Regulation ofwaaHby PhoB during PiStarvation Promotes Biofilm Formation byEscherichia coliO157:H7

ABSTRACTIn open environments such as water, enterohemorrhagicEscherichia coliO157:H7 responds to inorganic phosphate (Pi) starvation by inducing the Pho regulon controlled by PhoB. This activates the phosphate-specific transport (Pst) system that contains a high-affinity Pitransporter. In the Δpstmutant, PhoB is constitutively activated and regulates the expression of genes in the Pho regulon. Here, we show that Pistarvation and deletion of thepstsystem enhanceE. coliO157:H7 biofilm formation. Among differentially expressed genes of EDL933 grown under Pistarvation conditions and in the Δpstmutant, we have found that a member of the PhoB regulon,waaH, predicted to encode a glycosyltransferase, was highly expressed. Interestingly, WaaH contributed to biofilm formation ofE. coliO157:H7 during both Pistarvation and in the Δpstmutant. In the Δpstmutant, the presence ofwaaHwas associated with lipopolysaccharide (LPS) R3 core type modifications, whereas inE. coliO157:H7,waaHoverexpression had no effect on LPS structure during Pistarvation. Therefore,waaHparticipates inE. coliO157:H7 biofilm formation during Pistarvation, but its biochemical role remains to be clarified. This study highlights the importance of the Pistarvation stress response to biofilm formation, which may contribute to the persistence ofE. coliO157:H7 in the environment.IMPORTANCEEnterohemorrhagicEscherichia coliO157:H7 is a human pathogen that causes bloody diarrhea that can result in renal failure. Outside of mammalian hosts,E. coliO157:H7 survives for extended periods of time in nutrient-poor environments, likely as part of biofilms. InE. coliK-12, the levels of free extracellular Piaffect biofilm formation; however, it was unknown whether Piinfluences biofilm formation byE. coliO157:H7. Our results show that upon Pistarvation, PhoB activateswaaHexpression, which favors biofilm formation byE. coliO157:H7. These findings suggest that WaaH is a target for controlling biofilm formation. Altogether, our work demonstrates how adaptation to Pistarvation allowsE. coliO157:H7 to occupy different ecological niches.

Escherichia coliO157:H7 responds to phosphate starvation by modifying LPS involved in biofilm formation

ABSTRACTIn open environments such as water, enterohemorrhagicEscherichia coliO157:H7 responds to inorganic phosphate (Pi) starvation by inducing the Pho regulon controlled by PhoB. The phosphate-specific transport (Pst) system is the high-affinity Pi transporter. In the Δpstmutant, PhoB is constitutively activated and regulates the expression of genes from the Pho regulon. InE. coliO157:H7, the Δpstmutant, biofilm, and autoagglutination were increased. In the double-deletion mutant ΔpstΔphoB, biofilm and autoagglutination were similar to the wild-type strain, suggesting that PhoB is involved. We investigated the relationship between PhoB activation and enhanced biofilm formation by screening a transposon mutant library derived from Δpstmutant for decreased autoagglutination and biofilms mutants. Lipopolysaccharide (LPS) genes involved in the synthesis of the LPS core were identified. Transcriptomic studies indicate the influence of Pi-starvation andpstmutation on LPS biosynthetic gene expression. LPS analysis indicated that the O-antigen was deficient in the Δpstmutant. Interestingly,waaH, encoding a glycosyltransferase associated with LPS modifications inE. coliK-12, was highly expressed in the Δpstmutant ofE. coliO157:H7. Deletion ofwaaHfrom the Δpstmutant and from the wild-type strain grown in Pi-starvation conditions decreased the biofilm formation but without affecting LPS. Our findings suggest that LPS core is involved in the autoagglutination and biofilm phenotypes of the Δpstmutant and that WaaH plays a role in biofilm in response to Pi-starvation. This study highlights the importance of Pi-starvation in biofilm formation of E. coli O157:H7, which may affect its transmission and persistence.IMPORTANCEEnterohemorrhagicEscherichia coliO157:H7 is a human pathogen responsible for bloody diarrhea and renal failures. In the environment, O157:H7 can survive for prolonged periods of time under nutrient-deprived conditions. Biofilms are thought to participate in this environmental lifestyle. Previous reports have shown that the availability of extracellular inorganic phosphate (Pi) affected bacterial biofilm formation; however, nothing was known about O157:H7 biofilm formation. Our results show that O157:H7 membrane undergoes modifications upon PhoB activation leading to increased biofilm formation. A mutation in the Pst system results in reduced amount of the smooth type LPS and that this could influence the biofilm composition. This demonstrates how theE. coliO157:H7 adapts to Pi starvation increasing its ability to occupy different ecological niches.

Altered Regulation of the Diguanylate Cyclase YaiC Reduces Production of Type 1 Fimbriae in a Pst Mutant of Uropathogenic Escherichia coli CFT073

ABSTRACT The pst gene cluster encodes the phosphate-specific transport (Pst) system. Inactivation of the Pst system constitutively activates the two-component regulatory system PhoBR and attenuates the virulence of pathogenic bacteria. In uropathogenic Escherichia coli strain CFT073, attenuation by inactivation of pst is predominantly attributed to the decreased expression of type 1 fimbriae. However, the molecular mechanisms connecting the Pst system and type 1 fimbriae are unknown. To address this, a transposon library was constructed in the pst mutant, and clones were tested for a regain in type 1 fimbrial production. Among them, the diguanylate cyclase encoded by yaiC (adrA in Salmonella) was identified to connect the Pst system and type 1 fimbrial expression. In the pst mutant, the decreased expression of type 1 fimbriae is connected by the induction of yaiC. This is predominantly due to altered expression of the FimBE-like recombinase genes ipuA and ipbA, affecting at the same time the inversion of the fim promoter switch (fimS). In the pst mutant, inactivation of yaiC restored fim-dependent adhesion to bladder cells and virulence. Interestingly, the expression of yaiC was activated by PhoB, since transcription of yaiC was linked to the PhoB-dependent phoA-psiF operon. As YaiC is involved in cyclic di-GMP (c-di-GMP) biosynthesis, an increased accumulation of c-di-GMP was observed in the pst mutant. Hence, the results suggest that one mechanism by which deletion of the Pst system reduces the expression of type 1 fimbriae is through PhoBR-mediated activation of yaiC, which in turn increases the accumulation of c-di-GMP, represses the fim operon, and, consequently, attenuates virulence in the mouse urinary tract infection model. IMPORTANCE Urinary tract infections (UTIs) are common bacterial infections in humans. They are mainly caused by uropathogenic Escherichia coli (UPEC). We previously showed that interference with phosphate homeostasis decreases the expression of type 1 fimbriae and attenuates UPEC virulence. Herein, we identified that alteration of the phosphate metabolism increases production of the signaling molecule c-di-GMP, which in turn decreases the expression of type 1 fimbriae. We also determine the regulatory cascade leading to the accumulation of c-di-GMP and identify the Pho regulon as new players in c-di-GMP-mediated cell signaling. By understanding the molecular mechanisms leading to the expression of virulence factors, we will be in a better position to develop new therapeutics.

Mycobacterium tuberculosis PhoY Proteins Promote Persister Formation by Mediating Pst/SenX3-RegX3 Phosphate Sensing

ABSTRACT The Mycobacterium tuberculosis phosphate-specific transport (Pst) system controls gene expression in response to phosphate availability by inhibiting the activation of the SenX3-RegX3 two-component system under phosphate-rich conditions, but the mechanism of communication between these systems is unknown. In Escherichia coli, inhibition of the two-component system PhoR-PhoB under phosphate-rich conditions requires both the Pst system and PhoU, a putative adaptor protein. E. coli PhoU is also involved in the formation of persisters, a subpopulation of phenotypically antibiotic-tolerant bacteria. M. tuberculosis encodes two PhoU orthologs, PhoY1 and PhoY2. We generated phoY single- and double-deletion mutants and examined the expression of RegX3-regulated genes by quantitative reverse transcription-PCR (qRT-PCR). Gene expression was increased only in the ΔphoY1 ΔphoY2 double mutant and could be restored to the wild-type level by complementation with either phoY1 or phoY2 or by deletion of regX3. These data suggest that the PhoY proteins function redundantly to inhibit SenX3-RegX3 activation. We analyzed the frequencies of antibiotic-tolerant persister variants in the phoY mutants using several antibiotic combinations. Persister frequency was decreased at least 40-fold in the ΔphoY1 ΔphoY2 mutant compared to the frequency in the wild type, and this phenotype was RegX3 dependent. A ΔpstA1 mutant lacking a Pst system transmembrane component exhibited a similar RegX3-dependent decrease in persister frequency. In aerosol-infected mice, the ΔphoY1 ΔphoY2 and ΔpstA1 mutants were more susceptible to treatment with rifampin but not isoniazid. Our data demonstrate that disrupting phosphate sensing mediated by the PhoY proteins and the Pst system enhances the susceptibility of M. tuberculosis to antibiotics both in vitro and during infection. IMPORTANCE Persister variants, subpopulations of bacteria that are phenotypically antibiotic tolerant, contribute to the lengthy treatment times required to cure Mycobacterium tuberculosis infection, but the molecular mechanisms governing their formation and maintenance are poorly characterized. Here, we demonstrate that a phosphate-sensing signal transduction system, comprising the Pst phosphate transporter, the two-component system SenX3-RegX3, and functionally redundant PhoY proteins that mediate signaling between Pst and SenX3-RegX3, influences persister formation. Activation of RegX3 by deletion of the phoY genes or a Pst system component resulted in decreased persister formation in vitro. Activated RegX3 also limited persister formation during growth under phosphate-limiting conditions. Importantly, increased susceptibility to the front-line drug rifampin was also observed in a mouse infection model. Thus, the M. tuberculosis phosphate-sensing signal transduction system contributes to antibiotic tolerance and is a potential target for the development of novel therapeutics that may shorten the duration of tuberculosis treatment. IMPORTANCE Persister variants, subpopulations of bacteria that are phenotypically antibiotic tolerant, contribute to the lengthy treatment times required to cure Mycobacterium tuberculosis infection, but the molecular mechanisms governing their formation and maintenance are poorly characterized. Here, we demonstrate that a phosphate-sensing signal transduction system, comprising the Pst phosphate transporter, the two-component system SenX3-RegX3, and functionally redundant PhoY proteins that mediate signaling between Pst and SenX3-RegX3, influences persister formation. Activation of RegX3 by deletion of the phoY genes or a Pst system component resulted in decreased persister formation in vitro. Activated RegX3 also limited persister formation during growth under phosphate-limiting conditions. Importantly, increased susceptibility to the front-line drug rifampin was also observed in a mouse infection model. Thus, the M. tuberculosis phosphate-sensing signal transduction system contributes to antibiotic tolerance and is a potential target for the development of novel therapeutics that may shorten the duration of tuberculosis treatment.

EMERGENCY SHUTDOWN VALVE RELIABILITY FUNCTION TEST BY AUTOMATED PARTIAL STROKE TESTING SYSTEM

Partial stroke testing (PST) is a technique that is regularly practiced in oil and gas industries to test the emergency shutdown (ESD) valve by closing a certain percentage of the valve position and stop any flow through the pipeline. Generally, it only functions when there is an emergency occurring in the production system. When the ESD valve remains in one position for a long period, there is a risk and potential of fail on demand which is, the ESD valve fail to operate during the emergency shutdown. This testing can reveal approximately 75% of unrevealed failures in valves. It can also provide predictive maintenance data that can contribute to the extension of the preventive maintenance for the ESD valve. The objectives of this paper are to design, simulate, build and test the performance of the automated PST system based on PLC. Four guidelines and methodology are used in this work. First, understanding the operation of the PST system. Then, the utilization of the capability of MATLAB-Simulink software as the simulation tool for the PST design system. Next, designing the PST automated system based on PLC design and lastly, testing the performance of the PST design system using lab scale PST system prototype that has been built. Results of the project shows that the PST system is successfully designed and simulated via MATLAB-Simulink and the PLC programming is working in the correct order as performed on the prototype.

Mycobacterium tuberculosis Requires Phosphate-Responsive Gene Regulation To Resist Host Immunity

Mycobacterium tuberculosispersists in the tissues of mammalian hosts despite inducing a robust immune response dominated by the macrophage-activating cytokine gamma interferon (IFN-γ). We identified theM. tuberculosisphosphate-specific transport (Pst) system component PstA1 as a factor required to resist IFN-γ-dependent immunity. A ΔpstA1mutant was fully virulent in IFN-γ−/−mice but attenuated in wild-type (WT) mice and mice lacking specific IFN-γ-inducible immune mechanisms: nitric oxide synthase (NOS2), phagosome-associated p47 GTPase (Irgm1), or phagocyte oxidase (phox). These phenotypes suggest that ΔpstA1bacteria are sensitized to an IFN-γ-dependent immune mechanism(s) other than NOS2, Irgm1, or phox. In other species, the Pst system has a secondary role as a negative regulator of phosphate starvation-responsive gene expression through an interaction with a two-component signal transduction system. InM. tuberculosis, we found that ΔpstA1bacteria exhibited dysregulated gene expression during growth in phosphate-rich medium that was mediated by the two-component sensor kinase/response regulator system SenX3-RegX3. Remarkably, deletion of theregX3gene suppressed the replication and virulence defects of ΔpstA1bacteria in NOS2−/−mice, suggesting thatM. tuberculosisrequires the Pst system to negatively regulate activity of RegX3 in response to available phosphatein vivo. We therefore speculate that inorganic phosphate is readily available during replication in the lung and is an important signal controllingM. tuberculosisgene expression via the Pst-SenX3-RegX3 signal transduction system. Inability to sense this environmental signal, due to Pst deficiency, results in dysregulation of gene expression and sensitization of the bacteria to the host immune response.

Increased Pho Regulon Activation Correlates with Decreased Virulence of an Avian Pathogenic Escherichia coli O78 Strain

ABSTRACT Avian pathogenic Escherichia coli (APEC) strains are associated with respiratory infections, septicemia, cellulitis, peritonitis, and other conditions, since colibacillosis manifests in many ways. The Pho regulon is jointly controlled by the two-component regulatory system PhoBR and by the phosphate-specific transport (Pst) system. To determine the specific roles of the PhoBR regulon and the Pst system in the pathogenesis of the APEC O78 strain χ7122, different phoBR and pst mutant strains were tested in vivo in chickens and in vitro for virulence traits. Mutations resulting in constitutive activation of the Pho regulon rendered strains more sensitive than the wild type to hydrogen peroxide and to the bactericidal effects of rabbit serum. In addition, production of type 1 fimbriae was also impaired in these strains. Using a chicken competitive infection model, all PhoB constitutive mutants were outcompeted by the wild-type parent, including strains containing a functional Pst system. Cumulative inactivation of the Pst system and the PhoB regulator resulted in a restoration of virulence. In addition, loss of the PhoB regulator alone did not affect virulence in the chicken infection model. Interestingly, the level of attenuation of the mutant strains correlated directly with the level of activation of the Pho regulon. Overall, results indicate that activation of the Pho regulon rather than phosphate transport by the Pst system plays a major role in the attenuation of the APEC O78 strain χ7122.

Increased Transcription of the Phosphate-Specific Transport System of Escherichia coli O157:H7 after Exposure to Sodium Benzoate

Sodium benzoate is a widely used food antimicrobial in drinks and fruit juices. A microarray study was conducted to determine the transcriptional response of Escherichia coli O157:H7 to 0.5% (wt/vol) sodium benzoate. E. coli O157:H7 grown in 150 ml of Luria-Bertani broth was exposed to 0% (control) and 0.5% sodium benzoate. Each treatment was duplicated and sampled at 0 (immediately after exposure), 5, 15, 30, and 60 min. Total RNA was extracted and analyzed with E. coli 2.0 Gene Chips. Significant ontology categories affected by sodium benzoate exposure were determined with JProGO software. The phosphate-specific transport (Pst) system transports inorganic phosphate into bacterial cells, under phosphate-limited conditions. The Pst system was found to be highly upregulated. Increased expression of the Pst system was observed after the short 5 min of exposure to sodium benzoate; pstS, pstA, pstB, and pstC genes were upregulated more than twofold (linear scale) at 5, 15, 30, and 60 min. Increased expression of several other efflux systems, such as AcrAB-TolC, was also observed. The Pst system may act as an efflux pump under these stress-adapted conditions, as well as increase transport of phosphorus to aid in DNA, RNA, ATP, and phospholipid production. Understanding adaptations of Escherichia coli O157:H7 under antimicrobial exposure is essential to better understand and implement methods to inhibit or control its survival in foods.

The high-affinity phosphate transporter Pst in Proteus mirabilis HI4320 and its importance in biofilm formation

Proteus mirabilis causes urinary tract infections (UTIs) in individuals requiring long-term indwelling catheterization. The pathogenesis of this uropathogen is mediated by a number of virulence factors and the formation of crystalline biofilms. In addition, micro-organisms have evolved complex systems for the acquisition of nutrients, including the phosphate-specific transport system, which has been shown to be important in biofilm formation and pathogenesis. A functional Pst system is important during UTIs caused by P. mirabilis HI4320, since transposon mutants in the PstS periplasmic binding protein and the PstA permease protein were attenuated in the CBA mouse model of UTI. These mutants displayed a defect in biofilm formation when grown in human urine. This study focuses on a comparison of the proteomes during biofilm and planktonic growth in phosphate-rich medium and human urine, and microscopic investigations of biofilms formed by the pst mutants. Our data suggest that (i) the Δpst mutants, and particularly the ΔpstS mutant, are defective in biofilm formation, and (ii) the proteomes of these mutants differ significantly from that of the wild-type. Therefore, since the Pst system of P. mirabilis HI4320 negatively regulates biofilm formation, this system is important for the pathogenesis of these organisms during complicated UTIs.