(sigma 32 homolog) from Pseudomonas aeruginosa

onirrg the gene for the heat shock response positive regulator sigma 32 from Pseudomonas aerugirrosa

Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Health Canada on 11/19/17 For personal use only.

Zerlina M. Naczynski, Chris Mueller, and Andrew

M. Kropinski

Abstract: A 3 1 base pair synthetic oligonucleotide based on the genes for the Eschcricl~iacuEi heat shock D ) employed in sigma factor (rpo86)and tlae Pseudomoiaas nerugincpscs housekeeping sigma factor ( I - ~ ~ Iwas conjunction with the Tanaka et al. (K. Tmaka, T. Skiina, and H. Takahashi, 1988. Science (Washington, D.C.), 242: 1040-1042) RpoD box probe to identify the location of the rpoH gene in B. oen6glizo~ugenomic digests. This gene was cloned ii~tcbplasmid pGEM3Z(f+), sequenced, and found to share 67% nucleoticle identity and 77% amino acid homology with the yoPb gene and its product (d2) of E. coli. The plasmid containing the lpnH gene complemented the function of e~~~ in an E. coli rpoH deletion mutant. Furthemore. this plasn-niddirected the synthesis of a 32-kDa protein in an E. coli S-30 in vitro transc~ption-translation system. Primer extension studies were used to identify the transcriptional start sites under control and heat-stressed (45 and 50°C) conditions. Two promoter sites were identified having sequence homology to the E. c ~ luTO i and nz4 consensns sequences. Kc~ywordL~: heat shock, Bseucio~lzonasaeruginu~a,sigma factor, transcription, oligonucleotide pmbe.

RQornC : Un oligonuclkotide synthktique de 3 I paires de bases. bas6 SUH les gknes pow le facteur de choc themique sigma ( I ~ C Pde H EscFaerickzic~coli et Be facteur sigma m6nager (rpoD) de Pseuldome~nasut'rugino~l~ a kt6 utilisC de concert avec la sonde pour la boite RpoD de Tanakra et coll. (K. Tanka, T. Shiina, et H. TakaBnashi, 1988. Science (Washington. D.C.), 242: 1040-1042) afin d'identifier Ba position du gkne ~ p o H dans des digestions g6na~miquesde P. caeruginnsa. Ce gkne a kt6 clonk dans Ic plasmide pGEM3ZCf-k). Le skquengage a r6vklC qu'il poss6dait 67% d'identitk nuclCotidique et 77% d9homologieen acides arnia~Csavec le gkne rpoH de E. ccpli et son produit t ~ Le ~plasmide ~ . contenant le g h e rpoH complCn~entaitla fonction de d2dans ran mutant de dklktion ~ p o Hde E. coli. Be plus. ce plasmide dirigeait la synthkse d'une protkine de 32 kDa dans un essai de transcription-tradktction in vitro avec des extraits S-30 de E. c-oli.Des Ctudes d'extensicsns d9amsrsesont kt6 utiliskcs pour identifier les sites d'initiation de la tmnscription dans des co~aditiasnscontr8les ek de choc thedqane (45 et 50°@).Deux sites pmmoteurs c~i.at6t6 identifiks cornme ayant des homologies de sCqueaace avec des skqanences consensus de aT0et a24de E. calk.

MOLYcE4s : chsc themique. Pseudomonns. uel-ugiiaosu,facterar sigma, transcription, sonde oligonuclkotidiqbae. [Trad~ritpar Ia RCdractic~nl

lntaoductien The heat shock response is generally defined as a rapid and transient increase in the synthesis of a small number of proteins (heat shock proteins, HSPs) in response to hyperthemic conditions. While heat is the most efficient inducer sf HSPs, a number of other stressors can induce HSP synthesis. Bacterial

Received August 11, 1994. Revision received October 25, 1994. Accepted October 26, 1994.

%.kf.Nsczynski and A.M. Brspinskii Department of Microbiology and Immunology, Faculty of Medicine, Queen's University, Kingston, OW K7L 3N6, Canada. C. Mneller Department of Biochelraistry, Faculty of Mediciiae. Queen's University, Kiiagston, ON K7L 3N6, Crinada. Author to whom all correspondence shc~uldhe addressed. Cantn. J. Microbid. 41:75-87 (1995) Printed in C a n d a 1 Imprim6 au Canada

stressors include recovery from a~aoxia,M202, some metals (Cd"), canavanine, purornycin, extremes of pH, ethanol, nalidixic acid, and proteins (No\Ier 1991a). One of the earliest and most striking discsve~eswas the universality of the response. This response has been observed in eukaryotes as well as thermophilic, rnesophilic, and psychrophilic eubacte6a. In addition, a heat shock response has been characterized in members of the Archaea meb be^ et al. I991 ; Nover li 99 1b).Furthemore, when the nucleotide sequences of the heat shock genes (or their proteilms) from Escherichia ~ ' o l i , Drosopfziln sp,, yeast, plants, and humans were compared, many of the HSPs were revealed to be highly homologous (Bardwell and Craig 1984, 1987; McMullin and Mallberg 1988). The heat shock response of E. coli has been thoroughly studied (for recent ree7iewssee Craig et al. 1993; Gross et al. 1990; hforirnoto et al. L 990: Nover L 99 1b Parsell and Linquist 1993;Yura et al. 1993). The heat shock proteins were identified as members of a regulon, since synthesis of these proteins was


Can. J. Micrsbi~I.V d . 41, 1995 coordinately regulated by a single gene (htpR). The latter gene, also known as rpoW, encodes a protein of approximately 32 kDa that displays the characteristics of a sigma factor including association with core ((BPtar2)RNA polymerase and trmanscripticsn of heat shock genes from unique promoter sequences. Pseudomonads are clinically and ecomrnically important as causative agents of human, animal, and plmt diseases. Many Bseeadomo?zas species are also environmentally m d biotechnolsgically significant. The heat shock response of Pseudomoraas asruginssa was characterized in 1988 (Allm et al. 1988). The responses of am-uginosa and E. codi show similar characteristics. Within 2 min of a temperature shift from 30 to 45"C, an increase in a small number of proteins was detected. After 10 min, 17 heat shock proteins were discerned by one-dimensional SDS-polyacrylamide gel electrophoresis. The synthesis of non-heat shock proteins was markedly s~appressedfor 60 min. Five major proteins were heat induced (86.4, 76, '30.4, 68.6, and 15.7 ma).Unlike the situation with E. colk, in which ethanol induces all the HSPs, ethanol induced aei-uginosa. the synthesis of only seven proteins in Furthemore, only three of these could be identified as HSPs. Antibodies against the E. codi GroEL and BnaK proteins reacted with the 60.6- and 76-kDa proteins, respectively, indicating the consemation of these two MSPs in this bacterium. This has been confirmed by sequence analysis. S i p s et al. (1991) demonstrated 86% similarity between the GroEL proteins of E. coki and I? aeruginosa. Allm and Kropinski (1987) isolated RNA polymerase from f? aeruginosa and determined it to have a typical eubacterial structure (PPta2a). A major sigma factor of 87 kDa was detected by SBS-polyacrylamide gel electrophoresis, Tanaka et al. (1988) utilized the amino acid homology between the major sigma factors of E. coli m d B. subtili~to design an oligonucleotide probe and reported two hybridization signals in chromosomal digests of I? ~asrmginssa.Tanaka and Tkkahashi (199 1) cloned one (rpoDA) of these two genes and demonstrated complementation of a temperature-sensitive mutation in E. coIi ypoDe %twas determined that the B. aeruginosa rpoDA gene functioned as a homolog of the E. coli g;ppoDgene product. The other signal was reported to correspond to the rpnS homolog in P. aaerugisrosa (Ronald et al. 1992). RpoS functions as a significant sigma factor in gene expression during stationary phase in E. codi (Tmaka et a]. 2993). To determine if the major sigma factor of P. aeruginssa was a heat-induced protein, a monoclonal antbody against the 87-kDa sigma factor was used to probe Western blots (Allm m d Kropinski 1984). The results indicated that the major sigma factor is also an HSP?as it is in E. cobi. The addition of a transcriptional inhibitor (rifampicin) prior to heat shock prevented the synthesis of the HSPs. Therefore? induction sf the HSPs in P. aerugialosa, as in E. cedi, is regulated at the level of transcription. It was postulated (Allm et al. 1988) ghat antibodies ag&nst purified RNA polymerase might copreelpitate Ecr3"rom heat-shocked cells. Using this approach a 40-kBa protein was identified in heat-shocked but not control cells. Furthemore, a protein with a similar molecular mass was found to copt~rifywith RNA polymerase in heat-shocked cells. Another protein with rn apparent molecular maass of 38.5 kDa was also found associated with the WNNP$polymerase from heat-shocked cells. These proteins were

not purified so it was not determined if either was the heat shock sigma factor. As outlined above, it has been detemined that the heat shock response in P. aeruginosa is regulated, at least in part, at the level of transcription. This response was initially examined in I? aeruginosw to gain a greater understanding of the regulation of gene expression in this bacterium. Cloning of the I ~ O H homolog from P. aerugknosu, together with identification of heat shock promoter sequences upstream from genes such as mopB (GroES) m d dnbrK, should greatly enhance our understanding of transcriptional regulation in pseudomonads. The former goal, that of cloning the rpoH homolog from P.~eru,ykkaosa,has been acheived. It was shown to be highly homologous to the d2protein of E. colk m d indeed would complement an E. colt Mrpolf~ mutant.

Materials and methods Bacterial strains and growth conditions Pseudomonas aerngi~~osa AKl012 (Jarrell m d Kropinski 1977), a lipopolysaccharide-defective mutant of strain PA0 1 , was utilized for chromosomal DNA preparations. Escbterichka codi BH5a (F-cb8OdlacZUI5 A(lacZYA-argF)U 169 deoR r e d l en&/ bz~dRI7(rk-'mk+) supE44 h- thi-l gyrA96 relAb) (Gibco-BRC, Burlington. Ont.) was utilized in the initial cloning of the rpoH gene from I? aerugkraosa. Escherichia codi strain TKY45 (ATOH) (Kogoma and Ybra 1992) was utilized as a recipient in rpoH complementation studies. Bacterial cultures were g o w n in Luria broth (LB; Difco Laboratories, Detroit, Michigan) or tryptic soy broth (TSB; Difco), with shaking at 37°C. LB or TSB plates were prepared by the addition of Bacto agar (Difco) to a concentration of 1.5% (w/v). Ampicillin (Sigma Chemical Co., St. Louis, Mo.) was added to a final concentration of 100 pg/mL to select and maintain BHSa trmsfomants. Cultures were stored in TSB in the presence of 7.7% (v/v) dimethyl sulphoxide in 1.8-mL Nunc Cryovials (Gibco-BRL) at -70°C.

Plasmids pGEM-3Zf(+) (Promega Corporation, Madison, Wis.)? a 3199 base pair (bp) plasmid derived from the pUC vector series, was used in the cloning and DNA sequencing experiments. In E. cokf BH5a, pGEM-PZf(+) is capab%eof a-complemenhtion, allowing visual screening for recombinant transfomants when plated on ampicillin and X-Gal (5-brsmo-4-chlors-3-indoyl- f3-D-galactoside;Gibcs-BRk). Plasmid prpoH, a derivative of pBR322 carrying the E. cokk K12 rpoH gene, was kindly supplied by A. Nolte (University of California, Berkeley, Calif*..).The clone described in this study, p29A#%1, contains the f? a~rugl'lzosarpoH gene inserted into the SwlI site in the multiple cloning site of plasmid pGEM-3Zf(+).

Isolation of DNA CIaromossmal DNA was isisslated using a modification sf M m u r 7 s procedure (Johnson 1981). The purity and concentration of the DNA were determined by spectrophotometry (Sambrook et al. 1989). Plasmid DNA was isolated using a rno&fication (Sambrook et al. 1989) of the alkaline lysis method (Birnboim and Boly 1979). For larger scale (>I50 mL; Maxipreps) isolations of


plasmid DNA the Qiagen plasmid midi kit (Qiagen Inc., Chatsworth, Calif.) was employed.

Cloning the rpsH gene from R aaeruginosa Bseudnmonas ~sruginosugenomic DNA was digested with restriction endonucleases (New England Biolabs, Mississauga, Ont.) following procedures outlined in Sambrook et al. (1989) and according to the nnanufacturer9ssuggestions. Gea~omic digestions were performed at 37°C for 6 h with the addition of enzyme every 2 h. DNA was electrophoresed tlmrough 1% agarose gels, in khe presence of 0.5 pg/mL etl~idium bromide, as outlined by Sambrook et al. (1989). Aker electrophoresis, the DNA was visualized under ultraviolet light, and photographed using Polaroid 667 film (Polaroid Canada, Wexdale, Ont.). DNA fragments sf interest were isolated directly from the gel using a silica-based DNA purification matrix (Prep-A-Gene purification kit, Bio-Bad Laboratories, Mississauga, Owt.). Fragments were ligated into pGEM-3Zf(+) and transformed inlo competent E. co&i DH5a using standard methodologies (Sambrook et al. 1989). Clones were colorimetricalIgr identified on plates containing ampicillin (100 pg/mL) and X-Gal(40 pg/mL). Oligonucleotide probes and hybridization protocols Synthetic oligonucleotide probes for hybridization studies m d for DNA sequencing were screened for hairpin and dimer foranation using Primer 2 (Primer Designer Program; Scientific & Educational Software, State Line, Pa.). They were purchased from the Oligonucleotide Synthesis Laboratory, Core Facility for Prstein/DWA Chemistry at Queen's University. The probes were 5'-radiolabelled using y["'P]A'P (167 TBq/mmol, 370 M B q / d ; Dugont Canada Ins., Markham, Ont.) and T4 polynucleotide kinase (New England Biolabs) following the manufacturers ' protocols. The 1.%-kilobase(kb) EcoRV fragment, containing a portion of the E. coki rpoH gene, was isolated from plasanid psp08, as described above, and nick translated using the nick translation system of Gibco-BWL. The rpoH hybridization studies utilized a rarely employed hybridization technique first reported by Shinnick et al. ( 1 975). DNA was electrophoresed in agarose gels containing ethidiaam bromide (0.5 p,g/mL) as described above and photographed. The gels were then immersed in 0.5 M NaBH, 0.15 M NaCl, with gentle shaking, on a rocker platform (Bellco Biotechnology, Vineland. N.J.) for 30 min, to denature the DNA. After a 30-min neutralization step in 0.5 M Tris-HCl (pH 8),0.15 h4 NaCl at 4"C, gels were placed upside down on a sheet of Whatman 3MM paper (Whatman International, Maidstone, U.R.). A sheet of GelBond film (EMC BioProducts, Rockand, Maine) was overlaid, hydrophilie side down. This was placed in a slab gel drier and subjected to vacuum for 30 min. The temperature was raised stabsequently to 60°C for an additional 40 min. The supported gel was hybridized immediately or stored in a plastic bag (Naczynski and Kropinski 1993). Wemoval from filter paper was hcilitated by soaking the gels in distilled water and gently rubbing away particulate matter vI1ith a gloved finger. Routinely, 2-10 mL of hybridization buffer (6 X SSC (1 >( SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 0.017 M NaH2P04,0.033 M Wa2HPQ4,0,05 % (w/v)

SDS, 50 pg/mL denatured salmon sperm DNA, 5 >( Denhardt9s solution (1% ((w/v) Ficoll 408; 0.1% (w/v) polyvinylpymolidincy 0.1% (w /v) bovine semm albumin)) (Sambrook et al. 19868) were added to the hybridization bag. End-labelled or nick-translated DNA probes were acldeci to the bag and distributed evenly. Hybridization of probe to embedded nucleic acids was allowed to continue overnight. Probe and hybridization solution were removed after hybridizabiom for future use. Gels were initially washed in 6 X SSC. 0,1%1 SSDS at 42°C to remove nonspecifically bound probe. The concentration of salt in the wash was decreased and the temperature raised to increase the st-lngenacy of the wash conditions. Gels, which were sealed in hybridization bags, were exposed to DrtPont Cronex film (Bicker International, Brampton, $141.) at -70°C overnight and developed in the automated developing system (Department of Biochemistay, Queen's University).

DNA sequencing and sequence analysis Fluorescent dye dideoxy chain-terminating DNA sequencing was perfomed by the DNA Sequencing Ltiboratory (Core Facility for Protein/DNA Chemistry, Queen's University) on an Applied Biosystems automated sequencer. The DNA sequence data were analyzed using PC/GENE software (IlnteliGenetics, Inc.. mountain vie^^ Calif.). The sequence was deposited with GenBank under accession number UCB9560. rpoH complementation studies Plasmid p29A# 1 l was electroporated (Gene Pulser, Bio-Wad) into E. coit TKY45. Transformed clones were selected on ampicillin (100 pg/mL) plates that had been incubated at room temperature. Individual colonies of TKY45(p29A#1 I ) and the parental strain were inoculated into 5 mL of TSB and incubated at 20°C h r 48 h. These cultures were serially diluted in TSB and 0.1-mL aliquots were pklated on TSBA ( o p t i c soy broth agar) plates. The plates were incubated at 20, 28, and 37°C. After the colonies had reached sufficient size (1-3 days), they were counted. The results are expressed as efficiency of plating (EOP), that is, the colony-forming units (cfu)/mL, at a given temperature, divided by the cfu/mL at 20°C.

In vitro transcription and translation In vitro synthesis of proteins was accomplished using a modified Zubay cel-free coupled transc~ptian-translati011 system (prokaryotic DNA-directed translation kit, Amersham Life Science, Bakville, Ont.). An E. c-oliMRE 680 (WNaseI-1 S-30 extract (containing WNA polymerase and ribosomes), a supplement solution (containing n~acleotidesfor transcription, tWNAs for translation, an energy generating system, and inorganic salts), amino acids minus methionine, and radiohbelled L-[S"]methionine (Amersham) were i~mcubated together for 30 min at 37°C to allow for synthesis of encoded proteins. A 5-axin chase with unlabelled methionine alloweti the completion of protein chains that may lrave been terminated owing to am insufficient supply of radiolabelled rnethionine. Protein synthesis was terminated by placing the reaction tubes on ice. Two controls vI1ere included: no added DNA, and the vector, pGEM3Zf(+), without insert. Protein samples were denatured by boiling for 5 min in an equal volume of 2 X electrophoretic sample buffer (0.125 M Tris (pH 6.8),


Can. J. Mierobiol. VoI. 41, 1995

2% (w/v) SDS, 20% (wlv) glycerol, 4% 2-mercaptoethanol, 0.002% (w/v) brornophenol blue, 2063 pM phenylmethylsulfonylflusride). Protein samples were concentrated in a polyacrylamide (Bio-Rad) stacking gel (4.5%; w/v) and separated in a resolving gel (10%; w/v). The denaturing gel was electrsphoresed at room temperature and a constant 100 V until the tracking dye entered the resolving gel, at which time the voltage was increased to 125 %/'. Gels were electrophoresed in Laemmli running buffer (0.025 M Tris (pH 8.3), 0.192 M glycine, 0,1% (wlv) SDS) (kaemmli 1970). Protein molecular m a s markers (Boehringer Mmnhein Canada, Laval. Que.) with a range of 170 800 to 39 200 Da were also included on the gel. Following electrophoresis, the gel was stained with Coomassie Brilliant Blue R-250 dye (Bis-Rad) in 50% methanol - 7% acetic acid and destained in 25% ethanol - 8% acetic acid. It was then impregnated with EWWANCE (New England Nuclear, Boston, Mass.) for 1 h at room temperature and the fluor was precipitated?in situ, by soaking the gel in cold water for 1 h. The 1.5-mm gel was then dried on a gel dryer and subsequently exposed to DuPont Cronex film (Picker International) at --70QC for 48 h.

Transcriptional studies Escherichia coIl DH5a (p29A#11) was grown in 50-mE samples of TSB at 30°Cwith shaking to an of 0.5. Two sf the flasks were transferred to water baths at 45 and 50°C, respectivelqh while the other was maintained at 30°C. After 10 min the cells were chilled on ice, and harvested by centrifugation, and the total cellular RWNA was isolated using the TRIzol reagent (Gibco-BRk). A 27-base oligonucleotide (5'-CCTGGAACCAAGGCATGTACAGGTTGC) corresponding to the region around the 5' end of the rpoH gene was labelled with [ s ~ - ~ ~ B ] Aand TP T4 pslynucleotide kinase. Two nanograms of this primer was precipitated with 10 pg of the various WNAs described above, as well as 18 r~lgof tRNA. The pellet was dissolved in 8 FL of water and 2 pL of 5X buffer (50 rnM piperazine-N, NF-bis(2-ethanesulfonicacid (PIPES), pH 6.4, and 2 M NaCl) was added. This mixture was overlaid with mineral oil and heated to $O0C for 5 min then overnight at 60°C. Forty microlitres of a reverse transcriptase mix consisting of 10 mbf Tris-HCI (pH $.8 at 25"C), 58 mM KCZ, 0.1% Triton %-100, I mk1 dNTPs, 30 units RWasin, 5 anM dithiothreitol (DTT), and 50 units MbIuLV reverse transcriptase was then added and incubated at 37OC for 2 h. This reaction was then extracted with phenol-chlorofom and ethanol precipitated. The pellet was resuspended in 10 pL of 80% fomamide - 1X Tris-borateEDTA buffer and heated to 90°C for 3 min, and 2 FL was separated on an 8% sequencing gel. Dideoxysequencing using the same primer and the cloned gene was also carried out 2nd run in parallel with the extension product to locate the specific start sites.

Results Identification of the R aaeragi~osar p d gene using hybridization analysis of genomic digests Hybridization analysis was employed to identify the rpoH gene in P. a~rugilznsaPA8 chmmssomal digests. A 1.2-kb EsoRV fragment containing the E. coCi rp& gene did not hybridize to

the I? a ~ r u g i n o ~DNA a at the stringencies tested. To circumvent this problem a series of synthetic sligonucleotides were made on the basis sf comparisons, at the nucleotide s r amino acid level, of rpsH from Eo ~011'and rpoD from HP aeruginosa (Tables 1-31. The first of these potential probes detects the a-$oDand rpsS genes in aaerugiaigosu (Ronald et al. 1992; Tdndca et al. 1988, 1993; Tanaka and Takahashi 1991). This ~poD-)poS specific probe was based on amino acid homology within the principal sigma factors (rpoD gene products) of E. coIi, P. aeruginosa, and B. s~hfl'lis(Table I). The E. coli WpoS (aM)also contains what Tanaka et al. (1988) refer to as the RpoD (TYATWWIRQA) box sequence. The second probe (4356) was based on consenred nucleotide sequences between the minor heat shock sigma factor gene (rpodd) sf E. coli and the principal sigma factor gene (rpoD) of P. ael-u,ginos&cand in theory should be ~poD-rpoHspecific for aerugil~o~a (Table 2). A third synthetic oligonucleotide (rpoH#2) was designed on the basis of conserved nucleotides between the E. coli r-poH and the HP aeragino~arpoD genes seven bp upstream from the region used in the 4356 probe. Therefore, this ol igonucleotide overlaps the sequence of probe 4356. The two mismatched bases are made to be degenerate in this probe. As a result, probe rpoH#2 is an exact match for the E. coli rpoH gene and the aendginosu r p d gene*The fourth synthetic probe (~poH#3) was designed on the basis of conserved amino acids (DLHQEGN) between E. coCi a" and aceerlaginosa d % n d biased for Pseudomo~aascodon usage (Table 9; West and Iglewski 1988), The results of hybridization with the Tanaka probe and probe 4356 are show11 in Figs. IA and 1B. In each case two bands hyba-idized to the probe. It was reasoned that the common band represented hybridization to the P. wrugi~zosarpoD gene. On the basis of probe design it was proposed that the rpoS and rpoH genes of I? ueruginos&care within the other bands in each digest detected by the Tc~na.&a probe and the 4356 probe, respectively. The probe also hybridized to the E. co%irpoN gene probe (data not shown). Probe rpoH#3 also hybridized to the same two P. ckerugiraosce chromosomal bands in each digest as were detected with probe 4356 (data not shown). Therefore? this probe potentially also detects the rpoH and rpoD genes sf P. aerugilzosa. No hybridization to the E. coli rpoH gene was detected at the stringency employed. It should be noted that this 2 1-bp prsbe had four mismatches for the E. coli rpoH gene at positions 3, 4,9, and 18. Probe rpoH#2 hybridized to E. coli rpoH DNA and to only ael-uginos&cchrsmossmal DNA digest. one band in each This band comesponded to the rpoD gene (data not shown). In summq7, therefore, only three probes (4356, spoN#2, and E. coli g ~ o H ) were capable of hybridizing to the E. cold rpoH gene. Probe 4356 replaces mismatches with inssines and prsbe rpoH#2 is degenerate; therefore, both are exact matches for E. c'oli rp0B-B. It should be recalled that rpoH#3 also hybridized to two bands in aasrugiimsa chromosomal digests but not tc~ the E. coli rpoH gene. On the basis of this analysis, it was inferred that the 4356 probe might well be useful for isolation of the rp0N gene from P. aeruginosw.

Table 1. Alignment of ammo acid sequences in thee principal sigma factors (E. colk a 3 $ (rpoS gene product); E. coEP a 7 0 ( r p d gene product); B. subtilis d ( r p d gene homolog)) demonstrating regions of homology and the resulting synthetic oligonucleotideprobe for the ~poD-rpc15'gene (the Tanaka probe) (Tanaka el al. 1988). h i n o acid or nucleotide sequence


Sigma factor or probe

13'" RGHRFS- TYATWIRQShIEWMhf 42"G~mS-TYATWIRQA-ITRSIA 444 S27 RGYKFS-TYAWWLRQA-ITWSIA448

E. coti a 3 $ E. coEi u 7 0 P. aeruginosa a 7 0 B. subrilis w4 Synthetic probe

~~"SYKKFS-TYATWWIRQA-HTRAIA 203 3'- TGI ATI CGI TGI ACC ACC TAE ICI GTT CG -5'

NOTE: I indicates inosine residues that are capable of forming two hydrogen bonds with adenine, thymine, and cytosine residues. The superscripted numbers associated with the m i n o acid sequence are the residue numbers in the respective proteins.

Table 2. Nucleotide sequence alignment of a portion of the E. coli ~ p o Hgene (Landick et al. 1984) and the aeruginosca rpoD gene (Fanaka and Takahashi 1991) and the resulting synthetic oiigonuclestide probe designed to be specific for the rpsD and vpoN genes of P.aeruginosa (probe 4356).

'

Sene or probe

Nucleotide sequence

""'-

GGA AGG TAA CAT CGG CCT GAT GAA AGC AGT G 270 5'- GGA AGG CAA CAT CGG GCT GAT GAA GGC GGT G lZ6l 3'- CCT TCC ITT GTA GCC GGA CTA CTT ICG ICA C -5'

E. coli rpoH P.auerrlginos~rpoD Synthetic probe 4356

1231

NOTE:Only 3 of 31 base pairs me not identical. These are substituted with inosine (T) residues in the probe. The superscripted numbers associated with the nucleotide sequences refer to the position of the sequence in the respective genes.

Table 3. Other probes utilized in this study. Probe vpoH #2

OSL number

6589

Description and sequence Based on conservd nucleotides between the E. coli rpoH (bases 233-253) and P. aaevugirzosn rpsD (bases 1224 -1244) genes, aapstream from region used in 4354 probe: rpoH 5 '-TGA TTC AGG AAG GTA ACA TCG rpoD 5 '- TGA TCC AGG AAG GCA ACA TCG Synthetic probe 5'-TGA TT/C@AGG AAG GT/CA ACA TCG-3' Based on conserved amino acids between E. calk u32and P.aerteginosa a 7 0 using Pseucfomonas codon usage: 77 BLIQEGN 83 E. coli a32 407 BLIQEGN 413 R aerugii.iosa a 7 0 Synthetic probe 5'-GAC CTG/C ATC CAG GAG/A GGC AAC-3'

No=: T/C, G/C , and G/A indicate that the oligonucleotide is degenerate at that position with two possible nucleotides present.

Cloning the rpsH gene of R aer~gdnosa Bseudomonas aarugitaosa DNA SwB fi~gmentsof approximately 4 kb were cloned into %all-digested pGEM3Zf(+). Approximately '7064 white colonies were pooled into groups of 1% in 5 mL of LB supplemented with ampicillin (100 p,g/mL) m d incubated overnight at 3'7OC. Plasmid DNA was isolated from these culture pools, mn on agaose gels, and screened by hybridization with the 4356 probe. Six pools were found to react with the probe under the appropriate stringencies. Clone 29A#lf was purified from one sf these pools and retained for further study.

Sequence analysis Using the 4356 probe as a primer, an attempt was made to obtain sequence data directly from the insert rather than via subcloning. This method was successful m d the sequencing of clone 29AWl l continued with the use of synthetic primers. The 1025-bp sequence has the following base composition: 21.2 mol% adenine, 16.5 mol% thymine, 31.4 mol% guanine, md 30.9 mol% cytosine. This translates to a G+G content of 62.3 mol% for this region o%Paerugknosa DNA, which is fairly close to the overall base composition of genomic DNA from this bacterium (6'7 m01%). The sequence data have been

80

Can. J. Micrsbiol. Vsl. 41, 1995


Fig. 1. (A) Autoradiograph of I? a e r u ~ k o s achromosomal D N A digests hybridized to the Tanaka probe. PsruckiPrno~zcds aeruginoscr chromosomal DNA was digested to completion with a variety of resthetion eadonucleases. DNA was run in a I % agaose gel at 30 V overnight. This gel was dried and probed with the Emaka probe. The gel was hybridized overnight with the probe at 37OC. The find washes were carried out in 6 X SSC at 52°C. Lane bI displays the position of a few size markers (in kb) derived from StyI-digested X DNA. Lanes 1-8 represent the enzymes Avnl, Avall, BatzHT, P,yfI.Son, S I ~ ~ I , S r d , and Sty%,respectvely The arrows indicate the bands that are in common with bands in B. (B) Autoradiograph of I? aerugino,~nchromosomal DNA digests hybridized to the new (4354) probe. Ps~udonznnascrernginosa chomosomal DNA was digested to completion with a variety of restriction enclonucleases. After electrophoresis this gel wm dried and probed with the 4356 probe. The gel was hykricdized overnight with the probe at 37°C. The final wash was carried out at 59°C in 6 X SSC. The lanes are identical to those in A. The arrows indicate the bands that are in common with bands in A.

submitted to GenBank (accession No. U03560), and is presented in Fig. 2. The 1825 bp of sequence was subjected to a sezrch for direct and inverted repeats using the PCIGENE REPEATS program. Four direct and six indirect repeats of 1 9 bp were identified. While the significance of most of these repeats is not Sunown, one of the inverted repeats bears striking similarity t s a rho-independeast teminatsr. An 8-bp stem (8625°C = -13.6 kcal/mol; 1 cal = 4.184 J), with a five base loop is followed, in the case of the potential rnRNA, by seven uridine residues. A cornpatepized restriction site analysis (PCIGENE RESTRI) was performed on the sequence data. A few enzymes that have unique sites in the putative sequence and in the putative P.aerugknosa ~.psoHgene are indicated in Fig. 3. The restriction sites for SQI are not shown on this figure. The latter restriction endsnuclease generates small fragments that are

presa~rnablylost from the gel, which would explain the lack of 4356 probe hybridization to the putative 1pc1H band in the Stjd cl~omssomaldigests. The nucleotide sequence was translated in both orientations, and in all three reading frames. One of these open reading frdmes (OWFs) could encode a protein of 284 amino acids. This is identical to the sizes of the E. cobi ( T (Landick ~ ~ et al. 1984) and the Citrsbartsr~~oundii a3"roteins (Daggett Gamin and Hardies 5989). This putative 8RH (852 bp) was trimmed of upstream and downstream sequence data and aligned with the nucleotide sequence of the E. r(~bi rp~H gene (852 bp), revealing an identity s f 66.6%. The putative I? aeluginosa rgoN gene was translated and the protein sequence displayed a molecular mass of 32.5 kDa. This compares well with the molecular mass of the E. coli 0-32

Fig. 2. Nucleotide sequence of the P. ~ueruginoscp.genomic fragment carrying the rpoH gene. The initiation (ATG) and termination (TGA) codcanas of the rpoH gene are indicated with a I+ and a B,


respectively. The likely Shine Dalgarno sequence (heavy line), rho-independent terminator (wavy line), transcriptional initiation points (arrows), and putative binding sites for Dna-4 protein (9) EaS"Cbroken line), a d ~u~~( triple line) are illustrated.

protein (32.3 kDa) (Landick et ale 1984). The C. fienndii d2 protein has a mass of 32 500 Da (Daggett Gamin and Hadies 1989). The calculated isoelectric point of the putative P. aerugiailosa ax protein is 6, while those of E. coki and G.fkeundki are 5.5 and 5.71, respectively. Codon usage analysis and the csdsn statistics of the sequence are displayed in 'Fable 4. A f? ueruginosa codon usage table generated from available I? aea-kigtnosa sequence data by West and Hglekvski (1988) revealed a G+C content of nucleotides 1,

2, and 3 of the coding triplets to be 64.8, 43.5 and 8 8 . 7 % ~ ~ respectively. Therefore, P. ma-uginosa, an organism with a high G+C content, prefers G or C in the wobble position. En the case of the r ~ o Hgene (62.7 msl% G+@)sf this bacterium, with the exception of glutamic acid, every ;mino acid displayed csdsn bias to\vards one ending in a G or @. Approximately 83.2% of the time there was a G or C in position 3. These data provide additional evidence that the 8 R F encodes a protein in several pseudomonads.

Can. J. Microbial. VoI. 41, 1995

Table 4. Codon usage analysis of the putative

Table 4. (concluded)

I? aeruginosa rpoH sequence. Amino acid

Codon

No. of residues

Amino acid Tyr

Ala


A%

Asn Asp Cys GIn Glu G~Y

His Ile

Leu

Met Phe

GCT GCC GCA GCG CGT CGC CGA CGG AGA AGA AAT AAC GAT GAC TGT TGC CAA CAG GAA GAG GGT GGC GGA GCG CAT CAC ATT ATC ATA TTA TTG CTT CTC CTA CTG AAA AAG ATG TTT

nc

Ser

Thr

TV

CCT CCC CCA CCG AGT AGC TCT TCC TCA TCG ACT ACC ACA ACG TGG

Val

Codon

No. of residues

TAT TAC GTT GTC GTA GTG T M TAG TGA

NOTE: This sequence wwassubjected to a codon usage statistics analysis (PCIGENE). A G+C content of 62.3 msl% was found for this putative gene. Note that for evzay amino acid (except gltatamic acid) the preferred codon is the one ending in a G or C.

Fig. 3. Restriction map of the 1025-bp fragment cmying the putative P. aeruginssa rpoH gene. A restriction map was generated on the basis of a PCIGENE restriction analysis of the sequence data. A few enzymes tkat have unique sites in the putative P.aerugiiaczsu rpoH gene are indicated. There were nno sites for BctmHL, CluI, EcoRI, HirzdIII, H'aI, KplzI, NarI, NdrI, PstI, Pa~ul,Scefl,Smal, SprI, SphI, XbaI, or XhoI.

This 284 amino acid sequence was aligned (PCIGENE, CLUSTAL) with the protein sequence of the E. coEi sigma 32 protein and the C. freundii sigma 32 protein (Fig. 4). These proteins showed strong homology (59%identity, 33% similarity), which was p a t i c ~ l a l yevident from Leu-56 to Arg-141. Alignment between the E. codi sigma 32 and the putative b" ael-kagirzosa sigma 32 m i n o acid sequences revealed an overall homology of 76.8%. Interestingly, I1 bp upstream from the ATS start site of the rpoH gene is the sequence GGAGGA (Fig. 2). This sequence should provide a good ribosome binding site (Shine D a l g m o box) for the P. avvuginosa rpoH gene (Shine and D a l g m o 1974). As mentioned previously722 bp downstream from the lasf codon exists a sequence tkat resembles a rho-independent transcription ternillation signal.

CsmpEementatiasn analysis To determine if the I? aevuginosa rpoH gene could complement E. coki lpoH functions, p29A#ll was electroporated into an P'P~OH deletion mutant (TKY45). Cultures s f TKY45 and TKY45(p29A#ll) were grown at 20°C overnight, diluted in TSB, and plated on TSBA plates. The plates were incubated at 28,28, and 37°C. The results of this complementation study are

Fig. 4. Multiple sequence alignment (CLUSTAL) of the amino acid sequences of the E. coli a" (ECOLI$IG32), the C. fieuradii u" ((CFHTPR),and the putative $. cdierncginosa KT" (PAHTPRS). Positions in the alignment that are perfectly conserved are indicated by m askbask, and positions that are well camserved are indicated by a period. The 284 amino acid protein sequences showed an overall hsmslsgy of 91.9%. This is due to an identity of 59.3%. PAHTPRS ECOkISIG32 CFHTPR

MTTSLQPVHALVPGWNLEAYVHSVNSPPLLSPEQE~ MTDK~QSL-A~PVGNLDSYI~NAWPMLSADEERWLAEKLHYHGDLEAWKI


ECOLISIG32 CFMTPR

aim* * Q A ~ , a * , , ~ * ,

ECOLISPG32 CFWTPR

**

a****,

. *

LAHLWFWHI~SYSGYGLAQADLIQEGNVGLMKbaVKRFPEMGVRLVSFAVWI 110 ESHLRPWHIWRNYWGYGLPQWDLIQEGNPGE~AVRRFNPEVG~LVSFAVHWI 189 LSH&RFWMVWWMYAGYGEWQWBLIQEGNHGLMKAV~FNPEVG~LVV PO9 *,*itAQ*Q*,*eva.A*A*

PAHTPRS

54

MTKEMQNL-WLWPVGNLESYI~MWWPMLSWDEE~LAEKLHYQGBLE~KTL 54 **,,*a

PAHTPRS

55

k i t * a A a k X * m * t * * * * a a k . A Q * m * * k

KWEIBEFILRNWRIVKVATTKAQRKLFFI9LRSQKKRLAWLEVAELGV 165 KWEIHEYVERNWR%VKVhTTUQRKLFF%kLRKTKQRLGWFPHQ6>E$IE~~GT164 M W E H H E Y V E W N W R % V K V W T T ~ Q R % ( L F F M L R K T K Q R E G W E V 164

* , * * , Q . * ~ ~ + *a~t *~+ ,+ *

*a~a*~~~aftkftka**$*r~ar~a*****a***~

PAHTPRS

PREVREMESRL%GQD~FDP~DADDESWYQSPAHLEDHRDPARQLEDADWSD 220 DMTFDLSSBDBSDSQP~PVLYLQBKSSNFABGEEBDM&dEE 219 D M T F D M S S D D E S B S Q P ~ P V L Y L Q D K S S N F A B G I E E D N D 219

CFHTPW

m . * * * * * * * * a e a Q * * , .*. * * - a m o * o

****,,

*trn

.*

a * m o a * a o

Identity : 1 6 9 ( 5 9 . 3 8 ) Similarity: 9 3 ( 3 2 . 6 % )

Table 5. Complernentatio~~ analysis of an rpoH deletion mutant. Temperature

COc)

E. c-oliTKY45 cfu/mL

EOP

E. colb TKY45(p29A#11) cfu/mL

EOP

NOTE:Escherichia coli strains TKY45 and TKY45(p29A#11) were plated on TSBA plates and incubated at 20,28, and 37°C.

shown in Table 5. We noted that at 20°C there is little diEerence in the number of cfu/mL between the parental strain, TKY45, and strain TKY45(p29A#ll). Since these cultures were inoculated from a single colony it would appear that at 20°C there is no significant difference in their growth rates. However, at 28 and 37°C there are significantly more viable cells in strain TPKY45(p29A#ll) than in the parental strain, TKY45, as indicated by colony formation. This strongly suggests that the gene is transcribed and translated in E. roii. The incomplete suppression of the temperatue sensitivity at 37°C in E. roii TKY45 by plasmid p29A#11 was unexpected.

In vitro transcription-translatio~~studies On the basis of the positive results of the complem~entation analysis:in E. roli. m effort was made to determine if p29A# I 1 could direct the synthesis of the P3. ak~rugiaosac~~~ protein. The

pGEM3Zf(+) plasmid was added to the E. soli cell-free system as an additional control. In addition, a reaction containing no DNA was included as a negative control. The production of newly synthesized proteins from p29A#11 was examined by SDS-polyacrylamide gel electrophoresis (Fig. 5). The difference between the pGEM3Zf(+) vector and p29A#11 is the production of a 32-kDa protein by p29A#1 I . This size agrees well with the predicted molecular mass of 32.5 kBa. The dense band below the putative RgoH polypeptide in lanes 2 and 3 presumably corresponds to processed p-lactarnase (28 kDa). This experiment provides evidence for p29A#11 directed I? aerugino~au42synthesis in E. ecoli.

Transcriptional studies Two potential promoter sequences were visually identified in the 122 bp of upstream nucleotide sequence. A sequence @@9"rGCA(N)17TACACT extending from -59 to - 30 bears strong sequence homology to the Pseudo~a;eoa;ias u7~-dependent promoter consensus sequence sf YSTTGRC(N)17-raYRTAAT (note: Y = C or T; R = A or G; S = G or C) derived by Ronald et al. (1992). Overlapping this sequence, one observes GAACT'T(N)16TCAGA, whicln bears striking homology to the E. solb opB consensus sequence of GAACTT(N)16-17 TCTGA (Delany et al. 1992; Erickson m d Gross 1989; Marow et al. 1991; Raina m d Georgopolous 1990; Raina et a1. 1991). In addition, this upstream sequence contains, as it does in the case of the E. coli rpoH gene, a sequence (TTATACACC) highly reminiscent of a DnaA binding site. The consensus sequence for the BnaA protein binding is TTAT(C/A)@A(C/A)A(Wang

Can. J. Microbial. MI. 41, 1995


Fig. 5. Autoradiogram of prokaryotic DNA-directed protein synthesis in ths in a7it1-o transcription-translation experiment. Lane M displays the position of size makers (in kDa). Lane I represents the negative control (no DNA). Lane 2 represents the reaction that included p29A# 1 1 DNA. Lane 3 represents the pGEMJZf(+) (vector alone) reaction. The gel was dried and exposed to film for 48 h.

and Kaguni 1989bj. This nucleotide motif is located dkectly between the putative -35 promoter regions for the EerSoand Eu2. RNA transcriptional studies employing primer extension were used to determine the transcriptional start sites under control (30°C) and two heat shock regimes (45 and 5UQC),The results are presented in Fig. 6. The major initiation site in E. coIi grown at 30°C occurrecl 23 bp upstream of the translational initiation codon. At 45'C this transcript increased by 2.7 times, while at 50°C9the i17erease was only by 1.7 times. IIPadditic~n,at the latter temperature9the predominant transcript occured from a point six nuclecrtides further upstream, and del~sitometricdeterminantions indicated these transcripts were 3 times more common than those initiated at 30sC.

Discussion Employing a synthetic oligonucleotide probe, based on the consenled sequences between the E. coli t-poH gene P. ~ac.rrlginos.arj70D gene. we have identified the rpoS and

Fig. 6. Primer extension studies with RNA extracted from E. coti DH5a (p29A# l l ) grown at JO°C and exposed to a 10-min heat shock regime at 45 and 50°C. The predicted transcriptional start sites are indicated with arrows.

Temperature

rpoH, together with the rpoD, genes in chromosomal digests of the latter bacterium. Furthemore, the B ~ O Hgene, with associated upstream and downstream regulatory sequences, has been succesfbaBly cloned from this bacterium. Using the 1.2-kb EcoRV fragment of prpoH, containing the E. cnbi rpoH gene, in hybridization studies did not give any infomation regarding the Iscation of the ~ p o H gene in aaeruginosa chromosomal digests. This method has also proved unsuccessful for P. Hoffman (Balhousie University) in his search for the rpoH gene of Legionella spp. (personal cornmarnication).Oligonucleotide probes proved effective. All three of the oligonucleotide probes used in this study are clustered around the nucleotides 23 1-272 of the f? aerugtnosa rpoH gene, which code for the amino acid sequence DLIQEGNVGLMKAV The sequence of DLIQEGN is consea-ved in minor m d major sigma factors and is involved in binding to the core RWApolymerase (Lonetto et al. 1992). It is interesting that the rpoH#3 probe did not bind to the P- ael-ugi~~r?osu 1yoS gene, since the probe is biased for Pseudomoraas csdon usage and this principal sigma factor gene contains the sequence DLIQEGW. The Tanaka probe is based


on the amino acid sequence TYATWWIRQA (the RpoD box), which is approximately 20 amino acids downstream from the sequence DLIQEGN, which is only conserved in the major sigma factors (Lonetto et al. 1992). In summary, two probes (4356 and rpoH#3) we now available that are capable of hybridizing to the v o H gene of I? weruginnso. These have to be used in conjunction with the WpoD box probe of Tanaka et al. (1 988) to identify the unique signal of rpoH. Clone 29A#11 was isolated using probe 4356, and sequencing studies were initiated using this probe as a pri-imea: A region of 1025 bp was sequenced and translated in both orientations and all three OWFs. An ORF of 284 amino acids, the size of the C. fr~landiiand E. coli a%, was detected. The nucleotide sequence was trimmed to the coding region and found to be 66.6% homologous to the coding region of the E. c.c~lirpoH gene. The amino acid sequences of the E, cobi u32 and the putative aaerugincssa &"ere aligned m d found to be 76.8% similar (60.6% identity, m d 16.2% conservative changes). Amino acid sequence alignment of the E. C O / ~d 2 , C. fi-eua;~$iia35 and P. aaeruginosa a3"roteins revealed an overall identity of 59.3%. Analysis of the a 7 0 family of transcriptional Fdctors has shown four regions that are highly conserved (Gribskov and Burgess 1986; Helmmn and Chamberlin 1988; Lonetto et al. 1992; Stragier et aH. 1985). These in turn have been subdivided (Lonetto et al 1992). Region I, which is a consenled domain near the N-terminus of group 1 and group 2 sigma factors is, as expected, poorly conserved in RpoH proteins. With the exception of the IMVREMESR motif, region 3.1 showed overall pow (51 96) identity. Perhaps this motif is associated with binding of the sigma factor to the core (a2Pbi) polymerase. Region 2 showed the highest (289%) identity in subregions 2.2, 2.3, and 2.4, that is between 72Tyrand 83t)Thr. Subregion 2.1, which has been implicated in core polymerase binding, showed only 67% amino acid identity between the three RpoH proteins. On the other hand it contained the following highiy conserved motif: "HLRFVVH. Subregions 2.3 and 2.4 have been implicated in strand separation and recognition of the 10 promoter region, respectively (Daniels et al. 1990; Helmmn rand Chamberlin 1988; Kahn and Ditta 1991; Lonetto et al. 1992; Siegele et al. 1989; Wdburger et al. 19961; Zuber et al. 1989). The recognition of the -35 promoter region is associated with subregion4.2, which shows an overall sequence identity of $1996, but contains a highly conserved 14-mer: """21 to 275Met,Gamier (1990) secondary structure analysis suggests that this region and the adjacent conserved sequence in subregion 4.1 (23%ly-B7Ser) form two helical regions. Lastly, the region between 2.4 and 3.1, that is residues 131-140, is perfectly preserved in the three factors. This area. which has been termed region C (Yura et al. 1993), may mediate association with BnaK, J, and GpE, which in turn may mediate translational repression or stimulate degradation of this unstable protein. Plasmid p29A#11 was electropsrated into an E. colk rpoH deletion mutant (TKY45) to determine if the P. aeruginosa rpoM gene could complement E. coli I ~ Q Hfunctions. No significant difference existed between the growth rrates sf the parental strain, TKY45, and TKY45(p29A#1%)at 20°C. This was expected as rpoH is not required at temperatures ~284°C. However, at 37°C strain TKY45 failed to form colonies whereas the plasmid-containing strain (TKY45(p29A#ll))

displayed colony-forming ability. These complementation studies suggest that the I? aerugino,sn rpoH gene is transcribed and translated in E. soIi and able to replace RpoH functions in E. coli Arp& at 37°C. They are complicated by the observation that at 37°C there are fewer colonies than there are at 28"C, where complementation is 100%. Possibilities to explain this unexpected result include plasmid instability at the higher temperature (Tolker-Nielsen and Boe 1994). In addition, at the elevated temperature, transcription of the gene or its translati011 may be affected, resulting in reduced or no induction of essential HSPs. In vitro transcription-translation studies provided further evidence for the expression of the P. caerugikaosce rpsH gene. A protein of 32 kDa was synthesized in the presence of p29A# 11 that was not seen in the presence of vector alone. One may argue that this protein is not P. a e r u g i n s s ~a3Qut rather represents expression of mother gene located on the insert DNA. M i l e this is possible, the evidence of complementation of an E. coli rpoH mutant, the presence of enough upstream sequence to accommodate a promoter sequence, m d the agreement in protein size argue against this hypothesis. Using caerugknosa RNA polyclonal antibodies against purified polymerase, Allan et al. (1988) found proteins of 40 and 38.5 kDa that copurified with RNA polymerase from heat-shocked cells. It was postulated that one of these proteins could be the P. neruginosa heat shock sigma factor. This appears not be the case, since the predicted m d observed molecular weights of the I? aeruginosca a3"rotein are 32 567 m d 32 800, respectively. Transcriptional studies, in E. coli, have indicated at least three start sites, two of which are associated with sequences that bear striking homology to promoters recognized by Ea70 and Ea2f also known as aE (Eriekson and Gross 1989; E ~ c k s o net aI. 1987). These promoters are analogous to the P4 and P3 promoters of the E. coki rpoH gene (Yura et al. 1993). a24plays a role in transcription of this gene at lethal temperatures (>4S0C) (Erickson and Gross 1989; Erickson et al. 1987; Wmg and Kaguni 1989ce), and these studies suggest that a homolog should be present in I? caeruginosa. Attempts to identify a CWP-dependent site in the P. aeruginosa upstream sequence were not found, as expected, since this protein is not ink701vedin regulation in this bacterium. Lastly, as occurs in the upstream region s f the rpoH gene of E. coli (Wmg and Kaguni 1989b; Yura et al. 8993), we have found sequence evidence to suggest that DnaA protein may be involved in modulating transcription. The nucleotide and amino acid sequence results, the complementation analysis, and the prokaryotic DNA-directed translation studies support the conclusion that the rpoH gene of P.caerugiraosa has been cloned and is able to express the ~ protein in E. coli. These studies will pemit us to study the transcriptional regulation of the heat shock regulon in P. aeruginosa. We have recently cloned the rnspBA operon (GroES, GroEL) from P.~erugiaaosawith associated promoters (Went 1994). The availability of cloned and expressed RpoH will pemit the in vitro transcriptional studies of this m d other heat shock loci from aeruginosa.

3

~

Can. J. Microbial. VoB. 41, 1995

Acknowledgments


This work was supported by a grant to A.M.K. from the Natural Sciences and Engineering Research Council sf Canada. G.M. is a Research Scientist of the Natisnd Cancer institute of Canada. The E. c-oli A(rpc~H)strain was kindly donated by Dr. T. Kogoma.

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