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Microbiology (1996),142,347-357
Printed in Great Britain
The sigA gene encoding the major G factor of
RNA polymerase from the marine
cyanobacterium Synechococcus sp. strain PCC
7002 : cloning and characterization
Laurie F. Caslaket and Donald A. Bryant
Author for correspondence: Donald A. Bryant. Tel:
e-mail : [email protected]
Department of
Biochemistry and
Molecular Biology, and
Center for Biomolecular
Structure and Function,
The Pennsylvania State
University, U niversity Park,
PA 16802,USA
+ 1 814 865 1992. Fax: + 1 814 863 7024.
The gene encoding the principal a factor from Synechococcus sp. strain PCC
7002 was isolated and characterized. The Synechococcus sp. strain PCC 7002
sigA gene encodes a protein of 375 amino acids (43.7 kDa) that is required for
viability under normal growth conditions. The SigA protein was overproduced
in Escherichia coli and the purified protein was used to raise polyclonal
antiserum in rabbits. This antiserum was used in immunoblot analyses of
partially purified RNA polymerase from Synechococcus sp. strain PR6000. The
probable in wivo translational start site was identified by a comparison of
amino acid sequencing results obtained with SigA proteins overproduced in E.
coli with immunoblot analyses of SigA protein in crude preparations of RNA
polymerase from the cyanobacterium. The sigA gene is encoded on a transcript
of 1700 bases that initiates 496 nucleotidesupstream from the probable in
wiwo translational start site. The abundance of sigA transcripts decreases
rapidly after the removal of combined nitrogen from the growth medium.
~~
Keywords: Jynechococcz/s sp., RNA polymerase, sigma factors, cyanobacteria
INTRODUCTION
Gene expression is controlled, in part, by the interaction
between DNA-dependent RNA polymerase (EC
2.7.7.6) and the promoter regions of genes and operons.
Bacterial RNA polymerase exists in two forms : the core
enzyme, responsible for DNA binding and possessing
catalytic activity ; and the holoenzyme, comprising the
core enzyme plus one of a family of dissociable CJ factors.
The 0 subunit confers on the RNA polymerase core
enzyme the ability to recognize specific promoter sequences (for reviews, see Helmann & Chamberlin, 1988;
Lonetto e t al., 1992). Many species of bacteria contain
multiple forms of RNA polymerase holoenzyme that
differ only in the CJ subunit. These alternate 0 factors play
a major role in Bacilltls stlbtilis sporulation (Stragier &
Losick, 1990), the response to environmental stress in
Escherichia coli (Grossman e t al., 1984; Erickson & Gross,
1989; Mulvey & Loewen, 1989), cell development and
differentiation in M?jxococctls xanthzrs (Inouye, 1990 ;
t Present address: Department of Microbiology, 527 Biological Sciences
Building, University of Georgia, Athens, GA 30602-2605, USA.
The GenBank accession number for the nucleotide sequence reported in
this paper is U15574.
0002-0201 0 1996SGM
Apelian & Inouye, 1990, 1993), and the transcription of
genes involved in a multitude of physiological roles in
enteric bacteria (Kustu et al., 1989). In each case the
alternate o factor directs transcription from a different set
of promoters and thus provides a means to regulate
coordinately a large set of genes.
Synecbococcm sp. strain PCC 7002 (Pasteur Culture Collection) is a unicellular, marine cyanobacterium that does
not fix nitrogen and does not differentiate heterocysts,
akinetes or hormogonia. In its natural environment this
organism is probably exposed to repeated fluctuations in
incident light intensity as well as nutrient levels. Environmental changes have been shown to alter the
transcription of a large number of genes in several species
of cyanobacteria (Tandeau de Marsac & Houmard, 1993).
For example, Northern blot hybridization analyses of
RNAs extracted from cells of Synechococczrs sp. strain PCC
7002 starved for various nutrients gave particularly
striking results when simultaneously hybridized with
gene probes for p s a A B and p s b A (Gasparich, 1989).
Transcripts encoding PsaA and PsaB are present among
RNAs extracted from cells grown in replete medium,
from cells starved for iron or sulfur for 24 h and from cells
starved for phosphorus for 12 h. However, p s a A B
transcripts are not detectable in RNAs extracted from
347
L. F. C A S L A K E a n d D. A. B R Y A N T
cells starved for nitrogen for 5 h or from cells starved for
carbon for 12 h. In contrast, p s b A transcripts are present
at approximately the same levels in RNAs extracted from
cells grown under all conditions tested (Gasparich, 1989).
Alignment of sequences upstream of mapped 5’ endpoints of many Synecbococct/ssp. strain PCC 7002 genes has
revealed that the promoter regions of several genes
contain sequences similar to the consensus promoter
sequence of the E. coli RNA polymerase principal o factor,
0 7 0 (Curtis & Martin, 1994). Other mapped 5’ end-points
have promoter sequences that resemble either the 0 7 0
consensus -35 hexamer or the -10 hexamer, while
others have promoter sequences that do not resemble the
consensus sequence for any known o factor (Rosenberg &
Court, 1979; Wiggs e t al., 1981; Cowing e t a!., 1985;
Erickson & Gross, 1989; Moran, 1993). Such alignments,
along with evidence for differential gene expression, led
us to postulate that Synecbococctlssp. strain PCC 7002 might
possess multiple o factors that could account for differential patterns of gene expression.
Genes encoding CJ factors have been reported from only
three cyanobacteria thus far. The unicellular cyanobacterium Synecbococczls sp. strain PCC 7942 has been
reported to possess four genes encoding CJ factors (Tanaka
e t al., 1992a, b). The complete sequence of only one of
these genes, denoted rpoDI, was reported (Tanaka e t al.,
1992b), but no further characterization of the transcription of these genes, nor characterization of the structure
and functions of their protein products, has been reported.
The sigA gene, encoding the principal vegetative-cell o
factor from Calotbrix sp. PCC 7601 encodes a polypeptide
with an apparent mass of 55 kDa. The SigA protein and
its interaction with RNA polymerase core enzyme was
biochemically characterized, but no studies on the possible
transcriptional or post-transcriptional regulation of o
factor gene(s) in this organism were reported (Schyns e t
al., 1994). Three genes (@A,sigB and sigC) encoding o
subunits have been cloned and sequenced from the
filamentous, heterocy stous cyanobacterium Anabaena sp.
strain PCC 7120 (Brahamsha & Haselkorn, 1991, 1992).
While the sigA gene was shown to be transcribed during
growth in nutrient-replete medium as well as during
nitrogen starvation, sigB and sigC are only transcribed
from 6 h to 24 h after removal of combined nitrogen from
the growth medium. Transcription of these genes could
not be detected again at 30 h after the onset of nitrogen
starvation. Neither sigB nor sigC seems to be critical for
growth under standard photoautotrophic conditions,
since interruption of these genes does not affect viability,
nitrogen fixation or heterocyst differentiation. The deduced amino acid sequences of all of these cyanobacterial
genes show significant similarity to the major o factors of
E. coli and B. stlbtilis. Nonetheless, while it is clear that
even non-differentiating cyanobacteria contain multiple o
factors of the 0’’family, the physiological roles of such o
factors remains completely unknown.
In this paper we report that Synechococcm sp. strain PCC
7002 DNA encodes multiple B factors for RNA polymerase (at least five 0 7 0 proteins of Group 1 and Group 2
have been identified to date). It is our long-term goal to
348
understand the physiological roles of the multiple o
factors of the unicellular marine cyanobacterium Synechococc~~s
sp. strain PCC 7002. It is our hope that thorough
analyses of these o factors, and mutants lacking certain
of these proteins, will provide insights into the molecular
basis for transcriptional responses to environmental
changes in cyanobacteria. As a first step in analysing the
structure and function of these B factors, it was essential to
identify the o factor protein encoding the principal (Group
1; Lonetto e t al., 1992) o factor. The molecular cloning,
nucleotide sequence, and transcriptional characterization
of the Synecbococctls sp. strain PCC 7002 sigA gene is
reported in this paper. Mutational analysis has established
that this gene is the principal B factor of this cyanobacterium. The SigA protein was overproduced in E. coli,
purified to electrophoretic homogeneity and used to raise
polyclonal rabbit antibodies. Initial studies to examine the
abundance of SigA in partially purified Synecbococczis sp.
strain PCC 7002 RNA polymerase preparations are
described.
METHODS
Bacterial strains, plasmids and growth conditions. The
unicellular marine cyanobacterium S.’nechococcus sp. strain PCC
7002 (formerly Agmenellum quadruplicatum strain PR-6 ; Rippka
e t al., 1979) was maintained on 1.5 YO(w/v) agar plates and in
liquid culture bubbled with 1 YOC02/99 % air (v/v) at 39 “C in
Medium A (Stevens & van Baalen, 1973) supplemented with 1 g
sodium nitrate 1-l. The wild-type isolate in our laboratory has
been designated Jynechococcus sp. strain PR6000. Standard
illumination conditions were approximately 250 pE m-2 s-l of
cool fluorescent light. The effect of nitrogen starvation on gene
expression was analysed by growing cells in replete medium to
a mean OD,,, of 0.8. The cells were centrifuged, washed twice
with Medium A, resuspended at the original cell density in
Medium A and incubated under standard illumination and
aeration conditions for the indicated times.
E. coli strain DH5a (BRL) was used for most recombinant DNA
manipulations except protein overproduction. Plasmid vector
pUC19 was used for all routine cloning and sequencing
procedures (Yanisch-Perron e t al., 1985). E. coli strain HBlOl
was used as host for a S’nechococcus sp. strain PRGOOO cosmid
library. E. coli strains were grown on LB medium, supplemented, when appropriate, with ampicillin (100 mg 1-1 in LB),
chloramphenicol (30 mg 1-1 in LB) or kanamycin (40 mg 1-1 in
LB, 200 mg 1-1 in Medium A). E. coli strain BL21(DE3) was
used for overproduction of the SigA protein (Studier e t al.,
1990).
DNA and RNA isolation, DNA sequence analysis and transcript analysis. Small-scale preparations of plasmid DNA from
E. coli were extracted by the alkaline lysis method (Birnboim &
Doly, 1979) or the rapid-boiling procedure (Holmes & Quigley,
1981). Large-scale DNA preparations were performed by the
alkaline lysis method and further purified by CsC1-ethidium
bromide equilibrium density-gradient ultracentrifugation.
Plasmid DNA used in nested deletion reactions was purified
through two successive density-gradients. S’nechococcus sp. PCC
7002 strain PRGOOO chromosomal DNA was prepared as
described by de Lorimier e t al. (1984). Southern blots were
transferred to nitrocellulose membranes and hybridized overnight at the temperatures indicated (Bryant & Tandeau de
Marsac, 1988). DNA fragments used in subcloning or labelling
were purified by electrophoresis on agarose gels followed by
Synecbococctls sp. strain PCC 7002 sigA
silica-gel chromatography (Geneclean, Bio-101). Labelling of
DNA probes with [a-32P]dATP (1.11 x 1014Bq mmol-l, New
England Nuclear) was performed using DNA polymerase I
Klenow fragment and a Random Primed DNA Labelling Kit
(Boehringer Mannheim). DNA sequencing was performed
using the chain termination method (Sanger e t a/., 1977) on
base-denatured templates (Hattori & Sakaki, 1986). DNA
fragments were labelled with [a-35S]-thio-dATPwith Sequenase
2.0 (USB). Nucleotide sequence data were analysed with
MacVector Sequence Analysis Programs Version 3.5 (EastmanKodak) and HIBIO MacDNAsis Pro (Version 2.0, Hitachi
Software Engineering). Protein sequence alignments were
generated with IntelliGenetics FastDB Release 5.4. Codon
usage was analysed with the CodonUse 3.1 program (a gift of
Drs Conrad Halling and Robert Haselkorn, University of
Chicago, Chicago, IL, USA).
RNA from Synechococcus sp. strain PRGOOO was extracted by
mechanical cell disruption as described by Golden e t al. (1987).
RNA for Northern hybridizations was separated on
formaldehyde-containing agarose gels with buffer recirculation
and transferred to Hybond-N membrane according to manufacturer’sinstructions (Amersham). Northern blots were hybridized overnight at 42 OC under conditions recommended by the
manufacturer. The 5’ end-point of the sigA mRNA was mapped
using the primer extension protocol described by Ausubel e t al.
(1987) except that 100 pg total RNA was used in the extension
reaction. The oligonucleotide used was 5’ CGCTTGGGTCATGCCTATTTCCTC 3’ (complementary to nt 923-946 in Fig.
3). The primer extension product was denatured and subjected
to electrophoresis on a standard DNA sequencing gel alongside
DNA sequencing ladders generated using the same primer on a
plasmid template containing sigA and using a universal primer
on single-stranded M13 phage DNA. Oligonucleotide primers
were synthesized and purified by reverse-phase HPLC at The
Biotechnology Institute (The Pennsylvania State University,
University Park, PA, USA).
Cloning, insertional inactivation and transformation procedures. The 1.4 kb HindIII-HincII fragment from the
Anabaena sp. strain PCC 7120 sigA gene (Brahamsha &
Haselkorn, 1991) was radiolabelled and used as a hybridization
probe for a Southern blot prepared with Synechococcus sp. strain
PRGOOO chromosomal DNA digests. The strongest hybridization signals corresponded to a 3.3 kb HindIII fragment and a
20 kb EcoRI fragment. The sigA probe was hybridized to a
genomic library of Synecbococcus sp. strain PRGOOO DNA
fragments produced by partial digestion with EcoRI and cloned
into the EcoRI site of the cosmid vector pHC79 (Hohn &
Collins, 1980). Screening of the cosmid library resulted in the
isolation of a single clone containing the 3.3 kb HindIII
fragment that was subcloned into pUC19.
For insertional inactivation of the Sjwzechococcussp. strain PRGOOO
s&4 gene, a 1-32kb KpnI fragment containing the apbII gene
was inserted into the unique KpnI site (nt 1025-1030) (see Figs
1 and 3). The aphII gene encodes aminoglycoside 3’phosphotransferase 11, an enzyme that confers resistance to the
antibiotic kanamycin. Transformation of Synechococcus sp. strain
PRGOOO was performed as described by Buzby e t al. (1983).
Synthesis and purification of the SigA protein. The sigA gene
was modified by oligonucleotide-mediated mutagenesis in M13
to introduce a unique NdeI restriction site and an ATG start
codon at nt 884-889 (see Results and Fig. 3); the sequence
modification was verified by DNA sequence analysis. The
modified sigA gene was excised as an NdeI-EcoRI fragment and
cloned into pET3a (Studier et al., 1990). This plasmid,
pET3aSigA, was unable to support high levels of protein
production in E. coli BL21(DE3) or BL21(DE3)pLysS ex-
pression cell lines after induction with 0.5 mM IPTG. This may
have been due to the continuous production of low levels of
SigA that interfered with transcription and growth of E. coli. To
reduce background levels of protein production, sigA was
inserted into the PET1l a expression vector and transformed
into the E. cofi BL21(DE3) expression cell line (Studier et al.,
1990).
E. coli strain BL21(DE3) cells containing the pETllaSigA
plasmid were grown in NZCYM medium at 30 OC with shaking.
Protein production was induced when the ODeooof the cell
culture reached 0.6. Cells from 500 ml medium, 5 h after
induction, were centrifuged, resuspended in 25 mM Tris/HCl,
pH 7*5,20mM NaCl, and lysed by two passages through a cold
French pressure cell at 1-38x lo5 kPa. Inclusion bodies were
pelleted by centrifugation at 1900g for 8 min. The inclusion
bodies were resuspended in 10 mM HEPES, pH 6.8, 50 mM
NaCl, 1.0 mM EDTA, 0.1 mM DTT, 5 % (v/v) glycerol, 6.8 M
urea. This protein suspension was centrifuged at 7500g for
20 min at 4 OC and loaded on a 17 cm x 2.5 cm CM-Sepharose
column. The column was washed with 10 mM HEPES, pH 6.8,
50 mM NaC1,l mM EDTA, 0.1 mM DTT, 5 % glycerol, 6.8 M
urea, and eluted with a linear gradient of 50-400 mM NaCl in
the same buffer. Column fractions containing SigA were
identified by SDS-PAGE, and the protein was subjected to
preparative electrophoresis on a 7.5 % (w/v) acrylamide gel.
SigA was excised from the gel, electroeluted into 25 mM
Tris/HCl, 0.2 M glycine, 0.1 % SDS and precipitated by
addition of ice-cold acetone to 80 % (v/v) final concentration.
The precipitated protein was sent to The Antibody Core Facility
at The University of Nebraska (Lincoln, NE, USA) for antibody
production.
Partial purification of Synechococcus sp. strain PR6000 RNA
polymerase. For immunoblot analysis of Synechococcus sp. strain
PRGOOO CJ factors, cells were grown in nitrogen-replete medium
(2 1) to a mean OD,,, of 0.8 and were harvested by centrifugation
at 7000g for 15 min. Cells were washed with 20 mM Tris/HCI,
pH 7.5,20 mM NaCl, resuspended in 10 mM Tris/HCl, pH 8.0,
100 mM NaC1, 1 mM EDTA, 0.3 g PMSF 1-’, 10% glycerol,
and lysed by two passages through a cold French pressure cell at
137.9 MPa. The cell lysates were centrifuged at 50000g for
30 min at 4 OC and loaded onto a 1 cm x 3 cm heparin-agarose
column; the columns were washed with 5 column vols of the
same buffer. Crude RNA polymerase was eluted with 1 column
vol. of 10 mM Tris/HCl, pH 8.0, 1 M NaC1, 1 mM EDTA,
0.3 g PMSF l-l, 10 % glycerol.
SDSPAGE, immunoblot analysis and protein sequencing.
SDS-PAGE was performed as described by Laemmli (1970).
Proteins separated by electrophoresis were either stained with
Coomassie Brilliant Blue or transferred electrophoretically onto
nitrocellulose membranes for 400 mA h. Protein transfer was
verified by monitoring the transfer of prestained molecular mass
standards. Rabbit antibodies against the SigA protein were
raised and isolated by the Antibody Core Facility (see above).
Immunodetection of specific polypeptides on blots was performed with alkaline phosphatase conjugated to goat anti-rabbit
IgG as described by Harlow & Lane (1988).
Amino-terminal and carboxyl-terminal protein sequencing was
performed by D r Jindong Zhao (Applied Biosystems).
RESULTS
Cloning and sequence analysis of the sigA gene
A physical map of the 2.3 k b HindIII-EcoRV fragment
containing the Synecbococczrs sp. strain PRGOOO sigA gene is
shown in Fig. 1. Subsequent use of the 820 bp KpnI-BghI
349
L. F. C A S L A K E a n d D. A. B R Y A N T
I
UDhZzI
R
HC C
..........................................................................................................................................................
Fig. 1. A physical map of the 2.3 kb HindIII-EcoRV fragment
containing the sigA gene encoding the principal o factor of the
RNA polymerase of Synechococcus sp. strain PR6000. Arrows in
the boxes indicate the direction of transcription. Arrows
beneath the diagram indicate the sequencing strategy; plain
arrows indicate sequence obtained from subclones with either
M13 forward or M13 reverse sequencing primers. The circle
represents the position of a synthetic oligonucleotide used in
primer extension analysis. The aphll gene, that encodes
aminoglycoside 3'-phosphotransferase II conferring resistance
to kanamycin, was inserted into the Kpnl site of the sigA gene
in a mutational study. HI Hindlll; C, Hincll; Y, Styl; 5, Spel; K,
Kpnl; GI Bglll; R, EcoRV.
12345
kb
-21
-5.1
-4.3
- 3.5
sigA +
-2.0
-1.9
- 1.4
..........................................................................................................................................................
Figrn 2. Chromosomal Southern blot hybridization of
Synechococcus sp. strain PR6000 DNA using an 820 bp
Bglll-KpnI sigA gene-internal fragment as probe. The blot was
hybridized overnight a t 55 "C. The 3.3 kb Hindlll fragment
containing the sigA gene is indicated. The DNA was digested
with Hindlll (lane l), EcoRl (lane 2), Hincll (lane 3), Hindlll and
EcoRl (lane 4), and Hindlll and Hincll (lane 5).
fragment of sigA as a hybridization probe for a Synecbococcus sp. strain PRGOOO chromosomal Southern blot at
reduced stringency revealed multiple hybridizing fragments in all restriction digests (Fig. 2). Cloning and
sequencing studies (L. F. Caslake, T. Gruber & D. A.
Bryant, unpublished data) of these other hybridizing
fragments indicates that the Synecbococczts sp. strain PRGOOO
chromosome contains at least five genes with strong
sequence similarity to bacterial o factors of Types I and I1
as defined by Lonetto e t al. (1992).
Sequence analysis of the 2289 bp HindIII-EcoRV fragment revealed several ORFs. The largest ORF encoded a
polypeptide of 391 amino acids with a predicted molecular
3 50
mass of 45561 Da (Fig. 3). In the original subclone
obtained from the cosmid library, a TAA stop codon was
found at codon 80. The correct genomic sequence at this
position was determined by cloning the SpeI-EcoRV
fragment from a partial chromosomal library. Sequence
analysis of a positive clone revealed a GAA codon at
position 80. The polypeptide was initially assumed to
begin at the TTG codon at nt 887 due to sequence
similarity between the deduced amino acid sequence of
Synecbococczts sp. strain PRGOOO SigA and the sequenced
amino terminus of Anabaena sp. strain PCC 7120 SigA
(Fig. 4; Brahamsha & Haselkorn, 1991). A potential
ribosome binding site (5' AGAAG 3') precedes this
presumed start site by 6 bp (bold type in Fig. 3). However,
the possibility that translation begins at the methionine
at codon 17 (Fig. 3) could not be excluded (see below).
A very strong, potential ribosome binding site (5'
AAGAGGAAA 3') also precedes this translation initiation start point (underlined in Fig. 3 ) . Two imperfect
inverted repeats follow the stop codon of sigA (arrows in
Fig. 3); the second of these resembles a p-independent
terminator (Rosenberg & Court, 1979) and could form an
energetically favourable hairpin with a GC-rich stem of
8 bp followed by a run of 6 thymidine residues.
An O RF potentially encoding a protein of 69 amino acids
occurs upstream from the sigA gene and is cotranscribed
with the sigA gene (see below). Database searches have
failed to detect proteins with significant homology to the
protein predicted by this ORF, whose significance is
presently unknown.
Analysis of the deduced amino acid sequence for SigA of
Synecbococczts sp. strain PRGOOO revealed regions with
strong sequence similarity to the four conserved regions
of o"-type o factors defined by Helmann & Chamberlin
(1988). The deduced amino acid sequence is also highly
similar to major o factors from the cyanobacteria Anabaena
sp. strain PCC 7120 and Synecbococczts sp. strain PCC 7942,
and B. stlbtilis and E. coli (Fig. 4). Region 2.2, which is
believed to be involved in binding to the core RNA
polymerase, is 100 % identical to Synecbococcus sp. strain
PCC 7942 RpoD1, 100 YOidentical to Anabaena sp. strain
PCC 7120 SigA, 55% identical to 043of B. sztbtilis and
65 % identical to 0" of E. cob. Region 2.4, which overlaps
the ' RpoD box' (Tanaka e t al., 1988) and is believed to be
involved in binding to the - 10 region of the promoter, is
loo%, lOO%, 86% and 82% identical to the major o
factors from Synecbococcus sp. strain PCC 7942, Anabaena
sp. strain PCC 7120, B. subtilis and E. coli, respectively.
Region 4.2, implicated in recognition of the -35 region
of the promoter, is 97 YO,100 YO,81 YOand 81 YOidentical
to Synecbococczts sp. strain PCC 7942 RpoD1, Anabaena sp.
strain PCC 7120 SigA, B. sztbtilis 043and E. coli o",
respectively.
Transcript analyses
Total RNAs isolated from Sjnecbococczts sp. strain PRGOOO
cells grown in replete medium and from cells deprived of
nitrogen for increasing time were hybridized with the
820 bp KpnI-BgnI fragment (Fig. 1) of the sigA gene. The
Jjwechococcus sp. strain PCC 7002 sigA
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D
R
A
A
E
A
A
G
R
M
K
T
L
K
T
E E E I E L A
P G A A G A A G A A A W
G M P L K D P
GGGATGCCCCTCAAAOATIT
K P S T Y A T
W W I R Q A
I T R A I A D
TAAGTWXTACCPACGCGA C C ' I G G ~ T X G ' X A G G C T ATTACGCGGGCGATCGCCGA
S
Q
E L G
AAGAMT-CC
R
K
P
T E E E I
ACCGAGGAAGAGA-
A
E
K
L
R
P
I
A
K
S
A
Q
L
P
I
S
L
E
T
P
I
G
K
E
E
D
S
R
L G D P I E A
TPGGG'XACTNXTfXAAGC
Q
V
S
K
S
L
L
R
E
D
L
E
N
V
L
D
T
L
S
A
R
E
R
D
V
L
R L R Y G
CGTCTCCGTTA-
-
1801 CGATGGGGAAACTCCAGAAG ATCMG'KTCCAAGAGCCTC CVXGGGAAGATCTAGAAAA CC.K;CITC;ATACCC'ICAGTG C C C G % A G G T G A T G m
D
A
K Y M N R G
L S P Q D L I
AAAAATATATGAACCGCGW CTR'CGTITCAGGACTIGAT
1681 GCGCAlGGAAATCACCATCG AAAMClGCGCTTCATCGCC AAGTCTOCCCAGTTACCGAT TTCCCTCGAAAmCCGAllG GT-TRXCGC!
D
L
K
E
E
I
G
Q
I
P
N
V
T
R
E
R
I
R
Q
I
E
A
K
A
L
R
K
L
R
H
P
N
R
N
L
D
S
I
1921 TGATCGCCGCATGAllAACCC TCGAAGMAT'SGGCCAAATT TlTAACGTAACCCGTGAACG GATKGCCAGAXGAAGCAA AAGCGCPGCGTAAACmCOT CACCCGAACCGCAACAGCAT
2041
L K E Y L E '
CCNAAAGAATATATCCGCT AGCCATATPCAGCCTCAATC ' M T ~ T A C ' I G A GAACATAMATTXTAQM~A CCC~CCAGlKX3G'IGGGGTT TITTATN%ICAATIT'ICCC
2161
GAAGAAATAWTGTAAAAA AACCGTAAAAATlnXAlGG WACAGTAGCATAGCGGCA ACAGCAf3T"T-C
2281
TACGATATC
+
CA'lGGCAAAClCTt2CCCn;A CGATCACGAmATCCGGCA
Fig. 3. Nucleotide sequence of the 2.3 kb HindIII-EcoRV fragment containing sigA. Deduced amino acid sequences are
shown in single letter code. The ORF, designated orfA, found in the transcribed leader region of sigA is shown. The
unique Kpnt site into which the aphl gene is inserted is underlined (nt 1025-1030). The major 5' end-point of sigA
mapped by primer extension analysis is indicated by an arrow at nt 440. The putative ribosome binding sites are also
underlined, and the inverted repeats that are located 3' to the sigA gene and that are capable of forming stem-loop
structures are indicated by paired arrows. A probable in vivo translation initiation start site at codon 17 is indicated by
bold type (see text for details). The amino acids underlined were determined by amino- and carboxyl-terminal
sequencing of overproduced SigA protein.
probe hybridizes to a smear of transcripts (Fig. 5a), the
largest of which is approximately 1700 bases. To ensure
that the apparent instability of the sigA mRNA was not
due to general RNA degradation, this blot was also
hybridized with a DNA fragment encoding the Synecbococcus sp. strain PR6OOO glnB gene. No evidence of
degradation of glnB transcripts was detected (data not
shown), indicating that the sigA transcript is probably
inherently unstable. The abundance of all transcripts
hybridizing to the sigA probe decreases rapidly after more
than 1 h of nitrogen starvation (Fig. 5a, lanes 2-6).
Primer extension analyses were performed with total
RNA extracted from Sjmecbococctls sp. strain PR6OOO cells
grown under nitrogen-replete conditions to map the 5'
end of the sigA transcript (Fig. 5b, lane 7). The major 5'
end-point of the sigA transcript occurs 446 nt upstream
from the putative TTG translation start site, or 496 nt
upstream from the alternative ATG start site at nt 935
(Fig. 3). The predicted size of a transcript beginning at nt
440 and ending at the last T of the downstream inverted
repeat is 1696 bases, in excellent agreement with the
results obtained by Northern hybridization. Upstream of
this mapped 5' end-point is a good match to the E. coli
consensus -35 hexamer; however, a sequence similar to
the -10 hexamer is not present. As noted above, the
leader sequence contains an ORF encoding a polypeptide
of 69 amino acids (Fig. 3) with no significant similarity to
sequences in databases.
Primer extension analyses were also performed with
RNAs isolated from cells deprived of nitrogen for 2 , 4 and
8 h (Fig. 5b, lanes 8-10). The major 5' end-point observed
in all cases occurred 446 nt upstream from the putative
351
L. F. C A S L A K E a n d D. A. B R Y A N T
Synechococcus s p . PR6000 SigA
Synechococcus sp. 7942 RpoDl
Anabaene s p . PCC 7120 SigA
Bacillus subtilis 043
Escherichia coli U70
r
w 1
MNVWLVAFLNEQEEIGWTQATNW--LDQTRNEGDIDYSALAEAQIKEGTDYVELTLPTKKSRK
MTQATEL:DPALKPAE:K:KRSSRKKAT:AVVEPAT-TIAPT:DVDAID:EDSVGEDEDAAA:
MNQA"V:DSIYQPDLEI~Q:EIE::DLLI:E:E:~~D~D:D:F~P~DEDDA:SG~
MADKQTHETELTFDQ~LTESGKItRG:LTYE:IA:RMSSFEIESDQMDEWEFL:EQGVELIS:NEET:DPNIQQ:AKAEEEFDL
MEQNPQSQLKL------LVTRGKEQOYLTYA:VNDHLPEDIVDSDQIE:IIQMIN:MGIQVME::PDADDLMLA:N:ADEDAAEA
................................................................................................................................................................................................................................................................E
Fig. 4. Alignment of the predicted amino acid sequences of the major Q factors from Synechococcus sp. strain PR6000,
Synechococcus sp. strain PCC 7942, Anabaena sp. strain PCC 7120, B. subtilis, and E. coli. The overlined regions correspond
to conserved regions 1, 2, 3 and 4 of 0 factors as defined by Helmann & Chamberlin (1988). Colons indicate that the
amino acid at that position is identical to that of Synechococcus sp. strain PR6000 SigA; dashes indicate gaps introduced
to align the protein sequence optimally. Percentage values following the polypeptides are percentage identity values
when compared to the Synechococcus sp. strain PR6000 SigA polypeptide.
TTG translational start site. Although the relative abundance of sigA transcripts detected by primer extension
appears to decrease after 4 h of nitrogen starvation,
transcripts are still detectable even after starvation for 8 h.
The apparent contradiction between the results obtained
from the Northern and primer extension analyses is
believed to be due to three factors. Firstly, the 'perception ' of nitrogen limitation through depletion of
intracellular nitrogen stores typically does not occur until
3 h after removal of nitrate from the growth medium
(Gasparich, 1989). Secondly, the primer extension analyses are significantly more sensitive than the Northern
hybridization method. Finally, some transcription of sigA
apparently continues during nitrogen limitation but
mRNA stability might be substantially reduced leading to
an overall decrease in the steady-state levels of sigA
transcripts detectable by Northern hybridization.
Attempted interposon mutagenesis of the sigA gene
Synechococczls sp. strain PR6OOO cells were transformed
with the construction shown in Fig. 1, and kanamycinresistant transformants were repeatedly streaked on selective medium (at least six times) to allow segregation of the
mutant and wild-type alleles. Chromosomal DNAs were
purified from several independent kanamycin-resistant
352
transformants, digested with HindIII, size-fractionated by
agarose gel electrophoresis and transferred to a nitrocellulose filter. This Southern blot was hybridized with
two radiolabelled DNA probes: an 820 bp KpnI-BgAI
fragment of the sigA gene and a 1.32 kb fragment
encoding the aphII gene (Fig, 1). Hybridization with the
820 bp KpnI-BgAI fragment of sigA showed that the
transformants, but not the wild-type strain, contained a
4-6 kb HindIII fragment (the size of the wild-type
fragment, 3.3 kb, plus 1-32kb from the interposon
cartridge) as well as the 3.3 kb HindIII fragment. When
the same blot was hybridized with the 1-32 kb apbII gene,
only the 4-6 kb HindIII fragment present in the DNAs
from the transformed cells hybridized (data not shown).
These results indicate that stable merodiploids were
generated and strongly suggest that the sigA gene is
required for viability of Synecbococctrs sp. strain PR6OOO
cells.
Purification and amino acid sequencing of
overproduced SigA
The SigA protein was overproduced in E. cob after
modification of the putative T T G start codon described
above (see Fig. 3 and Methods). The protein was purified
by chromatography on CM-Sepharose after being solu-
Syrzechococcus sp. strain PCC 7002 sigA
1
(a)
1
kb
2.9
2.3
2
3
4
5
6
106.5
1.5-
0.5 -
(b)
7
8
9
3
4
5
6
kDa
-
ACGTA
2
10
c-446 nt
........................................ ......................................... ....................................... ..................................
Fig. 5. Northern blot (a) and primer extension (b) analyses of
Synechococcus sp. strain PR6000 sigA transcripts. (a) The
Northern blot was hybridized with a radiolabelled 820 bp
Bglll-Kpnl sigA gene-internal fragment (see Fig. 1). Lanes: 1,
total RNA extracted from Synechococcus sp. strain PR6000 cells
grown under standard, nutrient-replete conditions; 2-6, RNAs
from cells deprived of nitrogen for 1 h (2), 2 h (31, 4 h (4), 6 h
(5) and 8 h (6). Transcript sizes were estimated by comparison
with 235 (Kumano et a/., 1983) and 165 (Tomioka & Sugiura,
1983) rRNAs and their breakdown products (Doolittle, 1972). (b)
Primer extension product from the sigA gene. Lanes: 7, primer
extension products with RNA extracted from Synechococcus sp.
strain PR6000 cells grown under standard, nutrient-replete
conditions; 8-10, primer extension products with RNAs from
cells deprived of nitrogen for 2 h (8), 4 h (9) and 8 h (10). Primer
extension products were denatured and subjected to
electrophoresis alongside a DNA sequencing ladder generated
using a universal sequencing primer on single-stranded M13
phage DNA. The major mapped 5’ end-point is 446 nt upstream
of the TTG translation start site or 496nt upstream from the
ATG translational start site (nt 440 in Fig. 3). Additional minor
primer extension products, corresponding to potential
transcript end-points mapping closer to to the translational
start site, were also observed (lanes 8 and 9). The amounts of
these products varied considerably in replicate experiments,
and it is possible that these represent non-specific termination
artifacts produced by reverse transcriptase.
-
80
-
49.5
-
32.5
-
Fig. 6. SDS-PAGE analyses of fractions obtained during the
overproduction and purification of the Synechococcus sp. strain
PR6000 SigA polypeptide. Proteins were electrophoresed on a
10% acrylamide gel and were stained with Coomassie Brilliant
Blue. Lanes: 1, pre-stained low molecular mass standards; 2,
whole-cell extracts of BLZl(DE3) cells; 3, whole-cell extracts of
BLZl(DE3) cells transformed with the PET1laSigA plasmid; 4,
whole-cell extracts of BL2l (DE3) cells transformed with the
pETl1aSigA plasmid after 4 h induction in the presence of
0.5 mM IPTG; 5, BL21(DE3) SigA inclusion bodies solubilized in
8 M urea; 6, purified SigA protein after CM-Sepharose
chromatography.
bilized from inclusion bodies. Fig. 6 shows SDS-PAGE
analysis of samples from various stages during the
purification of the SigA polypeptide; two forms of the
protein were usually detected (not completely resolved in
Fig. 6, lane 4 , but see also Fig. 7a and b, lane 3). Aminoand carboxyl-terminal amino acid sequencing was performed on purified, overproduced SigA. Amino-terminal
sequencing of overproduced SigA revealed amino-terminal heterogeneity ; three different amino-terminal
amino acids were obtained (underlined in Fig. 3). The
majority of polypeptides (approximately 80 YO)had methionine as the amino-terminal amino acid with the sequence
MNVHL ... Minor fractions had asparagine (about 10 YO;
NVHLV ...) or threonine (about 10 % ; TQATN ...) at
their amino termini. The presence of a portion of the
overproduced SigA polypeptides beginning with threonine indicates that the methionine at codon 17 is used in
E. coli as a translation start site ;this suggests that this start
codon could also be used in Synecbococczls sp. strain PR6OOO
(see below), Carboxyl-terminal amino acid sequencing of
overproduced SigA revealed a unique sequence, KEKIR,
as the last five amino acids of the overproduced protein.
This differs from the predicted carboxyl-terminal amino
acid sequence for SigA: KEYIR; the reason for this
discrepancy is presently unknown.
lmmunoblot analysis of SigA
An antibody against SigA was produced for analyses of
Synechococcus sp. strain PR6OOO Q factors. Fig. 7, lane 2
shows Coomassie-stained (Fig. 7a) and immunoblot (Fig.
7b) analyses of a crude RNA polymerase fraction from
3 53
L. F. C A S L A K E a n d D. A. B R Y A N T
(a)
kDa
80
-
49.5
-
32.5
-
1
2
3
DISCUSSION
(b)
kDa
80
1
2
3
-
32.5 -
Figrn7. SDS-PAGE (a) and immunoblot (b) analyses of a crude
RNA polymerase fraction from Synechococcus sp. strain PR6000.
Lanes: 1, pre-stained low molecular mass standards; 2, crude
RNA polymerase fraction from Synechococcus sp. strain PR6000
grown in nitrogen-replete medium; 3, a mixture of whole-cell
extracts from E. coli BLZl(DE3) cells transformed with
PET1laSigA and PET1laSigB, and BLZl(DE3)pLysS cells
transformed with pET3dSigC after 4 h induction with 0.5 mM
IPTG. (a) SDS-PAGE (10% acrylamide) analysis of proteins
stained with Coomassie Brilliant Blue. (b) lmmunoblot analysis
of crude RNA polymerase preparation and Q factor proteins
overproduced in E. coli with an antiserum to Synechococcus sp.
strain PR6000 SigA. The arrows on the right side of the figure
identify the two forms of overproduced SigA, SigB and SigC
derived from the appropriate E. coli strains.
Synecbococcus sp. strain PRGOOO grown in nitrogen-replete
medium. Fig. 7, lane 3 contains a mixture of whole-cell
extracts from E. coli BL21(DE3) cells overproducing
SigA and the alternate Synechocacczls sp. strain PRGOOO Q
factors SigB and SigC (L. F. Caslake & D. A. Bryant,
unpublished data). Two forms of the SigA protein
overproduced in E. coli are detected with apparent
molecular masses of 52 and 49 kDa. These probably
correspond to two forms of the protein produced using
the alternate translation initiation start sites (Fig. 3) as
detected by amino-terminal sequence analysis (see above).
These proteins would be expected to differ in mass by
approximately 2 kDa. The major cross-reacting protein in
lane 2 (Fig. 7b) has an apparent mass of 49 kDa and is
presumed to be the in vivo form of SigA due to its
molecular mass and recognition by the SigA antibody.
This polypeptide comigrates with the smaller overproduced polypeptide (Fig. 7b, lane 3). These results
strongly imply that the methionine at codon 17 (nt
935-937 in Fig. 3) is the in vivo translation initiation start
site in Synecbococcus sp. strain PR6000. As shown in Fig. 7b,
lane 3, the SigA antiserum weakly cross-reacts with SigB
and SigC proteins as detected by immunoblotting. However, polypeptides corresponding to SigB and SigC (or
any other Q factors) were not detected in partially purified
RNA polymerase from Synechococcus sp. strain PRGOOO
cells grown under nitrogen-replete conditions. A complete analysis of SigA protein levels as a function of
nutrient/growth conditions has been undertaken as part
of the description of a functional analysis of the sigB and
sigC genes and their products (L. F. Caslake & D. A.
Bryant, unpublished data).
3 54
The sigA gene encoding the principal Q factor from the
cyanobacterium Synecbococcus sp. strain PRGOOO was isolated and sequenced, and the SigA protein was overproduced in E. coli BL21(DE3) cells and purified.
Translation was initially assumed to begin at the TTG
codon (labelled +1 in Fig. 3) due to the presence of an
acceptable ribosome binding site upstream and because of
sequence similarity between the deduced protein sequence
from Synecbococcus sp. strain PRGOOO sigA and the sequenced amino terminus of SigA from Anabaena sp.
strain PCC 7120 (Brahamsha & Haselkorn, 1991). However, several lines of evidence suggest that translation
begins at the internal methionine (codon 17, nt 935-937;
Fig. 3) in Synecbococcus sp. strain PRGOOO. A strong,
consensus ribosome binding site (5’ AAGAGGAAA 3’)
precedes this ATG by 5 bp and approximately 10% of
the purified, overexpressed protein had threonine as the
amino-terminal amino acid. Additionally, the major
protein species recognized by the SigA antibody in
partially purified RNA polymerase preparations comigrates with the smaller overproduced SigA polypeptide
that is believed to originate from the ATG start codon at
position 17. The predicted molecular mass of SigA
(43737 Da) is significantly smaller than that calculated
from the electrophoretic mobility of SigA on SDS-PAGE
(Figs 6 and 7; Schneider & Haselkorn, 1988). This
discrepancy between electrophoretic mobility and molecular mass is attributed to the large percentage of charged
residues (36 %) in SigA and also has been observed for the
principal o factors from Anabaena sp. strain PCC 7120
(Brahamsha & Haselkorn, 1991), B. sHbtilis (Chang &
Doi, 1990), E. coli (Burton et al., 1981; Lesley & Burgess,
1989) and M. xantbus (Inouye, 1990).
Transcription of sigA appears to be influenced by the
nutrient status of the cell. Northern blot hybridization
analyses indicate that the sigA mRNA either becomes
unstable during nitrogen starvation or is no longer
transcribed at the same rate (Fig. 5a). The apparent
accumulation of a hybridizing 500 base RNA species
could be explained by partial degradation of the message
to a stable secondary structure. Results from Northern
blot and primer extension analyses suggest that the sigA
gene is transcribed as a monocistronic unit (if orfA in the
5’ leader sequence is not a gene). This is similar to the
organization of the sigA gene in Anabaena sp. strain PCC
7120 (Brahamsha & Haselkorn, 1991) but differs from
both E. coli and B. subtilis. In E. coli, the rpoD gene is the
promoter-distal gene in an operon containing dnaG (DNA
primase) and rpsU (S21; Burton e t al., 1983). In B. subtilis
the rpoD gene is the promoter-distal gene in an operon
containing dnaE (DNA primase) and an ORF of unknown
function (P23; Wang & Doi, 1986). It should be noted
that the oligonucleotide used in primer extension reactions to map the 5’ end of sigA transcripts was chosen
prior to the protein analyses described above. The 3’ end
of this primer is complementary to the ribosome binding
site of the putative internal start site. However, multiple
primer extension reactions with this primer failed to
reveal any transcripts initiating near the predicted trans-
s_Ynechococcs/ssp. strain PCC 7002 sigA
lation start site. Since it is unlikely that the sigA mRNA
would not include a ribosome binding site, it is unlikely
that the use of this primer could have failed to detect a
transcription start site near the probable translational start
at nt 935 (see Fig. 3).
The upstream untranslated region of the sigA transcript
contains an ORF of 69 amino acids, designated orf., that
is not homologous to any sequence presently in databases.
This ORF has unusual codon usage for Synecbococctls sp.
strain PRG000. For example, within this 210 nt ORF there
are three ATA (isoleucine) codons that occur very
infrequently in Synechococcz/s sp. strain PRGOOO genes
(CodonUse 3.1). If orfA is expressed in Synecbococctls sp.
strain PRG000, it is probably not a highly expressed gene
due to the number of unusual codons present.
Strong evidence that sigA encodes the principal Q factor
of Synecbococcz/s sp. strain PRGOOO is provided by the
inability to insertionally inactivate this gene. In contrast,
insertional inactivation of genes encoding four additional
0 factors (sigB, sigC, sigD and sigE; L. F. Caslake, T.
Gruber & D. A. Bryant, unpublished data) has been
successful, indicating that these other o factor genes are
not required for viability. These observations are consistent with the idea that SigA is the principal, or Group
1,Q factor (Lonetto e t al., 1992).Among Group 1 Q factors
three regions show almost complete conservation : 1,the
‘RpoD box’ (Tanaka e t al., 1988), that overlaps Region
2.4 (Helmann & Chamberlin, 1988) and is implicated in
recognition of the -10 hexamer of promoters
(Waldburger e t al., 1990); 2, an amino acid motif of 14
amino acids just carboxyl-terminal to the ‘RpoD box’;
and 3, a conserved 20 amino acid stretch that overlaps
Region 4.2 (Lonetto e t al., 1992) that is implicated in
recognizing the - 35 hexamer of promoters (Siegele et al.,
1989). In these three regions, encompassing 54 amino
acids, Synecbococcus sp. strain PRGOOO SigA is 100%
identical to Anabaena sp. strain PCC 7120 SigA, 100%
identical to S’necbococczrs sp. strain PCC 7942 RpoD1,94 YO
identical to B. stlbtilis 043and 91 % identical to E. coli 070.
Several lines of evidence lead to the speculation that the
Synechococctls sp. strain PRGOOO SigA protein confers
holoenzyme specificity for a promoter sequence similar to
the consensus sequence for E. coli doand B. subtilis 043.(i)
In the two regions implicated in binding the consensus
promoter, Synechococcus sp. strain PRGOOO SigA is identical
with the major Q factors from B. subtilis and E. coli which
have been shown to recognize identical consensus promoter sequences (Moran e t al., 1982; Rosenberg & Court,
1979). (ii) Promoter sequences upstream of several
mapped 5’ end-points in Synecbococcus sp. strain PR6OOO
resemble the consensus sequence for the E. coli and B.
st/btilis major 0 factors (Curtis & Martin, 1994; Gasparich,
1989). In Synechococcm sp. strain PR6000, the level ofpsbA
mRNA does not fluctuate in response to nitrogen, carbon,
phosphorus or sulfate deprivation (Gasparich, 1989). The
putative promoter region of thepsbA gene contains -35
(TTTACA) and - 10 (TAATAT) recognition hexamers
similar to the E. coli doconsensus promoter sequence.
This suggests that the potential Synecbococcm sp. strain
PRGOOO ‘housekeeping ’ promoter sequence may resemble
the Q~~ consensus sequence. (iii) Several Synecbococctls sp.
strain PRGOOO genes have been shown to be expressed in
E. coli, and several E. coli genes can be expressed in
Synecbococcus sp. strain PRGOOO (Murphy e t al., 1987).
Studies using crude extracts or purified RNA polymerase
from Anabaena sp. PCC 7120 revealed that the strongest
transcription occurred from a cyanophage promoter with
consensus
a strong resemblance to the E. coli
(Schneider et al., 1991). Recent transcriptional analyses
with purified RNA polymerase from Calotbrix sp. strain
PCC 7601 revealed that the strongest cpcl promoter used
in vim (Pa) had -35 (TAAACA) and - 10 (TATAAA)
recognition elements that are similar to the consensus
sequence for E. coli 0 7 0 (Schyns e t al., 1994). Further, the
addition of Calothrix sp. strain PCC 7601 SigA to E. coli
core RNA polymerase conferred recognition of P, (as
above) and P, (- 35, TAAAGC; - 10, TGTTAA) in in
vitro transcription assays of the cpcl promoter. However,
no specific transcription of the cpc7 promoter occurred
with E. coli RNA polymerase holoenzyme, indicating that
the recognition elements for Calothrix sp. strain PCC 7601
SigA may not be identical to those for do or that
additional factors are required for transcription from this
promoter.
Southern blot hybridization analyses at reduced stringency with a gene-internal fragment of Synecbococcz4s sp.
strain PRGOOO sigA, revealed five or more hybridizing
Hind111 fragments (Fig. 2). This indicated the presence of
multiple genes that could encode alternate 0 factors
involved in responses to environmental changes. Synecbococct~s sp. strain PRGOOO is a marine species which is
exposed to variations in incident light as well as nitrogen
and carbon availability in its native environment. As with
all photosynthetic organisms, an inverse relationship
exists between the amount of light harvesting proteins
and the amount of light that reaches the cell. Earlier
studies by Gasparich e t al. (1987) found a reduction in the
amounts of transcripts for light harvesting proteins when
Synecbococcus sp. strain PRGOOO cells were deprived of
nitrogen or carbon, and it is known that cyanobacteria use
phycobiliproteins as a storage form for reduced nitrogen
(Daley & Brown, 1973; de Vasconcelos & Fay, 1974;
Tandeau de Marsac & Cohen-Bazire, 1977; Boussiba &
Richmond, 1980; Stevens e t al., 1981). It is possible that
such changes in gene expression are controlled by
alternate Q factors, that are known to play important roles
during B. subtilis sporulation (Stragier & Losick, 1990);
differentiation in Streptomyes coelicolor (Chater e t al., 1989)
and M. xantbus (Apelian & Inouye, 1990) ;the response to
stress (Grossman e t al., 1984) and carbon starvation
(McCann e t al., 1991) in E. coli; the expression of the
flagellar, chemotaxis and motility regulon in E. coli
(Amosti & Chamberlin, 1989) and B. st/btilis (Helmann e t
al., 1988); and the response to nitrogen starvation in
Salmonella gphimurium (Hirschman et al., 1985).
In bacteria the ability to initiate RNA synthesis at specific
sites is an intrinsic property of RNA polymerase holoenzyme and requires the presence of a 0 subunit. To begin
to understand the role of the multiple Q factors in the nondifferentiating, unicellular cyanobacterium Synecbococcus
355
L. F. C A S L A K E a n d D. A. B R Y A N T
sp. strain PR6000, it was first necessary to identify the
principal CT factor of this organism. The sigA gene is
essential and apparently belongs to the Group 1 family of
o"-type CT factors that are responsible for most RNA
synthesis in the cell. Due to its amino acid sequence
similarity to E. coli '
0 and B. szlbtilis ag3,it is anticipated
that this CT factor recognizes a promoter sequence similar
to the E. coli consensus promoter sequence. Characterization of other CT factors of Sjnechococczls sp. strain
PR6OOO is in progress. The ability to analyse in vivo
fluctuations in CF factor protein levels through the use of
the antibodies to SigA produced in this work should
provide important information concerning the possible
function(s) of alternate CT factors in this cyanobacterium.
ACKNOWLEDGEMENTS
This work was supported by USPHS grant GM31625 awarded
to D.A.B. The authors would like to thank D r Jindong Zhao
for performing amino- and carboxyl-terminal amino acid
sequence analyses, and Drs B. Brahamsha and R. Haselkorn for
providing the cloned sigA gene of Anabaena sp. strain PCC
7120. The authors thank Drs Stephen Wagner and B. Tracy
Nixon for the cosmid library of Synecbococcus sp. strain PR6000.
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Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman,
J. G., Smith, J. A. & Struhl, K. (eds) (1987). Current Protocols in
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Received 11 July 1995; revised 8 September 1995; accepted
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