Gallibacterium elongation factor-Tu possesses amyloid-like protein characteristics, participates in cell adhesion, and is present in biofilms.

Gallibacterium, which is a bacterial pathogen in chickens, can form biofilms. Amyloid proteins present in biofilms bind Congo red dye. The aim of this study was to characterize the cell-surface amyloid-like protein expressed in biofilms formed by Gallibacterium strains and determine the relationship between this protein and curli, which is an amyloid protein that is commonly expressed by members of the Enterobacteriaceae family. The presence of amyloid-like proteins in outer membrane protein samples from three strains of G. anatis and one strain of Gallibacterium genomospecies 2 was evaluated. A protein identified as elongation factor-Tu (EF-Tu) by mass spectrometric analysis and in silico analysis was obtained from the G. anatis strain F149T. This protein bound Congo red dye, cross-reacted with anti-curli polyclonal serum, exhibited polymerizing properties and was present in biofilms. This protein also reacted with pooled serum from chickens that were experimentally infected with G. anatis, indicating the in vivo immunogenicity of this protein. The recombinant EF-Tu purified protein, which was prepared from G. anatis 12656-12, polymerizes under in vitro conditions, forms filaments and interacts with fibronectin and fibrinogen, all of which suggest that this protein functions as an adhesin. In summary, EF-Tu from G. anatis presents amyloid characteristics, is present in biofilms and could be relevant for the pathogenesis of G. anatis.

Staphylococcal species heterogeneity in the nasal microbiome following antibiotic prophylaxis revealed by tuf gene deep sequencing.

BACKGROUND: Staphylococci are a major constituent of the nasal microbiome and a frequent cause of hospital-acquired infection. Antibiotic surgical prophylaxis is administered prior to surgery to reduce a patient's risk of postoperative infection. The impact of surgical prophylaxis on the nasal staphylococcal microbiome is largely unknown. Here, we report the species present in the nasal staphylococcal microbiome and the impact of surgical prophylaxis revealed by a novel culture independent technique. Daily nasal samples from 18 hospitalised patients, six of whom received no antibiotics and 12 of whom received antibiotic surgical prophylaxis (flucloxacillin and gentamicin or teicoplanin +/- gentamicin), were analysed by tuf gene fragment amplicon sequencing. RESULTS: On admission to hospital, the species diversity of the nasal staphylococcal microbiome varied from patient to patient ranging from 4 to 10 species. Administration of surgical prophylaxis did not substantially alter the diversity of the staphylococcal species present in the nose; however, surgical prophylaxis did impact on the relative abundance of the staphylococcal species present. The dominant staphylococcal species present in all patients on admission was Staphylococcus epidermidis, and antibiotic administration resulted in an increase in species relative abundance. Following surgical prophylaxis, a reduction in the abundance of Staphylococcus aureus was observed in carriers, but not a complete eradication. CONCLUSIONS: Utilising the tuf gene fragment has enabled a detailed study of the staphylococcal microbiome in the nose and highlights that although there is no change in the heterogeneity of species present, there are changes in abundance. The sensitivity of the methodology has revealed that the abundance of S. aureus is reduced to a low level by surgical prophylaxis and therefore reduces the potential risk of infection following surgery but also highlights that S. aureus does persist.

Translation elongation factor EF-Tu modulates filament formation of actin-like MreB protein in vitro.

EF-Tu has been shown to interact with actin-like protein MreB and to affect its localization in Escherichia coli and in Bacillus subtilis cells. We have purified YFP-MreB in an active form, which forms filaments on glass slides in vitro and was active in dynamic light-scattering assays, polymerizing in milliseconds after addition of magnesium. Purified EF-Tu enhanced the amount of MreB filaments, as seen by sedimentation assays, the speed of filament formation and the length of MreB filaments in vitro. EF-Tu had the strongest impact on MreB filaments in a 1:1 ratio, and EF-Tu co-sedimented with MreB filaments, revealing a stoichiometric interaction between both proteins. This was supported by cross-linking assays where 1:1 species were well detectable. When expressed in E. coli cells, B. subtilis MreB formed filaments and induced the formation of co-localizing B. subtilis EF-Tu structures, indicating that MreB can direct the positioning of EF-Tu structures in a heterologous cell system. Fluorescence recovery after photobleaching analysis showed that MreB filaments have a higher turnover in B. subtilis cells than in E. coli cells, indicating different filament kinetics in homologous or heterologous cell systems. The data show that MreB can direct the localization of EF-Tu in vivo, which in turn positively affects the formation and dynamics of MreB filaments. Thus, EF-Tu is a modulator of the activity of a bacterial actin-like protein.

Reprint of "Assessment of marker proteins identified in whole cell extracts for bacterial speciation using liquid chromatography electrospray ionization tandem mass spectrometry".

Staphylococcal strains (CoNS) were speciated in this study. Digests of proteins released from whole cells were converted to tryptic peptides for analysis. Liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI MS/MS, Orbitrap) was employed for peptide analysis. Data analysis was performed employing the open-source software X!Tandem which uses sequenced genomes to generate a virtual peptide database for comparison to experimental data. The search database was modified to include the genomes of the 11 Staphylococcus species most commonly isolated from man. The number of total peptides matching each protein along with the number of peptides specifically matching to the homologue (or homologues) for strains of the same species were assessed. Any peptides not matching to the species examined were considered conflict peptides. The proteins typically identified with the largest percentage of sequence coverage, number of matched peptides and number of peptides corresponding to only the correct species were elongation factor Tu (EF Tu) and enolase (Enol). Additional proteins with consistently observed peptides as well as peptides matching only homologues from the same species were citrate synthase (CS) and 1-pyrroline-5-carboxylate dehydrogenase (1P5CD). Protein markers, previously identified from gel slices, (aconitate hydratase and oxoglutarate dehydrogenase) were found to provide low confidence scores when employing whole cell digests. The methodological approach described here provides a simple yet elegant way of identification of staphylococci. However, perhaps more importantly the technology should be applicable universally for identification of any bacterial species.

Design and properties of efficient tRNA:EF-Tu FRET system for studies of ribosomal translation.

Formation of the ternary complex between GTP-bound form of elongation factor Tu (EF-Tu) and aminoacylated transfer RNA (aa-tRNA) is a key event in protein biosynthesis. Here we show that fluorescently modified Escherichia coli EF-Tu carrying three mutations, C137A, C255V and E348C, and fluorescently modified Phe-tRNA(Phe) form functionally active ternary complex that has properties similar to those of the naturally occurring (unmodified) complex. Similarities include the binding and binding rate constants, behavior in gel retardation assay, as well as activities in tRNA protection and in vitro translation assays. Proper labeling of EF-Tu was demonstrated in MALDI mass spectroscopy experiments. To generate the mutant EF-Tu, a series of genetic constructions were performed. Two native cysteine residues in the wild-type EF-Tu at positions 137 and 255 were replaced by Ala and Val, respectively, and an additional cysteine was introduced either in position 324 or 348. The assembly FRET assay showed a 5- to 7-fold increase of Cy5-labeled EF-Tu E348C mutant fluorescence upon formation of ternary complex with charged tRNA(Phe)(Cy3-labeled) when the complex was excited at 532 nm and monitored at 665 nm. In a control experiment, we did not observe FRET using uncharged tRNA(Phe)(Cy3), nor with wild-type EF-Tu preparation that was allowed to react with Cy5 maleimide, nor in the absence of GTP. The results obtained demonstrate that the EF-Tu:tRNA FRET system described can be used for investigations of ribosomal translation in many types of experiments.

Identification of Rack1, EF-Tu and Rhodanese as aging-related proteins in human colonic epithelium by proteomic analysis.

The aging process of human colonic epithelium involves a slow decline in physiological vigor and an increasing susceptibility to age-related diseases, especially, colon cancer, but the mechanisms still remain to be elucidated. To reveal the molecular bases of colonic epithelial aging, a proteomic approach was used to screen for differential proteins in the human normal colonic epithelial tissues from young and old people. As a result, 17 differential proteins were identified by two-dimensional electrophoresis and mass spectrometry, and the partial differential proteins were confirmed by immunohistochemistry. Rack1, EF-Tu and Rhodanese, three validated differential proteins, were further investigated for their role in the in vitro cell senescence. Western blot showed that the expression of all the three proteins was downregulated in the senescent NIH/3T3 cells induced by D-galactose as compared to the control cells. Furthermore, knockdown of Rack1 by siRNA could promote NIH/3T3 cell senescence. Taken together, our results suggest that Rack1, EF-Tu and Rhodanese are aging-related proteins in human colonic epithelium, and injury of mitochondrial function and decline of antioxidant capability are important reasons for the aging of human colonic epithelium.

The surface-associated elongation factor Tu is concealed for antibody binding on viable pneumococci and meningococci.

Proteome analyses revealed that elongation factor-Tu (EF-Tu) is associated with cytoplasmic membranes of Gram-positive bacteria and outer membranes of Gram-negative bacteria. It is still debatable whether EF-Tu is located on the external side or the internal side of the membranes. Here, we have generated two new monoclonal antibodies (mAbs) and polyclonal rabbit antibodies against pneumococcal EF-Tu. These antibodies were used to investigate the amount of surface-exposed EF-Tu on viable bacteria using a flow cytometric analysis. The control antibodies recognizing the pneumococcal surface protein A and phosphorylcholine showed a significant binding to viable pneumococci. In contrast, anti-EF-Tu antibodies did not recognize pneumococcal EF-Tu. However, heat killing of pneumococci lacking capsular polysaccharides resulted in specific antibody binding to EF-Tu and, moreover, increased the exposure of recognized phosphorylcholine epitopes. Similarly, our EF-Tu-specific antibodies did not recognize EF-Tu of viable Neisseria meningitidis. However, pretreatment of meningococci with ethanol resulted in specific antibody binding to EF-Tu on outer membranes. Importantly, these treatments did not destroy the membrane integrity as analysed with control mAbs directed against cytoplasmic proteins. In conclusion, our flow cytrometric assays emphasize the importance of using viable bacteria and not heat-killed or ethanol-treated bacteria for surface-localization experiments of proteins, because these treatments modulate the cytoplasmic and outer membranes of bacteria and the binding results may not reflect the situation under physiological conditions.

Palaeotemperature trend for Precambrian life inferred from resurrected proteins.

Biosignatures and structures in the geological record indicate that microbial life has inhabited Earth for the past 3.5 billion years or so. Research in the physical sciences has been able to generate statements about the ancient environment that hosted this life. These include the chemical compositions and temperatures of the early ocean and atmosphere. Only recently have the natural sciences been able to provide experimental results describing the environments of ancient life. Our previous work with resurrected proteins indicated that ancient life lived in a hot environment. Here we expand the timescale of resurrected proteins to provide a palaeotemperature trend of the environments that hosted life from 3.5 to 0.5 billion years ago. The thermostability of more than 25 phylogenetically dispersed ancestral elongation factors suggest that the environment supporting ancient life cooled progressively by 30 degrees C during that period. Here we show that our results are robust to potential statistical bias associated with the posterior distribution of inferred character states, phylogenetic ambiguity, and uncertainties in the amino-acid equilibrium frequencies used by evolutionary models. Our results are further supported by a nearly identical cooling trend for the ancient ocean as inferred from the deposition of oxygen isotopes. The convergence of results from natural and physical sciences suggest that ancient life has continually adapted to changes in environmental temperatures throughout its evolutionary history.

Functional metaproteome analysis of protein extracts from contaminated soil and groundwater.

Using proteins from soil or groundwater as functional biomarkers requires efficient extraction. We developed an extraction method in which the separation of proteins from the inorganic and organic constituents of the soil matrix was achieved by a combination of 0.1 M NaOH treatment and phenol extraction. Incubation with NaOH released humic acids and proteins from soil minerals, and simultaneously, disrupted microorganisms. The subsequent phenol extraction separated the proteins from the humic organic matter. Protein extracts were applied to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 2D-electrophoresis (2-DE). Spots and bands were excised and individual proteins identified by liquid chromatography online linked to mass spectrometry (MS) via electrospray ionization source (LC-ESI-MS). To assess the suitability of the method for the functional analysis of environmental metaproteomes, it was applied to soil that had been enriched in chlorophenoxy acid-degrading bacteria by incubation with 2,4-dichlorophenoxy acetic acid (2,4-D) for 22 days. The method was also used to analyze groundwater from the aquifer of a chlorobenzene-contaminated site. The identification of enzymes such as chlorocatechol dioxygenases was consistent with bacterial metabolic pathways expected to be expressed in these samples. The protocol enabled thus the analysis of the metaproteome of soil and groundwater samples. It thereby provides a means to study the diversity of environmental microbial communities while addressing functional aspects more directly than metagenome or even metatranscriptome analysis.

Immune evasion of the human pathogen Pseudomonas aeruginosa: elongation factor Tuf is a factor H and plasminogen binding protein.

Pseudomonas aeruginosa is an opportunistic human pathogen that can cause a wide range of clinical symptoms and infections that are frequent in immunocompromised patients. In this study, we show that P. aeruginosa evades human complement attack by binding the human plasma regulators Factor H and Factor H-related protein-1 (FHR-1) to its surface. Factor H binds to intact bacteria via two sites that are located within short consensus repeat (SCR) domains 6-7 and 19-20, and FHR-1 binds within SCR domain 3-5. A P. aeruginosa Factor H binding protein was isolated using a Factor H affinity matrix, and was identified by mass spectrometry as the elongation factor Tuf. Factor H uses the same domains for binding to recombinant Tuf and to intact bacteria. Factor H bound to recombinant Tuf displayed cofactor activity for degradation of C3b. Similarly Factor H bound to intact P. aeruginosa showed complement regulatory activity and mediated C3b degradation. This acquired complement control was rather effective and acted in concert with endogenous proteases. Immunolocalization identified Tuf as a surface protein of P. aeruginosa. Tuf also bound plasminogen, and Tuf-bound plasminogen was converted by urokinase plasminogen activator to active plasmin. Thus, at the bacterial surface Tuf acts as a virulence factor and binds the human complement regulator Factor H and plasminogen. Acquisition of host effector proteins to the surface of the pathogen allows complement control and may facilitate tissue invasion.

Two-dimensional differential gel electrophoresis of rat heart proteins in ischemia and ischemia-reperfusion.

Ischemia-reperfusion injury occurs in acute myocardial infarction, cardiopulmonary bypass surgery, and heart transplantation. However the precise mechanisms still remain unclear. In order to identify proteins that are involved in ischemia-reperfusion injury, we compared precipitated 100,000g fractions of normal, ischemic, and ischemic-reperfused rat hearts using two-dimensional (2D) difference gel electrophoresis (2D-DIGE). 2D-DIGE is reliable method to define quantitative protein differences, especially when subtle protein changes are under investigation. In this study, six spots that changed more than twofold and two additional spots related to these spots were detected. Five of the spots were identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry as protein disulfide isomerase, one as 60 kDa heat-shock protein, and two as elongation factor Tu.

Quantitative analysis of representative proteome components and clustering of Helicobacter pylori clinical strains.

BACKGROUND: Several Helicobacter pylori proteins have been reported to be associated with severe symptoms of gastric disease. However, expression levels of most of these disease-associated proteins require further evaluation in order to clarify their relationships with gastric disease patterns. Representative proteome components of 71 clinical isolates of H. pylori were analyzed quantitatively to determine whether the protein expression levels were associated with gastric diseases and to cluster clinical isolates. METHODS: After two-dimensional electrophoresis (2-DE) of H. pylori isolates, spot intensities were analyzed using pdquest 2-D Gel Analysis Software. The intensities of 10 representative protein spots, identified by peptide fingerprinting using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) or peptide sequencing using quadrupole TOF MS, were subjected to the nonparametric Mann-Whitney test and hierarchical agglomerative cluster analysis. The relationship between clusters and gastric diseases was analyzed by the chi-squared test. RESULTS: Although the spot intensities of the 10 representative proteins were highly variable within each gastric disease group, the expression levels of CagA, UreB, GroEL, EF-Tu, EF-P, TagD, and FldA showed some significant differences among the gastric disease patterns. On the basis of the 10 target protein intensities, hierarchical agglomerative cluster analysis generated a dendrogram with clusters indicative of chronic gastritis/gastric cancers and gastric/duodenal ulcers. CONCLUSION: These results indicated that quantitative analysis of proteome components is a feasible method for examining disease-associated proteins and clustering clinical strains of H. pylori.

[Effects of the Radix Ranuncoli Ternati extracts on Mycobacterium tuberculosis proteome profiling revealed by 2D electrophoresis].

Radix Ranuncoli Ternati is clinically effective traditional Chinese medicine for multidrug resistant tuberculosis. Its active components and mechanism of action remain unsolved. Two dimensional gel electrophoresis (2-DE) was employed to address this problem. Globlal proteome of Mycobacterium tuberculosis untreated and treated with Radix Ranuncoli Ternati were compared, and 22 protein spots were found to be expressed differentially. 3 protein spots which remarkably decreased in Mycobacterium tuberculosis treated with Radix Ranuncoli Ternati were subjected to matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis. The data obtained from peptide mass finger printing were used for database search. The 3 protein spots in gel were identified as cysA2 (thiosulfate sulfurtransferase), tsf (elongation factor EF-Ts) and hspX (heat shock protein X). These data provide insights into the changed global protein patterns of Mycobacterium tuberculosis treated with Radix Ranuncoli Ternati and may prove useful for further study in the mechanism in how Radix Ranuncoli Ternati influence the life of Mycobacterium tuberculosis. The differentially expressed proteins may be potential novel antituberculosis drug targets.

Molecular detection of Enterococcus species in root canals of therapy-resistant endodontic infections.

OBJECTIVES: The objectives were to identify Enterococcus spp in nonhealing endodontic cases using PCR amplification and molecular sequencing, and to determine if the prevalence of enterococci is increased in diabetic patients. STUDY DESIGN: Specimens from 40 cases undergoing retreatment were incubated in prereduced thioglycollate broth at 37 degrees C. Extracted DNA had PCR amplification using primers that target the tuf gene of 14 Enterococcus spp. PCR products were directly sequenced and identified phylogenetically. RESULTS: Three cases were eliminated because the patients were on antibiotics or the tooth did not have a periradicular radiolucency. The remaining 37 specimens included 6 from diabetic patients. Eight specimens were positive for Enterococcus spp. Of these, 6 (19%) were from nondiabetic and 2 (33%) from diabetic patients (odds ratio = 2.1; chi squared, P = .45). Phylogenetically, all sequences from positive specimens matched E faecalis V583 (AE016947). Conclusion E faecalis was the only enterococcal species detected, with an overall prevalence of 22%.

Comparative proteome analysis of Mycobacterium tuberculosis grown under aerobic and anaerobic conditions.

Data are presented from two-dimensional (2-D) PAGE analysis of Mycobacterium tuberculosis strain Harlingen grown during aerobic and anaerobic culture, according to a modified Wayne dormancy model. M. tuberculosis cultures were grown to the transition point between exponential growth and stationary phase in the presence of oxygen (7 days) and then part of the cultures was shifted to anaerobic conditions for 16 days. Growth declined similarly during aerobic and anaerobic conditions, whereas the ATP consumption rapidly decreased in the anaerobic cultures. 2-D PAGE revealed 50 protein spots that were either unique to, or more abundant during, anaerobic conditions and 16 of these were identified by MALDI-TOF. These proteins were the alpha-crystalline homologue (HspX), elongation factor Tu (Tuf), GroEL2, succinyl-CoA : 3-oxoacid-CoA transferase (ScoB), mycolic acid synthase (CmaA2), thioredoxin (TrxB2), beta-ketoacyl-ACP synthase (KasB), l-alanine dehydrogenase (Ald), Rv2005c, Rv2629, Rv0560c, Rv2185c and Rv3866. Some protein spots were found to be proteolytic fragments, e.g. HspX and GroEL2. These data suggest that M. tuberculosis induces expression of about 1 % of its genes in response to dormancy.

Analysis of conserved non-rRNA genes of Tropheryma whipplei.

The causative agent of Whipple's disease, Tropheryma whipplei, is a slow-growing bacterium that remains poorly-understood. Genetic characterization of this organism has relied heavily upon rRNA sequence analysis. Pending completion of a complete genome sequencing effort, we have characterized several conserved non-rRNA genes from T. whipplei directly from infected tissue using broad-range PCR and a genome-walking strategy. Our goals were to evaluate its phylogenetic relationships, and to find ways to expand the strain typing scheme, based on rDNA sequence comparisons. The genes coding for the ATP synthase beta subunit (atpD), elongation factor Tu (tuf), heat shock protein GroEL (groEL), beta subunit of DNA-dependent RNA polymerase (rpoB), and RNase P RNA (rnpB) were analyzed, as well as the regions upstream and downstream of the rRNA operon. Phylogenetic analyses with all non-rRNA marker molecules consistently placed T. whipplei within the class, Actinobacteria. The arrangement of genes in the atpD and rpoB chromosomal regions was also consistent with other actinomycete genomes. Tandem sequence repeats were found upstream and downstream of the rRNA operon, and downstream of the groEL gene. These chromosomal sites and the 16S-23S rRNA intergenic spacer regions were examined in the specimens of 11 patients, and a unique combination of tandem repeat numbers and spacer polymorphisms was found in each patient. These data provide the basis for a more discriminatory typing method for T. whipplei.

Proteome analysis of highly immunoreactive proteins of Helicobacter pylori.

BACKGROUND: Identification of the immunoreactive proteins of Helicobacter pylori is important for the development of both diagnostic tests and vaccines relating to the organism. Our aim was to determine whether there are significant differences between human IgG and IgA reactivities to individual H. pylori proteins, and whether patterns of immunoreactivity are sustained across different strains of H. pylori. METHOD: The total complement of protein from seven strains of H. pylori was resolved by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Proteins were transferred electrophoretically onto polyvinylene difluoride (PVDF) membranes, which were probed with sera pooled either from H. pylori-infected patients, or noninfected (control) patients. Highly immunoreactive proteins were detected using chromogenic enzyme-antibody conjugates recognising either serum IgG or IgA. These proteins were then characterised by tryptic peptide-mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). RESULTS: Highly immunoreactive proteins were detected which were common to all seven strains, and recognised by both immunoglobulin subclasses. The proteins appear to be localised in five groups. Protein analysis established that these groups encompass multiple isoforms of chaperonin HspB (two subgroups); urease beta-subunit UreB; elongation factor EF-Tu; and flagellin FlaA. The pattern of highly immunoreactive proteins was strongly conserved across the seven strains. CONCLUSION: These results suggest that within a tightly defined region on the H. pylori proteome map there are five groups of proteins that are highly reactive to both IgG and IgA. Our analysis suggests it is unlikely that the highly immunoreactive clusters harbour any significant proteins other than isoforms of HspB, UreB, EF-Tu and FlaA, and that, with the partial exception of FlaA, these clusters are strongly conserved across all seven strains.

The effect of mutations in EF-Tu on its affinity for tRNA as measured by two novel and independent methods of general applicability.

Elongation factor Tu is essential for binding and a correct delivery of aminoacyl-tRNA during protein biosynthesis. For a good characterization of its interaction with tRNA in terms of structure-function relationship, determinations of kinetic equilibrium parameters are of great value. We describe two novel methods for that purpose. One method is based on EF-Tu protection of the tRNA 3' acceptor end against RNase A cleavage and yields the Kd value together with the corresponding dissociation and association rate constants from one single set of experiments. The other is a rapid method for screening relative affinities of mutant EF-Tus for tRNA. It is based on competition between EF-Tu species with and without a (His)6 extension for the same aminoacyl-tRNA and yields a relative Kd value. The method can be of general importance for the measuring of ligand affinities of all sorts of His-tagged proteins. Both methods are illustrated by their application in the analysis of mutant EF-Tus with changed interactions with tRNA and antibiotics. Raising the assay temperature from 4 to 37 degrees C causes a 30-fold increase of Kd for EF-Tu x GTP x Phe-tRNA complexes. The mutation K237E leads to rapid inactivation at the latter temperature. A parallel is found between the order of increasing Kd values for EF-Tus with mutation G316D, A375T and Q124K, respectively, and their order of increasing resistance to kirromycin.

Defining protease specificity with proteomics: a protease with a dibasic amino acid recognition motif is regulated by a two-component signal transduction system in Salmonella.

Microbial proteases play diverse and important roles in bacterial virulence but their detection and characterisation is often hampered by their limited abundance or lack of expression in the absence of suitable environmental signals. We describe here a sensitive proteomic approach to detect proteases that are under the control of a virulence regulator and to characterise their recognition motifs. Using MG++-depleted growth media or a mutant strain of Salmonella in which the PhoP-PhoQ virulence regulatory system is constitutively active, truncated forms of DnaK, elongation factor G, elongation factor Tu and ribosomal protein S1 proteins were detected. Two other global regulatory mutants and cells exposed to acid or to oxidative stress failed to produce the truncated proteins, indicating specific control of the protease activity by the PhoP-PhoQ system. Our results suggest that at least two proteases are induced. To define the proteolytic cleavage sites of one of the proteases, peptides from each of the truncated proteins were identified by tryptic mass fingerprinting/nanoelectrospray mass spectrometry and mapped onto the sequence of the intact protein. Alignment of the regions around the cut site indicates that the protease recognises a dibasic amino acid motif characteristic of the omptin protease family. The induction of such proteases in bacteria depleted of Mg++ ions may contribute to the PhoPQ-mediated resistance of Salmonella to cationic antimicrobial peptides. Additionally, our results suggest it would be prudent to keep the concentration of this ion above micromolar levels during bacterial sample preparation for proteomic analyses.