School of Health Science, Griffith University, PMB 50 Gold Coast Mail Centre, Gold Coast, Qld 4217, Australia1
Author for correspondence: Ifor R. Beacham. Tel: +61 7 5594 8087. Fax: +61 7 5594 8908. e-mail: i.beacham{at}mailbox.gu.edu.au
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ABSTRACT |
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Keywords: protease, Pseudomonas fluorescens, ECF sigma factor, transmembrane activator, temperature regulation
The GenBank accession numbers for the sequences reported in this paper are AF228766 and AF228767.
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INTRODUCTION |
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The regulation by temperature of protease and lipase production in psychrotrophic strains of P. fluorescens has been previously documented. Lipase production is increased at temperatures below the optimum growth temperature (Andersson, 1980 ). In contrast, protease production is reduced by about 50% at 4 °C above the optimum growth temperature of 27 °C, at which the generation time is reduced by 26% (R. G. Woods and others, unpublished data), and is almost completely inhibited at 32 °C (McKellar & Cholette, 1987
). However, little is known about the regulatory mechanisms involved in the determination of protease production at different temperatures. In this report, we show that the production of protease by P. fluorescens LS107d2 at 29 °C, slightly above the optimal growth temperature of 25 °C, is dependent on PrtR, which we propose is a novel member of a group of anti-sigma factors and transmembrane activators which interact with ECF (extracytoplasmic function) sigma factors of the
70 family. ECF sigma factors are necessary for the transcription of genes which encode products with extracytoplasmic functions and are usually encoded in an operon which includes the gene encoding the anti-sigma factor or activator (Missiakas & Raina, 1998
; Hughes & Mathee, 1998
). Our data suggest that PrtR is a transmembrane activator, rather than an anti-sigma factor, and that prtR is translationally coupled to an upstream gene, prtI, which encodes an ECF sigma factor.
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METHODS |
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Recombinant DNA techniques and nucleotide sequence analysis.
Restriction enzyme digests, ligation of DNA fragments, end-filling with Klenow polymerase, agarose electrophoresis, transformation of E. coli and Southern blot analysis were performed as described by Sambrook et al. (1989) . Probes were labelled by nick translation using [
-32P]dCTP. Plasmid DNA was prepared using alkaline lysis (Sambrook et al., 1989
) or a column (Qiagen) and genomic DNA was prepared using the cetyltrimethylammonium bromide method (Wilson, 1987
). DNA sequencing was accomplished using an Applied Biosystems model 377 sequencer. Database comparisons of nucleotide sequences, following translation in six frames, utilized the BLAST program (Altschul et al., 1997
; Gish & States, 1993
).
Library construction and screening.
Genomic libraries from P. fluorescens strains LS107d2 and B52 were made in Zap Express (Stratagene) using DNA partially digested with Sau3A. Plaques were screened, following transfer to a positively charged nylon membrane (Bio-Rad), using a probe derived by inverse PCR (see below). From these
clones, plasmid clones were excised to give p62A and p53 containing the prtIR region from LS107d2 and B52, respectively (see Fig. 1
).
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Transposon mutagenesis.
Random transposon mutagenesis was accomplished by conjugal transfer of pUTmini-Tn5 Km from S17.1pir to P. fluorescens LS107d2 (de Lorenzo et al., 1990
; Herrero et al., 1990
). Protease-negative mutants were detected following screening of exconjugants on media containing 2·5% skimmed milk.
Inverse PCR.
Inverse PCR, used to identify DNA sequence flanking the transposon insertion into prtR, was accomplished using an adaptation of the method of Rich & Willis (1990) . Genomic DNA from the mutant of interest was cut with EagI and religated with T4 DNA ligase. PCR using divergent primers (AAGGCGAATCACCAAGGTAGTCGGCA and GATAGCTAGACTGGGCGGTTTTATGG) within the mini-Tn5Km cassette (de Lorenzo et al., 1990
) was performed with the following PCR program for 35 cycles: 60 s at 95 °C, 30 s at 50 °C and 90 s at 74 °C. The resulting PCR product was gel-extracted, sequenced and compared to sequences in databases.
Chromosomal insertional mutagenesis.
To construct a prtI insertion mutant, a 300 bp SalIXhoI internal prtI fragment from p53 was purified and cloned into the SalI site of the suicide vector pJP5603 (Penfold & Pemberton, 1992 ), which carries the kan gene, to give pJP5603-1. Following transformation into JM109
pir, the construct was conjugated into P. fluorescens LS107d2 using triparental mating (Herrero et al., 1990
). Kanamycin-resistant exconjugants arising from a single crossover event were isolated on Luria broth media containing kanamycin and then screened on 2·5% skimmed milk media for protease activity at 29 and 23 °C. Colonies which were AprX- at 29 °C but not at 23 °C were presumed to be prtI::kan and were confirmed by Southern blot analysis.
Complementation analysis.
To confirm that prtI and prtR were required for protease expression at 29 °C, DNA fragments from p53 containing either prtI, prtR or the entire prtIR region were cloned into the broad-host-range cloning vector pBBR1MCS (Kovach et al., 1995 ), to give pBBR4, pBBR14 and pBBR53, respectively. The DNA fragments, derived from p53, were a 2·4 kb PstIKpnI fragment containing prtIR; a 1·4 kb XhoIKpnI fragment containing prtR; and a 1·1 kb PstIBclI fragment containing prtI (see Fig. 1
). The three resulting pBBR1MCS derivatives were each transformed into TG1 and conjugated into the P. fluorescens LS107d2 target strains by triparental mating. The phenotypes of the exconjugants were tested for protease activity on 2·5% skimmed milk agar plates.
Transcription mapping.
The transcriptional start point of aprX was mapped using S1 nuclease protection. A 456 bp DNA fragment was derived by PCR from plasmid pLS51 (see above) using primers LS186 (3'-CTGCCAACTAGTGAAACTT-5') and PRTMb270 (3'-AAGACGACCCGCAACTTGAC-5'), which bind at positions -186 to -167, and 250 to 270, respectively (where position 1 is the aprX ATG start codon). A 32P-labelled single-stranded antisense probe using 150 pmol primer prtmS1 (3'-CATACTGGCACCGCCGTTGGA-5' nt 82103) was used in a linear amplification with 1 µg of the above purified PCR product as template. The PCR reaction, containing 62 µmol each of dATP, dTTP and dGTP, 18 µmol dCTP, 3 µl [-32P]dCTP (1014 Bq mmol-1; 370 Bq µl-1) and 2·5 units Taq DNA polymerase (Promega), was cycled under the following conditions: 96 °C for 40 s, 50 °C for 40 s and 72 °C for 90 s for a total of 40 cycles. The probe was purified by excision from a 6% denaturing polyacrylamide gel following identification by autoradiography. The probe was eluted from the polyacrylamide slice by soaking in 300 µl elution buffer (0·5 M ammonium acetate, 1 mM EDTA and 0·2% SDS) overnight at 37 °C, ethanol-precipitated, resuspended in 100 µl diethyl-pyrocarbonate-treated water and stored at -20 °C until required. Total RNA (5 µg), purified as described by Aiba et al. (1981)
, was mixed with the purified 32P-labelled probe, ethanol-precipitated and dissolved in 10 µl hybridization buffer (80% deionized formamide, 100 mM sodium citrate pH 6·4, 300 mM sodium acetate pH 6·4, 1 mM EDTA). The DNARNA mix was denatured at 85 °C for 10 min and then allowed to hybridize at 37 °C for 3 h. The reaction was then made up to 200 µl with 1x S1 nuclease buffer (Promega), 50 units S1 nuclease were added and incubated at 37 °C for 3 h. The digested products were ethanol-precipitated, resuspended in 8 µl gel loading buffer (95% formamide, 20 mM EDTA, 0·05% bromophenol blue and 0·05% xylene cyanole FF), and 2 µl was electrophoresed on a 6% denaturing polyacrylamide gel alongside dideoxy sequencing reactions. The latter were derived using modified T7 polymerase (United States Biochemical) and labelled with [
-32P]dCTP. The gel was dried and visualized by autoradiography.
Protease assays.
Due to the relatively low levels of protease produced by strain LS107d2, a well assay was used (Lawrence et al., 1967 ): wells were cut into 2·5% skimmed milk agar plates using a 4 mm cork borer, 40 µl supernatant from liquid cultures was placed into each well and the plates were incubated at 37 °C for 25 h. Supernatant from a parallel culture of P. fluorescens B52 was assayed using both the azocasein method (Wassif et al., 1995
) and the well assay in order to generate a standard curve of protease activity in units ml-1 versus the diameter (in mm) of the hydrolysed skimmed milk halo produced around the wells; protease activity for the LS107d2 cultures in equivalent units ml-1 for the azocasein assay (defined as
A450 min-1) was then calculated from the standard curve.
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RESULTS AND DISCUSSION |
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Sequence analysis of the region from LS107d2 and B52 revealed a dicistronic operon, which we designated prtIR (Fig. 1). The first gene clearly encodes an ECF sigma factor related protein (PrtI), with algU (Martin et al., 1993
) and sigE (Martin et al., 1994
) being its closest homologues, with 43% and 38% identity, respectively. Further, PrtI acts at the level of transcription since, in contrast to the wild-type strain (see below), no aprX transcript is detectable in a prtI mutant strain (prtI::kan; see Methods) at 29 °C by S1 nuclease analysis (data not shown). The second gene encodes a protein with no substantial amino acid sequence similarity to other sequences in databases. However, ECF sigma factors are all cotranscribed with their cognate regulators, a group of membrane-associated anti-sigma factors and transmembrane transcriptional activators (Hughes & Mathee, 1998
), suggesting that PrtR might be such a protein. In agreement with this suggestion, the hydrophobicity profile of PrtR (Fig. 2
) shows a centrally placed single membrane-spanning domain typical of these regulators (Mathee et al., 1997
; Hughes & Mathee, 1998
). Furthermore, the stop codon of prtI and the start codon of prtR overlap, indicating the existence of translational coupling. Evidence from the CarQRS system suggests that this facilitates interaction between the C-terminal end of the sigma factor (CarQ) and the N-terminal end of anti-sigma factor (CarR), allowing both to be inserted into the inner membrane concurrently upon the completion of translation (Gorham et al., 1996
). Thus although we have not demonstrated directly that PrtR is membrane-associated, the evidence strongly suggests that it is a transmembrane regulator.
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Interestingly, the amino acid sequence of PrtR shows no significant similarity to the two other transmembrane activators currently reported, as foreshadowed by the initial sequence analysis of the region flanking the transposon insertion (see above): amino acid sequence alignment of PrtR with FecR (Van Hove et al., 1990 ) and PupR (Chi & Bartlett, 1995
) using CLUSTAL (Thompson et al., 1994
) showed that PrtR is only 18% identical to PupR and 18·5% to FecR (data not shown), although FecR and PupR are 38% identical to each other.
The corresponding genes from P. fluorescens strain B52 encode proteins which are 89% identical (PrtI) and 87% (PrtR) identical to those from LS107d2, and also show translational coupling (see Fig. 1).
Insertional mutagenesis of prtIR and complementation analysis
In order to determine the function of prtI in the expression of protease activity, a chromosomal mutant of LS107d2 was constructed (prtI::kan; see Methods). As in the case of the prtR::Tn mutant, the prtI::kan mutant is protease-deficient at 29 °C but not at 23 °C (Fig. 3). We have determined whether this mutation is complemented by prtI and/or prtR following transfer of multicopy plasmids containing each of these genes and testing for protease production at 29 °C either phenotypically or by assay of culture supernatants. The results indicate that prtI::kan is complemented by prtI and that prtR::Tn is complemented by prtR (Figs 3
and 4
), as expected if both are required for a protease-positive phenotype at 29 °C. It would be expected that the prtI::kan mutation would have a polar effect on prtR expression and hence not be complemented by prtI alone; the fact that it is complemented by prtI alone indicates that expression of prtR, in the prtI::kan mutant strain, is occurring from a promoter within pJP5603-1 since the entire construct is integrated into the chromosome as a result of a single recombinational event (see Methods).
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We conclude that both PrtI and PrtR are essential for protease production at 29 °C, and further suggest that PrtR can interact with other sigma factors (when in excess of normal levels) whereas PrtI specifically requires active PrtR to form a functional RNA polymerase at 29 °C.
The aprX promoter
In view of the involvement of PrtI in expression of aprX at 29 °C, we sought to determine whether the aprX promoter possesses sequence features in common with other ECF promoters. We have therefore determined the start site of transcription in strain LS107d2 using S1 nuclease analysis (see Methods and Fig. 5).
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In conclusion, this work strongly suggests that prtI and prtR encode a sigma factor and a transmembrane activator, respectively, and that both are required for protease expression at 29 °C. The requirement for such a transmembrane signalling system and an alternative sigma factor at 29 °C is consistent with a stress response to higher temperature. The nature of the signal that is presumably transduced by PrtR is not known; it may be temperature directly, perhaps inducing a conformational change in PrtR, or it may be unfolded periplasmic or outer-membrane proteins, even at the relatively moderate temperature of 29 °C; some precedent for the latter notion is the ECF sigma factor encoded by rpoE in E. coli, whose transcription is increased by misfolded periplasmic and outer-membrane proteins (see Hughes & Mathee, 1998 ; Missiakas & Raina, 1998
; Pogliano et al., 1997
). Support for this suggestion comes from the analysis of a transposon mutant of LS107d2 with a very similar phenotype to LS107d2prtR::Tn (unpublished data). This mutant has an insertion in a gene encoding the regulator component of a two-component regulatory system with amino acid sequence similarity to CpxR which, like RpoE, responds to misfolded proteins in the E. coli model (Pogliano et al., 1997
).
A related question is why the target promoter is inactive at 29 °C. It is possible that the relevant sigma factor is rendered inactive at 29 °C; differing, optimum temperatures for in vitro transcription by several sigma factors in a single species, including different ECF sigma factors, has been demonstrated (Maeda et al., 2000 ).
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ACKNOWLEDGEMENTS |
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Received 17 March 2000;
revised 11 July 2000;
accepted 5 September 2000.