identification of multiple rna polymerase sigma factor homologs

JOURNAL OF BACrERIOLOGY, Nov. 1992, p. 7273-7282 Vol. 174, No. 220021-9193/92/227273-10$02.00/0Copyright 0) 1992, American Society for Microbiology

Identification of Multiple RNA Polymerase Sigma FactorHomologs in the Cyanobacterium Anabaena sp. StrainPCC 7120: Cloning, Expression, and Inactivation of

the sigB and sigC GenesB. BRAHAMSHAt* AND R. HASELKORN

Department of Molecular Genetics and Cell Biology, The University of Chicago,Chicago, Illinois 60637

Received 4 June 1992/Accepted 31 August 1992

The sigA gene ofAnabaena sp. strain PCC 7120, encoding the principal RNA polymerase sigma factor, andthe complement of the rpoD oligonucleotide (K. Tanaka, T. Shiina, and H. Takahashi, Science 242:1040-1042,1988) were used as probes to isolate two genes, sigB and sigC, which encode two putative sigma factorsexhibiting high degrees of similarity to SigA, to HrdA, -B, -C, and -D of Streptomyces coelicolor, and to KatFof Escherichia coli. sigB and sigC code for polypeptides of 332 and 416 amino acids with predicted molecularweights of 38,431 and 47,459, respectively. sigB and sigC mRNAs are detectable only under nitrogen-limitingconditions. Insertional inactivation of sigB and sigC indicates that neither gene alone is essential for nitrogenfixation or heterocyst differentiation.

The cyanobacteria are a diverse group of oxygen-evolvingphotosynthetic prokaryotes, many of which are capable ofbiological nitrogen fixation, a process sensitive to oxygen.The filamentous cyanobacterium Anabaena sp. strain PCC7120 responds to deprivation of combined nitrogen by dif-ferentiating specialized cells called heterocysts at regularintervals along its filaments. In these cells the enzymenitrogenase is produced and nitrogen fixation takes place.Unlike adjacent vegetative cells which carry out oxygenicphotosynthesis, the heterocysts are modified to maintain alow internal 02 concentration. During heterocyst develop-ment, a new cell envelope consisting of inner glycolipid andouter polysaccharide layers is synthesized, thus limiting thediffusion of 02 into the cell (28). The photosynthetic mem-branes of heterocysts are reorganized to contain only pho-tosystem I, thereby eliminating 02 evolution. The nitrogenfixed in the heterocysts is exported to the vegetative cells inthe form of glutamine, while the vegetative cells provide theheterocysts with carbohydrates that yield reductant fornitrogenase (24, 38).

This compartmentalization of functions results from dif-ferential gene expression which, at least in some cases, isregulated at the transcriptional level. Using promoter fusionsto luciferase, Elhai and Wolk demonstrated that the genesfor the large and small subunits of ribulose-1,5-bisphosphatecarboxylase, an enzyme whose activity is present only invegetative cells, are transcribed only in vegetative cells,while the nifHDK genes coding for the subunits of nitroge-nase are transcribed only in heterocysts (10).One strategy commonly employed by eubacteria to regu-

late the temporal and spatial expression of discrete sets ofgenes is the modification of RNA polymerase by alternativeforms of the sigma subunit, thereby coupling transcriptionalspecificity to particular physiological conditions. Alternative

* Corresponding author.t Present address: Marine Biology Research Division, 3135

Hubbs Hall, Scripps Institute of Oceanography, University ofCalifornia, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0202.

sigma subunits play important roles in differentiation inStreptomyces coelicolor (8), Bacillus subtilis (25), and Myxo-coccus xanthus (1), as well as in regulating the expression ofheat shock proteins in Escherichia coli (15) and the expres-sion of flagellar and chemotaxis genes in E. coli and B.subtilis (16) and in the response to nitrogen (18) and carbonstarvation (23) in Salmonella typhimurium and E. coli,respectively.As part of ongoing studies on the molecular basis for

differential gene expression during heterocyst differentia-tion, we sought to determine whether multiple RNA poly-merase sigma factors are present in Anabaena sp. strainPCC 7120. We previously isolated the sigA gene, whichencodes the principal vegetative cell sigma factor, and haveshown that the polypeptide which it encodes is homologousto the principal sigma factors of E. coli and B. subtilis (3).Recently, a number of approaches based on the fact thatsigma factors constitute a protein family have been used toisolate homologous genes. Tanaka et al. (35) used an oligo-nucleotide based on a sequence of 10 amino acids that iscompletely conserved in the principal sigma factors of E. coliand B. subtilis to demonstrate that Streptomyces coelicolorhas four homologs of this factor. Also, by using homology tothe M. xanthus sigA gene, Apelian and Inouye isolated sigB,which is involved in myxospore differentiation (1). In thisstudy, we demonstrate that Anabaena sp. strain PCC 7120encodes multiple homologs of sigA, the principal RNApolymerase vegetative cell sigma factor. In this paper, wedescribe the isolation, sequence analysis, expression, andinsertional inactivation of two of these homologs, sigB andsigC.

(This work was presented in part at the 1990 AnnualMeeting of the American Society for Microbiology, Ana-heim, Calif.)

MATERIALS AND METHODS

Bacterial strains and growth conditions. The bacterialstrains used in this study are described in Table 1. Anabaena

7273

on February 11, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

7274 BRAHAMSHA AND HASELKORN

TABLE 1. Bacterial strains and plasmids used in this study

Strain or plasmid Relevant characteristics Source and/orreference

E. coli strainsDH5a Host for pUC18, pUC19, and constructions BRLUHB101 Host for cosmid library and for plasmids to be conjugated intoAnabaena sp. strain PCC 7120 22MC1061 Host for plasmids to be conjugated into Anabaena sp. strain PCC 7120 7

Anabaena sp. strainsDR1 Anabaena sp. strain PCC 7120 in which the sigB gene is interrupted by a spectinomycin- This study

streptomycin resistance cassetteC9 Anabaena sp. strain PCC 7120 in which the sigC gene is interrupted by a spectinomycin- This study

streptomycin resistance cassettePlasmidspWB79 Cosmid vector; Apr Tetr W. J. BuikemapCPF3 pWB79 containing a 3.4-kb HincII fragment ofAnabaena sp. strain PCC 7120 DNA hybridiz- W. J. Buikema

ing to the 740-bp EcoRI-AccI fragment of the Anabaena sp. strain PCC 7120 sigA genepUC18,19 Apr 39pDW9 pUC19 derivative containing a spectinomycin-streptomycin resistance cassette 13pRL271 Cmr Emr; contains sacB Yuping Cai (6)pRL528 Helper plasmid used in conjugations; encodes AvaI and Eco47II methylase genes that 9

block restriction by AvaI and AvaIIpBH500 pUC19 containing a 4.7-kb EcoRI fragment of Anabaena sp. strain PCC 7120 DNA encod- This study

ing sigBpBH501 pBH500 with a 2-kb spectinomycin-streptomycin resistance cassette in the NheI site This studypBH502 pBR322 containing a 6.7-kb EcoRI fragment from pBH501 This studypBH503 pRL271 containing a 6.7-kb EcoRI fragment from pBH502 in the NnI site This studypBH700 pUC 19 containing a 3.4-kb HincIl fragment encoding sigC This studypBH701 pBH700 in which the 600-bp EcoRV fragment is replaced by a a spectinomycin-streptomy- This study

cin resistance cassettepBH702 pRL271 containing a 4.8-kb HincII fragment of pBH701 in the NruI site This studypBH720 pUC19 containing a 3.4-kb HincII fragment from pBH700 in the opposite orientation This studya BRL, Bethesda Research Laboratories, Inc., Gaithersburg, Md.

sp. strain PCC 7120 was maintained in BGll medium (29).Solid medium contained 1% purified agar (BBL). Whenrequired, streptomycin and spectinomycin were present atconcentrations of 2 ,ug/ml. To prepare medium for selectionagainst sacB-containing strains, filter-sterilized sucrose wasadded to autoclaved BG11 medium to a final concentrationof 5%. E. coli strains were grown in L broth (22). Ampicillin(100 ,ug/ml), spectinomycin (25 ,ug/ml), chloramphenicol (10,ug/ml), and kanamycin (50 ,ug/ml) were used, where appro-priate, for the selection and maintenance of plasmids. Ana-baena sp. strain PCC 7120 was subjected to heat shock,osmotic shock (60 mM sucrose), and salinity stress (60 mMNaCl) as previously described (2). Cold shock involvedexposure to 10°C for 30 min, while oxidative stress involvedexposure to 120 ,uM H202 for 30 min.

Molecular biological techniques. For general molecularbiological techniques we used standard procedures (22).Southern and Northern hybridizations were performed aspreviously described (3). When the oligonucleotide was usedas a probe, the hybridization and washing temperature was45°C. All other hybridizations and washes were performed at65°C. In Northern hybridizations, the sigB probe consistedof a 400-bp NheI-HindIII fragment internal to the codingsequence, while the sigC probe was a 600-bp EcoRV frag-ment internal to the coding sequence.

Nucleotide sequences were determined by the dideoxynu-cleotide chain termination method of Sanger et al. (30), usingSequenase reagents (U.S. Biochemical Corp., Cleveland,Ohio) and double-stranded plasmid templates prepared asdescribed by Kraft et al. (19). The deletions used in sequenc-ing the coding strand of sigB and flanking regions weregenerated by partial exonuclease III digestion of pBH500,using an Erase-a-base kit purchased from Promega, Madi-

son, Wis. The sequence of the noncoding strand of sigB wasdetermined by using oligonucleotide primers and pBH500 asthe template. The deletions used in sequencing sigC weregenerated as described above from pBH700 (noncodingstrand) and pBH720 (coding strand). Sequence analysis andalignment were performed as described previously (3).To determine the 5' ends of the sigB and sigC transcripts,

primer extension analyses were carried out as describedpreviously (3). For sigB, 100 ,ug of RNA prepared fromnitrogen-replete or nitrogen-starved cultures (12 h postin-duction) or 100 jig of yeast tRNA was used in each reaction.The oligonucleotide used was 5'-CTTGGGATGTIGAATTAGGC-3', which is complementary to nucleotides 539 to 558(see Fig. 3). A sequence ladder generated by using the sameprimer on pBH500 was coelectrophoresed. For sigC, 50 p,gof RNA prepared from nitrogen-replete or nitrogen-starvedcultures (18 h postinduction) or 50 p,g of yeast tRNA wasused. The oligonucleotide was 5'-GTCTCT'ATGGCTCCAGGTAA-3', which is complementary to nucleotides -7 to -26(see Fig. 4). pBH720 was used as the template in thegeneration of the sequencing ladder.

Isolation of the Anabaena sp. strain PCC 7120 sigB gene.TheAnabaena sp. strain PCC 7120 sigB gene was isolated byscreening a size-directed EcoRI library of Anabaena DNAby colony hybridization (22), using as a probe oligonucleo-tide 5'-ACITAIGCIACITGGTGGATIIGICAIGC-3', whichis the complement of the rpoD oligonucleotide described byTanaka et al. (35). A 40-,ug portion of total Anabaena sp.strain PCC 7120 DNA was digested to completion withEcoRI and separated by electrophoresis on a 0.7% agarosegel. The fragments migrating to the area between the 4.3- and4.8-kb X BstEII markers were excised, purified from theagarose by adsorption to glass beads (Gene-Clean; Bio 101,

J. BACrERiOL.


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

ANABAENA SIGMA FACTOR HOMOLOGS 7275

La Jolla, Calif.), and ligated into the EcoRI site of pUC18.The ligation mixture was used to transform E. coli DH5a.Approximately 400 ampicillin-resistant colonies werescreened by colony hybridization, using end-labelled oligo-nucleotide as the probe. Two hybridizing colonies werechosen for further analysis. The plasmids from these twocolonies both contained a 4.7-kb EcoRI fragment that hy-bridized with the oligonucleotide probe. One of these plas-mids, pBH500, was chosen for all further work.

Isolation of sigC. The Anabaena sp. strain PCC 7120 sigCgene was isolated by screening a cosmid library (consistingof Anabaena sp. strain PCC 7120 DNA partially digestedwith Cpfl and cloned into the BglII site of cosmid vectorpWB79) by colony hybridization, using as the probe a 740-bpAccI-EcoRI fragment of the Anabaena sp. strain PCC 7120sigA gene (3). This resulted in the isolation of a number ofreactive clones, one of which, pCPF3, contained a 3.4-kbHincII fragment that hybridized with the probe. The 3.4-kbHincII fragment of pCPF3 was ligated into the HincII site ofpUC19 to yield pBH700. pBH720, containing the insertion inthe orientation opposite the orientation in pBH700, wasgenerated by cutting pBH700 with HincHI, religating, andscreening by restriction enzyme digestion.

Disruption of sigB and sigC. Anabaena sp. strain DR1, inwhich the genomic sigB gene is interrupted by a spectino-mycin-streptomycin resistance cassette, was constructed asdescribed below. The 2-kb spectinomycin-streptomycin re-sistance cassette of pDW9 (13) was excised with HincII andligated into the end-repaired NheI site of pBH500 to yieldpBH501. pBH502 was constructed by ligating the 6.7-kbEcoRI fragment of pBH501 into the EcoRI site of pBR322.The 6.7-kb EcoRI fragment from pBH502 was end repairedand ligated into the NruI site of pRL271 (6) to yield pBH503.pBH503 was used to transform E. coli HB101(pRL528) (9)for conjugation into Anabaena sp. strain PCC 7120. Fortriparental matings we used standard procedures (9).

Similar methods were used to construct Anabaena sp.strain C9, in which the genomic sigC gene is interrupted bya spectinomycin-streptomycin resistance cassette. pBH701,in which the 2-kb spectinomycin-streptomycin resistancecassette replaces a 600-bp EcoRV internal fragment of sigC,was constructed by cutting pBH700 with EcoRV, isolatingthe large fragment, and ligating into it the spectinomycin-streptomycin resistance cassette from pDW9 cut withHincII. The 4.8-kb HincII fragment of pBH701 was thenligated into the NruI site of pRL271 to yield pBH702, whichwas used to transform E. coli MC1061(pRL528) for conju-gation into Anabaena sp. strain PCC 7120.

Exconjugants were selected by plating the mating mixtureonto BG11 plates containing 2 Fg of spectinomycin per mland 2 ,g of streptomycin per ml. Single recombinant colo-nies in which all of pBH503 or all of pBH702 had integratedinto the chromosomal copy of sigB or sigC, respectively,appeared within 10 to 14 days. Double recombinants wereselected as described previously (6) on BGll plates contain-ing 5% sucrose, 2 ,g of spectinomycin per ml, and 2 ,ug ofstreptomycin per ml. Presumptive single and double recom-binants were screened by Southern hybridization to confirmthe expected restriction pattern that was indicative of twocopies of sigB or sigC (one interrupted) and plasmid se-quences in the single recombinant or one copy of theinterrupted sigB or sigC gene in the double recombinants(data not shown).

Nucleotide sequence accession numbers. The DNA se-quences reported in this paper are available from GenBankunder accession numbers M95760 (sigB) and M95759 (sigC).

RESULTS

Isolation of the sigB and sigC genes. The complement of the"rpoD" box oligonucleotide corresponding to a conservedsequence found in the principal sigma factors of a number oforganisms (35) was used to probe HindIlI and EcoRI chro-mosomal digests of Anabaena sp. strain PCC 7120 DNA.Two very strongly hybridizing bands and two or threeweaker ones are evident in each digest (Fig. 1). One of thestrongly hybridizing bands in each digest, the 15-kb HindIIIfragment and the 2.5-kb EcoRI fragment, corresponded tothe previously characterized Anabaena sp. strain PCC 7120sigA gene (3). The strongly hybridizing 4.6-kb EcoRI frag-ment was cloned into the EcoRI site of pUC18 to yieldpBH500. The open reading frame (ORF) encoded by thehybridizing region (see below) was designated sigB.To determine whether additional sequences homologous

to sigA are present in the Anabaena sp. strain PCC 7120genome, a 740-bp EcoRI-AccI fragment of sigA was used toprobe a Southern blot of Anabaena sp. strain PCC 7120genomic DNA digested with HindIII, EcoRI, HincII, XbaI,and NheI under conditions of high stringency. In all cases, inaddition to the major hybridizing band corresponding tosigA, a number of other bands (from three to four, dependingon the digest) are present (Fig. 2). The 3.4-kb HincIIfragment which hybridized to the probe (Fig. 2) was identi-fied in a cosmid library of Anabaena sp. strain PCC 7120genomic DNA. This 3.4-kb HincII fragment was cloned intothe HincII site of pUC19 to yield pBH700. The ORFencoded by the hybridizing region (see below) was desig-nated sigC.

SigB and SigC are homologous to principal sigma factors. Asequence analysis of 1,610 bp of pBH500 containing theregion hybridizing to the complement of the rpoD oligonu-cleotide revealed an ORF (sigB) that codes for a polypeptideof 332 amino acids with a predicted molecular mass of 38,431Da (Fig. 3). A potential ribosome binding site, GAGG,precedes the ORF by 11 bp. Inspection of the sequencedregion upstream of sigB revealed a partial ORF with atermination codon 90 bp upstream of the sigB ATG. Nosignificant similarities were found in comparisons of thepredicted protein sequence of this partial ORF (147 aminoacids) with GenPept (release 71). A sequence analysis of2,019 bp of pBH700 containing the region hybridizing to thesigA probe indicated the presence of an ORF (sigC) of 416amino acids with a predicted molecular mass of 47,459 Da(Fig. 4). A potential ribosome binding site, GAGA, precedesthe sigC ORF by 7 bp. Following the sigC stop codon is a setof 28-bp inverted repeats that could form a stem-and-loopstructure with a calculated free energy of formation of -23.3kcal/mol (ca. -9.75 x 104 J/mol) resembling a rho-indepen-dent terminator (Fig. 4).The polypeptides predicted by the sigB and sigC ORFs are

similar to each other and to the Anabaena sp. strain PCC7120 SigA protein, the principal sigma factor (Fig. 5). LikeSigA (3), SigB and SigC contain sequences with significantsimilarity to the four conserved regions of sigma factors (14,17) (Fig. 5). Furthermore, SigB and SigC exhibit a muchgreater degree of homology to SigA, to other principal sigmafactors that have been described previously (4, 12) (data notshown), to HrdA, -B, -C, and -D of Streptomyces coelicolor(32, 36), and to KatF of E. coli (27) (Fig. 5) than they do toalternative forms of sigma, such as od2 (20) or the alternativesigma subunits of B. subtilis (25) (data not shown).Over the most conserved regions, regions 2 to 4, SigB and

VOL. 174, 1992


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


1A.

2 3 4.70E. cOli CS

B. subtilis SigA

Anabaena PCC7120 SigA

E. coliB. subtilis TYATWWIRQA

Anabaena P

B.

Oligonucleotide probe 5'

C.

(1 5 kb) sigA -p

'CC7120 TYATWWIRQA

T Y A T W W I R Q AACI TAI GCI ACI TGG TGG ATI IGI CAI GC 3'

"0-:

* 0

(4 kb) sigB >4L-ISi siglB (4.6 kb)_ w

d 4_ sigA (2.5 kb)

FIG. 1. Southern analysis of restriction digests of total genomic Anabaena sp. strain PCC 7120 DNA probed with an oligonucleotidespecific for a conserved region of bacterial sigma factors. (A) Amino acid sequence alignment of Anabaena sp. strain PCC 7120 SigA, B.

subtilis SigA, and E. coli or" (shown as deleted for a nonconserved 245-amino-acid stretch separating region 1 from region 2). The solid boxescorrespond to conserved regions 1 through 4 of sigma factors (14, 17). Below the alignment is the sequence of the 10 completely conservedamino acids that was used to design the oligonucleotide probe. (B) Sequence of the oligonucleotide probe which is the complement of the rpoDoligonucleotide described by Tanaka et al. (35). (C) Southern analysis.

SigC are more similar to one another than they are to SigA;they are 80% identical in region 2, 36% identical in region 3,and 44% identical in region 4. SigC is more similar to SigAthan SigB is, exhibiting 69% identity to SigA in region 2, 40%identity in region 3, and 48% identity in region 4. SigBexhibits 71% identity to SigA in region 2, 28% identity inregion 3, and 39% identity in region 4. SigA and SigC are

more similar in region 1. Unlike SigA (3) and SigC (Fig. 4),SigB lacks a stretch of acidic amino acids at its N terminus

(Fig. 3). The deduced SigB and SigC polypeptides havecalculated net charges of +7 and +3, respectively, and thusare unlike SigA, which has a net charge of -6. SigB containsa relatively high proportion of glutamines (11%).

Expression of sigB and sigC. To determine whether sigBand sigC are expressed and whether this expression issubject to regulation by combined nitrogen, Northern blotswere made in which restriction fragments internal to the sigBand sigC genes were used to probe totalAnabaena sp. strain

J. BACTERIOL.

M-


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


Anabaena PCC7120 SigA1 2 3 4

Probe

14.2

8.5

5.7-

3.7 -

2.3-

1.9

FIG. 2. Southern analysis of restriction digests of total genomic Anabaena sp. strain PCC 7120 DNA probed with a 740-bp EcoRI-AccIrestriction fragment internal to the sigA gene. The top line represents SigA; the solid boxes correspond to conserved regions 1 through 4 ofsigma factors (14, 17). Below this line is the restriction fragment used as the probe, aligned with respect to the region which it encompasses.The asterisk indicates the position of the 3.4-kb HincII fragment encoding sigC.

PCC 7120 RNA prepared from cells grown under nitrogen-replete conditions and from cells harvested at various timesfollowing combined nitrogen deprivation. Neither the sigBmessage nor the sigC message is detectable under conditionsof combined nitrogen sufficiency (Fig. 6). However, bothsigB and sigC are expressed following the removal of com-bined nitrogen. A message hybridizing to the sigB probe isfirst detectable at 6 h, increases in abundance at 12 and 18 h,and then disappears. The hybridizing RNA appears repro-ducibly as a smear, suggesting that the message is unstable.The 1.4- and 1.7-kb transcripts hybridizing to sigC are firstdetectable at 6 h following combined nitrogen stepdown.From 6 to 12 h the level of the sigC message increases two-to threefold, and then it decreases after 18 h. The presence ofmultiple bands suggests either that the gene is transcribedfrom multiple promoters, that the transcripts terminate atdifferent sites, or that the mRNA is processed. A Northernblot probed with a noncoding region upstream of sigC gaveidentical results (data not shown), indicating that the multi-plicity of bands seen is not due to a signal from anothersigma-like gene homologous to sigC. To determine whetherthe expression ofsigB and sigC could be induced by stressesother than nitrogen deprivation, RNA was prepared fromAnabaena sp. strain PCC 7120 subjected to heat shock, saltshock, osmotic shock, cold shock, and oxidative stress andprobed with sigB and sigC in a Northern analysis. None ofthese treatments resulted in the appearance of sigB or sigCmessage (data not shown), suggesting that the expression ofsigB and sigC is confined to conditions where there isnitrogen deprivation and/or heterocyst differentiation.

Primer extension experiments were carried out to deter-mine the 5' ends of the sigB and sigC transcripts. We wereunable to detect a primer extension product for sigB. Thiscould have been due to several possibilities. Because sigB is

preceded by another ORF, it is possible that it is transcribedas part of an operon with the transcriptional start site lyingfar upstream of the sigB translational start. It is also possiblethat a product was not detected because of the low level andapparent instability of the sigB message.For sigC, one major 5' end was detected at position -328

in RNA prepared from cells that had been starved forcombined nitrogen but not in RNA from cells that had beengrown in the presence of ammonia (Fig. 7). This finding isconsistent with the results of the Northern experiments (Fig.6). The predicted size of a message beginning at position-328 and ending at the last T of the inverted repeat is 1,665nucleotides, which is consistent with the observed 1.7-kbmessage. We also detected much less abundant RNAs with5' ends at positions -186 and -206, which were present onlyin cells starved for combined nitrogen (data not shown) andmay represent the 5' ends of the observed less abundant1.4-kb message. The putative -10 and -35 regions preced-ing the position -328 start site do not resemble the regions ofpromoter sequences recognized byAnabaena sp. strain PCC7120 vegetative cell RNA polymerase (31), nor do theyexhibit similarity to sequences preceding the start sites ofseveral Anabaena sp. strain PCC 7120 nitrogen-regulatedgenes that are expressed relatively late in the differentiationprocess (26). sigC is first expressed within 6 h of combinednitrogen stepdown, and at present its start site is the onlyone that has been determined for an early nitrogen-regulatedgene.

Insertional inactivation of sigB and sigC. To understandtheir possible function in transcriptional regulation in Ana-baena sp. strain PCC 7120, the sigB and sigC genes wereinactivated in vivo by inserting a spectinomycin-streptomy-cin resistance cassette in the coding sequence of each gene(Fig. 3 and 4). Double recombinants in which the wild-type

ME

VOL. 174, 1992


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

7278 BRAHAMSHA AND HASELKORN J. BACTERIOL.

1 CCGTGTAGAAGCTGCTGTCAAACTTTATAAAATGGGAAAGATTAGTAAACTCTTGATGACAGGAGATAACAGTACAGTTTCCTATAATGAAGTCAGGTCTR V E A A V K L Y K M G K I S K L L M T G D N S T V S Y N E V R S

101 ATGCTGAAATATGCCCATGATCTGGGTGTACCGATGAAAAACATTACCCTAGACTATGCAGGTTTCAGCACTTATGAGAGTTGTTATCGCGCTCATAAAGM L K Y A H D L G V P M K N I T L D Y A G F S T Y E S C Y R A H K V

201 TTTTTGGTGTACATAAAGCTGTTGTCATAACTCAAAATTATCATCTTCCCCGCACTGTTTACACCTGTCGCCAATTAGGACTAAAGACAGTTGGTTTAGGF G V H K A V V I T Q N Y H L P R T V Y T C R Q L G L K T V G L G

301 AACTCCAGATATAGAAATTTATGGACTACGAGGAATGATTCCTGATTTATTACGGGAATCATTAGCGAATGTTAAGGCTTTGTGGGAAGTTGACATTACCT P D I E I Y G L R G M I P D L L R E S L A N V K A L W E V D I T

401 CGTCCCCGACCTACTTTTTTAGGACAGTTTGAACCAATTAATTGATGAAGTTTTTAAAATTTATCATTTCGATTATTTTATGCTTATTTATTTTTATGTAR P R P T F L G Q F E PIN *

501 ACCATTTGATTTTATTCAATTGAGGCTAACATATTTATGCCTAATTCAACATCCCAAGTGACTAAACTTAAGAGCAAGAATAACGAATTTTCATATACAGM P N S T S Q V T K L K S K N N E F S Y T A

601 CAGATGCAGTAAGTATCTATCTTCATAAAATTGGACGTGTACCTCTGTTAAGCCATGAGCAAGAAATTTTTTTTGCTCAACAAGTTCAGCAAATGATGGTD A V S I Y L H K I G R V P L L S H E Q E I F F A Q Q V Q Q M M V

701 GATGTTTACTGCAAAAGAAGAfiCTAaQTGAAAAATTACAGCGTGAACCGACTCTGCAAGAATGGGCTGACAAGATGCAGTTGAAAGAAGATGTGCTGCTGM F T A K E E L A E K L Q R E P T L Q E W A D K M Q L K E D V L L

801 CAACAACTAAGTCAAGGACAAATTGCCAAGCAGAAGATGATTCAGGCTAATCTGCGGTTAGTGGTATCTATTGCTAAAAAATACCAGAAACGCAATCTGGQ Q L S Q G Q I A K Q K M I Q A N L R L V V S I A K K Y Q K R N L E

901 AGTTTCTAGACTTAATTCAAGAAGGTGCATTGGGGCTAGAACGAGGGGTAGAGAAATTTGATCCAACTCTTGGATACAAGTTTTCCACTTATGCTTACTGF L D L I Q E G A L G L E R G V E K F D P T L G Y K F S T Y A Y W

1001 GTGGATTCGTCAGGGAATTACACGAGCAATAGCACAACAATCCCGCACTATTCGTTTACCCATTCACATGGCTGATAAGCTGAACAAAATTAAGTGTGTTW I R Q G I T R A I A Q Q S R T I R L P I H M A D K L N K I K C V

1101 CAGCGAGAGTTATCTCAAAAGCTTGGTTACATTGCTGGCGTAACGGAAATCGCCCAAGCCCTCAATTTGGAACCTAGTCAGATTCGAGAATATTTGCAGCQ R E L S Q K L G Y I A G V T E I A Q A L N L E P S Q I R E Y L Q L

1201 TTGTTCGTCAACCTGTTTCCTTAGATATGCGAATTGGGTTTGAGCAGGATACCCAATTACAGGATCTGCTGAAGGATGATGGAATGTCTCCCGAACGCTAV R Q P V S L D M R I G F E Q D T Q L Q D L L K D D G M S P E R Y

1301 CGCCGAACGAGAATTGCTCTATCAAGACATTCACAACCTATTAGCAAAGCTGACTCCCCAGCAAAAAGAAGTCTTAATCTTGCGCTTTGGTTTAGCAGGTA E R E L L Y Q D I H N L L A K L T P Q Q K E V L I L R F G L A G

1401 GGATGCGAACTGACCCTAGTACAGATTAGTCAACGGATGGGTATTAGTCGGGAACGAGTACGACAAGTGGAAAAACAAGCTCTCACGCTTCTACGAAGATG C E L T L V Q I S Q R M G I S R E R V R Q V E K Q A L T L L R R Y

1501 ATGGAATTGACTCACGCAGCTATCTAGCTGACTGATACTCTTTAATTTTGAAAGCTTGTAGTGTCAACTGGTAAGTGAAAAACCGCCTGCTTGCTGTCACG I D S R S Y L AD *

1601 TGGTCTAAAT

FIG. 3. Nucleotide sequence of the Anabaena sp. strain PCC 7120 sigB gene and 5' and 3' flanking regions. The predicted amino acidsequence of sigB is shown in single-letter code and begins at nucleotide 537. The underlined sequence 11 bp upstream of the initiation codonrepresents a putative ribosome binding site. The underlined hexanucleotide beginning at nucleotide 722 represents the NheI site into whicha cassette encoding resistance to spectinomycin and streptomycin was ligated in order to interrupt the sigB gene.

copy of each gene was replaced by the mutated version DISCUSSIONcontaining the cassette were obtained by the method of Cai RNA polymerase sigma factors constitute a homologousand Wolk (6). BothAnabaena sp. strain DR1, in which sigB protein family, sharing at least two, and in some cases four,is inactivated, and strain C9, in which sigC is interrupted, regions of similarity (14, 17). Using an approach based onare viable, indicating that neither sigB norSmgC is an this similarity, we have isolated two genes, sigB and sigC,essential gene. Furthermore, the sigB and sigC mutants were from the cyanobacteriumAnabaena sp. strain PCC 7120 thatnot impaired in heterocyst differentiation or in growth in exhibit high degrees of homology to sigma factors. sigB wasliquid medium under nitrogen-limiting conditions. On solid isolated by using an oligonucleotide probe complementary tomedia lacking combined nitrogen, however, there were the rpoD oligonucleotide described by Tanaka et al. (35),subtle differences in colony morphology. Strain DR1 formed which is based on a 10-amino-acid stretch that is completelycolonies that were more diffuse than those of the wild type, conserved between the principal sigma factors of E. coli andwhile strain C9 formed slightly smaller colonies than the wild B. subtilis. This oligonucleotide has been used to detecttype. principal sigma factors and related genes in a number of


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

VOL. 174, 1992 ANABAENA SIGMA FACTOR HOMOLOGS 7279

-467 AACTGACAACAAGTCCCCAATTTAACTCATCACTACTCATCATCCCCATTACTCCACACCATACTGAGTCCTCAAAAATCCTGAGAAAAAAATTAGAAAA* . . ......I--->

-267 TAATAGCCAACAAGGTGAAAACGCTGACTAAGTAATTCGGAAATTAAAATCTAATCGTAAATTTGAATAGCTATTGGTTCAATCAGACTCTCAACAGCCA

-167 ACTATAGTTAGGCTGTAATGTCTGAAAACAACCACTAAGGTTTTTTTGTAAAAAAAATTTTAGTGAAAACCTTAACCGTGTTAGTTCATCTAGAATCACG+1 .

-67 GGAACAATAGCAACAGTTAAATAGCTGACCGCAATCTATTGTTACCTGGAGCCATAGAGACGATCTTATGCCAGCAACATCTTTTTACGCAGATGCCGCCM P A T S F Y A D A A

34 TACAATACCCAAAAATCCCGCCAGGCTTTAGACCCTGATATCGCCATTGATGACAGTGATTTGTCGGTGGATGAGATCCAAGAATTAGAGATAGCTGCTGY N T Q K S R Q A L D P D I A I D D S D L S V D E I Q E L E I A A A

134 CTGATCCCGCTACTTTTGGTCGTAGCGCTAACCGTCGTAGTACAGACTTAGTACGTCTATACCTTCAGGAAC-..CGGTCGAGTCCGTTTGTTAGGACGGGAD P A T F G R S A N R R S T D L V R L Y L Q E I G R V R L L G R D

234 CGAAGAAGTTTCTGAAGCTCAAAAAGTCCAGCGTTACTTAAAGTTGCGGATAGTGCTAGCTAATGCTGTCAAACAGGGAGATGAAGTTGCTACTCCTTATE E V S E A Q K V Q R Y L K L R I V L A N A V K Q G D E V A T P Y

334 CTACATTTAATCGAAGTTCAAGAGCGTCTAGCATCAGAACTTGGCCATCGTCCTTCCTTGGAAAGATGGGCTGCTACTGCTGGTATCAACTTATGTGACCL H L I E V Q E R L A S E L G H R P S L E R W A A T A G I N L C D L

434 TGAAGCCAATTTTATCGGAAGGTAAACGTCGCTGGGCAGAAATTGCCAAGATGACGGTGGAAGAATTGGAGAAAATGCAATCTCAAGGTCTTCAGTCAAAK P I L S E G K R R W A E I A K M T V E E L E K M Q S Q G L Q S K

534 AGAACACATGATTAAGGCTAATCTGCGCTTAGTCGTGTCTGTTGCCAAAAAATACCAAAATCGTGGTTTAGAATTATTAGACTTAGTTCAAGAAGGCACTE H M I K A N L R L V V S V A K K Y Q N R G L E L L D L V Q E G T

634 CTCGGCTTAGAACGAGCTGTAGAGAAATTTGATCCAACTAAAGGAZAT.CGTTTTAGTACCTATGCCTACTGGTGGATTCGCCAAGGGATCACAAGAGCGAL G L E R A V E K F D P T K G Y R F S T Y A Y W W I R Q G I T R A I

734 TCGCTACTTCTAGTCGCACAATTCGCCTCCCTGTTCATATTACAGAAAAATTAAACAAAATTAAAAAGGCTCAACGCAAAATCGCTCAAGAAAAAGGTCGA T S S R T I R L P V H I T E K L N K I K K A QR K I A Q E K G R

834 CACTCCTACTTTAGAAGACCTAGCAATTGAGTTAGATATGACCCCTACCCAAGTGCGGGAAGTTTTACTAAGAGTACCCCGTTCTGTTTCTTTGGAAACCT P T L E D L A I E L D M T P T V R E V L L R V P R S V S L E T

934 AAGGTAGGTAAAGATAAAGACACTGAGTTGGGAGAATTATTAGAGACTGACGGTGTAACTCCTGAAGAAATGTTAATGCGAGAATCTTTACAAAGAGACTK V G K D K D T E L G E L L E T D G V T P EEEM L M R E S L R D L

1034 TGCAACATTTATTAGCTGATTTAACTAGCCGTGAACGTGATGTCATCTTGATGCGTTTCGGTTTAGCTGATGGTCATCCTTACTCATTGGCAGAAATTGGO H L L A D L T S R E R D V I L M R F G L A D G H P Y S L A E I G

1134 CCGCGCTCTGGATTTATCA'CGGGAGCGAGTACGCCAAATTGAATCTAAGGCCTTGCAAAAACTCCGCCAACCCAAGCGTCGCAACCTTATCCGTGACTATR A L D L S R E R V R Q I E S K A L Q K L R Q P K R R N L I R D Y

1234 TTGGAATCTCTGAGTTAGATAGTTAATAGTCAATAGTCAATAGTCAATAGTCAATAATCAATAGTAAACAAAATCCATACTATAGATTATTGACTCTGGAL E S L S *---------------------------> <----------------------

1334 CTATTTATTTCATCACAACCAATTAAGCATAACGGCAGATTTTGCCCCTGGTCACGGAGGAAAACTCATCACCTTGTGATTGGAGTTTATACATTAATTA

1434 ACATAATGTGATTTATTCTCGCCTAGCAATTTAACAACTACTTGTTACTTATAGGAAACTTTTGCATTTAATTAAAATGTATTTTTGCAAAGCATTGCTT

1534 GGAAACATAAAATAGTAAC

FIG. 4. Nucleotide sequence of the Anabaena sp. strain PCC 7120 sigC gene and 5' and 3' flanking regions. The predicted amino acidsequence ofsigC is shown in single-letter code. The underlined sequence 7 bp upstream of the initiation codon represents a putative ribosomebinding site. The underlined hexanucleotides at nucleotides 70 and 677 represent the EcoRV sites into which a cassette encoding resistanceto spectinomycin and streptomycin was ligated, replacing the EcoRV fragment, in order to interrupt the sigC gene. The boldface nucleotideat position -328 indicates the 5' end of the sigC transcript mapped by primer extension; the dashed arrow beginning at this nucleotideindicates the direction of transcription. The dashed arrows beginning at nucleotides 1272 and 1311 indicate inverted repeats.

diverse bacterial species (35), including the unicellular cya- Anabaena sp. strain PCC 7120 genome, suggesting thenobacterium Synechococcus sp. strain PCC 7942 (34). sigC presence of more sigma-like genes.was identified by using a fragment of the Anabaena sigA SigB and SigC exhibit higher degrees of similarity to thegene encoding the principal vegetative cell sigma factor as Anabaena sp. strain PCC 7120 SigA protein, to other prin-the probe (3). The use of the sigA gene as a probe also cipal sigma factors, and to the principal sigma-like proteinsrevealed that a number of other homologous regions, in HrdA, HrdC, HrdD of Streptomyces coelicolor (36) andaddition to those attributed to sigB and sigC, exist in the KatF of E. coli (27) than they do to other alternative forms of


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

7280 BRAHAMSHA AND HASELKORN J. BACrERIOL.

Region lbSigA 80 DSIRLYLQEIGRIRLLRADEEIELARKIADLL---------------------ELERVRERLSEKLERDPSigB 23 DAVSIYLHKIGRVPLLSHEQEIFFAQQVQQMM---------------------VMFTAKEELAEKLQREPSigC 60 DLVRLYLQEIGRVRLLGRDEEVSEAQKVQRYLKLRIVLANAVKQGDEVATPYLHLIEVQERLASELGHRPHrdA 98 DLFRQYLREIGRIPLLSAAEEVDLARRVEAGLFAEEKLRCS------------------PGLDDRLAL--HrdB 144 DPVKDYLKQIGKVPLLNAEQEVELAKRIEAGLFAEDKLANS------------------DKLAPKLKR--HrdC 37 DLLGQYLTQIGATPLLTAEDEVRLATRIEAGVRAREELETA--------------DTGEPAPTPRRRR--HrdD 34 DLVGMYLDEIARTPLLDAAKEVELSQTIEAGVFARQVLEGY------------EETGADAT---RE--KatF 68 DATQLYLGEIGYSPLLTAEEEVYFARRA-------------------------------------LRG--

* ** * . ** *.

SigA 129 RDSEWAEAVQLPLPAFRYRLHIGRR----------------------AKDKMVQSNLRLVVSIAKKYMNRSigB 72 TLQEWADKMQLKEDVLLQQLSQGQI----------------------AKQKMIQANLRLVVSIAKKYQKRSigC 130 SLERWAATAGINLCDLKPILSEGKRRWAEIAKMTVEELEKMQSQGLQSKEHMIKANLRLVVSVAKKYQNRHrdA 148 ------------------------------------ DLDRLVVLGRLAKRRLIEANLRLVVSVAKRYVGRHrdB 194 ------------------------------------ ELEIIAEDGRRAKNHLLEANLRLVVSLAKRYTGRHrdC 91 ------------------------------------ TLEETVHDGQEAKDHMVRANLRLVVSMAKRHAHRHrdD 85 ------------------------------------ ELQALIDESERAKDVFIRSNLRLVVAVARRYPRSKatF 99 ------------------------------------DVA---SRRRMIESNLRLVVKIARRYGNR

*******

Region 2SigA 177 GLSFQDLIQEGSLGLIRAAEKFDHEKGYKFSTYATWWIRQAITRAIADQSRTIRLPVHLYETISRIKKTTSigB 120 NLEFLDLIQEGALGLERGVEKFDPTLGYKFSTYAYWWIRQGITRAIAQQSRTIRLPIHMADKLNKIKCVQSigC 200 GLELLDLVQEGTLGLERAVEKFDPTKGYRFSTYAYWWIRQGITRAIATSSRTIRLPVHITEKLNKIKKAQHrdA 182 GLTMLDLVQEGNLGLIRAVEKFDYARGYKFSTYATWWIRQAMSRALADQARTIRVPVHVVELINRVVRVQHrdB 228 GMLFLDLIQEGNLGLIRAVEKFDYTKGYKFSTYATWWIRQAITRAMADQARTIRIPVHMVEVINKLARVQHrdC 125 GLPLLDVIQEGNLGLIRAVEKFDHTKGFKFSTYATWWIRQAIERGLATHARTVRLPVHVVEQLQKLAKVEHrdD 119 GLPLLDLIQEGNAGLVRAVEKFDYRKGFKFSTYATWWIRQAITRSIADQSRTIRLPVHLVEELGRIRRVQKatF 125 GLALLDLIEEGNLGLIRAVEKFDPERGFRFSTYATWWIRQTIERAIMNQTRTIRLPIHIVKELNVYLRTA

***** ** *. **** *..***** ***** * **.. *****

-10 recognitionRegion 3

SigA 247 KLLSQEMGRKPTEEEIATRMEMTIEKLRFIAKSAQLPISLETPIGKEEDSRLGDFIE-SDGETPEDQVSKSigB 190 RELSQKLGYIAGVTEIAQALNLEPSQIREYLQLVRQPVSLDMRIGFEQDTQLQDLLKD-DGMSPERYAERSigC 270 RKIAQEKGRTPTLEDLAIELDMTPTQVREVLLRVPRSVSLETKVGKDKDTELGELLE-TDGVTPEEMLMRHrdA 252 RRMLQERGCEPTPQEVAAHLDLAPERVGEVLRLAQEPVSLHAPVGEEDDVALGDLIEDGDAASPVESAAFHrdB 298 RQMLQDLGREPTPEELAKELDMTPEKVIEVQKYGREPISLHTPLGEDGDSEFGDLIEDSEAVVPADAVSFHrdC 195 RKLRAGLDREPTTEEVAAESGIDVDKVVWLRRVGRDAVSLDTPVDETGDTVVGDLIPDTEVLRAPEVAEFHrdD 189 REFNREHGREPEPAEIAAELGSTPERVHDVLDWARDPVSLNMSVDDEGETQFGDLLEDTSAV-SPEQSVLKatF 195 RELSHKLDHEPSAVEIAEQLDKPVDDVSRMLRLNERITSVDTPLGGDSEKALLDILADEKENGPEDTTQD

__________________________Region 4SigA 316 NLLR-EDLEKVLDSLSPRERDVLRLRYGLDDGRMKTLEEIGQIFNVTRERIRQIEAKALRKLRHPNRNSVSigB 259 ELL-YQDIHNLLAKLTPQQKEVLILRFGLAGGCELTLVQISQRMGISRERVRQVEKQALTLLRRYGIDS-SigC 339 ESLQ-RDLQHLLADLTSRERDVILMRFGLADGHPYSLAEIGRALDLSRERVRQIESKALQKLRQPKRRNLHrdA 322 LLLR-QHLEAVLSTLGERERKVVQLRYGLADGRPRTLEEIGRLGGVTRERIRQIESKTLSKLRDHAYADQHrdB 368 TLLQ-EQLHSVLDTLSEREAGVVSMRFGLTDGQPKTLDEIGKVYGVTRERIRQIESKTMSKLRHPSRSQVHrdC 265 QAL-AAELREAVGTLAPRESLILSLRYGLHDGRPRTLQQVAQHVGLTRERVRQLEKESLAHLRAPENRERHrdD 258 TLLRSEELDDLIGRLDPRTASIIKMRYGIDDGRERTLTEVGKEHGLTRERIRQIEKHALLELKKLA-RDTKatF 265 DDMKQSIVKWLFE-LNAKQREVLARRFGLLGYEAATLEDVGREIGLTRERVRQIQVEGLRRLREILQTQG.~~~ . .. * . . . . ... ..... ***.**........ *.

-35 recognitionSigA 385 LK--EYIRSigB 327 -R--SYLADSigC 408 IR--DYLESLSHrdA 391 LR--GYLDHrdB 437 LR--DYLDHrdC 334 LL--DWASHrdD 327 GF--EAAAKatF 334 LNIEALLPRVSKHLSERPVSSEAGFFCAQ

FIG. 5. Amino acid sequence alignment ofAnabaena sp. strain PCC 7120 SigA, SigB, and SigC, Streptomyces coelcolor HrdA, HrdB,HrdC, and HrdD, and E. coli KatF. The asterisks indicate amino acids that are identical in all eight sequences; the dots indicate amino acidsthat are similar in all eight sequences. The overlined regions correspond to conserved regions lb, 2, 3, and 4 of sigma factors (14, 17). Boldfaceresidues have been implicated in interactions with the -10 and -35 sequences of promoters (11, 33, 37).

the sigma subunit. In particular, both SigB and SigC are promoters, respectively (11, 33, 37). Residues which areconserved in amino-terminal region lb (14, 17), which is thought to interact with the -35 hexamer (11, 33) arefound only in principal sigma and principal sigma-like fac- conserved in SigB (Arg-305 and Arg-309) and SigC (Arg-385tors. LikeAnabaena sp. strain PCC 7120 SigA (3), SigB and and Arg-389), as they are in SigA (3), as well as two thoughtSigC are also conserved in regions 2.4 and 4, which have to contact the -10 hexamer (33, 37) (Gln-159 and Thr-162 forbeen implicated in binding to the -10 and -35 sequences of SigB and Gln-239 and Thr-242 for SigC). This conservation


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


sigB sigC

0 6 12 18 24 30 0 6 121 8 24 30

1.7 kh1.4 kb- <-- 1.4 kb

FIG. 6. Northern analyses of the Anabaena sp. strain PCC 7120sigB and sigC genes. Total RNAs prepared from cells grown on

combined nitrogen (lanes 0) and from cells harvested at 6, 12, 18, 24,and 30 h following the removal of combined nitrogen (lanes 6, 12, 18,24, and 30, respectively) were probed with restriction fragmentsinternal to sigB or sigC.

suggests that SigB and SigC interact with promoters that are

very similar in sequence to the promoters recognized bySigA.The presence in Anabaena sp. of at least two genes that

are very similar to the one encoding the principal RNApolymerase sigma factor represents a situation analogous tothe situation seen in Streptomyces coelicolor, which hasthree homologs of the principal sigma factor, hrdB (32). Atpresent, the function of the majority of principal sigma-likegenes is not understood. In the case ofAnabaena sp. strainPCC 7120, sigB and sigC are not essential. Although both of

- ~

-

_ _

z z X

_ <4(- -328

FIG. 7. Identification of the 5' end of the Anabaena sp. strainPCC 7120 sigC transcript. An oligonucleotide complementary tonucleotides -7 to -26 was annealed to 50-,ug portions of RNAprepared from cells grown in the presence of combined nitrogen(lane N+) and from cells deprived of combined nitrogen for 18 h

(lane N-) or to yeast tRNA (lane tRNA) and extended with reverse

transcriptase. The primer-extended product was coelectrophoresedwith a sequencing ladder generated with the same oligonucleotideprimer. The arrow indicates the position of the product, numberedwith respect to the translation start of sigC.

these genes are induced specifically under conditions ofnitrogen limitation, neither is required alone for growth orfor heterocyst differentiation. Likewise, in Streptomycescoelicolor, hrdA, hrdC, and hrdD mutants have no obviousphenotype (5, 36). Although hrdD is transcribed, it cannotcompensate for mutations in the gene for the principal sigmafactor, hrdB, suggesting that it is not functionally redundant(5). At least one of these principal sigma-like genes isfunctional. In E. coli, katF is required for the expression ofgenes that are induced during stationary phase and carbonstarvation (21, 23, 27). It is possible that sigB and sigC, aswell as hrdA, hrdC, and hrdD, are required for growth orsurvival under conditions that have not been investigatedyet.

ACKNOWLEDGMENTSWe thank Paul Gardner for the synthesis of oligonucleotides,

W. J. Buikema for the use of his cosmid library, Jennifer Moran forhelp with sequencing, and Conrad Halling and Brian Palenik forcritically reading the manuscript.

This work was supported by Public Health Service research grantGM-21823 from the National Institutes of Health. B.B. was sup-ported by an American Cancer Society postdoctoral fellowship andby training grant HD-07136 in developmental biology from theNational Institutes of Health.

ADDENDUM IN PROOF

Since this article was submitted, we have constructed adouble mutant of Anabaena sp. strain PCC 7120 in whicboth the sigB and sigC genes are inactivated. The doublmutant is capable of both heterocyst differentiation an Jgrowth in the absence of combined nitrogen. We have als:determined that sigC, but not sigB, is strongly induced unde rconditions of sulfur starvation. Recently, J. L. Collier an JA. R. Grossman (J. Bacteriol. 174:4718-4726, 1992) havecharacterized the bleaching response induced by nutrientdeprivation in the unicellular cyanobacterium Synechococ-cus sp. strain PCC 7942. They have isolated genes involvedin this response which are expressed during both N and Sstarvation. Perhaps sigC and its counterpart in unicellularcyanobacteria, are involved in the expression of such genes.

REFERENCES1. Apelian, D., and S. Inouye. 1990. Development-specific a-factor

essential for late-stage differentiation of AMyxococcus xanthus.Genes Dev. 4:1396-1403.

2. Bhagwat, A. A., and S. K. Apte. 1989. Comparative analysis ofproteins induced by heat shock, salinity, and osmotic stress inthe nitrogen-fixing cyanobacteriumAnabaena sp. strain L-31. J.Bacteriol. 171:5187-5189.

3. Brahamsha, B., and R. Haselkorn. 1991. Isolation and charac-terization of the gene encoding the principal sigma factor of thevegetative cell RNA polymerase from the cyanobacterium An-abaena sp. strain PCC 7120. J. Bacteriol. 173:2442-2450.

4. Burton, Z., R R Burgess, J. Lin, D. Moore, S. Holder, and C.Gross. 1981. The nucleotide sequence of the cloned rpoD genefor the RNA polymerase sigma subunit for E. coli K12. NucleicAcids Res. 9:2889-2903.

5. Buttner, M. J., K. F. Chater, and M. J. Bibb. 1990. Cloning,disruption, and transcriptional analysis of three RNA polymer-ase sigma factor genes of Streptomyces coelicolor A3(2). J.Bacteriol. 172:3367-3378.

6. Cai, Y., and C. P. Wolk 1990. Use of a conditionally lethal genein Anabaena sp. strain PCC 7120 to select for double recombi-nants and to entrap insertion sequences. J. Bacteriol. 172:3138-3145.

7. Casadaban, M. J., and S. N. Cohen. 1980. Analysis of genecontrol signals by DNA fusion and cloning in Escherichia coli.

VOL. 174, 1992


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


J. Mol. Biol. 138:179-207.8. Chater, K. F., C. J. Bruton, K. A. Plaskitt, M. J. Buttner, C.

Mendez, and J. D. Helmann. 1989. The developmental fate of S.coelicolor hyphae depends upon a gene product homologouswith the motility v factor of B. subtilis. Cell 59:133-143.

9. Elhai, J., and C. P. Wolk 1988. Conjugal transfer of DNA tocyanobacteria. Methods Enzymol. 167:747-754.

10. Elhai, J., and C. P. Wolk. 1990. Developmental regulation andspatial pattern of expression of the structural genes for nitroge-nase in the cyanobacterium Anabaena. EMBO J. 9:3379-3388.

11. Gardelia, T., H. Moyle, and M. M. Susskind. 1989. A mutantEscherichia coli a70 subunit of RNA polymerase with alteredpromoter specificity. J. Mol. Biol. 206:579-590.

12. Gitt, M. A., L.-F. Wang, and R. H. Doi. 1985. A strong sequencehomology exists between the major RNA polymerase a factorsof Bacillus subtilis and Escherichia coli. J. Biol. Chem. 260:7178-7185.

13. Golden, J. W., and D. R. Wiest. 1988. Genome rearrangementand nitrogen fixation in Anabaena blocked by inactivation ofxisA gene. Science 242:1421-1423.

14. Gribskov, M., and R. R. Burgess. 1986. Sigma factors fromEschenichia coli, phage SPOl, and phage T4 are homologousproteins. Nucleic Acids Res. 14:6745-6763.

15. Grossman, A. D., J. W. Erickson, and C. A. Gross. 1984. ThehtpR gene product of E. coli is a sigma factor for heat-shockpromoters. Cell 38:383-390.

16. Helmann, J. D. 1991. Alternative sigma factors and the regula-tion of flagellar gene expression. Mol. Microbiol. 5:2875-2882.

17. Helmann, J. D., and M. Chamberlin. 1988. Structure andfunction of bacterial sigma factors. Annu. Rev. Biochem. 57:839-872.

18. Hirschman, J., P.-K. Wong, K. Sei, J. Keener, and S. Kustu.1985. Products of nitrogen regulatory genes ntrA and ntrC ofenteric bacteria activate glnA transcription in vitro: evidencethat the ntrA product is a sigma factor. Proc. Natl. Acad. Sci.USA 82:7525-7529.

19. Kraft, R., J. J. Tardiff, K. S. Krauter, and L. A. Leinwand.1988. Using mini-prep plasmid DNA for sequencing doublestranded templates with Sequenase. BioTechniques 6:544-547.

20. Landick, R., V. Vaughan, E. T. Lau, R. VanBogelen, J. W.Erickson, and F. C. Neidhardt. 1984. Nucleotide sequence of theheat-shock regulatory gene of Escherichia coli suggests itsprotein product may be a transcription factor. Cell 38:175-182.

21. Lange, R., and R. Hengge-Aronis. 1991. Identification of acentral regulator of stationary-phase gene expression in Esche-richia coli. Mol. Microbiol. 5:49-59.

22. Maniatis, T., E. F. Fritsch, and J. Sambrook 1982. Molecularcloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

23. McCann, M. P., J. P. Kidwell, and A. Matin. 1991. The putativea factor KatF has a central role in development of starvation-mediated general resistance in Eschenichia coli. J. Bacteriol.173:4188-4194.

24. Meeks, J. C., C. P. Wolk, W. Lockau, N. Schilling, P. W.Shaffer, and W.-S. Chien. 1978. Pathways of assimilation of[13N]N2 and 13NH4+ by cyanobacteria with and without hetero-cysts. J. Bacteriol. 134:125-130.

25. Moran, C. P. J. 1989. Sigma factors and the regulation oftranscription, p. 167-184. In I. Smith, R. A. Slepecky, and P.Setlow (ed.), Regulation of procaryotic development. AmericanSociety for Microbiology, Washington, D.C.

26. Mulligan, M. E., and R. Haselkorn. 1989. Nitrogen fixation (nif)genes of the cyanobacterium Anabaena species strain PCC7120. The nifB-fdxN-nifS-nifU operon. J. Biol. Chem. 264:19200-19207.

27. Mulvey, M. R., and P. C. Loewen. 1989. Nucleotide sequence ofkatF of Eschenichia coli suggests KatF protein is a novel sigmatranscription factor. Nucleic Acids Res. 17:9979-9991.

28. Murry, M. A., A. J. Horne, and J. R. Benemann. 1984. Physi-ological studies of oxygen protection mechanisms in the hetero-cysts of Anabaena cylindrica. Appl. Environ. Microbiol. 47:449-454.

29. Rippka, R., J. Deruelles, J. B. Waterbury, M. Herdman, andR. Y. Stanier. 1979. Genetic assignments, strain histories andproperties of pure cultures of cyanobacteria. J. Gen. Microbiol.111:1-61.

30. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequenc-ing with chain-terminating inhibitors. Proc. Natl. Acad. Sci.USA 74:5463-5467.

31. Schneider, G. J., J. D. Lang, and R. Haselkorn. 1991. Promoterrecognition by the RNA polymerase from vegetative cells of thecyanobacteriumAnabaena 7120. Gene 105:51-60.

32. Shiina, T., K. Tanaka, and H. Takahashi. 1991. Sequence ofhrdB, an essential gene encoding sigma-like transcription factorof Streptomyces coelicolor A3(2): homology to principal sigmafactors. Gene 107:145-148.

33. Siegele, D. A., J. C. Hu, W. A. Walter, and C. A. Gross. 1989.Altered promoter recognition by mutant forms of the a70 subunitofEscherichia coli RNA polymerase. J. Mol. Biol. 206:591-603.

34. Takahashi, H. Personal communication.35. Tanaka, K., T. Shiina, and H. Takahashi. 1988. Multiple prin-

cipal sigma factor homologs in eubacteria: identification of the"rpoD box." Science 242:1040-1042.

36. Tanaka, K., T. Shiina, and H. Takahashi. 1991. Nucleotidesequence of genes hrdA, hrdC, and hrdD from Streptomycescoelicolor A3(2) having similarity to rpoD genes. Mol. Gen.Genet. 229:334-340.

37. Waldburger, C., T. Gardella, R. Wong, and M. M. Susskind.1990. Changes in conserved region 2 of Eschenichia coli F70affecting promoter recognition. J. Mol. Biol. 215:267-276.

38. Wolk, C. P. 1968. Movement of carbon from vegetative cells toheterocysts inAnabaena cylindrica. J. Bacteriol. 96:2138-2143.

39. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. ImprovedM13 phage cloning vectors and host strains: nucleotide se-quences of the M13mpl8 and pUC19 vectors. Gene 33:103-119.

J. BACTERIOL.


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

identification of multiple rna polymerase sigma factor homologs

Documents