-nisms. Obviously, it is important that moreunderstanding be gained about cyanide assimi-lation by microorganisms, both from an evolu-tionary standpoint and from the light it canshed on the metabolism of one-carbon com-pounds.Cyanide inhibits not only cytochrome oxi-

dases but also a wide range of other enzymes(although usually at somewhat higher concen-trations; 41). Hence, cyanide would be expectedto inhibit growth of anaerobes as well asaerobes, unless they contain detoxication mech-anisms. Because of the presence of cyanide in


the primordial soup, it seems probable that thedevelopment of primitive anaerobic organismswas paralleled by evolution of mechanisms ofdetoxication of cyanide; these could have eitherbeen precursors of the pathways of cyanide as-similation found in contemporary microorga-nisms or the latter could have developed in-dependently. Schievelbein et al. (147) havepointed out that cyanide now occurs relativelyseldom in nature (although the reader of thisarticle will be aware that it occurs less seldomthan might otherwise be imagined), yet rho-danese activity is found in a wide range ofmicroorganisms, plants, and animals. Theyhave proposed that the original function ofrhodanese was detoxication of cyanide ratherthan sulfur metabolism and, partially at least,that it is a biochemical relic.Although the function of nitrogenases is to

fix dinitrogen, they are also able to carry out arange of other reductive processes, includingconversion of cyanide to ammonia and methane(67). If nitrogenase emerged at the time of evo-lution of primitive anaerobes, it would seemunlikely that its original function was to fixnitrogen: there would have been a lack of selec-tive pressure in the ammonia-rich primitiveuniverse. Silver and Postgate have pointed out(155) that the nitrogenase system might haveinitially evolved for reduction of some othersubstrate and only recently adapted to fix nitro-gen; detoxication of cyanide is one such possibleearly function. On the other hand, nitrogenasemight have evolved more recently and the abil-ity to reduce cyanide, etc., is coincidental to itsdevelopment (129).At the time the atmosphere of the earth

changed from anaerobic to aerobic and cyto-chrome oxidases evolved, cyanide was presum-ably still prevalent in the environment andprimitive types ofoxidase would have had to berelatively cyanide tolerant. Therefore, an un-derstanding of the mechanisms of cyanide-re-sistant respiration could give some insightsinto the evolution of early aerobes, in additionto aiding our comprehension ofthe biochemicalmechanism of oxidase function. It is not yetclear whether any particular one of the knowncytochrome oxidases is specifically cyanide re-sistant, and hence possibly a direct descendantof a primitive oxidase, although cytochrome dcould be a candidate since it is (i) the cyanide-resistant oxidase ofAchromobacter (5, 123), (ii)the least cyanide-sensitive oxidase ofE. coli (6,130, 131) and Azotobacter vinelandii (1, 78), and(iii) induced in C. violaceum under cyanide-evolving growth conditions (122). It remains tobe seen whether these d-type cytochromes areunusually cyanide insensitive or whether d-


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674 KNOWLES

type cytochromes in general are more cyanideresistant than other oxidases. Unlike cyto-chrome d, which is a bacterial oxidase, thecyanide-resistant alternate oxidases (which arenot cytochromes) have so far been found exclu-sively in eukaryotes (70) and are, therefore,unlikely to be descendents of early oxidases.Although it is self-evident why some microor-

ganisms assimilate or grow on cyanide, it is notso immediately clear why the same or othermicroorganisms (and plants) produce cyanide.After all, production of so toxic a chemical willresult in self-inhibition unless cyanide-resist-ant oxidases and other enzymes are concomi-tantly produced.Cyanide excretion could be used to destroy or

inhibit growth of neighboring organisms,thereby (i) increasing the competitiveness ofthe excreting organism, (ii) preventing possiblepredatory activity of the affected organisms, or(iii) enabling parasitism of other cells. In thesnow mold, copperspot, and fairy ring diseases(page 653), a varient of (iii) occurs, wherebythe parasitic fungi excrete an enzyme (0-glu-cosidase) that causes destructive release of cya-nide from the host plant cyanogens, possiblywith coordinate release of cyanide from thepathogens themselves. Traces of cyanide couldalso be produced by many bacteria and fungi asa convenient form ofnitrogen and/or carbon (inthe same way that heterotrophic microorga-nisms produce and utilize CO2). Only undercertain unusual conditions, such as growth inmedia containing an excess of the precursor(glycine), would relatively large and inhibitoryconcentrations of cyanide be formed. Finally,cyanide could be produced as a secondary me-tabolite, as proposed by Castric (27), actingsolely to remove an excess of a substance (pre-sumably glycine) that would otherwise be toxicto the cell ifpresent in too high a concentration.

ACKNOWLEDGMENTSI would like to thank Frode Wissing for his hospi-

tality and particularly for the helpful discussionsand ideas that arose during my short visit to hislaboratory, which was supported by the EuropeanMolecular Biology Organisation. I am grateful toElinor Meynell and Donald Niveh for their criticalreading of the manuscript, to Barbara Kerry, SadieDawes, and Sadie Loughlin for typing the manu-script, and to Pauline Collins for checking the man-uscript.

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