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Natamycin is a poorly soluble, polyene macrolide antifungal agent used in the food industry for the surface treatment of cheese and sausages. This use is not of safety concern. However, highly soluble natamycin–cyclodextrin inclusion complexes have been developed for the protection of beverages. This practice leads to high drug exposures exceeding the safety level. Apart from the definition of an acceptable daily dietary exposure to natamycin, its effect on the faecal flora as a reservoir for resistance has to be examined. Consumption of food to which natamycin has been added and mixed homogeneously, such as yoghurt, and in particular the addition of cyclodextrin inclusion complexes to beverages and wine generates high faecal natamycin concentrations resulting in high drug exposures of faecal Candida spp . Development of natamycin resistance has been observed in Candida spp . colonising the intestinal tract of patients following natamycin treatment of fungal infections. Horizontal gene transfer among different Candida spp. and within Aspergillus fumigatus spreads resistance. Therefore, it cannot be denied that use of natamycin for preservation of yoghurt and beverages may foster development of resistance to polyenes in Candida spp. [ABSTRACT FROM AUTHOR]

Objectives Multidrug efflux pumps mediate resistance to antibiotics and other toxic compounds. We studied the role of AcrAB-TolC, the main efflux pump in Escherichia coli, in regulating gene expression. Methods Deletion mutants, an acrABp-lacZ fusion and reverse transcription–real-time quantitative PCR experiments were used to study the role of AcrAB-TolC and metabolism in regulating gene expression of the acrAB operon and its transcriptional regulators. Results Deletion of the acrB gene increased the expression of the acrAB operon. A similar induction of acrAB was found when acrA or tolC was deleted, and when the pump function was inhibited using phenylalanine-arginine-β-naphthylamide. The induction of acrAB in the ΔacrB strain was totally (AcrR or SoxS) or partially (SoxR or MarA) prevented when the genes for these acrAB regulators were also deleted. The expression of soxS and marA, but not of acrR, was increased in the ΔacrB strain, which also showed altered expression of many other genes related to different cellular processes, including motility. Deletion of the metabolic genes entA and entE (enterobactin biosysnthesis), glpX (gluconeogenesis), cysH (cysteine biosynthesis) and purA (purine biosynthesis) also prevented activation of the acrAB promoter in the ΔacrB strain. Addition of the enterobactin biosynthesis intermediate metabolite 2,3-dihydroxybenzoate induced the expression of acrAB. Conclusions These results together suggest a model in which the AcrAB-TolC pump effluxes cellular metabolites that are toxic and/or have a signalling role. If the pump is inactivated or inhibited, these metabolites would accumulate, inactivating AcrR and/or up-regulating soxS and marA expression, ultimately triggering the up-regulation of acrAB expression to restore homeostasis. [ABSTRACT FROM PUBLISHER]

MarR is the dedicated autorepressor of the mar RAB operon found in seven genera of the Enterobacteraceae. The MarA transcriptional regulator directly activates numerous genes involved in multidrug resistance and other environmental responses. MarR is inactivated by certain phenolic ligands, such as salicylate, by an unknown mechanism. Our recent work has shown that several amino acid residues of Escherichia coli MarR affecting ligand binding are located between the dimerization and DNA-binding domains. To further characterize the ligand-binding region of MarR, we have now examined 7 point mutants generated by random mutagenesis and 11 site-directed alanine replacement mutants for inactivation by three ligands: salicylate, 2,4-dinitrophenol, and plumbagin. Inactivation of MarR was quantitated in intact cells by loss of MarR-mediated repression of a chromosomal mar-lacZ transcriptional fusion. The results showed that most of the residues important for ligand effectiveness lay in the α1 and α2 helices of MarR, between the putative DNA-binding domain and the dimerization domain of MarR, reinforcing our earlier findings. Moreover, the three ligands had different, but overlapping, sets of residues impacting their effects on MarR. [ABSTRACT FROM AUTHOR]

Paralogous transcriptional regulators MarA, Rob, and SoxS act individually and together to control expression of more than 80 Escherichia coli genes. Deletion of marA, rob, and soxS from an E. coli clinical isolate prevents persistence beyond 2 days postinfection in a mouse model of pyelonephritis. We used microarray analysis to identify 242 genes differentially expressed between the triple deletion mutant and its parent strain at 2 days postinfection in the kidney. One of these, znuC of the zinc transport system ZnuACB, displayed decreased expression in the triple mutant compared to that in the parental strain, and deletion of znuC from the parental strain reduced persistence. The mart rob soxS triple deletion mutant was less viable in vitro under limited-Zn and Zn-depleted conditions, while disruption of znuC caused a reduction in the growth rates for the parental and triple mutant strains to equally low levels under limited-Zn or Zn-depleted conditions. Complementation of the triple mutant with soxS, but not marA or rob, restored the parental growth rate in Zn-depleted medium, while deletion of only soxS from the parental strain led to low growth in Zn-depleted medium. Both results suggested that SoxS is a major regulator responsible for growth under Zn-depleted conditions. Gel shift experiments failed to show direct binding of SoxS to the znuCB promoter, thus suggesting indirect control of znuCB expression by SoxS. While SoxS expression in the triple mutant fully restored persistence, increased expression of znuACB via a plasmid in this mutant only partially restored wild-type levels of persistence in the kidney. This work implicates SoxS control of znuCB expression as a key factor in persistence of E. coli in murine pyelonephritis. [ABSTRACT FROM AUTHOR]

The history of the tetracyclines involves the collective contributions of thousands of dedicated researchers, scientists, clinicians, and business executives over the course of more than 60 years. Discovered as natural products from actinomycetes soil bacteria, the tetracyclines were first reported in the scientific literature in 1948. They were noted for their broad spectrum antibacterial activity and were commercialized with clinical success beginning in the late 1940s to the early 1950s. The second-generation semisynthetic analogs and more recent third-generation compounds show the continued evolution of the tetracycline scaffold toward derivatives with increased potency as well as efficacy against tetracycline-resistant bacteria, with improved pharmacokinetic and chemical properties. Their biologic activity against a wide spectrum of microbial pathogens and their uses in mammalian models of inflammation, neurodegeneration, and other biological systems indicate that the tetracyclines will continue to be successful therapeutics in infectious diseases and as potential therapeutics against inflammation-based mammalian cell diseases. [ABSTRACT FROM AUTHOR]

Bacterial two-hybrid studies of randomly cloned Escherichia coli DNA identified a physical interaction between GyrA, subunit A of gyrase, and MarR, a repressor of the marRAB operon. GyrA-His immobilized on Ni-nitrilotriacetic acid (NiNTA) resin bound MarR, while MarR alone did not bind. GyrA interfered with MarR binding to marO, as detected by electrophoretic mobility assays. In a strain bearing the marRAB operon and a marO-lacZ reporter, overexpression of GyrA increased LacZ activity, indicating decreased repression of marO-lacZ by MarR. These results were confirmed by an increased survival of cells treated with quinolones and other antibiotics when GyrA was overexpressed. This work, like a previous study examining TktA (12), shows that unrelated proteins can regulate MarR activity. The findings reveal an unexpected regulatory function of GyrA in antibiotic resistance. [ABSTRACT FROM AUTHOR]

The article presents a study regarding cationic antimicrobial peptides (CAMPs) and the susceptibility of Escherichia coli to these peptides considering transcriptional regulators including soxS, rob, and marA. It states that overexpression of marA decreased the susceptibility to CAMPs while overexpression of soxS yielded a different result. It notes that susceptibility was not highly affected by rob and that CAMP functions in achieving marA drug resistance.

The annotation process of a newly sequenced bacterial genome is largely based on algorithms derived from databases of previously defined RNA and protein-encoding gene structures. This process generally excludes the possibility that the two strands of a given stretch of DNA can each harbor a gene in an overlapping manner. While the presence of such structures in eukaryotic genomes is considered to be relatively common, their counterparts in prokaryotic genomes are just beginning to be recognized. Application of an in vivo expression technology has previously identified 22 discrete genetic loci in Pseudomonas fluorescens Pf0-1 that were specifically activated in the soil environment, of which 10 were present in an antisense orientation relative to previously annotated genes. This observation led to the hypothesis that the physiological role of overlapping genetic structures may be relevant to growth conditions outside artificial laboratory media. Here, we examined the role of one of the overlapping gene pairs, iiv19 and leuA2, in soil. Although iiv19 was previously demonstrated to be preferentially activated in the soil environment, its absence did not alter the ability of P. fluorescens to colonize or survive in soil. Surprisingly, the absence of the leuA2 gene conferred a fitness advantage in the soil environment when leucine was supplied exogenously. This effect was determined to be independent of the iiv19 gene, and further analyses revealed that amino acid antagonism was the underlying mechanism behind the observed fitness advantage of the bacterium in soil. Our findings provide a potential mechanism for the frequent occurrence of auxotrophic mutants of Pseudomonas spp. in the lungs of cystic fibrosis patients. [ABSTRACT FROM AUTHOR]

The annotated genome sequences of prokaryotes seldom include overlapping genes encoded opposite each other by the same stretch of DNA. However, antisense transcription is becoming recognized as a widespread phenomenon in eukaryotes, and examples have been linked to important biological processes. Pseudomonas fluorescens inhabits aquatic and terrestrial environments, and can be regarded as an environmental generalist. The genetic basis for this ecological success is not well understood. In a previous search for soil-induced genes in P. fluorescens Pf0-1, ten antisense genes were discovered. These were termed 'cryptic' genes, as they had escaped detection by gene-hunting algorithms, and lacked easily recognizable promoters. In this communication, we designate such genes as 'non-predicted' or 'hidden'. Using reverse transcription PCR, we show that at each of six non-predicted gene loci chosen for study, transcription occurs from both 'sense' and 'antisense' DNA strands. Further, at least one of these hidden antisense genes, iiv14, encodes a protein, as does the sense transcript, both identified by poly-histidine tags on the C-terminus of the proteins. Mutational and complementation studies showed that this novel antisense gene was important for efficient colonization of soil, and multiple copies in the wildtype host improved the speed of soil colonization. Introduction of a stop codon early in the gene eliminated complementation, further implicating the protein in colonization of soil. We therefore designate iiv14 ''cosA''. These data suggest that, as is the case with eukaryotes, some bacterial genomes are more densely coded than currently recognized. [ABSTRACT FROM AUTHOR]

Treatment of infections is compromised worldwide by the emergence of bacteria that are resistant to multiple antibiotics. Although classically attributed to chromosomal mutations, resistance is most commonly associated with extrachromosomal elements acquired from other bacteria in the environment. These include different types of mobile DNA segments, such as plasmids, transposons, and integrons. However, intrinsic mechanisms not commonly specified by mobile elements—such as efflux pumps that expel multiple kinds of antibiotics—are now recognized as major contributors to multidrug resistance in bacteria. Once established, multidrug-resistant organisms persist and spread worldwide, causing clinical failures in the treatment of infections and public health crises. [Copyright &y& Elsevier]

The Escherichia coli transcriptional regulator MarA affects functions that include antibiotic resistance, persistence, and survival. MarA functions as an activator or repressor of transcription utilizing similar degenerate DNA sequences (marboxes) with three different binding site configurations with respect to the RNA polymerase-binding sites. We demonstrate that MarA down-regulates rob transcripts both in vivo and in vitro via a MarA-binding site within the rob promoter that is positioned between the -10 and -35 hexamers. As for the hdeA and purA promoters, which are repressed by MarA, the rob marbox is also in the ‘backward’ orientation. Protein-DNA interactions show that SoxS and Rob, like MarA, bind the same marbox in the rob promoter. Electrophoretic mobility shift analyses with a MarA-specific antibody demonstrate that MarA and RNA polymerase form a ternary complex with the rob promoter DNA. Transcription experiments in vitro and potassium permanganate footprinting analysis show that MarA affects the RNA polymerase-mediated closed to open complex formation at the rob promoter. [ABSTRACT FROM AUTHOR]

Abstract: While previously recognized antibiotic-resistant bacteria continue to increase in frequency and numbers globally, new resistance problems have recently emerged which further complicate and impede treatment of critical infectious diseases. Vancomycin resistance among Staphylococcus aureus, extended spectrum β-lactamases among gram-negative pathogens, and fluoroquinolone resistance among already multidrug-resistant Escherichia coli and Neisseria gonorrheae are adding to the toll of resistance on patients suffering with infectious diseases. [Copyright &y& Elsevier]

Cysteine replacement of Asp190, Gtu192 and Ser201 residues in the cytoplasmic interdomain loop of the TetA(B) tetracycline efflux antiporter from Tn10 reduces tetracycline resistance [Tamura, N., Konishi, S., Iwaki, S., Kimura-Someya, T., Nada, S. & Yamaguchi, A. (2001). J Biol Chem 276, 20330–20339]. It was found that these Cys substitutions altered the substrate specificity of TetA(B), increasing the relative resistance to doxycycline and minocycline over that to tetracycline by three- to sixfold. Substitutions of Asp190 and Glu192 by Ala, Asn and Gin also impaired the ability of TetA(B) to mediate tetracycline resistance while Ser201 Ala and Ser201 Thr substitutions did not. A Leu9Phe substitution in the first transmembrane helix of TetA(B) suppressed the Ser201Cys mutation, undoing the alterations in resistance and specificity. That the interdomain loop might contact substrate during transport, as is suggested from its role in substrate specificity, is unexpected considering that the primary sequence in the loop is not conserved among a group of otherwise homologous TetA proteins. However, in the interdomain loop of 11 of 14 homologous TetA efflux proteins, computational analysis revealed a short α-helix, which includes some residues affecting activity and substrate specificity. Perhaps this conserved secondary structure accounts for the role of the non-conserved interdomain loop in TetA function. [ABSTRACT FROM AUTHOR]

The optimism of the early period of antimicrobial discovery has been tempered by the emergence of bacterial strains with resistance to these therapeutics. Today, clinically important bacteria are characterized not only by single drug resistance but also by multiple antibiotic resistance-the legacy of past decades of antimicrobial use and misuse. Drug resistance presents an ever-increasing global public health threat that involves all major microbial pathogens and antimicrobial drugs. [ABSTRACT FROM AUTHOR]

Studies were undertaken to determine the genetic needs for the survival of Pseudomonas fluorescens Pf0-1, a gram-negative soil bacterium potentially important for biocontrol and bioremediation, in soil. In vivo expression technology (IVET) identified 22 genes with elevated expression in soil relative to laboratory media. Soil-induced sequences included genes with probable functions of nutrient acquisition and use, and of gene regulation. Ten sequences, lacking similarity to known genes, overlapped divergent known genes, revealing a novel genetic organization at those soil-induced loci. Mutations in three soil-induced genes led to impaired early growth in soil but had no impact on growth in laboratory media. Thus, IVET studies have identified sequences important for soil growth and have revealed a gene organization that was undetected by traditional laboratory approaches. [ABSTRACT FROM AUTHOR]

The serine 202 to phenylalanine substitution within the cytoplasmic interdomain loop of Tet(C) greatly reduces tetracycline resistance and efflux activity (Saraceni-Richards, C. A., and Levy, S. B. (2000) J. Biol. Chem. 275, 6101-6106). Second-site suppressor mutations were identified following hydroxylamine and nitrosoguanidine mutagenesis. Three mutations, L11F in transmembrane 1 (TM1), A213T in the central interdomain loop, and A270V in cytoplasmic loop 8-9, restored a wild type level of resistance and an active efflux activity in Escherichia coli cells bearing the mutant tet(C) gene. The Tet S202F protein with the additional A270V mutation was expressed in amounts comparable with the original mutant, whereas L11F and A213T Tet(C) protein mutants were overexpressed. Introduction of each single mutation into the wild type tet(C) gene by site-directed mutagenesis did not alter tetracycline resistance or efflux activity. These secondary mutations may restore resistance by promoting a conformational change in the protein to accommodate the S202F mutation. The data demonstrate an interaction of the interdomain loop with other distant regions of the protein and support a role of the interdomain loop in mediating tetracycline resistance. [ABSTRACT FROM AUTHOR]

Summary MarA is a global regulator that mediates resistance to multiple environmental hazards such as antibiotics, disinfectants and oxidative stress agents by modulating the expression of a large number of genes in the Escherichia coli chromosome. Two E. coli MarA homologues, SoxS and Rob also control, to different extents, genes in the mar/sox/rob regulon. The controlling element for these proteins is a 20 bp ‘marbox’ sequence in the promoter region of regulated genes. Using in vitro assays and mutagenesis of promoter fusions in whole cells, we identified the cis regulatory element involved in MarA upregulation of the oxygen-insensitive nitroreductase nfnB gene. MarA binds to a marbox that is highly divergent from the previously proposed consensus (eight differences out of 14 specified nucleotides). Although purified SoxS and Rob proteins, like MarA, activated nfnB transcription in vitro , only constitutive expression of chromosomal marA , but not of soxS and rob genes, affected nfnB expression in whole cells. Increased expression, but limited as compared with MarA, was only achieved by plasmid-mediated overexpression of SoxS and Rob. This study shows that MarA can regulate gene expression through a functional marbox that is considerably divergent from the current consensus sequence. The data suggest that MarA is preferred over SoxS and Rob in upregulating nfnB . The findings imply that other different but physiologically important marbox DNA–MarA interactions take place in the regulation of still uncharacterized members of the mar regulon. [ABSTRACT FROM AUTHOR]

Bacterial resistance presents therapeutic dilemmas to clinicians worldwide. The warnings were there long ago, but too few people heeded them. Thus an emerging problem has grown to a crisis. Resistance is an ecological phenomenon stemming from the response of bacteria to the widespread use of antibiotics and their presence in the environment. While determining the consequences of inaction on the present and future public health, we must work to remedy the lack of action in the past. By improving antibiotic use and decreasing resistance gene frequency at the local levels, we can move towards reversing the resistance problem globally. [ABSTRACT FROM AUTHOR]

MarR is a regulator of multiple antibiotic resistance in Escherichia coli. It is the prototypical member of the MarR family of regulatory proteins found in bacteria and archaea that play important roles in the development of antibiotic resistance, a global health problem. Here we describe the crystal structure of the MarR protein, determined at a resolution of 2.3 Å. This is the first reported crystal structure of a member of this newly-described protein family. The structure shows MarR as a dimer with each subunit containing a winged-helix DNA binding motif. [ABSTRACT FROM AUTHOR]

The recent entry of products containing antibacterial agents into healthy households has escalated from a few dozen products in the mid-1990s to more than 700 today. Antibacterial products were developed and have been successfully used to prevent transmission of disease-causing microorganisms among patients, particularly in hospitals. They are now being added to products used in healthy households, even though an added health benefit has not been demonstrated. Scientists are concerned that the antibacterial agents will select bacteria resistant to them and cross-resistant to antibiotics. Moreover, if they alter a person's microflora, they may negatively affect the normal maturation of the T helper cell response of the immune system to commensal flora antigens; this change could lead to a greater chance of allergies in children. As with antibiotics, prudent use of these products is urged. Their designated purpose is to protect vulnerable patients. [ABSTRACT FROM AUTHOR]

Studies differential expression of chromosomal genes in Escherichia coli by constitutive expression of MarA protein. Genes affected by constitutive MarA expression; Confirmation of previously identified MarA-regulated genes; Effects of constitutive expression on operons and cotranscribed units.

Examines the changes of the repressor activity of MarR, the negative regulator of the Escherichia coli marRAB locus. Function of mar in controlling susceptibility to multiple antibiotics; Use of a restriction enzyme site protection assay; Inducer interference with MarR function in vitro.

Reports on the generation and characterization of MarR superrepressor mutants in Escherichia coli. Bacterial strains, plasmids, and genetic techniques; Assay of repressor activity by Beta-galactosidase; Selection of MarR superrrepressors.

Discusses the marRAB operon in the mar locus of Escherichia coli. Intrinsic resistance or susceptibility to multiple antibiotics; Inducible by structurally unrelated compounds such as tetracycline and chloramphenicol; Transcription studied under different conditions to clarify role in response to environmental signals.

Discusses the identification of multiple antibiotic resistance (marRAB) operon regulated genes in Escherichia coli. Regulated mapping by the mar operon; Sequencing and gene identification of cloned junctional fragments; Antibiotic susceptibility phenotypes; Expression of mar-regulated gene fusions.

Focuses on second-site suppressor mutations of inactivating substitutions at Gly247 of the tetracycline efflux protein, Tet(B). Eight different classes of Tet proteins; Secondary amino acid substitutions which restore tetracycline resistance to Tet(B) Asp247 or Asn247 mutants; Topological model of Tet(B).

Opinion. Argues efforts at all levels of drug discovery and use are needed to control and reverse the problem of drug resistance. Awareness and acknowledgment of problem's reality; Shorter treatment schedules for certain diseases; Epidemiological spread of resistance genes and their host bacteria; Individual as gatekeeper for antibiotic use; Supporting research in molecular and genetic basis of resistance.

Presents an introduction to the August 16, 2005 issue of the journal "Clinical Infectious Diseases."

Presents a correspondence on infectious diseases. Carriage of antibiotic-resistant fecal bacteria; Detection of antibiotic resistance in the fecal flora; Diagnosis of tuberculosis.

Discusses a study by Telenti, et al, in the March 13, 1993 issue of `Lancet.' Detection of a specific mutation site in Mycobacterium tuberculosis that is associated with resistance to rifampicin; Details.

Looks at antimicrobial resistance, focussing on the behavior of bacterial agents. Introduction of resistant bacteria from hospital; Results of studies of emerging resistant bacteria; Results of studies concerning resistance in bacteria.

A correction to the article "GyrA Interacts with MarR to Reduce Repression of the marRAB Operon in Escherichia coli" that was published in a previous issue is presented.

The MarA protein of Escherichia coli can both activate and repress the initiation of transcription, depending on the position and orientation of its degenerate 20-bp binding site ("marbox") at the promoter. For all three known repressed genes, the marbox overlaps the promoter. It has been reported that MarA represses the rob promoter via an RNA polymerase (RNAP)-DNA-MarA ternary complex. Under similar conditions, we found a ternary complex for the repressed purA promoter also. These findings, together with the backwards orientation of repressed marboxes, suggested a unique interaction of MarA with RNAP in repression. However, no repression-specific residues of MarA could be found among 38 single-alanine replacement mutations previously shown to retain activation function or among mutants from random mutagenesis. Mutations Thr12Ala, Arg36Ala, Thr95Ile, and Pro106Ala were more damaging for activation than for repression, some up to 10-fold, so these residues may play a specific role in activation. We found that nonspecific binding of RNAP to promoterless regions of DNA was presumably responsible for the ternary complexes seen previously. When RNAP binding was promoter specific, MarA reduced RNAP access to the rob promoter; there was little or no ternary complex. These findings strongly implicate steric hindrance as the mechanism of repression of rob by MarA. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented in response to the article "Sampling the Antibiotic Resistome," by V.M. D'Costa in the January 20, 2006 issue.

A letter to the editor is presented in response to a study on the prevalence and outcome of bacteremia among rural children in Kenya by researcher James A. Berkley and associates in the January 6, 2005 issue.

This editorial asserts that resistance to antibiotics is a problem that confronts all of us, thwarting treatment of inpatients and outpatients and compromising therapy for animals, fish, and agricultural crops. A discussion is presented about a strain of Salmonella enterica serotype typhimurium, known as definitive type 104 (DT104), in the United States that is resistant to five drugs. the strain's presence in Europe and the United States is mentioned.

Previous data from our laboratory suggest a relationship between increased pmrAB expression and virulence in an Escherichia coli mouse infection model of pyelonephritis. Competitive infections with wild type and pmrAB mutants showed that disruption of pmrAB caused decreased persistence of E. coli within the mouse kidney. these results were confirmed with plasmid-mediated complementation of the pmrAB mutant. Additionally, increased expression of pmrAB from this complementing plasmid in a previously attenuated marA-rob-soxS triple mutant displayed increased bacterial persistence in the infection when compared with the triple mutant alone. these findings suggest a role for this two- component regulatory system in the virulence of E. coli in a murine pyelonephritis model. [ABSTRACT FROM AUTHOR]

Pseudomonas species can exhibit phenotypic variation resulting from gacS or gacA mutation. P. fluorescens Pf0-1 is a gacA mutant and exhibits pleiotropic changes following the introduction of a functional allele. GacA enhances biofilm development while reducing dissemination in soil, suggesting that alternative Gac phenotypes enable Pseudomonas sp. to exploit varied environments. [ABSTRACT FROM AUTHOR]

The marRAB operon is conserved in seven genera of enteric bacteria ( Escherichia, Shigella, Klebsiella, Enterobacter, Salmonella, Cronobacter, and Citrobacter). MarA is a transcriptional regulator affecting many genes involved in resistance to stresses, and MarR is an autorepressor of the operon, but a role for the marB gene has been unclear. A recent work reported that deletion of marB causes resistance to certain stresses and increases the amount of marA transcript. We show here that the small (216 bp) marB gene encodes a protein, not an sRNA, because two different stop codons within the predicted open reading frame of marB prevented plasmid-borne marB from complementing Δ marB::Kan. The Δ marB::Kan mutation did not increase the stability of the marA transcript, suggesting that MarB does not destabilize the marA transcript but rather reduces its rate of transcription. Placing the putative signal sequence of MarB upstream of signal-sequence-less alkaline phosphatase guided the phosphatase to its normal periplasmic location. We conclude that MarB is a small periplasmic protein that represses the marRAB promoter by an indirect mechanism, possibly involving a signal to one of the cytoplasmic regulators of that promoter. [ABSTRACT FROM AUTHOR]

Pseudomonas spp. adapt rapidly to environmental fluctuations. Loss or overproduction of polyphosphate reduces the fitness of Pseudomonas fluorescens Pf0-1, indicating the importance of the fine-tuning of polyphosphate production. An antisense RNA was investigated and shown to regulate the polyphosphate kinase gene (ppk) by a posttranscriptional mechanism reducing ppk transcript abundance. [ABSTRACT FROM AUTHOR]

Knowledge of the genetic basis for bacterial survival and persistence in soil is a critical component in the development of successful biological control strategies and for understanding the ecological success of bacteria. We found a locus specifying polyphosphate kinase (ppk) and a nonpredicted antisense RNA (iiv8) in Pseudomonasfluorescens Pf0-1 to be necessary for optimal competitive fitness in LB broth culture and sterile loam soil. Pf0-1 lacking ppk and iiv8 was more than 10-fold less competitive against wild-type Pf0-1 in sterile loam soil low in inorganic phosphate. Studies indicated that ppk, and not iiv8, was required for competitive fitness. No role for iiv8 was identified. While a ppk and iiv8 mutant of Pf0-1 did not have increased sensitivity to osmotic, oxidative, and acid stress, it was more sensitive to elevated temperatures in laboratory medium and during growth in sterile soil. ppk was shown to be part of the Pho regulon in P. fluorescens, being upregulated in response to a low external Pi concentration, Of importance, overproduction of polyphosphate in the soil environment appears to be more deleterious than production of none at all. Our findings reveal a new role for polyphosphate (and the need for proper regulation of its production) in competitive fitness of P. fluorescens in laboratory and soil environments. [ABSTRACT FROM AUTHOR]

The Escherichia coli marRAB operon specifies two regulatory proteins, MarR (which represses) and MarA (which activates expression of the operon). The latter controls expression of multiple other chromosomal genes implicated in cell physiology, multiple drug resistance and virulence. Using randomly cloned E. coli DNA fragments in the bacterial adenylate cyclase two-hybrid system, we found that transketolase A (TktA) interacts with MarR. Purified (6H)-TktA immobilized on NiNTA resin-bound MarR. Overexpression or deletion of tktA showed that TktA interfered with MarR repression of the marRAB operon. Deletion of tktA increased antibiotic and oxidative stress susceptibilities, while its overexpression decreased them. Hydrogen peroxide induced tktA at 1 h treatment, while an increase in marRAB expression occurred only after 3 h exposure. This increase was dependent on the presence of tktA. Two MarR mutations which eliminated MarR binding to the marRAB operator and one which decreased dimerization of MarR had no effect on MarR interaction with TktA in the two-hybrid system. However, the interaction was disrupted by one of the three tested superrepressor mutant MarR proteins known to increase MarR binding to DNA. TktA inhibition of repression by MarR demonstrates a previously unrecognized level of control of the expression of marRAB operon. [ABSTRACT FROM AUTHOR]

Background. Much has been written recently about the potential hazards versus benefits of antibacterial (biocide)—containing soaps. The purpose of this systematic literature review was to assess the studies that have examined the efficacy of products containing triclosan, compared with that of plain soap, in the community setting, as well as to evaluate findings that address potential hazards of this use—namely, the emergence of antibiotic-resistant bacteria. Methods. The PubMed database was searched for English-language articles, using relevant keyword combinations for articles published between 1980 and 2006. Twenty-seven studies were eventually identified as being relevant to the review. Results. Soaps containing triclosan within the range of concentrations commonly used in the community setting (0.1%–0.45% wt/vol) were no more effective than plain soap at preventing infectious illness symptoms and reducing bacterial levels on the hands. Several laboratory studies demonstrated evidence of triclosan-adapted cross-resistance to antibiotics among different species of bacteria. Conclusions. The lack of an additional health benefit associated with the use of triclosan-containing consumer soaps over regular soap, coupled with laboratory data demonstrating a potential risk of selecting for drug resistance, warrants further evaluation by governmental regulators regarding antibacterial product claims and advertising. Further studies of this issue are encouraged. [ABSTRACT FROM AUTHOR]

We examined whether household use of antibacterial cleaning and hygiene products is an emerging risk factor for carriage of antimicrobial drug-resistant bacteria on hands of household members. Households (N = 224) were randomized to use of antibacterial or nonantibacterial cleaning and hygiene products for 1 year. Logistic regression was used to assess the influence of antibacterial product use in homes. Antibacterial product use did not lead to a significant increase in antimicrobial drug resistance after 1 year (odds ratio 1.33, 95% confidence interval 0.74-2.41), nor did it have an effect on bacterial susceptibility to triclosan. However, more extensive and longer term use of triclosan might provide a suitable environment for emergence of resistant species. Further research on this issue is needed. [ABSTRACT FROM AUTHOR]

Escherichia coli strain TP985, which has an insertional mutation in the gene for the periplasmic murein tripeptide binding protein MppA, was previously reported to overproduce MarA and exhibit a multipleantibiotic resistance (Mar) phenotype (H. Li and J. T. Park, J. Bacteriol. 181:4842-4847, 1999). We found that TP985 contained a previously unrecognized marR mutation which was responsible for the Mar phenotype. Transduction of the mppA mutation from TP985 to another wild-type strain did not affect antibiotic susceptibility. Overproduction of MppA repressed marA transcription in TP985 but not in other mppA or marR mutants. Therefore, TP985 contains an additional unknown mutation(s) that facilitates the repression of marA expression by MppA. [ABSTRACT FROM AUTHOR]

TetA specified by Tn10 is a class B member of group of related bacterial transport proteins of 12 transmembrane alpha helices that mediate resistance to the antibiotic tetracycline. A tetracycline-divalent metal cation complex is expelled from the cell in exchange for an entering proton. The site(s) where tetracycline binds to this export pump is now known. We found that, when cheated to tetracycline, Fe[sup 2+] cleaved the backbone of TetA predominantly at a single position, glutamine 225 in transmembrane helix 7. The related class D TetA protein from plasmid RA1 was cut at exactly the same position. There was no cleavage with glycylcycline, an analog of tetracycline that does not bind to TetA. The Fe[sup 2 +] -tetracycline complex was not detectably transported by TetA. However, cleavage products of the same size as with Fe[sup 2+] occurred with Co[sup 2+], known to be cotransported with tetracycline. The known substrate Mg[sup 2+] -tetracycline interfered with cleavage by Fe[sup 2+]. These findings suggest that cleavage results from binding at a substrate-specific site. Fe[sup 2+] is known to be able to cleave amide bonds in proteins at distances up to approximately 12 Å. We conclude that the α carbon of glutamine 225 is probably within 12 Å of the position of the Fe[sup 2+] ion in the Fe[sup 2+] -tetracycline complex bound to the protein. [ABSTRACT FROM AUTHOR]

Analyzes the genetic sequences of Escherichia coli affecting intrinsic susceptibility to inhibitors of peptidoglycan synthesis. Conditions creating of membrane-bound PhoaA fusion protein; Increase in intrinsic susceptibility to beta-lactam antibiotics; Structure restoring resistance to susceptibility in the parental level.