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INTEGRATION, AT H A G OR ELSEWHERE, OFH2 (PHASE-2 FLAGELLIN) GENES TRANSDUCED FROM SALMONELLA

TO ESCHERICHIA COL11y2

MASATOSHI ENOMOT03 AND BRUCE A. D. STOCKER

Department of Medical Microbiology, Stanford University School of Medicine, Stanford, California 94305

Manuscript received June 2, 1975

ABSTRACT

A f la mutant of E. coli K12 was given fla+ and HI-i by phage Plkc cotrans- duction from S. typhimurium, then made Fla- by transduction of ah1 from S. typhimurium. Motile clones expressing a Salmonella phase-2 antigen, e,n,z or 1,2, were obtained from the K12 i ah1 (therefore Fla-) line by Plkc transduction of flagellin-specifying genes, H2-e,n,x or H2-1,2, from Salmonella donors. Of eighteen such transductants sixteen failed to show phase variation, and on transduction back to Salmonella each structural gene for a phase-2 flagellin (or at least for its antigenically determinant part) now behaved as an allele of HI , presumably in consequence of incorporation in the hag region of the K12 recipient, in place of HI-i ahl. The e,n,x- and I,2-specifying genes were shown to have been integrated in the K12 chromosome without the linked HI-repressor gene or the adjacent uh2 gene (controlling rate of phase-variation) and they responded to the repressing activity of an H2 allele elsewhere in the cell, in this respect resembling H I alleles of Salmonella or hag alleles of E . coli. TWO K12 e,n,x transductants had flagellin-specifying genes which when transduced back to Salmonella were integrated at H2; they are inferred to have resulted from integration of HZ-e,n,x in the K12 chromosome elsewhere than the hag region. These two clones showed phase variation, between a Fla+ phase, with antigen e,n,x, and a Fla- phase (with e,n,x determinant in the nonactive state and the determinant of antigen i inactivated by a h l ) . The two integrated e,n,z genes when in the “active” state retained the ability to repress expression of exogenote HI alleles, which indicates that the closely linked H I - repressor gene also was integrated. One of the two exceptional transductants derived its e,n,r gene from a Salmonella donor with the linked vh2- gene, which in Salmonella almost entirely prevents change of phase, and transduction of this e,n,z gene back to Salmonella recipients proved that vh2- had been incorporated into the E . coli chromosome along with the e,n,z determinant and the HI-repressor gene. The high frequency of change of phase (F la+eFla- ) in the K12 e,n,z vh2- transductant concerned suggests that vh2- fails to prevent frequent change of state of the phase-determinant part of H 2 when vh2- and H2 are incorporated in the E. coli chromosome.

ROSSES of Salmonella Hfr donors to E. coli F- recipients, and of E . coli Hfr Cdonors to Salmonella recipients, and transduction to Salmonella recipients by

1This project was supported by Grant No, AI 071G8 awarded by the National Institute of Allergy and Infectious Diseases, DHEW.

Contribution No. 1062 from the National Institute of Genetics, Mishima, Japan. a Present address: Department of Biology, Faculty of Science, Okayama University, Okayama 700, Japan.

Genetics 81: 595-614 December. 1975

596 M. ENOMOTO A N D B. A. D. STOCKER

phage P22 of genes of E . coli origin fro” hybrids obtained by the latter type of conjugational cross have confirmed the close similarity of the genetic maps of the two genera, and shown that genes derived from a strain of one genus function normally when transferred to bacteria of the other genus (for reviews see BARON et el. 1968; SANDERSON 1971; MIDDLETON and MOJICA-A 1971). One striking difference concerns the genes controlling flagellar antigen (s) in the two genera. In Salmonella the many known antigens are controlled by allelic series at two loci, H I , determining phase-1 flagellar antigens, and H2, determining phase-2 flagellar antigens ( LEDERBERG and EDWARDS 1953). These are the structural genes for the corresponding flagellins (constituent protein of the filamentous part of the flagellum), as shown by differences in primary structure of antigenically distinct flagellins determined by allelic genes ( MCDONOUGH 1965 ; ENOMOTO and IINO 1966). H I and H2 are well separated on the chromosome, H I within the cluster of genes concerned with flagellar and motility characters, near his, and H2, not precisely mapped but between purC and strA (SMITH and STOCKER 1962). Most Salmonella species possess two flagellar antigens (and are described as diphasic) and manifest phase variation, i.e., the expression of either the H I allele o r the H 2 allele in any given bacterium. By contrast E. coli strains have only one flagellar antigen, determined at a flagellin-specifying locus, now termed hag (ARMSTRONG and ADLER 1969) which corresponds to HZ of Salmonella (MAKELA 1964); no phase variation has been observed in this genus. (For review of genetics of flagellar characters see IINO 1969.) The genetic elements (known or postulated) concerned in phase variation are shown in Figure 1, whose footnote provides a glossary of the gene symbols used in this paper.

/ /

/

\

/ /

\

\ \

/

\ I

hls 1 I \

/ \

I

\ \ /

/ \ \

\

/ \

-strA-(vh2,[(PD,ah2) -b (H2,rh l)])-purC- -his- fla-[H l e (H 1 Op,ah ?)I- fla-trp-

H l region H2 region

FIGURE 1.-Linkage map of Salmonella (symbols outside circle) and of E. coli (symbols inside circle), and of H l and H2 segments of Salmonella map, showing genetic elements (proven o r postulated) lnvolved in phase variation. Distances within H I and H 2 regions arbitrary, and irrelevant genes within H I region omitted. Meaning of symbols: -

Order of loci within parentheses, o r orientation relative to outside markers, not known. Regulation unit, with + from regulator element(s) to regulated element(s).

( ) : [ 1.

H2 OF SALMONELLA TRANSDUCED TO K12 597

In diphasic Salmonella, such as S. typhimurium or S. abony, a bacterium in a given phase produces progeny in the same phase; or in the opposite phase, with a probability in the range 1 0-3-1 O-5/bacterium/generation (STOCKER 1949; MXKELA 1964). Analysis by transduction (LEDERBERG and IINO 1956) showed that the phase of a given bacterium is determined by the “state” of its H 2 locus: when H2 (cr a hypothetical “phase-determinant” closely linked to it) is in the active state, H 2 is expressed, Hi‘ is repressed, the cell is in phase 2 and, if flagellate, manifests its phase-2 flagellar antigen. When H2 is in its alternate, non-active state, H 2 is not expressed, HI is not repressed, the cell is in phase 1 and, if flagellate, shows its phase-1 flagellar antigen.

flu: H i :

Genes for production of flagella. f7a- bacteria are non-flagellate. Structural gene for phase-I flagellin of Salmonella. Hl-i , the wild-type allele of S. typhimurium, specifies flagellin of antigenic character i . The wild-type allele of S. abony, HI-b, specifies flagellin of antigenic character b. (Symbols i and b are used in this paper to indicate genes specifying flagellins of corresponding antigenic character, originally alleles at HI of Salmonella, after their transfer to unspecified sites in the E . coli chromosome.) Structural gene for flagellin of E. coli, antigenically unrelated to any Salmonella flagellin. Corresponds to HI of Salmonella. Structural gene for phase-2 flagellin of Salmonella. H2-1,2, the wild-type allele of S. typhimun‘um, specifies flagellin of antigenic character 1,2. The wild-type allele of S. abony and S. abortus-equi, Ha-e,n,x, specifies flagellin of antigenic character e,n,x. (Symbols 1,2 and e,n,z are used in this paper to indicate genes specifying flagellins of indicated antigenic character, originally alleles at H2 of Salmonella, after their transfer to unspecified sites in the chromosome of E. coli.) “Activator” of gene H I . ahi- bacteria cannot express adjacent (cis) HI gene and therefore are non-flagellate when in phase 1. ah1 may be a separate gene, or identical with HI-Op, or may be an operator-proximal site within H I .

HI-Op: Used in this diagram to indicate postulated HI operator site, which binds repressor

hag:

H2:

ah l :

rhl:

PD:

ah2:

vh2:

H2on:

protein specified by rhl and prevents expression of H l adjacent (cis) to Hi-Op. May be the same as ahl . Repressor of H l . Close to H2 and regulated coordinately with it by the state of phase determinant adjacent (cis) to H2 and rh l . Used in this diagram for postulated phase-determinant element. In bacteria in phase 2 element PD is in “on” or “active” state and the adjacent (cis) H2 and rhl genes are then expressed; in bacteria in phase 1 PD is in “off’ or “inactive” state and adjacent H 2 and rhl are not expressed. May be a separate gene, or part of gene H2, or the same as ah2. “Activator” of gene H2 or of H2 operon. Presence of gene ah2- (derived from some naturally occurring phase-I monophasic strains) prevents expression of H2 and of the Hi-repressor, rhl . May be a separate genetic element, or part of H2, o r the same as PD. Phase-stabilizer locus. In Salmonella with uh2+ (wild-type allele of S. typhimurium, etc.) change of phase occurs at frequencies of 10-3-1 0-5/bacterium/generation. In Salmonella with uh2- (allele from S. abortus-equi) change of phase is almost entirely prevented (frequency < 10-7/bacterium/generation). Used in this paper to indicate active (= on) state of H2 region of phase-2 bacteria carrying vh2-, and therefore unable to change phase.

H20ff: Used in this paper to indicate inactive (= off) state of H2 region in phase-1 bacteria carrying vh2- and therefore unable to change phase.


An active H2 gene prevents expression not only of a chromosomal HI allele but also of an HZ allele forming part of an abortively transduced chromosome fragment (PEARCE and STOCKER 1965) : this and other evidence indicates that the HZ locus controls expression of H1 by determining production of an Hl-repressor substance. There is now good evidence (FUJIT~, YAMAGUCHI and IINO 1973; ENOMOTO 1974) that the repressor substance is distinct from the flagellin specified by gene H2 itself. It therefore seems that the H2 region comprises at least two protein-specifying genes, one for phase-2 flagellin and the other for a repressor of HI, and that both genes are inactive, or both active, according to the “state” of the postulated “phase-determinant” (which may perhaps be part of one or the other of these two genes). The symbol rhl has been proposed for the HI-repressor gene (FUJITA, YAMAGUCHI and IINO 1973). Experiments on expression of N I and H2 in H2 abortive transductants have shown that a phase determinant controls the expression only of the flagellin-specifying gene cis to it, and is without effect on the activity o r inactivity of another H2 flagellin- specifying gene. in trans (PEARCE and STOCKER 1967). Phase variation thus seems to result from random changes of a phase determinant in the H2 region between alternative metastable states, one resulting in activity of the immediately adjacent unit (operon?) comprising H2 and rhl, the other determining inactivity of these two genes. The molecular mechanism of the two alternative metastable states of the phase determinant is not known: various hypotheses have been proposed (IINO 1969; LEDERBERG and STOCKER 1970). Several additional, regula- torv, genes are concerned with phase variation. Gene uh2- (phase-stabilizer) , found in several strains of S. abortus-equi, is closely linked to H2: its presence almost entirely prevents change of phase, in either direction, so that a Salmonella strain which is diphasic but ~112- is “fixed” in one phase or the other (in this paper we indicate the stable state of H2 in uh2- strains by a superscript symbol; thus, H2-e,n,xorf or H2-e,n,xon). Mutation at ah2 (activator of H Z ) , closely linked to (perhaps part of) HI. prevents expression of H2: diphasic strains which are ahl- when they are in phase 1 do not manufacture flagellin and are therefore non-flagellate. Phase variation in such strains appears as an alternation between a flagellate phase 2 and a non-flagellate phase 1 (Fla+@Fla- o r H-0 variation) (IINO 1961a). Gene ah2- (activator of H2), so far encountered only in some naturally occurring phase-1 monophasic strains, seems to prevent expression of both the N2 flagellin-specifying gene and the HI-repressor gene, rhZ (IINO 1969; FUJITA. YAMAGUCHI and IINO 1973). It should be noted that some of the genes, in particular the Hl-repressor, a receptor for it (H1 operator region?) and a phase-determinant (which might be identical with nh2) postulated to account for experimental results, are not as yet fully proven, for instance. by isolation of gene product.

MAKELA (1964) isolated E. coli recombinants with the H2 region of Salmonella from crosses of S. abony Hfr with E. coli F-: they showed phase variation, at rates comparable to those observed in the Salmonella parent. The hybrids when in one phase expressed only their hag gene. of E. coli origin, and when in the other phase expressed only their Salmonella-derived H2 allele. The absence of co-expression

H2 OF' SALMONELLA TRANSDUCED TO K12 599

of hag with H 2 showed that their E , coli gene hag responded to the HI-repressor substance specified by the rhI component of their H 2 region, from Salmonella. It would be expected that most such recombinants would obtain from the donor not only the whole of the H2 region but probably a fairly long chromosomal segment on each side of it. Recently transduction by phage PI between E. coli and S. typhimurium has become practicable, by the use of Salmonella strains made restriction-negative by mutation and making galactose-deficient lipopolysaccharide, through mutation at galE, so that they absorb phage P1 at an adequate rate (ENOMOTO and STOCKER 1974). In the present study several Salmonella flagellin-specifying genes, either of the H I or the H2 series, have been transduced to E. coli, with the object of clarifying the mechanism of phase variation and perhaps the evolutionary origin of the H2 locus. Two of the Salmonella flagellin genes used, HI-i and HI-b, specify, respectively, flagellins of antigenic types i and b, found only as phase-1 antigens in Salmonella. Another two flagellin genes, H2-1,2 and H2-e,n,z, determine flagellins of antigenic types, respectively, I ,2 and e,n,z, found only as phase-2 antigens. One cannot be sure, a priori, as to where in the E. coli chromosome a flagellin-specifying gene transferred from Salmonella will be integrated. For this reason (and to avoid having to decide whether to denote a Salmonella gene of the HI series integrated at the hag locus of E . coli by an H;I or hag symbol) we shall hereinafter refer to flagellin-specifying genes of Salmonella origin integrated into the E. coli chromosome only by the symbol for their antigenic character: thus the genes olf the H I series concerned are each indicated by a single-letter symbol, i or b, whereas those of the H2 series are each represented by compound symbols (referring to serological cross-reactivity with other allelic forms), I ,2 or e,n,x.

MATERIALS A N D M E T H O D S

Bacterial strains and phages: The main strains used are listed in Table 1. Phages Plkc and Plvir were used fo r transduction; all the S. typhimurium strains used as donors or recipients have gi1E mutations, causing production of galactose-deficient LPS, apt for adsorption of phage PI (ORNELLAS and STOCKER 1374; ENOMOTO and STOCKER 1974). In experiments involving transduction from E . coli to S. typhimurium the recipients were derivatives of an LT2 line made restriction-negative by mutations at hspLT and hspS (COLSON and COLSON 1971; ENOMOTO and STOCKER 1974). Phage x (MEYNELL 1961) was used t o select non-motile mutants.

Media: The nutrient broth, nutrient agar and semisolid nutrient gelatin agar, used for selection of motile transductants, were as described by ENOMOTO and STOCKER (1974). Flagellar antigens were determined by slide agglutination.

Phage and transduction methods: These were as previously described (ENOMOTO and STOCKER 1974). Phage P I was first grown on a restriction-negative but modification-positive strain o f S. typhimurium, e.g., SL4213, to make it able to attack restriction-positive S. typhimurium. Phage Pluir was used to make lysates of strains lysogenic f o r Plkc: strains to be used as recipients for Pluir transduction were first made lysogenic for Plkc.

Measurement of frequency of uariation from Flu+ to Flcc-: To measure the rate of production of non-motile variants (either by phase change, in the case of strains with HI inactivated by ahl, o r by fla mutation) a swarm in semisolid medium (i.e., a clone selected for high motility) of the strain was streaked out on nutrient agar: after ca. 18 hr a t 37" a block of agar bearing a single colony was cut out and transferred to 10 ml broth in a centrifuge tube. After ca. 4 hrs at 37", the proportion of non-motile variants in the clone, now numbering 1-2 x 139 bacteria in


TABLE 1

Main strains used

Strain no. Relevant genotype' Origin and referencesf

(a) : Derivatives of S. typhimurium LT2 . , ELI0

SL873

SL4013

SL4202

SL4204

SL4213

SL4.235

SL4262

SL4263

SL4266

SL4271

SL4273

SJ2468

SJ2470

HI-i ahl-I , H2-2,2 vh2+, galE702

HI-i ahl-I , H2-I,2 uh2+

HI-i , HZ-e,n,xon vh2-

H1-i motA453, H2-e,n,xOff uh2-, galEl62 HI-i, H2-2,Z vh2+, galEI63

HI-b, H2-e,n,x v h l f , hspLT6, hspS29, galE496 H I - i ahl-I8 motA453, HZ-e,n,xoff vh2-, galE162 Hl-b, HZ-e,n,zoff vhZ-, galE162

HI-b, HZ-e,n,xon uh2; galEl62

HI - i ahI-1, HZ-e,n,zOn vh2-, galE702

HI- i ahI-I, H2-I,2 vh2+, galE70I

HI-b, H2-1,Z vh2+, hspLT6, hspS29, galE496

HI-b, HZ-1,2 vh2+, hspLT6, hspS29, galE496 (Plkc)

HI-b, HZ-e,n,xOn uh2-, hspLT6, hspS29, galE496 (Plkc)

SL870 (below) made nml, mal, leu and gal by successive mutations (ref. h ) his mutant (ref. e ) of SW1061 (ref. b ) : shows Fla+* Fla- phase variation

LT2 given H2-e,n,xon vh2- from S. abortus-equi (refs. c and f ) : fixed in phase 2 (antigen e,n,x) SL4131 (ref. a ) made galE by mutation: fixed in phase 1 (antigen i ) From LT2 by galE mutation: normal phase variation

Derived from CL4419 (refs. a and d ) : normal phase variation

Spontaneous ah1 mutant of SL4202 (above): fixed in phase 1 (Fla-)

Derived from SL4202 (above) by transduction of mot+ HI-b from SL4213: fixed in phase 1 (antigen b ) Derived from SL4262 (above) by transduction of HZ-e,n,xon from SL4013: fixed in phase 2 (antigen e,n,x) Derived from ELI0 (above) by transduction of H2-e,n,xon vh2- from SL4013: fixed in phase 2 (antigen e,n,z) galE mutant of SL870 (above): shows Flaf c3 Fla- phase variation

Derived from SL4213 (above) by transduction of HI-I,2 from SL870: normal phase variation

PI kc-lysogenic derivative of SL4273 (above) : normal phase variation

Derived from SL42.73 (above) by transduction of HZ-e,n,xon vh2- from SL4013, then lysogenization: fixed in phage 2 (antigen e,n,x)

(b) : Derivatives of Escherichia coli K12 WI485 A s Wild type except "cured' of A. Flaf,

EJ26 flc Spontaneous mutant of WI485, selected by

antigen H48 (ref. g)

phage x : Fla-, corresponding to flaAZZ or PaAZZZ of Salmonella

from SL42M EJ31 flu+ i EJ26 made Fla+, antigen i , by transduction

H2 O F SALMONELLA TRANSDUCED TO K I 2

TABLE l-Continued

Strain no. Relevant genotype* Origin and referencest

601

EJ32 Pa+ b

EJ34 i ahl-1

EJ35 Pa-

EJ26 made Fla+, antigen b, by transduction from SL4213 EJ31 made Fla- by transduction of ahl-1 from ELI0 Spontaneous mutant of EJ31 (above): Fla-, corresponding to fZaK of Salmonella

* Nutritional, antibiotic-resistance and other irrelevant mutations not listed. Gene symbols not separated by commas can be co-transduced by phage P22. U*: allele of S. abortus-equi NCTC5727, which prevents phase variation (IINO 196lb). The “state”, active or inactive, of H2 alleles cis to vh2- is indicated by superscript, “on” or “off”. Hl-b derived from S. abony SW803. HZ-e,n,x vh2- derived from S. abortus-equi NCTC5727, H2-e,n,x vh2+ from S. abony SW803.

I. References. a = ENOMOTO and STOCKER (1974) ; b = I I N O (1961a) ; c = IINO (1961b) ; d = ORNELLAS and STOCKER (1974) ; e = PEARCE and STOCKER (1967) ; f = VARY and STOCKER (1973) ; g = LEDERBERG and LEDERBERG (1953) ; h = E. M . LEDERBERG, personal communication.

total, was determined by an enrichment method. The 10-ml culture was centrifuged, 8 ml of supernatant discarded and the bacteria resuspended. A sample was plated for viable count. Antiflagellar serum, ca. 0.01 ml, was added, the mixture gently shaken at 37“ for 10 min, then centrifuged at ca. 30 G, so as to deposit only the agglutinated bacteria. The supernatant, containing Fla- cells and a much-reduced number of Fla+, was plated on semisolid medium containing serum against the flagellar antigen of the motile starting strain. After overnight incubation at 37” colonies of type L P (large, pale), characteristic of Fla- cells growing in semisolid medium ( ENOMOTO and IINO 1963), were easily recognized, and counted, by low-power stereomicroscopy, among the type SD (small, dense) colonies produced by Fla+ cells growing on semisolid medium but prevented from spreading by the serum. The calculated proportion of Fla- variants in the clone, divided by the number of generations (log to base 2 of the clone size), was taken as a measure of the probability of change from Fla+ to Fla-/bacterium/generation (STOCKER 1949; MAKELA 1964). In the case of strains showing a high rate of variation, the proportion of type LP colonies was determined directly, on suitably diluted samples from the 10-ml broth culture, without use of the serum enrichment method. Essentially the same procedure was used to estimate rate of change from one motile phase to the alternate phase, also motile: clones of changed phase were then recognized as swarms, instead of as L P (i.e., Fla-) colonies.

RESULTS

Transduction of H1 from Salmonella to E. coli: Five non-motile mutants, three of them mot (flagellate but non-motile) and two fla (non-flagellate) , were isolated from E . coli K12 W1485 (=K12 cured of X prophage, otherwise wild- type) by selection with x phage (MEYNELL 1961; ENOMOTO and IINO 1963). Phage Plkc grown on SL4204, a galE (therefolre Pl-sensitive) derivative of S. typhimurium LT2 (HI- i , H2-1,2), was applied to the non-motile K12 strains and selection made for motility on semisolid medium. After two days’ incubation at 37” many trails (motile abortive transductants) were visible on all the transduction plates. In addition two swarms (motile complete transductants) were obtained from Es26, one of the K12 fla recipients. Both clones proved to have antigen i, indicating transduction of the Salmonella phase-1 flagellin determinant H-I-i, along with f la f . One of these i transductants was labeled EJ31. A fZa+ transductant with antigen b, designated EJ32, was similarly obtained


from EJ26 (K12 fla-) by treatment with Plkc grown on SL4213 (LT2, HI-b, galE, etc.). These two transductants, if each arose by only two crossovers, must have incorporated Salmonella genetic material extending at least from HI to the fla gene affected in the K12 fla mutant, EJ26: complementation tests suggest that this E. coli gene corresponds to flaAIII or flaAII of Salmonella (YAMAGUCHI et al. 1972).

An attempt was next made to introduce a known ahZ- mutation (which is perhaps an operator-negative o r nonsense mutation in HI-i; IINO 1969) into the K12 f1.f i stock, EJ31. Plkc grown on EL10 (S . typhimurium LT2, HZ-i ahl-I , galE, etc.) was mixed with a broth culture of EJ31, and non-motile transductants were selected by addition of x phage to the transduction mixture, and plating in soft semisolid medium with x phage (at a final concentration of ca. 10* pfu/ml). Colonies of type LP (=large. pale) , characteristic of nonflagellate cells growing on semisolid medium (ENOMOTO and IINO 1963) were picked and purified. To distinguish ahl- transductants from fla- mutants among the 131 LP clones thus obtained they were treated with phage P lkc grown on SL4235, an LT2 HI-i ahl- line in phase 1 and unable to change phase because of presence of gene uh2- (IINO 1961b; VARY and STOCKER 1973); two of the 131 clones gave no trails and no swarms, as expected for K12 derivatives carrying flu+ HI-i ahl- of S. typhimurium origin. (All the other 129 LP clones gave trails in this test, and are therefore presumed to be fla- mutants, not ahl- transductants.) One of the two nhI- transductant clones was labeled EJ34.

Expression of Salmonella Hl and H2 exogenote genes in an E. coli recipient: E. coli has no gene corresponding to gene H2 of Salmonella and the availability of a K12 derivative carrying i ahl- of S typhimurium origin therefore made it possible to test for the expression of exogenote HI and H2 alleles in a non-motile recipient without the complication of the presence of an H2 (endogenote) allele (or of regulator genes linked to H2) , a situation which cannot be achieved when using a Salmonella recipient since even monophasic phase-I strains may possibly carry a stably repressed H2 allele, rather than a deletion of the H2 region. To test expression of exogenote HI-b and H2-e,n,x in the Kl9 i ahI- stock EJ34, it was treated with P1 kc grown on S. typhimurium lines of constitution HI-b, H2-e,n7x vh2-, fixed either in phase 1 (=SL4262, N2"ff) or in phase 2 (=SL4263, H2"") ; samples of the transduction mixtures were plated on semisolid medium, unsupplemented or with anti-b or anti-e,n,x serum (Table 2) . Antiserum against the antigen determined by the donor HI allele. HI-b, entirely prevented trail formation by cells treated with phage grown on the donor fixed in phase 1, whereas anti-e.n,x serum had no such effect. By contrast the number of trails evoked by phage grown on the donor fixed in phase 2 was reduced by about 26% by the presence of anti-b serum, and by about 55% by anti-e.n,x serum. This indicates: (i) that some of the motile abortive transductants evoked by phage grown on a donor fixed in phase 2 make flagella of antigenic character e,n,x; i.e., they express the donor H2 allele, H2-e,n,x-which thus replaces the flagellin- specifying function of the recipient i allele, inactivated by ahl-; (ii) that others of the motile abortive transductants evoked by phage gro.im on the donor fixed

H2 OF SALMONELLA TRANSDUCED TO K I Z 603

TABLE 2

Complete and abortive transduction of Salmonella flagellin-specifying genes to an i ah1 derivative of E. coli

Selection on semisolid medium with:

N o serum Anti-b serum Anti-e,n,x serum

Donor Swarms Trails Swarms Trails Swarms Trails

SL4262 (HI-b, H2-e,n,xQff) 85 470 (=103%) 1 0 85 540 (= 115%) SL4263 (Hl-b, H2-e,n,xon) 81 789 (=loo%) 2 581 (=74%) 84 355 (=45%)

Phage Plkc grown on SL4262 (LT2, HI-b, HZ-e,n,xoffvh2-) or SL 4263 (LT2, HI-b, H2-e,n,xon vh2-, i.e. as SL 4262 but fixed in phase 2 instead of phase 1) was mixed, at phage: bacterium ratio of ca. 2:1, with late log phase culture of EJ34 (K12 i ahl) and 0.1 ml of mixture was streaked on each selection plate. Numbers shown are numbers of swarms and of trails observed on two plates.

in phase 2 have flagella of antigenic character b, resulting from expression, in the heterogenote, of an Hi allele which in the donor was repressed by the action of its active H2 allele. Similar results were obtained by the use of P lkc grown on phase-1 and phase-2 clones of a diphasic (Hl-by H2-e,n,x vh2+) strain, SL4213.

The presence of anti-e,n,z serum had no detectable effect on the number of swarms evoked by either kind of lysate, whereas the presence of anti-b serum almost entirely prevented the appearance of swarms-the result expected if nearly all complete motile transductants result from replacement of the i ahl- genes, located at the hag region of the K12 recipient, by the donor HI-b ah1+ segment. Two swarms whose spread was not inhibited by anti-b serum were obtained by treatment with Plkc grown on SL4263, the donor fixed in phase 2. One of these clones had antigen i, and must therefore have resulted either from reversion of the recipient ahl- to ahI + or from incorporation of the donor ah1+ without its H I - b . The other had antigen e,n,z, presumably resulting from incorporation of the donor H2-e,n,x allele, as discussed below.

Complete transduction of H2 alleles to E. coli K12: The just-mentioned K12 derivative with antigen eynyz resulted from transduction of an e,n,z-specifying gene from an S. typhimurium donor carrying vh2-, and fixed in phase 2, that is with an H2 region permanently in the “active” state. An attempt was next made to obtain further K12 transductants with flagellar antigens determined by H2 alleles transduced by phage P I from either vh2+ (i.e., phase-variable) or vh2- S. typhimurium donors. The recipient used was, as before, EJ34 (=K12 i ahl-) . Four strains were used as donors: two of them, EL10 and SL4271, are S. typhimurium of constitution Hl- i ahl-, H2-1,2 VU+ and selection was then made on semisolid medium plus anti-i serum; a third donor was a phase-2 culture of SL4213, which is LT2 HI-by H2-eyn,z vh2+-for this combination selection was made an medium with both anti-i and anti-b sera; the fourth donor was SL4266, which is LT2 HI-i, H2-e,nYz0* vh2- (i.e., fixed in phase 2)-here selection was on medium with anti-i serum. A few swarms (frequency 1 to 5 x lO-’/pfu) were


obtained from all four combinations and on subculture all of eighteen such clones proved to have the phase-2 antigen of the donor concerned, in eight instances e,n,x and in ten 1,2 (Table 3 ) .

It seemed likely that variation between active and inactive states of an H2 allele transduced from Salmonella and incorporated into the K12 chromosome would manifest itself as rapid alternation between a flagellate and a non-flagellate phase. The eighteen Kl2 transductants with antigen e,n,x or 1,2 were therefore tested for rate of production of non-flagellate variants during growth in broth (see MATERIALS AND METHODS). For all except two of them the rate of production of Fla- variants was in the range 1 x I O + to 6 x 10-5/cell/generation, which is lower than the expected rate of variation from phase 2 to phase 1 in diphasic (U&?+) Salmonella, and no greater than the rate of production of flu- mutants by the motile K12 i ancestor, EJ31, tested as a control (Table 4). Of the two exceptional transductants one was EJ38, with antigen e,n,x derived from an

TABLE 3

Origin and properiles of motile transductants with antigen e,n,x or 1,2 derived from EJ34* ( K l 2 i ahl) by application of Plkc lysates of S. typhimurium deriuatiues

K12 e.n.z or 1.2 transductant

S. typhimurium donor (strain, Fla+ + Fla- Inferred site genotype, phase var. character) Strain Antigen rate+ of integration

EJ36 e,n,x 1.6 x IC-5 hag SL4213: HI-b, HZ-e,n,z vh2+: EJ37 e,n,x 4.4 x 10-6 hag

EJ39 e,n,z 1.4 x hag normal phase var., phase 2 culture EJ38 e,n,z 6.8 x not hag

EJ40 I,2 6.1 x 10-5 hag EJ43 l>2 1.4 x 10-5 hag

EL10: HI- i ahl, H2-1,2 uh2+: Fla- e Fla+ phase var., phase 2 culture

EJ44 e,n,x 8.0 x IO-" not hag SL4266: Hi-i ahl , H2-e,n,xon uh2-: EJ4S e,n,z 2.6 x 1 ~ - 5 hag fixed in phase 2, Fla+ EJ46 e,n,z 1.1 x 1 ~ ~ 5 hag

hag 1.4 x 10-5 EJ47 e,n,x

EJ48 1 2 3.9 x 10-5 han EJ49 1,2 2.1 x 10-5 hag EJ5O 12 5.2 x 10-6 hag EJ51 1 2 3.3 x 10-5 hag EJ52 1,2 2.2 x 10-6 hag EJ53 7.5 x 10-6 haa

SL4271: HI-i ahl , H2-1,2 vh2+: Fla- Fla+ phase var., phase 2 culture

~~~~~

* The recipient, EJ34 (=K12 i ahl, non-motile because of inactivation of its flagellin-specifying gene i by a h i ) was treated with Pltc grown on the indicated S. typhimurium donor strains. Motile transductants were selected on semisolid medium with anti-i serum, o r with anti-i + anti-b serum when the donor was SL4213, Hi-b. Yield of swarms was between 1 and 5 per ID9 pfu, in all four combinations. + Rate of variation from Fla+ to Fla-/bacterium/generation.

H2 O F SALMONELLA TRANSDUCED TO K12 605

H2-e,n,x vh2+ donor; it produced Fla- clones at a frequency of ca. 6.8 X bacterium/generation. The other exceptional clone was EJ44, with antigen e,n,x derived from an H2-e,n,x vh2- donor; its rate of production of non-flagellate variants was ca. 8 X 104/bacterium/generation. The rates of variation from phase 2 to phase 1 in two diphasic S. typhimurium stxains, examined as controls, were 1.6 X IO4 for SL4213 (HI-b , H2-e,n,x vh2+) and 1.9 X lo4 for SL870 (HI- i ahl; H2-1,2 v h 2 + ) . The exceptional e,n,x transductants, EJ38 and EJM, produced Fla- variants at such a high rate that they were easily detectable as LP-type colonies among the predominance of SD (small, dense, because flagellate) colonies when samples of broth cultures were plated directly on semisolid medium containing anti-e,n,x serum-that is, without use of the procedure for enrichment of Fla- cells (see MATERIALS AND METHODS). Table 4 records the rate of production of non-flagellate variants by EJ38 and ET44, and, for comparison, rate of production of phase-I clones by a phase-2 clone of a diphasic S. typhimurium, SL4213, determined by such direct plating; and also the rates of production of Fla- clones (measured by the enrichment method) for the K12 i parent, EJ31, and representatives of the other sixteen K12 lines with antigen e,n,x or 1,2. From these observations it seems that two transductants, EJ38 and W44, have each incorporated not only a flagellin-specifying gene but also the linked determinant (or possibly part of the same gene) which confers the ability to alternate between “active” and “inactive” states, that is to manifest phase-variation; but that the other transductants, six with antigen e , n j , and ten with antigen 1,2,

TABLE 4

Measurement of rate of variation from Flu+ to Fla- in representative K12 e.n.x and 1,2 transductants, and in K12 i and diphasic S. typhimurium as controls

Strain Number Fla-/Number tested

EJ38*, clone 1 11 7/448 EJ38, clone2 94/448 EJ38, clone 3 124/427 EJ44*, clone 1 37/640 EJ44, clone 2 28/640 EJ44, clone 3 17/620

EJ48t 2.6 X 106/1.46 x l O Q SL4213$ (HI-b Ha-e,n,x) 5/1309 SL87W (HI-iahl H2-1,2) 6.6 X 106/1.12 X l O Q EJ31-t (K12 i) 4.0 x 105/1.35 x IO9

EJ39-f 1.0 x 105j2.22 x 109

Number of

31.6 31.5 30.9 29.6 30.0 29.0 32.1 31.4 32.0 31.0 31.3

Rate, Flat + Fla-

8.3 x 10-3 6.7 x 10-3 9.4 x 10-3 2.0 x 10-3 1.5 x 10-3 9.5 x 10-4

5.7 x 10-5 1.6 x lo“ 1.9 x 10-4 9.5 x 10-6

1.4 X

* In experiments on EJ38 and EJ44, Fla- colonies (detected by LP appearance) were counted on diluted samples plated directly on semisolid medium with anti-e,n,x serum, without preliminary serum treatment to remove most Fla+ cells. t In experiments on these strains numbers shown are number of Fla- colony-forming units/ml

in sample treated with serum to remove most Fla+ and total number of colony-forming units/ml in sample taken before serum treatment.

$ In the experiment on SL4213 (diphasic and motile in each phase) rate of change from phase 2 (antigen e,n,x) to phase 1 (antigen b ) was measured by direct plating on semisolid medium with anti-e,n,x serum. Phase-1 clones were recognized as swarms.


have each incorporated a flagellin structural gene but not the factor required fo r phase variation.

Genetic analysis of Fla- variants of K12 e,n,x and 1,2 transductants: Fla- variants of EJ38 and EJ44, the two K12 e,n,x lines thought to show phase variation, were stabbed to semisolid medium; all of them produced swarms. The Flaf “revertants” thus obtained in turn gave Fla- variants at high frequency, detectable as LP colonies when broth cultures were streaked on semisolid medium containing anti-e,n,x serum. Fla- variants of the other 16 transductants similarly tested either produced no swarms o r gave swarms at obviously lower frequency than the Fla- derivatives of EJ38 and EJ94. Fire Fla- variants from each of EJ38 and EJ44 and from seven of the other sixteen K12 e,n,x and I,2 transductants were tested as transductional recipients, to see if they behaved as expected for cells lacking flagella only because their flagellin-specifying gene was “inactive.” or like cells containing an “active” flagellin gene but non-flagellate because of a flu- mutation. In one experiment the non-flagellate recipients were treated with P1 kc grown on EJ34 (=K12 i ahl-) and plated on semisolid medium: none of ten Fla- clones derived from EJ38 and EJ44 produced trails, as expected if their lack of flagella resulted from inactivity of their flagellin-specifying gene, since the only flagellin-specifying gene in the K12 donor, HI-i, is also inactive, because of presence of ahl-. By contrast all the Fla- clones derived from other K12 e,n,z or 1,Z lines (five each from EJ39, EJ45, EJ48, EJ49 and EJ53) produced trails when treated with the same lysate, as expected if all these Fla- variants resulted from mutation at flu loci. In another experiment the same set of Fla- variants were treated with phage grown on W31, which is K12 i, motile; samples of the transduction mixtures were plated on semisolid medium without serum or with antiserum against the antigen, e,n,x or 1,2, of the motile parent of the Kl2 Fla- recipient concerned. The ten Fla- clones derived from EJ38 and EJ44 all produced trails even in medium with anti-e,n,z. serum; this is the result expected if, in these recipients, the e,n,x-specifying region is inactive, since no flagellin of specificity e,n,x will then be manufactured by abortive transductants containing the i-specifying gene of the donor. By contrast the 25 Fla- clones obtained from five of the other sixteen K12 e,n,x or 1,2 transductants all produced trails on semisolid medium, but not on the same medium with anti-e,n,x or anti-1,2 serum; this is the result expected if these Fla- clones result from flu mutation, since abortive /la+ transductants will express both their endogenote e,n,x or 1,2 gene and the exogenote i-specifying gene (PEARCE and STOCKER 1965; ENOMOTO 1967). Thus, both in respect of reversibility of change between Fla+ and Fla- and of behavior as transductional recipients, the Fla- clones produced at high frequency by two K12 e,n,z transductants seem to result from spontaneous, reversible loss of activity of their gene specifying flagellin e,n,r; but those obtained at low frequency from the other K12 e,n.x and 1,2 transductants behave like flu- mutants.

Return of e,n,x and 1,2-specifying genes from K12 to Salmonella by transduction: It should be noted that EJ44, one of the K12 e,n,x transductants showing high frequency of Fla+eFla- variation, had been obtained by treatment of EJ34

H2 OF SALMONELLA TRANSDUCED TO K i 2 607

(K12 i ahl-) with Plkc grown on an LT2 H2-e,n,xon vh2- strain, SL4266, i.e., one fixed in phase 2. The high frequency of Fla+*Fla- variation in EJ44 might have resulted either from incorporation of the e,n,x-specifying gene without incorporation of the linked phase-stabilizer, vh2-; or from failure of a co-transduced vh2- allele to prevent alternation of the adjacent e,n,x gene between active and inactive states when integrated with it into the K12 "chromosome". Phage Plkc grown on EJ44 was therefore applied to SL4273 (LT2, restriction-negative, HI-b, H2-1,2 vh2+ and therefore showing normal frequency of phase variation); selection was made for motility in the presence of antiserum against the recipient flagellar antigens, b and 1,2. Two sorts of swarms with antigen e,n,x were obtained (Table 5 ) ; two of the 20 tested showed normal frequency of phase variation between phase 1 (antigen b) and phase 2 (antigen e,n,x) and were inferred to be vh2+ and to have incorporated the e,n,z gene of the K12 donor in place of the recipient H2 allele, H2-1,2. The other 18 had antigen e,n,x and changed to phase 1 (antigen b) either at very low frequency (1 1 clones) or not at all (7 clones) ; they are inferred to have incorporated both an e,n,x gene and a linked vh2- from the K12 donor, EJ44, which must therefore possess vh2- even though it shows a high frequency of phase variation (Fla+*Fla-). EJ38, the other K12 e,n,x line showing frequent Fla+*Fla- variation, was also inferred to carry both an e,n,x gene and a vh2 allele, because phage grown on it transduced (i) an e,n,x-specifying gene to an HI-b, H2-1,2 vh2+ recipient, SJ2468, and (ii) ability to show a normal frequency of phase variation (and by inference gene vh2+) to SJ2470, which LT2 HI-b H2-e,n,xon vh2-.

Phage P1 grown on three K12 e,n,x or 1,2 transductants not showing rapid Fla+*Fla- variation was applied to diphasic S. typhimurium recipients with phase-2 antigen 1,2 or e,n,x and selection was made on semisolid medium with sera against each of the recipient H antigens; transductants with the antigen of the K12 donor, e,n,x or 1,2, were obtained. These transductants changed phase at a normal rate; but, contrary to expectation, the second antigen then revealed was not the recipient phase-I antigen, b, but its original phase-2 antigen, either 1,2 or e,n,x according to the recipient used (Table 5 ) . This indicated that the flagellin-specifying gene transduced from any of these three E. coli K12 e,n,x or 1,2 donors had replaced not the H2 but the HI allele, HI-b, of the diphasic recipient-even though both the e,n,x and the 1,2 gene had been originally H2 alleles in Salmonella. A probable explanation was that the e,n,x or I,2 gene in each of the three K12 transductants concerned was located at the hag region of the K12 chromosome, where it had replaced the i ahl- of EJ31, and that on transduction back to S. typhimurium the homology of neighboring genes with the corresponding genes in the HI region of the Salmonella recipient resulted in incorporation there, rather than at the H2 region with which the flagellin- specifying gene itself would be homologous. If this explanation was correct it would suggest that the e,n,x gene in each of the two K12 e,n,x clones which did show phase variation (Fla+eFla-) was located in the K12 chromosome elsewhere than the hag region, so that on transduction back to S. typhimurium it would be incorporated at H2, because of the homology of the transduced flagellin structural


c 7

I , + + - c


gene itself and of the co-transduced vh2 allele with the chromosomal H2-vh2 region of the diphasic S. t yphimurium recipient.

To test this possibility Plkc (or Pivir) was grown on all the eight K12 e,n,x and ten K12 1,2 transductional clones (Table 3) and the lysates were applied to EJ35, a fZa mutant (by complementation corresponding to fZaK of Salmonella) of EJ31 (=K12 i). All eighteen mixtures gave both trails and swarms, as expected considering that all eighteen donors are fZa+. On semisolid medium with anti-i serum none of the mixtures produced trails, again as expected since fZa+ abortive transductants normally express their chromosomal flagellin-specifying gene, in this case i. Swarms, shown on subculture to have the H antigen of the donomr, e,n,x or 1,2, were obtained from sixteen of the mixtures, but not from those in which the donor was EJ38 or EJ44, the K12 e,n,x clones showing phase variation (Fla+eFla-) and suspected to have e,n,x integrated elsewhere than the K12 hag region. In the sixteen crosses in which fZa+ complete transductants with the donor flagellar antigen were obtained, the donor flagellin-specifying gene must have been co-transduced with the fZa gene concerned, as expected if located at hag, for in S. typhimurium crosses HI is co-transduced (at a frequency ca. 69%) with fZaK+ by phage Plkc (ENOMOTO and STOCKER 1974). For the same reason the failure of the lysates of EJ38 and EJM to evoke fZa+ e,n,x swarms suggests that in each of these two K12 derivatives the e,n,x gene is located elsewhere than the hag region, so that it cannot be co-transduced with the hag-linked fZa gene.

Test of repressing ability and repressibility of 1,2 and e,n,x genes integrated into K12 chromosome: As mentioned above, anti-i serum prevented trail formation by EJ35, the “fZaK’ mutant (Salmonella terminology) of K12 i, when treated with P1 grown on any of the eighteen K12 e,n,x or 1,2 clones. In such crosses, all or nearly all the fZa+-containing chromosome fragments transduced from the sixteen K12 lines with an e,n,x-specifying or 1,Z-specifying gene integrated at hag would be expected to contain also the flagellin-specifying gene; the preven- tion of trail formation by anti-i serum therefore indicates that the inferred excr- genote e,n,x or 1,2 gene lacks the HI- (and hag-) repressing ability characteristic of “active” H2 alleles in diphasic Salmonella and Salmonella-Escherichia hybrids (MAKELA 1964). For a more direct test of the repressing activity, lysates of all eighteen K12 e,n,x and Kl2 1,2 clones were applied to EJ31 (=K12 i, motile) and the mixtures were streaked on semisolid medium with anti-i serum. Some swarms but no trails were obtained from the mixtures made with lysates of the sixteen K12 clones not showing Fla+*Fla- phase variation and inferred to have an e,n,x or 1,2 flagellin gene integrated at hag; this result confirms the inferred inability of these integrated flagellin-specifying genes to repress the endogenote i. By contrast the same recipient treated with phage grown on EJ38 or E344 produced many trails on semisolid medium with anti-i serum; thus the e,n,x-specifying gene in each of these two K12 lines (or, more probably, a closely linked HI- repressor gene, rhl) when present as exogenote repressed the endogenote i gene, located at hag. Similarly phage grown on EJ38 and EJ44 when applied to the ten K12 1,2 clones not showing Fla+eFla- phase variation evoked formation of trails by all of them on semisolid medium with anti-i,2 serum; this shows that


the e,n,x alleles (or closely linked Hl-repressor gene) of EJ38 and EJ44 can repress l ,2 genes integrated at hag. It thus appears that in Kl2 derivatives EJ38 and EJ44 the e,n,x gene (or an HI-repressor gene, rhl, closely linked to it), incorporated in the K12 chromosome elsewhere than at hag, retains ability to repress a flagellin-specifying gene located at hag; but that the e,n,x or l ,2 genes, incorporated at hug in the other sixteen K12 derivatives, are unable to repress an allele at hug and are themselves susceptible to repression by a product of the e,n,x gene cluster incorporated somewhere in the chromosome of derivatives EJ38 and EJ44.

DISCUSSION

Phage Plkc grown on S. typhimurium derivatives was applied to a K12 line given, by successive transductions from S. typhimurium, first a f l d gene and the linked determinant for the phase-1 antigen i, then gene uh1-, preventing expression of i and so causing loss ol motility. Selection for motile transductants expressing a phase-2 Salmonella flagellar antigen ( l ,2 or e,n,z, according to donor) yielded 18 such clones, eight with antigen, e,n,x and ten with antigen 1.2 (Table 3). Two of them, EJ38 and EJ44, produced Fla- variants at frequencies > 1 O-3/bacterium/generation (Table 4) : these variants reverted at high frequency and in genetic tests behaved as though their lack of flagella resulted only from inability to synthesize flagellin, because of loss of activity of their e,n,x determinant. It was inferred that EJ38 and EJ44 were showing phase variation, manifest as Fla+ -+ Fla- variation because in them passage of the phase determinant into the nonactive state by preventing synthesis of e,n,x prevents synthesis of any flagellin. This showed that the Salmonella genes incorporated in clones EJ39 and EJ44 include the postulated phase determinant, very closely linked to (perhaps part of) the flagellin-specifying gene. On transduction of the e,n,z determinants of EJ38 and EJ44 back to diphasic S. typhimurium recipients of appropriate vh2 character, they were incorporated at H2, corresponding to their ancestral location, in each case accompanied by the vh2 allele of the original Salmonella donor. Phage Plkc grown on EJ38 and EJ44 evoked fla+ abortive transductants (trails) not expressing the endogenote i from a flu mutant (by complementation corresponding to fZaK of Salmonella) of the E. coli K12 i line; this shows that the H2 region incorporated in EJ38 and EJ44. when in “active” state. prevented expression of a flagellin-specifying gene in the hug region. Thus the genes from the H2 region of Salmonella incorporated in EJ38 and EJ44 include rhl. the HI repressor gene.

The evidence thus suggests that in the K12 e,n,x transductants EJ38 and EJ44 the incorporated genetic material of Salmonella origin comprises all the known components of the H2 region, viz. the flagellin-specifying gene, the HI-repressor rhl and the phase determinant whose ‘Lstate” determines activity or inactivity of these two genes, and also the adjacent phase-stabilizer gene (or region) vh2. The site of integration of e,n,x in the E. coli chromosome was not investigated in the present examination. Preliminary results ( ENOMOTO 1974) indicate a location near 50 min on the standard E. coli map, between lys and nalA, in both clones;

H2 O F SALMONELLA TRANSDUCED TO Ki2 61 1

in the case of EJ44 but not of EJ38 the flagellin-specifying gene is co-transduced at detectable frequency with tyr or pheA.

In EJ38 and EJ44 the rate of variation from Fla+ to Fla-, attributed to change of integrated phase determinant from active to nonactive state, was greater than the corresponding rate of variation from phase 2 to phase 1 in the Salmonella strain from which the e,n,x gene and phase determinant were derived. Thus in EJ38 (K12 e,n,r: vh2+) the rate of Fla+ 4 Fla- change, ca. 8 X 10-3/bacterium/ generation, was about fifty times greater than the rate of change from phase 2 to phase 1, ca. 1.6 X 10-4/bacterium/generation, in the diphasic vh2+ donor parent, SL4213. The molecular basis of change of state of the phase determinant is unknown, so that many possibilities might be considered to account for the difference in rate according to bacterial host. If change from active to inactive state results from gradual accumulation of a hypothetical H2 repressor substance the substance might accumulate more rapidly in E. coli cells than in Salmonella cells. If change of phase results from a crossing over event between more or less homologous segments of an inverted repeat (LEDERBERG and STOCKER 1970) or of insertion sequences such as Zsl, then an altered frequency of such events according to genetic background would not be surprising: compare the high frequency with which a large R plasmid converts from one circle into two smaller circles in Proteus cells. despite its stability in E. coti (see review by CLOWEE 1972). The rate of Ma+ 4 Fla- change in clone EJ44 (Kl2 e,n,x vh2-) was about 1.5 x 1 0-3/bacterium/generatic4n. about one-fifth the rate in EJ38, but a t least X104 the just detectable rate of change from phase 2 to phase 1 in Salmonella strains which are uh2- (IINO 1961b, 1969; VARY and STOCKER 1973). As the way in which the vh2- gene or region (of S. abortuse-equi origin) prevents phase variation in Salmonella is unknown, it would be premature to speculate as to possible reasons for the almost complete failure of uh2- to prevent change of state (in either direction) of the phase determinant integrated with it into the K12 chromosome in clone EJ44. In a comparable investigation MXKELA (1964) measured the rates of change of phase, in each direction, for several diphasic hybrids resulting from integration of the H 2 region of an S. abony Hfr donor into the chromosome of an E. coZi 0100 F- recipient. In two hybrids the rates of change were about the same as in the Salmonella parent, i.e. ca. 5 x IO-*/bacterium/ generation from phase 2 to phase 1 and about one-tenth this rate for the reverse change; in two other hybrids the rates were lower, 0.16-0.4 of the rates in the S. abony parent.

The only two K12 transductants with a gene specifying a phase-2 flagellin integrated elsewhere than at hag were both from crosses in which the donor carried HZ-e,n,x, either H2-e,n,x uh2- from S. abortus-equi or HS-e,n,x vh2+ from S. abony. No K12 transductants showing phase variation (as Fla+ 4 Fla- variation) were observed in tests of ten K12 1,2 clones from crosses in which the donor had the 1,2 region of S. typhimurium (nor among an additional 13 clones of the same sort, obtained in a repeat experiment, not included in Table 3). However more clones would have to be analyzed, to permit decision as to the significance of the different results according to H2 allele involved.


Most of the K12 transductants acquiring a Salmonella phase-2 antigen (all of ten with antigen I,2, six of the eight with antigen e,n,x) failed to show the rapid Fla+ 4 Fla- variation observed in clones EJ38 and EJ44 and attributed to change of state of an incorporated phase determinant from active to inactive. Further- more the Fla- variants which they did produce, at low frequency, behaved, in respect of reversion frequency and in genetic analysis by abortive transduction, in the way expected for f l n mutants. A probable explanation of absence of frequent reversible change of activity of the incorporated e,n,x or 1,2 genes is that the phase-determinant element has not been incorporated into the chromosome along with the flagellin structural gene. On transfer back to diphasic S. typhimurium recipients (Table 5 ) the ten 1,2 and six e,n,x genes all behaved as alleles of HI, not of H 2 as they originally were. This indicates that in these 16 clones the structural gene for I , Z or e,n,x flagellin has been incorporated at hag, replacing the i ah1 genes which themselves had replaced the original hag gene of K12. Independent proof of the incorporation of the 1,2 o r e,n,x genes in the vicinity of hag was the observation of their co-transduction with a flu gene which by complementation corresponds to the HI-linked flaK of Salmonella. The sixteen I,2 or e,n.x genes as exogenotes in abortive transductants did not repress an endogenote gene specifying flagellin of type i, known to be susceptible of repression by an active H2 region: this suggests that the HI-repressor gene, rhl , was not incorporated with the flagellin determinant in the 16 clones. Moreover the ten I ,2 determinants as endogenote were susceptible to repression by an active e,n.x region. introduced by abortive transduction. This suggests that the crossover events by which the I,2 gene was incorporated did not displace the postulated receptor for HZ-repressor substance. Presumably the hug region of E . coli (and the HI region of Salmonella) includes a proximal element (HI-operator?) which binds a repressor protein specified by rhI and, distal to it, a segment which includes ah1 (perhaps a promoter element, o r perhaps merely a site in the flazellin structural gene outside the part of it which specifies the serologically determinant part of the polypeptide) and also “hug” (or “HI”), in the operational sense of the determinant of the serological character of the flagellar protein. If so, then the sixteen transductants with 1,2 o r e,n,x integrated at hag could have arisen by replacement of only the ahl-i segment of the K l 2 i ah1 recipient by a functionally corresponding segment from the H2 region of the donor, without either replacement of the recognition site for the HI -repressor substance or integration of any of the other elements of the H2 region, i.e., phase-determinant and rhl and vh2 genes.

The usual incorporation of the HZ (Salmonella) alleles specifying flagellins e,n,x or I,2 at the hag region of the recipient, with replacement of the determinant for flagellin i and of ah2 indicates that these H2 alleles still possess some synaptic homology with the HI allele determining flagellin of type i. The functional equivalence of HI and H 2 of Salmonella is evident both from the phenomenon of phase variation and from the equal ability of exogenote HI genes (if not repressed by recipient H 2 region) and exogenote H2 (if an ‘‘active’’ state) to confer motility on recipients deficient only in ability to synthesize flagellin


(PEARCE and STOCKER 1967; this paper). A region or regions of the flagellin structural genes, specifying segments of the polypeptide chain important for polymerization into helical filaments, may have remained relatively unaltered during the evolutionary divergence of these genes; such a region or regions would presumably facilitate crossing over between synapsed H I and H 2 genes. In the present work the identity of the flagellin specified at the hag region as of type i or e,n,rr: or 1,2 rests only on agglutination reactions. All of the serological specificity of Salmonella flagellin of type f,g has been shown to reside in a single cyanogen-bromide-generated fragment, made up of about half the flagellin molecule (PARISH, WISTAR and ADA 1969). It is therefore possible that each of the H2 alleles incorporated in the hag region of the K12 chromosome consists of only a part of the original allele, inserted by crossovers one or both of which are within the flagellin structural gene. The same considerations would apply to other known or suspected examples of replacement of an H2 allele by an H1 allele, or vice versa: viz. the isolate of S. paratyphi B type iava carrying an HI allele specifying flagellin of antigenic type I ,2 (normally encountered only as a phase2 antigen) (LEDERBERG 1961) and the exceptional transductant in which the H I allele of the donor, specifying antigen b, has been incorporated at the H 2 locus of the diphasic S. abony recipient (IINO 1 9 6 1 ~ ) .

The phenomena here reported may have two applications. The availability of Salmonella with flagellin structural genes originally derived from H2 but now located at H I , in the well-mapped Hl,fZa,mot,nmZ region should facilitate fine- structure mapping of HZ. The E. coli K12 lines each with a single Salmonella flagellar antigen, i o r 1,2 or e,n,z, may prove useful in production of anti- flagellin sera, since the somatic antigen of the rough E. coli K12 will not evoke antibodies affecting smooth Salmonella of any 0 group.

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