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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 A A G C T t t t 1 c t 4 t C CGAGGAAAGAA?iAAT"CC GGCAGCGTCACAAGTmrGc CCCCr0cTPC;ACAOCCMCT t t OAATCAM;TCITCPGCTIW; CCCAAGTl'G~TCTAAGTC 121 GGGCTl'ACTGGTCATlTGGA TCGCTGCCGCGAGATAGGAT TTCATAlGGCC'IGAGAmTI' T l T A G A A A T A G G G A W AAMCCGTCMCCAGAAGTT T'lGATCGlTAAA-A 241 ATACCATAGCAGAATC"C CCCACCAACA'XGGGGCGATC GCl"2GAGA-A 'IGATCAAAM- GCATACI-- 361 AWTlGCGGCGATCGCATI'A CCAAAGCACAGATGTCTCGG 'ITPCAAAACCITIAGCGCAC AAMTIllQCCCAlGG481 ZWGAATCATlGGCAlGGAA CATRTCAGAAMGATCAGT TAACCTATTAGCATCAAGTT OrfA>> M R L A M L E A I A I H Q S L Y L G T R I P V L N TC'XGGATAGCCCTTGACAM G G T E C A T C C C A C W T m G A C K G C G A T G CTAGAGOCCATCGCCAlCCA TCAOTCOC'FITACTIGGGCA CTAGAATAmTACTTAAC K K T Y G W A K P T D P A I T M W A I K D K P T K M A K D L L Q A I S S E K L I 601 AAAAAAACCTAlGGGTGGGC GAAGTlTACAGA'ITITXTA TAACCATCTGGGCAATAAAA GACAAATXACTAAAASGC TAAGGATTXCTCCAAGCTA "CG'XGGAGAAGCTCATI' 721 T D F D * ACAGATITPGACTAG'MTTA GAGACGGAATAAAACCCCCA AAAGGTAGCAGGAAAGTPCC CATCTACTAGTACMTCAAA ACCCACAGTXTWFXTATC A A A C A T A G C C m T A G G G SigA>> H I V H L V A P L N E Q E E I G M T Q A T N P V L 841 AAAACCTCGACTKGACAGA A T C A T T C T C r r r O C T M TCTCCATKAATGTCCATIT AGTIGCTI'ITITAAACOMC ~ T A G G C A ' I G A C CCAAGCGACGAACCCCGTACT D Q T R N E G D I D Y S A L A E A Q I K E G T D Y V E S R R K E T A T K K K P Y T E D S I R I Y L Q E I G R 961 G G A T C A G A C m ' E A G G G'XGATATEATTACXG'XCC CTlGCGOMOCGCAMTTAA A-CTAmAG L T L P T K K S R AACTTACCCTCCCCACGAM -AUMC 1081 CAGTCGTCGTAAGGAAACTG CCACCAAGAAAAAACCCTAC ACCGAAGACTCGATPCGGAT TTACCPCcAAGAMlToocC G 1201 I R G L R K I A D L L E L E R M R E Q L T E H E S R V P T D K E W A C C G G A A A A ~ T m T ; CTCGAACTAGAGCGGAlGCGG GAACAACTCACCGAGCA'KA G"ICGCG1TcCTACGGACA -CGAAGCCGCT R R R L P H G 1441 Q E G S L CCAAGAGGGTTC- G L 1561 P V H L Y E T I S R I K K T T K I L Q S R T I R L TCAATCCCGGACCATPCGTC lGCCTGTTCACCTCTACGAA A C C A ' I T X C C G C A T C W AACCACCAAGA-CC R R A K D K M V Q S N L R I R A A E K P D H E K 'XGATCCGTGCCGCCGAAAAG TTIGACCAC-lTA G Y L V V S I A K 1321 TCGGCGIwjcTpGTPCCATC GTCGCCGlGCCAAAGACAAA AlGGTWAATCAAACCTCCG TC'IGG'IGGTC"ATIGCGA R M E M T I E G E T P E 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. 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