TheevolutionofdevelopmentinStreptomyces ......which development is initiated, or to changes in cellular structure or morphology. Introduction: morphology, ecology, phylogenyand evolutionary

Theevolutionofdevelopment inStreptomyces analysedbygenomecomparisonsKeith F. Chater & Govind Chandra

John Innes Centre, Norwich Research Park, Colney, Norwich, UK

Correspondence: Keith F. Chater,

Department of Molecular Microbiology, John

Innes Centre, Norwich Research Park, Colney,

Norwich NR4 7UH, UK. Tel.: 144 1603

450297; fax: 144 1603 450778; e-mail:

[email protected]

Received 21 February 2006; revised 10 April

2006; accepted 12 April 2006.

First published online 22 June 2006.

DOI:10.1111/j.1574-6976.2006.00033.x

Editor: Simon Cutting

Keywords

comparative genomics; evolution of

development; sporulation; actinobacteria;

Streptomyces .

Abstract

There is considerable information about the genetic control of the processes by

which mycelial Streptomyces bacteria form spore-bearing aerial hyphae. The recent

acquisition of genome sequences for 16 species of actinobacteria, including two

streptomycetes, makes it possible to try to reconstruct the evolution of Strepto-

myces differentiation by a comparative genomic approach, and to place the results

in the context of current views on the evolution of bacteria. Most of the

developmental genes evaluated are found only in actinobacteria that form

sporulating aerial hyphae, with several being peculiar to streptomycetes. Only four

(whiA, whiB, whiD, crgA) are generally present in nondifferentiating actinobacter-

ia, and only two (whiA, whiG) are found in other bacteria, where they are

widespread. Thus, the evolution of Streptomyces development has probably

involved the stepwise acquisition of laterally transferred DNA, each successive

acquisition giving rise either to regulatory changes that affect the conditions under

which development is initiated, or to changes in cellular structure or morphology.

Introduction: morphology, ecology,phylogeny and evolutionary history ofstreptomycetes

Streptomycetes are among the most complex of bacteria

(Chater & Losick, 1997). They grow, usually in soil, as

branching thread-like hyphae to form a vegetative or

substrate mycelium. This growth habit, coupled with the

activities of an abundance of extracellular hydrolytic en-

zymes, helps them to gain access to the nutrients locked up

in the insoluble polymers of soil. As the substrate mycelium

becomes nutrient-limited, it goes on to manifest two

properties for which streptomycetes are particularly famous:

the production of antibiotics and other secondary metabo-

lites; and the formation of reproductive aerial branches,

which are at least partially parasitic on the mycelium from

which they emerge. The aerial hyphae form aseptate tip

compartments up to c. 100 mm long, often containing many

tens of copies of the genome. When such a compartment

stops growing, it undergoes synchronous multiple septation

to give a string of unigenomic prespore compartments. The

cell walls of prespore compartments undergo thickening and

rounding up, and usually accumulate spore pigment, as they

turn into chains of spores. Unlike the highly resistant

endospores of bacilli, Streptomyces spores do not show

conspicuous resistance to heat, but they do survive well in

including dry conditions, and they presumably facilitate

dispersal by wind and water, and via animals. Although

most streptomycetes do not seem to sporulate in submerged

culture, a few can (including some strains of Streptomyces

griseus and Streptomyces venezuelae). Presumably, differ-

ences in the detailed developmental regulatory circuitry are

involved in this departure from the norm, but at the time of

writing, there is insufficient published information for us to

consider this interesting question here.

Phylogenetically, streptomycetes are part of the Actino-

bacteria, the class of gram-positive and morphologically

diverse bacteria that have DNA comparatively rich in G1C.

Among free-living actinobacteria, G1C content ranges from

54% in some corynebacteria to more than 70% in strepto-

mycetes, but in the obligate pathogen Tropheryma whipplei it

is less than 50%. Figure 1 is a phylogenetic tree of sequenced

actinobacteria based on 16S rRNA gene sequence compar-

isons. It appears that the last common ancestor of the

actinobacteria lived about 2–1.5 Ga, when evidence suggests

that the atmosphere first began to be oxygenated (Embley &

Stackebrandt, 1994). Streptomycetes are believed to have

originated about 440 Ma, apparently immediately following

the invasion of the land by green plants, and coinciding with

a rapid rise of oxygen in the atmosphere towards present

FEMS Microbiol Rev 30 (2006) 651–672 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

levels (Heckman et al., 2001). Reflecting this timing, strep-

tomycetes are particularly well adapted to the utilization of

refractory plant remains, are aerobic, and have a great

number of genes and systems associated with oxidative

stress (Lee et al., 2005). Earthworms and arthropods such

as springtails that consume decaying plant remains, includ-

ing the associated microorganisms, appeared soon after the

first land plants, so it is quite possible that they exerted other

major selective pressures on Streptomyces evolution. For

example, the frequent passage of most soil through earth-

worms (Darwin, 1881) must have selected for resistance of

Streptomyces spores to the chemically and enzymically harsh

conditions in worm guts. The frequent consumption of

small soil animals by vertebrates (such as reptiles, small

mammals and birds) in more recent evolutionary time,

while it may have contributed to the dispersal and diversi-

fication of streptomycetes, could not have been a funda-

mental element in their early evolution.

Comparative genomics of actinobacteria:the bigger picture

In the last few years, genome sequences have been obtained

for diverse actinobacteria (19 complete and annotated

genomes, 16 species, 10 genera at the time of writing), as

indicated in Table 1. The analysis in this review is largely

confined to these sequenced genomes, about which more

comprehensive statements can be made. Of the two se-

quences from streptomycetes, one is from the academic

model organism Streptomyces coelicolor A3(2) (note that this

strain is improperly named, being more similar to the

Streptomyces violaceoruber clade; but the name is too deeply

enshrined in the literature to justify making the correction),

and the other is from Streptomyces avermitilis, an important

antibiotic producer (Bentley et al., 2002; Ikeda et al., 2003).

These two species are phylogenetically fairly well separated

from each other and from Streptomyces griseus, another

species that has been important in the study of development,

and whose genome sequence is currently being determined

(S. Horinouchi, pers. commun.). The genome sequence of

another species, the plant pathogen Streptomyces scabies,

is also available, though currently not annotated (ftp://

ftp.sanger.ac.uk/pub/pathogens/ssc/Ssc.dbs). This makes it

possible to use comparative genomics to attempt to trace the

evolution of Streptomyces development, and in doing this,

perhaps to deepen our understanding of developmental

mechanisms.

For such an approach, criteria must first be adopted for

whether related genes can be considered to be orthologues

(i.e. are the direct, functionally equivalent derivatives of a

gene present in the last common ancestor of the species

being compared) or are merely paralogues (related through

ancestral gene duplication). A first criterion is sequence

relatedness between gene products. One would expect most

(though not all) orthologous genes to show a fairly consis-

tent degree of amino-acid identity in comparisons between

particular organisms. This ‘orthology value’ could be deter-

mined by initially making assumptions about obvious

candidates for orthology, such as genes for RNA polymerase

core enzyme subunits, or genes for the biosynthesis of

amino acids etc. (as long as such candidate genes are

represented only once in the genomes). We compared seven

such gene products among S. coelicolor, S. avermitilis,

Thermobifida fusca, Corynebacterium glutamicum and My-

cobacterium tuberculosis (Table 2). Within this set of

orthologues, comparisons between the two streptomycetes

indicated a range of 88–97% amino-acid identity, while

identities between S. coelicolor and the other organisms

ranged from 69–82% (T. fusca) to 52–71% (C. glutamicum).

On the other hand, when sets of multiple paralogues

are encoded within one organism, members of each

set are generally well under 50% identical. We therefore

used 50% as a threshold value when considering

orthology in the developmental genes discussed in this

article.

The second criterion for orthology takes advantage of

similarity of gene arrangement along the chromosome

(‘synteny’), which is usually extensive between members of

the same genus. Across different genera of actinobacteria

there are many patches of local synteny, and there is a

0.10

100 Mtb

MboMav

MleNfa

CdiCefCgxCgl

Pac

TfuSco

Sav

BloSth

Bsu

100100

100

100

1009970

97

75100

100

7987

98

LxyTwaTwx

Fig. 1. Phylogeny of 16S rRNA gene of sequenced actinobacterial

genomes. The analysis used the Phylip package. Abbreviations are as

follows: Mtb, Mycobacterium tuberculosis; Mbo, Mycobacterium bovis;

Mav, Mycobacterium avium; Mle, Mycobacterium leprae; Nfa, Nocardia

farcinica; Cdi, Corynebacterium diphtheriae; Cef, Corynebacterium effi-

ciens; Cgx, Cgl, Corynebacterium glutamicum; Tfu, Thermobifida fusca;

Pac, Propionibacterium acnes; Sco, Streptomyces coelicolor; Sav, Strep-

tomyces avermitilis; Lxy, Leifsonia xyli; Twa, Twx, Tropheryma whipplei;

Blo, Bifidobacterium longum; Sth, Symbiobacterium thermophilum; Bsu,

Bacillus subtilis (outgroup).

FEMS Microbiol Rev 30 (2006) 651–672c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

652 K.F. Chater & G. Chandra

Table 1. Sequenced actinobacterial genomes consulted in this article

Organism

Morphology,

special features Accession number Database Genome attributes

Streptomyces

coelicolor

Mycelial, spore

chains on aerial

hyphae

AL645882 http://streptomyces.org.uk 8.66 Mb, 72.1% GC,

7825 genes,

146 (2%) with TTA

Streptomyces

avermitilis

Mycelial, spore

chains on aerial

hyphae

BA000030 http://www.bio.nite.go.jp/dogan/

MicroTop?GENOME_ID=sav_G1

9.02 Mb, 70.7% GC,

7575 genes,

260 (3%) with TTA

Thermobifida

fusca

Mycelial, spores

clumped on aerial

hyphae

CP000088 http://genome.jgi-psf.org/

draft_microbes/thefu/

thefu.home.html

3.64 Mb, 67.95% GC,

3419 genes,

449 (13%) with TTA

Nocardia farcinica Mycelial, substrate

and aerial hyphae

form fragments

AP006618 http://nocardia.nih.go.jp/ 6.02 Mb, 70.8% GC,

5683 genes,

242 (4%) with TTA

Mycobacterium

avium

Irregular rods AE016958 http://www.cbc.umn.edu/

ResearchProjects/AGAC/Mptb/

Mptbhome.html

4.83 Mb, 69.2% GC,

4350 genes

589 (14%) with TTA

Mycobacterium

bovis

Irregular rods BX248333 http://www.sanger.ac.uk/Projects/

M_bovis/

4.35 Mb, 65.6% GC,

3953 genes

1442 (36%) with TTA

Mycobacterium

tuberculosis

Irregular rods AL123456

AE000516

http://www.sanger.ac.uk/Projects/

M_tuberculosis/

http://www.tigr.org/tigr-scripts/

CMR2/

GenomePage3.spl?database=gmt

4.41 Mb, 65.6% GC,

3999/4189 genes

1462/1489 (36%) with TTA

Leifsonia xyli Rods AE016822 http://leifsonia.lbi.ic.unicamp.br 2.58 Mb, 67.6% GC,

2030 genes

109 (5%) with TTA

Bifidobacterium

longum

Irregular rods,

sometimes

branched

AE014295 2.26 Mb, 60.1% GC,

1727 genes

450 (26%) with TTA

Corynebacterium

efficiens

Rods BA000035 http://www.bio.nite.go.jp/dogan/

MicroTop?GENOME_ID=ce__G1

3.15 Mb, 63.1% GC,

2942 genes

717 (24%) with TTA

Corynebacterium

glutamicum

Rods BA000036

BX927147

http://genome.ls.kitasato-u.ac.jp/

esequenced.html

http://www.uni-bielefeld.de/

3.31/3.28 Mb, 53.8% GC,

3099/3058 genes

1942/1885 (62%) with TTA

Corynebacterium

diphtheriae

Rods BX248353 http://www.sanger.ac.uk/Projects/

C_diphtheriae/

2.49 Mb, 53.4% GC,

2320 genes

1535 (66%) with TTA

Propionibacterium

acnes

Pleomorphic rods AE017283 http://www.g2l.bio.uni-

goettingen.de/projects/

f_projects.html

2.56 Mb, 60.0% GC, 2297

genes

1249 (53%) with TTA

Symbiobacterium

thermophilum

Obligate symbiont;

possibly not

actinobacterial

AP006840 http://genome.ls.kitasato-u.ac.jp/

esequenced.html

3.57 Mb, 68.6% GC,

3337 genes

272 (8%) with TTA

Tropheryma

whipplei

Obligate pathogen BX072543

AE014184

http://www.sanger.ac.uk/Projects/

T_whipplei/

http://www.genoscope.cns.fr/

externe/English/Projets/

projets.html

0.93 Mb, 46.3% GC,

784/808 genes

687/718 (88%) with TTA

Mycobacterium

leprae

Obligate pathogen AL450380 http://www.sanger.ac.uk/Projects/

M_leprae/

3.27 Mb, 57.7% GC,

2720 genes

1972 (73%) with TTA


653Evolution of Streptomyces development

recognizable tendency for a substantial number of genes to

retain a similar relative distance from the replication origin,

even though the overall organization of the chromosomes

has usually undergone many rearrangements (Fig. 2). The

rearrangements mostly involve deletions, insertion of newly

acquired DNA, and more or less symmetrical exchanges

from opposite sides in relation to the replication origin

(Bentley et al., 2002).

Table 2. Percentage amino-acid identity of representative gene products from four actinobacteria to their orthologues in Streptomyces coelicolor

Gene or enzyme

Streptomyces avermitilis Mycobacterium tuberculosis Corynebacterium glutamicum Thermobifida fusca

(70.7% GC) (65.6% GC) (53.8% GC) (70% GC)

Amino-acid biosynthesis

hisA 96.2 68.2 59.9 74.3

hisB 99.0 57.4 52.5 75.8

leuB 93.1 67.8 67.8 69

Intermediary metabolism

Aconitase 92.7 70.4 68.3 78.5

Malate dehydrogenase 94.8 70.8 64.6 82

Transcription

rpoB 95.1 75.3 71.2 80.6

rpoC 96.8 71.6 63.6 78.2

Mean (‘orthology value’) 95.4 68.8 64.0 77.0

S. coelicolor

S. coelicolor

S. coelicolor

S. coelicolor vs S. avermitilis S. coelicolor vs M. tuberculosis

`S. a

verm

itilis

T. fu

sca

S. a

verm

itilis

T. fu

sca

T. fu

sca

M. t

uber

culo

sis

M. tuberculosis

8000

7000

6000

5000

4000

3000

2000

1000

0

3500

3000

2500

2000

1500

1000

500

0

3500

3000

2500

2000

1500

1000

500

0

3500

4000

3000

2500

2000

1500

1000

500

00 1000 2000 3000 4000 5000 6000 7000 8000

0 1000 2000 3000 4000 5000 6000 7000 8000 0 500 1000 1500 2000 2500 3000 3500 4000

0 1000 2000 3000 4000 5000 6000 7000 8000

S. coelicolor

S. coelicolor

S. coelicolor vs T. fusca M. tuberculosis vs T. fusca

M. tuberculosis

S. coelicolor

Fig. 2. Plots of synteny between some actinobacterial chromosomes. The points represent positions of genes whose products reciprocally show closest

relatedness in Blast comparisons between the chromosomes. A simple diagonal line would indicate fully conserved gene arrangement in plots

comparing chromosomes aligned from a comparable point (i.e. the end of linear chromosomes, which have an roughly centrally located replication

origin, in the case of Streptomyces avermitilis against Streptomyces coelicolor; and the origin of replication of circular chromosomes, in the case of

Mycobacterium tuberculosis against Thermobifida fusca). The extent to which the diagonal becomes a cross reflects the occurrence of symmetrical

exchanges on either side of the replication origin. When circular chromosomes are compared with linear ones, synteny is expected to give inflected

plots, because the coordinates of the chromosomes start from different positions. Symmetrical exchanges on either side of the replication origin are

then expected to give a diamond-shaped plot.



Most of the genes that are conserved between streptomy-

cetes and other genera are located in a central core region of

the linear Streptomyces chromosomes. Thus, of the order of

1–2 Mb at each end consists mostly of laterally transferred,

comparatively recently acquired, genes. The core regions of

the S. coelicolor and S. avermitilis chromosomes show

remarkable synteny, involving some 4000 genes. Only a very

small number of exchanges between chromosome arms have

taken place, all more or less symmetrically in relation to the

origin of replication (Ikeda et al., 2003). The synteny is

punctuated by about 2000 nonhomologous genes. This

indicates that the two genomes have been repeatedly ex-

posed to foreign DNA over evolutionary time, with the cores

acquiring new genes or losing old ones on average several

times in every million years in the course of the estimated

220 Myr since the last ancestor common to the two species

(A. Ward, pers. commun.). Nevertheless, despite the numer-

ous insertions and deletions that separate the species, the

conserved gene set has almost entirely retained its organiza-

tion. Some powerful selection must exist to maintain this, so

it is interesting that there are marked differences in genome

organization between different genera. Possibly, new genera

arise initially in response to dramatic or cataclysmic novel

selective circumstances (such as were briefly discussed in the

previous section), when some major rearrangement

of the genome confers an adaptive value that would not be

favourable in gradual adaptation to slowly changing envir-

onmental circumstances. The extensive preservation of

genomic arrangement within streptomycetes suggests that

their subsequent evolution has largely involved microadap-

tation to environments that have changed little until the

present.

Although the 1–2 Mb end regions of Streptomyces

chromosomes are evidently a major primary depot for

receiving horizontally transferred segments of DNA,

their well-documented instability is not well-suited to

long-term retention of particular genes; so those genes

whose selective benefits are repeatedly acted upon may

well find their way into the more stable central region.

There are experimental accounts of unequal exchange

between chromosome ends, and between the ends of

chromosomes and linear plasmids (Chen et al., 2002;

Wenner et al., 2003), such as might permit the effective

migration of valuable DNA towards the central region.

Once a gene has migrated inwards far enough to be

closer to the core than some other selectively advantageous

gene, it will become less susceptible to loss involving

chromosome ends. This mechanism does not preclude

other routes for chromosomal gene acquisition, particularly

those involving transposition or site-specific integration

of specialized elements such as prophages (see final section

for a discussion of the possible roles of phages in gene

acquisition).

The genetic basis of developmentalcomplexity in streptomycetes

Over several decades, some progress has been made in the

genetic dissection of the complex multicellular development

briefly outlined above, particularly in S. coelicolor and S.

griseus (Chater, 2001; Chater & Horinouchi, 2003; Flardh &

van Wezel, 2004). Several tens of genes have been identified

whose products play regulatory, catalytic, organizational or

structural roles in development, and there is a moderate

body of information about the integrated action of these

genes and gene products. Much of this information ema-

nates from the isolation of S. coelicolor mutants that fail to

complete normal development, most being defective either

in aerial growth (‘bald’, or bld, mutants) (Hopwood, 1967;

Merrick, 1976) or in the formation of mature grey spores on

the fluffy aerial mycelium (‘white’, or whi, mutants) (Hop-

wood et al., 1970; Chater, 1972). Most of these mutants are

affected in genes with regulatory character. The details of

these genes and their interactions are explored in several

recent reviews (Chater & Losick, 1997; Chater, 1998, 2001;

Chater & Horinouchi, 2003; Chater, 2006), and are only

briefly summarized here.

When grown near to each other on certain rich media,

most of an early collection of bld mutants exhibit directional

interactions that lead to aerial growth (Willey et al., 1993;

Kelemen & Buttner, 1998). These interactions have been

interpreted as a signalling cascade that is transmitted in the

sequence bldJ/bldK,L/bldAH/bldG/bldC/bldD,M/ram (Fig.

3). Although some of the more recently isolated bld mutants

do not fit in this cascade, its features have helped in the

formulation of developmental models (Chater, 2001). It has

also been shown that some mutants that fall outside the

cascade can be phenotypically corrected by pH buffering,

indicating that their morphological defects are a secondary

SapB

Chaplins, rodlins

Signal 1(oligopeptide)

Signal5

Signal2 Signal

3

Signal4

bldC(regulatory)

bldG(regulatory)

bldH(regulatory)

bldA (tRNA forrare codon

UUA)

bldK(Oligopeptidetransporter)

bldD, M(regulatory)

bldJ(unknown)

bldL(unknown)

ramStartHere!

Fig. 3. Extracellular signalling dependent on bld genes leading to the

production of surface proteins involved in aerial growth in Streptomyces

coelicolor. The figure has been updated from that of Chater (1998).



consequence of the secretion of organic acids (Viollier et al.,

2001). In other species, different extracellular interactions

leading to development have been described. One example,

the restoration of sporulation to a bald mutant of S.

alboniger by the addition of an antibiotic, pamamycin, has

recently been interpreted in terms of calcium signalling

(Danilenko et al., 2005); but the most extensively studied is

the dependence of development in some S. griseus strains on

A-factor, a member of the species-specific g-butyrolactone

signalling molecules that are often involved in switching on

antibiotic biosynthesis in streptomycetes (Horinouchi,

2002). The variety of extracellular signals used during

Streptomyces development may reflect some selective benefit

of preventing competitors from responding to each other’s

signals, and thus be a cause or reflection of speciation

(Chater & Horinouchi, 2003).

Seven of the whi loci (whiA,B,D,E,G,H,I) identified in

early studies of S. coelicolor sporulation (Chater, 1972) have

been studied further [two ‘loci’ have not: whiF99 proved to

be a whiG allele; and whiC mutants did not survive

prolonged storage (C.J. Bruton and K.F. Chater, unpub-

lished)]. Figure 4 indicates the present understanding of the

regulatory interactions of some of these genes. The most

clear-cut gene-to-gene interactions are the requirements of

the whiH and whiI promoters for an RNA polymerase

holoenzyme that contains the s factor specified by whiG.

The only known gene-to-gene interaction between the bld

and whi gene cascades is the repression of whiG by BldD

(Elliot et al., 2001).

In this article we mainly focus on the genes shown in Figs

3 and 4, because it has been possible to fit them into

interaction networks. We also include several other devel-

opmentally and morphogenetically important genes, such as

the ssgA-like gene family, mutations in some of which give a

white colony phenotype. (Keijser et al., 2003; Traag et al.,

2004; Noens et al., 2005). This anthology of genes was used

to search the genomes of actinobacteria for likely ortholo-

gues.

Genes implicated in the regulation offormation of aerial hyphae

bldA -- the determinant of the tRNA for a rarecodon

In streptomycetes, the very high GC content of the DNA

makes it inevitable that GC-rich codons are preferentially

used. As a consequence, the third position in codons is a G

or a C in about nine out of every 10 codons, providing the

basis for one of the main tools used to delineate probable

coding sequences in Streptomyces DNA sequences (Bibb

et al., 1984; Ishikawa & Hotta, 1999). In the standard genetic

code, one of six codons for leucine (UUA) has no G or C

residues. Not surprisingly, this codon is rarely used in

streptomycetes – in fact, it is by far the rarest codon in S.

coelicolor, being used in only 2% (145/7825) of chromoso-

mal genes (260, i.e. 3%, of the 7575 chromosomal genes of S.

avermitilis contain TTA codons) (Fig. 5). These observations

are of more than anecdotal interest, because the only tRNA

capable of reading UUA codons efficiently is the product of a

gene that can be completely deleted without diminishing

growth (Lawlor et al., 1987; Leskiw et al., 1991). However, as

Growth stops(glycogendeposition)

Growth of longapical compartment

Multiple septation(FtsZ, ParAB, SALPs,DNA condensation,glycogen degraded)

Rounding ofpresporecompartments,wall thickening,spore pigmentdeposition

σ WhiG

FtsZ

ParAB

GlgBI etc DNA condensation

WhiB (I) WhiB (II)

WhiA (I) WhiA (II)

WhiI (I) WhiI (II)

WhiH (I) WhiH (II)

σ F

WhiD

MaturationFig. 4. Regulation of aerial hyphal differen-

tiation into spore chains in Streptomyces

coelicolor. The regulatory scheme uses infor-

mation from Kelemen et al. (1996, 1998),

Ryding et al. (1998), Tan et al. (1998), Flardh

et al. (1999), Ainsa et al. (1999), Noens et al.

(2005) and Jakimowicz et al. (2006).



indicated by this gene’s designation (bldA), such mutations

do have phenotypic effects – most obviously, they give rise

to very easily seen bald colonies that are devoid of pigmen-

ted secondary metabolites on most media (Merrick, 1976).

The complexity of this phenotype suggests that bldA may

have a regulatory role in aspects of developmental and

stationary phase biology. The bldA tRNA, with its 30-AAU-

50 anticodon, should also be able to read the alternative

leucine codon UUG via ‘wobble’ interaction, whereas a

tRNA with the 30-AAC-50 anticodon cognate with the UUG

codon is not expected to be able to translate UUA codons

(Crick, 1966). Both tRNAs are present in streptomycetes,

perhaps in part because of the need to translate UUG codons

under conditions in which translation of UUA codons

would be disadvantageous.

A bldA-like tRNA locus is present in all actinobacteria. In

the current absence of any attempts to inactivate it in any

nonstreptomycete, it is not experimentally ruled out that

UUA codons are developmentally significant beyond strep-

tomycetes. However, TTA codons are more frequent in the

genomes of other actinobacteria, though those with high GC

content generally have the lowest frequency (Table 1).

Nocardia farcinica, with 70.8% GC, has TTA codons in only

4% of genes, but these include at least one important gene of

primary metabolism (hisA), so we doubt whether the

developmental role of bldA in streptomycetes has a close

parallel in N. farcinica. Moreover, the distribution of TTA

codons within annotated N. farcinica genes shows less

positional bias than in S. coelicolor and S. avermitilis (Fig.

5), suggesting that they do not fulfil a similar role to that of

TTA codons in streptomycetes: in S. coelicolor, 22% of TTA

codons occur in the first 10 codons, compared to 9% in N.

farcinica (Fuglsang, 2005). Since bldA mutants defective in

aerial mycelium formation have been obtained in species as

diverged as S. coelicolor (Merrick, 1976), S. griseus (Kwak

et al., 1996)and S. clavuligerus (Trepanier et al., 2002), bldA

was probably adopted as an element of the developmental

biology of streptomycetes very early in their evolution.

Although the focus of this article is on morphological

development, we note in passing that the frequently ob-

served effects of bldA mutations on antibiotic production

are mostly exerted directly via UUA codons in the mRNAs of

pathway-specific regulatory genes (Chater & Horinouchi,

2003; Chater, 2006).

In a comparison between the genomes of S. coelicolor and

S. avermitilis, it turns out that only 11 orthologous genes

contain TTA codons in both species (while a further 10 TTA-

containing S. coelicolor genes have TTA-free orthologues in

S. avermitilis. Seven of those 11 have some kind of functional

annotation. One, SCO2792 (bldH), is dealt with in the next

section. The others, and their predicted functions, are:

SCO5495 (cyclic di-GMP phosphodiesterase); SCO1980

and 3423 (both abaA orfA-like, see section on whiJ below);

SCO1434 (cbxX-, cbqX-like); SCO1242 (DNA-binding pro-

tein, WhiJ-like; see below); and SCO4114 (a sporulation-

associated protein; Babcock & Kendrick, 1990). The func-

tion-unknown genes are SCO4395, 5040, 6623 and 7251.

Thus, although a small number of the TTA-containing genes

found in modern streptomycetes were already present early

in the genus, the great majority (83% in S. coelicolor) have

been acquired by lateral transfer in the comparatively recent

evolutionary past.

bldH , SCO2792 (= SAV5261), is the main targetthrough which bldA affects differentiation

One of the 11 TTA-containing genes common to S. coelicolor

and S. avermitilis is adpA, or bldH (Nguyen et al., 2003;

0.35

0.30

0.25

0.20

0.15

0.10

0.05

0.000.1 0.2 0.3 0.4 0.5 0.6

Relative position of the first TTA codon in the genes

Per

cent

of T

TA

con

tain

ing

gene

s

0.7 0.8 0.9 1.0

ScoSavNfar

Fig. 5. The relative positions of TTA codons in

TTA-containing genes of Streptomyces coelico-

lor (black), Streptomyces avermitilis (dark grey)

and Nocardia farcinica (pale grey). Positions of

TTA codons are plotted as a fraction of total

number of codons. Note that in the Strepto-

myces genes the TTA codons are much more

likely to be close to the start of the gene.



Takano et al., 2003). This gene was first studied in S. griseus

(Ohnishi et al., 1999), in which it also contains a TTA codon,

in the same position within the gene as in the other two

orthologues. In S. coelicolor, this TTA codon is the major

route through which bldA influences development, and

AdpA is apparently not involved in regulating antibiotic

production; while in S. griseus the influence of bldA on

antibiotic production is partially mediated via adpA. This

suggests that the way in which adpA is integrated into the

whole physiology of its particular host has some species-

specificity, a notion reinforced by the regulation of adpA

itself. Thus, in S. griseus, adpA expression is directly

repressed by a protein called ArpA (‘A-factor receptor

protein’), repression being lifted in response to an extra-

cellular accumulation of the g-butyrolactone A-factor, a

small membrane-diffusible signalling molecule, which dif-

fuses back into cells and interacts with ArpA. In S. coelicolor,

a comparable g-butyrolactone signalling system seems to

have no marked effects on morphological differentiation or

adpA expression (Horinouchi, 2002; Kato et al., 2002;

Takano et al., 2003; Takano et al., 2005). The differences in

regulation of adpA are possibly reflected in divergences in

upstream DNA sequences; an overall similarity between

these regions of S. coelicolor and S. griseus is interrupted at

the region known to bind ArpA and impart A-factor-

dependence in S. griseus (Takano et al., 2003; Kato et al.,

2005).

From these observations, it seems that the cascade from

bldA via adpA to differentiation is a more ancient aspect of

Streptomyces evolution than the integration of g-butyrolac-

tones into their biology. g-Butyrolactones are themselves

mainly confined to streptomycetes, among which they are

widespread. They exhibit considerable diversity, and may

well have coevolved with antibiotic biosynthetic gene sets

that have been laterally transferred among streptomycetes

(Takano et al., 2005).

In S. griseus, adpA is autoregulatory, contributing to its

own transcriptional repression (Kato et al., 2005). Four out

of six of the AdpA-binding sites of the adpA promoter of

S. griseus are conserved in the adpA promoter regions of

S. coelicolor and S. avermitilis, so autoregulation may be a

general and ancient function of AdpA (Kato et al., 2005).

Although AdpA orthologues are known only in strepto-

mycetes, AdpA belongs to a subfamily within the family of

activator/repressor proteins whose best-known member is

AraC of E. coli (Yamazaki et al., 2004; Kato et al., 2005).

AraC-like proteins are characterized by the features of a dual

helix-turn-helix DNA-binding domain typically located far

from the N-terminus. In the case of AdpA, this domain is

from aminoacids 240–332. Residues 57–195 conform to a

Pfam domain DJ-1 PfpI that is involved in dimerization

(Yamazaki et al., 2004). The rather large size of AdpA

(around 400 aa) suggests that it may respond to some other

molecule, but there are no experimental data addressing this

possibility.

Extensive studies in the laboratory of S. Horinouchi have

revealed a substantial regulon of target genes for AdpA in S.

griseus, and a consensus AdpA binding site has been defined.

Among the target genes are several for proteases and

protease inhibitors (Ohnishi et al., 2005; Kim et al., 2005).

It seems likely that one of the main ways in which AdpA

influences development is via the extracellular interactions

of proteases and protease inhibitors (Chater, 2006). Many

streptomycetes produce extracellular proteases related to

trypsin, the activity of which is held in check by binding to

small proteinaceous inhibitors, such as leupeptin (Taguchi

et al., 1993; Kim & Lee, 1995). In some cases it has been

found that specific proteolysis of the inhibitor by a second

protease is used to activate the trypsin-like protease, which

is then involved in activating development (Kim & Lee,

1995). Since AdpA-dependent extracellular protease: inhibi-

tor interactions are found in phylogenetically diverse spe-

cies, it is likely that they are an ancient feature of

streptomycetes (but among other sequenced actinobacterial

genomes, only that of T. fusca apparently encodes such a

protease inhibitor). The interactions may be important in

the reuse of the biomass of the vegetative mycelium that

provides nutrients for growth of the aerial mycelium (Man-

teca et al., 2006). It will be interesting to find out whether

these interactions have any connection with the extracellular

signalling cascade discussed earlier for S. coelicolor (Fig. 3),

and whether species-specificity is involved, as is often the

case for extracellular signalling.

Several other bld genes appear to be confinedto actinomycetes with sporulating aerialmycelium

In the first two steps in the cascade shown in Fig. 3, bldJ,

which is still uncharacterized, is required for the production

of signal 1, an extracellular oligopeptide that has been

partially characterized (Nodwell & Losick, 1998). Signal 1 is

believed to be imported by a multicomponent ATP-depen-

dent transport system specified by the genes of the bldK

locus (SCO5112–5116; Nodwell et al., 1996). The bldK locus

is absent from other actinobacterial genomes. However, the

next three bld genes ‘downsteam’ of the bldA/adpA stage in

the extracellular signalling cascade are found not only in

streptomycetes, but also in T. fusca, the only other actino-

mycete among those with known genome sequences that has

a well-developed sporulating aerial mycelium (Fig. 6). This

is surprising, in view of the rather wide apparent phyloge-

netic distance between these two genera based on 16S rRNA

gene (Fig. 1), though the comparatively high orthology

value for S. coelicolor and T. fusca (Table 2) suggests that

the two genera may be more closely related than Fig. 1



indicates. There is currently no information bearing on

whether the T. fusca bld gene orthologues play a role in

aerial growth.

bldG , SCO3549 ( = SAV4614)

This gene encodes a member of a protein family found

exclusively in gram-positive bacteria, and with multiple

representatives in many of them (there are 13 in S. coelicolor)

(Bignell et al., 2000). In the characterized cases, these

proteins are known as antagonists of anti-s factors. The

interaction between the two proteins depends on the

phosphorylation state of the anti-anti-s factor. When

phosphorylated, it does not bind to the anti-s factor

efficiently, so the latter protein is free to bind to, and hold

inactive, its target s factor, whereas the unphosphorylated

anti-anti-s sequesters the anti-s, thus releasing the s factor

to interact with RNA polymerase and activate a specific set

of promoters. s factors of a particular subclass are regulated

in this way, the best characterized being sB of B. subtilis

(Chen et al., 2003).

The phosphorylation state of the anti-anti-s is deter-

mined both by the kinase activity of the anti-s factor and by

dephosphorylation by a sensor phosphatase; and a phos-

phorylated form of BldG has been demonstrated (Hesketh

et al., 2002; Bignell et al., 2003). In sporulating bacilli and

clostridia, some of these systems are involved in forespore

development (Errington, 2003) and others, notably sB, in

stress responses (Chen et al., 2003). In those cases, the bldG-

like gene is clustered with the target anti-s factor gene and

the cognate s factor determinant; there are six examples of

such clusters in the S. coelicolor genome. However, although

bldG is adjacent to a gene (SCO3548, = SAV4615) for a

probable anti-s factor, the two genes are not located close

to a recognizable s factor determinant. It remains comple-

tely unknown whether BldG is involved in regulating

activity of any of the nine s B-like proteins encoded

in the S. coelicolor genome. In view of the evidence of

interplay between stress responses and differentiation in

S. coelicolor (Chater, 2001; Kelemen et al., 2001; Viollier

et al., 2003; Lee et al., 2005), it may well be that particular sfactors are subject to regulation by more than one anti-ssystem.

The T. fusca orthologues of bldG (Tfus0984, 77% iden-

tity) and its partner gene are at one end of a region

containing 12 syntenously arranged genes. Several of those

adjacent genes (but not the bldG pair) show widespread

synteny among actinobacteria.

bldC , SCO4091 ( = SAV4130)

This gene encodes a small protein, identical in S. coelicolor

and S. avermitilis, consisting of little more than a MerR

family DNA-binding domain (Hunt et al., 2005). The bldC

noncoding upstream region is conserved for more than

400 bp between S. coelicolor and S. avermitilis (4 90%

identity, interrupted by one nonrelated segment about

310 bp upstream of the coding region in each case). The

400 bp includes (at least in S. coelicolor) 145 bp of tran-

scribed leader sequence. The length of this conserved region

is quite surprising in view of the apparently constitutive

expression of bldC (Hunt et al., 2005). Although there are

other genes encoding single-domain MerR-like proteins in

these and other genomes, none of them is closely related to

bldC except the bldC orthologue (Tfus1491, 75% aminoacid

identity) in T. fusca (Hunt et al., 2005). Tfus1491 is located

at one end of a stretch of six genes that show synteny

with a region of the S. coelicolor genome (with two

additional genes in the streptomycete). Three of these

adjacent genes are present and syntenous in most actino-

bacteria. Figure 7 shows the extent of synteny in this

genomic region, as a typical example of the relative genome

strucures of S. coelicolor, S. avermitilis, T. fusca and other

actinobacteria.

bldD , SCO1489 ( = SAV6861)

BldD is a regulatory protein that possesses a putative DNA-

binding domain similar to those of Xre-like regulators, but

bldJ BldK1

2bldA

AdpA

AdpA*

3

BldG-P

BldG:anti-σ

releasesunknown σ

4 BldC

5

BldD

BldD*

ramRRamRRamR*RamCSAB

SapB

Aerialgrowth

Alsopresent inT. fusca

Peculiar toStreptomyces

BldC*σBldN

BldM

BldM*

bldN

bldMchpA-H

chaplins

Fig. 6. Distribution among sequenced actinobacteria of gene functions

involved in the extracellular cascade for aerial mycelium formation.

Except for blbD (see text) none of the genes for the proteins in this

diagram has orthologues in any characterized genomes apart from those

of streptomycetes and Thermobifida fusca (the latter are indicated on

yellow backgrounds). Gene products remaining inside the cell are inside

coloured areas. The numbered dotted arrows correspond to the extra-

cellular signals in Fig. 3, and unbroken lines or arrows represent likely

sequential events based on the authors’ interpretation of the literature

(see text). Asterisks indicate hypothetical activated forms of proteins.



is not otherwise closely similar to any known class of

regulators (Elliot et al., 1998). It represses its own expression

and that of several other developmental genes including

bldN and whiG (Elliot & Leskiw, 1999; Elliot et al., 2001). It

has been suggested that it may play a role similar to that of

the transition-state regulators SinR and AbrB of B. subtilis

(Elliot et al., 2001). The T. fusca orthologue (Tfus1058; 77%

identity) is located at the end of an 11-gene segment

showing synteny with the bldD region of streptomycetes,

much of which (but not including bldD) is widespread

among actinobacteria. Surprisingly, an apparent orthologue

is present in Kineococcus (Deinococcus) radiotolerans, an

outlying member of the actinobacteria (Accession no.

NZ_AAEF01000025). The bldD orthologues in S. coelicolor,

S. avermitilis and S. griseus show considerable conservation

in the promoter region , including complete identity in a

sequence near the � 10 promoter element shown to bind

BldD in vitro in S. coelicolor and S. griseus (Elliot et al., 2001;

Ueda et al., 2005). There is no such similarity upstream of

the T. fusca bldD orthologue, perhaps indicating that the

gene is regulated differently.

bld genes at the downstream end of theextracellular signalling cascade are peculiar tostreptomycetes

The bldG, bldC and bldD genes, which are also found in T.

fusca (see above), may have roles in the stress responses of

mycelial organisms. Such a role was previously suggested for

Streptomyces bld genes, in a model in which the extracellular

signal cascade was considered as a succession of indications

that all possible alternative responses to hard times had been

tried before commitment to reproductive aerial growth,

which is the ‘last resort’ (Chater, 2001). However, like the

genes involved in the start of the extracellular cascade (bldK,

bldA, bldH/adpA), those at its finish (bldM, bldN, ramC-

SABR) appear to be confined to streptomycetes (Fig. 6).

Conceivably, then, they fulfil roles that are specifically

appropriate to the particular features of Streptomyces aerial

growth.

bldM , SCO4768 ( = SAV4998), and bldN , SCO3323( = SAV4735)

BldM is an aberrant orphan response regulator (Molle &

Buttner, 2000), and BldN an ECF s factor (Bibb et al., 2000).

It appears that sBldN is directly required for the transcrip-

tion of one of two promoters of bldM (Bibb et al., 2000). An

important consequence of the expression of bldM is the

activation of the chp genes encoding the morphogenetic

chaplin proteins (Elliot et al., 2003; see below). Interestingly,

some bldM and bldN alleles give rise to a white aerial

mycelium phenotype (Ryding et al., 1999), suggesting that

they are both needed at more than one stage of develop-

ment. Both of the S. avermitilis orthologues show consider-

able conservation in their upstream noncoding regions with

their S. coelicolor equivalents: in the case of bldM this

extends for about 350 bp, showing 88% identity with the

exception of one short mismatched segment (20 bp in S.

avermitilis, 10 bp in S. coelicolor); while the homology

upstream of bldN extends for about 160 bp of 94% identity.

The same segment is about 84% identical in the S. griseus

orthologue of bldN, called adsA, which is a direct target of

AdpA ( = BldH) (Yamazaki et al., 2000); and bldN transcrip-

tion is also bldH-dependent in vivo in S. coelicolor (Bibb

et al., 2000). Curiously, much of the divergence is in the

segment shown in vitro to be protected by AdpA in S. griseus,

which, unusually, is within the transcribed region upstream

of adsA (Yamazaki et al., 2000). In S. coelicolor, bldN loses its

temporal control in a bldD mutant, being strongly expressed

even early in vegetative growth; and the equivalent segment

of the bldN upstream region of S. coelicolor binds to BldD

protein in vitro (Elliot et al., 2001). However, it is not clear if

AdpA and BldD both interact with the bldN upstream region

of either organism. A second BldD-binding site for bldN

corresponds to the segment of the promoter region expected

to interact with RNA polymerase (Elliot et al., 2001), which

is extensively conserved among the three species. We sup-

pose that the temporal expression of bldN reflects both

repression by BldD and activation by AdpA in all three

species, and that the regulation of bldN by these two

proteins is likely to be an ancient trait of streptomycetes.

However, divergence in the shared BldD-/AdpA-binding

DNA segment may cause the fine details of any competition

or interaction between the two proteins to differ in S. griseus

from those in S. coelicolor and S. avermitilis.

Most actinobacteria

bldC

S. avermitilis

T. fusca

S. coelicolor

Fig. 7. Synteny in the bldC region of actinobacterial genomes, com-

pared with Streptomyces coelicolor. Connecting bridges indicate the

absence of a gene in an otherwise syntenous series of genes; gaps

indicate that no likely orthologue is present in the genome; and genes in

brackets represent apparent orthologues located elsewhere in the

genome.



The ramCSABR genes, SCO6681--6685(= SAV7499--7503)

A second important morphogenetic endpoint of the cas-

cade, that seems to bypass bldN and bldM and the produc-

tion of chaplins, is the five-gene ramCSABR cluster

( = amfTSBAR in S. griseus) (Chater & Horinouchi, 2003;

Ueda et al., 2005; Willey et al., 2006). [There is a paradox in

the literature here, since it was reported that a bldM mutant

behaved the same as a bldD mutant in the extracellular

complementation cascade (Molle & Buttner, 2000); but

these experiments may be complicated by the fact that some

of the mutants used to define the cascade have different

genetic backgrounds.] The first step in ram cluster expres-

sion is the transcription of ramR, which encodes a response

regulator that contains most of the residues conserved in

phosphorylation pockets that are the targets of histidine

protein kinases in bacteria. However, ramR is not located

close to a sensor kinase gene, and evidence for RamR

phosphorylation has proved elusive (Keijser et al., 2002;

Nguyen et al., 2002; O’Connor & Nodwell, 2005). Never-

theless, it seems that a change in the state of RamR results in

full activation of the ramCSAB operon. The membrane-

bound RamC, previously thought to be a serine-threonine

protein kinase, has now been found to have another role, in

processing RamS peptide (Kodani et al., 2004). RamA/AmfB

and RamB/AmfA are subunits of an ABC transporter believed

to transport processed RamS/AmfS out of the cell (Ueda

et al., 2002; Willey et al., 2006). The primary RamS product

(38 amino acids) undergoes both cleavages and amino-acid

modifications to end up as a 17-mer amphipathic oligopep-

tide, called SapB in S. coelicolor, that contains lanthionine

cross-bridges, and hence resembles lantibiotics, though it has

not yet been found to have antibiotic properties (Kodani

et al., 2004). The name SapB originates from its first discovery

as one of several ‘spore associated proteins’ (Guijarro et al.,

1988). Remarkably, the extracellular addition of SapB can

bring about aerial growth of any of the bld mutants that can

participate in the extracellular cascade, except bldN (Willey

et al., 1993; Bibb et al., 2000).

Interestingly, the ram/amf cluster shows lower sequence

conservation (just over 50% identity for every gene product)

between Streptomyces species than do any of the other

components of the bld gene cascade. This leads to the

hypothesis that SapB may have a species-specific aspect – in

other words, that there may have been positive selection for

changes in SapB-like oligopeptides in the course of evolu-

tion. Arguing against this, the simple morphogenetic activ-

ity of SapB lacks specificity, since aerial growth of bld

mutants can be stimulated equally by certain other amphi-

pathic proteins (Kodani et al., 2004, 2005). Future work with

the equivalent systems of a wider variety of species may help

to resolve this paradox.

The differentiation of aerial hyphae intospores

A section of the sporulation regulatory networkis found only in streptomycetes

Four of the regulatory genes shown in Fig. 4 that are needed

for aerial hyphae to form mature grey spores are absent from

other actinobacteria (though one of them, whiG, is found in

many nonactinobacterial bacteria, including E. coli and B.

subtilis). Three of the four genes (whiG, H, I) form a

particular regulatory subsection of the early whi gene net-

work, and the fourth (sigF) encodes a s factor for late gene

expression.

whiG , SCO5621 ( = SAV2630)

When aerial hyphae begin to grow, it appears that they are

developmentally indeterminate. They acquire a specific

developmental fate when the sporulation-specific WhiG sfactor becomes active (Mendez & Chater, 1987; Chater et al.,

1989) (perhaps partially as a result of the release of whiG

from repression by BldD; see above). WhiG is an orthologue

of the s factors that, in many bacteria, direct transcription

of some of the genes associated with motility and chemo-

taxis (Tan et al., 1998). Although the S. coelicolor chromo-

some contains a prophage-like insertion upstream of whiG

that is absent from S. avermitilis, the genes downstream are

conserved and syntenous between the species. The whiG

genes of several other streptomycetes have been shown to

play a similar role in committing aerial hyphae to sporula-

tion, and, where sequenced, all are highly conserved in their

coding regions and in their promoters at least from � 1 to

� 45, the region containing one of the two characterized

BldD-binding sites in the whiG upstream region of S.

coelicolor (Soliveri et al., 1993; Kormanec et al., 1994; Elliot

et al., 2001; Catakli et al., 2005).

whiH , SCO5819 ( = SAV2445) and whiI , SCO6029( = SAV2230)

Two sporulation regulatory genes, whiH and whiI, are well-

established targets for sWhiG RNA polymerase holoenzyme

(Ryding et al., 1998; Ainsa et al., 1999; Kormanec et al.,

1999). There are significant matches between S. coelicolor

and S. avermitilis in the upstream regions for each of these

genes. Each gene encodes a member of a well known and

very widespread family of regulatory DNA-binding proteins.

WhiH is in the family containing GntR, many of whose

members respond to small organic acids, prompting spec-

ulation that WhiH responds to a change in concentration of

such a metabolite during aerial mycelium growth (Ryding

et al., 1998). WhiH appears to induce the strong



developmentally controlled ftsZp2 promoter to bring about

sporulation septation (Flardh et al., 2000).

WhiI resembles the response regulators associated with

bacterial two-component systems (but has important differ-

ences in the normally conserved phosphorylation pocket,

which are likewise changed in the S. avermitilis orthologue)

(Ainsa et al., 1999). There is circumstantial evidence that

different genes are induced or repressed by different forms of

WhiI, and it is postulated that WhiI (and perhaps other

orphan response regulators, including BldN and RamR; see

above) responds to a phosphorylated intermediary metabo-

lite rather than being phosphorylated by a histidine protein

kinase (Y. Tian, K. Fowler, K.C. Findlay and K.F. Chater, in

preparation). WhiI may induce the chromosome condensa-

tion that accompanies sporulation (Ainsa et al., 1999).

sigF, SCO4035 ( = SAV4185)

The s factor encoded by sigF is required for the late stages of

sporulation (Potuckova et al., 1995; Kormanec et al., 1996;

Kelemen et al., 1996). Thus, a sigF mutant undergoes

sporulation septation, but the resulting spore chains are

irregular, thin-walled and more or less unpigmented, and

contain uncondensed DNA. There is no detectable tran-

scription of sigF in whiA, B, G, H, I or J mutants, but the

reason for this is unknown (Kelemen et al., 1996). The S.

aureofaciens orthologue is also dependent on at least whiG

(Kormanec et al., 1996), indicating that sigF fulfils the same

role in diverse species, and therefore probably acquired this

role early in Streptomyces evolution. Interestingly, sF is a

member of the same subgroup of gram-positive-specific sfactors as the two forespore ss of B. subtilis and the B. subtilis

stess-responsive sB, two of which are controlled by anti-s/

anti-anti-s cascades (see bldG above). However, no genes

for such a cascade are located in the vicinity of sigF or its

orthologue in S. avermitilis. No direct targets for sF have yet

been identified, but one of the whiE promoters (for orfVIII)

is inactive in a sigF mutant (Kelemen et al., 1998; Kelemen &

Buttner, 1998).

A section of the sporulation regulatory networkinvolves genes that are found inmorphologically simple actinobacteria

whiA , SCO1950 ( = SAV6294), whiB , SCO3034( = SAV5042) and whiD , SCO4767 ( = SAV3216)

whiA and whiB mutants both have unusually long and curly

aerial hyphae that completely lack sporulation septa (Flardh

et al., 1999). A whiB mutant of S. aureofaciens had a similar

phenotype (Kormanec et al., 1998), and the whiB ortholo-

gue of Streptomyces griseocarneum complemented a whiB

mutant of S. coelicolor (Soliveri et al., 1993). Thus, WhiA

and WhiB are thought to be required to stop the continued

extension of aerial hyphae. Probably, sporulation septation

can take place only in nongrowing hyphae. It is likely that

the two gene products are closely intertwined in their

actions, either by an intimate regulatory interplay (they

certainly influence each other’s expression) or by virtue of

some protein–protein interaction (Jakimowicz et al., 2006).

The developmentally controlled expression of whiA and

whiB in S. coelicolor (Soliveri et al., 1992; Ainsa et al., 2000)

and at least of whiB in S. aureofaciens (Soliveri et al., 1992;

Kormanec et al., 1998) appears to be largely independent of

whiG.

whiA is located in a gene cluster that shows significant

conservation across all gram-positive bacteria. For example,

homologues of whiA and the two upstream genes are

similarly clustered in B. subtilis (Ainsa et al., 2000). A whiA-

free version of the cluster is found in many gram-negative

bacteria. There appear to be no reports of the physiological

role or molecular structure and function of WhiA-like

proteins in any organisms except streptomycetes. Perhaps

in other bacteria too it is required to bring growth to an

orderly halt, but the elimination of this function might not

give rise to an obvious phenotype in unicellular organisms.

whiA is expressed at a low level throughout growth by

readthrough transcription extending from the upstream

transcription unit, but is strongly upregulated during aerial

hyphal development, from an additional promoter of its

own within the immediately upstream coding sequence

(Ainsa et al., 2000). Presumably whiA acquired its sporula-

tion-specific function by mutations affecting its regulation

that occurred very early in the Streptomyces lineage. The

distribution of whiB-like (‘wbl’) genes is quite different from

that of whiA homologues. WhiB orthologues are omnipre-

sent in free-living actinobacteria, but absent from all other

organisms. They are members of a substantial family of

generally small (80–120 aa) proteins whose major conserved

features are a set of four cysteine residues and a short

glycine- and tryptophan-rich segment (Soliveri et al.,

2000). Several of these proteins, like WhiB itself, have

orthologues widespread among actinobacteria, but others

show species-specificity, implying that members of the

family have often been acquired laterally. This is consistent

with the frequent occurrence of wbl genes on phages and

plasmids of actinomycetes – for example, the large linear

plasmid SCP1 present in S. coelicolor A3(2) carries three wbl

genes (Bentley et al., 2004). Several wbl genes and their

products have been studied experimentally in streptomy-

cetes and mycobacteria. One of these, whiD, is a late

sporulation gene in S. coelicolor (Chater, 1972; Molle et al.,

2000), while its orthologue (whmB, or whiB3) in mycobac-

teria is involved in a physiologically ill-defined but impor-

tant role in the pathogenicity of M. tuberculosis and M. bovis

(Steyn et al., 2002). Studies of the whiD/whmB/whiB3



orthologues have provided insights into the function and

structure of WhiB-like proteins. Evidence that WhiB3 inter-

acts directly with the principle s factor of M. smegmatis

(Steyn et al., 2002) may have broad relevance to the action of

Wbl proteins, particularly since another wbl gene (the wblP

gene of plasmid SCP1) encodes a fusion of a Wbl domain

with an ECF s factor domain (Bentley et al., 2004); and four

other wbl genes of S. coelicolor and its plasmid SCP1 are

located very close to s factor determinants. The physiologi-

cal stimulus for the activity of Wbl proteins has been

postulated to be some kind of internally generated redox

change associated with the switch from growth to non-

growth, based on the four conserved cysteine residues

(Soliveri et al., 2000); and the idea of redox sensing has

received support from evidence that WhiD contains an

oxygen-sensitive 4Fe,4S cluster (Jakimowicz et al., 2005).

Other developmental genes peculiar to complexactinobacteria

The ssg gene family

The ssgA gene (SCO3926, = SAV4267) was identified first in

S. griseus as a high copy-number suppressor of a hyperspor-

ulating mutant (Kawamoto & Ensign, 1995; Kawamoto

et al., 1997), and was shown to be required for correct

sporulation septation of S. griseus (Jiang & Kendrick, 2000),

and of S. coelicolor on most media (the exception being

minimal medium with mannitol as carbon source) (Van

Wezel et al., 2000). It has been suggested that the different

abilities of S. griseus and S. coelicolor to sporulate in

submerged culture may reflect differences in ssgA regulation

in the two organisms, since it is a target of AdpA (and hence

is A-factor-dependent) in S. griseus, but is AdpA-indepen-

dent in S. coelicolor, in which it is regulated solely by the

adjacent ssgR gene (SCO3925, = SAV4268, encoding an

IclR-like protein) (Traag et al., 2004). There also seem to be

functional differences between SsgR and its S. griseus

orthologue, SsfR, since ssfR did not complement an ssgR

mutant.

The whi genes described above have little obvious influ-

ence on ssgA expression, but, on the other hand, whiH

appears to be overexpressed in ssgA or ssgR mutants. This is

an interesting observation in view of the implied involve-

ment of ssgA in septation, since whiH has been tentatively

implicated in the sporulation-specific regulation of the key

cell-division gene ftsZ (see above).

Streptomyces coelicolor has six other ssgA paralogues, and

S. avermitilis five (hence the term ‘SALP’ for SsgA-like

proteins). In addition, at least two ssgA-like genes are

present in T. fusca, and one in K. radiodurans, the gene

family being otherwise absent from the database (Noens

et al., 2005). One of the paralogues in S. coelicolor, ssgB

(SCO1541), is also involved in sporulation: a knockout

mutant was white and completely defective in sporulation

septation, and, interestingly, had a striking large-colony

phenotype (Keijser et al., 2003). It appears that ssgB is not

needed for ssgA expression. In a comprehensive gene knock-

out analysis of the ssgA-like genes of S. coelicolor, it has

emerged that all of the SALPs have roles in the precise

determination of the pattern of peptidoglycan biosynthesis

and hydrolysis that is needed for proper sporulation septa-

tion and spore morphogenesis (Noens et al., 2005).

samR , SCO2935 ( = SAV5141)

The Beijing laboratory of H. Tan has studied several white

colony mutants of Streptomyces ansochromogenes. Most of

these have proven to be defective in orthologues of known

whi genes of S. coelicolor, providing confirmation (together

with similar work on S. aureofaciens from the laboratory of J.

Kormanec in Bratislava) that these genes (whiG, B, A)

control development in a similar manner in diverse species.

In the course of this work, Tan et al. (1998) also discovered a

previously unknown developmental gene, samR, mutations

in which had a bald colony phenotype; but when they went

on to disrupt the orthologous gene of S. coelicolor, the

mutant colonies produced fluffy, nonsporulating aerial

mycelium similar to that of a whiG mutant. The product of

samR is a member of the IclR family of transcriptional

regulators, and has no orthologues outside of streptomy-

cetes.

whiJ , SCO4543 (SAV3425), and its relatives andtheir satellite gene families, including bldB

The literature on whiJ is complicated in several ways. The

term whiJ was first applied when Ryding (1995) succeeded in

complementing the whi-77 mutant analysed by Chater and

Merrick (1976) and several other previously little-studied

whi mutants from the collection first isolated by Hopwood

et al. (1970), from which the whiA, B, D, E, G, H and I loci

had been discovered (Chater, 1972). Complementation was

attributable to one gene, SCO4543, which was therefore

defined as whiJ in the EMBL database entry (AF106004) (J.

Ainsa, N.J. Ryding and K.F. Chater, unpublished). The

product of this gene has a lambda repressor-like DNA-

binding domain at its N-terminus. Subsequently, a transpo-

son-induced whi mutant was found to harbour a transposon

insertion in SCO4543, and it was pointed out that this gene

and its two neighbours (SCO4542 and 4544) are all mem-

bers of apparently Streptomyces-specific extensive gene fa-

milies (Gehring et al., 2000). These are found in various

combinations, often in combination with members of

further gene families. One of these families includes the bldB

gene cloned by Piret & Chater (1985). All of these three gene



families are peculiar to streptomycetes and, it now emerges,

T. fusca. One of the clusters containing members of these

families, termed abaA, was discovered in a search for genes

that, at high copy number, increased antibiotic production

(Fernandez-Moreno et al., 1992). The extent of the confu-

sion surrounding these genes is illustrated by the fact that

whiJ has inadvertently been used to refer to SCO4542

instead of the correct SCO4543, and abaA to refer to just

one of the five genes (ORFs1–5) in the abaA locus

(SCO0700–0704), in a comparison of BldB with some of its

paralogues (Eccleston et al., 2002).

The phenotypes of mutations in the genes flanking whiJ

are not yet known. Interestingly, a TTA-containing para-

logue of one of the genes flanking whiJ (SCO4544) , and of

abaA orfA (SCO0702), is present in both long terminal

inverted repeats of the linear plasmid SCP1 of S. coelicolor

(SCP1.58c, SCP1.295), where it is associated with the genes

for three spore-associated proteins (SapC, D and E) (Bentley

et al., 2004). It will be rewarding to subject these gene

families to more extensive analysis.

Genes directly involved in morphogenesis andspore structure

We have already discussed the SapB morphogenetic oligo-

peptide in some detail, because of its special place as an

endpoint of the bld gene cascade; but several other surface

components also play special – perhaps unique – roles in

morphogenesis (Elliot & Talbot, 2004).

chpA-H [in order, SCO2716, 7257, 1674, 2717,1800, 2705, 2699, 1675; there are Streptomycesavermitilis orthologues only of chpC (SAV6636),chpE (SAV6478), chpH (SAV6635)]

The growth of aerial hyphae into the air has turned out to

require a partially functionally redundant family of highly

surface-active proteins, called ‘chaplins’, encoded by the chp

genes (Claessen et al., 2003; Elliot et al., 2003). Some of the

chaplins are thought to be covalently linked by sortase

enzymes to the aerial hyphal peptidoglycan, and others

probably attach noncovalently to the anchored chaplins.

The only known occurrence of chaplins outside streptomy-

cetes is in the aerial mycelium-forming T. fusca, which has a

single chp gene. It is considered that chaplins enable aerial

hyphae to break through the surface tension barrier of a

water layer that separates the substrate mycelium from

direct contact with the air (Claessen et al., 2003; Elliot

et al., 2003; Elliot & Talbot, 2004; Gebbink et al., 2005); but

we think it is also possible that the chaplin layer acts to

capture a column of water between the aerial hyphal

membrane and the chaplin-containing layer, to provide an

aqueous environment within which cellular growth of aerial

hyphae can take place. This alternative theory might also

apply to the analogous hydrophobins of mycelial fungi. The

production of chaplins depends on the integrity of the

extracellular cascade involving bld genes (see above).

rdlA and rdlB (SCO2718, 2719)

The surface of aerial hyphae of S. coelicolor also contains

another class of amphipathic proteins, the rodlins, which

possibly interact with chaplins, and are encoded by two

adjacent rdl genes (Claessen et al., 2002; Claessen et al.,

2004). Although they are made only by aerial hyphae,

rodlins are not essential for aerial growth, and indeed they

are absent from S. avermitilis, apparently because of a

deletion of a genomic segment including the rdl genes (they

are present in many other streptomycetes, but are unknown

in other organisms) (Claessen et al., 2004). Interestingly,

mutants lacking rodlins (Claessen et al., 2002) or chaplins

(Gebbink et al., 2005) attach less well to hydrophobic

surfaces, loosely supporting the idea that aerial hyphae may

serve as exploratory hyphae as well as sporophores (Yeo &

Chater, 2005).

whiE (SCO5314–5321)

This is a cluster of eight genes, most of which encode

recognizable enzymes of polyketide biosynthesis (Davis &

Chater, 1990). Mutations in whiE genes cause a loss of spore

pigment or a change in spore colour, and there is consider-

able information about the roles of these enzymes in the

synthesis of a 24-carbon chain-length polyketide (Shen

et al., 1999; Moore & Piel, 2000). However, the mature

WhiE polyketide has not been characterized because it seems

to be covalently attached to the spore wall. The whiE genes

form two diverging transcription units, both of which are

switched on only during sporulation. Both promoters are

dependent on all the early whi genes, but only the one

reading into orfVIII is also dependent on the late sporula-

tion-specific SigF s factor (Kelemen et al., 1998).

The S. avermitilis cluster (SAV2837–2844) and the par-

tially sequenced cluster of S. halstedii are colinear with that

of S. coelicolor. Although the clusters are flanked on both

sides by quite different genes in S. avermitilis and S.

coelicolor, their overall positions in the genome are con-

served. The conservation of genomic position suggests that

the flanking genes have been subjected to extensive flux

(insertion and deletion differences), while the whiE genes

themselves have remained anchored. This interpretation

may be over-simple, in view of the results of a large-scale

survey of whiE ORFII genes in diverse streptomycetes

(Metsa-Ketela et al., 2002), which showed that the phylo-

geny of these strains obtained by ribosomal DNA sequen-

cing differed substantially from that obtained by analysis of



the whiE genes. This surprising finding could mean that

whiE genes have intermittently been transferred laterally

between genomes, raising the question – yet to be addressed

by further genomic comparisons – of whether other seg-

ments of the core genome might also have been exchanged

between streptomycetes over evolutionary time.

Remarkably, the genome of S. griseus contains no whiE

gene cluster (Y. Ohnishi and S. Horinouchi, pers. com-

mun.). Instead, the pigmentation of both spores and myce-

lium is attributable to a form of melanin that results from

the action of a cytochrome P-450 (P-450mel) on 1,3,6,8-

tetrahydroxynaphthalene, itself made by the condensation

of five molecules of malonyl-CoA by a type III polyketide

synthase (RppA) (Funa et al., 2005). P-450mel and rppA

genes are also present in S. coelicolor (albeit somewhat

diverged fron their S. griseus counterparts), but they do not

appear to contribute to pigmentation (Izumikawa et al.,

2003).

crgA , SCO3854 (SAV4331)

Like samR, crgA (Del Sol et al., 2003) affects differentiation

differently in different species. It encodes a small, mem-

brane-bound protein that inhibits premature sporulation

septation in S. coelicolor. A crgA mutant showed premature

aerial mycelium growth and sporulation, as well as early

production of the blue antibiotic actinorhodin. On the other

hand, a crgA mutant of S. avermitilis had white, coiled,

mostly nonsporulating aerial mycelium. Multiple copies of

crgA in both species gave white colonies in which the aerial

hyphae did not undergo sporulation septation. The gene is

present (and shows local synteny) in all actinomycete

genomes, and it would be interesting to know whether it

always affects septation.

Other genes important for development

The morphological changes associated with development

involve several proteins that determine the location and

components of events associated with sporulation septation.

These include FtsZ (cell-division) and ParAB (chromosome

segregation), both of which have complex promoter regions,

with separate segments being responsible for expression

during vegetative growth or differentiation (Flardh et al.,

2000; Jakimowicz et al., 2006). On the other hand, a

different solution to the need for expression at more than

one developmental stage has been found for at least one set

of enzymes active at different developmental stages: glyco-

gen deposition at the end of vegetative growth or at the end

of aerial growth involves differently regulated but almost

identical operons for isoforms of four enzymes that include

the glycogen branching enzyme (Schneider et al., 2000; Yeo

& Chater, 2005).

Conclusion: an overview of the evolutionof Streptomyces development

The pattern of occurrence of the genes discussed in this

article in different genomes can be used in an attempt to

reconstruct the evolution of Streptomyces development (Fig.

8). It seems that the aerial growth-stimulating system

involving both SapB/AmfS-like lanthionine-containing oli-

gopeptides and chaplins was established very early in the

evolutionary emergence of streptomycetes, presumably pro-

viding a means by which a mycelium could proceed to aerial

Low GC Gram-positives

Other actinobacteria?

Gram-negatives

whiG, whiA, in ancient bacterial progenitor

Loss of whiA

Loss of whiG; acquisition of crgA;acquisition and diversification of whiB -like genes

mycobacteria corynebacteria

Thermobifida

Streptomyces

speciation

ram; bldA/adpA;whiG,H,I,E; samR; ssgA, other ssg genes

bldC,D,G; bldB/whiJ-like; chp; protease inhibitor; ssgB

rodlins, antibioticsγ-butyrolactones

First oxygenation of atmosphere

Second oxygenation of atmosphere

Aerial growth

Fig. 8. Proposed pathway for the evolution of

Streptomyces development. The scheme was

drawn to minimize the number of gene acquisi-

tion and gene loss events.



growth in a coordinated manner. The production of these

molecules in S. coelicolor is itself dependent on other

extracellular signals, most of which are still uncharacterized;

but at least one of them (signal 1) is an incompletely defined

oligopeptide (Nodwell & Losick, 1998). These signals within

the colony indicate activity of the BldH, G, C and D

regulatory proteins, as well as of the uncharacterized bldJ

gene product that is needed to generate signal 1. It has been

proposed that these gene products are involved in responses

to different stresses that would otherwise limit continued

growth (Chater & Horinouchi, 2003): thus, only when such

responses have all been activated would the ‘last resort’ of

terminal differentiation (aerial growth at the expense of the

accumulated substrate mycelial biomass) be undertaken. At

least in this sense, the signal cascade ending in SapB

accumulation might be seen to be equivalent to the intra-

cellular sporulation phosphorelay in Bacillus spp. and their

relatives, which culminates in the accumulation of phos-

phorylated Spo0A regulatory protein, the trigger for com-

mitment to endospore formation (Errington, 2003; Fujita &

Losick, 2005).

It seems likely that AdpA (BldH) played a key role in

development early in Streptomyces evolution (from some-

where between 440 and 220 Ma), and that even then the

UUA codon of adpA mRNA was being used as a route

through which bldA influenced development. However,

certain other details of adpA regulation show species-

specificity, such as its A-factor-sensitive repressibility by

ArpA in S. griseus. The nature and range of signal inputs

affecting AdpA levels and/or activity have probably devel-

oped as part of the adaptation of different species to

different ecological niches, or their adoption of different

strategies for responding to a shared niche. For example,

colonies of one species might sporulate comprehensively

and more or less synchronously at a hint of imminent hard

times, while those of another could have evolved to delay

sporulation as long as possible in case an environmental

change restores conditions favourable for growth, making it

possible to achieve greater biomass before commitment to

sporulation. Since there are orthologues of bldG, bldC and

bldD in T. fusca, but apparently not in other completely

sequenced actinobacteria, it is likely that any stress responses

in which these genes take part predated the emergence of

streptomycetes. T. fusca and streptomycetes are alone among

sequenced actinobacteria in being obligately mycelial and

reproducing principally via a sporulating aerial mycelium,

so these stresses might conceivably be associated with this

life-style. The absence of the amf system and of multiple

SALPs, chaplins and rodlins from all organisms except

streptomycetes suggests that the acquisition of these features

was associated with the development of the Streptomyces

aerial mycelium, and that T. fusca may have a different

means of solving the problems associated with reproduction

via aerial growth, albeit probably using its single chaplin and

two SALPs. It has been suggested that Streptomyces aerial

hyphae may serve not only as sporophores, but alternatively

as the means by which a mycelial organism can extend across

the air space of tiny cavities in the soil habitat to reach and

colonize other nearby surfaces (Yeo & Chater, 2005). This

suggests, in turn, that exploratory aerial growth could have

preceded the acquisition by aerial hyphae of their reproduc-

tive function, and provided the evolutionary opportunity

for it. The capture of whiG, whiH and whiI, followed by their

adaptation to become increasingly precise regulators of the

fragmentation process that became sporulation, presumably

happened around the time of the birth of the first ancestor

peculiar to streptomycetes. In this light, it is curious that

whiH and whiI are located about 200 and 400 genes away

from whiG, in both S. coelicolor and S. avermitilis, and that

the intervening regions, although not showing extensive

synteny with other actinobacterial genomes, include a

number of genes for which there are orthologues in most

actinobacteria. If whiG and its targets were acquired in a

single event, for example as a regulon on a plasmid, there

must have been several changes in chromosomal organiza-

tion in the whiG,H,I region quite soon after that initial

acquisition event.

If many of the genes for development have been horizon-

tally acquired, what is their source? Most of them are absent

from other known bacteria, but a huge pool of novel genes is

present in bacteriophages. Presumably, many of these genes

have evolved under extreme selective pressures in the face of

the constant evolution of host organisms to resist death by

phage infection, as well as under occasional competition

between phages infecting the same host (Hendrix, 2005).

This means that phages are potentially a rich source both of

novel regulatory genes and of genes for surface-acting and

other structural proteins. Both of these gene classes are

important for the evolution of developmental and morpho-

logical complexity. Moreover, phages provide the means by

which new genes can be efficiently introduced into bacterial

hosts (Hendrix, 2005). At least in the case of whiB-like genes,

there are examples in phage genomes. One can push the idea

of phages as a source of developmental possibilities further,

in considering how the use of the bldA-specified tRNA for a

rare codon might have acquired specific developmental

significance. Thus, a drift towards high GC content could

be associated with selection against phages through codon

bias, and could eventually permit a loss of the UUA-reading

tRNA, and an associated complete inability to translate UUA

codons that would confer resistance to phages that employ

this codon. Some phages would counter this by acquiring a

copy of the tRNA (tRNA genes are often present in phage

genomes). By this means, the tRNA might be reintroduced

into a proto-streptomycete as part of a prophage genome.

Because of continued selection for phage resistance, the



newly acquired tRNA might not be expressed abundantly

during vegetative growth (the main phage-sensitive stage of

bacteria), but it could be expressed without disadvantage in

stationary phase, and come to be exploited by genes that are

specifically useful in stationary phase, as seems to be the case

for TTA-containing genes of streptomycetes (Chater, 2006).

Spores (as we know them) presumably evolved from

mycelial fragments, under selective pressures that may have

included the need to survive inside the guts of worms and

other invertebrates that consumed the plant remains on

which the early streptomycetes were growing. The ability of

spores to survive in such a hostile environment would have

been enhanced by the cross-linking of the aromatic spore

pigment to the spore wall in a lignification-like process; it is

not surprising, therefore, that whiE genes appear to have

been acquired early in the Streptomyces lineage (notwith-

standing their absence from S. griseus: possibly the melanin

pigment responsible for spore colour in that organism also

‘lignifies’ spore walls). Most antibiotics, including many

other aromatic polyketides, are much more strain-specific,

and have presumably been acquired more recently. Two

sporulation-specific gene sets, for SapB and spore pigment,

have some relatedness to genes for antibiotic production

(lantibiotics and aromatic polyketides, respectively). Since

their acquisition by Streptomyces appears to have preceded

the acquisition of antibiotic production genes, it is an

intriguing possibility that they may have been important in

the origin of such genes.

The whiAB part of the sporulation regulatory cascade

seems to have had a different evolution, involving vertically

inherited (rather than laterally acquired) genes whose initi-

ally nondevelopmental regulation and regulatory roles came

to be integrated into sporulation. The presence of whiA

orthologues in all gram-positive bacteria indicates a parti-

cularly ancient lineage, preceding the divergence of the low

and high GC gram-positive bacteria some 2 Ga. No role for

WhiA-like proteins has been found in any nonstreptomy-

cetes, but the evidence connecting WhiA with the orderly

shut-down of growth may provide a clue for studies of

orthologues in other bacteria. On the other hand, whiB

orthologues and their paralogues (generally termed wbl

genes) are confined to actinobacteria. In view of evidence

that the products of such genes may be redox-sensitive

regulators, it is interesting that actinobacteria share another

special feature of their redox regulation: they use mycothiol

as a thiol-disulphide buffer, a role played by glutathione in

many other bacteria (Fahey, 2001). Possibly, there is a

Wbl–mycothiol association, which could have been fixed at

the time when the actinobacteria emerged (about 1.5–2 Ga),

coinciding with the first appearance of oxygen at significant

levels in the atmosphere. Five wbl genes (whiB, whiD, wblA,

wblC and wblE) are present in most actinobacteria, so wbl

genes must have diversified very early in actinobacterial

evolution. One of them, wblC, is involved in the expression

of multiple antibiotic resistance in both mycobacteria and

streptomycetes (Morris et al., 2005), so, by extrapolation, it

is likely that each of the five had acquired a specific

physiological role at this early stage of evolution. We there-

fore suppose that whiB and whiD were subsequently (and

probably sequentially) sequestered for sporulation in strep-

tomycetes, rather than evolving by duplication and diver-

gence from a common sporulation-specific progenitor

during a gradual increase in the complexity of sporulation

control. Thus, the evolution of the later stages of sporula-

tion, which might be equated with an increase in the

effectiveness of mycelial fragments as survival and dispersal

agents, was probably associated with the acquisition of

successively later-acting regulons; hence the adoption of sigF

and whiD, each a member of a paralogous gene family, as

Streptomyces sporulation-specific controlling elements. This

process may have interesting differences from the evolution

of developmental complexity that has emerged from other

systems. For example, in B. subtilis endospore formation,

each of the two initial compartment-specific s factors, sE

and sF, is replaced in the later stages of sporulation by a

closely related paralogous s factor (sK and sG, respectively)

(Errington, 2003), strongly suggesting that the evolution of

the late stages of sporulation, during which the spores

acquire their resistance properties, involved the duplication

of the sporulation-specific progenitors of the sE and sF

genes, and their subsequent divergence. This theme of

developmental evolution by the duplication and divergence

of developmentally specific master control genes is also

found in the homeobox genes of vertebrates and the

MADS-box genes that determine floral organ identity in

plants (Cohen, 1999). We note here that the multiple SALPs

of streptomycetes, which play important roles in consecutive

stages of cell wall reorganization during sporulation, seem to

have evolved by duplication and divergence as the process of

sporulation of aerial hyphae evolved (Noens et al., 2005).

Unlike most other whi genes, both whiA and whiB have

two promoters, one of which is low-level and constitutive,

while the other is associated with development (Soliveri

et al., 1992; Flardh et al., 2000). The developmental promo-

ters appear to be part of an autoactivating circuit in which

both gene products affect the expression of both genes

(though it is not known if this regulation is direct) (Jakimo-

wicz et al., 2006). Although the constitutive promoters may

simply serve to provide a priming element in the autoregu-

latory developmental circuit, it is also possible that WhiA

and WhiB serve some other physiological purpose in

vegetative hyphae (perhaps similar to whatever their role is

in nondifferentiating actinobacteria). Likewise, the ftsZ and

parAB genes, both of which have somewhat different roles in

vegetative growth and sporulation, also have dual promo-

ters, one for vegetative functioning and one for sporulation



(Flardh et al., 2000; Jakimowicz et al., 2006). Thus, it is quite

common to find that genes have acquired additional RNA

polymerase interaction sites as developmental complexity

has evolved. In the cases of ftsZ and parAB, it seems that

WhiH and WhiA/WhiB are, respectively, closely involved in

this developmental activation (Flardh et al., 2000; Jakimo-

wicz et al., 2006). No doubt many other examples of the

evolutionary plasticity of promoter regions will emerge in

future analysis of Streptomyces development.

Note added in press

The genome sequence of a representative of the mycelial

nitrogen-fixing symbiont Frankia has very recently become

available (http://genome.jgi-psf.org/draft_microbes/fra_c/

fra_c.home.html). Although members of this genus are not

reported to form aerial mycelium, orthologues of several bld

genes are present (bldC, D, G, N), as well as an ssgB-like

gene. This profile is interestingly similar to that of T. fusca.

Acknowledgements

This work was supported by grant IGF12432 from the

BBSRC, a Competitive Strategic Grant to the John Innes

Centre from the BBSRC, and the John Innes Foundation. We

thank Alan Ward for discussions on Streptomyces phylogeny,

Sueharu Horinouchi and Yasuo Ohnishi for communicating

unpublished results, and David Hopwood for helpful com-

ments on the manuscript.

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