The truA gene of Pseudomonas aeruginosa is ... - Semantic Scholar

Microbiology (2004), 150, 539–547

DOI 10.1099/mic.0.26652-0

The truA gene of Pseudomonas aeruginosa is required for the expression of type III secretory genes Kyung-Seop Ahn,1,2 Unhwan Ha,1 Jinghua Jia,1 Donghai Wu3 and Shouguang Jin1 1

Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610, USA

Correspondence Shouguang Jin

2

[email protected]

Immunomodulator Laboratory, Korea Institute of Bioscience and Biotechnology, Taejon 305-600, Republic of Korea

3

Institute for Nutritional Sciences, SIBS, Chinese Academy of Sciences, Shanghai 200031, China

Received 17 July 2003 Revised 5 November 2003 Accepted 28 November 2003

Invasive strains of Pseudomonas aeruginosa can cause rapid host cell apoptosis by injecting the type III effector molecule ExoS. A transposon insertional mutant bank of P. aeruginosa was screened to identify P. aeruginosa genes that contribute to the ability of the bacteria to trigger host cell apoptosis. Several isolated mutants had disruptions in the fimV gene. A fimV mutant was unable to induce the expression of exoS, exoT and exsA genes under type III inducing conditions, thus exhibiting a defect in type III protein secretion. Furthermore, this mutant was defective in twitching motility, although type IV pili were present on the bacterial surface. Complementation by a fimV-containing cosmid clone restored both phenotypes to the wild-type levels. However, expression of the type III genes in the fimV mutant was not restored by the introduction of a fimV gene alone, although it restored the twitching motility. A gene downstream of fimV, encoding a tRNA pseudouridine synthase (truA) homologue, was able to complement the type III gene expression defect of the fimV mutant. Thus fimV and truA form an operon and fimV mutation has a polar effect on truA. Indeed, a truA mutant is defective in type III gene expression while its twitching motility is unaffected, and a truA clone is able to complement the type III secretion defect. Pseudouridination of tRNAs is important for tRNA structure, thereby improving the fidelity of protein synthesis and helping to maintain the proper reading frame; thus the results imply that truA controls tRNAs that are critical for the translation of type III genes or their regulators.

INTRODUCTION Pseudomonas aeruginosa is an opportunistic human pathogen that primarily infects patients with burn wounds or cystic fibrosis, or immunocompromised individuals (Asboe et al., 1998; Bodey et al., 1983; Davies, 2002; Pier, 2002). Clinical isolates of P. aeruginosa have been grouped into invasive and noninvasive (cytolytic) strains based on their interactions with non-phagocytic corneal epithelial cells (Fleiszig et al., 1997). The invasive and noninvasive strains encode different sets of exoenzymes that are translocated into the host cells via a type III secretion machinery (Fleiszig et al., 1997; Frithz-Lindsten et al., 1997). The type III system is a common virulence machinery of Gram-negative animal and plant pathogens, such as enterohaemorrhagic Escherichia coli, Camphylobacter, Salmonella, Shigella, Yersinia, Erwinia and Xanthomonas species (Cornelis & Wolf-Watz, 1997; Galan & Collmer, 1999; Greenberg & Vinatzer, 2003; Groisman & Ochman, 1993; Hueck, 1998; Van Gijsegem 0002-6652 G 2004 SGM

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et al., 1993). The type III secretion system of P. aeruginosa encodes about 20 proteins, including components of a secretory apparatus which is devoted to the direct translocation of effectors into the host cell cytoplasm, and four effector molecules, ExoS, -T, -U and -Y, which alter normal host cell processes (Frank, 1997; Hauser et al., 1998; Yahr et al., 1996a, 1997). The noninvasive strains contain exoT and exoU genes, whereas the invasive strains contain exoS and exoT genes (Fleiszig et al., 1997). ExoS and ExoT are bifunctional exotoxins with C-terminal ADP-ribosylation activities. ExoS and ExoT share 75 % amino acid identity, but ExoT possesses a lower catalytic activity, with only 0?2 % of the ADP-ribosyltransferase activity of ExoS (Yahr et al., 1996b). The ADP-ribosyltransferase activity of ExoS requires a eukaryotic cofactor termed factor activating ExoS (FAS), which is a member of the highly conserved, multifunctional 14-3-3 family of proteins, whose primary function involves the regulation of eukaryotic 539

K.-S. Ahn and others

enzyme activities (Aitken et al., 1995; Fu et al., 1993). The requirement of a eukaryotic cofactor for activity and the functional importance of the in vivo target proteins suggest that ExoS contributes to pathogenesis by disrupting normal cellular processes. ExoS preferentially ADP-ribosylates a number of proteins, including vimentin and several Ras family GTP-binding proteins that regulate intracellular vesicle transport, cell proliferation and differentiation (Bourne et al., 1990; Coburn & Gill, 1991). The ADP-ribosylating activity of ExoS was also shown recently to cause apoptosis in various tissue culture cells (Kaufman et al., 2000; Jia et al., 2003). Both ExoS and ExoT also cause severe cell rounding by disrupting the actin cytoskeleton with their N-terminal GTPase-activating protein domain (GAP) (Goehring et al., 1999; Krall et al., 2000; Pederson et al., 1999). Expression of these exoenzymes is coordinately regulated by a transcriptional activator, ExsA, in response to various environmental signals, including low calcium and direct contact with tissue culture cells (Vallis et al., 1999). P. aeruginosa expresses type IV pili on the cell surface which not only function as an adhesin but also as a motility apparatus enabling the bacterium to glide on solid surfaces, a motion called twitching motility. Such motility is achieved by the retractile movement of the type IV pili (Merz et al., 2000). There are separate sets of P. aeruginosa genes devoted to pilin gene expression, processing, assembly of the pilin subunits on the bacterial surface and twitching motility (Alm & Mattick, 1997; McBride, 2001). In the course of screening P. aeruginosa genes that affect bacterial ability to inject type III effector molecules into the host cell, we have identified fimV mutants that are defective in both type III secretion and twitching motility. Complementation assay results are consistent with the notion that the fimV gene forms an operon with a downstream gene, truA, and that the fimV gene is required for twitching motility while the truA gene is required for the type III secretory gene expression. Since pseudouridination of tRNAs is known to be required for maturation of tRNAs from their precursors, aminoacylation and stabilization of the stem–loop structure through improved intramolecular base-pairing (Auffinger & Westhof, 1998; Davis, 1995; Durant & Davis, 1999; Price & Gray, 1998), our observations imply that truA controls tRNAs that are critical for the expression of type III genes or their regulators.

METHODS Bacterial strains and plasmids. The bacterial strains and plas-

mids used in this study are listed in Table 1. P. aeruginosa was grown on Luria (L) agar or in L broth at 37 uC. Antibiotics were used at the following final concentrations. For P. aeruginosa: carbenicillin, 150 mg ml21; spectinomycin, 200 mg ml21; streptomycin, 200 mg ml21; and tetracycline, 100 mg ml21. For E. coli: ampicillin, 100 mg ml21; spectinomycin, 50 mg ml21; streptomycin, 25 mg ml21; and tetracycline, 20 mg ml21. The pppB gene was PCR amplified using primer sets pppB-1 (59-GCC GCA CCA GCC TGG TGC TGG AGA AGA TGG-39) and pppB-2 (59-CCT GAC GGC CGT GGT GAT CCG CCA AAG ATC TA-39). PCR products were first cloned 540

into pCR2.1-TOPO, and then the inserts were reisolated as EcoRI– BglII fragments for cloning into C-terminal FLAG tag fusion vector pFLAG-CTC (Sigma). The pppB-FLAG clones were further isolated as HindIII–ScaI fragments and subcloned into HindIII–SmaI digests of the shuttle vector pUCP19 to generate pPppB-FLAG. Resulting plasmids were confirmed for in-frame fusions by sequencing. Screen of mutants defective in HeLa cell lifting. A Tn5G transposon insertional mutant library was generated as described earlier (Wang et al., 1996). Individual mutants were grown overnight in 96-well plates with L broth containing gentamicin. The bacterial cells (0?1 ml) were used to infect freshly grown HeLa cells on 24-well plates, incubated for 6 h and washed with water to remove lifted HeLa cells. Crystal violet staining was used to visualize HeLa cells that remained adhered to the plates. Bacterial mutants that were unable to lift HeLa cells within the 6 h infection period were further characterized. First, slow-growing mutants were eliminated by testing their growth curves, then a HeLa cell adherence assay was conducted to eliminate those having mutations leading to significantly low binding (Ha & Jin, 2001). Chromosomal DNA of the mutants was digested with EcoRI and the Tn5G insertions together with the neighbouring chromosomal DNA were cloned into pTZ18R. DNA sequencing analysis was further conducted to identify the transposon insertion sites. Construction of isogenic mutants of fimV and truA. The fimV

and truA genes were PCR amplified from the P. aeruginosa PAK chromosome using the following two pairs of primers: fimV-1 (59GTC TCT TCG ACC GCC AGA TCG CCT TCA ACC TGC TC-39) with fimV-2 (59-CAT CGT TCA TGA GGG TCG CTT CAT TTC CGG ATC AG -39); and truA-1 (59-TCC TCG ACG AAG TCC TGG CCG AAG GTA ATG ACA GC-39) with truA-2 (59-TCT CGA TGG TAG CAA AAG CCC GAT TCG ACG TCA GC-39), respectively. The fimV and truA genes were first cloned into pCR2.1-TOPO, resulting in pHW0035 and pKS0206, respectively, and then further subcloned into the suicide vector pEX18Tc to generate pHW0199 and pKS0212, respectively. The spectinomycin/streptomycin-resistance gene cartridge (2 kb V cassette) was inserted into the BamHI site within the fimV gene of pHW0199 and also into the NdeI site of the truA gene in pKS0212, giving pHW01100 and pKS0204, respectively. For a non-polar fimV mutant, oligonucleotides fimV-sd-f (59-GAT ATT AAA AGG GAT TAC ACT GCA GTT CGG CTT CGT ACA CTG-39) and fimV-sd-b (59-CAG TGT ACG AAG CCG AAC TGC AGT GTA ATC CCT TTT AAT ATC-39) were used to mutate the start codon ATG into GCA while generating a PstI restriction site on pHW0199, resulting in pKS0304. The three plasmids were used to generate fimV and truA null mutants following the sucrose selection method as described by Schweizer (1992). The resulting fimV and truA mutants were confirmed by Southern hybridization. Detection of ExoS and ExoT proteins in culture medium.

Bacteria were grown overnight in L broth with 5 mM EGTA to a cell density of OD600 4?0 and 0?5 ml culture supernatants were concentrated to 10 ml using a Centricon-30, mixed with an equal volume of 26 loading buffer, boiled and subjected to separation by 12 % SDS-PAGE (Laemmli, 1970). Protein bands were visualized either directly by Coomassie staining or following Western blotting. For Western blot analysis, proteins on the gel were transferred electrophoretically onto Hybond-C nitrocellulose (Amersham) in the Tris/glycine system as described by Towbin et al. (1979). FLAGtagged ExoS and ExoT were detected with anti-FLAG antiserum (Sigma) followed by goat anti-mouse immunoglobulin G conjugated to horseradish peroxidase (Amersham) and an enhanced chemiluminescence (ECL) detection kit from Amersham. Twitching motility assay. Twitching motility was assayed as

described by Alm & Mattick (1995). Briefly, the P. aeruginosa strain to be tested was stab-inoculated through a 1 % agar plate, grown Microbiology 150

Y synthase affects type III secretion system

Table 1. Bacterial strains and plasmids used in this study Strain or plasmid P. aeruginosa PAK PAKexoSexoT PAKfimV : : V PAKtruA : : V PAKexsA : : V Plasmids pCR2.1-TOPO pDN19lacV pHW0005 pHW0006 pHW0032 pUCP19 pHW0027 pHW0029 pPppB-FLAG pHW0035 pHW0199 pHW01100 pKS0304 pHW01116 pKS0206 pKS0207 pKS0209 pKS0212 pKS0214 pMO010631

Description*

Source

Clinical isolate of wild-type invasive strain PAK with chromosomal disruption of exoS and exoT; Spr Smr Gmr PAK with chromosomal disruption of the fimV locus; Spr Smr PAK with chromosomal disruption of the truA locus; Spr Smr PAK with chromosomal disruption of the exsA locus; Spr Smr

D. BradleyD Kaufman et al. (2000) This study This study Frank et al. (1994)

PCR cloning vector; Apr Kmr Promoterless lacZ fusion vector; Spr Smr Tcr PAK exoS promoter fused to lacZ in pDN19lacV; Spr Smr Tcr PAK exoT promoter fused to lacZ in pDN19lacV; Spr Smr Tcr PAK exsA promoter fused to lacZ in pDN19lacV; Spr Smr Tcr Broad host range shuttle vector; Apr ExoT-FLAG fusion in pUCP19; Apr ExoS-FLAG fusion in pUCP19; Apr PppB-FLAG fusion in pUCP19; Apr fimV clone in pCR2.1-TOPO; Apr Kmr fimV clone in pEX18Tc; Tcr fimV gene disrupted by insertion of V in pHW0199; Spr Smr Tcr ATG of fimV mutated to GCA in pHW0199 fimV gene of PAK cloned in pUCP19; Apr truA clone in pCR2.1-TOPO; Apr Kmr truA gene of PAK cloned in pUCP19; Apr fimV–truA clone (5 kb StuI fragment) of PMO010631 in pUCP19; Apr truA clone in pEX18Tc; Tcr truA gene disrupted by insertion of V in pKS0212; Spr Smr Tcr Cosmid clone containing fimV and truA genes in pLA2917; Tcr

Invitrogen Totten & Lory (1990) Ha & Jin (2001) Ha & Jin (2001) Ha & Jin (2001) Schweizer (1991) Ha & Jin (2001) Ha & Jin (2001) Ha & Jin (2001) This study This study This study This study This study This study This study This study This study This study Croft et al. (2000)

*Apr, ampicillin resistance marker; Kmr, kanamycin resistance marker; Smr, streptomycin resistance marker; Spr, spectinomycin resistance marker; Tcr, tetracycline resistance marker. DFaculty of Medicine, Memorial University of Newfoundland, Canada A1B 3V6.

overnight at 37 uC, followed by a 2 day incubation at room temperature. The zone of twitching motility between the agar and the Petri dish interface was then visualized by staining with Coomassie brilliant blue R250. Other methods. A standard b-galactosidase assay (Miller, 1972) was

conducted to determine the expression of the exoS : : lacZ, exoT : : lacZ and exsA : : lacZ fusion genes. Standard methods were used for plasmid DNA preparation, restriction enzyme digestion and cloning (Sambrook et al., 1989). DNA sequence analysis was performed by PCR-mediated Taq DyeDeoxy Terminator Cycle sequence using an Applied Biosystems model 373A DNA sequencer. DNA restriction enzyme sites and open reading frame analyses were conducted using the DNA Strider program. Southern hybridizations were carried out using the ECL labelling and detection kit from Amersham.

RESULTS Identification of P. aeruginosa mutants defective in HeLa cell lifting Invasive strains of P. aeruginosa can cause efficient host cell apoptosis by injecting the type III effector molecule ExoS http://mic.sgmjournals.org

(Kaufman et al., 2000). In order to identify P. aeruginosa genes that affect its ability to inject ExoS into the host cell, a Tn5G transposon insertional mutant library of P. aeruginosa was screened. Since one of the hallmarks of HeLa cell apoptosis is cell lifting, we initially screened for mutants that are defective in causing HeLa cell lifting. At m.o.i. 20, wildtype PAK caused 100 % cell lifting by 4 h of incubation whereas a type III defective mutant strain (PAKexsA2) did not cause cell lifting even after 24 h incubation. An initial screen of 5000 individual mutants identified 60 mutants that were unable to cause HeLa cell lifting by 6 h. Further analysis of those mutants revealed that 47 had a significant defect in either growth or host cell binding whereas the remaining 13 had normal growth and attachment. Sequence analysis of the transposon insertion sites revealed five different genetic loci, including genes of fimV (PA3115), pilL (PA0413), hepA (PA3308), cyaA (PA5272) and nuoE (PA2640). cyaA encodes a protein that shares 54 % similarity with adenylate cyclase of E. coli (Aiba et al., 1983) and was recently reported to be essential for efficient expression of the type III genes in response to the low-calcium signal 541


A fimV mutant is defective in type III gene expression In order to confirm that mutation in fimV is responsible for the observed defect in HeLa cell lifting, a new defined fimV mutant was generated in the PAK background by inserting an V fragment, encoding resistance to streptomycin and spectinomycin, into the fimV locus by allelic exchange utilizing the sacB counterselection marker (Schweizer, 1992). The fimV mutant grew as well as wild-type PAK in L broth with (type III inducing condition) or without 5 mM EGTA. As reported by Semmler et al. (2000), the fimV mutant was completely defective in twitching motility (Fig. 1), although type IV pili were observed on the surface by scanning electron microscopy. The new fimV mutant also showed a defect in HeLa cell lifting, just like the original transposon insertional mutants (data not shown). Fig. 1. Twitching motility assays. Bacteria were inoculated on L agar, incubated overnight at 37 6C and placed at room temperature for a further 2 days. Strains: wt, wild-type PAK; fimV ”, fimV mutant; truA”, truA mutant. Plasmids: /fimV and /truA represent complemention by fimV clone pHW01116 and truA clone pKS0207, respectively.

(Wolfgang et al., 2003); thus its effect on cell lifting is understandable. FimV is a twitching motility protein (Semmler et al., 2000) and PilL is a probable component of the chemotactic signal transduction system, with its C-terminal half showing 51 % similarity to chemotaxis protein CheA of Synechocystis sp. (Mattick et al., 1996); thus twitching motility might affect the type III secretion as previously suggested (Comolli et al., 1999, Kang et al., 1997). HepA is 67 % similar to a probable ATP-dependent RNA helicase HepA of E. coli (Bork & Koonin, 1993) and NuoE is 81 % similar to NADH dehydrogenase I chain E of E. coli (Weidner et al., 1993). The involvement of the latter two proteins in the HeLa cell lifting is not clear at this point. Since the fimV gene is relatively well described and four of the 13 mutants had a Tn5G insertion in the fimV gene, we further characterized this genetic locus.

Next, we tested whether the secretion of the type III effector molecules is defective in the fimV mutant. Plasmids with exoS-FLAG or exoT-FLAG fusions (pHW0029 or pHW0027) were transformed into wild-type strain PAK, PAKfimV : : V and PAKexsA : : V, respectively. These strains were grown under type III inducing conditions (L-broth plus 5 mM EGTA) and Western blot analysis was conducted on both culture supernatants for secreted form and bacterial cell extracts for intracellular level using antibody against the FLAG-tag. As a control, FLAG-tag fusion to the C-terminal end of a known cytoplasmic protein, PppB, was used (Ha & Jin, 2001) to monitor cell-lysis-mediated protein release. As shown in Fig. 2, in both supernatant and cellular fractions, the ExoS and ExoT protein levels were much lower in the fimV mutant compared to that of wild-type PAK, suggesting that type III expression might be defective in the fimV mutant. Absence of PppB-FLAG in the supernatant eliminated the possibility of cell-lysis mediated protein release. To further test if the expression of the type III genes is defective in the fimV mutant, plasmid constructs containing transcriptional fusions between promoters of the exoS, exoT and exsA genes and a promoterless lacZ reporter gene, pHW0005, pHW0006 and pHW0032, respectively, were

Fig. 2. Secretion of type III proteins is defective in the fimV mutant. Bacteria were grown overnight in L broth with 5 mM EGTA and both culture supernatants and cell extracts were separated by SDS-PAGE. The proteins were transferred onto nitrocellulose membrane and blotted with anti-FLAG antibody. Strains: wt, wild-type strain PAK; fimV ”, fimV mutant; exsA”; exsA mutant. Plasmids: S, T and pppB are exoS, exoT and pppB with FLAG-tag, respectively. 542

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Fig. 3. Comparison of the type III gene expression in wild-type PAK (wt), fimV mutant (fimV ”) and exsA mutant (exsA”) backgrounds. Bacteria were grown in L broth (white bars) or in L broth supplemented with 5 mM EGTA (black bars) and b-galactosidase activities were measured (see Methods). Results are means of at least three replicates±SE. Plasmids: V, pDN19lacV vector; S, exoS : : lacZ fusion in pDN19lacV; T, exoT : : lacZ fusion in pDN19lacV.

used. The three fusion constructs were transformed into PAK, PAKfimV 2, and a type III mutant strain PAKexsA2, and b-galactosidase activities were monitored following induction in L broth with 5 mM EGTA. As shown in Fig. 3, the induction of exoS, exoT and exsA was abolished in the fimV mutant background, similar to that in the exsA mutant background, confirming that the fimV mutant is indeed defective in the expression of the whole type III secretion system. Furthermore, since these reporter constructs were transcriptional fusions, the results indicate that type III gene expression is affected at the transcriptional level.

A non-polar fimV mutant strain of PAK was further generated by double crossing into the chromosome a fimV mutant whose translational start codon ATG was changed into CTG. The resulting non-polar fimV mutant strain was completely defective in twitching motility while its type III gene expression was unaffected (data not shown). To further confirm the role of truA in type III gene expression, a truA null mutant was generated in the PAK background by the insertion of the V fragment. This mutant did not show a significant difference in growth from that of the wild-type. As expected, the truA mutant did not show a

The truA gene is required for the induction of type III secretion genes A cosmid clone containing a 27 kb insert covering the fimV region of the P. aeruginosa chromosome (pMO010631) was able to complement the fimV mutant for both twitching motility and type III expression. However, a PCR clone of the intact fimV gene, pHW01116, was only able to complement the twitching motility defect (Fig. 1) but not the type III secretion defect (Fig. 4), suggesting the requirement of additional genes for rescuing type III expression. We searched for neighbouring genes that might be affected by a polar effect upon insertional disruption of the fimV. An open reading frame (truA) with high homology to the hisT gene of E. coli, encoding tRNA pseudouridine synthase, was identified downstream of the fimV gene on the P. aeruginosa genome sequence (http://www.pseudomonas. com). Since there is no obvious promoter sequence 59 to truA, it is likely that the truA gene forms an operon with the upstream fimV gene. To test if the truA gene is required for complementation of the type III defect in the fimV mutant, plasmids harbouring truA, alone or together with fimV, pKS0207 and pKS0209, were constructed. As shown in Fig. 4, in contrast to the fimV-only clone, secretion of the ExoS and ExoT proteins in the fimV mutant was restored by the introduction of truA-containing clones (pKS0207 and pKS0209). However, the truA-only clone (pKS0207) was unable to complement the twitching motility defect of the fimV mutant (Fig. 1). These results demonstrate that fimV is required for the twitching motility while truA is required for the type III gene expression. The two genes are likely to form an operon and disruption of fimV interferes with truA expression through a polar effect. http://mic.sgmjournals.org

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Fig. 4. Complementation of the type III secretion defect of the fimV mutant by various gene clones. Bacteria were grown overnight in L broth with 5 mM EGTA to the same cell density (OD600 4?0); the same amount of culture supernatant for each strain was concentrated with a Centricon-30 and the samples were subjected to 12 % SDS-PAGE and stained with Coomassie blue. Lanes 1, wild-type PAK; 2, exoS and exoT double mutant; 3, fimV mutant; 4, fimV mutant harbouring fimV clone pHW01116; 5, fimV mutant harbouring truA clone pKS0207; 6, fimV mutant harbouring pKS0209, which contains both fimV and truA. 543


Fig. 5. Expression of type III genes in a truA mutant background. Bacteria were grown in L broth with (black bars) or without (white bars) 5 mM EGTA and b-galactosidase activities were measured (see Methods). Results are means of at least three replicates±SE. Strains: wt, wild-type PAK; truA”, truA mutant; exsA”, exsA mutant. Plasmids: V, pDN19lacV vector; S, exoS : : lacZ fusion in pDN19lacV; T, exoT : : lacZ fusion in pDN19lacV.

twitching motility defect (Fig. 1), but the expression of type III secretory genes was almost non-responsive to type III inducing conditions, similar to the phenotype of the fimV mutant, as shown by b-galactosidase activities (Fig. 5). Consistent with this, we failed to detect the ExoS and ExoT from culture supernatants of PAKtruA2 under type III inducing conditions (data not shown). Furthermore, the truA mutant showed the same defect as the fimV mutant in the HeLa cell lifting assay. These type III defect phenotypes were corrected to that of wild-type by introducing a truAcontaining clone (pKS0207) (Fig. 6), but not by the fimVonly clone (pHW01116). Taken together, these results clearly demonstrate that the truA gene is indeed a novel modulator regulating the expression of type III secretion genes in P. aeruginosa.

DISCUSSION Semmler et al. (2000) previously demonstrated that the fimV gene is required for twitching motility in P. aeruginosa. The FimV protein has a peptidoglycan-binding domain, predicted transmembrane domains, a highly acidic C terminus and unusual primary or secondary structure. Overexpression of fimV resulted in an unusual phenotype where the cells were massively elongated and migrated in large convoys at the periphery of the colony. It was suggested that FimV may be involved in remodelling of the peptidoglycan layer to enable assembly of the type IV fimbrial structure and machinery (Semmler et al., 2000). In the current study, a fimV mutant was found to be also defective in type III protein secretion, a defect which could not be complemented by the fimV clone, although the clone complemented the twitching 544

Fig. 6. Complementation of the type III gene expression defect of the truA mutant by truA clone pKS0207. Bacteria were grown in L broth with (black bars) or without (white bars) 5 mM EGTA and b-galactosidase activities were measured (see Methods). Results are means of at least three replicates±SE. Plasmids: V, pDN19lacV vector; exoS, exoS : : lacZ fusion in pDN19lacV; exoT, exoT : : lacZ fusion in pDN19lacV; exsA, exsA : : lacZ in pDN19lacV.

motility defect. This prompted us to test the downstream gene truA, which turned out to be the gene required for the type III gene expression under inducing conditions, both under low calcium conditions and upon contact with host cells. The complementation test results as well as the nonpolar mutant phenotype clearly demonstrated that fimV is required for twitching motility while truA is required for type III gene expression. As summarized in Fig. 7, fimV and truA form an operon; therefore disruption of fimV has a polar effect on the truA gene – the fimV mutant is defective in both twitching motility and type III gene expression while the truA mutant has a normal twitching motility but is defective in type III gene expression. In P. aeruginosa, the fimV gene is positioned between two genes, usg-1 and truA (hisT). Interestingly, in E. coli, usg-1 is located directly upstream of truA and the two genes form an operon (Stover et al., 2000). It is likely that the fimV gene in P. aeruginosa was acquired through horizontal gene transfer, and its insertion site as well as direction ensured the coregulation of the twitching motility and type III secretion apparatus; therefore, these two functions might be closely related. In support of this, all of the mutants that have been identified from the Tn5G insertion bank in the current study, fimV, pilL, cyaA, hepA and nuoE, seem to have defects in twitching motility. Despite the lack of twitching motility, none of these mutants are defective in the HeLa cell binding; in fact the fimV mutant seems to have a higher binding activity than the wild-type. Efforts are under way to elucidate the mechanism underlying this observation. tRNA pseudouridine synthase (Y synthase I) catalyses the conversion of uridine in tRNA to its C-glycoside isomer, pseudouridine (Y) (Ramamurthy et al., 1999). Pseudouridine (5-ribosyluracil) is a ubiquitous yet enigmatic constituent of structural RNAs (transfer, ribosomal, small Microbiology 150


Fig. 7. Gene organization and functional complementation of the fimV and truA mutants for twitching motility and type III expression defects.

nuclear and small nucleolar). Although pseudouridine was the first modified nucleotide to be discovered in RNA, and is the most abundant, its biosynthesis and biological roles have remained poorly understood since its identification as a ‘fifth nucleoside’ in RNA (Charette & Gray, 2000). Y is found in almost all tRNAs, notably as the nearly universal Y 55, after which the TYC stem–loop is named. Y also occurs at the D stem, the anti-codon stem and loop in all three domains of life (archaea, eubacteria and eukaryotes) as well as in organelles (mitochondria and chloroplasts) (Auffinger & Westhof, 1998). Pseudouridinylation plays an important biological role in fine-tuning the structure of those tRNAs in which it occurs, thereby influencing their decoding activity, improving the fidelity of protein biosynthesis, and helping to maintain the proper reading frame (Harrington et al., 1993). In the context of translation, the stability of a tRNA population is conferred in part by the broad range of known tRNA nucleoside modifications, which affect tRNA structure and function in a number of subtle ways. Recently, a strong and specific involvement of tRNA modifications in the adaptation of virulence gene expression to the nutritional quality of the growth medium has been reported (Durand & Bjork, 2003; Urbonavicius et al., 2002). The presence of modified nucleosides in tRNA was also shown to be important for virulence of Shigella (Durant & Davis, 1997), Agrobacterium tumefaciens (Gray et al., 1992), Pseudomonas aeruginosa (Sage et al., 1997; Urbonavicius et al., 2002) and Pseudomonas syringae (Kinscherf & Willis, 2002). Isolation of mutants with defective tRNA modification activity has frequently been associated with altered pathogenicity or metabolic activity phenotypes. The genes miaA and tgt, encoding tRNA isopentyladenosine synthase and tRNA-guanine transglycosylase, respectively, were identified as pathogenicity modulators in Shigella (Durand et al., 1994, 1997). The tgt mutant of Shigella flexneri lacks epoxyQ nucleoside in its subset of tRNA, showing an altered virulence gene expression pattern. The lack of Q in tRNA does not influence the synthesis of virF mRNA but reduces its translation capacity by an unknown mechanism, resulting in a reduced expression of the downstream genes in the cascade required for virulence (Durand et al., 1994). In the http://mic.sgmjournals.org

orp mutant of P. aeruginosa (a homologue of E. coli truB mutant), which exhibits temperature-sensitive growth on solid media and reduced salt tolerance, experimental data suggested that tRNA modification may play a role in potentiating the translation of specific mRNA molecules in osmotically stressed cells (Sage et al., 1997; Urbonavicius et al., 2002). Clearly, a fully modified tRNA seems to be required for the pathogenicity of at least some organisms by affecting the translation of specific mRNAs, the products of which are key elements in the expression of virulence. It has been proposed that the common function of the tRNA pseudouridinylation is to improve reading frame maintenance. The improvement occurs in two principal ways: by promoting the recruitment of the ternary complex to the Asite codon and thereby shortening the pause in the A-site; or by preventing slippage of the peptidyl-tRNA (Urbonavicius et al., 2001). Recently, the pseudouridination of tRNA molecules was implicated in the function and stability of certain tRNA molecules in plant mitochondria, where it is required for processing into mature tRNA, aminoacylation and stabilization of the secondary structure through improved pairing in the stem–loop structures (Auffinger & Westhof, 1998; Durant & Davis, 1999; Fey et al., 2001). Based on the proposed functions of tRNA pseudouridination in plant mitochondria and the fact that TruA affects type III genes at the transcriptional level, it is likely that the TruA of P. aeruginosa is essential for the stability and/or function of certain tRNA molecules that are critical for the translation of a transcriptional regulator which is required for the expression of type III genes. Identification of this unknown transcriptional regulator as well as the target tRNA of the TruA will help us to better understand the mechanism by which the truA gene affects the type III gene expression.

ACKNOWLEDGEMENTS We would like to thank Dr Paul Phibbs of the Pseudomonas Genetic Stock Center for providing the cosmid clone pMO010631. Work in the authors’ laboratory is supported by grants from the American Cancer Society and Cystic Fibrosis Foundation. 545


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