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Prodigiosin is a tripyrrole red secondary metabolite synthesized by many microorganisms, including Serratia marcescens. In this study, we found that the deletion of the gene of sensor kinase CpxA dramatically decreased the prodigiosin production, while the deletion of the gene of the response regulator CpxR or both genes of CpxRA has no effect on prodigiosin production, the kinase function of CpxA is not essential for its regulation on prodigiosin production while the phosphorylation site of CpxR is required. We further demonstrated that the CpxA regulates the prodigiosin biosynthesis at the transcriptional level and the phosphatase activity of CpxA plays vital roles in the regulation of prodigiosin biosynthesis. Finally, we proposed that CpxR/A regulates the prodigiosin biosynthesis by negative control and the phosphorylation level of CpxR may determine the positive or negative control of the genes it regulated., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Salmonella outer protein D (SopD) is secreted into a host during the first stages of the Salmonella infection and contributes to the systemic virulence of the bacterium. SopD2 is a SopD homolog and possesses GTPase activating protein (GAP) activity towards Rab32. Here, we identified Rab-proteins as putative SopD-targets using a yeast two-hybrid approach. In vitro investigations subsequently revealed Rab8a as an exclusive SopD substrate in contrast to SopD2, which has a broader specificity targeting Rab29, Rab32 and Rab38 in vitro. Additionally, we determined the catalytic efficiencies of SopD and SopD2 towards their physiologically relevant substrates. Moreover, mutagenesis studies provided insights into possible key residues of the Rab-protein and the GAP involved in the conversion of active to inactive GTPase. In conclusion, we demonstrate that Salmonella SopD and SopD2 act as RabGAPs and can inactivate Rab signaling., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Aim: Chlamydia trachomatis has evolved various strategies to alleviate oxidative stress of host cells to maintain their intracellular survival. However, the exact mechanism of anti-oxidative stress of C. trachomatis is still unclear. The activation of nuclear factor erythroid 2-related factor 2/quinone oxidoreductase (Nrf2/NQO1) signal pathway has been identified as an efficient antioxidant defensive mechanism used by host cells to counteract oxidative stress. Pgp3 is a pivotal virulence factor of C. trachomatis involved in intracellular survival. The aim of this study is to explore the role of Pgp3 on Nrf2/NQO1 signal pathway against oxidative stress., Main Methods: After HeLa cells were stimulated with Pgp3 protein, Nrf2 location and the inclusion bodies of C. trachomatis were detected by indirect immunofluorescence, western blotting and Oxidative stress assay kits were used to separately determine the protein expression and the content of malondialdehyde (MDA), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) before and after the interference of Nrf-2 and NQO1., Key Findings: Pgp3 promoted the nuclear translocation of Nrf2 to increase NQO1 expression and reduced oxidative stress induced by LPS to contribute to the survival of C. trachomatis. Inhibition of Nrf2/NQO1 signal pathway with Nrf2 inhibitor and down-regulation of NQO1 with siRNA-NQO1 suppressed oxidative stress resistance induced by Pgp3., Significance: Here we found that Pgp3 alleviated oxidative stress to promote the infectivity of C. trachomatis through activation of Nrf2/NQO1 signal pathway, which provided a novel understanding of the effects of Pgp3 in the pathogenesis of C. trachomatis., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The exposure of Bacteroides fragilis to highly oxygenated tissues induces an oxidative stress due to a shift from the reduced condition of the gastrointestinal tract to an aerobic environment of host tissues. The potent and effective responses to reactive oxygen species (ROS) make the B. fragilis tolerant to atmospheric oxygen for several days. The response to oxidative stress in B. fragilis is a complicated event that is induced and regulated by different agents. In this review, we will focus on the B. fragilis response to oxidative stress and present an overview of the regulators of responses to oxidative stress in this bacterium., Competing Interests: Declaration of competing interest There is no conflict of interest., (Copyright © 2021 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

RyeA/SraC is a cis-encoded small RNA (sRNA), which act as an anti-toxin to RpoS-regulated RyeB toxin in Escherichia coli. Ectopic expression of RyeA was reported to diminish the RyeB accumulation by serving as a RNA trap. Lower abundance of RyeA in the early exponential growth phase turned out to be the outcome of its degradation by RNase BN/Z. In the current study, we show that RyeA is an acid stress inducible sRNA, and global stress responsive factor RpoS appeared to be inessential in RyeA induction. Although, ryeB-pphA dicistronic transcript at low pH condition was stimulated by ∼4-fold, however, RyeB population was found to be decreased by > 50% under the same condition by the decoy action of enhanced RyeA accumulation. Investigation of the mechanism of RyeA induceduction at low pH in the exponential phase, revealed that RNase BN/Z, which catabolizes RyeA in the exponential phase, appeared to be highly sensitive to low pH stress. Both mRNA and protein level of RNase BN transpired to be decreased to <10% of their initial population. The expression of RyeA under acid stress is regulated by a feed-forward mechanism to normalize the RyeB profusion., Competing Interests: Declaration of competing interest No potential conflicts of interest were disclosed., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Bacteria sense and respond to their environment through a highly conserved assembly of transmembrane chemoreceptors (MCPs), the histidine kinase CheA, and the coupling protein CheW, hereafter termed "the chemosensory array". In recent years, great strides have been made in understanding the architecture of the chemosensory array and how this assembly engenders sensitive and cooperative responses. Nonetheless, a central outstanding question surrounds how receptors modulate the activity of the CheA kinase, the enzymatic output of the sensory system. With a focus on recent advances, we summarize the current understanding of array structure and function to comment on the molecular mechanism by which CheA, receptors and CheW generate the high sensitivity, gain and dynamic range emblematic of bacterial chemotaxis. The complexity of the chemosensory arrays has motivated investigation with many different approaches. In particular, structural methods, genetics, cellular activity assays, nanodisc technology and cryo-electron tomography have provided advances that bridge length scales and connect molecular mechanism to cellular function. Given the high degree of component integration in the chemosensory arrays, we ultimately aim to understand how such networked molecular interactions generate a whole that is truly greater than the sum of its parts. This article is part of a Special Issue entitled: Molecular biophysics of membranes and membrane proteins., (Copyright © 2019. Published by Elsevier B.V.)

The role of arginines R64 and R89 at non-annular lipid binding sites of KcsA, on the modulation of channel activity by anionic lipids has been investigated. In wild-type (WT) KcsA reconstituted into asolectin lipid membranes, addition of phosphatidic acid (PA) drastically reduces inactivation in macroscopic current recordings. Consistent to this, PA increases current amplitude, mean open time and open probability at the single channel level. Moreover, kinetic analysis reveals that addition of PA causes longer open channel lifetimes and decreased closing rate constants. Effects akin to those of PA on WT-KcsA are observed when R64 and/or R89 are mutated to alanine, regardless of the added anionic lipids. We interpret these results as a consequence of interactions between the arginines and the anionic PA bound to the non-annular sites. NMR data shows indeed that at least R64 is involved in binding PA. Moreover, molecular dynamics (MD) simulations predict that R64, R89 and surrounding residues such as T61, mediate persistent binding of PA to the non-annular sites. Channel inactivation depends on interactions within the inactivation triad (E71-D80-W67) behind the selectivity filter. Therefore, it is expected that such interactions are affected when PA binds the arginines at the non-annular sites. In support of this, MD simulations reveal that PA binding prevents interaction between R89 and D80, which seems critical to the effectiveness of the inactivation triad. This mechanism depends on the stability of the bound lipid, favoring anionic headgroups such as that of PA, which thrive on the positive charge of the arginines., (Copyright © 2019. Published by Elsevier B.V.)

Introduction: Salmonella species are gram-negative facultative intracellular bacteria that are common causes of foodborne illness in North America. Infections by Salmonella during pregnancy are a significant cause of fetal loss in domestic livestock, and fetal and maternal mortality in mice. Furthermore, Salmonella infection is associated with miscarriage, stillbirth and preterm birth in pregnant women. Despite these collective associations, the extent to which Salmonella can infect the human placenta has not been investigated., Methods: Human placental villous explants from several gestational ages were exposed to Salmonella enterica serovar Typhimurium (STm) ex vivo. Infection was assessed by colony forming unit assay and whole mount immunofluorescence (WMIF)., Results: Viable bacteria were recovered from placental villous explants of all gestational ages tested, but the bacterial burden was highest in 1st trimester explants. Bacterial numbers did not change appreciably with time post-infection in explants from any gestational age examined, suggesting that STm does not proliferate in placental villi. Exposure of villous explants to STm strains defective for the type III secretion systems revealed that Salmonella pathogenicity island 1 is essential for optimal invasion. In contrast to placental explants, STm infected and proliferated within villous cytotrophoblast cells isolated from term placentas. WMIF demonstrated that STm was restricted primarily to the syncytiotrophoblast layer in infected placentas., Discussion: Our study demonstrates that STm can invade into the syncytiotrophoblast but does not subsequently proliferate. Thus, the syncytiotrophoblast may function as a barrier to STm infection of the fetus., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Drug resistance bacteria produce various proteins to combat xenobiotic, physical, and environmental stresses. These proteins play a pivotal role in bacterial cell survival, and thus are the focus of extensive research as potential targets for anti-bacterial drug discovery. During the current study, the MRSA252 (Methicillin-resistant Staphylococcus aureus) anti-σ B factor antagonist (RsbV) protein was expressed to study its structure, and function in vitro. RsbV and anti-σ B factor (RsbW) are involved in the transcription of σ B factor under stress conditions to help bacterial cell survival. The production of recombinant RsbV protein is important for ligand binding studies, and for new inhibitor identification. In order to have sufficient yield of RsbV protein for structural and functional analysis, the MRSA252 rsbV:pET25b clone was transformed into the Escherichia coli BL21(DE3). Purified RsbV protein was analyzed for structure and functions by using circular dichroism (CD) spectroscopy, and reactive oxygen species (ROS) activity assay, respectively. The CD analysis demonstrated that MRSA252 RsbV has a globular nature with α-helices, and β-strands. It is stable above the ambient temperature. RsbV protein in a cell-based antioxidant assay showed >20-fold more potent inhibition, as compared to drug ibuprofen. The structural element and functional analysis of RsbV protein indicated that RsbV protein is produced in MRSA252 probably as an active regulatory factor under physical, and chemical stresses., (Copyright © 2019. Published by Elsevier B.V.)

Objectives: Carbapenem resistance in Pseudomonas aeruginosa is growing and results from variable mechanisms. The objectives of the current study were to investigate mechanisms of carbapenem resistance and genetic relatedness of P. aeruginosa isolates recovered in Dubai hospitals., Methods: From June 2015 through June 2016, carbapenem-nonsusceptible P. aeruginosa were collected from 4 hospitals in Dubai, and subjected to antimicrobial susceptibility testing, molecular investigation of carbapenemases by PCR-sequencing, analysis of outer membrane porin OprD2 and multidrug efflux channel MexAB-OprM levels by qPCR, and fingerprinting by ERIC-PCR., Results: Out of 1969 P. aeruginosa isolated during the study period, 471 (23.9%) showed reduced carbapenem susceptibility. Of these, 37 were analyzed and 32% of them produced VIM-type metallo-β-lactamases, including VIM-2, VIM-30, VIM-31, and VIM-42, while GES-5 and GES-9 co-existed with VIM in 5.4% of isolates. Outer membrane impermeability was observed in 73% of isolates and 75.6% displayed overproduced MexAB-OprM. ERIC-PCR revealed one large clone including most carbapenemase-producing isolates indicating clonal dissemination., Conclusion: This is the first study on carbapenem-nonsusceptible P. aeruginosa from Dubai, incriminating VIM production as well as outer membrane permeability and efflux systems as resistance mechanisms. Further studies on carbapenem-nonsusceptible P. aeruginosa in Dubai are warranted for containment of such health hazard., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Tannerella forsythia is a gram-negative anaerobic bacterium that is associated with the development of destructive periodontal disease. T. forsythia secretes the metalloprotease-like enzyme karilysin. Using in vitro systems karilysin has been shown to modulate the host immune response by degradation of complement system proteins and by inactivation of the antimicrobial peptide LL-37 by proteolytic cleavage. This makes karilysin a highly interesting virulence factor to study in the framework of drug development and diagnostics. However, to date the presence of karilysin in clinical samples has not been demonstrated due to the lack of specific probes. In the present work, a high titer and stable affinity-purified avian IgY antibody against karilysin was developed. By surface plasmon resonance imaging the IgY affinity was found to be in the low nanomolar range. The antibody could be used to detect karilysin in saliva samples by immuno-blotting and was specific when tested towards human MMP-3. Furthermore, an avian IgY-based immunoassay was developed, which demonstrated low intra- and interday assay variability (CV's below 10%). Application of the immunoassay on a well-characterized set of saliva samples from adolescents with or without signs of periodontitis showed that it was possible to detect karilysin in saliva. A significant difference in karilysin concentration was found between saliva from participants with signs of periodontitis and saliva from healthy controls (p = .0024). The median of karilysin levels among periodontitis cases was 957 pg/ml (IQR, 499-2132 pg/ml) and the median for controls was 569 pg/ml (IQR, 210-1343 pg/ml). Collectively our data confirm the presence of karilysin in clinical samples. The described IgY-based immunoassay may prove useful as part of protein-based biomarker screenings in the clinic or in point-of care settings., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The availability of complete genome sequence of Mycobacterium tuberculosis has provided an important tool to understand the mycobacterial biology with respect to host-pathogen interaction, which is an unmet need of the hour owing to continuous increasing drug resistance. Hypothetical proteins are often an overlooked pool though half the genome encodes for such proteins of unknown function that could potentially play vital roles in mycobacterial biology. In this context, we report the structural and functional characterization of the hypothetical protein Rv3272. Sequence analysis classifies Rv3272 as a Family III CoA transferase with the classical two domain structure and conserved Aspartate residue (D175). The crystal structure of the wild type protein (2.2 Å) demonstrated the associated inter-locked dimer while that of the D175A mutant co-crystallized with octanoyl-CoA demonstrated relative movement between the two domains. Isothermal titration calorimetry studies indicate that Rv3272 binds to fatty acyl-CoAs of varying carbon chain lengths, with palmitoyl-CoA (C16:0) exhibiting maximum affinity. To determine the functional relevance of Rv3272 in mycobacterial biology, we ectopically expressed Rv3272 in M. smegmatis and assessed that its expression encodes significant alteration in cell surface with marked differences in triacylglycerol accumulation. Additionally, Rv3272 expression protects mycobacteria from acidic, oxidative and antibiotic stress under in vitro conditions. Taken together, these studies indicate a significant role for Rv3272 in host-pathogen interaction., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Introduction: Enterococcus faecalis is considered a predominant pathogen for persistent periapical infections and in addition is reportedly resistant to calcium hydroxide medication. The WalRK 2-component system of E. faecalis is essential for environmental adaptation, survival, and virulence. The goal of this study was to investigate the potential roles of walR in the regulation of biofilm aggregation, alkaline stress, and susceptibility to calcium hydroxide (CH) medication., Methods: Antisense walR RNA (aswalR) overexpression strains were constructed. Exopolysaccharide (EPS) production and bacterial viability of E. faecalis biofilms were evaluated by confocal laser scanning microscopy. Quantitative real-time polymerase chain reaction was used to investigate the expressions of virulent factor genes. The proportion of viable bacteria and EPS production in dentin were assessed after CH medication., Results: We showed that walR interference by aswalR RNA leads to a reduction in the dextran-dependent aggregation in E. faecalis biofilm. The overexpression of aswalR reduced the transcripts of the virulence genes and alkaline stress tolerance ability. Furthermore, the down-regulation of walR sensitized E. faecalis in infected canals to CH medication associated with inhibiting EPS synthesis., Conclusions: The data suggest a role for the walR regulator in the susceptibility to CH associated with dispelling the EPS matrix, which could be explored as a potential supplementary therapy for the management of root canal infection., (Copyright © 2018 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

The phycobilisome (PBS) is a giant highly-structured pigment-protein antenna of cyanobacteria and red algae. PBS is composed of the phycobiliproteins and several linker polypeptides. The large core-membrane linker protein (L CM or ApcE) influences many features and functions of PBS and consists of several domains including the chromophorylated PB-domain. Being homologous to the phycobiliprotein α-subunits this domain includes a so-called PB-loop insertion whose functions are still unknown. We have created the photoautotrophic mutant strain of the cyanobacterium Synechocystis sp. PCC 6803 with lacking PB-loop. Using various spectral techniques we have demonstrated that this mutation does not destroy the PBS integrity and the internal PBS excitation energy transfer pathways. At the same time, the deletion of the PB-loop leads to the decrease of connectivity between the PBS and thylakoid membrane and to the compensatory increase of the relative photosystem II content. Mutation provokes the violation of the thylakoid membranes arrangement, the inability to perform state transitions, and diminishing of the OCP-dependent non-photochemical PBS quenching. In essence, even such a minute mutation of the PBS polypeptide component, like the PB-loop deletion, becomes important for the concerted function of the photosynthetic apparatus., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The siroheme-containing subunit from the multimeric hemoflavoprotein NADPH-dependent sulfite reductase (SiR/SiRHP) catalyzes the six electron-reduction of SO 3 2- to S 2- . Siroheme is an iron-containing isobacteriochlorin that is found in sulfite and homologous siroheme-containing nitrite reductases. Siroheme does not work alone but is covalently coupled to a Fe 4 S 4 cluster through one of the cluster's ligands. One long-standing hypothesis predicted from this observation is that the environment of one iron-containing cofactor influences the properties of the other. We tested this hypothesis by identifying three amino acids (F437, M444, and T477) that interact with the Fe 4 S 4 cluster and probing the effect of altering them to alanine on the function and structure of the resulting enzymes by use of activity assays, X-ray crystallographic analysis, and EPR spectroscopy. We showed that F437 and M444 gate access for electron transfer to the siroheme-cluster assembly and the direct hydrogen bond between T477 and one of the cluster sulfides is important for determining the geometry of the siroheme active site., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The bacterial acyl protein thioesterase (APT) homologue FTT258 from the gram-negative pathogen Francisella tularensis exists in equilibrium between a closed and open state. Interconversion between these two states is dependent on structural rearrangement of a dynamic loop overlapping its active site. The dynamics and structural properties of this loop provide a simple model for how the catalytic activity of FTT258 could be spatiotemporally regulated within the cell. Herein, we characterized the dual roles of this dynamic loop in controlling its catalytic and membrane binding activity. Using a comprehensive library of loop variants, we determined the relative importance of each residue in the loop to these two biological functions. For the catalytic activity, a centrally located tryptophan residue (Trp66) was essential, with the resulting alanine variant showing complete ablation of enzyme activity. Detailed analysis of Trp66 showed that its hydrophobicity in combination with spatial arrangement defined its essential role in catalysis. Substitution of other loop residues congregated along the N-terminal side of the loop also significantly impacted catalytic activity, indicating a critical role for this loop in controlling catalytic activity. For membrane binding, the centrally located hydrophobic residues played a surprising minor role in membrane binding. Instead general electrostatic interactions regulated membrane binding with positively charged residues bracketing the dynamic loop controlling membrane binding. Overall for FTT258, this dynamic loop dually controlled its biological activities through distinct residues within the loop and this regulation provides a new model for the spatiotemporal control over FTT258 and potentially homologous APT function., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Thermotoga maritima Arginine Binding Protein (TmArgBP) is a valuable candidate for arginine biosensing in diagnostics. This protein is endowed with unusual structural properties that include an extraordinary thermal/chemical stability, a domain swapped structure that undergoes large tertiary and quaternary structural transition, and the ability to form non-canonical oligomeric species. As the intrinsic stability of TmArgBP allows for extensive protein manipulations, we here dissected its structure in two parts: its main body deprived of the swapping fragment (TmArgBP 20-233 ) and the C-terminal peptide corresponding to the helical swapping element. Both elements have been characterized independently or in combination using a repertoire of biophysical/structural techniques. Present investigations clearly indicate that TmArgBP 20-233 represents a better scaffold for arginine sensing compared to the wild-type protein. Moreover, our data demonstrate that the ligand-free and the ligand-bound forms respond very differently to this helix deletion. This drastic perturbation has an important impact on the ligand-bound form of TmArgBP 20-233 stability whereas it barely affects its ligand-free state. The crystallographic structures of these forms provide a rationale to this puzzling observation. Indeed, the arginine-bound state is very rigid and virtually unchanged upon protein truncation. On the other hand, the flexible ligand-free TmArgBP 20-233 is able to adopt a novel state as a consequence of the helix deletion. Therefore, the flexibility of the ligand-free form endows this state with a remarkable robustness upon severe perturbations. In this scenario, TmArgBP dissection highlights an intriguing connection between destabilizing/stabilizing effects and the overall flexibility that could operate also in other proteins., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Polychlorinated biphenyls (PCBs) are xenobiotic compounds that persists in the environment for long-term, though its productivity is banned. Abatement of the pollutants have become laborious due to it's recalcitrant nature in the environment leading to toxic effects in humans and other living beings. Biphenyl degrading bacteria co-metabolically degrade low chlorinated PCBs using the active metabolic pathway. bph operon possess different genetic arrangements in gram positive and gram negative bacteria. The binding ability of the genes and the active sites were determined by PCB docking studies. The active site of bphA gene with conserved amino acid residues determines the substrate specificity and biodegradability. Accumulation of toxic intermediates alters cellular behaviour, biomass production and downturn the metabolic activity. Several bacteria in the environment attain unculturable state which is viable and metabolically active but not cultivable (VBNC). Resuscitation-promoting factor (Rpf) and Rpf homologous protein retrieve the culturability of the so far uncultured bacteria. Recovery of this adaptive mechanism against various physical and chemical stressors make a headway in understanding the functionality of both environmental and medically important unculturable bacteria. Thus, this paper review about the general aspects of PCBs, cellular toxicity exerted by PCBs, role of unculturable bacterial strains in biodegradation, genes involved and degradation pathways. It is suggested to extrapolate the research findings on extracellular organic matters produced in culture supernatant of VBNC thus transforming VBNC to culturable state., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Given their involvement in catalysis, infection, and biofilm formation, Fe and Mn are essential for bacterial survival and virulence. In this study, we found that Streptococcus pneumoniae (S. pneumoniae) could grow in the Mn-deficient medium (MDCM). Furthermore, findings showed that the Fe concentration in the bacterium increased when the Mn concentration decreased. In addition, it was noted that supplementing MDCM with Fe resulted in the recovery of bacterial growth. Quantitative proteomics using stable-isotope dimethyl labeling was performed to investigate the adaptive growth mechanism of S. pneumoniae under Mn-deficient conditions. It was found that the expression levels of 25 proteins were downregulated, whereas those of 54 proteins were upregulated in S. pneumoniae grown in MDCM. It was also noted that several of the downregulated proteins were involved in cell energy metabolism, amino acid synthesis, and reduction of oxidation products. More importantly, several ATP-binding cassette transporters related to Fe uptake, such as PiuA, PiaA, PitA, and SPD&#95;1609, were overexpressed for increased Fe uptake from the MDCM. The results suggest that Mn deficiency disturbs multiple metabolic processes in S. pneumoniae. Furthermore, it causes a compensatory effect of Fe for Mn, which is beneficial for the survival of the bacterium in extreme environments., Significance: The relationship between manganese and iron metabolism in S. pneumoniae has not been clearly revealed. In this paper, we suggest that Mn limitation disturbs multiple metabolic processes and evidently decreases the ATP level in the bacterium. In order to survive in this extreme environment, bacteria upregulated three type of Fe ion transporters PiuABC (heme), PiaABC (ferrichrome) and PitABC (Fe 3+ ) to uptake enough Fe ions to response to Mn deficiency. Therefore, this study reveals a bacterial mechanism of Fe compensation for Mn, and provides new insight for investigating the relativeness of Fe and Mn metabolism of bacteria., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Multiple secretion pathways are known for export of protein(s) forming the S-layer in bacteria. The unicellular model cyanobacterium Synechocystis sp. strain PCC 6803 (hereafter S. 6803) also possesses a well-defined S-layer composed of Sll1951 protein. However, the mechanism of its secretion is not completely understood. In the present study, the putative T1SS (Type I secretion system) components, Sll1180 and Sll1181 [inner membrane ABC transporter and membrane fusion protein (MFP), respectively] were characterized for their role in Sll1951 secretion. The corresponding ORFs i.e. sll1180 and sll1181 were inactivated by insertion of a spectinomycin resistance gene. The viability of the homozygous mutants of both the genes indicated dispensability of the corresponding proteins under the experimental conditions. Interestingly, the culture supernatants of the mutants i.e. Δsll1180 and Δsll1181, lacked Sll1951 as observed on SDS-PAGE and confirmed by mass spectrometry. Immunofluorescence delineated a distinct outer ring of Sll1951 in S. 6803 cells only that was further iterated by transmission and scanning electron microscopy. The loss of S-layer imparted an aggregative phenotype to both the mutants. Surprisingly, Δsll1181 cells showed increased sensitivity to different antibiotics indicating a role in multidrug efflux. This is the first report establishing Sl1180 and Sll1181 proteins as partners of the previously characterized Slr1270, for Sll1951 secretion and thus S-layer biogenesis in S. 6803. Sll1181 (in conjunction with Slr1270) also acts as MFP in multidrug efflux along with a yet uncharacterized inner membrane protein., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Streptococcus suis is a major swine pathogen worldwide and causes considerable economic losses in the swine industry. S. suis is also an emerging zoonotic agent, mainly in Asia. In pigs and humans, S. suis can cause septicemia, pneumonia, endocarditis, arthritis, and meningitis with irreversible sequelae. Identification and characterization of the virulence factors produced by S. suis are major advances in the understanding of the pathogenesis of S. suis infections and has therefore opened promising avenues for vaccine development against this pathogen. This literature review aimed to update the current knowledge of the virulence mechanisms of S. suis and of the vaccination strategies tested until now., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Prokaryotic and eukaryotic cytosolic aminoacyl-tRNA synthetases (aaRSs) are essentially known for their conventional function of generating the full set of aminoacyl-tRNA species that are needed to incorporate each organism's repertoire of genetically-encoded amino acids during ribosomal translation of messenger RNAs. However, bacterial and eukaryotic cytosolic aaRSs have been shown to exhibit other essential nonconventional functions. Here we review all the subcellular compartments that prokaryotic and eukaryotic cytosolic aaRSs can reach to exert either a conventional or nontranslational role. We describe the physiological and stress conditions, the mechanisms and the signaling pathways that trigger their relocation and the new functions associated with these relocating cytosolic aaRS. Finally, given that these relocating pools of cytosolic aaRSs participate to a wide range of cellular pathways beyond translation, but equally important for cellular homeostasis, we mention some of the pathologies and diseases associated with the dis-regulation or malfunctioning of these nontranslational functions., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Staphylococcus aureus biofilms pose a serious clinical threat as reservoirs for persistent infections. Despite this clinical significance, the composition and mechanism of formation of S. aureus biofilms are unknown. To address these problems, we used solid-state NMR to examine S. aureus (SA113), a strong biofilm-forming strain. We labeled whole cells and cell walls of planktonic cells, young biofilms formed for 12-24h after stationary phase, and more mature biofilms formed for up to 60h after stationary phase. All samples were labeled either by (i) [ 15 N]glycine and l-[1- 13 C]threonine, or in separate experiments, by (ii) l-[2- 13 C, 15 N]leucine. We then measured 13 C- 15 N direct bonds by C{N} rotational-echo double resonance (REDOR). The increase in peptidoglycan stems that have bridges connected to a surface protein was determined directly by a cell-wall double difference (biofilm REDOR difference minus planktonic REDOR difference). This procedure eliminates errors arising from differences in 15 N isotopic enrichments and from the routing of 13 C label from threonine degradation to glycine. For both planktonic cells and the mature biofilm, 20% of pentaglycyl bridges are not cross-linked and are potential surface-protein attachment sites. None of these sites has a surface protein attached in the planktonic cells, but one-fourth have a surface protein attached in the mature biofilm. Moreover, the leucine-label shows that the concentration of β-strands in leucine-rich regions doubles in the mature biofilm. Thus, a primary event in establishing a S. aureus biofilm is extensive decoration of the cell surface with surface proteins that are linked covalently to the cell wall and promote cell-cell adhesion., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Vip3Aa insecticidal protein is produced from Bacillus thuringiensis and exerts a broad spectrum of toxicity against lepidopteran insect species. Although Vip3Aa has been effectively used as part of integrated pest management strategies, the mechanism of the toxin remains unclear. Here, we investigated the effect of pH in a range from 5.0 to 10.0 on the pore-forming activity of the trypsin activated Vip3Aa (actVip3Aa) by in vitro pore-forming assays. Based on calcein release assay, actVip3Aa could permeabilize the artificial neutral liposomes under all the pH tested, except pH10.0. The maximum membrane permeability of actVip3Aa was detected at pH8.0 and the permeability decreased and abolished when exposing to acidic and alkaline conditions, respectively. The planar lipid bilayer experiment revealed that actVip3Aa formed ion channels at pH5.0-8.0 but no current signals were detected at pH10.0, consistent with the observation from calcein release assay. The toxin formed ion channels with a diameter of 1.4nm at pH8.0 and pore size was gradually decreased when reducing the pH. This study provided a view of the molecular mechanism of Vip3Aa by which the pore-forming activity is regulated by pH., (Copyright © 2017 Elsevier B.V. All rights reserved.)

DnaA protein is the initiator of genomic DNA replication in prokaryotes. It binds to specific DNA sequences in the origin of DNA replication and unwinds small AT-rich sequences downstream for the assembly of the replisome. The mechanism of activation of DnaA that enables it to bind and organize the origin DNA and leads to replication initiation remains unclear. In this study, we have developed double-labeled fluorescent DnaA probes to analyze conformational states of DnaA protein upon binding DNA, nucleotide, and Soj sporulation protein using Fluorescence Resonance Energy Transfer (FRET). Our studies demonstrate that DnaA protein undergoes large conformational changes upon binding to substrates and there are multiple distinct conformational states that enable it to initiate DNA replication. DnaA protein adopted a relaxed conformation by expanding ~15Å upon binding ATP and DNA to form the ATP·DnaA·DNA complex. Hydrolysis of bound ATP to ADP led to a contraction of DnaA within the complex. The relaxed conformation of DnaA is likely required for the formation of the multi-protein ATP·DnaA·DNA complex. In the initiation of sporulation, Soj binding to DnaA prevented relaxation of its conformation. Soj·ADP appeared to block the activation of DnaA, suggesting a mechanism for Soj·ADP in switching initiation of DNA replication to sporulation. Our studies demonstrate that multiple conformational states of DnaA protein regulate its binding to DNA in the initiation of DNA replication., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In bacterial photosynthesis reaction center-light-harvesting 1 (RC-LH1) complexes trap absorbed solar energy by generating a charge separated state. Subsequent electron and proton transfers form a quinol, destined to diffuse to the cytochrome bc 1 complex. In bacteria such as Rhodobacter (Rba.) sphaeroides and Rba. capsulatus the PufX polypeptide creates a channel for quinone/quinol traffic across the LH1 complex that surrounds the RC, and it is therefore essential for photosynthetic growth. PufX also plays a key role in dimerization of the RC-LH1-PufX core complex, and the structure of the Rba. sphaeroides complex shows that the PufX C-terminus, particularly the region from X49-X53, likely mediates association of core monomers. To investigate this putative interaction we analysed mutations PufX R49L, PufX R53L, PufX R49/53L and PufX G52L by measuring photosynthetic growth, fractionation of detergent-solubilised membranes, formation of 2-D crystals and electron microscopy. We show that these mutations do not affect assembly of PufX within the core or photosynthetic growth but they do prevent dimerization, consistent with predictions from the RC-LH1-PufX structure. We obtained low resolution structures of monomeric core complexes with and without PufX, using electron microscopy of negatively stained single particles and 3D reconstruction; the monomeric complex with PufX corresponds to one half of the dimer structure whereas LH1 completely encloses the RC if the gene encoding PufX is deleted. On the basis of the insights gained from these mutagenesis and structural analyses we propose a sequence for assembly of the dimeric RC-LH1-PufX complex., (Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.)

To deal with fluctuating light condition, cyanobacteria have developed a photoprotective mechanism which, under high light conditions, decreases the energy arriving at the photochemical centers. It relies on a photoswitch, the Orange Carotenoid Protein (OCP). Once photoactivated, OCP binds to the light harvesting antenna, the phycobilisome (PBS), and triggers the thermal dissipation of the excess energy absorbed. Deactivation of the photoprotective mechanism requires the intervention of a third partner, the Fluorescence Recovery Protein (FRP). FRP by interacting with the photoactivated OCP accelerates its conversion to the non-active form and its detachment from the phycobilisome. We have studied the interaction of FRP with free and phycobilisome-bound OCP. Several OCP variants were constructed and characterized. In this article we show that OCP amino acid F299 is essential and D220 important for OCP deactivation mediated by FRP. Mutations of these amino acids did not affect FRP activity as helper to detach OCP from phycobilisomes. In addition, while mutated R60L FRP is inactive on OCP deactivation, its activity on the detachment of the OCP from the phycobilisomes is not affected. Thus, our results demonstrate that FRP has two distinct activities: it accelerates OCP detachment from phycobilisomes and then it helps deactivation of the OCP. They also suggest that different OCP and FRP amino acids could be involved in these two activities., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Tumor necrosis factor (TNF)-α-inducing protein (Tipα) is a newly identified carcinogenic factor secreted by Helicobacter pylori (H. pylori). Although it has been proved that Tipα is a strong inducer of epithelial-mesenchymal transition (EMT), a crucial process of migration, the exact molecular mechanism is unknown. Current evidence indicates that the oncogenic transcription factor signal transducers and activators of transcription 3 (STAT3) is inappropriately activated in multiple malignancies, including gastric cancer. In this study, we showed that Tipα significantly down-regulated the expression of EMT-related markers E-cadherin as well as up-regulated N-cadherin and vimentin in SGC7901 cells, with typical morphological changes of EMT. Tipα also promoted proliferation and migration of SGC7901 cells. Furthermore, Tipα activated interleukin-6 (IL-6)/STAT3 signaling pathway in SGC7901 cells. The effects of Tipα treatment observed was abolished when we block IL-6/STAT3 signaling pathway. Altogether, our data demonstrated that Tipα may accelerate tumor aggressiveness in gastric cancer by promoting EMT through activation of IL-6/STAT3 pathway., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Unlabelled: Post-translational modifications such as acetylation are an essential regulatory mechanism of protein function. Spiroplasma eriocheiris, with no cell wall and a helical structure, is a novel pathogen of freshwater crustacean. There is no other evidence of acylation (such as succinylation and propionylation) except acetylation genes in S. eriocheiris concise genome. So the acetylation may play an important role in S. eriocheiris. Here, we conducted the first lysine acetylome in S. eriocheiris. We identified 2567 lysine acetylation sites in 555 proteins, which account for 44.69% of the total proteins in this bacterium. To date, this is the highest ratio of acetylated proteins that have been identified in bacteria. Fifteen types of acetylated peptide sequence motifs were revealed from the acetylome. Forty-five lysine-acetylated proteins showed homology with acetylated proteins previously identified from Escherichia coli, Vibrio parahemolyticus and Mycobacterium tuberculosis. Notably, most proteins in glycolysis and all proteins in the arginine deiminase system were acetylated. Meanwhile, the cell skeleton proteins (Fibril and Mrebs) were all acetylated the observed acetylation also played an important role in cell skeleton formation. The results imply previously unreported hidden layers of post-translational regulation in lysine acetylation that define the functional state of Spiroplasma., Biological Significance: This is the first time to analyze PTM of Spiroplasma. This is the highest ratio of acetylated proteins that have been identified in bacteria. S. eriocheiris lysine acetylome reveals acetylated proteins related to metabolism and helical structure. These data provide an important resource to elucidate the role of acetylation in Spiroplasma cellular physiology., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Brucella spp. are known to avoid host immune recognition and weaken the immune response to infection. Brucella like accomplish this by employing two clever strategies, called the stealth strategy and hijacking strategy. The TIR domain-containing protein (TcpB/Btp1) of Brucella melitensis is thought to be involved in inhibiting host NF-κB activation by binding to adaptors downstream of Toll-like receptors. However, of the five TIR domain-containing adaptors conserved in mammals, whether MyD88 or MAL, even other three adaptors, are specifically targeted by TcpB has not been identified. Here, we confirmed the effect of TcpB on B.melitensis virulence in mice and found that TcpB selectively targets MAL. By using siRNA against MAL, we found that TcpB from B.melitensis is involved in intracellular survival and that MAL affects intracellular replication of B.melitensis. Our results confirm that TcpB specifically targets MAL/TIRAP to disrupt downstream signaling pathways and promote intra-host survival of Brucella spp., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Unlabelled: Acidithiobacillus ferrooxidans is used in industrial bioleaching of minerals to extract valuable metals. A. ferrooxidans strain ATCC 53993 is much more resistant to copper than other strains of this microorganism and it has been proposed that genes present in an exclusive genomic island (GI) of this strain would contribute to its extreme copper tolerance. ICPL (isotope-coded protein labeling) quantitative proteomics was used to study in detail the response of this bacterium to copper. A high overexpression of RND efflux systems and CusF copper chaperones, both present in the genome and the GI of strain ATCC 53993 was found. Also, changes in the levels of the respiratory system proteins such as AcoP and Rus copper binding proteins and several proteins with other predicted functions suggest that numerous metabolic changes are apparently involved in controlling the effects of the toxic metal on this acidophile., Significance: Using quantitative proteomics we overview the adaptation mechanisms that biomining acidophiles use to stand their harsh environment. The overexpression of several genes present in an exclusive genomic island strongly suggests the importance of the proteins coded in this DNA region in the high tolerance of A. ferrooxidans ATCC 53993 to metals., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Unlabelled: Antimicrobial resistance is a growing public health threat worldwide that is still far from a complete understanding. Salmonella Typhimurium DT104 multiresistant strains with additional quinolone resistance are highly adaptive and have been responsible for global outbreaks and high mortality. In order to give new insights about the resistance mechanisms involved, the developed work aimed to point out subproteome changes between a DT104B clinical strain (Se20) that acquired quinolone resistance after patient treatment and an in vitro induced clonally related highly-resistant mutant (Se6-M). The intracellular subproteomes were compared by a 2-DE/LC-MS/MS approach and a total of 50 unique proteins were identified (32 more abundant in Se20 and 18 more abundant in Se6-M). The membrane subproteomes were analysed by a shotgun LC-MS/MS approach, where 7 differentially abundant proteins were identified (5 more abundant in Se6-M and 2 more abundant in Se20). Several proteins known to be directly related to quinolone resistance mechanisms (AAC(6')-Ib-cr4, OmpC, OmpD, OmpX, etc.) and MipA, recently reported as novel antibiotic resistance-related protein, were identified. Other proteins (Fur, SodA, SucB, AtpD/AtpG, OmpC, GltI, CheM/CheB, etc.) reflecting the metabolic re-adjustments occurred in each strain in order to acquire quinolone resistance were also identified. Moreover, proteins involved in lipopolysaccharide biosynthesis (RfbF, RfbG, GmhA) and export (LptA) were detected, supporting the importance of exploring these proteins as targets for the development of new antimicrobial agents. In conclusion, this study provides new insights into the mechanisms involved in the acquisition of antibiotic resistance, which can be highly valuable for the development of improved therapeutic strategies., Biological Significance: This comparative proteomic study revealed a large number of differentially regulated proteins involved in antibiotic resistance which can be of great value to drug discovery, research and development programmes., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Unlabelled: Diurnal rhythms are recurring 24h patterns such as light/dark cycles that affect many natural environmental and biological processes. The cyanobacterium Synechococcus elongatus PCC 7942 (S. elongatus) produces its energy through photosynthesis and therefore its internal molecular machinery is strongly influenced by these diurnal rhythms. Moreover, it has one of the simplest, self-sustained, circadian rhythms, extensively studied functionally and structurally. These characteristics together with the relatively small genome of S. elongatus, make it an ideal model system for the study of diurnal and circadian rhythms. Although expression of many gene transcripts has been shown to fluctuate in phase with the circadian rhythm, fluctuations at the protein level were less pronounced. This led us to hypothesize that the diurnal adaptation occurs at the level of higher organization of protein complexes. Therefore, we probed the abundance and constituency of S. elongatus protein complexes during the day and night. Following several well-known complexes such as the RNA polymerase, the ribosome and photosynthetic protein complexes, we observe for the first time that these complexes change not only in abundance but also in constituency. Therefore, we conclude that the dynamic assembly of protein complexes is indeed also a key-player in the processes governing the diurnal rhythm., Significance: The succession of day and night periods imposes drastic changes in all living organisms. Cyanobacteria produce their energy through photosynthesis and are therefore strongly influenced by diurnal rhythms. The cyanobacteria, Synechococcus elongatus PCC 7942 (S. elongatus), also exhibit a self-sustained biological clock. The connection between the central circadian oscillator and its output to the rest of the cell is not completely known. It has been shown that the expression of many gene transcripts heavily fluctuates in phase with the circadian rhythm; however, our recent global proteomics investigation revealed that the diurnal fluctuations seemed to be less pronounced at the protein level. As many known regulatory functions depend on protein-protein interactions (PPIs) and/or protein assemblies and the fact that so few fluctuations in protein abundances were observed earlier, here we investigated the diurnal adaptation at the level of dynamic changes in protein assembly. The paper demonstrates that the combination of native protein complex fractionation and high-resolution proteomics provides insight in the regulation of megadalton protein assemblies in cyanobacteria, including the ribosomal and photosynthetic complexes. The differences observed between the light and dark conditions in these complexes indicate a cyclic regulation of essential cellular processes., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Unlabelled: Ferric uptake regulator (Fur) is an important transcriptional regulator of Gram-negative bacteria. Edwardsiella tarda is a severe fish bacterial pathogen with a broad host range that includes humans. In this study, we examined the regulatory function of Fur in E. tarda via a proteomic approach. Compared to the wild type TX01, the fur mutant TX01Δfur exhibited (i) retarded growth, (ii) enhanced siderophore production, (iii) increased acid tolerance, which is in contrast to observations in other bacterial species, (iv) decreased survival against oxidative stress and host serum, (v) impaired ability to inhibit host immune response, (vi) attenuated tissue infectivity and overall virulence. The deficiency of TX01Δfur was rescued by introduction of an exogenous fur gene. iTRAQ-based comparative proteomic analysis of TX01Δfur and TX01 identified 89 differentially expressed proteins that cover a wide range of functional categories including those affected by fur mutation. In addition, 16 proteins were identified for the first time to be regulated by Fur in Gram-negative bacteria. These results provide the first protein-based interpretation of the global impact of Fur on the physiology and infectivity of E. tarda., Significance: This study demonstrates that in E. tarda, Fur controls multiple aspects of bacterial life, including growth, metabolism, iron acquisition, stress response, and host infection. In line with these observations, proteomics analysis identified a large amount of proteins affected in expression by Fur, which are involved in bacterial physiology and infectivity. Hence, these results link for the first time the pleiotropic effect of Fur with global protein expression and shed new light on the function and regulatory mechanism of Fur in pathogenic bacteria., (Copyright © 2016. Published by Elsevier B.V.)

Rickettsia parkeri is an Amblyomma-associated, spotted fever group Rickettsia species that causes an eschar-associated, febrile illness in multiple countries throughout the Western Hemisphere. Many other rickettsial species of known or uncertain pathogenicity have been detected in Amblyomma spp. ticks in the Americas, including Rickettsia amblyommii, "Candidatus Rickettsia andeanae" and Rickettsia rickettsii. In this study, we utilized an immunoproteomic approach to compare antigenic profiles of low-passage isolates of R. parkeri and R. amblyommii with serum specimens from patients with PCR- and culture-confirmed infections with R. parkeri. Five immunoreactive proteins of R. amblyommii and nine immunoreactive proteins of R. parkeri were identified by matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry. Four of these, including the outer membrane protein (Omp) A, OmpB, translation initiation factor IF-2, and cell division protein FtsZ, were antigens common to both rickettsiae. Serum specimens from patients with R. parkeri rickettsiosis reacted specifically with cysteinyl-tRNA synthetase, DNA-directed RNA polymerase subunit alpha, putative sigma (54) modulation protein, chaperonin GroEL, and elongation factor Tu of R. parkeri which have been reported as virulence factors in other bacterial species. Unique antigens identified in this study may be useful for further development of the better serological assays for diagnosing infection caused by R. parkeri., (Copyright © 2015 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Extra-chromosomal DNA maintenance (EDM) as an important process in the propagation and genetic engineering of microbes. Bacillus subtilis EdmS (formerly PgsE), a protein comprising 55 amino acids, is a mediator of the EDM process. In this study, the effect of mutation of global regulators on B. subtilis EDM was examined. Mutation of the swrA gene abolished EdmS-mediated EDM. It is known that swrA predominantly regulates expression of the fla/che operon in B. subtilis. We therefore performed EDM analysis using fla/che-deletion mutants and identified an EDM-mediated EDM cooperator in the flgB-fliL region. Further genetic investigation identified the flagellation factor FliF is a crucial EDM cooperator. To our knowledge, this is the first observation of the moonlighting function of FliF in DNA maintenance., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: Prior reports suggested that infection with Helicobacter pylori was associated with respiratory diseases; pathogenetic mechanisms however, were not defined. We tested the hypothesis that VacA, an exotoxin of H. pylori, a gastric pathogen, was aspirated into the lung and could stimulate secretion of inflammatory cytokines by lung epithelial cells., Methods: The presence of VacA was determined by immunohistochemistry in surgical lung biopsy tissue samples from 72 patients with interstitial pneumonia. The effects of VacA on A549 human alveolar epithelial adenocarcinoma cells and normal human bronchial epithelial cells were determined. After incubation with VacA, the secretions of cytokines were measured by Multiplex Luminex(®) Assays., Results: VacA was detected with anti-VacA antibodies in bronchial epithelial cells and alveolar epithelial cells from 10 of 72 patients with interstitial pneumonia. VacA was more prevalent in lungs of patients with collagen vascular disease-associated interstitial pneumonia than in those of patients with idiopathic pulmonary fibrosis, nonspecific interstitial pneumonia and cryptogenic organizing pneumonia. Incubation of A549 cells and normal human bronchial epithelial cells with VacA for 24 h was cytotoxic, and resulted in vacuolation. VacA induced interleukin-8 production by A549 cells and normal human bronchial epithelial cells and interleukin-6 production by A549 cells. Based on multiplex screening, interleukin-8 and interleukin-6 were the primary secretory products induced by VacA., Conclusions: H. pylori VacA is present in human lung and can induce interleukin-8 and interleukin-6 production by human lung cells. VacA could have a role in the pathogenesis of respiratory diseases by its cytotoxic effects and by inducing the secretion of interleukin-8 and interleukin-6 by targeted airway epithelial cells., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Purpose: We determined the prevalence of ESBL Enterobacteriaceae in urinary tract infections among inpatients, identified risk factors of acquisition, and evaluated the effectiveness of alternatives to carbapenems., Methods: The clinical, microbiological, and therapeutic data as well as the outcomes were recorded for all ESBL-E positive urine samples for three months., Results: Thirty-one (4%) of the 762 Enterobacteriaceae positive cultures were ESBL producers. The predisposing conditions for being infected with those strains were: immunodepression (61%), recent hospitalization (52%), recent antibiotic therapy (52%), and urinary catheterization (61%). 19% of infections were community acquired. The seven cases of acute pyelonephritis and five of prostatitis were treated with piperacillin-tazobactam (5), fluoroquinolones (4), ceftazidime (2), or carbapenems (only 1) after specialized advice. Four (33%) patients relapsed at week 10: three were immunodepressed and three presented with bacteremia., Conclusions: Alternatives to carbapenems (especially piperacillin-tazobactam) seem to be a good option for non-bacteremic UTI in immunocompetent patients., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

WalRK Two Component System (TCS) of Bacillus anthracis forms a functional TCS. This report elaborates upon the WalRK genomic architecture, promoter structure, promoter activity and expression under various stress conditions in B. anthracis. 5' RACE located the WalRK functional promoter within 317 bp region upstream of WalR. Reporter gene assays demonstrated maximal promoter activity during early growth phases indicating utility in exponential stages of growth. qRT-PCR showed upregulation of WalRK transcripts during temperature and antibiotic stress. However, WalR overexpression did not affect the tested antibiotic MIC values in B. anthracis. Collectively, these results confirm that WalRK responds to cell envelope stress in B. anthracis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Iron is an essential metal for almost all forms of life, but potentiates oxidative stress via Fenton catalysis. During microbial lag phase there is a rapid influx of iron with concomitant oxidative hypersensitivity. How and why iron accumulation occurs remains to be elucidated. Iron homeostasis in prokaryotes is mediated by the ferric uptake regulator (Fur), an iron-activated global regulator that controls intracellular iron levels by feedback inhibition with the metal. Herein it is postulated, based on the expression profiles of antioxidant enzymes within the Fur regulon as observed in wild type and Δfur mutants, that iron accumulation is mediated by a transitively low concentration of the Fur protein during lag phase. Vertebrate hosts sequester iron upon 'sensing' an infection in order to retard microbial proliferation through a process known as 'nutritional immunity'. It is herein argued that the purpose of iron accumulation is not principally a preparative step for the replicative phase, as suggested elsewhere, but an evolved behavior that counteracts host iron sequestration. This interpretation is supported by multiple clinical and animal studies that demonstrate that iron surplus in hosts advances progression and susceptibility to infection, and vice versa. Contextualizing iron accumulation as a counter-immune behavior adds impetus to the development of antibiotics targeting pathogenic modes of iron acquisition., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Virulent infections are expected to impair learning ability, either as a direct consequence of stressed physiological state or as an adaptive response that minimizes diversion of energy from immune defense. This prediction has been well supported for mammals and bees. Here, we report an opposite result in Drosophila melanogaster. Using an odor-mechanical shock conditioning paradigm, we found that intestinal infection with bacterial pathogens Pseudomonas entomophila or Erwinia c. carotovora improved flies' learning performance after a 1h retention interval. Infection with P. entomophila (but not E. c. carotovora) also improved learning performance after 5 min retention. No effect on learning performance was detected for intestinal infections with an avirulent GacA mutant of P. entomophila or for virulent systemic (hemocoel) infection with E. c. carotovora. Assays of unconditioned responses to odorants and shock do not support a major role for changes in general responsiveness to stimuli in explaining the changes in learning performance, although differences in their specific salience for learning cannot be excluded. Our results demonstrate that the effects of pathogens on learning performance in insects are less predictable than suggested by previous studies, and support the notion that immune stress can sometimes boost cognitive abilities., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Low pH is recognized as a major environmental stress to cyanobacteria that play a pivotal role in the global carbon cycling. Although several cellular mechanisms in response to acid stress were proposed, the regulatory mechanism related to acid stress has not been fully elucidated. By screening gene knockout mutants for all 44 putative response regulator (RR)-encoding genes of Synechocystis sp. PCC 6803 grown under acid stress, we found that a mutant of slr1909 (previously known as rre9), which encoded an orphan RR, grew poorly in BG11 medium at pH6.2-6.5 when compared with the wild type. Using a quantitative iTRAQ-LC-MS/MS proteomics approach coupled with GC-MS based metabolomics and quantitative real-time reverse transcription-PCR (RT-qPCR), we further determined the possible acid response network mediated by Slr1909. The results showed that the signal transduction pathway mediated by Slr1909 may be independent from that mediated by SphS-SphR previously discovered, as none of the proteins and their coding genes regulated by SphS-SphR were differentially regulated in the ∆slr1909 mutant grown under acid stress. Only 24 and 10 proteins were up- and down-regulated in the ∆slr1909 mutant when compared with the wild type under acid stress condition, respectively. Notably, three proteins, Slr1259, Slr1260 and Slr1261 whose encoding genes seem located in an operon, were down-regulated upon the knockout of the slr1909 gene, suggesting their roles in acid tolerance. In addition, metabolomic analysis allowed identification of a dozen metabolites important for the discrimination of the ∆slr1909 mutant and the wild type under acid stress, including several monosaccharide and fatty acids. The study provided a proteomic and metabolomic characterization of the acid-response network mediated by an orphan regulator Slr1909 in Synechocystis., Biological Significance: Low pH is recognized as a major environmental stress to cyanobacteria that play a pivotal role in the global carbon cycling. Although several cellular mechanisms in response to acid stress were proposed, the regulatory mechanism related to acid stress is still far from being fully elucidated. In a previous work, one two-component signal transduction system SphS-SphR was found involved in acid stress in Synechocystis. In this work, by screening gene knockout mutants for all 44 putative response regulator (RR)-encoding genes grown under acid stress, we found that a novel two-component response regulator Slr1909 was also involved in acid tolerance in Synechocystis. Moreover, the analysis showed that the signal transduction pathway mediated by Slr1909 may be independent from that mediated by SphS-SphR. Using a quantitative iTRAQ-LC-MS/MS proteomics and coupled with GC-MS based metabolomics and quantitative real-time reverse transcription-PCR (RT-qPCR), we further determined the possible acid response network mediated by Slr1909. The study provided a proteomic and metabolomic characterization of a novel acid-response network mediated by an orphan regulator Slr1909 in Synechocystis, and valuable new insight for better understanding of stress responses to acidity in cyanobacteria., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Salmonella enterica is a Gram-negative bacterium that causes gastroenteritis, bacteremia and typhoid fever in several animal species including humans. Its virulence is greatly dependent on two type III secretion systems, encoded in pathogenicity islands 1 and 2. These systems translocate proteins called effectors into eukaryotic host cell. Effectors interfere with host signal transduction pathways to allow the internalization of pathogens and their survival and proliferation inside vacuoles. SteA is one of the few Salmonella effectors that are substrates of both type III secretion systems. Here, we used gene arrays and bioinformatics analysis to study the genetic response of human epithelial cells to SteA. We found that constitutive synthesis of SteA in HeLa cells leads to induction of genes related to extracellular matrix organization and regulation of cell proliferation and serine/threonine kinase signaling pathways. SteA also causes repression of genes related to immune processes and regulation of purine nucleotide synthesis and pathway-restricted SMAD protein phosphorylation. In addition, a cell biology approach revealed that epithelial cells expressing steA show altered cell morphology, and decreased cytotoxicity, cell-cell adhesion and migration., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Studies have been reported on genomic analysis to explain virulence of MTB H37Rv vs. MTB H37Ra strain. Proteomic comparison analysis at our centre has shown variability of 244 proteins in MTB H37Rv. A single amino acid mutation in protein sequence may cause alteration in protein structure and function that may account for virulence and drug resistance properties of pathogenic organisms., Aim: To find the effect of single amino acid mutations on Mycobacterium tuberculosis H37Rv and H37Ra using computational approaches, Methods: Proteins with single amino acid mutation were analysed by different mutation analysis systems such as SIFT, PolyPhen, PROVEAN and HOPE. Subsequently, structure comparison of proposed modelled structure of mutant proteins of MTB H37Ra with native proteins of MTB H37Rv was performed., Results: We observed that amongst 40 single amino acid mutated proteins of MTB H37Ra, five were found to be damaged by all the mutation analysis systems. Upon structure comparison, the RMSD values between the native and mutant type proteins were found to be significantly higher. All the five proteins showed important biological function in MTB H37Rv. Further, when these five proteins of MTB H37Ra were compared with corresponding proteins of other virulent strains of MTB (i.e. F11 and CDC 1551), similar observation was made., Conclusion: The data suggests the important role of single amino acid mutation in five proteins in causing changes in the virulence and pathogenicity of clinical strains of MTB.

Propionibacterium acnes colonizes the lipid-rich sebaceous glands of the skin. This preferential anaerobic bacterium is easily identified if cultures are prolonged. It is involved in the inflammation process of acne, but until recently, it was neglected in other clinical presentations. Despite a reported low virulence, the new genomic, transcriptomic, and phylogenetic studies have allowed better understanding of this pathogen's importance that causes many chronic and recurrent infections, including orthopedic and cardiac prosthetic, and breast or eye implant-infections. These infections, facilitated by the ability of P. acnes to produce a biofilm, require using anti-biofilm active antibiotics such as rifampicin. The antibiogram of P. acnes is not systematically performed in microbiology laboratories because of its susceptibility to a wide range of antibiotics. However, in the last 10 years, the rate of antibiotic-resistant bacteria has increased, especially for macrolides and tetracyclines. Recently, rpoB gene mutations conferring resistance to rifampicin have been also reported. Thus in case of a biofilm growth mode, the therapeutic strategy should be discussed, according to the resistance phylotype and phenotype so as to optimize the treatment of these severe infections., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Antibiotic resistance has become a major public health problem in Algeria. Indeed the past decade, we have seen a significant increase in resistance to antibiotics especially in Gram-negative bacilli. Resistance to β-lactams in enterobacteria is dominated by the production of ESBL CTX-M-3 and CTX-M-15. The strains producing these enzymes are often the cause of potentially serious infections in both hospital and community settings. Identified plasmid cephalosporinases are CMY-2, CMY-12 and DHA-1. The isolation of strains of Enterobacteriaceae and Pseudomonas aeruginosa producing carbapenemases is rare in Algeria. Some Enterobacteriaceae producing OXA-48 or VIM-19 have been reported; so far, only VIM-2 has been identified in P. aeruginosa. However, the situation regarding the strains of Acinetobacter baumannii resistant to carbapenemases seems to be more disturbing. The carbapenemase OXA-23 is the most common and seems to be endemic in the north. The carbapenemase NDM-1 has also been identified. Resistance to aminoglycosides is marked by the identification armA gene associated with blaCTX-M genes in strains of Salmonella sp. Several other resistance genes have been identified sporadically in strains of Enterobacteriaceae, P. aeruginosa and A. baumannii. Resistance genes to fluoroquinolones are more recent identification in Algeria. The most common are the Qnr determinants followed by the bifunctional enzyme AAC[6']-Ib-cr. Resistance to sulfonamides and trimethoprim was also reported in Enterobacteriaceae strains in the west of the country., (Copyright © 2014. Published by Elsevier SAS.)

Microbial populations fulfil a critical role in the soil sustainability and their functionality can be ascertained by proteomics based on high-performance mass spectrometry (MS) measurements. However, soil proteomics is compromised by methodological issues, among which extraction is a limiting factor, and still has not been adequately applied in semiarid soils, which usually are nutrient limited. We aim to evaluate the functional and phylogenetic information retrieved from three semiarid soils with distinct edaphic properties and degradation levels. Three extraction methods with different physico-chemical bases were tested [1-3]. The HPLC-amino acid quantification of the extracted protein pellets revealed a tremendous inefficiency of the extraction methods, with a maximally 6.8% of the proteinaceous material being extracted in comparison with the protein content in the bulk soil. The composition of the proteomes extracted was analysed after SDS-PAGE and liquid chromatography coupled with electrospray-MS/MS. Chourey's method, based on boiling and DTT, yielded a high diversity of bacterial proteins and revealed differences in the community composition at the phylum level among the three soils. The overall metabolic information obtained by both extraction methods was similar, but Chourey's method provided additionally valuable bio-geochemical insights which suggest an ecological adaptation of microbial communities from semiarid soils for carbon and nitrogen fixation., Biological Significance: Microbial communities inhabiting the soil perform critical reactions for the sustainability of the planet. At biochemical level, soil proteomics is starting to provide incipient insights into the microbial functionality of soils. However, methodological comparisons are needed to assess which methods are more suitable. Precisely, such information under arid and semiarid environments is missing. By using amino acid quantification of extracted proteomes and LC-MS/MS based proteomics, we provide a novel methodological evaluation of the functional, phylogenetic and bio-geochemical information obtained by three extraction methods in semiarid soils with distinct edaphic properties., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Use of penicillin acylases for the production of semi-synthetic penicillins is well-known. Escherichia coli penicillin G acylase (EcPGA) has been extensively used for this purpose; however, Kluyvera citrophila penicillin G acylase (KcPGA) is assumed to be a better substitute, owing to its increased resilience to extreme pH conditions and ease of immobilization. In the present article we report a new dimension for the amidase activity of KcPGA by demonstrating its ability to cleave bacterial quorum sensing signal molecules, acyl homoserine lactones (AHL) with acyl chain length of 6-8 with or without oxo-substitution at third carbon position. Initial evidence of AHL degrading capability of KcPGA was obtained using CV026 based bioassay method. Kinetic studies performed at pH 8.0 and 50 °C revealed 3-oxo-C6 HSL to be the best substrate for the enzyme with V(max) and K(m) values of 21.37+0.85 mM/h/mg of protein and 0.1+0.01 mM, respectively. C6 HSL was found to be the second best substrate with V(max) and K(m) value of 10.06+0.27 mM/h/mg of protein and 0.28+0.02 mM, respectively. Molecular modeling and docking studies performed on the active site of the enzyme support these findings by showing the fitting of AHLs perfectly within the hydrophobic pocket of the enzyme active site., (Copyright © 2013 Elsevier Inc. All rights reserved.)

PhoB/PhoR is a two-component system originally described as involved in inorganic phosphate (Pi) transport and metabolism under Pi limitation. In order to disclose other roles of this system, a proteomic analysis of Vibrio cholerae 569BSR and its phoB/phoR mutant under high Pi levels was performed. Most of the proteins downregulated by the mutant have roles in energy production and conversion and in amino acid transport and metabolism. In contrast, the phoB/phoR mutant upregulated genes mainly involved in adaptation to atypical conditions, indicating that the absence of a functional PhoB/PhoR caused increased expression of a number of genes from distinct stress response pathways. This might be a strategy to overcome the lack of RpoS, whose expression in the stationary phase cells of V. cholerae seems to be controlled by PhoB/PhoR. Moreover, compared to the wild-type strain the phoB/phoR mutant presented a reduced cell density at stationary phase of culture in Pi abundance, lower resistance to acid shock, but higher tolerance to thermal and osmotic stresses. Together our findings show, for the first time, the requirement of PhoB/PhoR for full growth under high Pi level and for the accumulation of RpoS, indicating that PhoB/PhoR is a fundamental system for the biology of V. cholerae., Biological Significance: Certain V. cholerae strains are pathogenic to humans, causing cholera, an acute dehydrating diarrhoeal disease endemic in Southern Asia, parts of Africa and Latin America, where it has been responsible for significant mortality and economical damage. Its ability to grow within distinct niches is dependent on gene expression regulation. PhoB/PhoR is a two-component system originally described as involved in inorganic phosphate (Pi) transport and metabolism under Pi limitation. However, Pho regulon genes also play roles in virulence, motility and biofilm formation, among others. In this paper we report that the absence of a functional PhoB/PhoR caused increased expression of a number of genes from distinct stress response pathways, in Pi abundance. Moreover, we showed, for the first time, that the interrelationship between PhoB-RpoS-(p)ppGpp-poly(P) in V. cholerae, is somewhat diverse from the model of inter-regulation between those systems, described in Escherichia coli. The V. cholerae dependence on PhoB/PhoR for the RpoS mediated stress response and cellular growth under Pi abundance, suggests that this system's roles are broader than previously thought., (Copyright © 2013. Published by Elsevier B.V.)

All organisms rely on integrated networks to repair DNA double-strand breaks (DSBs) in order to preserve the integrity of the genetic information, to re-establish replication, and to ensure proper chromosomal segregation. Genetic, cytological, biochemical and structural approaches have been used to analyze how Bacillus subtilis senses DNA damage and responds to DSBs. RecN, which is among the first responders to DNA DSBs, promotes the ordered recruitment of repair proteins to the site of a lesion. Cells have evolved different mechanisms for efficient end processing to create a 3'-tailed duplex DNA, the substrate for RecA binding, in the repair of one- and two-ended DSBs. Strand continuity is re-established via homologous recombination (HR), utilizing an intact homologous DNA molecule as a template. In the absence of transient diploidy or of HR, however, two-ended DSBs can be directly re-ligated via error-prone non-homologous end-joining. Here we review recent findings that shed light on the early stages of DSB repair in Firmicutes., (Copyright © 2012 Elsevier B.V. All rights reserved.)