Antibiotic Bactericidal Activity Is Countered by Maintaining pH Homeostasis in Mycobacterium smegmatis.

Antibiotics target specific biosynthetic processes essential for bacterial growth. It is intriguing that several commonalities connect the bactericidal activity of seemingly disparate antibiotics, such as the numerous conditions that confer broad-spectrum antibiotic tolerance. Whether antibiotics kill in a manner unique to their specific targets or by a universal mechanism is a critical and contested subject. Herein, we demonstrate that the bactericidal activity of diverse antibiotics against Mycobacterium smegmatis and four evolutionarily divergent bacterial pathogens was blocked by conditions that worked to maintain intracellular pH homeostasis. Single-cell pH analysis demonstrated that antibiotics increased the cytosolic pH of M. smegmatis, while conditions that promoted proton entry into the cytosol prevented intracellular alkalization and antibiotic killing. These findings led to a hypothesis that posits antibiotic lethality occurs when antibiotics obstruct ATP-consuming biosynthetic processes while metabolically driven proton efflux is sustained despite the loss of proton influx via ATP synthase. Consequently, without a concomitant reduction in respiratory proton efflux, cell death occurs due to intracellular alkalization. Our findings indicate the effects of antibiotics on pH homeostasis should be considered a potential mechanism contributing to antibiotic lethality. IMPORTANCE Since the discovery of antibiotics, mortality due to bacterial infection has decreased dramatically. However, infections from difficult to treat bacteria such as Mycobacterium tuberculosis and multidrug-resistant pathogens have been on the rise. An understanding of the cascade of events that leads to cell death downstream of specific drug-target interactions is not well understood. We have discovered that killing by several classes of antibiotics was stopped by maintaining pH balance within the bacterial cell, consistent with a shared mechanism of antibiotic killing. Our findings suggest a mechanism of antibiotic killing that stems from the antibiotic's ability to increase the pH within bacterial cells by disrupting proton entry without affecting proton pumping out of cells. Knowledge of the core mechanism necessary for antibiotic killing could have a significant impact on the development of new lethal antibiotics and for the treatment of recalcitrant and drug-resistant pathogens.

A Burkholderia pseudomallei colony variant necessary for gastric colonization.

UNLABELLED: Diverse colony morphologies are a hallmark of Burkholderia pseudomallei recovered from infected patients. We observed that stresses that inhibit aerobic respiration shifted populations of B. pseudomallei from the canonical white colony morphotype toward two distinct, reversible, yet relatively stable yellow colony variants (YA and YB). As accumulating evidence supports the importance of B. pseudomallei enteric infection and gastric colonization, we tested the response of yellow variants to hypoxia, acidity, and stomach colonization. Yellow variants exhibited a competitive advantage under hypoxic and acidic conditions and alkalized culture media. The YB variant, although highly attenuated in acute virulence, was the only form capable of colonization and persistence in the murine stomach. The accumulation of extracellular DNA (eDNA) was a characteristic of YB as observed by 4',6-diamidino-2-phenylindole (DAPI) staining of gastric tissues, as well as in an in vitro stomach model where large amounts of eDNA were produced without cell lysis. Transposon mutagenesis identified a transcriptional regulator (BPSL1887, designated YelR) that when overexpressed produced the yellow phenotype. Deletion of yelR blocked a shift from white to the yellow forms. These data demonstrate that YB is a unique B. pseudomallei pathovariant controlled by YelR that is specifically adapted to the harsh gastric environment and necessary for persistent stomach colonization. IMPORTANCE: Seemingly uniform populations of bacteria often contain subpopulations that are genetically identical but display unique characteristics which offer advantages when the population is faced with infrequent but predictable stresses. The pathogen Burkholderia pseudomallei is capable of forming several reversible colony types, and it interconverted between one white type and two yellow types under certain environmental stresses. The two yellow forms exhibited distinct advantages in low-oxygen and acidic environments. One yellow colony variant was the only form capable of chronic stomach colonization. Areas of gastric infection were marked by bacteria encased in a DNA matrix, and the yellow forms were able to produce large amounts of extracellular DNA in vitro. We also identified the regulator in control of yellow colony variant formation. These findings demonstrate a role in infection for colony variation and provide a mechanism for chronic stomach colonization-a frequently overlooked niche in melioidosis.

Mycobacterium tuberculosis Lsr2 is a global transcriptional regulator required for adaptation to changing oxygen levels and virulence.

UNLABELLED: To survive a dynamic host environment, Mycobacterium tuberculosis must endure a series of challenges, from reactive oxygen and nitrogen stress to drastic shifts in oxygen availability. The mycobacterial Lsr2 protein has been implicated in reactive oxygen defense via direct protection of DNA. To examine the role of Lsr2 in pathogenesis and physiology of M. tuberculosis, we generated a strain deleted for lsr2. Analysis of the M. tuberculosis Δlsr2 strain demonstrated that Lsr2 is not required for DNA protection, as this strain was equally susceptible as the wild type to DNA-damaging agents. The lsr2 mutant did display severe growth defects under normoxic and hyperoxic conditions, but it was not required for growth under low-oxygen conditions. However, it was also required for adaptation to anaerobiosis. The defect in anaerobic adaptation led to a marked decrease in viability during anaerobiosis, as well as a lag in recovery from it. Gene expression profiling of the Δlsr2 mutant under aerobic and anaerobic conditions in conjunction with published DNA binding-site data indicates that Lsr2 is a global transcriptional regulator controlling adaptation to changing oxygen levels. The Δlsr2 strain was capable of establishing an early infection in the BALB/c mouse model; however, it was severely defective in persisting in the lungs and caused no discernible lung pathology. These findings demonstrate M. tuberculosis Lsr2 is a global transcriptional regulator required for control of genes involved in adaptation to extremes in oxygen availability and is required for persistent infection. IMPORTANCE: M. tuberculosis causes nearly two million deaths per year and infects nearly one-third of the world population. The success of this aerobic pathogen is due in part to its ability to successfully adapt to constantly changing oxygen availability throughout the infectious cycle, from the high oxygen tension during aerosol transmission to anaerobiosis within necrotic lesions. An understanding of how M. tuberculosis copes with these changes in oxygen tension is critical for its eventual eradication. Using a mutation in lsr2, we demonstrate that the Lsr2 protein present in all mycobacteria is a global transcriptional regulator in control of genes required for adaptation to changes in oxygen levels. M. tuberculosis lacking lsr2 was unable to adapt to both high and very low levels of oxygen and was defective in long-term anaerobic survival. Lsr2 was also required for disease pathology and for chronic infection in a mouse model of TB.

The DosR regulon of M. tuberculosis and antibacterial tolerance.

Adaptation of Mycobacterium tuberculosis to an anaerobic dormant state that is tolerant to several antibacterials is mediated largely by a set of highly expressed genes controlled by DosR. A DosR mutant was constructed to investigate whether the DosR regulon is involved in antibacterial tolerance. We demonstrate that induction of the regulon is not required for drug tolerance either in vivo during a mouse infection or in vitro during anaerobic dormancy. Thus, drug tolerance observed in these models is due to other mechanisms such as the bacilli simply being in a non-replicating or low metabolic state. Our data also demonstrate that the DosR regulon is not essential for virulence during chronic murine infection. However, decreased lung pathology was observed in the DosR mutant. We also show that the DosR regulon genes are more highly conserved in environmental mycobacteria, than in pathogenic mycobacteria lacking a latent phase or environmental reservoir. It is possible that the DosR regulon could contribute to drug tolerance in human infections; however, it is not the only mechanism and not the primary mechanism for tolerance during a mouse infection. These data suggest that the regulon evolved not for pathogenesis or drug tolerance but for adaptation to anaerobic conditions in the environment and has been adapted by M. tuberculosis for survival during latent infection.

The Mycobacterium tuberculosis sigma factor sigmaB is required for full response to cell envelope stress and hypoxia in vitro, but it is dispensable for in vivo growth.

The numerous sigma (sigma) factors present in Mycobacterium tuberculosis are indicative of the adaptability of this pathogen to different environmental conditions. In this report, we describe the M. tuberculosis sigma(B) regulon and the phenotypes of an M. tuberculosis sigB mutant strain exposed to cell envelope stress, oxidative stress, and hypoxia. The sigB mutant was especially defective in survival under hypoxic conditions in vitro, but it was not attenuated for growth in THP-1 cells or during mouse and guinea pig infection.

A predicted operon map for Mycobacterium tuberculosis.

The prediction of operons in Mycobacterium tuberculosis (MTB) is a first step toward understanding the regulatory network of this pathogen. Here we apply a statistical model using logistic regression to predict operons in MTB. As predictors, our model incorporates intergenic distance and the correlation of gene expression calculated for adjacent gene pairs from over 474 microarray experiments with MTB RNA. We validate our findings with known examples from the literature and experimentation. From this model, we rank each potential operon pair by the strength of evidence for cotranscription, choose a classification threshold with a true positive rate of over 90% at a false positive rate of 9.1%, and use it to construct an operon map for the MTB genome.

Mycobacterium tuberculosis gene expression during adaptation to stationary phase and low-oxygen dormancy.

The innate mechanisms used by Mycobacterium tuberculosis to persist during periods of non-proliferation are central to understanding the physiology of the bacilli during latent disease. We have used whole genome expression profiling to expose adaptive mechanisms initiated by M. tuberculosis in two common models of M. tuberculosis non-proliferation. The first of these models was a standard growth curve in which gene expression changes were followed from exponential growth through the transition to stationary phase. In the second model, we followed the adaptive process of M. tuberculosis during transition from aerobic growth to a state of anaerobic non-replicating persistence. The most striking finding from these experiments was the strong induction of the entire DosR "dormancy" regulon over approximately 20 days during the long transition to an anaerobic state. This is contrasted by the muted overall response to aerated stationary phase with only a partial dormancy regulon response. From the results presented here we conclude that the respiration-limited environment of the oxygen-depleted NRP model recreates at least one fundamental factor for which the genome of M. tuberculosis encodes a decisive adaptive program.

Regulation of the Mycobacterium tuberculosis PE/PPE genes.

The genome of Mycobacterium tuberculosis encodes approximately 170 members of the unique mycobacterial PE and PPE gene families. Evidence suggests members of these families are surface-associated cell wall proteins that may provide a diverse antigenic profile and affect immunity. To determine if the expression patterns of PE/PPE genes are consistent with a role in antigenic variability, we analyzed microarray data from 132 experimental conditions for expression of PE/PPE genes. Whole genome expression patterns show that the PE/PPE genes are regulated in a variable and largely independent manner. Gene expression profiling of 15 unique conditions identified differential regulation of 128 of the 169 PE/PPE genes. Expression of the PE/PPE genes appears to be controlled by a variety of independent mechanisms. These data indicate that differential expression of the PE/PPE genes has the potential to provide a dynamic antigenic profile during the course of changing microenvironments within the host.

The Mycobacterium tuberculosis ECF sigma factor sigmaE: role in global gene expression and survival in macrophages.

In previously published work, we identified three Mycobacterium tuberculosis sigma (sigma) factor genes responding to heat shock (sigB, sigE and sigH). Two of them (sigB and sigE) also responded to SDS exposure. As these responses to stress suggested that the sigma factors encoded by these genes could be involved in pathogenicity, we are studying their role in physiology and virulence. In this work, we characterize a sigE mutant of M. tuberculosis H37Rv. The sigE mutant strain was more sensitive than the wild-type strain to heat shock, SDS and various oxidative stresses. It was also defective in the ability to grow inside both human and murine unactivated macrophages and was more sensitive than the wild-type strain to the killing activity of activated murine macrophages. Using microarray technology and quantitative reverse transcription-polymerase chain reaction (RT-PCR), we started to define the sigmaE regulon of M. tuberculosis and its involvement in the global regulation of the stress induced by SDS. We showed the requirement for a functional sigE gene for full expression of sigB and for its induction after SDS exposure but not after heat shock. We also identified several genes that are no longer induced when sigmaE is absent. These genes encode proteins belonging to different classes including transcriptional regulators, enzymes involved in fatty acid degradation and classical heat shock proteins.

NADP, corepressor for the Bacillus catabolite control protein CcpA.

Expression of the alpha-amylase gene (amyE) of Bacillus subtilis is subject to CcpA (catabolite control protein A)-mediated catabolite repression, a global regulatory mechanism in Bacillus and other Gram-positive bacteria. To determine effectors of CcpA, we tested the ability of glycolytic metabolites, nucleotides, and cofactors to affect CcpA binding to the amyE operator, amyO. Those that stimulated the DNA-binding affinity of CcpA were tested for their effect on transcription. HPr-P (Ser-46), proposed as an effector of CcpA, also was tested. In DNase I footprint assays, the affinity of CcpA for amyO was stimulated 2-fold by fructose-1,6-diphosphate (FDP), 1.5-fold by oxidized or reduced forms of NADP, and 10-fold by HPr-P (Ser-46). However, the triple combinations, CcpA/NADP/HPr-P (Ser-46) and CcpA/FDP/HPr-P (Ser-46) synergistically stimulated DNA-binding affinity by 120- and 300-fold, respectively. NADP added to CcpA specifically stimulated transcription inhibition of the amyE promoter by 120-fold. CcpA combined with HPr (Ser-46) inhibited transcription from the amyE promoter, but it also inhibited several control promoters. FDP did not stimulate transcription inhibition by CcpA nor did the triple combinations. The finding that NADP had little effect on CcpA DNA binding but increased the ability of CcpA to inhibit transcription suggests that catabolite repression is not simply caused by CcpA binding amyO but rather a result of interactions with the transcription machinery enhanced by NADP.

The -16 region of Bacillus subtilis and other gram-positive bacterial promoters.

The Bacillus subtilis alpha-amylase promoter amy P contains an essential TGTG motif (-16 region) upstream of the -10 region. Mutations of this region significantly reduced in vitro promoter strength. A -15 G-->C transversion eliminated transcription from amy P by both B.subtilis and Escherichia coli RNA polymerase (RNAP). A second alpha-amylase promoter ( amy P2) also required the -16 region for function. To determine conserved sequences in promoters containing -16 region elements, sequences of 64 B.subtilis promoters with the second TG motif of the -16 region were aligned and analyzed. Unlike the E.coli class of 'extended -10 promoters', with a similar TG motif but lacking a -35 region, the -16 region promoters contain highly conserved -35 regions. They also contain conserved A n and T n tracts upstream of the -35 region. In addition, we analyzed all available gram-positive bacterial promoter compilations to determine the generality of the -16 region. From this analysis, the -16 region TRTG motif (R = purine) appears to be a basic element found in a large portion of gram-positive bacterial promoters and is, in the case of at least the alpha-amylase promoters, necessary for transcription by the major form of B.subtilis and E.coli RNAP.

Significance of HPr in catabolite repression of alpha-amylase.

CcpA and HPr are presently the only two proteins implicated in Bacillus subtilis global carbon source catabolite repression, and the ptsH1 mutation in the gene for the HPr protein was reported to relieve catabolite repression of several genes. However, alpha-amylase synthesis by B. subtilis SA003 containing the ptsH1 mutation was repressed by glucose. Our results suggest HPr(Ser-P) may be involved in but is not required for catabolite repression of alpha-amylase, indicating that HPr(Ser-P) is not the sole signaling molecule for CcpA-mediated catabolite repression in B. subtilis.

The -16 region, a vital sequence for the utilization of a promoter in Bacillus subtilis and Escherichia coli.

The promoter (amyP) of the Bacillus subtilis alpha-amylase gene, which is recognized by E sigma A, has a three out of six match to the consensus promoter in both the -35 and -10 hexamers. Oligonucleotide-directed mutagenesis was used to identify important bases for promoter utilization in the spacer sequence between the hexamers. Mutations in the sequence TGTG extending from positions -18 to -15 (the -16 region) caused a 5-94-fold decrease in alpha-amylase production. A G-C transversion at position -15 was the most detrimental mutation: it essentially eliminated amyP utilization in B. subtilis and in Escherichia coli. Mutating the -35 and -10 hexamers to the E sigma A consensus promoter increased alpha-amylase production 56-fold in B. subtilis and fivefold in E. coli. Introducing the -15 G to C transversion into the consensus promoter reduced alpha-amylase production threefold, in contrast to the 94-fold reduction for the wild-type promoter in B. subtilis. The -15 G to C transversion did not reduce alpha-amylase synthesis directed by the consensus promoter in E. coli. The alpha-amylase gene is subject to two forms of transcriptional regulation: catabolite repression and temporal regulation. None of the mutants constructed in this study had any effect on either type of regulation. The -16 region, especially the G at position -15, appears to be moderately conserved in B. subtilis and in other Gram-positive organisms and weakly conserved in E. coli. The evidence suggests that the -16 region is an additional region of E sigma A promoters in B. subtilis and E sigma 70 promoters in E. coli, essential in some weak promoters such as the alpha-amylase promoter but, of little benefit to very strong promoters.

Rapid isolation and sequencing of purified plasmid DNA from Bacillus subtilis.

We report two methods for isolation of plasmid DNA from the gram-positive bacterium Bacillus subtilis. The protoplast alkaline lysis procedure was developed for general use, and the protoplast alkaline lysis magic procedure was developed for isolation of DNA for sequencing. Both procedures yielded large amounts of high-quality DNA in less than 1 h, while current protocols require 4 to 7 h to perform and give lower yields and quality. Plasmid DNA was obtained from strains containing either high- or low-copy-number plasmids. In addition, the procedures were easily adapted to yield large amounts of plasmid DNA suitable for sequencing from another gram-positive organism, Staphylococcus aureus. Further, we demonstrated that neither chloramphenicol, used for plasmid selection, nor the mutation recE4 reduced plasmid DNA yield from the strains we examined.