Functional replacement of the Escherichia coli hfq gene by the homologue of Pseudomonas aeruginosa

Elisabeth Sonnleitner1, Isabella Moll1 and Udo Bläsi1

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


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
The 102 aa Hfq protein of Escherichia coli (HfqEc) was first described as a host factor required for phage Qß replication. More recently, Hfq was shown to affect the stability of several E. coli mRNAs, including ompA mRNA, where it interferes with ribosome binding, which in turn results in rapid degradation of the transcript. In contrast, Hfq is also required for efficient translation of the E. coli and Salmonella typhimurium rpoS gene, encoding the stationary {sigma} factor. In this study, the authors have isolated and characterized the Hfq homologue of Pseudomonas aeruginosa (HfqPa), which consists of only 82 aa. The 68 N-terminal amino acids of HfqPa show 92% identity with HfqEc. HfqPa was shown to functionally replace HfqEc in terms of its requirement for phage Qß replication and for rpoS expression. In addition, HfqPa exerted the same negative effect on E. coli ompA mRNA expression. As judged by proteome analysis, the expression of either the plasmid-borne hfqPa or the hfqEc gene in an E. coli Hfq- RpoS- strain revealed no gross difference in the protein profile. Both HfqEc and HfqPa affected the synthesis of approximately 26 RpoS-independent E. coli gene products. These studies showed that the functional domain of Hfq resides within its N-terminal domain. The observation that a C-terminally truncated HfqEc lacking the last 27 aa [HfqEc(75)] can also functionally replace the full-length E. coli protein lends further support to this notion.

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


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Escherichia coli host factor I (HF1/Hfq), the product of the hfq gene, was first described as a factor required for phage Qß replication (de Fernandez et al., 1968 ). More recent experiments have suggested that Hfq stimulates Qß replication by melting out the 3' end of Qß plus-strand RNA, which appears to facilitate access of the replicase (Schuppli et al., 1997 ).

Hfq has been reported to positively regulate the rpoS gene encoding the {sigma}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 {Omega} 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.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and growth conditions.
The strains and plasmids used in this study are listed in Table 1. E. coli IM1101 was constructed by transducing rpoS-354::Tn10 from E. coli RH90 to E. coli AM111, using phage P1 and standard procedures (Miller, 1972 ). Unless indicated otherwise, bacterial cultures were grown at 37 °C in Luria–Bertani (LB) medium (Miller, 1972 ), supplemented with the appropriate antibiotics. Antibiotics were added to the following final concentrations: 100 µg ampicillin ml-1, 25 µg kanamycin ml-1, 10 µg chloramphenicol ml-1, 15 µg gentamicin ml-1 and 20 µg tetracycline ml-1.


View this table:
[in this window]
[in a new window]
 
Table 1. Bacterial strains and plasmids

 
Construction of plasmids used in this study.
pESE102, pESE75 and pESP82 (Table 1) were constructed as follows. The hfqEc gene [nucleotide -16 to +310 with regard to the A (position +1) of the start codon] was amplified by PCR using pHFQ607 and the forward primer E15 (5'-TTTTTTGAATTCGGATCCGTGAGGAAAAGAGAGAATGGCTAAGGGG-3'), containing a BamHI site (bold), together with the reverse primer 5'-CCCGAATTCTTATTCGGTTTCGCTGTCC-3', containing an EcoRI site (bold). The DNA fragment was cleaved with BamHI and EcoRI, and then cloned into the corresponding sites of pUC19, resulting in pESE102.

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 BamHI–PstI sites of vector pUC18.

The putative hfqPa gene (position 5548396–5548644 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 EcoRI–HindIII 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 EcoRI–BamHI 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 pACYC–hfq 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 antibody–antigen 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 3–10 (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 manufacturer’s 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.


   RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Sequence similarity between HfqPa and (putative) Hfq homologues from other bacteria
After identification of the putative HfqPa protein (see Methods), we performed a search for homologous proteins using the sequence alignment algorithm BLASTP. As shown in Fig. 1, HfqPa shows considerable amino acid similarity to previously identified Hfq homologues from other Gram-negative bacteria (73 to 96%) (Brown & Elliott, 1996 ; Chuang et al., 1999 ; Deckert et al., 1998 ; Durand et al., 1997 ; Fleischmann et al., 1995 ; Kajitani & Ishihama, 1991 ; Kaminski et al., 1994 ; Nakao et al., 1995 ; Robertson & Roop, 1999 ), whereas lower amino acid similarity (41 to 45%) was found with putative Hfq homologues from three Gram-positive bacteria. The multiple alignment (Fig. 1) revealed a high conservation of the N-terminal amino acids. There is considerable variation in the C-terminal amino acids, suggesting that the functional domain of Hfq resides within the first 70 aa. This suggestion is supported by the fact that insertion of an {Omega} cassette approximately in the middle of hfqEc (nucleotide +117, after codon 39) rendered the protein inactive, whereas insertion of the cassette at the 3' end of the gene (nucleotide +236, after codon 78) did not affect the activity of the protein (Tsui et al., 1994 ). In addition, further support for this assertion stems from the fact that the 82 aa HfqPa is only conserved within its first 68 aa. Finally, in the Hfq homologues of the different Gram-negative bacteria, and to a somewhat reduced extent in that of Gram-positive bacteria, positively charged amino acids are found at invariant positions within the first 70 aa (Fig. 1), suggesting their functional importance in nucleic acid binding.



View larger version (115K):
[in this window]
[in a new window]
 
Fig. 1. Sequence of HfqPa and (putative) Hfq homologues from Gram-positive and -negative bacteria. Approximately the first 70 aa of the N-terminal of Hfq are highly homologous. Identical amino acids are highlighted black, whereas conservative exchanges are highlighted grey. Positively charged residues, at invariant positions, are indicated by an arrow.

 
Both HfqPa and HfqEc(75) can functionally replace HfqEc in phage Qß replication
Because of the identity between their N-terminal amino acids, we asked whether hfqPa could functionally replace hfqEc. Therefore, pESE102 and pESP82 harbouring hfqEc and hfqPa, respectively, were constructed. The Hfq proteins of E. coli, S. typhimurium, Y. entercolitica and Pectobacterium carotovorum are nearly identical up to amino acid 75 (Fig. 1). pESE75, encoding the first 75 aa (hfqEc(75)), was therefore created with the aim of determining whether the C terminus of HfqEc was functionally irrelevant. The expression of hfqEc, hfqPa and hfqEc(75) was verified by Western blot analysis with antibodies against HfqEc. As judged by quantitative Western blotting, the apparent level of production of HfqPa was reduced by approximately threefold when compared to that of both HfqEc and HfqEc(75) (data not shown), which could result either from a reduced cross-reactivity with the antibodies raised against HfqEc or from a reduced rate of expression of hfqPa in E. coli.

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. 2a, lanes 1, 2 and Fig. 2b) 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.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 2. Both HfqPa and HfqEc(75) stimulate the expression of rpoS in E. coli. (a) Western blot analysis was carried out as described in Methods. Only the relevant section of the immunoblot showing the RpoS-specific band is depicted. Lanes 1–5 are visual representations of the RpoS levels in E. coli AM111F’ harbouring the following plasmids. Lanes: 1, pESE102 (hfqEc); 2, pESE75 (hfqEc(75)); 3, pESP82 (hfqPa); 4, pESPGm (hfqPa::aacC1); 5, pUC18 (control). Lane 6, RpoS strain RH90. No RpoS signal was detected for this strain. (b) Graphical representation of the results presented in (a). Quantification of the Western blot signals was done with the ImageQuant software. Values were normalized to the RpoS signal obtained from AM111F’(pUC18), which was set at 1. Results represent data from triplicate experiments; the error bars represent SD.

 
HfqPa and HfqEc show the same regulatory effects on RpoS-independent genes
Several studies have suggested that Hfq acts as global regulator, which affects the expression of RpoS-dependent as well as RpoS-independent genes (Muffler et al., 1997 ; Tsui et al., 1997 ; Vytvytska et al., 2000 ). To test whether HfqPa and HfqEc show the same alteration in the expression of RpoS-independent genes, we compared the two-dimensional gel electrophoretic patterns of total-cellular-protein synthesis in IM1101(pESE102) and IM1101(pESP82) with that of the RpoS- Hfq- control strain IM1101(pUC18). As mentioned above, although the protein levels of HfqEc and HfqPa seem to vary in strains IM1101(pESE102) and IM1101(pESP82) no gross difference was observed in their protein profile. (Fig. 3b–c). This again showed that HfqPa can functionally replace HfqEc and that the C-terminal end of HfqEc is not necessary for its function. When compared to the RpoS- Hfq- control strain, the synthesis of at least five proteins was found to be enhanced in the presence of Hfq (indicated by solid arrows, Fig. 3a–c), whereas Hfq appears to be essential for the synthesis of four proteins (marked by open arrows, Fig. 3a–c). In contrast, the synthesis of at least six proteins was found to be turned off in the presence of Hfq (indicated by open arrowheads, Fig. 3a–c) and the synthesis of 11 proteins was decreased in the presence of Hfq (indicated by solid arrowheads in Fig. 3a–c). In accordance with a recent study (Vytvytska et al., 2000 ), OmpA was found among these 11 proteins (Table 2; protein n). Using the E. coli Swiss-2D PAGE, we have attempted to identify the proteins whose synthesis was affected by Hfq. Among the 26 proteins, 11 proteins were tentatively assigned (Table 2, a–z).



View larger version (40K):
[in this window]
[in a new window]
 
Fig. 3. Two-dimensional protein synthesis pattern of E. coli IM1101 bearing pUC18 (a), pESE102 (b) and pESP82 (c), respectively. Total cellular protein extracts were analysed. The pH gradient in the first dimension ranged from 3 to 10 (from left to right). Proteins whose synthesis was decreased in the presence of Hfq are depicted by solid arrowheads; those proteins whose synthesis was turned off in the presence of Hfq are indicated by open arrowheads. The proteins whose synthesis was enhanced in the presence of Hfq are indicated by solid arrows. Proteins which were absent in IM1101(pUC18) and present in the strains containing the plasmid-encoded HfqPa or HfqEc are indicated by open arrows.

 

View this table:
[in this window]
[in a new window]
 
Table 2. Regulatory effects by Hfq on the synthesis of RpoS-independent genes

 
Suh et al. (1999) have reported that RpoS mediates the resistance of P. aeruginosa to heat shock, H2O2 and osmotic stress. Additionally, RpoS affects the accumulation of exotoxin A, pyoverdin and alginate, which seem to be important for P. aeruginosa to survive adverse conditions during infection. Given that HfqPa affected the expression of E. coli rpoS (see Fig. 2) in a positive manner it remains to be seen whether it has the same role in P. aeruginosa. In addition, studies are currently under way to test whether HfqPa directly affects the expression of P. aeruginosa genes, as was indicated in this study for a number of E. coli genes.


   ACKNOWLEDGEMENTS
 
We would like to thank Dr A. Hengge-Aronis (University of Berlin, Germany), Dr A. Ishihama (National Institute of Genetics, Shizuoka, Japan) and Dr F. Norel for their gifts of bacterial strains and materials. Preliminary sequence data were obtained from the Institute for Genomic Research (http://www.tigr.org). Sequencing of P. aeruginosa PAO1 was accomplished with support from the Cystic Fibrosis Foundation, the University of Washington Genome Center and the PathoGenesis Cooperation. This work was supported within the framework of the Special Research Program (017) on ‘Modulators of RNA Fate and Function’ by grant F1715 from the Austrian Science Fund (FWF) to U.B.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990). Basic local alignment search tool. J Mol Biol 215, 403-410.[Medline]

Azam, T. A., Hiraga, S. & Ishihama, A. (2000). Two types of localization of the DNA-binding proteins within the Escherichia coli nucleoid. Genes Cells 5, 613-626.[Abstract/Free Full Text]

Bodey, G. P., Bolivar, R., Fainstein, V. & Jadeja, L. (1983). Infections caused by Pseudomonas aeruginosa. Rev Infect Dis 5, 279-313.[Medline]

Brown, L. & Elliott, T. (1996). Efficient translation of the RpoS sigma factor in Salmonella typhimurium requires host factor I, an RNA-binding protein encoded by the hfq gene. J Bacteriol 178, 3763-3770.[Abstract]

Brown, L. & Elliott, T. (1997). Mutations that increase expression of the rpoS gene and decrease its dependence on hfq function in Salmonella typhimurium. J Bacteriol 179, 656-662.[Abstract]

Brückner, R. (1992). A series of shuttle vectors for Bacillus subtilis and Escherichia coli. Gene 122, 187-192.[Medline]

Carmichael, G. G., Weber, K., Niveleau, A. & Wahba, A. J. (1975). The host factor required for RNA phage Qß RNA replication in vitro. J Biol Chem 250, 3607-3612.[Abstract]

Chuang, D. Y., Kyeremeh, A. G., Gunji, Y., Takahara, Y., Ehara, Y. & Kikumoto, T. (1999). Identification and cloning of an Erwinia carotovora subsp. carotovora bacteriocin regulator gene by insertional mutagenesis. J Bacteriol 181, 1953-1957.[Abstract/Free Full Text]

Cunning, C., Brown, L. & Elliott, T. (1998). Promotor substitution and deletion analysis of upstream region required for rpoS translational regulation. J Bacteriol 180, 4564-4570.[Abstract/Free Full Text]

Deckert, G., Warren, P. V., Gaasterland, T. & 12 other authors (1998). The complete genome of the hyperthermophilic bacterium Aquifex aeolicus. Nature 392, 353–358.[Medline]

de Fernandez, M. T. F., Eoyang, L. & August, J. T. (1968). Factor fraction required for the synthesis of bacteriophage Qß-RNA. Nature 219, 588-590.[Medline]

de Fernandez, M. T. F., Hayward, W. S. & August, J. T. (1972). Bacterial proteins required for replication of Qß ribonucleic acid. J Biol Chem 247, 824-831.[Abstract/Free Full Text]

Durand, J. M., Bjork, G. R., Kuwae, A., Yoshikawa, M. & Sasakawa, C. (1997). The modified nucleoside 2-methylthio-N6-isopentenyladenosine in tRNA of Shigella flexneri is required for expression of virulence genes. J Bacteriol 179, 5777-5782.[Abstract]

Fleischmann, R. D., Adams, M. D., White, O. & 37 other authors (1995). Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269, 496–512.[Medline]

Holloway, B. W. (1955). Genetic recombination in Pseudomonas aeruginosa. J Gen Microbiol 13, 572-581.

Kajitani, M. & Ishihama, A. (1991). Identification and sequence determination of the host factor gene for bacteriophage Qß. Nucleic Acids Res 19, 1063-1066.[Abstract]

Kajitani, M., Kato, A., Wada, A., Inokuchi, Y. & Ishihama, A. (1994). Regulation of the Escherichia coli hfq gene encoding the host factor for phage Qß. J Bacteriol 176, 531-534.[Abstract]

Kaminski, P. A., Desnoues, N. & Elmerich, C. (1994). The expression of nifA in Azorhizobium caulinodans requires a gene product homologous to Escherichia coli HF-I, an RNA-binding protein involved in the replication of phage Qß RNA. Proc Natl Acad Sci USA 91, 4663-4667.[Abstract]

Laemmli, U. K. (1970). Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 227, 680-685.[Medline]

Lange, R. & Hengge-Aronis, R. (1991). Identification of a central regulator of stationary-phase gene expression in Escherichia coli. Mol Microbiol 5, 49-59.[Medline]

Lanzer, M. & Bujard, H. (1988). Promoters largely determine the efficiency of repressor action. Proc Natl Acad Sci USA 85, 8973-8977.[Abstract]

Majdalani, N., Cunning, C., Sledjeski, D. D., Elliott, T. & Gottesman, S. (1998). DsrA RNA regulates translation of RpoS message by an anti-antisense mechanism, independent of its action as an antisilencer of transcription. Proc Natl Acad Sci USA 95, 12462-12467.[Abstract/Free Full Text]

Miller, J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Muffler, A., Fischer, D. & Hengge-Aronis, R. (1996). The RNA-binding protein HF-I, known as a host factor for phage Qß RNA replication, is essential for rpoS translation in Escherichia coli. Genes Dev 10, 1143-1151.[Abstract]

Muffler, A., Traulsen, D. D., Fischer, D., Lange, R. & Hengge-Aronis, R. (1997). The RNA-binding protein HF-I plays a global regulatory role which is largely, but not exclusively, due to its role in expression of the {sigma}S subunit of RNA polymerase in Escherichia coli. J Bacteriol 179, 297-300.[Abstract]

Nakao, H., Watanabe, H., Nakayama, S. & Takeda, T. (1995). yst gene expression in Yersinia enterocolitica is positively regulated by a chromosomal region that is highly homologous to Escherichia coli host factor 1 gene (hfq). Mol Microbiol 18, 859-865.[Medline]

Ried, G., Koebnik, R., Hindennach, I., Mutschler, B. & Henning, U. (1994). Membrane topology and assembly of the outer membrane protein OmpA of Escherichia coli K12. Mol Gen Genet 243, 127-135.[Medline]

Robertson, G. T. & Roop, R. M. (1999). The Brucella abortus host factor I (HF-I) protein contributes to stress resistance during stationary phase and is a major determinant of virulence in mice. Mol Microbiol 34, 690-700.[Medline]

Rose, R. E. (1988). The nucleotide sequence of pACYC184. Nucleic Acid Res 16, 355.[Medline]

Schuppli, D., Miranda, G., Tsui, H. T., Winkler, M. E., Sogo, J. M. & Weber, H. (1997). Altered 3'-terminal RNA structure in phage Qß adapted to host factor-less Escherichia coli. Proc Natl Acad Sci U S A 94, 10239-10242.[Abstract/Free Full Text]

Sledjeski, D. D., Gupta, A. & Gottesman, S. (1996). The small RNA, DsrA, is essential for the low temperature expression of RpoS during exponential growth in Escherichia coli. EMBO J 15, 3993-4000.[Abstract]

Sledjeski, D. D., Whitman, C. & Zhang, A. (2001). Hfq is necessary for regulation by the untranslated RNA DsrA. J Bacteriol 183, 1997-2005.[Abstract/Free Full Text]

Stover, C. K., Pham, X. Q., Erwin, A. L. & 28 other authors (2000). Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406, 959–964.[Medline]

Suh, S., Silo-Suh, L., Woods, D. E., Hassett, D. J., West, S. E. H. & Ohman, D. E. (1999). Effect of rpoS mutation on the stress response and expression of virulence factors in Pseudomonas aeruginosa. J Bacteriol 181, 3890-3897.[Abstract/Free Full Text]

Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673-4680.[Abstract]

Tsui, H. T., Leung, H. E. & Winkler, M. E. (1994). Characterization of broadly pleiotropic phenotypes caused by an hfq insertion mutation in Escherichia coli K-12. Mol Microbiol 13, 35-49.[Medline]

Tsui, H. T., Feng, G. & Winkler, M. E. (1997). Negative regulation of mutS and mutH repair gene expression by the Hfq and RpoS global regulators of Escherichia coli K-12. J Bacteriol 179, 7476-7487.[Abstract]

Vytvytska, O., Jakobsen, J. S., Balcunaite, G., Andersen, J. S., Baccarini, M. & von Gabain, A. (1998). Host factor I, Hfq, binds to Escherichia coli ompA mRNA in a growth rate-dependent fashion and regulates its stability. Proc Natl Acad Sci USA 95, 14118-14123.[Abstract/Free Full Text]

Vytvytska, O., Moll, I., Kaberdin, V. R., von Gabain, A. & Bläsi, U. (2000). Hfq (HF1) stimulates ompA mRNA decay by interfering with ribosome binding. Genes Dev 14, 1109-1118.[Abstract/Free Full Text]

Wassarman, K. M., Repoila, F., Rosenow, C., Storz, G. & Gottesman, S. (2001). Identification of novel small RNAs using comparative genomics and microarrays. Genes Dev 15, 1637-1651.[Abstract/Free Full Text]

West, S. E. H., Schweizer, H. P., Dall, C., Sample, A. K. & Runyen-Janecky, L. J. (1994). Construction of improved EscherichiaPseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa. Gene 128, 81-86.

Yanisch-Perron, C., Vieira, J. & Messing, J. (1985). Improved M13 cloning vectors and host strains: nucleotide sequence of M13mp18 and pUC19 vectors. Gene 33, 103-119.[Medline]

Zhang, A., Altuvia, S., Tiwari, A., Argaman, L., Hengge-Aronis, R. & Storz, G. (1998). The OxyS regulatory RNA represses rpoS translation and binds the Hfq (HF-I) protein. EMBO J 17, 6061-6068.[Abstract/Free Full Text]

Received 6 August 2001; revised 16 October 2001; accepted 9 November 2001.