Triggering the stringent response enhances synthetic methanol utilization in Escherichia coli.

Synthetic methylotrophy aims to engineer methane and methanol utilization pathways in platform hosts like Escherichia coli for industrial bioprocessing of natural gas and biogas. While recent attempts to engineer synthetic methylotrophs have proved successful, autonomous methylotrophy, i.e. the ability to utilize methane or methanol as sole carbon and energy substrates, has not yet been realized. Here, we address an important limitation of autonomous methylotrophy in E. coli: the inability of the organism to synthesize several amino acids when grown on methanol. By activating the stringent/stress response via ppGpp overproduction, or DksA and RpoS overexpression, we demonstrate improved biosynthesis of proteinogenic amino acids via endogenous upregulation of amino acid synthesis pathway genes. Thus, we were able to achieve biosynthesis of several limiting amino acids from methanol-derived carbon, in contrast to the control methylotrophic E. coli strain. This study addresses a key limitation currently preventing autonomous methylotrophy in E. coli and possibly other synthetic methylotrophs and provides insight as to how this limitation can be alleviated via stringent/stress response activation.

The CXXC Motifs Are Essential for the Function of BosR in Borrelia burgdorferi.

BosR, a Fur family member, is essential for the pathogenesis of the Lyme disease pathogen, Borrelia burgdorferi. Unlike typical Fur proteins in which DNA binding represses gene expression, binding of BosR to the rpoS promoter directly activates rpoS transcription in B. burgdorferi. However, virtually nothing is known concerning potential structural features and amino acid residues of BosR that are important for protein function and virulence regulation in B. burgdorferi. Particularly, it remains unknown what structural motifs or residues of BosR coordinate Zn, although previous analyses have indicated that the function of BosR may depend on Zn. To address these information gaps, we herein introduced mutations into four conserved cysteine residues in two putative CXXC motifs of BosR. Our data showed that the ability of BosR to bind Zn was dramatically reduced when the CXXC motifs were mutated. Moreover, we found that the two CXXC motifs contributed to the ability of BosR to form dimers. By using a trans-complementation genetic approach, we additionally demonstrated that both CXXC motifs of BosR were essential for in vivo gene expression regulation. Mutation of any of the four cysteines abolished the transcriptional activation of rpoS. In contrast to wild type BosR, each mutant protein was incapable of binding the rpoS promoter in electrophoretic mobility shift assays. The combined data strongly support that the two CXXC motifs and four cysteines constitute the structural site essential for Zn-coordination, protein dimerization, and the unique regulatory activity of BosR.

Escherichia coli can survive stress by noisy growth modulation.

Gene expression can be noisy, as can the growth of single cells. Such cell-to-cell variation has been implicated in survival strategies for bacterial populations. However, it remains unclear how single cells couple gene expression with growth to implement these strategies. Here, we show how noisy expression of a key stress-response regulator, RpoS, allows E. coli to modulate its growth dynamics to survive future adverse environments. We reveal a dynamic positive feedback loop between RpoS and growth rate that produces multi-generation RpoS pulses. We do so experimentally using single-cell, time-lapse microscopy and microfluidics and theoretically with a stochastic model. Next, we demonstrate that E. coli prepares for sudden stress by entering prolonged periods of slow growth mediated by RpoS. This dynamic phenotype is captured by the RpoS-growth feedback model. Our synthesis of noisy gene expression, growth, and survival paves the way for further exploration of functional phenotypic variability.

Influences of epigallocatechin gallate and citric acid on Escherichia coli O157:H7 toxin gene expression and virulence-associated stress response.

Citric acid and EGCG at their minimum inhibitory concentrations were tested in this study. Logarithmic phase cells of Escherichia coli O157:H7 (ATCC 43895) were exposed to EGCG and citric acid respectively. The results of RT-real time PCR showed that both EGCG and citric acid increased stx2 and oxyR expression and decreased stx1, recA and Q expression. The result of Western blotting for RecA protein further indicated that both EGCG and citric acid decreased RecA production. Both EGCG and citric acid increased the level of intracellular reactive oxygen species and H2 O2 production and decreased superoxide dismutase activity. Therefore, EGCG and citric acid might induce stx2 production by increasing oxidative stress response and inhibit stx1 production by suppressing SOS response. In our study, the differential effects of the two antimicrobials were observed. EGCG reduced ompC and rpoS expression. However, citric acid caused an increase in ompC and rpoS expression. Membrane permeability is associated with toxin release. Citric acid increased the outer membrane permeability of E. coli O157:H7. However, the outer membrane of E. coli O157:H7 remained unaffected by EGCG. SIGNIFICANCE AND IMPACT OF THE STUDY: Shiga toxins are the major virulence factors of Escherichia coli O157:H7. The use of antimicrobials triggering Shiga toxin production is controversial. (-)-epigallocatechin-3-gallate (EGCG) citric acid are often used singly or in combination to prevent micro-organisms in some food products. This study evaluated toxin induction in E. coli O157:H7 in response to EGCG and citric acid and investigated the potential mechanism of action. The findings may contribute to the proper use of EGCG and citric acid as antimicrobials.

Transcription and translation of the sigG gene is tuned for proper execution of the switch from early to late gene expression in the developing Bacillus subtilis spore.

A cascade of alternative sigma factors directs developmental gene expression during spore formation by the bacterium Bacillus subtilis. As the spore develops, a tightly regulated switch occurs in which the early-acting sigma factor σF is replaced by the late-acting sigma factor σG. The gene encoding σG (sigG) is transcribed by σF and by σG itself in an autoregulatory loop; yet σG activity is not detected until σF-dependent gene expression is complete. This separation in σF and σG activities has been suggested to be due at least in part to a poorly understood intercellular checkpoint pathway that delays sigG expression by σF. Here we report the results of a careful examination of sigG expression during sporulation. Unexpectedly, our findings argue against the existence of a regulatory mechanism to delay sigG transcription by σF and instead support a model in which sigG is transcribed by σF with normal timing, but at levels that are very low. This low-level expression of sigG is the consequence of several intrinsic features of the sigG regulatory and coding sequence-promoter spacing, secondary structure potential of the mRNA, and start codon identity-that dampen its transcription and translation. Especially notable is the presence of a conserved hairpin in the 5' leader sequence of the sigG mRNA that occludes the ribosome-binding site, reducing translation by up to 4-fold. Finally, we demonstrate that misexpression of sigG from regulatory and coding sequences lacking these features triggers premature σG activity in the forespore during sporulation, as well as inappropriate σG activity during vegetative growth. Altogether, these data indicate that transcription and translation of the sigG gene is tuned to prevent vegetative expression of σG and to ensure the precise timing of the switch from σF to σG in the developing spore.

Salt stress affects global protein expression profiles of extracellular membrane-derived vesicles of Listeria monocytogenes.

Our previous study has suggested that Listeria monocytogenes produces extracellular membrane vesicles (MVs) and its general stress transcription factor sigma B (σB) affects the production of MVs under energy stress. The objective of this study was to evaluate the production of MVs and perform global protein profiling for MVs with or without salt stress to understand the function of MVs in the pathogenesis of L. monocytogenes. When cells of L. monocytogenes were grown under 0.5 M salt stress, protein concentrations of MVs derived from wild-type strain and its isogenic ΔsigB mutant were approximately doubled compared to those of MVs derived from cells without salt stress. Proteomic analyses showed that the number of MV proteins expressed in wild-type strain was similar to that in ΔsigB mutant under salt stress. However, global protein expression profiles were dramatically changed under salt stress compared to those without salt stress. Fifteen σB dependent proteins were expressed in MVs of wild-type strain under salt stress, including osmolyte transporter OpuCABCD. In addition, MVs produced by salt stressed wild-type and ΔsigB mutant inhibited biofilm formation abilities of both strains. Taken together, our results suggest that salt stress can promote the production of MVs involved in carnitine transporter proteins, with σB playing a pivotal role in biological event.

Expression of Two RpoH Sigma Factors in Sinorhizobium meliloti upon Heat Shock.

The plant symbiotic α-proteobacterium Sinorhizobium meliloti has two RpoH-type sigma factors, RpoH1 and RpoH2. The former induces the synthesis of heat shock proteins and optimizes interactions with the host. Using a Western blot analysis, we examined time course changes in the intracellular contents of these factors upon a temperature upshift. The RpoH1 level was relatively high and constant, suggesting that its regulatory role in the heat shock response is attained through the activation of the pre-existing RpoH1 protein. In contrast, the RpoH2 level was initially undetectable, and gradually increased. These differential patterns reflect the functional diversification of these factors.

Broad-scale redistribution of mRNA abundance and transcriptional machinery in response to growth rate in Salmonella enterica serovar Typhimurium.

We have investigated the connection between the four-dimensional architecture of the bacterial nucleoid and the organism's global gene expression programme. By localizing the transcription machinery and the transcriptional outputs across the genome of the model bacterium Salmonella enterica serovar Typhimurium at different stages of the growth cycle, a surprising disconnection between gene dosage and transcriptional output was revealed. During exponential growth, gene output occurred chiefly in the Ori (origin), Ter (terminus) and NSL (non-structured left) domains, whereas the Left macrodomain remained transcriptionally quiescent at all stages of growth. The apparently high transcriptional output in Ter was correlated with an enhanced stability of the RNA expressed there during exponential growth, suggesting that longer mRNA half-lives compensate for low gene dosage. During exponential growth, RNA polymerase (RNAP) was detected everywhere, whereas in stationary phase cells, RNAP was concentrated in the Ter macrodomain. The alternative sigma factors RpoE, RpoH and RpoN were not required to drive transcription in these growth conditions, consistent with their observed binding to regions away from RNAP and regions of active transcription. Specifically, these alternative sigma factors were found in the Ter macrodomain during exponential growth, whereas they were localized at the Ori macrodomain in stationary phase.

Persistent damaged bases in DNA allow mutagenic break repair in Escherichia coli.

Bacteria, yeast and human cancer cells possess mechanisms of mutagenesis upregulated by stress responses. Stress-inducible mutagenesis potentially accelerates adaptation, and may provide important models for mutagenesis that drives cancers, host pathogen interactions, antibiotic resistance and possibly much of evolution generally. In Escherichia coli repair of double-strand breaks (DSBs) becomes mutagenic, using low-fidelity DNA polymerases under the control of the SOS DNA-damage response and RpoS general stress response, which upregulate and allow the action of error-prone DNA polymerases IV (DinB), II and V to make mutations during repair. Pol IV is implied to compete with and replace high-fidelity DNA polymerases at the DSB-repair replisome, causing mutagenesis. We report that up-regulated Pol IV is not sufficient for mutagenic break repair (MBR); damaged bases in the DNA are also required, and that in starvation-stressed cells, these are caused by reactive-oxygen species (ROS). First, MBR is reduced by either ROS-scavenging agents or constitutive activation of oxidative-damage responses, both of which reduce cellular ROS levels. The ROS promote MBR other than by causing DSBs, saturating mismatch repair, oxidizing proteins, or inducing the SOS response or the general stress response. We find that ROS drive MBR through oxidized guanines (8-oxo-dG) in DNA, in that overproduction of a glycosylase that removes 8-oxo-dG from DNA prevents MBR. Further, other damaged DNA bases can substitute for 8-oxo-dG because ROS-scavenged cells resume MBR if either DNA pyrimidine dimers or alkylated bases are induced. We hypothesize that damaged bases in DNA pause the replisome and allow the critical switch from high fidelity to error-prone DNA polymerases in the DSB-repair replisome, thus allowing MBR. The data imply that in addition to the indirect stress-response controlled switch to MBR, a direct cis-acting switch to MBR occurs independently of DNA breakage, caused by ROS oxidation of DNA potentially regulated by ROS regulators.

Effect of subinhibitory concentrations of tigecycline and ciprofloxacin on the expression of biofilm-associated genes and biofilm structure of Staphylococcus epidermidis.

Staphylococcus epidermidis is a leading cause of foreign body-associated infections. This is related to the bacterium's ability to form biofilms on synthetic materials. Bacteria within a biofilm may be exposed to subinhibitory concentrations (sub-MICs) of antibiotics because of an agent's limited penetration into the biofilm core. Here, we investigated the effect of sub-MICs of tigecycline and ciprofloxacin on the expression of biofilm-associated genes, i.e. icaA, altE and sigB, and the biofilm structure of five clinical isolates of S. epidermidis. For most tested isolates, the expression of these genes increased after exposure to 0.25 MIC and 0.5 MIC tigecycline. A slight decrease in icaAmRNA levels was observed only in two isolates in the presence of 0.25 MIC tigecycline. The effect of ciprofloxacin exposure was isolate-dependent. At 0.5 MIC, ciprofloxacin induced an increase of sigB and icaAmRNA levels in three of the five tested isolates. At the same time, expression of the altE gene increased in all isolates (from 1.3-fold to 42-fold, depending on the strain). Confocal laser scanning microscopy analysis indicated that sub-MIC ciprofloxacin decreased biofilm formation, whereas tigecycline stimulated this process. Our data suggest that sub-MIC tigecycline may have bearing on the outcome of infections.

RpoS Affects Gene Expression in Salmonella enterica serovar Typhi Under Early Hyperosmotic Stress.

During the infectious procedure of Salmonella enterica serovar Typhi (S. Typhi), Salmonella would suffer from some severe environmental stresses, such as gastric acid stress, enteric hyperosmotic stress, bile acids stress, and oxidative stress. S. Typhi must regulate the expression of numerous genes through the complex regulatory network to adapt strict stresses. RpoS, which encodes sigma factor σs, was reported to be a very important regulator in the maximal survival of enteric pathogens including S. enterica in the stress conditions. However, the role of RpoS in S. Typhi under early hyperosmotic stress is not clear. In this study, we prepared the RpoS-deleted mutant (△RpoS) and compared the growth of △RpoS and wild-type strain. In addition, we analyzed its global transcription profile under early hyperosmotic stress by a microarray. The results showed that △RpoS grew significantly slower than wild-type strain and 24 genes displayed differential expression between the wild-type strain and ΔRpoS upon 30-min exposure in the high osmolarity. Most of these genes are associated with enzymes of metabolism. Taken together, our study firstly demonstrated that RpoS affects gene expression in S. Typhi under early hyperosmotic stress and may impact the growth of bacteria by regulating bacterial metabolic enzymes.

Negative Autogenous Control of the Master Type III Secretion System Regulator HrpL in Pseudomonas syringae.

The type III secretion system (T3SS) is a principal virulence determinant of the model bacterial plant pathogen Pseudomonas syringae T3SS effector proteins inhibit plant defense signaling pathways in susceptible hosts and elicit evolved immunity in resistant plants. The extracytoplasmic function sigma factor HrpL coordinates the expression of most T3SS genes. Transcription of hrpL is dependent on sigma-54 and the codependent enhancer binding proteins HrpR and HrpS for hrpL promoter activation. hrpL is oriented adjacently to and divergently from the HrpL-dependent gene hrpJ, sharing an intergenic upstream regulatory region. We show that association of the RNA polymerase (RNAP)-HrpL complex with the hrpJ promoter element imposes negative autogenous control on hrpL transcription in P. syringae pv. tomato DC3000. The hrpL promoter was upregulated in a ΔhrpL mutant and was repressed by plasmid-borne hrpL In a minimal Escherichia coli background, the activity of HrpL was sufficient to achieve repression of reconstituted hrpL transcription. This repression was relieved if both the HrpL DNA-binding function and the hrp-box sequence of the hrpJ promoter were compromised, implying dependence upon the hrpJ promoter. DNA-bound RNAP-HrpL entirely occluded the HrpRS and partially occluded the integration host factor (IHF) recognition elements of the hrpL promoter in vitro, implicating inhibition of DNA binding by these factors as a cause of negative autogenous control. A modest increase in the HrpL concentration caused hypersecretion of the HrpA1 pilus protein but intracellular accumulation of later T3SS substrates. We argue that negative feedback on HrpL activity fine-tunes expression of the T3SS regulon to minimize the elicitation of plant defenses. IMPORTANCE: The United Nations Food and Agriculture Organization has warned that agriculture will need to satisfy a 50% to 70% increase in global food demand if the human population reaches 9 billion by 2050 as predicted. However, diseases caused by microbial pathogens represent a major threat to food security, accounting for over 10% of estimated yield losses in staple wheat, rice, and maize crops. Understanding the decision-making strategies employed by pathogens to coordinate virulence and to evade plant defenses is vital for informing crop resistance traits and management strategies. Many plant-pathogenic bacteria utilize the needle-like T3SS to inject virulence factors into host plant cells to suppress defense signaling. Pseudomonas syringae is an economically and environmentally devastating plant pathogen. We propose that the master regulator of its entire T3SS gene set, HrpL, downregulates its own expression to minimize elicitation of plant defenses. Revealing such conserved regulatory strategies will inform future antivirulence strategies targeting plant pathogens.

Piecewise linear approximations to model the dynamics of adaptation to osmotic stress by food-borne pathogens.

Addition of salt to food is one of the most ancient and most common methods of food preservation. However, little is known of how bacterial cells adapt to such conditions. We propose to use piecewise linear approximations to model the regulatory adaptation of Escherichiacoli to osmotic stress. We apply the method to eight selected genes representing the functions known to be at play during osmotic adaptation. The network is centred on the general stress response factor, sigma S, and also includes a module representing the catabolic repressor CRP-cAMP. Glutamate, potassium and supercoiling are combined to represent the intracellular regulatory signal during osmotic stress induced by salt. The output is a module where growth is represented by the concentration of stable RNAs and the transcription of the osmotic gene osmY. The time course of gene expression of transport of osmoprotectant represented by the symporter proP and of the osmY is successfully reproduced by the network. The behaviour of the rpoS mutant predicted by the model is in agreement with experimental data. We discuss the application of the model to food-borne pathogens such as Salmonella; although the genes considered have orthologs, it seems that supercoiling is not regulated in the same way. The model is limited to a few selected genes, but the regulatory interactions are numerous and span different time scales. In addition, they seem to be condition specific: the links that are important during the transition from exponential to stationary phase are not all needed during osmotic stress. This model is one of the first steps towards modelling adaptation to stress in food safety and has scope to be extended to other genes and pathways, other stresses relevant to the food industry, and food-borne pathogens. The method offers a good compromise between systems of ordinary differential equations, which would be unmanageable because of the size of the system and for which insufficient data are available, and the more abstract Boolean methods.

Disruption of MiaA provides insights into the regulation of phenazine biosynthesis under suboptimal growth conditions in Pseudomonas chlororaphis 30-84.

Many products of secondary metabolism are activated by quorum sensing (QS), yet even at cell densities sufficient for QS, their production may be repressed under suboptimal growth conditions via mechanisms that still require elucidation. For many beneficial plant-associated bacteria, secondary metabolites such as phenazines are important for their competitive survival and plant-protective activities. Previous work established that phenazine biosynthesis in Pseudomonas chlororaphis 30-84 is regulated by the PhzR/PhzI QS system, which in turn is regulated by transcriptional regulator Pip, two-component system RpeA/RpeB and stationary phase/stress sigma factor RpoS. Disruption of MiaA, a tRNA modification enzyme, altered primary metabolism and growth leading to widespread effects on secondary metabolism, including reduced phenazine production and oxidative stress tolerance. Thus, the miaA mutant provided the opportunity to examine the regulation of phenazine production in response to altered metabolism and growth or stress tolerance. Despite the importance of MiaA for translation efficiency, the most significant effect of miaA disruption on phenazine production was the reduction in the transcription of phzR, phzI and pip, whereas neither the transcription nor translation of RpeB, a transcriptional regulator of pip, was affected. Constitutive expression of rpeB or pip in the miaA mutant completely restored phenazine production, but it resulted in further growth impairment. Constitutive expression of RpoS alleviated sensitivity to oxidative stress resulting from RpoS translation inefficiency in the miaA mutant, but it did not restore phenazine production. Our results support the model that cells curtail phenazine biosynthesis under suboptimal growth conditions via RpeB/Pip-mediated regulation of QS.

The Impact of 18 Ancestral and Horizontally-Acquired Regulatory Proteins upon the Transcriptome and sRNA Landscape of Salmonella enterica serovar Typhimurium.

We know a great deal about the genes used by the model pathogen Salmonella enterica serovar Typhimurium to cause disease, but less about global gene regulation. New tools for studying transcripts at the single nucleotide level now offer an unparalleled opportunity to understand the bacterial transcriptome, and expression of the small RNAs (sRNA) and coding genes responsible for the establishment of infection. Here, we define the transcriptomes of 18 mutants lacking virulence-related global regulatory systems that modulate the expression of the SPI1 and SPI2 Type 3 secretion systems of S. Typhimurium strain 4/74. Using infection-relevant growth conditions, we identified a total of 1257 coding genes that are controlled by one or more regulatory system, including a sub-class of genes that reflect a new level of cross-talk between SPI1 and SPI2. We directly compared the roles played by the major transcriptional regulators in the expression of sRNAs, and discovered that the RpoS (σ38) sigma factor modulates the expression of 23% of sRNAs, many more than other regulatory systems. The impact of the RNA chaperone Hfq upon the steady state levels of 280 sRNA transcripts is described, and we found 13 sRNAs that are co-regulated with SPI1 and SPI2 virulence genes. We report the first example of an sRNA, STnc1480, that is subject to silencing by H-NS and subsequent counter-silencing by PhoP and SlyA. The data for these 18 regulatory systems is now available to the bacterial research community in a user-friendly online resource, SalComRegulon.

Effect of Spermidine Analogues on Cell Growth of Escherichia coli Polyamine Requiring Mutant MA261.

The effects of spermidine analogues [norspermidine (NSPD, 33), spermidine (SPD, 34), homospermidine (HSPD, 44) and aminopropylcadaverine (APCAD, 35)] on cell growth were studied using Escherichia coli polyamine-requiring mutant MA261. Cell growth was compared at 32°C, 37°C, and 42°C. All four analogues were taken up mainly by the PotABCD spermidine-preferential uptake system. The degree of stimulation of cell growth at 32°C and 37°C was NSPD ≥ SPD ≥ HSPD > APCAD, and SPD ≥ HSPD ≥ NSPD > APCAD, respectively. However, at 42°C, it was HSPD ¼ SPD > NSPD > APCAD. One reason for this is HSPD was taken up effectively compared with other triamines. In addition, since natural polyamines (triamines and teteraamines) interact mainly with RNA, and the structure of RNA is more flexible at higher temperatures, HSPD probably stabilized RNA more tightly at 42°C. We have thus far found that 20 kinds of protein syntheses are stimulated by polyamines at the translational level. Among them, synthesis of OppA, RpoE and StpA was more strongly stimulated by HSPD at 42°C than at 37°C. Stabilization of the initiation region of oppA and rpoE mRNA was tighter by HSPD at 42°C than 37°C determined by circular dichroism (CD). The degree of polyamine stimulation of OppA, RpoE and StpA synthesis by NSPD, SPD and APCAD was smaller than that by HSPD at 42°C. Thus, the degree of stimulation of cell growth by spermidine analogues at the different temperatures is dependent on the stimulation of protein synthesis by some components of the polyamine modulon.

Expression of virulence and stress response genes in Aeromonas hydrophila under various stress conditions.

Aeromonas hydrophila has emerged as an important human pathogen as it causes gastroenteritis and extra-intestinal infections. Information regarding the influence of environmental stresses on gene expression profile of A. hydrophila is lacking. The impact of nutrient replenishment, nutrient deprivation, acid stress, and cold shock on housekeeping, general stress-response, and virulence genes was studied using quantitative real-time PCR in two A. hydrophila strains, CECT 839T , and A331. These sub-lethal stresses invoked significant changes in the expression of these genes in a strain-dependent manner. Overall, nutrient replenishment and deprivation significantly induced the expression of housekeeping (rpoD), general stress regulators (uspA and rpoS), and virulence (aer) genes, indicating their importance in regulating the survival and virulence of A. hydrophila under these stress conditions. rpoS gene was significantly induced under cold shock; whereas, acid stress significantly induced the expression of uspA gene. This is the first study to investigate the effect of environmental parameters on the expression of stress-response and virulence genes in A. hydrophila strains.

New Insights into the Antibacterial Activity of Hydroxycoumarins against Ralstonia solanacearum.

Coumarins are important plant-derived natural products with wide-ranging bioactivities and extensive applications. In this study, we evaluated for the first time the antibacterial activity and mechanisms of action of coumarins against the phytopathogen Ralstonia solanacearum, and investigated the effect of functional group substitution. We first tested the antibacterial activity of 18 plant-derived coumarins with different substitution patterns, and found that daphnetin, esculetin, xanthotol, and umbelliferone significantly inhibited the growth of R. solanacearum. Daphnetin showed the strongest antibacterial activity, followed by esculetin and umbelliferone, with MICs of 64, 192, and 256 mg/L, respectively, better than the archetypal coumarin with 384 mg/L. We further demonstrated that the hydroxylation of coumarins at the C-6, C-7 or C-8 position significantly enhanced the antibacterial activity against R. solanacearum. Transmission electron microscope (TEM) and fluorescence microscopy images showed that hydroxycoumarins may interact with the pathogen by mechanically destroying the cell membrane and inhibiting biofilm formation. The antibiofilm effect of hydroxycoumarins may relate to the repression of flagellar genes fliA and flhC. These physiological changes in R. solanacearum caused by hydroxycoumarins can provide information for integral pathogen control. The present findings demonstrated that hydroxycoumarins have superior antibacterial activity against the phytopathogen R. solanacearum, and thus have the potential to be applied for controlling plant bacterial wilt.

Staphylococcus aureus Strain USA300 Perturbs Acquisition of Lysosomal Enzymes and Requires Phagosomal Acidification for Survival inside Macrophages.

Methicillin-resistant Staphylococcus aureus (MRSA) causes invasive, drug-resistant skin and soft tissue infections. Reports that S. aureus bacteria survive inside macrophages suggest that the intramacrophage environment may be a niche for persistent infection; however, mechanisms by which the bacteria might evade macrophage phagosomal defenses are unclear. We examined the fate of the S. aureus-containing phagosome in THP-1 macrophages by evaluating bacterial intracellular survival and phagosomal acidification and maturation and by testing the impact of phagosomal conditions on bacterial viability. Multiple strains of S. aureus survived inside macrophages, and in studies using the MRSA USA300 clone, the USA300-containing phagosome acidified rapidly and acquired the late endosome and lysosome protein LAMP1. However, fewer phagosomes containing live USA300 bacteria than those containing dead bacteria associated with the lysosomal hydrolases cathepsin D and ß-glucuronidase. Inhibiting lysosomal hydrolase activity had no impact on intracellular survival of USA300 or other S. aureus strains, suggesting that S. aureus perturbs acquisition of lysosomal enzymes. We examined the impact of acidification on S. aureus intramacrophage viability and found that inhibitors of phagosomal acidification significantly impaired USA300 intracellular survival. Inhibition of macrophage phagosomal acidification resulted in a 30-fold reduction in USA300 expression of the staphylococcal virulence regulator agr but had little effect on expression of sarA, saeR, or sigB. Bacterial exposure to acidic pH in vitro increased agr expression. Together, these results suggest that S. aureus survives inside macrophages by perturbing normal phagolysosome formation and that USA300 may sense phagosomal conditions and upregulate expression of a key virulence regulator that enables its intracellular survival.

To Modulate Survival under Secondary Stress Conditions, Listeria monocytogenes 10403S Employs RsbX To Downregulate σB Activity in the Poststress Recovery Stage or Stationary Phase.

Listeria monocytogenes is a saprophytic bacterium that thrives in diverse environments and causes listeriosis via ingestion of contaminated food. RsbX, a putative sigma B (σ(B)) regulator, is thought to maintain the ready state in the absence of stress and reset the bacterium to the initial state in the poststress stage in Bacillus subtilis. We wondered whether RsbX is functional in L. monocytogenes under different stress scenarios. Genetic deletion and complementation of the rsbX gene were combined with survival tests and transcriptional and translational analyses of σ(B) expression in response to stresses. We found that deletion of rsbX increased survival under secondary stress following recovery of growth after primary stress or following stationary-phase culturing. The ΔrsbX mutant had higher expression of σ(B) than its parent strain in the recovery stage following primary sodium stress and in stationary-phase cultures. Apparently, increased σ(B) expression had contributed to improved survival in the absence of RsbX. There were no significant differences in survival rates or σ(B) expression levels in response to primary stresses between the rsbX mutant and its parent strain during the exponential phase. Therefore, we provide clear evidence that RsbX is a negative regulator of L. monocytogenes σ(B) during the recovery period after a primary stress or in the stationary phase, thus affecting its survival under secondary stress.