Analysis of the rpoD gene encoding the principal sigma factor of Pseudomonas putida

Gene. 167 (1995)93-98 © 1995 Elsevier,¢~cienceB,¥. All rightsreserved,0378-1119/95/509.50

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GENE 09379

Analysis of the rpoD gene encoding the principal sigma factor of P~eu:lo1:wnas putida (RNA polymerase; heat-shock response)

Masaya Fujita a, Yoshihiko Hanaura b and Axinori Amemura b aRadioisotope Center. National Institute of Genetics. Mishhna, Shizuoka 411. Japan:and bDepartmentof Biotechuolog3; Facultyof Engineering, Fukuyama Unirersit3;Fukuyama, Hiroshima729-02, Japan. Tel. (81-849) 362-I11. Received by A. Nakazawa:2 June 1995;Revised/Accepted:7 August1995;Receivedat publishers:21 September1995

SUMMARY The gene (rpoD) encoding the principal o factor of Pseadomonas putida (Pp) was cloned and sequenced. The aminoacid sequence deduced from the nucleotide sequence of rpoD contained sequences with significant similarity to th~ conserved region of the principal o factors. In vivo transcriptional analyses revezled that the Pp rpoD is transcribed as a monocistronic mRNA of 2.1 kl:. and that the transcription of rpoD is under control of the heat-shock (HS) response. The transcription start point (tsp) of the gene was determined and found to be different depending on either .normal growth (at 30°C) or HS (at 42°C) conditions. The possible promoter sequences for the principal (o 70) and the HS RNA polymerase of Pseudomonas were located in the upstream region of the tsp.

INTRODUCTION The Pseudomonas bacteria are found in large numbers in soil and aqueous environments, and metabolize various natural and synthetic compounds such as aliphatic and aromatic hydrocarbons. Their metabolic capacity is directed by gene clusters encoding catabolic enzymes (Silver et al., 1990). To survive and propagate, Pseudomonas spp. use a variety of compounds existing at low concentrations in their environment. Correspondence to: Dr. M. Fujita, Radioisotope Center, National Institute of Genetics, Mishima, Shizuoka 41I, Japan, Tel. and Fax (81-559) 81-6870; e-mail: [email protected] Abbreviations: an, amino acid[s);bp. base pair(s); Bs, Bacillus subtilis; dNTP, decxyribunucleosidetriphosphate;Ec, Escherichiacolt; kb, kilobase(s) or 1000bp; HS. heat shock; LB, Luria-Bertani (medium); M-MLV,Moloneymurineleukemiavirus;nt, nudeotideis);ORF,open reading frame;Pa, P. aeruginosa; PAGE,polyacrylamide-gelelcetrophorests; Pc and Pus, promoters;Pp, P. putida; RNAP, RNA polymerase; rpoD, geneencodingprincipal o factor; Rp D, gene product of rpoD; RT, reversetranseription(ase);So, Streptom)~es coelicolor, SD. ShineDal/~arno (sequence);SDS, sodium dodecyl sulfate; tsp, transcription star. point(s);UTR, untranslated region(s). SSDI 0378-1119(95100675-3

Transcription in bacteria is regulated at the level of functional RNAP and active promoters. Global switches in transcription pattern are known to be attributable mainly to modulation of promoter selectivity of multiple RNAP holoenzymes with a common core enzyme and several different 6 factors (Losick and Peru, 1981; McClure, 1985; Reznikoff et al., 1985). Multiple species of 6 subunits have been demonstrated in Escherichia coli (Ec) (Helmann and Chamberlin, 1988), Bacillus subtilis (Bs) (Errington, 1993), Streptomyces coelicolor (Sc) (Westpheling et al., 1985), P. aeruginosa (Pa) (Tanaka and Takahashi, 1991; 1994) and P. putida (Pp) (Inouye et al., 1989). Recently, accumulation of sequence data of the genes encoding o factors has revealed that the o factors fall into two broad classes: one family is similar to the o 7° subunit of Ec (Lonetto et al., 1992), the other is similar to the o s4 (NtrA, RpoN) subunit of Ec (Kustu et al., 1989). Analogs of these o factors actually exist in Pseudomonas spp. and play an important role to survive and propagate in various culture conditions (Inouye eta!., i989; Totten et al., 1990). Especially Pp utilizes

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many aromatic compounds as carbon and energy sources, and some functions depend on enzymes encoded by plasmids. Pp PpGI 1ATCC17453) was originally isolated by enrichment culture with o-camphor as the sole carbon source (Bradshow et al., 1959). Recently, the mechanism of,regulation of genes encoding enzymes involved in the pathway for catabolism of o-camphor has been characterized (Aramaki et al., 1993). Their expression is controlled in part by the principal ~ factor (670) of Pp. Moreover, we have purified the RNAP containing 07o from Pp and characterized biochemical properties and transcription specificities (Fujita and Amemura. 1992a,b; Fujita et al., 1993a). However, the primary structure of 07o has not yet been reported. in this study, we report the cloning, sequencing and

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(a) Cloning and sequence analysis of the Pp rpoD gene We used the 0.8-kb Sphl fragment obtained from pASB3 containing Pa rpoD as a probe to isolate two rpoD

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Fig. 2. The nt .sequence of rpoD and its deduced aa sequence. The numbering begins at the 5' end of nt. The tsp of Pc and P,s mRNAs arc indicated. Sequences that are homologous to the consensus sequence of promoters recognized ~7o and ~ n RNAP are boxed. The SD sequence is underlined. The aa sequence of RpoD is shown in single letters, The numbering begins at the start codon. Transcription stop is indicated by the vertical arrow. The horizontal arrows represent palindromic sequences. For the nt sequence analysis, the recombinant plasmid DNA was sequenced by the dideoxy chain-termination method, using the BcaBESTTM dit :oxy sequencing kit from Takara Shuzo after subeloning and:or creating deletions of 3.2-kb Sall-Kpnl fragment IFig. 1). The nt sequence data was deposited at DDBJ. EMBL and GenBank under accession number D3~45. The proposed conserved regions are shown below the aa sequences.

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Fig. 3. Cell growth and transcription of rpoD.(A) Pp PpGI was grown at 30C (lanes 1-5) and at 37C (lanes 6 10L The cells were harvested and total RNAs were extracted at the indicated times after temperature upshift by the hot-pbenol method as described previously (Fujita et al., 1993a). S1 nuclease mapping experiments were carried out as described previously (Fujita et aL 1993aLFor preparation of probe 1 [ Fig. I L pUCI9 carrying the 5' portion of rpoD was digested with EcuRV, and the 5' ends of the resulting DNA were labeled with [T)ZP]ATP by using T4 polynucleotide kinase after dephosphorylation with bacterial alkaline phosphatase. Then, the end-labeled DNA was digested with Sail. the produced fragments were separated by 5% PAGE, and the band corresponding to the 1344-bp labeled SalI-EcoRV fragment was cut out from the gel and eluted. Probe I was used for 5'-ends mapping of mRNA. The reaction products were analyzed by 8 M urea-8% PAGE and the gel was exposed to X-ray film. The positions of the fragments protected by Pc and Pus mRNAs are marked. (B) The cells were grown at 30"C (lanes 1-5) and at 42'C (lanes 6-10L The other conditions are the same as for panel A. (C) Growth curve of Pp PpGI in LB medium. The cells were cultured at 30 C until mid.log phase, as indicated by the arrowhead. Then. the culture was split and one half was cultured at 30:C as a control and the other was cultured at 37 or 42~C. clones from o u r XDASHII library. Restriction analysis indicated t h a t two of the isolates h a d the same insert. S o u t h e r n analysis of c h r o m o s o m a l D N A with the 5'- a n d 3"-UTR of Pa rpoD as a p r o b e showed t h a t the Pp g e n o m e in this p h a g e h a d the complete region of rpoD (data not shown). The nt sequence of the 3.2-kb Sall-KpnI fragment containing rpoD was determined b y subcloning a p p r o p r i a t e fragments from the ~.DASHII clone a n d m a k i n g a series of deletion derivatives c o n s t r u c t e d by the exonuclease III digestion method. The restriction m a p a n d the o r g a n i z a tion of rpoD are s h o w n in Fig. 1. The determined nt sequence of 3208 b p a n d the deduced a a sequence are s h o w n in Fig. 2. "fhe sequence c o n t a i n s a n O R F of 1842 b p starting from nt 1069 (ATG) a n d t e r m i n a t i n g at nt 2911 ( T G A ) t h a t encodes a protein of 614 a a corres p b n d i n g to 67°. A 69 567-Da protein could be translated from this O R F . The sequence 5 ' - G G A T A G G G , found

5 b p u p s t r e a m of the fitst nt of the start codon, is p r e s u m ably the SD sequence. The p r i m a r y structure of R p o D of Pp was c o m p a r e d with those of Pa ( T a n a k a a n d Takahashi, 1991) a n d Ec ( B u r t o n et al., 1981 ). C o m p a r i s o n of Pp R p o D sequence with Pa a n d Ec R p o D sequences shows 75 a n d 6 9 % identity, respectively (data not shown). The conserved regions in principal cr factors are f o u n d in Pp o 7° (Fig. 2). (b) H S response of rpoD transcription In Ec a n d Pa, transcription of rpoD is u n d e r c o n t r o l of H S response. Therefore, we tested the synthesis of rpoD m R N A in cultures of Pp at various t e m p e r a t u r e s by SI nuclease m a p p i n g using p r o b e 1 (Fig. 1 ). W h e n Pp was cultured at 30°C, a protected D N A c o r r e s p o n d i n g to a n R N A transcript (Pc m R N A ) was identified (Fig. 3A, lanes 1-5). W h e n cultures were shifted from 30 to 37°C, a n o t h e r m R N A (PHs m R N A ) increased (Fig. 3A, lanes 7

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Fig. 4. 5'- and Y-end mapping, and Northern analysis of the rpoD mRNA. Primer extension experiment was done to determine the 5' end of rpoD mRNA. (A) The 5' ends of Pc' mRNA. B) The 5' end of Pus mRNA. RNAs were extracted from cells at 30 C (lane 1) and I0 min after a HS from 30 to 4TC ( lane 2). Lanes G, A, T and C are the products of the dideoxy sequencing reaction with synthetic primer as markers. The primer-extension products are indicated by the arrowheads. The oligodeoxyribonucleotides used for primer extension and sequencing reactions had the following sequences: 5'-ACGAGACTGCTGTTGCGCITI~CCGAACATIrpoD-Pc), 5'-TGACGTGATTTCCTGAGCAG(rpoD-Pus). For the RT reaction, 4 p.l of 5 x RT buffer (250 mM Tris. HCI pH 8.3/375 mM KCFI5 mM MgCIz/50 mM dithiothreitol), 2 Ill of 3ZP-5'-end labeled primer (2 pmol) and 50 lag of RNA were combined in a total volume of 13 ttl. The mixture was incubated for 1 h at 8 0 C and then at 4 2 C for 1.5 h. Then, 5 lal of 2 mM dNTPs/2 pl of M-MLV-RT (200 units, US Biochemical) were added to the above mixture, and the reaction was continued another 1 h at 37°C, The RNA-primer extended hybrid was phenol-chloroform extracted, ethanol nreeipitated :mr ~'~!yzed by S ~?. urea-g% FAGE (sequencing gel). The lengths of the primer extension products were calculated by running products of the sequencing reaction with the same primer on the same gel. (C) Determination of the 3"-end of rpoD mRNA by SI nuclease mapping. For preparation of probe 2 1Fig. I ) pUCI9, carrying the 3' portion of rpoD, was digested with Psrl+Kpnl, the produced fragments were separated b3 5% PAGE. and the band cori'esponding to the 726-bp Pstl-Kpnl fragment was cut out from the gel and elnted. Then, the purified DNA was digested with Sau3Al. and the 630-bp Sau3AI-Kpnl fragment was purifed. The pnrified 630-bp Sau3A1-Kpnl fragment was the 3'.end labeled with [a-32P]dCTP by using Pollk. Resulting DNA was labeled at the 3' end of Sau3Al site, specifically since this site is 5' protruding (the Kpnl site is 3' protruding). Probe 2 was hybrid zed to RNA (40 fag)extracted from cells at 30:C (lane I ) and 10 rain after a HS from 30 to 42 C (lane 2). Lanes G + A and C + T are the products of chemical cleavage of the probe. The ir~sition of protected DNA is indicated by the arrow head. For high-resolution analysis, the probes were cleaved chemically and run with the products of SI nuclease mapping using the same probe on the ~:ame sequencing gel as described above. (D) Identification of the transcript of rpoD by Northern hybrid3zation. Samples of RNA from cells at 30C Ilanes: I, 5 pg; 2. 10 ttg: 3. 20 pgl and at 4 2 C for 5 rain (lanes: 4, 5 lag; 5, 10 lag: 6, 20 lag) were ¢lectrophoresed on a 2.2 M formaldehyde-I% agarose gel in a solution of 20raM 3qN-morpholinojpropanesulfonic acid (MOPS) pH 7.0/8 mM Na.aeetate/l mM EDTA pH 8.0 and transferred to a nylon membrane. GeneScreen Plus (NEN Research Products. Boston. MA. USA). Left panel is photographs before autoradiography. Right panel shows hybridization with the probe (451-bp Psrl fragment located the middle of rpoD). The arrow on the right indicates the position of the rpoD mRNA. The positions of rRNAs are indicated on :he left. Probe was obtained by labeling a purified 451-bp Pstl fragment located the middle part of the rpoD with [~t-32p]dCTP by using a BcaBESTTM random prime labeling kit from Takara Shuzo.

97 and 81. Pus m R N A appeared transiently during the early period after a temperature shift up to 37°C, but at later period (2ff 60 min), PHs m R N A disappeared, reflecting adaptation of the HS response (Fig. 3A). When the culture was shifted from 30 to 42°C, P , s m R N A was synthesized continuously and Pc m R N A disappeared (Fig. 3B, lanes 6-101. As shown in Fig. 3C, cell growth was inhibited by a shift to 42°C but not to 37:C. These results ir~dieate that the HS response at 42°C does not enter the adaptation phase as observed at 37~C.

(c) 5'- and 3'-ends mapping and Northern analysi~ of the rpoD m R N A The 5" ends of the rpoD transcripts were mapped by primer extension. As shown in Fig. 4A, Pc m R N A had at least two 5' ends as indicated by arrows. Pc m R N A was equally abundant under both the low (30'C) and high (42°C) temperature growth conditions. O n the other hand, a primer-extended band from Pns m R N A was detected only at the high (42:C) growth temperature (Fig. 4B). The putative - 10 and - 3 5 regions, preceding the 5' ends of mRNAs, resembled the regions of the promoter sequences of Pa rpoD gene. The sequences C T G G C G and TATAAT were found as - 3 5 and - 1 0 promoter sequences recognized by P ~7°-RNAP (Fujita et al., 1993b). For Pns, the sequences C C T T G A A and CACTATA, preceding the 5' end of PHs mRNA, were found as promoter sequences recognized by P HS cr-RNAP (Fujita et al., 1993b}. The spacings between the - 3 5 and - 10 sequences of Pc and PNs promoters were 19 and 14 bp, respectively. These sequences and spacings (Fig. 2) showed good homology to those of Pa rpoD. The 3' end of the rpoD transcript was determined by S1 nuclease mapping. The 3'-end-labeled probe 2 (Fig. 1 ) was hybridized with total RNA extracted from cells cultured at 30":C and shifted to 42~C for 5 min, and the probe was treated with SI nuclease. The gelclcctropho~esis pattern showed that the 3' end of tToD m R N A was located 10 bp downstream of the stop codon. not only at 3 0 C but also under the HS condition (Fig. 4C). The 3' end of rpoD m R N A was followed by T-stretches. This region contains a self-complementary structure for a potential p-independent termination signal for rpoD m R N A synthesis (Fig. 2). Thus, we conclude that this self-complementary structure is actually a terminator for synthesis of rpoD mRNA. In order to determine the transcriptional unit of the rpoD gene, total RNA was separated by electrophoresis on an agarose gel, blotted onto a filter and probed with a-'P-labeled D N A by Northern analysis. Total RNAs were prepared from Pp cells grown under low (30°C) and high (42°C) temperatures. A band corresponding to an RNA transcript of approx. 2.1 kb was observed in all the lanes

(Fig. 4D). Since the coding region of rpoD is 1842 bp, the gene appeared to be monocistronic. Taken together these results, in Pp, rpoD was transcribed from its own promoter (Pc or Pus), extended to the rpoD terminator, giving a m R N A of about 2.1 k b in size.

(d) Conclusions (1) The rpoD gene of Pp P p G I , encoding a 70-kDa protein, has been cloned and sequenced. The conserved aa regions in o 7° are found in Pp o v°. (2) The rpoD of Pp is transcribed as a monocistronic m R N A of 2.1 kb and the transcription of rpoD is under the cop, trol of HS response. (3) The promoter sequences of Pp rpoD for the principal and the HS R N A P were located in the upstream region of the tsp.

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