Quantitative mapping of mRNA 3' ends in Pseudomonas aeruginosa reveals a pervasive role for premature 3' end formation in response to azithromycin.

Pseudomonas aeruginosa produces serious chronic infections in hospitalized patients and immunocompromised individuals, including patients with cystic fibrosis. The molecular mechanisms by which P. aeruginosa responds to antibiotics and other stresses to promote persistent infections may provide new avenues for therapeutic intervention. Azithromycin (AZM), an antibiotic frequently used in cystic fibrosis treatment, is thought to improve clinical outcomes through a number of mechanisms including impaired biofilm growth and quorum sensing (QS). The mechanisms underlying the transcriptional response to AZM remain unclear. Here, we interrogated the P. aeruginosa transcriptional response to AZM using a fast, cost-effective genome-wide approach to quantitate RNA 3' ends (3pMap). We also identified hundreds of P. aeruginosa genes with high incidence of premature 3' end formation indicative of riboregulation in their transcript leaders using 3pMap. AZM treatment of planktonic and biofilm cultures alters the expression of hundreds of genes, including those involved in QS, biofilm formation, and virulence. Strikingly, most genes downregulated by AZM in biofilms had increased levels of intragenic 3' ends indicating premature transcription termination, transcriptional pausing, or accumulation of stable intermediates resulting from the action of nucleases. Reciprocally, AZM reduced premature intragenic 3' end termini in many upregulated genes. Most notably, reduced termination accompanied robust induction of obgE, a GTPase involved in persister formation in P. aeruginosa. Our results support a model in which AZM-induced changes in 3' end formation alter the expression of central regulators which in turn impairs the expression of QS, biofilm formation and stress response genes, while upregulating genes associated with persistence.

Effect of Vancomycin on Cytoplasmic Peptidoglycan Intermediates and van Operon mRNA Levels in VanA-Type Vancomycin-Resistant Enterococcus faecium.

Resistance in VanA-type vancomycin-resistant Enterococcus faecium (VREfm) is due to an inducible gene cassette encoding seven proteins (vanRSHAXYZ). This provides for an alternative peptidoglycan (PG) biosynthesis pathway whereby D-Ala-D-Ala is replaced by D-Ala-d-lactate (Lac), to which vancomycin cannot bind effectively. This study aimed to quantify cytoplasmic levels of normal and alternative pathway PG intermediates in VanA-type VREfm by liquid chromatography-tandem mass spectrometry before and after vancomycin exposure and to correlate these changes with changes in vanA operon mRNA levels measured by real-time quantitative PCR (RT-qPCR). Normal pathway intermediates predominated in the absence of vancomycin, with low levels of alternative pathway intermediates. Extended (18-h) vancomycin exposure resulted in a mixture of the terminal normal (UDP-N-acetylmuramic acid [NAM]-l-Ala-D-Glu-l-Lys-D-Ala-D-Ala [UDP-Penta]) and alternative (UDP-NAM-l-Ala-γ-D-Glu-l-Lys-D-Ala-D-Lac [UDP-Pentadepsi]) pathway intermediates (2:3 ratio). Time course analyses revealed normal pathway intermediates responding rapidly (peaking in 3 to 10 min) and alternative pathway intermediates responding more slowly (peaking in 15 to 45 min). RT-qPCR demonstrated that vanA operon mRNA transcript levels increased rapidly after exposure, reaching maximal levels in 15 min. To resolve the effect of increased van operon protein expression on PG metabolite levels, linezolid was used to block protein biosynthesis. Surprisingly, linezolid dramatically reduced PG intermediate levels when used alone. When used in combination with vancomycin, linezolid only modestly reduced alternative UDP-linked PG intermediate levels, indicating substantial alternative pathway presence before vancomycin exposure. Comparison of PG intermediate levels between VREfm, vancomycin-sensitive Enterococcus faecium, and methicillin-resistant Staphylococcus aureus after vancomycin exposure demonstrated substantial differences between S. aureus and E. faecium PG biosynthesis pathways. IMPORTANCE VREfm is highly resistant to vancomycin due to the presence of a vancomycin resistance gene cassette. Exposure to vancomycin induces the expression of genes in this cassette, which encode enzymes that provide for an alternative PG biosynthesis pathway. In VanA-type resistance, these alternative pathway enzymes replace the D-Ala-D-Ala terminus of normal PG intermediates with D-Ala-D-Lac terminated intermediates, to which vancomycin cannot bind. While the general features of this resistance mechanism are well known, the details of the choreography between vancomycin exposure, vanA gene induction, and changes in the normal and alternative pathway intermediate levels have not been described previously. This study comprehensively explores how VREfm responds to vancomycin exposure at the mRNA and PG intermediate levels.

Collateral Sensitivity to ß-Lactam Drugs in Drug-Resistant Tuberculosis Is Driven by the Transcriptional Wiring of BlaI Operon Genes.

The evolution of resistance to one antimicrobial can result in enhanced sensitivity to another, known as "collateral sensitivity." This underexplored phenomenon opens new therapeutic possibilities for patients infected with pathogens unresponsive to classical treatments. Intrinsic resistance to ß-lactams in Mycobacterium tuberculosis (the causative agent of tuberculosis) has traditionally curtailed the use of these low-cost and easy-to-administer drugs for tuberculosis treatment. Recently, ß-lactam sensitivity has been reported in strains resistant to classical tuberculosis therapy, resurging the interest in ß-lactams for tuberculosis. However, a lack of understanding of the molecular underpinnings of this sensitivity has delayed exploration in the clinic. We performed gene expression and network analyses and in silico knockout simulations of genes associated with ß-lactam sensitivity and genes associated with resistance to classical tuberculosis drugs to investigate regulatory interactions and identify key gene mediators. We found activation of the key inhibitor of ß-lactam resistance, blaI, following classical drug treatment as well as transcriptional links between genes associated with ß-lactam sensitivity and those associated with resistance to classical treatment, suggesting that regulatory links might explain collateral sensitivity to ß-lactams. Our results support M. tuberculosis ß-lactam sensitivity as a collateral consequence of the evolution of resistance to classical tuberculosis drugs, mediated through changes to transcriptional regulation. These findings support continued exploration of ß-lactams for the treatment of patients infected with tuberculosis strains resistant to classical therapies. IMPORTANCE Tuberculosis remains a significant cause of global mortality, with strains resistant to classical drug treatment considered a major health concern by the World Health Organization. Challenging treatment regimens and difficulty accessing drugs in low-income communities have led to a high prevalence of strains resistant to multiple drugs, making the development of alternative therapies a priority. Although Mycobacterium tuberculosis is naturally resistant to ß-lactam drugs, previous studies have shown sensitivity in strains resistant to classical drug treatment, but we currently lack understanding of the molecular underpinnings behind this phenomenon. We found that genes involved in ß-lactam susceptibility are activated after classical drug treatment resulting from tight regulatory links with genes involved in drug resistance. Our study supports the hypothesis that ß-lactam susceptibility observed in drug-resistant strains results from the underlying regulatory network of M. tuberculosis, supporting further exploration of the use of ß-lactams for tuberculosis treatment.

Alcohol stress on cyanobacterial membranes: New insights revealed by transcriptomics.

Cyanobacteria are model photosynthetic prokaryotic organisms often used in biotechnology to produce biofuels including alcohols. The effect of alcohols on cyanobacterial cell physiology and specifically on membrane fluidity is poorly understood. Previous research on various primary aliphatic alcohols found that alcohols with a short hydrocarbon chain (C1-C3) do not affect expression of genes related to membrane physical state. In addition, less water-soluble alcohols with a hydrocarbon chain longer than C8 are found to have a reduced ability to reach cellular membranes hence do not drastically change membrane physical state or induce expression of stress-responsive genes. Therefore, hexan-1-ol (C6) is suggested to have the most profound effect on cyanobacterial membrane physical state. Here, we studied the effects of hexan-1-ol on the cyanobacterium Synechocystis sp. PCC 6803 transcriptome. The transcriptome data obtained is compared to the previously reported analysis of gene expression induced by benzyl alcohol and butan-1-ol. The set of genes whose expression is induced after exposure to all three studied alcohols is identified. The expression under alcohol stress for several general stress response operons is analyzed, and examples of antisense interactions of RNA are investigated.

Monitoring arsenic using genetically encoded biosensors in vitro: The role of evolved regulatory genes.

Toxic pollutant (TP) detection in situ using analytical instruments or whole-cell biosensors is inconvenient. Designing and developing genetically coded biosensors in vitro for real-world TP detection is a promising alternative. However, because the bioactivity and stability of some key biomolecules are weakened in vitro, the response and regulation of reporter protein become difficult. Here, we established a genetically encoded biosensor in vitro with an arsenical resistance operon repressor (ArsR) and GFP reporter gene. Given that the wildtype ArsR did not respond to arsenic and activate GFP expression in vitro, we found, after screening, an evolved ArsR mutant ep3 could respond to arsenic and exhibited an approximately 3.4-fold fluorescence increase. Arsenic induced expression of both wildtype ArsR and ep3 mutant in vitro, however, only ep3 mutant regulated the expression of reporter gene. Furthermore, the effects of cell extracts, temperature, pH, incubation, and equilibrium time were investigated, and the equilibration of reaction mixtures for 30 min at 37 °C was found to be essential for in vitro arsenic detection prior to treatment with arsenic. Based on our data, we established a standard procedure for arsenic detection in vitro. Our results will facilitate the practical application of genetically encoded biosensors in TP monitoring.

Mapping the Role of AcrAB-TolC Efflux Pumps in the Evolution of Antibiotic Resistance Reveals Near-MIC Treatments Facilitate Resistance Acquisition.

Antibiotic resistance has become a major public health concern as bacteria evolve to evade drugs, leading to recurring infections and a decrease in antibiotic efficacy. Systematic efforts have revealed mechanisms involved in resistance. Yet, in many cases, how these specific mechanisms accelerate or slow the evolution of resistance remains unclear. Here, we conducted a systematic study of the impact of the AcrAB-TolC efflux pump on the evolution of antibiotic resistance. We mapped how population growth rate and resistance change over time as a function of both the antibiotic concentration and the parent strain's genetic background. We compared the wild-type strain to a strain overexpressing AcrAB-TolC pumps and a strain lacking functional pumps. In all cases, resistance emerged when cultures were treated with chloramphenicol concentrations near the MIC of their respective parent strain. The genetic background of the parent strain also influenced resistance acquisition. The wild-type strain evolved resistance within 24 h through mutations in the acrAB operon and its associated regulators. Meanwhile, the strain overexpressing AcrAB-TolC evolved resistance more slowly than the wild-type strain; this strain achieved resistance in part through point mutations in acrB and the acrAB promoter. Surprisingly, the strain without functional AcrAB-TolC efflux pumps still gained resistance, which it achieved through upregulation of redundant efflux pumps. Overall, our results suggest that treatment conditions just above the MIC pose the largest risk for the evolution of resistance and that AcrAB-TolC efflux pumps impact the pathway by which chloramphenicol resistance is achieved.IMPORTANCE Combatting the rise of antibiotic resistance is a significant challenge. Efflux pumps are an important contributor to drug resistance; they exist across many cell types and can export numerous classes of antibiotics. Cells can regulate pump expression to maintain low intracellular drug concentrations. Here, we explored how resistance emerged depending on the antibiotic concentration, as well as the presence of efflux pumps and their regulators. We found that treatments near antibiotic concentrations that inhibit the parent strain's growth were most likely to promote resistance. While wild-type, pump overexpression, and pump knockout strains were all able to evolve resistance, they differed in the absolute level of resistance evolved, the speed at which they achieved resistance, and the genetic pathways involved. These results indicate that specific treatment regimens may be especially problematic for the evolution of resistance and that the strain background can influence how resistance is achieved.

Burkholderia collagen-like protein 8, Bucl8, is a unique outer membrane component of a putative tetrapartite efflux pump in Burkholderia pseudomallei and Burkholderia mallei.

Bacterial efflux pumps are an important pathogenicity trait because they extrude a variety of xenobiotics. Our laboratory previously identified in silico Burkholderia collagen-like protein 8 (Bucl8) in the hazardous pathogens Burkholderia pseudomallei and Burkholderia mallei. We hypothesize that Bucl8, which contains two predicted tandem outer membrane efflux pump domains, is a component of a putative efflux pump. Unique to Bucl8, as compared to other outer membrane proteins, is the presence of an extended extracellular region containing a collagen-like (CL) domain and a non-collagenous C-terminus (Ct). Molecular modeling and circular dichroism spectroscopy with a recombinant protein, corresponding to this extracellular CL-Ct portion of Bucl8, demonstrated that it adopts a collagen triple helix, whereas functional assays screening for Bucl8 ligands identified binding to fibrinogen. Bioinformatic analysis of the bucl8 gene locus revealed it resembles a classical efflux-pump operon. The bucl8 gene is co-localized with downstream fusCDE genes encoding fusaric acid (FA) resistance, and with an upstream gene, designated as fusR, encoding a LysR-type transcriptional regulator. Using reverse transcriptase (RT)-qPCR, we defined the boundaries and transcriptional organization of the fusR-bucl8-fusCDE operon. We found exogenous FA induced bucl8 transcription over 80-fold in B. pseudomallei, while deletion of the entire bucl8 locus decreased the minimum inhibitory concentration of FA 4-fold in its isogenic mutant. We furthermore showed that the putative Bucl8-associated pump expressed in the heterologous Escherichia coli host confers FA resistance. On the contrary, the Bucl8-associated pump did not confer resistance to a panel of clinically-relevant antimicrobials in Burkholderia and E. coli. We finally demonstrated that deletion of the bucl8-locus drastically affects the growth of the mutant in L-broth. We determined that Bucl8 is a component of a novel tetrapartite efflux pump, which confers FA resistance, fibrinogen binding, and optimal growth.

In-silico approach to identify novel potent inhibitors against GraR of S. aureus.

With rising antibiotic resistance at alarming rates in S. aureus, a major human pathogen, it is important to identify targets for new antimicrobial therapies. A number of two-component systems (TCS) have been implicated in S. aureus resistance to several antibiotics. The glycopeptide-resistance associated TCS, GraSR, is involved in cationic antimicrobial peptides (CAMPs) resistance through the regulation of mprF, dltABCD, and vraFG operons. GraS is a sensor histidine kinase, while GraR is a response regulator transcription factor, which is potential drug target. In lieu of the significance of GraSR in antibiotic resistance and the lack of structural studies on GraR, we undertook to determine the GraR structure through homology modelling. A series of small molecules were virtually screened and the top-scored molecules were analyzed for different pharmacophore properties and assessed for their binding potency to GraR (IC50). Further, a molecular dynamics simulation study of GraR-ligand complexes revealed that the predicted molecules exhibited good binding affinities at the dimerization interface of GraR. Thus, these molecules could be suitable inhibitors for the GraR-mediated signalling processes, which may be further utilized to develop novel antimicrobial agents against S. aureus.

Oxytropis glabra DC. Inhibits Biofilm Formation of Staphylococcus epidermidis by Down-Regulating ica Operon Expression.

Staphylococcus epidermidis is one of the main causes of medical device-related infections and bovine mastitis owing to its biofilm-forming abilities. Oxytropis glabra DC. is one of the most widespread Fabaceae species and used as a Chinese herbal formulation in Western China. Our research investigated the effects of O. glabra on the biofilm formation of S. epidermidis and the possible inhibiting mechanism. The biofilm-forming reference strain, S. epidermidis SE-1 (ATCC 35,984), was employed as a model and semi-quantitative biofilm assay was performed to evaluate the antibiofilm activity of O. glabra. The exopolysaccharides (EPS) production and expression of ica operon were studied to explore the possible antibiofilm mechanism using thin-layer chromatography and quantitative real-time PCR assay, respectively. The results obtained indicated that O. glabra decoction at 7.5 mg mL-1 significantly inhibited biofilm formation by about 95% without affecting cell growth of S. epidermidis. Two hydrolysis productions of EPS were significantly decreased by 64% and 54% with the addition of 7.5 mg mL-1O. glabra and the expression of icaR was significantly up-regulated 2.2-times, whereas icaB was significantly down-regulated more than 50% by 7.5 mg mL-1O. glabra. These findings suggest a potential application for O. glabra as a promising candidate for the exploration of new drugs against S. epidermidis biofilm-associated infections.

Genomic insights on heterogeneous resistance to vancomycin and teicoplanin in Methicillin-resistant Staphylococcus aureus: A first report from South India.

Methicillin-resistant Staphylococcus aureus (MRSA) infection is an important clinical concern in patients, and is often associated with significant disease burden and metastatic infections. There is an increasing evidence of heterogeneous vancomycin-intermediate S. aureus (hVISA) associated treatment failure. In this study, we aim to understand the molecular mechanism of teicoplanin resistant MRSA (TR-MRSA) and hVISA. A total of 482 MRSA isolates were investigated for these phenotypes. Of the tested isolates, 1% were identified as TR-MRSA, and 12% identified as hVISA. A highly diverse amino acid substitution was observed in tcaRAB, vraSR, and graSR genes in TR-MRSA and hVISA strains. Interestingly, 65% of hVISA strains had a D148Q mutation in the graR gene. However, none of the markers were reliable in differentiating hVISA from TR-MRSA. Significant pbp2 upregulation was noted in three TR-MRSA strains, which had teicoplanin MICs of 16 or 32 µg/ml, whilst significant pbp4 downregulation was not noted in these strains. In our study, multiple mutations were identified in the candidate genes, suggesting a complex evolutionary pathway involved in the development of TR-MRSA and hVISA strains.

Comprehensive study of nitrofuroxanoquinolines. New perspective donors of NO molecules.

The goal of present work is the study of NO releasing mechanisms in nitrofuroxanoquinoline (NFQ) derivatives. Mechanisms of their structural non-rigidity and pathways of NO donation - spontaneous or under the action of sulfanyl radicals or photoirradiation - were considered in details, both experimentally and quantum chemically. Furoxan-containing systems of the discussed type are not capable of spontaneous or photoinduced decomposition under mild conditions, and sulfanyl (radical) induced processes are the most preferable. It was shown that appropriate modification of NFQ through [3 + 2] cycloaddition and subsequent aromatization is a powerful tool to design new prospective donors of NO molecule. Two newly obtained NFQ derivatives were proven to have unusually high NO activity in full accordance with the theoretical model. We hope that these examples will encourage community to seek for new NO active molecules among cycloadducts and modified furoxanes.

Role of the msaABCR Operon in Cell Wall Biosynthesis, Autolysis, Integrity, and Antibiotic Resistance in Staphylococcus aureus.

Staphylococcus aureus is an important human pathogen in both community and health care settings. One of the challenges with S. aureus as a pathogen is its acquisition of antibiotic resistance. Previously, we showed that deletion of the msaABCR operon reduces cell wall thickness, resulting in decreased resistance to vancomycin in vancomycin-intermediate S. aureus (VISA). In this study, we investigated the nature of the cell wall defect in the msaABCR operon mutant in the Mu50 (VISA) and USA300 LAC methicillin-resistant Staphylococcus aureus (MRSA) strains. Results showed that msaABCR mutant cells had decreased cross-linking in both strains. This defect is typically due to increased murein hydrolase activity and/or nonspecific processing of murein hydrolases mediated by increased protease activity in mutant cells. The defect was enhanced by a decrease in teichoic acid content in the msaABCR mutant. Therefore, we propose that deletion of the msaABCR operon results in decreased peptidoglycan cross-linking, leading to increased susceptibility toward cell wall-targeting antibiotics, such as ß-lactams and vancomycin. Moreover, we also observed significantly downregulated transcription of early cell wall-synthesizing genes, supporting the finding that msaABCR mutant cells have decreased peptidoglycan synthesis. More specifically, the msaABCR mutant in the USA300 LAC strain (MRSA) showed significantly reduced expression of the murA gene, whereas the msaABCR mutant in the Mu50 strain (VISA) showed significantly reduced expression of glmU, murA, and murD Thus, we conclude that the msaABCR operon controls the balance between cell wall synthesis and cell wall hydrolysis, which is required for maintaining a robust cell wall and acquiring resistance to cell wall-targeting antibiotics, such as vancomycin and the ß-lactams.

A versatile remote control system for functional expression of bacterial virulence genes based on the tetA promoter.

The expression of bacterial virulence factors is controlled in response to host or environmental factors and most virulence genes are not expressed under laboratory conditions. Investigations of molecular structures and cellular functions of bacterial virulence factors demand systems for experimentally controlled expression. We describe a simple and robust system that is based on the tetA promoter and the cognate repressor TetR. Expression under control of PtetA can be induced by non-antibiotic derivatives of tetracycline such as anhydrotetracycline (AHT). Tet-on expression cassettes can be used to replace native promoters of chromosomal genes or operons of interest. Tet-on plasmids allow episomal expression in homologous or heterologous host organisms. We demonstrate the application of Tet-on systems for the controlled induction of flagella assembly and motility, and for surface expression of adhesins of the chaperone/usher family of enteropathogenic Escherichia coli and autotransporter adhesins of Yersinia enterocolitica in Salmonella enterica and E. coli. Since inducer AHT can easily cross bacterial envelopes and mammalian cell membranes, the system can also be applied to control virulence genes in intracellular bacteria. We demonstrate the controlled synthesis, translocation and function of effector proteins of the type III secretion system of intracellular S. enterica.

Aminoglycoside-inducible expression of the mexAB-oprM multidrug efflux operon in Pseudomonas aeruginosa: Involvement of the envelope stress-responsive AmgRS two-component system.

Exposure of P. aeruginosa to the aminoglycoside (AG) paromomycin (PAR) induced expression of the PA3720-armR locus and the mexAB-oprM multidrug efflux operon that AmgR controls, although PAR induction of mexAB-oprM was independent of armR. Multiple AGs promoted mexAB-oprM expression and this was lost in the absence of the amgRS locus encoding an aminoglycoside-activated envelope stress-responsive 2-component system (TCS). Purified AmgR bound to the mexAB-oprM promoter region consistent with this response regulator directly regulating expression of the efflux operon. The thiol-active reagent, diamide, which, like AGs, promotes protein aggregation and cytoplasmic membrane damage also promoted AmgRS-dependent mexAB-oprM expression, a clear indication that the MexAB-OprM efflux system is recruited in response to membrane perturbation and/or circumstances that lead to this. Despite the AG and diamide induction of mexAB-oprM, however, MexAB-OprM does not appear to contribute to resistance to these agents.

Expression dynamics of the poly-γ-glutamic acid biosynthesis genes of Bacillus subtilis in response to glucose and glutamic acid-a pilot study.

Poly-γ-glutamic acid (PGA) is biosynthesized by various Bacillus species through PGA synthetase, encoded by the PGA operon comprised of the ywsC and ywtABC genes. Due to the minimal available knowledge, understanding the expression pattern of PGA operon genes is pivotal. In this study, the effect of glucose and glutamic acid on the global gene expression profile of Bacillus subtilis Natto3 was investigated using high throughput microarray, with an emphasis on the PGA operon and genes influencing PGA production. Two treatment groups (set1-in the presence of glutamic acid and set2-in the presence of glutamic acid + glucose) were analyzed against the control (in the presence of glucose). In the microarray, both the groups showed a trend of up-regulation for ywsC and ywtA genes (log2 fold change of 0.55, P = 0.0194, 0.92, P = 0.0069 in set1 and 0.78, P = 0.0023, 0.59, P = 0.0172 in set2, respectively) and down-regulation of ywtB and ywtC genes (log2 fold change of -1.83, P = 0.0001, -1.42, P = 0.0017 in set1 and -1.52, P = 0.0012, -0.55, P = 0.1112 in set2, respectively), supporting the indispensability of the ywsC and ywtA genes in PGA production. Interestingly, the ywtB and ywtC genes, belonging to the same operon, were down-regulated in both the conditions (set1 and set2). To the best of our knowledge, this expression pattern of PGA operon genes is a unique observation.

Sub-inhibitory concentrations of oxacillin modify the expression of agr locus in Staphylococcus aureus clinical strains belonging to different clonal complexes.

BACKGROUND: The ability of Staphylococcus aureus to invade tissues and cause an infectious disease is the result of a multi-factorial process supported by the huge number of virulence factors inherent to this microorganism tightly regulated by the accessory gene regulator (agr). During antimicrobial therapy bacteria may be exposed to sub-inhibitory concentrations (subMICs) of antibiotics that may trigger transcriptional changes that may have an impact on the pathogenesis of infection. The objective of this study was to investigate the effect of oxacillin sub-MICs on agr system expression as the key component in the regulation of virulence in methicillin-susceptible (MSSA) and -resistant S. aureus (MRSA) strains. Furthermore, we studied the genetic basis of the agr locus and their potential association with the expression levels. METHODS: We have examined the expression of RNAIII and agrA mRNA as biomarkers for agr expression in the presence and absence of oxacillin subMICs in 10 MSSA and 4 MRSA clinical strains belonging to 5 clonal complexes (CC45-agrI, CC8-agrI, CC5-agrII, CC15-agrII and CC30-agrIII) causing endovascular complications. The DNA sequences of agr locus were obtained by whole genome sequencing. RESULTS: Our results revealed that exposure to subMICs of oxacillin had an impact on agr locus expression modifying the relative levels of expression with increases in 11 strains and with decreases in 3 strains. Thereby, the exposure to subMICs of oxacillin resulted in higher levels of expression of agr in CC15 and CC45 and lower levels in CC30. We also observed the presence of mutations in agrC and agrA in 13/14 strains with similar mutation profiles among strains within individual CCs except for strains of CC5. Although, agr expression levels differed among strains within CCs, the presence of these mutations was associated with differences in agr expression levels in most cases. CONCLUSIONS: Changes in agr expression induced by exposure to oxacillin subMICs should be considered because they could lead to changes in the virulence modulation and have an adverse effect on the course of infection, especially in certain clonal complexes.

The consistent differential expression of genetic pathways following exposure of an industrial Pseudomonas aeruginosa strain to preservatives and a laundry detergent formulation.

Pseudomonas aeruginosa is a common contaminant associated with product recalls in the home and personal care industry. Preservation systems are used to prevent spoilage and protect consumers, but greater knowledge is needed of preservative resistance mechanisms used by P. aeruginosa contaminants. We aimed to identify genetic pathways associated with preservative exposure by using an industrial P. aeruginosa strain and implementing RNA-Seq to understand gene expression changes in response to industry relevant conditions. The consistent differential expression of five genetic pathways during exposure to multiple industrial growth conditions associated with benzisothiazolone (BIT) and phenoxyethanol (POE) preservatives, and a laundry detergent (LD) formulation, was observed. A MexPQ-OpmE Resistance Nodulation Division efflux pump system was commonly upregulated in response to POE, a combination of BIT and POE, and LD together with BIT. In response to all industry conditions, a putative sialic acid transporter and isoprenoid biosynthesis gnyRDBHAL operon demonstrated consistent upregulation. Two operons phnBA and pqsEDCBA involved in Pseudomonas quinolone signaling production and quorum-sensing were also consistently downregulated during exposure to all the industry conditions. The ability to identify consistently differentially expressed genetic pathways in P. aeruginosa can inform the development of future targeted preservation systems that maintain product safety and minimise resistance development.

Shikimate Induced Transcriptional Activation of Protocatechuate Biosynthesis Genes by QuiR, a LysR-Type Transcriptional Regulator, in Listeria monocytogenes.

Listeria monocytogenes is a common foodborne bacterial pathogen that contaminates plant and animal consumable products. The persistent nature of L. monocytogenes is associated with millions of dollars in food recalls annually. Here, we describe the role of shikimate in directly modulating the expression of genes encoding enzymes for the conversion of quinate and shikimate metabolites to protocatechuate. In L. monocytogenes, these genes are found within two operons, named qui1 and qui2. In addition, a gene named quiR, encoding a LysR-Type Transcriptional Regulator (QuiR), is located immediately upstream of the qui1 operon. Transcriptional lacZ-promoter fusion experiments show that QuiR induces gene expression of both qui1 and qui2 operons in the presence of shikimate. Furthermore, co-crystallization of the QuiR effector binding domain in complex with shikimate provides insights into the mechanism of activation of this regulator. Together these data show that upon shikimate accumulation, QuiR activates the transcription of genes encoding enzymes involved in shikimate and quinate utilization for the production of protocatechuate. Furthermore, the accumulation of protocatechuate leads to the inhibition of Listeria growth. Since protocatechuate is not known to be utilized by Listeria, its role is distinct from those established in other bacteria.

Tetracycline alters gene expression in Salmonella strains that harbor the Tn10 transposon.

In this report, we show that bacterial plasmids that harbor the Tn10 transposon (i.e., the IncHI1 plasmid R27) modify expression of different Salmonella regulons responding to the presence of tetracycline (Tc) in the medium. By using as a model the Tc-dependent upregulation of the ibpAB operon (which belongs to the heat shock regulon), we have identified Tn10-tetA (coding for a Tc efflux pump) and adjacent tetC sequences as required for ibpAB upregulation. Characterization of transcripts in the tetAC region showed that tetA transcription can continue into tetC sequences, generating a long 3'UTR sequence, which can protect transcripts from RNA processing, thus increasing the expression of TetA protein. In the presence of Tc, the DnaK and IbpA chaperones are overexpressed and translocated to the periplasm and to the membrane fraction respectively. DnaK targeting unfolded proteins is known to induce heat shock by avoiding RpoH proteolysis. We correlate expression levels of Tn10-encoded TetA protein with heat shock induction in Salmonella, likely because TetA activity compromises protein secretion.

Molecular basis of function and the unusual antioxidant activity of a cyanobacterial cysteine desulfurase.

Cysteine desulfurases, which supply sulfur for iron-sulfur cluster biogenesis, are broadly distributed in all phyla including cyanobacteria, the progenitors of plant chloroplasts. The SUF (sulfur utilization factor) system is responsible for Fe-S cluster biosynthesis under stress. The suf operon from cyanobacterium Anabaena PCC 7120 showed the presence of a cysteine desulfurase, sufS (alr2495), but not the accessory sulfur-accepting protein (SufE). However, an open reading frame (alr3513) encoding a SufE-like protein (termed AsaE, Anabaena sulfur acceptor E) was found at a location distinct from the suf operon. The purified SufS protein existed as a pyridoxal 5' phosphate (PLP)-containing dimer with a relatively low desulfurase activity. Interestingly, in the presence of the AsaE protein, the catalytic efficiency of this reaction increased 10-fold. In particular, for sulfur mobilization, the AsaE protein partnered only SufS and not other cysteine desulfurases from Anabaena. The SufS protein was found to physically interact with the AsaE protein, demonstrating that AsaE was indeed the missing partner of Anabaena SufS. The conserved cysteine of the SufS or the AsaE protein was essential for activity but not for their physical association. Curiously, overexpression of the SufS protein in Anabaena caused reduced formation of reactive oxygen species on exposure to hydrogen peroxide (H2O2), resulting in superior oxidative stress tolerance to the oxidizing agent when compared with the wild-type strain. Overall, the results highlight the functional interaction between the two proteins that mediate sulfur mobilization, in the cyanobacterial SUF pathway, and further reveal that overexpression of SufS can protect cyanobacteria from oxidative stress.