Institute of Microbiology and Genetics, Vienna Biocentre, Dr Bohrgasse 9, 1030 Vienna, Austria1
Author for correspondence: Udo Bläsi. Tel: +43 1 4277 54609. Fax: +43 1 4277 9546. e-mail: udo.blaesi{at}univie.ac.at
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ABSTRACT |
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Keywords: global regulator, Hfq, ompA, rpoS
Abbreviations: HfqEc, Hfq protein of Escherichia coli; HfqEc(75), C-terminally truncated HfqEc, lacking the last 27 aa; HfqPa, Hfq homologue of Pseudomonas aeruginosa; UTR, untranslated region
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INTRODUCTION |
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Hfq has been reported to positively regulate the rpoS gene encoding the S subunit of RNA polymerase (Brown & Elliot, 1996
; Muffler et al., 1996
). It has been suggested that Hfq activates rpoS translation by altering the secondary structure, sequestering its RBS (Brown & Elliot, 1997
; Cunning et al., 1998
; Muffler et al., 1996
). However, several other factors, including HNS and two small RNAs (DsrA and OxyS), are also involved in rpoS mRNA translation (Sledjeski et al., 1996
, 2001
; Wassarman et al., 2001
; Zhang et al., 1998
). Whereas DsrA RNA seems to stimulate the translation of rpoS by pairing with the mRNA and melting the secondary structure (Majdalani et al., 1998
), OxyS RNA represses rpoS translation by binding to Hfq (Zhang et al., 1998
). Although OxyS RNA does not prevent the binding of Hfq to the rpoS mRNA it apparently abrogates the stimulatory effect of Hfq on rpoS translation.
Hfq has been implicated in affecting the stability of the mRNAs of mutS, miaA, hfq (Tsui et al., 1997 ) and ompA (Vytvytska et al., 1998
). Vytvytska et al. (2000)
have shown that Hfq binds to the 5'-untranslated region (UTR) of ompA in the vicinity of the RBS, preventing translation and thereby indirectly subjecting the mRNA to degradation.
The E. coli Hfq protein (HfqEc) is heat-stable and consists of 102 aa. Immunofluorescence microscopy indicated that the majority of HfqEc is present in the cytosol and that it is most likely associated with the translation machinery; only a minor fraction appears to be associated with the nucleoid (Azam et al., 2000 ). Mutations in the E. coli hfq gene (hfqEc) caused pleiotropic effects. The insertion of an
cassette at the beginning of hfq resulted in a decreased growth rate, an increase in cell size, osmo-sensitivity and an increased sensitivity to UV light, and it also affected the supercoiling of plasmids (Tsui et al., 1994
). Furthermore, a hfq null-mutant did not synthesize glycogen, was starvation and multiple-stress sensitive and, as expected from its positive effect on rpoS translation, showed a down-regulation of RpoS-regulated genes (Muffler et al., 1997
). Taken together with the above-described effects on specific genes, these results suggested that Hfq acts as a global regulator involved in the regulation of RpoS-dependent and RpoS-independent genes. Moreover, Hfq homologues have been reported to stimulate synthesis of the heat-stable enterotoxin in Yersinia enterocolitica (Nakao et al., 1995
) and of the NifA protein in Azorhizobium caulinodans (Kaminski et al., 1994
). Also, the Hfq homologue of Brucella abortus appears to be a major determinant of virulence in mice (Robertson & Roop, 1999
).
As an opportunistic human pathogen Pseudomonas aeruginosa causes serious infections in immunocompromised hosts, but rarely infects healthy individuals (Bodey et al., 1983 ). It has been previously shown that RpoS of P. aeruginosa is required for the expression of severe-stress-resistance genes as well as for the expression of several virulence genes (Suh et al., 1999
). Since Hfq is required in both E. coli and Salmonella typhimurium for the efficient translation of rpoS, we were interested to determine whether a Hfq homologue is present in P. aeruginosa. Here, we report the isolation of the P. aeruginosa hfq gene (hfqPa). We also show that the Hfq protein of P. aeruginosa (HfqPa) can functionally replace HfqEc.
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METHODS |
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Plasmid pESE75 was constructed by first amplifying a fragment carrying the 3'-terminally truncated hfqEc gene [nucleotide -16 to +225; hfqEc(75)] by means of PCR using pHFQ607 and the forward primer E15 together with the reverse primer 5 '-TTTTTTTGGATCCCTGCAGttactaGGCGTTGTTACTGTGATGAG-3' containing a PstI site (bold) and two stop codons (lower case). This PCR fragment was then inserted into the BamHIPstI sites of vector pUC18.
The putative hfqPa gene (position 55483965548644 of the P. aeruginosa genomic sequence, www.pseudomonas.com; accession no. AE004091) was amplified by PCR from P. aeruginosa PAO1 genomic DNA using the forward primer X14 (5'-TTTTTTTTTTGGATCCCTATTCGACTAC-3'), containing a BamHI site (bold), and the reverse primer Y14 (5'-TTTTTTTTTTCTGCAGCCTGTTCCCACCACC-3'), containing a PstI site (bold). The hfqPa gene fragment (nucleotides -46 to +296) was digested with BamHI and PstI, and then cloned into the corresponding sites of pUC18, resulting in pESP82.
Plasmids pDLE102, pDLE75 and pDLP82 are derivatives of pACYC184 and were constructed as follows. pESE102, pESE75 and pESP82, respectively, were cleaved with PvuII. The resulting fragments, containing the lac promoter and the hfqEc, hfqEc(75) or hfqPa gene, were inserted into the EcoRV site of pACYC184, resulting in pDLE102, pDLE75 and pDLP82, respectively.
Plasmid pESPGm is a derivative of pESP82. The gentamicin cassette, aacC1, from pUCP24 was amplified by PCR using the primer pair B15 (5'-TTTTTTTTTTGATATCGGTACCTCTAGACCAGCGGCACCAGCGGC-3') and C15 (5'-TTTTTTTTTGATATCGGTACCGCGGCGTTGTGAC-3'); both of these primers contained a KpnI site (bold). The PCR fragment was digested with KpnI and cloned into the corresponding site of plasmid pUC19. The resulting pUC19 derivative was then cleaved with PvuII and HincII, and the fragment was inserted into the HincII site of pESP82 to generate pESPGm, which bore the insertionally inactivated hfqPa gene.
The ompAlacZ fusion plasmid, pIMZ, was constructed as follows. A constitutive lac promoter without operator sites was amplified by PCR from pUHE21-2 using the forward primer C10 (5'-AAATCTAGAATTCCCTTTCGTCTTCACCTCGAG-3'), containing a XbaI site (bold), and the reverse primer C12 (5'-AAAAAAGAATTCATCTAAGTATCATTGTTATCCG-3'), containing an EcoRI site (bold). The resulting fragment was cleaved with both EcoRI and XbaI and ligated to a 1300 bp EcoRIHindIII fragment isolated from pKSO325, containing the full-length ompA gene. This fragment was used to amplify the constitutive lac promoter together with the 5'-UTR of ompA up to nucleotide +69 by PCR with the forward primer C10 and the reverse primer E12 (5'-AAAAAAGGATCCGGAGCGGCCTGCGCTAGGG-3'), containing a BamHI site (bold). This fragment was then cloned into the XbaI and BamHI sites of pRB381 in-frame with lacZ, resulting in pRBompAlacZ. This plasmid was then digested with EcoRI and BamHI. The EcoRIBamHI fragment was cloned into plasmid pUHE21-2 downstream of the lac promoter. The resulting pUHE21-2 derivative was then cleaved with XhoI and BamHI, and the fragment containing the lac promoter and the 5' end of ompA was inserted into the corresponding sites of the single-copy, (28 °C)/transcriptional-fusion vector pOU251 in-frame with lacZ, resulting in pIMZ.
Qß plating assay.
Overnight cultures (100 µl) of E. coli AM111F' harbouring pUC18, pESE102, pESE75 or pESP82 were diluted in 5 ml LB and the corresponding hfq genes were induced by adding IPTG to a final concentration of 3 mM. The cultures were grown at 37 °C until they reached an OD600 of 0·4. Then 200 µl aliquots of the cultures and 10 µl of the Qß phage dilutions were added to 3 ml top agar. The agar was poured onto LB agar plates supplemented with the appropriate antibiotics and 3 mM IPTG. The p.f.u. ml-1 values were calculated from triplicate assays.
ß-Galactosidase assay.
E. coli AM111F' containing pIMZ and one of the pACYChfq derivatives (pDLE102, pDLE75, pDLP82 or pACYC184) was incubated at 28 °C. At an OD600 of 0·3, the pIMZ-encoded ompAlacZ gene and the respective hfq genes were induced by the addition of IPTG (3 mM). The ß-galactosidase activity (Miller, 1972 ) was determined from triplicate samples at 28 °C, to maintain a single copy of pIMZ.
Western blot analysis.
Cultures of AM111F', harbouring pESP82, pESPGm, pESE102, pESE75 or pUC18, and strain RH90 were grown at 37 °C until they reached an OD600 of 0·4, at which time IPTG was added to a final concentration of 3 mM. At an OD600 of 0·8 equal amounts of cells were withdrawn and boiled in Laemmli buffer. The proteins were separated on 12% SDS-polyacrylamide gels (Laemmli, 1970 ) and then transferred to a nitrocellulose membrane. The blots were blocked with 5% dry milk in TBS (8 g NaCl l-1, 0·2 g KCl l-1 and 3 g Tris-base l-1 in water, pH 7·5) and then probed with anti-RpoS antibodies (kindly provided by Dr F. Norel, Pasteur Institute, Paris). The antibodyantigen complex was visualized with goat-anti-rabbit immunoglobulin alkaline-phosphatase-conjugated antibodies (Sigma) using the chromogenic substrate nitro blue tetrazolium [2,2'-di-p-nitrophenyl-5,5'-diphenyl-3,3'-(3,3'-dimethoxy-4,4'-diphenylene)-ditetrazolium chloride; BIOMOL] and BCIP (5-bromo-4-chloro-3-indolyl phosphate) p-toluidine salt (BIOMOL) in alkaline phosphatase buffer (10 mM NaCl, 5 mM MgCl2, 100 mM Tris/HCl, pH 9·5). The quantification of the protein bands on the Western blots was performed with ImageQuant software (Molecular Dynamics, version 3.3).
Proteome anaylsis.
E. coli IM1101 harbouring pUC18, pESE102 or pESP82 was grown in M9 minimal medium containing 0·2% (v/v) glycerol to an OD600 of 0·8 and then labelled with 143 pM L-[35S]methionine (Amersham Pharmacia Biotech; >37 TBq mM-1) for 10 min. Total cellular protein extracts were analysed by two-dimensional gel electrophoresis. Equal amounts of cell material were dissolved in lysis buffer (8 M urea, 4%, w/v, CHAPS and 40 mM Tris-base) and the cells were disrupted by repeated freezing in liquid N2 and thawing at 37 °C. For the first dimension the Immobiline Dry strip pH 310 (18 cm) (Amersham Pharmacia Biotech) was used with the following IEF programme: 12 h rehydration, 1 h 500 V, 1 h 1000 V, 4 h 8000 V (IPGphor isoelectric focusing system). Resolution in the second dimension was performed on 12·5% SDS polyacrylamide gels for 15 min at 10 mA and then for 5 h at 20 mA. Buffers and conditions were used according to the manufacturers instructions. The gels were dried and then exposed to a Molecular Dynamics Phosphor Imager screen and analysed with PDQuest software (Bio-Rad).
Computer analysis.
The protein sequence of HfqPa was revealed by comparing the protein sequence of HfqEc with the proteins predicted to be encoded by the P. aeruginosa genome (Stover et al., 2000 ), using the sequence-alignment algorithm BLASTP (Altschul et al., 1990
). Preliminary sequence data were obtained from the Institute of Genomic Research (TIGR; http://www.tigr.org). Multiple alignments were done with CLUSTAL W (Thompson et al., 1994
) and visualized using BOXSHADE. The following (putative) Hfq sequences were compared: Pseudomonas aeruginosa (accession no. AE004091), Pseudomonas putida (NC002947), Pseudomonas syringae (NC002949), Escherichia coli (U00005), Salmonella typhimurium (U48735), Yersinia entercolitica (D28762), Pectobacterium carotovorum (AF039142), Haemophilus influenzae (U32724), Shigella flexneri (AB000785), Aquifex aeolicus (AE000674), Brucella abortus (AF154075), Acidithiobacillus ferrooxidans (NC002923), Azorhizobium caulinodans (X76450), Bacillus halodurans (BA000004), Bacillus subtilis (AL009126) and Clostridium acetobutylicum (AE001437). The name of the funding agency for each of the different bacterial genome projects can be found on the TIGR website.
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RESULTS AND DISCUSSION |
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Since Hfq is essential for phage Qß replication (Carmichael et al., 1975 ; de Fernandez et al., 1972
), we tested the functionality of HfqPa and HfqEc(75) in its replication. When compared to strain AM111F'(pESE102) (1·1x1010 p.f.u. ml-1), no significant difference in the p.f.u. ml-1 value was detected with strain AM111F'(pESE75) (1·0x1010p.f.u.ml-1) or strain AM111F'(pESP82) (1·0x1010 p.f.u. ml-1), whereas phage Qß was unable to replicate in the control strains AM111F' and AM111F'(pESPGm), the latter of which carries the inactivated hfqPa gene (Table 1
). Thus, the seemingly reduced HfqPa levels present in strain AM111F'(pESP82) were apparently not limiting for phage Qß replication.
Both HfqPa and HfqEc(75) affect the expression of E. coli ompA in a negative manner
It has been shown previously that HfqEc binds to the 5' UTR of E. coli ompA mRNA, which results in a decreased rate of expression for ompA (Vytvytska et al., 2000 ). To test whether the HfqPa and the HfqEc(75) protein exerted the same effect on the expression of ompA, expression of the pIMZ-encoded ompAlacZ fusion was monitored in AM111F' co-transformed with pDLE102 (hfqEc), pDLE75 (hfqEc(75)), pDLP82 (hfqPa) or pACYC184. Both ompAlacZ and hfq expression was induced at an OD600 of 0·3. At an OD600 of 0·6 the cells were harvested and the ß-galactosidase activities were determined. When compared to HfqEc, the presence of HfqPa or HfqEc(75) resulted in a similar decrease in the expression rate of the ompAlacZ fusion (data not shown), demonstrating that ompA mRNA is also a target for negative regulation by HfqPa and by HfqEc(75).
Both HfqPa and HfqEc(75) stimulate the expression of rpoS
Previous studies by Muffler et al. (1996) and by Brown & Elliott (1996)
have demonstrated that Hfq is required for the efficient translation of rpoS in E. coli and S. typhimurium, respectively. We therefore asked whether HfqPa and HfqEc(75) showed a similar positive effect on the translation of rpoS to that shown by HfqEc. The hfqEc, hfqPa and hfqEc(75) genes, encoded by pESE102, pESP82 and pESE75, respectively, were induced in AM111F' and the differences in the RpoS levels were determined by quantitative Western blotting. The induction of hfqEc and hfqEc(75) (Fig. 2
a, lanes 1, 2 and Fig. 2
b) resulted in a similar increase (
12-fold) in the RpoS concentration when compared to that present in the Hfq- strains AM111F'(pESPGm) and AM111F'(pUC18) (Fig. 2a
, lanes 4 and 5 and Fig. 2b
) and RH90 (Fig. 2a
, lane 6 and Fig. 2b
). The expression of hfqPa (Fig. 2a
, lane 3 and Fig. 2b
) resulted in an approximately sevenfold increase in the RpoS concentration when compared to the control strains. Whether this results from the apparently reduced levels of HfqPa present, when compared to those of HfqEc or HfqEc(75), remains to be determined.
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ACKNOWLEDGEMENTS |
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Received 6 August 2001;
revised 16 October 2001;
accepted 9 November 2001.