Eleonora Garcı́a Véscovi

The Salmonella typhimurium PhoP-PhoQ two-component regulatory system controls the expression of several genes, some of which are necessary for virulence. During a screening for PhoP-regulated genes, we identified the phoPQ operon as a PhoP-activated locus. beta-Galactosidase activity originating from phoPQ-lac transcriptional fusions required the presence of both the transcriptional regulator PhoP and its cognate sensor-kinase PhoQ. At low concentrations, PhoQ stimulated expression of phoPQ-lac transcriptional fusions. However, larger amounts of PhoQ protein without a concomitant increase in PhoP failed to activate phoPQ-lac fusions. Two different transcripts are produced from the phoPQ operon during exponential growth. These transcripts define two promoters: phoPp1, which requires both PhoP and PhoQ for activity and which is environmentally regulated, and phoPp2, which remains active in the absence of PhoP and PhoQ but which is slightly stimulated by these proteins. The pattern of transcriptional autoregulation was also observed at the protein level with anti-PhoP antibodies. In sum, autoregulation of the phoPQ operon provides several levels of control for the PhoP-PhoQ regulon. First, environmental signals would stimulate PhoQ to phosphorylate the PhoP protein that is produced at basal levels from the PhoP-PhoQ-independent promoter. Then, phospho-PhoP would activate transcription of phoPp1, resulting in larger amounts of PhoP and PhoQ and increased expression of PhoP-activated genes. A return to basal levels could be mediated by a posttranscriptional mechanism by which translation of the mRNA produced from phoPp1 is inhibited.

Outer membrane vesicles (OMVs) have been identified in a wide range of bacteria, yet little is known of their biogenesis. It has been proposed that OMVs can act as long-range toxin delivery vectors and as a novel stress response. We have found that the formation of OMVs in the gram-negative opportunistic pathogen Serratia marcescens is thermoregulated, with a significant amount of OMVs produced at 22 or 30°C and negligible quantities formed at 37°C under laboratory conditions. Inactivation of the synthesis of the enterobacterial common antigen (ECA) resulted in a hypervesiculation phenotype, supporting the hypothesis that OMVs are produced in response to stress. We demonstrate that the phenotype can be reversed to wild-type (WT) levels upon the loss of the Rcs phosphorelay response regulator RcsB, but not RcsA, suggesting a role for the Rcs phosphorelay in the production of OMVs. MS fingerprinting of the OMVs provided evidence of cargo selection within wild-type cells, suggesting a possible role for Serratia OMVs in toxin delivery. In addition, OMV-associated cargo proved toxic upon injection into the haemocoel of Galleria mellonella larvae. These experiments demonstrate that OMVs are the result of a regulated process in Serratia and suggest that OMVs could play a role in virulence.

The PhoP/PhoQ two-component system controls the expression of essential virulence traits in the pathogenic bacterium Salmonella enterica serovar Typhimurium. Environmental deprivation of Mg2+ activates the PhoP/PhoQ signal transduction cascade, which results in an increased expression of genes necessary for survival inside the host. It was previously demonstrated that the interaction of Mg2+ with the periplasmic domain of PhoQ promotes a conformational change in the sensor protein that leads to the down-regulation of PhoP-activated genes. We have now examined the regulatory effect of Mg2+ on the putative activities of the membrane-bound PhoQ. We demonstrated that Mg2+ promotes a phospho-PhoP phosphatase activity in the sensor protein. This activity depends on the intactness of the conserved His-277, suggesting that the phosphatase active site overlaps the H box. The integrity of the N-terminal domain of PhoQ was essential for the induction of the phosphatase activity, because Mg2+ did not stimulate the release of inorganic phosphate from phospho-PhoP in a fusion protein that lacks this sensing domain. These findings reveal that the sensor PhoQ harbors a phospho-PhoP phosphatase activity, and that this phosphatase activity is the target of the extracellular Mg2+-triggered regulation of the PhoP/PhoQ system. The PhoP/PhoQ two-component system controls the expression of essential virulence traits in the pathogenic bacterium Salmonella enterica serovar Typhimurium. Environmental deprivation of Mg2+ activates the PhoP/PhoQ signal transduction cascade, which results in an increased expression of genes necessary for survival inside the host. It was previously demonstrated that the interaction of Mg2+ with the periplasmic domain of PhoQ promotes a conformational change in the sensor protein that leads to the down-regulation of PhoP-activated genes. We have now examined the regulatory effect of Mg2+ on the putative activities of the membrane-bound PhoQ. We demonstrated that Mg2+ promotes a phospho-PhoP phosphatase activity in the sensor protein. This activity depends on the intactness of the conserved His-277, suggesting that the phosphatase active site overlaps the H box. The integrity of the N-terminal domain of PhoQ was essential for the induction of the phosphatase activity, because Mg2+ did not stimulate the release of inorganic phosphate from phospho-PhoP in a fusion protein that lacks this sensing domain. These findings reveal that the sensor PhoQ harbors a phospho-PhoP phosphatase activity, and that this phosphatase activity is the target of the extracellular Mg2+-triggered regulation of the PhoP/PhoQ system. Luria-Bertani broth isopropyl-β-d-thiogalactopyranoside polyacrylamide gel electrophoresis polyethyleneimine maltose binding protein Reversible protein phosphorylation is one of the most conspicuous mechanisms that regulate biological processes in cells, including modulation of enzymatic activities, protein-protein and protein-DNA interactions, and extracellular signal transduction. In prokaryotes and in lower eukaryotes and plants (1Perraud A.-L. Weiss V. Gross R. Trends Microbiol. 1999; 7: 115-120Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), the most widespread and efficient sensory-response devices rely on protein phosphotransfer. They are the so-called two-component regulatory systems that enable bacteria to monitor changes in their environment and adjust their structure and physiology accordingly to survive. The prototypical architecture of the two-component regulatory systems consists of a sensor protein and an associated effector protein (2Stock J.B. Surette M.G. Levit M. Park P. Hoch J.A. Silhavy T.J. Two-component Signal Transduction. ASM Press, Washington, DC1995: 25-51Google Scholar, 3Parkinson J.S. Cell. 1993; 73: 857-871Abstract Full Text PDF PubMed Scopus (614) Google Scholar). The sensor is generally a membrane protein whose C-terminal domain projects into the cytoplasm. This domain harbors a histidine autokinase activity and, in several sensors of this large family, a phosphatase activity (2Stock J.B. Surette M.G. Levit M. Park P. Hoch J.A. Silhavy T.J. Two-component Signal Transduction. ASM Press, Washington, DC1995: 25-51Google Scholar, 4Cavicchioli R. Schröder I. Constanti M. Gunsalus R.P. J. Bacteriol. 1995; 177: 2416-2424Crossref PubMed Google Scholar, 5Ninfa A.J. Neidhardt F.C. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia Coli and Salmonella: Cellular and Molecular Biology. 2nd Ed. 1. ASM Press, Washington, DC1996: 1246-1262Google Scholar, 6Hsing W. Silhavy T.J. J. Bacteriol. 1997; 179: 3729-3735Crossref PubMed Google Scholar, 7Hsing W. Russo F.D. Bernd K.K. Silhavy T.J. J. Bacteriol. 1998; 180: 4538-4546Crossref PubMed Google Scholar, 8Jiang P. Ninfa A.F. J. Bacteriol. 1999; 181: 1906-1911Crossref PubMed Google Scholar, 9Shi L. Liu W. Hulett F.M. Biochemistry. 1999; 38: 10119-10125Crossref PubMed Scopus (24) Google Scholar). The cognate response-regulator is most often a transcriptional regulator protein. Its N-terminal domain harbors a conserved aspartate that accepts the phosphate from the phosphohistidyl residue of the sensor protein. This modification is propagated to the C-terminal domain affecting its DNA binding properties (2Stock J.B. Surette M.G. Levit M. Park P. Hoch J.A. Silhavy T.J. Two-component Signal Transduction. ASM Press, Washington, DC1995: 25-51Google Scholar). Two major biochemical activities play an opposite role to balance the phosphorylation status of the response regulator: the autokinase activity of the sensor that defines the phosphotransfer availability and a specific phosphatase that dephosphorylates the response regulator. By modulating these reactions, the sensor component defines the phosphorylation status of the response regulator, which directs the expression of a specific set of target genes. This modulation results in the final adaptive response of the bacteria to its primary signal. Despite a considerable amount of research on different two-component systems, carried out to understand how the signal regulates the catalytic activities of the sensor to control the phosphorylation state in the effector molecule, a clear picture of this mechanism has not yet emerged. In Salmonella, the PhoP/PhoQ two-component system governs the adaptation to environmental Mg2+ deprivation and controls the expression of essential virulence factors (10Garcı́a Véscovi E. Soncini F.C. Groisman E.A. Cell. 1996; 84: 165-174Abstract Full Text Full Text PDF PubMed Scopus (675) Google Scholar, 11Soncini F.C. Garcı́a Véscovi E. Solomon F. Groisman E.A. J. Bacteriol. 1996; 178: 5092-5099Crossref PubMed Scopus (268) Google Scholar, 12Guo L. Lim K.B. Gunn J.S. Bainbridge B. Darveau R.P. Hackett M. Miller S.I. Science. 1997; 276: 250-253Crossref PubMed Scopus (480) Google Scholar, 13Guo L. Lim K.B. Poduje C.M. Daniel M. Gunn J.S. Hackett M. Miller S.I. Cell. 1998; 95: 189-198Abstract Full Text Full Text PDF PubMed Scopus (524) Google Scholar, 14Blanc-Potard A.-B. Groisman E.A. EMBO J. 1997; 16: 5376-5385Crossref PubMed Scopus (272) Google Scholar). It was previously demonstrated that PhoQ is the sensor of the system that specifically recognizes extracellular Mg2+. The interaction of the sensing periplasmic domain with the divalent cation promotes a conformational change in the protein that results in the repression of the transcription of at least 20 different PhoP-activated loci (10Garcı́a Véscovi E. Soncini F.C. Groisman E.A. Cell. 1996; 84: 165-174Abstract Full Text Full Text PDF PubMed Scopus (675) Google Scholar, 11Soncini F.C. Garcı́a Véscovi E. Solomon F. Groisman E.A. J. Bacteriol. 1996; 178: 5092-5099Crossref PubMed Scopus (268) Google Scholar,15Véscovi E.G. Ayala Y.M. DiCera E. Grosiman E.A. J. Biol. Chem. 1997; 272: 1440-1443Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). However, it was not clear which activity or activities that reside in PhoQ were affected by the Mg2+-induced conformational switch of the sensor. In this work we examined how Mg2+, as the specific regulatory signal, controls the phosphorylation state of PhoP. We demonstrated that the interaction of Mg2+ with PhoQ activates a phosphatase activity of the sensor protein that promotes the dephosphorylation of phospho-PhoP. Stimulation by Mg2+of this phosphatase activity required the N-terminal domain of the sensor, because no phosphatase was detected in a fusion protein lacking this domain while it retained the reversible autokinase and phosphotransfer activities. These results reveal that the sensor PhoQ harbors a phosphatase activity that acts on PhoP and that this phosphatase activity is the target of the Mg2+-triggered regulation of the PhoP/PhoQ system. Nitrocellulose membranes were from Bio-Rad. [γ-32P]ATP was obtained from NEN Life Science Products. The oligonucleotides were purchased from Bio-Synthesis, Inc. (Lewisville, TX). Cell culture media reagents were from Difco, and chemicals were from Sigma. Salmonella enterica serovar Typhimurium (S. typhimurium) strain EG5172 (phoQ::MudJ) was transformed with plasmid pEG9050 (16Soncini F.C. Garcı́a Véscovi E. Groisman E.A. J. Bacteriol. 1995; 177: 4364-4371Crossref PubMed Scopus (208) Google Scholar) or with the vector pUHE21-2lacI Q (16Soncini F.C. Garcı́a Véscovi E. Groisman E.A. J. Bacteriol. 1995; 177: 4364-4371Crossref PubMed Scopus (208) Google Scholar). These strains were used for the isolation of membranes harboring PhoQ or the control membranes, respectively. Escherichia colistrain PB1277 is BL21[DE3] carrying plasmid pPB1020. pPB1020 harbors a phoP His-tag fusion gene (full-length phoPfused to 6 His codons in its C terminus) under the control of the T7 φ10 promoter of the pT7-7 plasmid. This fusion gene was generated by polymerase chain reaction, using forward primer PhoP-NTF (5′-GAGGATCCATATGATGCGCGTACTGG-3′) and reverse primer PhoP-H6-CTR (5′-TCCAAGCTTAGTGGTGGTGGTGGTGGTGGCGCAATTCAAAAAGATATC-3′), and cloned between the NdeI and HindIII sites of plasmid pT7-7. The phoP His-tag fusion gene cloned into pUHE21-2lacI Q complemented aSalmonella phoP − strain to the same extent as the wild-type phoP. This shows that the addition of the His-tag to the PhoP C terminus does not affect its properties. 1M. E. Castelli, E. Garcı́a Véscovi, and F. C. Soncini, unpublished results. pPB1021 is a pMAL-c2 (New England Biolabs, Beverly, MA) derivative that encodes for the maltose binding protein fused to the C-terminal cytoplasmic region of PhoQ (MBP-Qc). The gene fusion was constructed by subcloning the phoQ fragment excised from pEG9050 encompassed between the StuI and the HindIII sites (which encodes from the Pro-220 to the C-terminal Glu-487 of PhoQ) into the vector pMAL-c2 between the XmnI and HindIII sites, respectively. Bacteria were grown at 37 °C in Luria-Bertani (LB)2 broth with shaking, and with the addition of 0.7 mmisopropyl-β-d-thiogalactopyranoside (IPTG) when indicated. Ampicillin was used at a final concentration of 50 μg/ml. Plasmid DNA was introduced into bacterial strains by electroporation using a Bio-Rad apparatus as recommended by the manufacturer. Recombinant DNA techniques were performed according to standard protocols (17Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). To replace His-277 with Val in PhoQ, and Asp-55 with Ala in PhoP, we used the site-directed mutagenesis protocol described by Deng et al. (18Deng W.P. Nickoloff J.A. Anal. Biochem. 1992; 200: 81-90Crossref PubMed Scopus (1079) Google Scholar). Polymerase chain reaction-derived constructions and site-directed mutagenesis were all confirmed by DNA sequence analysis performed using the femtomole DNA sequencing system as recommended by the manufacturer (Promega). Overnight cultures of S. typhimurium strains EG5172/pEG9050 and EG5172/pUHE21-2lacI Q were used to inoculate LB media containing 50 μg/ml ampicillin. They were then grown at 37 °C to logarithmic phase (OD 0.6) and induced by addition of 0.7 mm IPTG for an additional 3 h with shaking. Cells were collected, washed once with 10 mm Tris-HCl (pH 8.0), and resuspended in a solution containing 20 mm Tris-HCl (pH 8.0), 20% sucrose, 5 mm M EDTA, and 150 μg/ml lysozyme. After 40-min incubation at 4 °C, 20 mm MgCl2was added and cells were centrifuged for 20 min at 18,000 ×g. The pellet was resuspended in ice-cold 10 mmTris-HCl (pH 8.0) and subjected to sonication. Nonruptured spheroplasts were removed by a brief centrifugation at 7000 × g, and the membrane fraction was recovered after a 45-min centrifugation at 30,000 × g. The membranes were resuspended in 10 mm Tris-HCl (pH 8.0), 2 m KCl and centrifuged 30 min at 30,000 × g. The supernatant was discarded, and the pellet was resuspended in 10 mm Tris-HCl (pH 8.0), 5 mm EDTA. Finally, the membranes were washed twice with 10 mm Tris-HCl (pH 8.0) and resuspended in the same buffer at a final protein concentration of 10 mg/ml. All procedures were carried out at 4 °C. Protein concentration was determined by the bicinchoninic acid assay (Bio-Rad) using bovine serum albumin as standard. The His6-tagged fusion protein PhoP-H6 was purified from strain PB1277. Expression of PhoP-H6 was achieved by addition of 0.7 mm IPTG to induce the DE3-encoded T7 RNA polymerase. Cells were pelleted, resuspended in sonication buffer (50 mm sodium phosphate (pH 6.0), 300 mm NaCl) and subjected to sonication. Cell debris was removed by centrifugation, and the supernatant was passed through a Ni2+-NTA-agarose affinity column equilibrated with 50 mm sodium phosphate (pH 6.0), 300 mm NaCl, 10% glycerol. The PhoP-H6 protein was recovered by elution with 0.5m imidazole and dialyzed exhaustively against 25 mm Tris-HCl (pH 8.0), 50 mm KCl. The MBP-Qc fusion protein was purified from the E. coli strain TB1 (obtained from the pMAL Protein Fusion and Purification System, NEB, Inc.) transformed with plasmid pPB1021. Expression of MBP-Qc was achieved by addition of 0.3 mm IPTG to a log-phase cell culture in LB broth. After sonication and centrifugation, the soluble cellular extract containing the MBP-Qc protein was collected and subjected to amylose affinity chromatography analysis following the manufacturer's instructions (NEB, Inc.). All procedures were carried out at 4 °C. The protein profile of the purified proteins was determined by SDS-polyacrylamide gel electrophoresis (PAGE). For the autokinase assay, membranes (50 μg of total protein) harboring PhoQ were incubated with 50 μm[γ-32P]ATP (7500 cpm/pmol, NEN Life Science Products), in a 30-μl reaction mixture containing 20 mm Tris-HCl (pH 8.0), 50 mm KCl (buffer TK). Unless stated otherwise, 1 mm MgCl2 was added to the reaction medium. Reactions were started by addition of the membrane fraction, incubated 5 min at 37 °C, and stopped by addition of 6 μl of 5 × SDS-PAGE sample buffer (2.5% β-mercaptoethanol, 9% glycerol, 10% SDS, 600 mm Tris-HCl (pH 6.8), 0.006% bromphenol blue). The amount of radiolabeled PhoQ increased proportionally to the amount of membrane protein used in the range from 0.16 to 2.6 μg/μl; this indicated that PhoQ was not in excess at 1.66 μg/μl, the membrane concentration used in the autophosphorylation assay. To remove remnant Mg2+, ATP, and free inorganic phosphate, the membranes containing autophosphorylated PhoQ were washed once with buffer TK with 5 mm EDTA, followed by two washes with buffer TK. This fraction was analyzed by TLC to confirm absence of residual ATP and inorganic phosphate and used to test the stability of phosphorylated PhoQ and for the PhoQ → PhoP phosphotransfer assay. The phosphotransfer assay was carried out as follows: typically, membranes (50 μg of total protein) harboring phosphorylated PhoQ were coincubated in a 30-μl total volume with 10 μg of purified PhoP-H6 in a reaction buffer consisting of 20 mm Tris-HCl (pH 8.0) and 50 mm KCl for different periods of time. MgCl2 was added to the reaction as indicated in each individual assay. Reactions were stopped by addition of 5 × SDS-PAGE loading buffer. To obtain isolated phosphorylated PhoP-H6, 10 μg of purified PhoP-H6 was added to a 30-μl final volume containing membranes (50 μg of total protein) harboring PhoQ, 50 μm [γ-32P]ATP (7500 cpm/pmol), and 1 mm MgCl2 in buffer TK, and incubated for 5 min at 37 °C. This reaction mixture was centrifuged at 24,000 ×g for 45 min at 4 °C to remove the membrane fraction. The supernatant containing the phosphorylated PhoP-H6 was recovered, purified using a Ni2+-NTA agarose affinity column to eliminate remnant ATP, applied to a G-50 gel filtration column equilibrated with buffer TK to remove imidazole, and immediately used to test phosphorylated PhoP stability or for the phosphatase assay. For the phosphatase assay, phosphorylated PhoP-H6 (5 μg of protein) was incubated with control membranes, membranes enriched in PhoQ or in PhoQH277V (50 μg), or with purified MBP-Qc fusion protein (5 μg) in a 30-μl reaction mixture containing 20 mmTris-HCl (pH 8.0), 50 mm KCl, at 37 °C, in the presence or absence of 1 mm MgCl2 for different periods of time as indicated in each assay. The reaction was stopped by adding 5 × SDS-PAGE loading buffer. All reactions were analyzed by SDS-PAGE (12% polyacrylamide), transferred to nitrocellulose, and then subjected to autoradiography. When TLC analysis was performed, reactions were stopped by addition of 1% SDS, applied to a polyethyleneimine (PEI)-cellulose plate (J. T. Baker), and developed in 0.8 m LiCl, 0.8 m acetic acid as described (19Hess J.F. Bourret R.B. Simon M.I. Methods Enzymol. 1991; 200: 188-204Crossref PubMed Scopus (65) Google Scholar). The plates were air-dried and then exposed to autoradiography films. Additionally, the stability of phospho-PhoP-H6 or phospho-PhoQ was determined by incubating each phosphoprotein in buffer TK with addition of 1 mm EDTA or 20 mmMgCl2, at 37 °C. Aliquots were withdrawn at different time points, and the reaction was stopped by adding 5 × SDS-PAGE loading buffer. The samples were analyzed by SDS-PAGE and transferred to nitrocellulose membranes, followed by autoradiography. Autoradiographies from SDS-PAGE analysis or TLC assays were densitometrically scanned to perform quantitative determinations. For the stability assays, the PhoP or PhoQ bands were cut from the gel and the incorporation of 32P was determined using a Wallac 1209 Rackbetta liquid scintillation counter. Chemical stability assays of the phosphoproteins (alkali and acid lability) were performed as described (9Shi L. Liu W. Hulett F.M. Biochemistry. 1999; 38: 10119-10125Crossref PubMed Scopus (24) Google Scholar). Binding of Mg2+ to the sensor protein PhoQ was identified as the primary event in the signal transduction cascade that results in the down-regulation of the PhoP-activated genes (10Garcı́a Véscovi E. Soncini F.C. Groisman E.A. Cell. 1996; 84: 165-174Abstract Full Text Full Text PDF PubMed Scopus (675) Google Scholar, 15Véscovi E.G. Ayala Y.M. DiCera E. Grosiman E.A. J. Biol. Chem. 1997; 272: 1440-1443Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). To determine the activities of PhoQ that are affected in response to the signal, we sought to analyze sequentially the steps that are involved in the transduction mechanism. To examine in vitro the biochemical activities of the Mg2+ sensor PhoQ, this protein was expressed from the pEG9050 plasmid. Cellular fractionation was performed to isolate the membranes that harbor the sensor protein. The electrophoretic analysis of the membrane fraction revealed a major protein band (estimated to be 5–7% of the total protein by densitometric analysis) that corresponded to the predicted mobility of PhoQ. The identity of the protein was assessed also by Western blot analysis using polyclonal antibodies raised against the cytoplasmic C-terminal domain (data not shown). To avoid conformational distortions or artifacts that would result from the solubilization of the membrane protein with detergents, all experiments involving PhoQ were performed with the sensor protein in its native membrane environment. This approach ensures an accurate response to different variables in the reaction medium that mimic the environmental cues that control the system. According to the two-component paradigm, PhoQ would first undergo autophosphorylation in a conserved histidine residue, and the phosphate would in turn be transferred to a conserved aspartate residue in the cognate transcriptional regulator PhoP. Indeed, we showed that incubation of the membranes with [γ-32P]ATP resulted in autophosphorylation of the sensor protein. This autokinase activity was dependent on the presence of micromolar concentrations of Mg2+, because no phosphorylation was detectable in membranes devoid of the divalent cation after exhaustive washes with EDTA (Fig. 1 A). When Mg2+ concentrations above 200 μm(demonstrated to down-regulate the system (10Garcı́a Véscovi E. Soncini F.C. Groisman E.A. Cell. 1996; 84: 165-174Abstract Full Text Full Text PDF PubMed Scopus (675) Google Scholar, 11Soncini F.C. Garcı́a Véscovi E. Solomon F. Groisman E.A. J. Bacteriol. 1996; 178: 5092-5099Crossref PubMed Scopus (268) Google Scholar)) were used, the divalent cation had no inhibitory effect on the autokinase activity of PhoQ. This result showed that micromolar concentrations of Mg2+ are required for the autophosphorylation reaction, suggesting that Mg2+-ATP is the true substrate of the autokinase. Additionally, we determined that the acid lability and alkali resistance of the phosphorylated moiety corresponded to the chemical characteristics of a phosphohistidine residue (data not shown). Moreover, the PhoQH277V mutant protein, where the conserved His-277 is replaced by Val, was unable to undergo autophosphorylation, irrespective of the presence of Mg2+in the phosphorylation reaction (data not shown). This indicates that the phosphorylation takes place in the predicted conserved histidine residue. We decided to investigate the next step in the activation by phosphorylation of the response regulator PhoP. To uncouple the autokinase from the phosphotransfer reaction and to analyze the effect of Mg2+ exclusively on the latter, the membrane-bound PhoQ was first subjected to autophosphorylation. Immediately after, the membranes were exhaustively washed with EDTA to deplete the membranes of remnant Mg2+and ATP. The membranes harboring phospho-PhoQ were incubated with purified PhoP resulting in the phosphorylation of the response regulator. Chemical stability tests and site-specific mutagenesis identified amino acid Asp-55 of PhoP as the phosphorylated residue (not shown). As it is shown in the time course assays performed in the presence of different concentrations of Mg2+ (Figs.2 and 3), the initial rate of phosphotransfer is favored by increasing the concentration of the divalent cation. Strikingly, Mg2+concentrations higher than 250 μm stimulated the dephosphorylation of phospho-PhoP along the time course (Figs. 2 and3 B). A net loss of the protein-bound radiolabeled phosphate becomes evident when we compare the radioactivity associated to PhoQ (Figs. 2 and 3 A) and to PhoP (Figs. 2 and 3 B) as a function of time, for each Mg2+ concentration used. Moreover, the total protein-bound phosphate decreased up to 60% when concentrations of MgCl2 higher than 250 μmwere added to the phosphotransfer reaction and up to 90% at concentrations higher than 10 mm MgCl2 (Figs. 2and 3 C). On the other hand, when the stability of each individual phosphorylated protein was assayed, phospho-PhoQ retained a 72 ± 13% of the initial label over the 120-min period tested (irrespective of the MgCl2 concentration used), whereas phospho-PhoP showed a half-life of 64 min in the presence of 1 mm EDTA and of 60 min in the presence of 20 mmMgCl2 (Fig. 4).Figure 3Densitometric analysis of the PhoQ → PhoP phosphotransfer reaction. Autoradiographs from three independent assays like the one shown in Fig. 2 were densitometrically scanned, and the total amounts of phospho-PhoQ (A), phospho-PhoP (B), and protein-bound phosphate (C) were plotted. The average results are expressed as the ratio between the indicated phosphorylated protein and the total phospho-PhoQ present at the initial time of the phosphotransfer reaction (0 min).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Effect of Mg2+ on the stability of the phosphorylated PhoQ and PhoP proteins. Membranes harboring phosphorylated PhoQ depleted of Mg2+, ATP, and inorganic phosphate (0 min) were split into two equal fractions and incubated in buffer TK, with addition of 1 mm EDTA or 20 mmMgCl2, respectively. Aliquots were taken at 10, 30, 60, and 120 min, and 5× SDS-PAGE sample buffer was added to stop the reaction. Samples were analyzed by SDS-PAGE, followed by transfer to nitrocellulose and autoradiography (PhoQ-P). An identical protocol was carried out using phospho-PhoP free of ATP and inorganic phosphate as indicated under “Experimental Procedures” (PhoP-P).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The above results indicate that Mg2+ neither activates an autophosphatase activity of the response regulator nor promotes PhoQ dephosphorylation and strongly suggest that a Mg2+-induced phosphatase is present and acts on phosphorylated PhoP. The status of the environment as detected by the sensor is reflected by the phosphorylation state of the effector molecule. The phosphorylation status of PhoP is predicted to be due to the balance of three fundamental biochemical reactions: PhoQ autophosphorylation, the phosphotransfer from PhoQ to PhoP, and the dephosphorylation of phospho-PhoP that restores the effector protein to its original state. To examine if PhoQ was responsible for the Mg2+-induced dephosphorylation of PhoP, radiolabeled phospho-PhoP was incubated with membranes harboring unlabeled PhoQ. Surprisingly, in the absence of added Mg2+ we observed dephosphorylation of PhoP with the simultaneous phosphorylation of PhoQ, indicating that a reverse phosphotransfer from PhoP to PhoQ took place (Fig.5 A). The reverse phosphotransfer was partially inhibited by EDTA, showing that a low concentration of the divalent cation favors the reaction. However, when we added 1 mm MgCl2 to the reaction medium, dephosphorylation of phospho-PhoP took place but no phosphorylated PhoQ was detected, resulting in a net loss of the total label associated to proteins (Fig. 5 A). This effect was not due to a PhoQ-unrelated phosphatase activity present in the membrane fraction, because membranes lacking the sensor protein did not show Mg2+-stimulated phosphate release from phospho-PhoP (Fig.5 A). Moreover, this result was consistent with the PhoQ-mediated dephosphorylation of PhoP triggered in the presence of high concentrations of Mg2+ previously observed in the direct phosphotransfer assay (Figs. 2 and 3). Although controlled proteolysis of native PhoQ and binding assays performed using its purified sensing domain demonstrated that extracellular Mg2+ interacts with the periplasmic domain of the sensor, thereby triggering a conformational change in the protein (10Garcı́a Véscovi E. Soncini F.C. Groisman E.A. Cell. 1996; 84: 165-174Abstract Full Text Full Text PDF PubMed Scopus (675) Google Scholar, 15Véscovi E.G. Ayala Y.M. DiCera E. Grosiman E.A. J. Biol. Chem. 1997; 272: 1440-1443Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), it was still unclear how this change was transduced in the modulation of the biochemical activities of PhoQ. To assess the role of the periplasmic sensing domain of PhoQ in the Mg2+-induced phosphatase activity, we constructed a fusion protein containing its cytoplasmic domain fused to the maltose binding protein (MBP-Qc). MBP-Qc was able to undergo autophosphorylation (not shown) and to receive the phosphate from PhoP analogously to wild-type PhoQ (Fig.5 A). However, the addition of Mg2+ did not promote the dephosphorylation of PhoP. A comparative densitometric analysis of a reverse phosphotransfer time course from phospho-PhoP to either PhoQ or to MBP-Qc in the presence of 1 mmMgCl2 is shown in Fig. 5 B. A net loss of label associated to the proteins was evident only when wild-type PhoQ was used (Fig. 5 C). When we analyzed by TLC the dephosphorylation of phospho-PhoP in the presence of control membranes, membranes harboring PhoQ, or MBP-Qc, we confirmed that the detected loss of label shown in Fig. 5 corresponded to the release of inorganic phosphate (Fig.6). This release was significantly enhanced only when wild-type PhoQ and 1 mmMgCl2 were present in the reaction (Fig. 6 A). The densitometric analysis shows the relative distribution of the total label between the phosphate bound to the proteins and the inorganic phosphate released in each sample (Fig. 6 B). More than 80% of the total label was released as inorganic phosphate by membrane-bound PhoQ in the presence of MgCl2, whereas less than 15% of the label was detected as inorganic phosphate in the absence of the cation. On the other hand, regardless of the concentration of Mg2+ used in the assay, more than 80% of the total radioactivity remained protein-bound when phospho-PhoP was incubated with either control membranes or MBP-Qc. Cumulatively, these results demonstrate that Mg2+ triggers a specific PhoP-phosphatase activity in PhoQ that

ABSTRACT The enterobacterial common antigen (ECA) is a highly conserved exopolysaccharide in Gram-negative bacteria whose role remains largely uncharacterized. In a previous work, we have demonstrated that disrupting the integrity of the ECA biosynthetic pathway imposed severe deficiencies to the Serratia marcescens motile (swimming and swarming) capacity. In this work, we show that alterations in the ECA structure activate the Rcs phosphorelay, which results in the repression of the flagellar biogenesis regulatory cascade. In addition, a detailed analysis of wec cluster mutant strains, which provoke the disruption of the ECA biosynthesis at different levels of the pathway, suggests that the absence of the periplasmic ECA cyclic structure could constitute a potential signal detected by the RcsF-RcsCDB phosphorelay. We also identify SMA1167 as a member of the S. marcescens Rcs regulon and show that high osmolarity induces Rcs activity in this bacterium. These results provide a new perspective from which to understand the phylogenetic conservation of ECA among enterobacteria and the basis for the virulence attenuation detected in wec mutant strains in other pathogenic bacteria.

Serratia marcescens is a Gram-negative bacterium that thrives in a wide variety of ambient niches and interacts with an ample range of hosts. As an opportunistic human pathogen, it has increased its clinical incidence in recent years, being responsible for life-threatening nosocomial infections. S. marcescens produces numerous exoproteins with toxic effects, including the ShlA pore-forming toxin, which has been catalogued as its most potent cytotoxin. However, the regulatory mechanisms that govern ShlA expression, as well as its action toward the host, have remained unclear. We have shown that S. marcescens elicits an autophagic response in host nonphagocytic cells. In this work, we determine that the expression of ShlA is responsible for the autophagic response that is promoted prior to bacterial internalization in epithelial cells. We show that a strain unable to express ShlA is no longer able to induce this autophagic mechanism, while heterologous expression of ShlA/ShlB suffices to confer on noninvasive Escherichia coli the capacity to trigger autophagy. We also demonstrate that shlBA harbors a binding motif for the RcsB regulator in its promoter region. RcsB-dependent control of shlBA constitutes a feed-forward regulatory mechanism that allows interplay with flagellar-biogenesis regulation. At the top of the circuit, activated RcsB downregulates expression of flagella by binding to the flhDC promoter region, preventing FliA-activated transcription of shlBA. Simultaneously, RcsB interaction within the shlBA promoter represses ShlA expression. This circuit offers multiple access points to fine-tune ShlA production. These findings also strengthen the case for an RcsB role in orchestrating the expression of Serratia virulence factors.

Serratia marcescens strains are ubiquitous bacteria isolated from environmental niches, such as soil, water, and air, and also constitute emergent nosocomial opportunistic pathogens. Among the numerous extracellular factors that S. marcescens is able to produce, the PhlA phospholipase is the only described exoprotein secreted by the flagellar apparatus while simultaneously being a member of the flagellar regulon. To gain insight into the regulatory mechanism that couples PhlA and flagellar expression, we conducted a generalized insertional mutagenesis and screened for PhlA-deficient strains. We found that three independent mutations in the wec cluster, which impaired the assembly of enterobacterial common antigen (ECA), provoked the inhibition of PhlA expression. Swimming and swarming assays showed that in these strains, motility was severely affected. Microscopic examination and flagellin immunodetection demonstrated that a strong defect in flagellum expression was responsible for the reduced motility in the wec mutant strains. Furthermore, we determined that in the ECA-defective strains, the transcriptional cascade that controls flagellar assembly was turned off due to the down-regulation of flhDC expression. These findings provide a new perspective on the physiological role of the ECA, providing evidence that in S. marcescens, its biosynthesis conditions the expression of the flagellar regulon.

ABSTRACT The PhoP/PhoQ two-component system controls the extracellular magnesium depletion response in Salmonella enterica . Previous studies have shown that PhoP is unable to up-regulate its target genes in the absence of PhoQ function. In this work, we demonstrate that PhoP overexpression can substitute for PhoQ- and phosphorylation-dependent activation. Either a high concentration of PhoP or activation via phosphorylation stimulates PhoP self-association.

The Salmonella enterica serovar Typhimurium PhoP/PhoQ system has largely been studied as a paradigmatic two-component regulatory system not only to dissect structural and functional aspects of signal transduction in bacteria but also to gain knowledge about the versatile devices that have evolved allowing a pathogenic bacterium to adjust to or counteract environmental stressful conditions along its life cycle. Mg2+ limitation, acidic pH, and the presence of cationic antimicrobial peptides have been identified as cues that the sensor protein PhoQ can monitor to reprogram Salmonella gene expression to cope with extra- or intracellular challenging conditions. In this work, we show for the first time that long chain unsaturated free fatty acids (LCUFAs) present in Salmonella growth medium are signals specifically detected by PhoQ. We demonstrate that LCUFAs inhibit PhoQ autokinase activity, turning off the expression of the PhoP-dependent regulon. We also show that LCUFAs exert their action independently of their cellular uptake and metabolic utilization by means of the β-oxidative pathway. Our findings put forth the complexity of input signals that can converge to finely tune the activity of the PhoP/PhoQ system. In addition, they provide a new potential biochemical platform for the development of antibacterial strategies to fight against Salmonella infections.Background: The PhoP/PhoQ system governs crucial Salmonella typhimurium pathogenic traits.Results: A screening of natural compounds showed that long chain fatty acids present in the bacterial growth medium down-regulate the PhoP/PhoQ-dependent regulon.Conclusion: Long chain unsaturated fatty acids specifically inhibit PhoQ autokinase activity.Significance: The novel PhoQ input signal reveals a new scenario for the control of Salmonella virulence, providing a rationale for future antibacterial strategies. The Salmonella enterica serovar Typhimurium PhoP/PhoQ system has largely been studied as a paradigmatic two-component regulatory system not only to dissect structural and functional aspects of signal transduction in bacteria but also to gain knowledge about the versatile devices that have evolved allowing a pathogenic bacterium to adjust to or counteract environmental stressful conditions along its life cycle. Mg2+ limitation, acidic pH, and the presence of cationic antimicrobial peptides have been identified as cues that the sensor protein PhoQ can monitor to reprogram Salmonella gene expression to cope with extra- or intracellular challenging conditions. In this work, we show for the first time that long chain unsaturated free fatty acids (LCUFAs) present in Salmonella growth medium are signals specifically detected by PhoQ. We demonstrate that LCUFAs inhibit PhoQ autokinase activity, turning off the expression of the PhoP-dependent regulon. We also show that LCUFAs exert their action independently of their cellular uptake and metabolic utilization by means of the β-oxidative pathway. Our findings put forth the complexity of input signals that can converge to finely tune the activity of the PhoP/PhoQ system. In addition, they provide a new potential biochemical platform for the development of antibacterial strategies to fight against Salmonella infections. Background: The PhoP/PhoQ system governs crucial Salmonella typhimurium pathogenic traits. Results: A screening of natural compounds showed that long chain fatty acids present in the bacterial growth medium down-regulate the PhoP/PhoQ-dependent regulon. Conclusion: Long chain unsaturated fatty acids specifically inhibit PhoQ autokinase activity. Significance: The novel PhoQ input signal reveals a new scenario for the control of Salmonella virulence, providing a rationale for future antibacterial strategies.

Bacterial survival in diverse and changing environments relies on the accurate interplay between different regulatory pathways, which determine the design of an adequate adaptive response. The proper outcome depends on a precise gene expression profile generated from the finely tuned and concerted action of transcriptional factors of distinct regulatory hierarchies. Salmonella enterica serovar Typhimurium harbors multiple regulatory systems that are crucial for the bacterium to cope with harsh extra- and intracellular environments. In this work, we found that the expression of Salmonella RstA, a response regulator from the two-component system family, was able to downregulate the expression of three RpoS-controlled genes (narZ, spvA, and bapA). Furthermore, this downregulation was achieved by a reduction in RpoS cellular levels. The alternative sigma factor RpoS is critical for bacterial endurance under the most-stressful conditions, including stationary-phase entrance and host adaptation. Accordingly, RpoS cellular levels are tightly controlled by complex transcriptional, translational, and posttranslational mechanisms. The analysis of each regulatory step revealed that in Salmonella, RstA expression was able to promote RpoS degradation independently of the MviA-ClpXP proteolytic pathway. Additionally, we show that RstA is involved in modulating Salmonella biofilm formation. The fact that the RpoS-modulated genes affected by RstA expression have previously been demonstrated to contribute to Salmonella pathogenic traits, which include biofilm-forming capacity, suggests that under yet unknown conditions, RstA may function as a control point of RpoS-dependent pathways that govern Salmonella virulence.

The trend for material and energy recovery from residues along with the need to reduce greenhouse gases has led to an increased interest in the thermal exploitation of biomass and/or their wastes. Due to the enormous quantity generated every year, agro-industrial byproducts have an attractive potential to be recycled. One way to do this is by means of pyrolysis, a thermal decomposition of high molecular weight polymers into simpler compounds. In this study, we applied an autographic assay to analyze the biological effect of bio-oils produced by pyrolysis of soybean hulls. The discovery of a compound with antimicrobial activity validated this novel approach as a tool for the generation of bioactive compounds.

PrtA is the major secreted metalloprotease of Serratia marcescens Previous reports implicate PrtA in the pathogenic capacity of this bacterium. PrtA is also clinically used as a potent analgesic and anti-inflammatory drug, and its catalytic properties attract industrial interest. Comparatively, there is scarce knowledge about the mechanisms that physiologically govern PrtA expression in Serratia In this work, we demonstrate that PrtA production is derepressed when the bacterial growth temperature decreases from 37°C to 30°C. We show that this thermoregulation occurs at the transcriptional level. We determined that upstream of prtA, there is a conserved motif that is directly recognized by the CpxR transcriptional regulator. This feature is found along Serratia strains irrespective of their isolation source, suggesting an evolutionary conservation of CpxR-dependent regulation of PrtA expression. We found that in S. marcescens, the CpxAR system is more active at 37°C than at 30°C. In good agreement with these results, in a cpxR mutant background, prtA is derepressed at 37°C, while overexpression of the NlpE lipoprotein, a well-known CpxAR-inducing condition, inhibits PrtA expression, suggesting that the levels of the activated form of CpxR are increased at 37°C over those at 30°C. In addition, we establish that PrtA is involved in the ability of S. marcescens to develop biofilm. In accordance, CpxR influences the biofilm phenotype only when bacteria are grown at 37°C. In sum, our findings shed light on regulatory mechanisms that fine-tune PrtA expression and reveal a novel role for PrtA in the lifestyle of S. marcescensIMPORTANCE We demonstrate that S. marcescens metalloprotease PrtA expression is transcriptionally thermoregulated. While strongly activated below 30°C, its expression is downregulated at 37°C. We found that in S. marcescens, the CpxAR signal transduction system, which responds to envelope stress and bacterial surface adhesion, is activated at 37°C and able to downregulate PrtA expression by direct interaction of CpxR with a binding motif located upstream of the prtA gene. Moreover, we reveal that PrtA expression favors the ability of S. marcescens to develop biofilm, irrespective of the bacterial growth temperature. In this context, thermoregulation along with a highly conserved CpxR-dependent modulation mechanism gives clues about the relevance of PrtA as a factor implicated in the persistence of S. marcescens on abiotic surfaces and in bacterial host colonization capacity.

ABSTRACT Introduction The PhoP–PhoQ system from Salmonella enterica serovar Typhimurium controls the expression of factors that are critical for the bacterial entry into host cells and the bacterial intramacrophage survival. Therefore it constitutes an interesting target to search for compounds that would control Salmonella virulence. Localisation of such compounds in complex matrixes could be facilitated by thin‐layer chromatography (TLC) bioautography. Objective To develop a TLC bioautography to detect inhibitors of the PhoP–PhoQ regulatory system in complex matrixes. Methods The TLC plates were covered by a staining solution containing agar, Luria‐Bertani medium, 5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐galactopyranoside (X‐gal), kanamycin and a S. typhimurium strain that harbours a reporter transcriptional lacZ ‐fusion to an archetypal PhoP‐activated gene virK . After solidification, the plate was incubated at 37°C for 16 h. Results A bioautographic assay suitable for the localisation of inhibitors of the PhoP–PhoQ system activity in S. enterica serovar Typhimurium present in a complex matrix is described. The assay was used to analyse a series of hydrolysed extracts prepared by alkaline treatment of crude plant extracts. Bioassay‐guided analysis of the fractions by NMR spectroscopy and MS led to the identification of linolenic and linoleic acids as inhibitory input signals of the PhoP–PhoQ system. Conclusion A practical tool is introduced that facilitates detection of inhibitors of the Salmonella PhoP–PhoQ regulatory system. The assay convenience is illustrated with the identification of the first naturally occurring organic compounds that down‐regulate a PhoP–PhoQ regulatory system from a hydrolysed extract. Copyright © 2013 John Wiley & Sons, Ltd.

In the multidrug resistant (MDR) pathogen Acinetobacter baumannii the global repressor H-NS was shown to modulate the expression of genes involved in pathogenesis and stress response. In addition, H-NS inactivation results in an increased resistance to colistin, and in a hypermotile phenotype an altered stress response. To further contribute to the knowledge of this key transcriptional regulator in A. baumannii behavior, we studied the role of H-NS in antimicrobial resistance. Using two well characterized A. baumannii model strains with distinctive resistance profile and pathogenicity traits (AB5075 and A118), complementary transcriptomic and phenotypic approaches were used to study the role of H-NS in antimicrobial resistance, biofilm and quorum sensing gene expression. An increased expression of genes associated with β-lactam resistance, aminoglycosides, quinolones, chloramphenicol, trimethoprim and sulfonamides resistance in the Δhns mutant background was observed. Genes codifying for efflux pumps were also up-regulated, with the exception of adeFGH. The wild-type transcriptional level was restored in the complemented strain. In addition, the expression of biofilm related genes and biofilm production was lowered when the transcriptional repressor was absent. The quorum network genes aidA, abaI, kar and fadD were up-regulated in Δhns mutant strains. Overall, our results showed the complexity and scope of the regulatory network control by H-NS (genes involved in antibiotic resistance and persistence). These observations brings us one step closer to understanding the regulatory role of hns to combat A. baumannii infections.

Clofibric and ethacrynic acids are prototypical pharmacological agents administered in the treatment of hypertrigliceridemia and as a diuretic agent, respectively. They share with 2,4-dichlorophenoxyacetic acid (the widely used herbicide known as 2,4-D) a chlorinated phenoxy structural moiety. These aryloxoalcanoic agents (AOAs) are mainly excreted by the renal route as unaltered or conjugated active compounds. The relatedness of these agents at the structural level and their potential effect on therapeutically treated or occupationally exposed individuals who are simultaneously undergoing a bacterial urinary tract infection led us to analyze their action on uropathogenic, clinically isolated Escherichia coli strains. We found that exposure to these compounds increases the bacterial resistance to an ample variety of antibiotics in clinical isolates of both uropathogenic and nonpathogenic E. coli strains. We demonstrate that the AOAs induce an alteration of the bacterial outer membrane permeability properties by the repression of the major porin OmpF in a micF-dependent process. Furthermore, we establish that the antibiotic resistance phenotype is primarily due to the induction of the MarRAB regulatory system by the AOAs, while other regulatory pathways that also converge into micF modulation (OmpR/EnvZ, SoxRS, and Lrp) remained unaltered. The fact that AOAs give rise to uropathogenic strains with a diminished susceptibility to antimicrobials highlights the impact of frequently underestimated or ignored collateral effects of chemical agents.

ABSTRACT Serratia marcescens strains are ubiquitous bacteria isolated from environmental niches and also constitute emergent nosocomial opportunistic pathogens. Here, we report on the draft genome sequence of S. marcescens strain RM66262, which was isolated from a patient with urinary tract infection in the Bacteriology Service of the Rosario National University, Rosario, Argentina.

Serratia marcescens is an opportunistic bacterium that infects a wide range of hosts including humans. It is a potent pathogen in a septic injury model of Drosophila melanogaster since a few bacteria directly injected in the body cavity kill the insect within a day. In contrast, flies do not succumb to ingested bacteria for days even though some bacteria cross the intestinal barrier into the hemolymph within hours. The mechanisms by which S. marcescens attacks enterocytes and damages the intestinal epithelium remain uncharacterized. To better understand intestinal infections, we performed a genetic screen for loss of virulence of ingested S. marcescens and identified FliR, a structural component of the flagellum, as a virulence factor. Next, we compared the virulence of two flagellum mutants fliR and flhD in two distinct S. marcescens strains. Both genes are required for S. marcescens to escape the gut lumen into the hemocoel, indicating that the flagellum plays an important role for the passage of bacteria through the intestinal barrier. Unexpectedly, fliR but not flhD is involved in S. marcescens-mediated damages of the intestinal epithelium that ultimately contribute to the demise of the host. Our results therefore suggest a flagellum-independent role for fliR in bacterial virulence.

Isolated thylakoids, Photosystem I and Photosystem II particles were phosphorylated with [γ-32P]ATP at high specific radioactivity in the dark under nonreducing conditions and in the light in the absence of electron acceptor. The resulting phosphoproteins were compared by gel electrophoresis and autoradiography. Phosphorylation of thylakoids in the dark and in the light rendered distinct patterns of phosphoproteins. Some of the dark-phosphorylated proteins in thylakoids were diminished or not detected in the light-phosphorylated membranes. Phosphorylation of subthylakoid particles was insensitive to light and most of the phosphoproteins in these membranes were also observed in the dark-phosphorylated thylakoids. Dark phosphorylation rendered mostly phosphoserine in individual proteins of thylakoids, subthylakoid particles and lysine-rich histone phosphorylated by the particles. Conversely, phosphothreonine was prevalent in light-phosphorylated thylakoids. The results are consistent with the presence of a protein serine kinase activity that is distributed homogeneously within the thylakoid regions, is more active in the dark, does not require reducing conditions for activity and phosphorylates a number of endogenous substrates most of which belong to the stroma membranes.

Serratia marcescens is an opportunistic human pathogen involved in antibiotic-resistant hospital acquired infections. Upon contact with the host epithelial cell and prior to internalization, Serratia induces an early autophagic response that is entirely dependent on the ShlA toxin. Once Serratia invades the eukaryotic cell and multiples inside an intracellular vacuole, ShlA expression also promotes an exocytic event that allows bacterial egress from the host cell without compromising its integrity. Several toxins, including ShlA, were shown to induce ATP efflux from eukaryotic cells. Here, we demonstrate that ShlA triggered a non-lytic release of ATP from CHO cells. Enzymatic removal of accumulated extracellular ATP (eATP) or pharmacological blockage of the eATP-P2Y2 purinergic receptor inhibited the ShlA-promoted autophagic response in CHO cells. Despite the intrinsic ecto-ATPase activity of CHO cells, the effective concentration and kinetic profile of eATP was consistent with the established affinity of the P2Y2 receptor and the known kinetics of autophagy induction. Moreover, eATP removal or P2Y2 receptor inhibition also suppressed the ShlA-induced exocytic expulsion of the bacteria from the host cell. Blocking α5β1 integrin highly inhibited ShlA-dependent autophagy, a result consistent with α5β1 transactivation by the P2Y2 receptor. In sum, eATP operates as the key signaling molecule that allows the eukaryotic cell to detect the challenge imposed by the contact with the ShlA toxin. Stimulation of P2Y2-dependent pathways evokes the activation of a defensive response to counteract cell damage and promotes the non-lytic clearance of the pathogen from the infected cell.

Abstract Serratia marcescens is an opportunistic bacterium that infects a wide range of hosts including humans. It is a potent pathogen in a septic injury model of Drosophila melanogaster as five bacteria directly injected in the body cavity of the fly kill the host within a day. In contrast, flies do not succumb to ingested bacteria for days even though some bacteria traverse the intestinal barrier into the hemolymph within a couple of hours. The mechanisms by which S. marcescens attacks enterocytes and damages the intestinal epithelium remain uncharacterized. To better understand intestinal infections, we performed a genetic screen for loss of virulence of ingested S. marcescens in which we identified FliR, a structural component of the flagellum, as a virulence factor. Next, we compared the virulence of two flagellum mutants fliR and flhD using two Serratia strains. Both genes are required for S. marcescens to escape the gut lumen into the hemocoel indicating that the flagellum plays an important role for the passage of bacteria through the intestinal barrier. In contrast, fliR but not flhD is needed to severely damage the intestinal epithelium and ultimately kill the host. Our results therefore suggest a flagellum-independent role for fliR in bacterial virulence.

Huanglongbing (HLB), the current major threat for Citrus species, is caused by intracellular alphaproteobacteria of the genus Candidatus Liberibacter (CaL), with CaL asiaticus (CLas) being the most prevalent species. This bacterium inhabits phloem cells and is transmitted by the psyllid Diaphorina citri. A gene encoding a putative serralysin-like metalloprotease (CLIBASIA_01345) was identified in the CLas genome. The expression levels of this gene were found to be higher in citrus leaves than in psyllids, suggesting a function for this protease in adaptation to the plant environment. Here, we study the putative role of CLas-serralysin (Las1345) as virulence factor. We first assayed whether Las1345 could be secreted by two different surrogate bacteria, Rhizobium leguminosarum bv. viciae A34 (A34) and Serratia marcescens. The protein was detected only in the cellular fraction of A34 and S. marcescens expressing Las1345, and increased protease activity of those bacteria by 2.55 and 4.25-fold, respectively. In contrast, Las1345 expressed in Nicotiana benthamiana leaves did not show protease activity nor alterations in the cell membrane, suggesting that Las1345 do not function as a protease in the plant cell. Las1345 expression negatively regulated cell motility, exopolysaccharide production, and biofilm formation in Xanthomonas campestris pv. campestris (Xcc). This bacterial phenotype was correlated with reduced growth and survival on leaf surfaces as well as reduced disease symptoms in N. benthamiana and Arabidopsis. These results support a model where Las1345 could modify extracellular components to adapt bacterial shape and appendages to the phloem environment, thus contributing to virulence.

Incubation of plastids isolated from ripening tomato fruits (Lycopersicon esculentum) with [γ-32P]ATP resulted in the rapid phosphorylation of several polypeptides. The optimal substrate as Pi donor for the reaction was Mg2+-ATP, and the recovery of [32P]serine after acid hydrolysis indicated predominance of protein serine kinases. The endogenous phosphorylating patterns in chromoplasts isolated from ripe tomato fruits were distinguishable from those observed in green fruit plastids, which in turn were different from those found in leaf plastids. Dark-light transitions had no influence on the pattern of phosphorylation observed in these plastids. The overall results strongly indicate that plastid-specific protein phosphorylation systems are present at defined stages during fruit formation. We also found that dark/light transitions induce a clear stimulation on the phosphorylation in serine rather than in threonine residues in endogenous thylakoid polypeptides of chloroplasts from tomato leaves.

Abstract In the multidrug resistant (MDR) pathogen Acinetobacter baumannii the global repressor H-NS was shown to modulate the expression of genes related to quorum sensing, biosynthetic pathways, cell adhesion, virulence, and autotransporters. In addition, H-NS is involved in stress response and its inactivation results in an increased resistance to colistin, and in a hypermotile phenotype an altered stress response. To further contribute to the knowledge of this key transcriptional regulator in A. baumannii behavior, we studied the role of H-NS in antimicrobial resistance. Using two well characterized A. baumannii model strains with distinctive resistance profile and pathogenicity traits (AB5075 and A118), complementary transcriptomic and phenotypic approaches were used to study the role of H-NS in antimicrobial resistance, biofilm and quorum sensing gene expression. An increased expression of genes associated with β-lactam resistance, aminoglycosides, quinolones, chloramphenicol, trimethoprim and sulfonamides resistance in the Δhns mutant background was observed. Genes codifying for efflux pumps were also up-regulated, with the exception of adeFGH . The wild-type transcriptional level was restored in the complemented strain. In addition, the expression of biofilm related genes and biofilm production was lowered when the transcriptional repressor was absent. The quorum network genes aidA, abaI, kar and fadD were up-regulated in Δhns mutant strains. Overall, our results showed the complexity and scope of the regulatory network control by H-NS (genes involved in antibiotic resistance and persistence). These observations brings us one step closer to understanding the regulatory role of hns to combat A. baumannii infections.