Download An Inventory of Genes Encoding RNA Polymerase Sigma Factors in

J. Mol. Microbiol. Biotechnol. (2002) 4(1): 77–91.
JMMB Research Article
An Inventory of Genes Encoding RNA Polymerase
Sigma Factors in 31 Completely Sequenced
Eubacterial Genomes
Gerhard Mittenhuber*
Institut für Mikrobiologie und Molekularbiologie,
Ernst- Moritz-Arndt-Universität, F.-L. Jahnstr. 15,
D-17487 Greifswald, Germany
Abstract
Sigma factors are important elements involved in
transcriptional regulation of gene expression by
conferring promoter specificity to RNA polymerase. The number of sigma factor encoding genes in
31 completely sequenced bacterial genomes were
compared. Two unrelated families of sigma
factors, the s70 - and the s 54-family were identified
previously. The s70-family can be further subdivided into two distantly related groups: the s 70
subfamily and the poorly characterized ECF
subfamily. A total of 215 sigma factors could be
attributed to these subfamilies. The construction
of phylogenetic trees allows subclassifications of
sigma factor encoding genes within the subfamilies. With the exception of Deinococcus radiodurans, all species possess a housekeeping
primary sigma factor. Free-living species possess
a higher number of both s 7 0 -type and ECF
alternative sigma factors than pathogens or
symbionts associated with animals. Different
bacterial species exhibit large differences in the
number of alternative sigma factor encoding genes
and consequently huge flexibility in their transcriptional regulatory patterns. Transcriptional
regulation in terms of regulons controlled by
alternative sigma factors is a late evolving
phenomenon. The current nomenclature for sigma
factor encoding genes is confusing and should be
revised.
Introduction
In eubacteria, different sigma factors exhibiting
different promoter specificities provide a convenient
way for differential transcriptional regulation. Sigma
factors and genes under control of a specific sigma
factor (the regulon) have been primarily investigated in
the gram negative model organism Escherichia coli
and the gram positive model organism Bacillus subtilis.
The primary sigma factor (housekeeping sigma
*For correspondence. Email Gerhard.Mittenhuber@biologie.
uni-greifswald.de; Tel. +49-3834-864218; Fax. +49-3834-864202.
# 2002 Horizon Scientific Press
factor; s70) of E. coli was initially discovered as a
control element of bacteriophage T4 (Bautz et al., 1969).
In 1981, the gene encoding this sigma factor (rpoD) was
sequenced by Burton et al. (1981). The functionally
equivalent B. subtilis counterpart of s70 , named s A has
a lower molecular weight (43 KDa) but is very similar to
RpoD (Gitt et al., 1985). With the exception of Deinococcus radiodurans (see below), the gene encoding the
major sigma factor RpoD/SigA is present in every
completely sequenced eubacterial genome.
The first alternative sigma factor of E. coli
discovered was the heat shock regulatory gene
product HtpR (RpoH) of controlling the heat shock
regulon of this organism (Grossman et al., 1984).
Other minor sigma factors of E. coli include the
stationary phase sigma factor RpoS (KatF) (Mulvey
and Loewen, 1989; Lange and Hengge-Aronis, 1991;
McCann et al., 1991; Tanaka et al., 1993) and a sigma
factor required for flagella biosynthesis named FliA
(Liu and Matsumara, 1995).
Research on sigma factors in B. subtilis was driven
by an imaginative hypothesis of Losick and Pero (1981)
who postulated that minor sigma factors might play an
important role in the cellular differentation process of
endospore formation. A nomenclature for sporulation
sigma factors was established (Losick et al., 1986) and
it could be demonstrated finally that a cascade of five
sigma factors (sH , sF, sE , sG , and sK ) operates in a
coordinated fashion during the sporulation process
(Losick and Stragier, 1992). However, the first alternative sigma factor discovered in B. subtilis, s37
(Haldenwang and Losick, 1980) which was renamed
sB (Losick et al., 1986) does not participate in the
sporulation cascade. This sigma factor is involved in
control of the general stress response of B. subtilis (for
reviews see Hecker and Völker, 1998, 2001). B. subtilis
also possesses a FliA ortholog, which is named sD in
this organism (Marquez et al., 1990). The RpoD/SigA,
RpoH, RpoS, FliA/SigD proteins and the five sporulation sigma factors constitute the s70 subfamily of sigma
factors (Lonetto et al., 1992; Wösten, 1998).
Lonetto et al. (1992) defined three groups and
Wösten (1998) provides an update of this classification: Group 1 constitute the primary sigma factors
RpoD/SigA which are essential for cell growth. Group 2
and group 3 sigma factors are nonessential for growth
and are also called alternative sigma factors. Sigma
factors in Group 2 are similar in sequence to group 1
sigma factors. In group 2, RpoS-type sigma factors,
sigma factors from high CG gram positive bacteria and
cyanobacterial sigma factors are included. Group 3
consists of heat shock sigma factors (RpoH), flagella
sigma factors and sporulation sigma factors.
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78 Mittenhuber
The existence of a distantly related group 3
subfamily of sigma factors became evident, when a
careful analysis of the sigE gene of Streptomyces
coelicolor was performed (Lonetto et al., 1994).
Common features of this subfamily include regulation
of extracytoplasmic functions and function as effector
molecules responding to extracytoplasmic stimuli
(Missiakas and Raina, 1998). This group was therefore
named the ECF (extra cytoplasmic function) family.
Members of the ECF family of E. coli are a second heat
shock sigma factor (RpoE, Rouvière et al.,1995) and
FecI involved in transcriptional regulation of ferric
citrate transport (Angerer et al., 1995). B. subtilis
possesses seven ECF sigma factors (sM, sV , sW, sX ,
sY , sZ , YlaC) (Kunst et al., 1997). The functions of the
ECF sigma factors in B. subtilis are just being
elucidated (Horsburgh and Moir, 1999; Huang and
Helmann, 1998; Huang et al., 1998).
The s54 -family is functionally and structurally
distinct from the s70-family (for recent reviews, see
Buck et al., 2000, and Studholme and Buck, 2000). No
sequence simliarity can be detected between the
s70 - and the s54 -family. The first sigma factor
belonging to the s54 -family discovered was s54
(RpoN). It is specifically required for the transcription
of nitrogen-regulated and of nitrogen-fixation promoters (Hunt and Magasanik, 1985; Reitzer and Magasanik, 1986) in E. coli. The RpoN equivalent of
B. subtilis is called SigL (Débarbouillé et al.,1991).
With the advent of completely sequenced bacterial
genomes it becomes possible to perform comparative
genomic studies. This emerging discipline is a powerful tool for a variety of applications. For example,
potential virulence genes can be identified by comparing the genomes of a virulent and an avirulent strain of
the same species. In this paper, we compare the genes
encoding sigma factors in completely sequenced
bacterial genomes by a phylogenetic analysis. We
examine whether a relationship between the number of
sigma factors in the genome of a given organism and
the habitat of the bacterium exists and whether a
relationship between the number of sigma factors in
the genome of a given organism and its genome size
and/or the number of genes in the genome can be
established.
Results
A total of 215 amino acid sequences from genes
encoding sigma factors from 31 species were obtained
from the NCBI server (supplementary Table 1 at http://
www.jmmb.net)*. For some species (e.g. E.coli, Helicobacter pylori ) the complete sequences of two strains
are avilable. In such cases, only one genome was
examined for the sigma factor content. The individual
sigma factors were classified as either s70-type, ECF,
or s54-type sigma factors (Table 2).
Table 2 also shows that a correlation between
the number of ECF sigma factor encoding genes and
the habitat of the species possessing these sigma
*Table 1 can be viewed at http://Jmmb.net/supplementary.
factors may be deduced. Pathogenic species tend to
have no or only few ECF sigma factors. The versatile
species Pseudomonas aeruginosa possesses the
highest number of ECF sigma factors (19), followed
by the nitrogen fixing symbiont Mesorhizobium
loti (18) and the stalked bacterium Caulobacter
crescentus (14).
Information given in Table 2 also tries to determine
whether a correlation exists between genome size and
the number of sigma factors in the genome. For this
purpose, two quotients were calculated. The first
quotient is defined as ‘‘genome size (in Mb)’’ divided
by ‘‘total number’’ of sigma factors. The second
quotient is defined as ‘‘genome size (in Mb)’’ divided
by ‘‘total number of genes’’ divided by 100. The basis
for this idea is simple: Obligately pathogenic species
are associated with their host and are used to their
constant environment. In contrast, free-living species
and those who are associated with hosts but are also
able to survive in the environment (e.g. E. coli, Vibrio
species) are faced with frequent changes of the
environmental conditions. Since it is essential for the
bacterial cell to adjust its metabolism to changing
environmental conditions, it seems reasonable to
speculate that a species adapted to a constant
environment has a smaller repertoire of regulatory
mechanisms than a species which is forced to cope
with many changing environmental conditions. Quotients like ‘‘genome size/number of sigma factors’’ and
‘‘total gene number/number of sigma factors’’ might
provide an estimate for transcriptional complexity:
Free-living species tend to have lower values of these
quotients than those associated with animals
(pathogens).
The distribution pattern of the s70 -family members
(RpoD/SigA, RpoS, RpoH, FliA/SigD, SigB, sporulation specific sigma factors and others with poorly
charcterized functions) within the 31 species is shown
in Table 3. It is noteworthy that Mycobacterium species
possess two rpoD/sigA genes and that the cyanobacterium Synechocystis sp. PCC 6803 has five paralogs.
Also M. loti possesses two RpoN and RpoH paralogs.
The occurence of the heat shock sigma factor RpoH of
E. coli is restricted to the proteobacteria. The FliA/SigD
sigma factors are not restricted to motile bacteria; for
example, C. crescentus, which does not possess a fliA
gene, is a motile bacterium. Two general stress sigma
factors (RpoS and SigB) show a mutually exclusive
distribution pattern. The occurence of sporulation
specific sigma factors is restricted to spore-forming
bacteria.
An unrooted phylogenetic tree based on an
alignment of whole amino acid sequences of the
s70-family is shown in Figure 1, a bootstrapped tree
based on an alignment of the conserved C-terminal
regions is shown in Figure 2. As observed previously
(Lonetto et al., 1992; Gruber and Bryant, 1997;
Wösten, 1998), members of the individual families
form distinct clusters. SigI of B. subtilis (Zuber et al.,
2001) is related to the putative sigma factor SigI of
C. acetobutylicum, the only other species possessing
this sigma factor. Simlilarly, the putative competence
Aae
Bha
Bsu
Aquifex
aeolicus
Bacillus
halodurans
Bacillus
subtilis
Borrelia
burgdorferi
Buchnera sp.
APS
Campylobacter
jejuni
Eco
Escherichia
coli K12
g-subdivision of
proteobacteria
Thermus/
Deinococcus
group
Cac
Dra
Low GC
grampositive
Ctr
Chlamydia
trachomatis
MoPn
Clostridium
acetobutylicum
Deinococcus
radiodurans
a-subdivision of
proteobacteria
Chlamydiales
Ccr
a-subdivision of
proteobacteria
e-subdivision of
proteobacteria
Low GC
gram positive
Spirochaetales
Low GC gram
positive
Aquificales
Phylogenetic
position
Caulobacter
crescentus
Cje
Bsp
Bbu
Abbreviation
Name
soil,
anaerobic
spore former
isolated from
environments rich in
organic
nutrients; as
well as from
nutrient poor
habitats
colonizes
lower gut of
animals,
survives release into
environment
marine, deep
sea vents;
chemolithoautotroph,
hyperthermophilic
(96 ! C )
alkaliphilic
organism from deep
sea sediment
soil, association
with plants
arachnid and
mammalian hosts
symbiont of
pea aphid
involved in
food poisoning,
especially in
contaminated
poultry; causes
inflammatory
enterocolitis
nutrient poor
aquatic
habitats
mouse pathogen
(pneumonitis)
Habitat and
features
Yes
Yes
Yes
No
Yes
No
No
No
4.64 Mb;
4290
2.65 Mb;
3174
3.94 Mb;
4023
1.04 Mb;
825
4.02 Mb;
3794
4.21 Mb;
4221
0.91 Mb;
903
0.64 Mb;
564
1.64 Mb,
1731
4.2 Mb;
4066
Yes
Yes
1.55 Mb;
1522
Genome
size;
number of
genes
Yes
free
living
7
2
14
3
17
3
2
3
18
19
4
Total
number
of sigma
factors
4
1
10
2
2
2
2
2
10
8
3
Number of
sigma
factors
in s70 family
1
0
1
1
1
1
0
1
1
1
1
Number of
sigma
factors
in s54 family
2
1
3
0
14
0
0
0
7
10
0
Number of
ECF sigma
factors
0.66; 0.61
1.33; 1.59
0.28; 0.29
0.35; 0.28
0.24; 0.22
0.55; 0.58
0.32; 0.28
0.30; 0.30
0.23; 0.23
0.22; 0.21
0.39; 0,38
Quotients
1; 2
Blattner et al., 1997
White et al.,
1999
Nölling et al., 2001
Read et al.,
2000
Nierman
et al., 2001
Kunst et al.,
1997
Fraser et al.,
1997
Shigenobu et al.,
2000
Parkhill et al.,
2000a
Takami et al.,
2001
Deckert et al.,
1998
Reference
(for completely
sequenced
genome)
Table 2. Distribution pattern of sigma factors belonging to the s70-family, the ECF subfamily and the s 54 -family within 31 bacterial species. Additionally, information on the phylogenetic position, habitat and
the genome size of each species is given. Two quotients ‘‘genome size divided by number of sigma factors in genome’’ (quotient 1) and ‘‘total number of genes divided by number of sigma factors in genome
divided by 100’’ (quotient 2) were calculated.
Inventory of Sigma Factors 79
Hin
Hpy
Lla
Mlo
Mle
Mtu
Mge
Haemophilus
influenzae
Helicobacter
pylori J99
Lactococcus
lactis
Mesorhizobium
loti
Mycobacterium
leprae
Mycobacterium
tuberculosis
Mycoplasma
genitalium
Table 2. Continued
Low GC
gram positive
High GC
gram positive
High GC
gram positive
a-subdivision
of proteobacteria
Low GC gram
positive
e-subdivision of
proteobacteria
g-subdivision of
proteobacteria
opportunistic
pathogen;
colonizes
the upper
respiratory
mucosa of
children and
adults
causes peptic
ulcer;
colonizes
gastric
environment
lactic acid
bacterium
closely
related to
Streptococcus;
used in
diary industry;
cheese
starter
culture
soil and
rhizophere
bacteria;
nitrogen-fixing
symbioses with
leguminous
plants
accumulation
in extremities of
the human body,
resides within
macrophages,
infection of
Schwann cells
of the periferous
nerve system;
causes
tuberculosis,
state of dormancy
in infected tissue
parasitic
association with
ciliated epithelial
cells of primate
genital and
respiratory
tracts
4.41 Mb;
3923
0.58 Mb;
468
No
3.27 Mb;
2770
7.04 Mb;
6804
2.37 Mb;
2310
1.64 Mb;
1491
1.83 Mb;
1707
No
No
Yes
Yes
No
No
1
13
4
23
3
3
4
1
3
2
3
2
2
2
0
0
0
2
0
1
0
0
10
2
18
1
0
2
0.58; 0.47
0.34; 0.30
0.82; 0.69
0.31; 0.30
0.79; 0.77
0.55; 0.5
0.46; 0.43
Fraser
et al., 1995
Cole
et al., 1998
Cole et al.,
2001
Kaneko
et al., 2000
Bolotin
et al., 2001
Tomb
et al., 1997
Fleischmann
et al., 1995
80 Mittenhuber
Mpn
Mpu
Nme
Pmu
Pae
Mycoplasma
pneumoniae
M129
Mycoplasma
pulmonis
Neisseria
meningitidis
Z2491
Pasteurella
multocida
Pseudomonas
aeruginosa
Table 2. Continued
g-subdivision
of
proteobacteria
g-subdivision of
proteobacteria
b-subdivision
of
proteobacteria
low GC
gram positive
Low GC gram
positive
versatile
bacterium;
found in soil,
marshes,
coastal
marine
habitats and
on plant
and animal
tissues;
forms biofilms
on wet
surfaces
(rocks, soil);
opportunistic
pathogen
(urinary tract
infections, burn
wounds, pneumonia;
cystic fibrosis)
human
pathogenic
bacterium
causing
tracheobronchitis
and primary
atypical
pneumonia
causal agent of
murine
respiratory
mycoplasmosis
opportunistic
pathogen causing
bacterial
meningitis
colonizing
nasopharynges and
oropharynges
multispecies
pathogen;
causes
serious diseases in
food animals
and humans
Yes
No
6.26 Mb;
5640
2.26 Mb;
2077
2.18 Mb;
2230
0.96 Mb;
814
No
No
0.82 Mb;
677
No
24
4
4
1
1
4
2
2
1
1
1
0
1
0
0
19
2
1
0
0
0.26; 0.24
0.56; 0.52
0.55; 0.56
0.96; 0.81
0.58; 0.68
Stover
et al., 2000
May
et al., 2001
Parkhill
et al., 2000b
Chambaud
et al., 2001
Himmelreich
et al., 1996
Inventory of Sigma Factors 81
Vch
Xfa
Tpa
Treponema
pallidum
Vibrio cholerae
Xylella
fastidiosa
Tma
Thermotoga
maritima
Uur
g-subdivision
of proteobacteria
Ssp
Synechocystis
sp.
PCC6803
Ureaplasma
urealyticum
Spirochaetales
Spy
Streptococcus
pyogenes
g-subdivision
of proteobacteria
Low GC
gram positive
Thermotogales
Cyanobacteria
Low GC
gram positive
Sau
Staphylococcus
aureus
Mu50
a-subdivision
of proteobacteria
Low GC
gram positive
Rpr
Rickettsia
prowazekii
Table 2. Continued
obligate
intracellular
parasite
nasal
membrane and
skin of
warm-blooded
animals
strict human
pathogen
associated
with a wide variety
of diseases
photoheterotrophic
growth,
facultative glucoseheterotrophic
optimum growth
temperature 80 ! C,
isolated from
geothermally
heated marine
sediment;
metabolizes
simple and
complex carbohydrates
causative
agent of syphilis
aetiological
agent of
cholera;
free-living species
in marine
environments;
pathogen via
horizontal gene
transfer
opportunistic
pathogen of the
human
urogenital tract
Plant
pathogen
transmitted by
leafhoppers;
embedded in
extracellular
translucent
matrix in the plant
No
No
Yes
2.68 Mb;
2830
0.75 Mb;
611
1.14 Mb;
1031
4.03 Mb;
3828
1.86 Mb;
1877
Yes
No
3.54 Mb;
3168
1.85 Mb;
1769
No
Yes
2.81 Mb;
2673
1.11 Mb;
834
No
No
4
1
8
5
4
8
3
3
2
2
1
4
3
3
6
3
3
2
1
0
1
1
0
0
0
0
0
1
0
3
1
1
2
0
0
0
0.67; 0.71
0.75; 0.61
0.5; 0.48
0.23; 0.21
0.47; 0.47
0.44; 0.40
0.62; 0.59
0.94; 0.89
0.56; 0.42
Simpson
et al., 2000
Glass
et al., 2000
Fraser
et al., 1998
Heidelberg
et al., 2000
Nelson
et al., 1999
Kaneko
et al., 1996
Ferretti
et al., 2001
Kuroda
et al., 2001
Andersson
et al., 1998
82 Mittenhuber
Inventory of Sigma Factors 83
Table 3. Distribution pattern of the (s70 -family and the (s54-family within the 31 species. The housekeeping sigma factor DR0804 of D. radiodurans
is not related to the other rpoD/SigA genes; this is indicated by the parantheses. The category "Other" includes specialized sigma factors unique to
several species: SigI (CAC1770) of C. acetobutylicum and B. subtilis, a heat shock inducible sigma factor in B. subtilis (Zuber et al., 2001), a
prophage-related sigma factor from B. subtilis (Xpf, McDonnell et al., 1994), a putative competence sigma factor from L. lactis (Bolotin et al., 2001), a
putative sigma factor similar to SigH of B. subtilis from Staphylococcus aureus (accession numbers AAK15307, BAB41722), and two putative
competence sigma factors from S. pyogenes (ComX1, ComX2; Lee and Morrison, 1999). Two putative sigma factors from C. acetobutylicum
(CAC1226, CAC2052) have unknown functions and are not related to any other sigma factor investigated in this study.
Abbreviation
Aae
Bha
Bsu
Bbu
Bsp
Cje
Ccr
Ctr
Cac
Dra
Eco
Hin
Hpy
Lla
Mlo
Mle
Mtu
Mge
Mpn
Mpu
Nme
Pmu
Pae
Rpr
Sau
Spy
Ssp
Tma
Tpa
Vch
Uur
Xfa
RpoD/SigA
RpoS
1
1
1
1
1
1
1
1
1
(1)
1
1
1
1
1
2
2
1
1
1
1
1
1
1
1
1
5
1
2
1
1
1
1
RpoH
FliA/SigD
SigB
Sporulation
Other
1
1
1
1
1
5
5
2
5
3
1
1
1
1
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
1
sigma factor ComX is present only in Lactococcus
lactis and Streptococcus pyogenes (2 copies). The
putative sigma factor SigH of Staphylococcus aureus
is only distantly related to the SigH cluster. An
alignment of the SigH, SigI proteins and of Xpf_Bsu,
CAC2052_Cac and DR0804_Dra was performed. A
similar condensed tree as in Figure 2 was obtained
(data not shown). The putative sigma factor DR0804 of
Deinococcus radiodurans is not related to any other
sigma factor and serves as outgroup for rooting the
tree.
An unrooted phylogenetic tree based on an
alignment of whole amino acid sequences of the ECF
subfamily is shown in Figure 3, a bootstrapped tree
based on an alignment of the conserved regions is
shown in Figure 4. The sequence of DR0804 of
D. radiodurans was used again as outgroup for rooting
the bootstrapped tree. It seems evident that many of
the genes encoding ECF sigma factors in the genomes
of C. crescentus, M. loti and P. aeruginosa evolved by
gene duplication. It will be a challenge for experimental
studies to determine the stimuli resulting in activation
of the sigma factors and to identify the corresponding
regulons.
An unrooted phylogenetic tree based on an
alignment of whole amino acid sequences of the s54
family is shown in Figure 5, a bootstrapped tree
based on an alignment of the conserved regions is
shown in Figure 6. Interestingly, the putative RpoN
sigma factor NMA0049 of Neisseria meningitidis is not
present in the cluster of the other proteobacterial
species.
Discussion
The number of sigma factor encoding genes in
completely sequenced bacterial genomes exhibits a
broad variation. With the exception of D. radiodurans, all eubacteria investigated in this study
possess a housekeeping sigma factor. This may
be surprising; however very little is known about
transcriptional regulation in this species (Meima et al.,
2001). The two RpoD paralogs in M. tuberculosis
fulfill different functions: SigA performs housekeeping
84 Mittenhuber
Figure 1. Unrooted phylogenetic tree of the s70-subfamily of sigma factors.
functions, whereas SigB seems to be involved in
stress responses, similar to RpoS (Hu and Coates,
1999). This result may indicate that M. tuberculosis
(and perhaps also Mycobacterium leprae) possess
both the RpoS mediated stress response typical for
E. coli and the SigB mediated general stress
response typical for B. subtilis (this sigma factor is
named SigF in M. tuberculosis (Chen et al., 2000)).
The gram-positive anaerobic bacterium Clostridium
acetobutylicum as well as Lactococcus lactis and
Streptococcus pyogenes which tolerate low oxygen
doses do not possess a homologous gene encoding
the general stress response sigma factor sB of B.
subtilis (Mittenhuber, 2002). This observation supports a conclusion that the presence of oxygen might
have contributed to the evolution of the sB depen-
dent general stress response in B. subtilis and
aerobic gram positive bacteria (Hecker and Völker,
2001).
In E. coli, the sigma factor RpoS controls a regulon
which is functionally similar to the sB regulon of
B. subtilis (Hengge-Aronis, 2000). Several studies
have been also performed on the RpoS regulons in
other bacteria besides E. coli: A connection between
an intact rpoS gene in Borrelia burgdorferi and
stationary phase gene expression was established
recently (Elias et al., 2000). In P. aeruginosa, RpoS
plays a role in regulating stress responses and
virulence (Suh et al., 1999), quorum sensing (Whiteley
et al., 2000) and biofilm formation (Xu et al., 2001). In
Vibrio cholerae, RpoS is required for stress resistance
(Yildiz and Schoolnik, 1998) and colonization of the
Inventory of Sigma Factors 85
Figure 2. Phylogenetic tree of the s70 -subfamily of sigma factors. The
tree was rooted using DR0804_Dra.
intestine (Merrell et al., 2000) but not for in vivo
survival (Yildiz and Schoolnik, 1998). Also, the
absence of an rpoS gene in the genome of Campylobacter jejuni has been proposed as explanation for the
absence of a stationary phase associated stress
response (Kelly et al., 2001).
A few interesting observations supporting some
aspects of this study have been reported previously:
In cyanobacteria, multiple paralogous genes encoding RpoD variants have been described previously
(Tanaka et al., 1992). Similar to M. loti, the
nitrogen fixing bacterium Bradyrhizobium japonicum
carries two RpoN paralogs (Kullik et al., 1991) but
three RpoH paralogs (Narberhaus et al., 1997).
C. acetobutylicum possesses a complete set of
sporulation specific sigma factors supporting previous work which indicated that clostridia may use
the same pathway as bacilli for spore formation
(Sauer et al., 1995).
However, the presence of a sigma factor in a
genome does not indicate that the same set of genes
as in model organisms forms the regulon controlled
by the individual sigma factor in different species.
For example, in Caulobacter crescentus RpoN controls a regulon involved in biosynthesis of polar
structures (flagellum, stalk) and disruption of rpoN
results in cell division defects (Brun and Shapiro,
1992). A combination of the RpoN and FliA regulons
is involved in flagellar biosynthesis in Vibrio cholerae
(Prouty et al., 2001) and Campylobacter jejuni
(Jagannathan et al., 2001). In B. subtilis, SigL is
required for certain catabolic reactions (use of
branched amino acids and argininine and ornithine
as nitrogen source; (Débarbouillé et al., 1991). As
reviewed by Studholme and Buck (2000), individual
RpoN regulons are quite diverse in the microbial
world.
The use of group 1 sigma factors as marker
molecules for molecular systematics has been
reported (Gruber and Bryant, 1997). To obtain additional information on the relationships between the
sigma factors themselves, group 2 sigma factors were
also used by Gruber and Bryant for the construction of
phylogenetic trees. Group 1 sigma factors are the only
suitable marker molecules for inferring phylogenetic
relationships simply because the content of alternative
sigma factors varies between individual species. This
variation in sigma factor content might indicate
immense flexibility of transcriptional regulation in
individual species. Taken together, all the observations discussed above strongly indicate that regulation
of gene expression in terms of regulons controlled by
specialized alternative sigma factors was a late
evolving phenomenon. Support for this point of view
was recently obtained in a phylogenomic study of the
stress response sigma factor sB of B. subtilis and its
regulatory proteins. Occurence of sB is restricted to a
86 Mittenhuber
Figure 3. Unrooted phylogenetic tree of the ECF subfamily of sigma factors.
small group of grampositive bacteria, whereas genes
encoding the regulatory proteins are also found in
species which do not possess a gene related to sB
(Mittenhuber, 2002). In an interesting contrast, regulation of another stress response, namely the stringent
response conferred by the regulatory molecule
(p)ppGpp is common to all eubacteria (Mittenhuber,
2001).
Different bacterial species use a wide variety of
mechanisms for transcriptional regulation which is of
course not restricted to alternative sigma factors
(Vicente et al., 1999). This transcriptional versality
conferred by alternative sigma factors can be even
observed in different laboratory stocks of the same
species: In different E. coli stocks, heterogeneity was
discovered with respect to the two sigma subunits FliA
and RpoS (Jishage and Ishihama, 1997). Sequence
variability in the rpoS gene of E. coli (called katF in this
paper) was also observed by Ivanova et al. (1992).
An attempt to relate genome size and/or total
gene number to the number of sigma factors provided
an unconvincing result. No threshold value can be
Inventory of Sigma Factors 87
Figure 4. Phylogenetic tree of the ECF subfamily of sigma factors. The
tree was rooted using DR0804_Dra.
determined which definitely distingues free-living from
pathogenic bacteria. However, free-living bacteria
tend to have lower values than pathogenic species.
Eisen (2000) stated that the number of currently
available completely sequenced genomes does not
reflect biodiversity. As more complete genome
sequences from a variety of closer and more distantly
related bacteria become available, time will tell
whether these quotients might be a useful measure.
For example, these quotients might be only useful for
a unique phylum or for a group of closely related
bacteria.
The present nomenclature for bacterial sigma
factors is quite confusing. For example, orthologous
sigma factors carry different names in different organisms: The general stress sigma factor SigB of the
Bacillus group is called SigF in Mycobacterium
tuberculosis and in Synechocystis sp PCC6803. In
Bacillus species, however, SigF is a sporulation sigma
factor. Another example: RpoE is a heat shock ECF
sigma factor in E. coli (Rouvière et al., 1995).
However, this designation has been also used for the
delta subunit of RNA polymerase in gram positive
bacteria (Lampe et al., 1988). This has resulted in an
error in the annotation of the S. pyogenes genome
(Ferretti et al., 2001): It is stated that S. pyogenes
possesses a sigma factor encoding gene similar to
rpoE of E. coli. The rpoE gene of S. pyogenes
(accession number AAK34603), however encodes
the delta subunit of RNA polymerase and not a sigma
factor. In order to avoid similar errors in future genome
projects, development of a new nomenclature system
for bacterial sigma factors would be very desirable.
However, finding consensus gene names is not an
easy task since changes in gene names are commonly
only accepted by the scientific community with some
reluctance (Pearson, 2001).
Experimental Procedures
Individual amino acid sequences of sigma factors
were obtained at the NCBI server (http://
www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?db=Protein).
The complete genome sequence of C. acetobutylicum
was obtained from Genome Therapeutics, Inc. (http://
www.genomecorp.com/programs/sequence_data_clost.
shtml). Alignments were generated using CLUSTALW
(Thompson et al., 1994) at the EBI server (http://
www.ebi.ac.uk/clustalw/). The construction of rooted
phylogenetic trees and statistical tests of tree topologies (bootstrapping) was performed using MEGA 2
(Kumar et al., 2001; http://www.megasoftware.net).
The neighbor-joining method (Saitou and Nei, 1987)
was used for tree construction. Bootstrapping
(Felsenstein, 1985) was performed on the basis of
the p-distance (proportion of different amino acids)
88 Mittenhuber
Figure 5. Unrooted phylogenetic tree of the s54 family of sigma factors.
Figure 6. Phylogenetic tree of the s54 family of sigma factors. The tree was not rooted.
with 1000 replicates. Unrooted phylogenetic trees
were constructed using the data from the alignment
with the help of the program TreeView (Page, 1996)
(http://taxonomy.zoology.gla.ac.uk/rod/treeview.html)
and edited using the program Metafile Companion
(http://www.companionsoftware.com/).
Acknowledgements
This work was performed in the laboratory of Michael Hecker, whose
support is gratefully acknowledged. I thank Doug Smith (Genome
Therapeutics, Inc.) for the permission to perform analyses using the
unpublished C. acetobutylicum genomic sequence. I thank Michael
Hecker and Ulrich Zuber for comments on the manuscript and Ulrich
Zuber and Knut Büttner for helpful discussions.
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