1 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610, USA
2 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
Correspondence
Shouguang Jin
sjin{at}mgm.ufl.edu
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ABSTRACT |
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INTRODUCTION |
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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 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 stemloop 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.
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METHODS |
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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 (5'-GTC TCT TCG ACC GCC AGA TCG CCT TCA ACC TGC TC-3') with fimV-2 (5'-CAT CGT TCA TGA GGG TCG CTT CAT TTC CGG ATC AG -3'); and truA-1 (5'-TCC TCG ACG AAG TCC TGG CCG AAG GTA ATG ACA GC-3') with truA-2 (5'-TCT CGA TGG TAG CAA AAG CCC GAT TCG ACG TCA GC-3'), 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 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 (5'-GAT ATT AAA AGG GAT TAC ACT GCA GTT CGG CTT CGT ACA CTG-3') and fimV-sd-b (5'-CAG TGT ACG AAG CCG AAC TGC AGT GTA ATC CCT TTT AAT ATC-3') 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 µl using a Centricon-30, mixed with an equal volume of 2x 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)
. FLAG-tagged 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 overnight at 37 °C, 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 -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.
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RESULTS |
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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 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).
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DISCUSSION |
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tRNA pseudouridine synthase ( synthase I) catalyses the conversion of uridine in tRNA to its C-glycoside isomer, pseudouridine (
) (Ramamurthy et al., 1999
). Pseudouridine (5-ribosyluracil) is a ubiquitous yet enigmatic constituent of structural RNAs (transfer, ribosomal, small 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
).
is found in almost all tRNAs, notably as the nearly universal
55, after which the T
C stemloop is named.
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 epoxy-Q 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 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 A-site 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 stemloop 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.
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ACKNOWLEDGEMENTS |
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Received 17 July 2003;
revised 5 November 2003;
accepted 28 November 2003.
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