RhgKL and CcpA directly regulate the rhiLFGN-rhgR-yesTUV operon involved in the metabolism of rhamnogalacturonan type I in Bacillus subtilis.

The Bacillus subtilis rhiLFGN-rhgR-yesTUVWXYZ (formerly yesOPQRSTUVWXYZ) gene cluster includes genes for metabolizing rhamnogalacturonan type I (RG-I), a major pectin constituent, and the rhgR gene encoding an AraC/XylS transcriptional activator. The yesL-rhgKL (formerly yesLMN) operon, adjacent to the rhiL gene, includes the rhgKL genes encoding a two-component regulatory system. The reporter analyses showed that 3 promoters immediately upstream of the rhiL, yesW, and yesL genes were induced by RG-I and repressed by glucose in the medium. The reporter analyses also showed that RhgL and RhgR contribute to the RG-I-dependent induction of the rhiL promoter and that CcpA mediates the catabolite repression of the rhiL and yesL promoters. The in vitro experiments demonstrated that the RhgL response regulator and the CcpA complex bind to each site in the rhiL promoter region. The RT-PCR analysis and the different properties of the rhiL and yesW promoters suggested the rhiLFGN-rhgR-yesTUV genes as an operon.

Investigating the rus and petI operon expression patterns in exposed Acidithiobacillus ferrooxidans sp. FJ2 to different doses of gamma irradiation.

The bioleaching process is developing as an economic and successful biotechnology method in the metallurgy industry. Acidithiobacillus ferrooxidans is one of the most important bacteria involved in uranium bioleaching which converts insoluble U4+ to soluble U6+ by oxidation of Fe2+ to Fe3+ using several periplasmic proteins encoded by the genes in rus and petI operons in its electron transport pathway. Accordingly, the purpose of this study was to consider the expression of these genes through exposed A. ferrooxidans sp. FJ2 to γ-ray in 17 different doses targeting uranium extraction yield. Acidithiobacillus ferrooxidans sp. FJ2 was irradiated by gamma rays at 25, 50, 75, 100, 150, 300, 450, 600, 750 Gy and 1, 2, 5, 10, 15, 20, 25 and 30 kGy doses. Moreover, the Eh value of 9k culture media was measured as special screening criteria to select the four treatments. The selected bacteria were cultured in 9k media, containing 50% uranium ore powder in the bioleaching process. Then, the value of pH & Eh of culture media, Fe2+ and uranium concentrations in 4, 8 and 13 day's period of incubation were measured. In followings, the expression levels of cyc1, cyc2, rus, coxB, petA, petB, petC and cycA genes at the end of each period were investigated by real-time PCR. Overall, all samples demonstrated a decrease in pH value and Fe2+ concentration and an increase in Eh value and U concentration in time intervals. The gamma irradiation in given doses raised the expression levels of all genes encoded in rus and petI operons, except petB gene during the bioleaching process, although, it had no effect either on the pH, Eh values or on Fe2+ and uranium concentrations. This result implies that during the oxidation of ferrous iron and formation of Jarosite sediment, the decreasing trend of pH and the increasing trend of Eh occurred in all samples. However, the differences in expression of the genes of rus and petI operons in the samples did not have an effect on uranium extraction.

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.

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.

Carbon metabolism modulates the efficacy of drugs targeting the cytochrome bc1:aa3 in Mycobacterium tuberculosis.

The influence of carbon metabolism on oxidative phosphorylation is poorly understood in mycobacteria. M. tuberculosis expresses two respiratory terminal oxidases, the cytochrome bc1:aa3 and the cytochrome bd oxidase, which are jointly required for oxidative phosphorylation and mycobacterial viability. The essentiality of the cytochrome bc1:aa3 for optimum growth is illustrated by its vulnerability to chemical inhibition by the clinical drug candidate Q203 and several other chemical series. The cytochrome bd oxidase is not strictly essential for growth but is required to maintain bioenergetics when the function of the cytochrome bc1:aa3 is compromised. In this study, we observed that the potency of drugs targeting the cytochrome bc1:aa3 is influenced by carbon metabolism. The efficacy of Q203 and related derivatives was alleviated by glycerol supplementation. The negative effect of glycerol supplementation on Q203 potency correlated with an upregulation of the cytochrome bd oxidase-encoding cydABDC operon. Upon deletion of cydAB, the detrimental effect of glycerol on the potency of Q203 was abrogated. The same phenomenon was also observed in recent clinical isolates, but to a lesser extent compared to the laboratory-adapted strain H37Rv. This study reinforces the importance of optimizing in vitro culture conditions for drug evaluation in mycobacteria, a factor which appeared to be particularly essential for drugs targeting the cytochrome bc1:aa3 terminal oxidase.

TfmR, a novel TetR-family transcriptional regulator, modulates the virulence of Xanthomonas citri in response to fatty acids.

The type III secretion system (T3SS) is required for Xanthomonas citri subsp. citri (Xcc) virulence by translocating effectors into host cytoplasm to promote disease development. The T3SS is controlled by the master transcriptional regulators HrpG and HrpX. While the function of HrpG and HrpX are well characterized, their upstream regulation remains elusive. By using transposon mutagenesis, we identified XAC3052, a TetR-family transcriptional regulator, which regulates T3SS gene expression. Deletion of XAC3052 caused significant reduction in the expression of T3SS and effector genes in vitro and in planta; as well as reduction of virulence in sweet orange (Citrus sinensis). Overexpression of hrpG restored the virulence of ∆XAC3052, suggesting that the loss of virulence is caused by reduction of T3SS gene expression. XAC3052 directly binds to the promoter region and represses the transcription of fadE, mhpC and fadH genes. FadE, MhpC and FadH are not involved in T3SS regulation, but involved in fatty acid catabolism. ∆XAC3052 displays altered fatty acid composition and retarded growth in environments limited in fatty acids. Exogenously supplemented long-chain fatty acids activate the fadE/mhpC promoter and suppress T3SS promoters in wild-type Xac but not in ∆XAC3052. Moreover, the binding of XAC3052 to its target promoter was disrupted by long-chain fatty acids in vitro. Herein, XAC3052 is designated as TfmR (T3SS and Fatty acid Mechanism Regulator). This study identifies a novel regulator of fatty acid metabolism and suggests that fatty acids play an important role in the metabolic control of virulence in Xcc.

Spatiotemporal expression of the putative MdtABC efflux pump of Phtotorhabdus luminescens occurs in a protease-dependent manner during insect infection.

Photorhabdus luminescens is an enterobacterium establishing a mutualistic symbiosis with nematodes, that also kills insects after septicaemia and connective tissue colonization. The role of the bacterial mdtABC genes encoding a putative multidrug efflux system from the resistance/nodulation/cell division family was investigated. We showed that a mdtA mutant and the wild type had similar levels of resistance to antibiotics, antimicrobial peptides, metals, detergents and bile salts. The mdtA mutant was also as pathogenic as the wild-type following intrahaemocoel injection in Locusta migratoria, but had a slightly attenuated phenotype in Spodoptera littoralis. A transcriptional fusion of the mdtA promoter (PmdtA) and the green fluorescent protein (gfp) encoding gene was induced by copper in bacteria cultured in vitro. The PmdtA-gfp fusion was strongly induced within bacterial aggregates in the haematopoietic organ during late stages of infection in L. migratoria, whereas it was only weakly expressed in insect plasma throughout infection. A medium supplemented with haematopoietic organ extracts induced the PmdtA-gfp fusion ex vivo, suggesting that site-specific mdtABC expression resulted from insect signals from the haematopoietic organ. Finally, we showed that protease inhibitors abolished ex vivo activity of the PmdtA-gfp fusion in the presence of haematopoietic organ extracts, suggesting that proteolysis by-products play a key role in upregulating the putative MdtABC efflux pump during insect infection with P. luminescens.

Positive and negative regulation of transferred nif genes mediated by indigenous GlnR in Gram-positive Paenibacillus polymyxa.

Ammonia is a major signal that regulates nitrogen fixation in most diazotrophs. Regulation of nitrogen fixation by ammonia in the Gram-negative diazotrophs is well-characterized. In these bacteria, this regulation occurs mainly at the level of nif (nitrogen fixation) gene transcription, which requires a nif-specific activator, NifA. Although Gram-positive and diazotrophic Paenibacilli have been extensively used as a bacterial fertilizer in agriculture, how nitrogen fixation is regulated in response to nitrogen availability in these bacteria remains unclear. An indigenous GlnR and GlnR/TnrA-binding sites in the promoter region of the nif cluster are conserved in these strains, indicating the role of GlnR as a regulator of nitrogen fixation. In this study, we for the first time reveal that GlnR of Paenibacillus polymyxa WLY78 is essentially required for nif gene transcription under nitrogen limitation, whereas both GlnR and glutamine synthetase (GS) encoded by glnA within glnRA operon are required for repressing nif expression under excess nitrogen. Dimerization of GlnR is necessary for binding of GlnR to DNA. GlnR in P. polymyxa WLY78 exists in a mixture of dimers and monomers. The C-terminal region of GlnR monomer is an autoinhibitory domain that prevents GlnR from binding DNA. Two GlnR-biding sites flank the -35/-10 regions of the nif promoter of the nif operon (nifBHDKENXhesAnifV). The GlnR-binding site Ⅰ (located upstream of -35/-10 regions of the nif promoter) is specially required for activating nif transcription, while GlnR-binding siteⅡ (located downstream of -35/-10 regions of the nif promoter) is for repressing nif expression. Under nitrogen limitation, GlnR dimer binds to GlnR-binding siteⅠ in a weak and transient association way and then activates nif transcription. During excess nitrogen, glutamine binds to and feedback inhibits GS by forming the complex FBI-GS. The FBI-GS interacts with the C-terminal domain of GlnR and stabilizes the binding affinity of GlnR to GlnR-binding site Ⅱ and thus represses nif transcription.

Alternative ribosomal proteins are required for growth and morphogenesis of Mycobacterium smegmatis under zinc limiting conditions.

Zinc is an essential micronutrient required for proper structure and function of many proteins. Bacteria regularly encounter zinc depletion and have evolved diverse mechanisms to continue growth when zinc is limited, including the expression of zinc-independent paralogs of zinc-binding proteins. Mycobacteria have a conserved operon encoding four zinc-independent alternative ribosomal proteins (AltRPs) that are expressed when zinc is depleted. It is unknown if mycobacterial AltRPs replace their primary paralogs in the ribosome and maintain protein synthesis under zinc-limited conditions, and if such replacements contribute to their physiology. This study shows that AltRPs from Mycobacterium smegmatis are essential for growth when zinc ion is scarce. Specifically, the deletion mutant of this operon (ΔaltRP) is unable to grow in media containing a high-affinity zinc chelator, while growth of the wild type strain is unaffected under the same conditions. However, when zinc is gradually depleted during growth in zinc-limited medium, the ΔaltRP mutant maintains the same growth rate as seen for the wild type strain. In contrast to M. smegmatis grown with sufficient zinc supplementation that forms shorter cells when transitioning from logarithmic to stationary phase, M. smegmatis deficient for zinc elongates after the expression of AltRPs in late logarithmic phase. These zinc-depleted bacteria also exhibit a remarkable morphology characterized by a condensed chromosome, increased number of polyphosphate granules, and distinct appearance of lipid bodies and the cell wall compared to the zinc-replete cells. However, the ΔaltRP cells fail to elongate and transition into the zinc-limited morphotype, resembling the wild type zinc-replete bacteria instead. Therefore, the altRP operon in M. smegmatis has a vital role in continuation of growth when zinc is scarce and in triggering specific morphogenesis during the adaptation to zinc limitation, suggesting that AltRPs can functionally replace their zinc-dependent paralogs, but also contribute to mycobacterial physiology in a unique way.

The Streptococcus agalactiae Stringent Response Enhances Virulence and Persistence in Human Blood.

Streptococcus agalactiae (group B Streptococcus [GBS]) causes serious infections in neonates. We previously reported a transposon sequencing (Tn-seq) system for performing genomewide assessment of gene fitness in GBS. In order to identify molecular mechanisms required for GBS to transition from a mucosal commensal lifestyle to bloodstream invasion, we performed Tn-seq on GBS strain A909 with human whole blood. Our analysis identified 16 genes conditionally essential for GBS survival in blood, of which 75% were members of the capsular polysaccharide (cps) operon. Among the non-cps genes identified as conditionally essential was relA, which encodes an enzyme whose activity is central to the bacterial stringent response-a conserved adaptation to environmental stress. We used blood coincubation studies of targeted knockout strains to confirm the expected growth defects of GBS deficient in capsule or stringent response activation. Unexpectedly, we found that the relA knockout strains demonstrated decreased expression of ß-hemolysin/cytolysin, an important cytotoxin implicated in facilitating GBS invasion. Furthermore, chemical activation of the stringent response with serine hydroxamate increased ß-hemolysin/cytolysin expression. To establish a mechanism by which the stringent response leads to increased cytotoxicity, we performed transcriptome sequencing (RNA-seq) on two GBS strains grown under stringent response or control conditions. This revealed a conserved decrease in the expression of genes in the arginine deiminase pathway during stringent response activation. Through coincubation with supplemental arginine and the arginine antagonist canavanine, we show that arginine availability is a determinant of GBS cytotoxicity and that the pathway between stringent response activation and increased virulence is arginine dependent.

Characterization of Pectobacterium carotovorum proteins differentially expressed during infection of Zantedeschia elliotiana in vivo and in vitro which are essential for virulence.

The identification of phytopathogen proteins that are differentially expressed during the course of the establishment of an infection is important to better understand the infection process. In vitro approaches, using plant extracts added to culture medium, have been used to identify such proteins, but the biological relevance of these findings for in planta infection are often uncertain until confirmed by in vivo studies. Here, we compared the proteins of Pectobacterium carotovorum ssp. carotovorum strain PccS1 differentially expressed in Luria-Bertani medium supplemented with extracts of the ornamental plant Zantedeschia elliotiana cultivar 'Black Magic' (in vitro) and in plant tissues (in vivo) by two-dimensional electrophoresis coupled with mass spectrometry. A total of 53 differentially expressed proteins (>1.5-fold) were identified (up-regulated or down-regulated in vitro, in vivo or both). Proteins that exhibited increased expression in vivo but not in vitro, or in both conditions, were identified, and deletions were made in a number of genes encoding these proteins, four of which (clpP, mreB, flgK and eda) led to a loss of virulence on Z. elliotiana, although clpP and mreB were later also shown to be reduced in growth in rich and minimal media. Although clpP, flgK and mreB have previously been reported as playing a role in virulence in plants, this is the first report of such a role for eda, which encodes 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, a key enzyme in Entner-Doudoroff metabolism. The results highlight the value of undertaking in vivo as well as in vitro approaches for the identification of new bacterial virulence factors.

Xanthoferrin, the α-hydroxycarboxylate-type siderophore of Xanthomonas campestris pv. campestris, is required for optimum virulence and growth inside cabbage.

Xanthomonas campestris pv. campestris causes black rot, a serious disease of crucifers. Xanthomonads encode a siderophore biosynthesis and uptake gene cluster xss (Xanthomonas siderophore synthesis) involved in the production of a vibrioferrin-type siderophore. However, little is known about the role of the siderophore in the iron uptake and virulence of X. campestris pv. campestris. In this study, we show that X. campestris pv. campestris produces an α-hydroxycarboxylate-type siderophore (named xanthoferrin), which is required for growth under low-iron conditions and for optimum virulence. A mutation in the siderophore synthesis xssA gene causes deficiency in siderophore production and growth under low-iron conditions. In contrast, the siderophore utilization ΔxsuA mutant is able to produce siderophore, but exhibits a defect in the utilization of the siderophore-iron complex. Our radiolabelled iron uptake studies confirm that the ΔxssA and ΔxsuA mutants exhibit defects in ferric iron (Fe3+ ) uptake. The ΔxssA mutant is able to utilize and transport the exogenous xanthoferrin-Fe3+ complex; in contrast, the siderophore utilization or uptake mutant ΔxsuA exhibits defects in siderophore uptake. Expression analysis of the xss operon using a chromosomal gusA fusion indicates that the xss operon is expressed during in planta growth and under low-iron conditions. Furthermore, exogenous iron supplementation in cabbage leaves rescues the in planta growth deficiency of ΔxssA and ΔxsuA mutants. Our study reveals that the siderophore xanthoferrin is an important virulence factor of X. campestris pv. campestris which promotes in planta growth by the sequestration of Fe3+ .

Investigation of Factors Affecting Aerobic and Respiratory Growth in the Oxygen-Tolerant Strain Lactobacillus casei N87.

Aerobic and respiratory cultivations provide benefits for some lactic acid bacteria (LAB). Growth, metabolites, enzymatic activities (lactate dehydrogenase; pyruvate and NADH oxidases, NADH peroxidase; catalase), antioxidant capability and stress tolerance of Lactobacillus casei N87 were evaluated in anaerobic, aerobic and respiratory (aerobiosis with heme and menaquinone supplementation) batch cultivations with different dissolved oxygen (DO) concentrations. The expression of pox (pyruvate oxidase) and cydABCD operon (cytochrome bd oxidase complex) was quantified by quantitative Real Time polymerase chain reaction. Respiration increased biomass production compared to anaerobiosis and unsupplemented aerobiosis, and altered the central metabolism rerouting pyruvate away from lactate accumulation. All enzymatic activities, except lactate dehydrogenase, were higher in respiratory cultures, while unsupplemented aerobiosis with 60% of DO promoted H2O2 and free radical accumulation. Respiration improved the survival to oxidative and freeze-drying stresses, while significant numbers of dead, damaged and viable but not cultivable cells were found in unsupplemented aerobic cultures (60% DO). Analysis of gene expression suggested that the activation of aerobic and respiratory pathways occurred during the exponential growth phase, and that O2 and hemin induced, respectively, the transcription of pox and cydABCD genes. Respiratory cultivation might be a natural strategy to improve functional and technological properties of L. casei.

Differential regulation of toxoflavin production and its role in the enhanced virulence of Burkholderia gladioli.

Burkholderia gladioli is a causal agent of bacterial panicle blight and sheath/grain browning in rice in many countries. Many strains produce the yellow pigment toxoflavin, which is highly toxic to plants, fungi, animals and microorganisms. Although there have been several studies on the toxoflavin biosynthesis system of B. glumae, it is still unclear how B. gladioli activates toxoflavin biosynthesis. In this study, we explored the genomic organization of the toxoflavin system of B. gladioli and its biological functions using comparative genomic analysis between toxoflavin-producing strains (B. glumae BGR1 and B. gladioli BSR3) and a strain not producing toxoflavin (B. gladioli KACC11889). The latter exhibits normal physiological characteristics similar to other B. gladioli strains. Burkholderia gladioli KACC11889 possesses all the genes involved in toxoflavin biosynthesis, but lacks the quorum-sensing (QS) system that functions as an on/off switch for toxoflavin biosynthesis. These data suggest that B. gladioli has evolved to use the QS signalling cascade of toxoflavin production (TofI/TofR of QS → ToxJ or ToxR → tox operons) similar to that in B. glumae. However, some strains may have evolved to eliminate toxoflavin production through deletion of the QS genes. In addition, we demonstrate that the toxoflavin biosynthetic system enhances the virulence of B. gladioli. These findings provide another line of evidence supporting the differential regulation of the toxoflavin system in Burkholderia strains.

Fitness cost of antibiotic susceptibility during bacterial infection.

Advances in high-throughput DNA sequencing allow for a comprehensive analysis of bacterial genes that contribute to virulence in a specific infectious setting. Such information can yield new insights that affect decisions on how to best manage major public health issues such as the threat posed by increasing antimicrobial drug resistance. Much of the focus has been on the consequences of the selective advantage conferred on drug-resistant strains during antibiotic therapy. It is thought that the genetic and phenotypic changes that confer resistance also result in concomitant reductions in in vivo fitness, virulence, and transmission. However, experimental validation of this accepted paradigm is modest. Using a saturated transposon library of Pseudomonas aeruginosa, we identified genes across many functional categories and operons that contributed to maximal in vivo fitness during lung infections in animal models. Genes that bestowed both intrinsic and acquired antibiotic resistance provided a positive in vivo fitness advantage to P. aeruginosa during infection. We confirmed these findings in the pathogenic bacteria Acinetobacter baumannii and Vibrio cholerae using murine and rabbit infection models, respectively. Our results show that efforts to confront the worldwide increase in antibiotic resistance might be exacerbated by fitness advantages that enhance virulence in drug-resistant microbes.

Vitamin B12 modulates the transcriptome of the skin microbiota in acne pathogenesis.

Various diseases have been linked to the human microbiota, but the underlying molecular mechanisms of the microbiota in disease pathogenesis are often poorly understood. Using acne as a disease model, we aimed to understand the molecular response of the skin microbiota to host metabolite signaling in disease pathogenesis. Metatranscriptomic analysis revealed that the transcriptional profiles of the skin microbiota separated acne patients from healthy individuals. The vitamin B12 biosynthesis pathway in the skin bacterium Propionibacterium acnes was significantly down-regulated in acne patients. We hypothesized that host vitamin B12 modulates the activities of the skin microbiota and contributes to acne pathogenesis. To test this hypothesis, we analyzed the skin microbiota in healthy subjects supplemented with vitamin B12. We found that the supplementation repressed the expression of vitamin B12 biosynthesis genes in P. acnes and altered the transcriptome of the skin microbiota. One of the 10 subjects studied developed acne 1 week after vitamin B12 supplementation. To further understand the molecular mechanism, we revealed that vitamin B12 supplementation in P. acnes cultures promoted the production of porphyrins, which have been shown to induce inflammation in acne. Our findings suggest a new bacterial pathogenesis pathway in acne and provide one molecular explanation for the long-standing clinical observation that vitamin B12 supplementation leads to acne development in a subset of individuals. Our study discovered that vitamin B12, an essential nutrient in humans, modulates the transcriptional activities of skin bacteria, and provided evidence that metabolite-mediated interactions between the host and the skin microbiota play essential roles in disease development.

Pseudomonas fluorescens ATCC 13525 containing an artificial oxalate operon and Vitreoscilla hemoglobin secretes oxalic acid and solubilizes rock phosphate in acidic alfisols.

Oxalate secretion was achieved in Pseudomonas fluorescens ATCC 13525 by incorporation of genes encoding Aspergillus niger oxaloacetate acetyl hydrolase (oah), Fomitopsis plaustris oxalate transporter (FpOAR) and Vitreoscilla hemoglobin (vgb) in various combinations. Pf (pKCN2) transformant containing oah alone accumulated 19 mM oxalic acid intracellularly but secreted 1.2 mM. However, in the presence of an artificial oxalate operon containing oah and FpOAR genes in plasmid pKCN4, Pf (pKCN4) secreted 13.6 mM oxalate in the medium while 3.6 mM remained inside. This transformant solubilized 509 µM of phosphorus from rock phosphate in alfisol which is 4.5 fold higher than the Pf (pKCN2) transformant. Genomic integrants of P. fluorescens (Pf int1 and Pf int2) containing artificial oxalate operon (plac-FpOAR-oah) and artificial oxalate gene cluster (plac-FpOAR-oah, vgb, egfp) secreted 4.8 mM and 5.4 mM oxalic acid, released 329 µM and 351 µM P, respectively, in alfisol. The integrants showed enhanced root colonization, improved growth and increased P content of Vigna radiata plants. This study demonstrates oxalic acid secretion in P. fluorescens by incorporation of an artificial operon constituted of genes for oxalate synthesis and transport, which imparts mineral phosphate solubilizing ability to the organism leading to enhanced growth and P content of V. radiata in alfisol soil.

Flavodiiron protein Flv2/Flv4-related photoprotective mechanism dissipates excitation pressure of PSII in cooperation with phycobilisomes in Cyanobacteria.

Oxygenic photosynthesis evolved with cyanobacteria, the ancestors of plant chloroplasts. The highly oxidizing chemistry of water splitting required concomitant evolution of efficient photoprotection mechanisms to safeguard the photosynthetic machinery. The role of flavodiiron proteins (FDPs), originally called A-type flavoproteins or Flvs, in this context has only recently been appreciated. Cyanobacterial FDPs constitute a specific protein group that evolved to protect oxygenic photosynthesis. There are four FDPs in Synechocystis sp. PCC 6803 (Flv1 to Flv4). Two of them, Flv2 and Flv4, are encoded by an operon together with a Sll0218 protein. Their expression, tightly regulated by CO2 levels, is also influenced by changes in light intensity. Here we describe the overexpression of the flv4-2 operon in Synechocystis sp. PCC 6803 and demonstrate that it results in improved photochemistry of PSII. The flv4-2/OE mutant is more resistant to photoinhibition of PSII and exhibits a more oxidized state of the plastoquinone pool and reduced production of singlet oxygen compared with control strains. Results of biophysical measurements indicate that the flv4-2 operon functions in an alternative electron transfer pathway from PSII, and thus alleviates PSII excitation pressure by channeling up to 30% of PSII-originated electrons. Furthermore, intact phycobilisomes are required for stable expression of the flv4-2 operon genes and for the Flv2/Flv4 heterodimer-mediated electron transfer mechanism. The latter operates in photoprotection in a complementary way with the orange carotenoid protein-related nonphotochemical quenching. Expression of the flv4-2 operon and exchange of the D1 forms in PSII centers upon light stress, on the contrary, are mutually exclusive photoprotection strategies among cyanobacteria.

Characterization of transcription factors that regulate the type IV secretion system and riboflavin biosynthesis in Wolbachia of Brugia malayi.

The human filarial parasite Brugia malayi harbors an endosymbiotic bacterium Wolbachia (wBm) that is required for parasite survival. Consequently, targeting wBm is a promising approach for anti-filarial drug development. The Type IV secretion system (T4SS) plays an important role in bacteria-host interactions and is under stringent regulation by transcription factors. In wBm, most T4SS genes are contained in two operons. We show the wBm is active since the essential assembly factor virB8-1, is transcribed in adult worms and larval stages, and VirB8-1 is present in parasite lysates. We also identify two transcription factors (wBmxR1 and wBmxR2) that bind to the promoter region of several genes of the T4SS. Gel shift assays show binding of wBmxR1 to regions upstream of the virB9-2 and wBmxR2 genes, whereas wBmxR2 binds to virB4-2 and wBmxR1 promoter regions. Interestingly, both transcription factors bind to the promoter of the ribA gene that precedes virB8-1, the first gene in operon 1 of the wBm T4SS. RT-PCR reveals ribA and virB8-1 genes are co-transcribed as one operon, indicating the ribA gene and T4SS operon 1 are co-regulated by both wBmxR1 and wBmxR2. RibA encodes a bi-functional enzyme that catalyzes two essential steps in riboflavin (Vitamin B2) biosynthesis. Importantly, the riboflavin pathway is absent in B. malayi. We demonstrate the pathway is functional in wBm, and observe vitamin B2 supplementation partially rescues filarial parasites treated with doxycycline, indicating Wolbachia may supply the essential vitamin to its worm host. This is the first characterization of a transcription factor(s) from wBm and first report of co-regulation of genes of the T4SS and riboflavin biosynthesis pathway. In addition, our results demonstrate a requirement of vitamin B2 for worm health and fertility, and imply a nutritional role of the symbiont for the filarial parasite host.

Secondary structure of antisense RNAß, an internal transcriptional terminator of the plasmid-encoded iron transport-biosynthesis operon of Vibrio anguillarum.

RNAß affects the transcription process of the iron transport-biosynthesis operon encoded in the pJM1 plasmid of Vibrio anguillarum at a stem-loop structure located in the intergenic region between the fatA and angR genes. The net result is a higher level of the fatD, fatC, fatB, and fatA moiety as compared with the longer transcript encoding those genes as well as the angR and angT genes. In this work we report the secondary structure of RNAß determined by treatment with single and double strand specific ribonucleases as well as lead acetate followed by sequencing. The generated in vitro structural data indicated that three of the four previously described loops are in agreement with the original model, however, the alteration of loop IV as well as several other structural differences in the overall shape of the molecule led to the necessity of creating a new in silico model. Using the sites of mutations in the various loops we modeled the change in the RNAß secondary structure induced by those mutations. Mutations of loops III and IV to their complementary bases alter the overall structure of the RNAß significantly and increase its function while mutations in loops I and II have the opposite effect, the structure is unchanged but the activity of RNAß decreases. This indicates that loops I and II are necessary for interaction with the target mRNA. It is possible that the structural rearrangement introduced by mutations in loops III and IV promote activity and binding in loops I and II through reducing steric hindrance or increased binding to the target. This result also indicates that the exact relative positions of the critical loops are unimportant for activity.