Bifunctional protein ArsRM contributes to arsenite methylation and resistance in Brevundimonas sp. M20.

BACKGROUND: Arsenic (As) with various chemical forms, including inorganic arsenic and organic arsenic, is the most prevalent water and environmental toxin. This metalloid occurs worldwide and many of its forms, especially arsenite [As(III)], cause various diseases including cancer. Organification of arsenite is an effective way for organisms to cope with arsenic toxicity. Microbial communities are vital contributors to the global arsenic biocycle and represent a promising way to reduce arsenite toxicity. METHODS: Brevundimonas sp. M20 with arsenite and roxarsone resistance was isolated from aquaculture sewage. The arsHRNBC cluster and the metRFHH operon of M20 were identified by sequencing. The gene encoding ArsR/methyltransferase fusion protein, arsRM, was amplified and expressed in Escherichia coli BL21 (DE3), and this strain showed resistance to arsenic in the present of 0.25-6 mM As(III), aresenate, or pentavalent roxarsone. The methylation activity and regulatory action of ArsRM were analyzed using Discovery Studio 2.0, and its functions were confirmed by methyltransferase activity analysis and electrophoretic mobility shift assays. RESULTS: The minimum inhibitory concentration of the roxarsone resistant strain Brevundimonas sp. M20 to arsenite was 4.5 mM. A 3,011-bp arsenite resistance ars cluster arsHRNBC and a 5649-bp methionine biosynthesis met operon were found on the 3.315-Mb chromosome. Functional prediction analyses suggested that ArsRM is a difunctional protein with transcriptional regulation and methyltransferase activities. Expression of ArsRM in E. coli increased its arsenite resistance to 1.5 mM. The arsenite methylation activity of ArsRM and its ability to bind to its own gene promoter were confirmed. The As(III)-binding site (ABS) and S-adenosylmethionine-binding motif are responsible for the difunctional characteristic of ArsRM. CONCLUSIONS: We conclude that ArsRM promotes arsenite methylation and is able to bind to its own promoter region to regulate transcription. This difunctional characteristic directly connects methionine and arsenic metabolism. Our findings contribute important new knowledge about microbial arsenic resistance and detoxification. Future work should further explore how ArsRM regulates the met operon and the ars cluster.

Cadmium stress efficiently enhanced meropenem degradation by the meropenem- and cadmium-resistant strain Pseudomonas putida R51.

The ß-lactam antibiotic meropenem (MEM) is widely used in infectious disease treatment and consequently can be released into the environment, causing environmental pollution. In this study, Pseudomonas putida strain R51 was isolated from the wastewater of a poultry farm and found to efficiently degrade MEM. The genome of strain R51 contains a variety of heavy metal and antibiotic resistance genes, including the metallo-ß-lactamase gene (JQN61_03315) and cadmium resistance gene cadA (JQN61_19995). Under cadmium stress, the degradation rate of MEM increased significantly in strain R51. Transcriptional analysis revealed that the expression of JQN61_03315 and cadA significantly increased under cadmium stress and that the expression of many genes associated with heavy metal and antibiotic resistance also changed significantly. Molecular docking analysis suggested that metallo-ß-lactamase JQN61_03315 binds to MEM. In addition, no plasmid was found in strain R51, and no mobile genetic elements were found nearby JQN61_03315. In conclusion. we proposed that JQN61_03315 was responsible for the degradation of MEM, that the expression of this gene was induced under cadmium stress, and that strain R51 can be used for bioremediation of MEM without the risk for the transmission of the MEM resistance gene. These findings will have importance for studying the microbial degradation of MEM in the presence of heavy metal pollutants.

The PolS-PolR Two-Component System Regulates Genes Involved in Poly-P Metabolism and Phosphate Transport in Microlunatus phosphovorus.

Microlunatus phosphovorus NM-1 is a polyphosphate (poly-P)-accumulating bacterium that accumulates poly-P under aerobic conditions and degrades poly-P under anaerobic conditions. In this study, the two-component system (TCS) PolS-PolR was identified in NM-1, and the response regulator PolR was found to directly bind to the promoters of genes related to phosphate transport (MLP_RS00235, MLP_RS23035, and MLP_RS24590); poly-P catabolism (MLP_RS12905) and poly-P synthesis (MLP_RS23025). RT-qPCR assays showed that ppgk (MLP_RS12905), ppk (MLP_RS23025), pstS (MLP_RS23035), and pit (MLP_RS24590) were down-regulated during the aerobic-anaerobic shift. The sequence GTTCACnnnnnGTTCaC was identified as a recognition sequence for PolR by MEME analysis and DNase I footprinting. EMSAs and ChIP-qPCR assays indicated that PolR binds to the promoters of pit (MLP_RS00235), ppgk (MLP_RS12905), ppk (MLP_RS23025), pstS (MLP_RS23035) and pit (MLP_RS24590), and ChIP-qPCR further suggested that the binding affinity of PolR was lower under anaerobic conditions than under aerobic conditions in vivo. These findings indicate that the PolS-PolR TCS in M. phosphovorus may be involved in the regulation of poly-P metabolism in response to levels of dissolved oxygen in the environment, and our results provide insights into new approaches for understanding the mechanisms of phosphorus accumulation and release.

Complete Genome Sequence of Actinosynnema pretiosum X47, An Industrial Strain that Produces the Antibiotic Ansamitocin AP-3.

Ansamitocins are extraordinarily potent antitumor agents. Ansamitocin P-3 (AP-3), which is produced by Actinosynnema pretiosum, has been developed as a cytotoxic drug for breast cancer. Despite its importance, AP-3 is of limited applicability because of the low production yield. A. pretiosum strain X47 was developed from A. pretiosum ATCC 31565 by mutation breeding and shows a relatively high AP-3 yield. Here, we analyzed the A. pretiosum X47 genome, which is ~8.13 Mb in length with 6693 coding sequences, 58 tRNA genes, and 15 rRNA genes. The DNA sequence of the ansamitocin biosynthetic gene cluster is highly similar to that of the corresponding cluster in A. pretiosum ATCC 31565, with 99.9% identity. However, RT-qPCR analysis showed that the expression levels of ansamitocin biosynthetic genes were significantly increased in X47 compared with the levels in the wild-type strain, consistent with the higher yield of AP-3 in X47. The annotated complete genome sequence of this strain will facilitate understanding the molecular mechanisms of ansamitocin biosynthesis and regulation in A. pretiosum and help further genetic engineering studies to enhance the production of AP-3.

Polyphosphate metabolic gene expression analyses reveal mechanisms of phosphorus accumulation and release in Microlunatus phosphovorus strain JN459.

The ability of Microlunatus phosphovorus to accumulate large amounts of polyphosphate (Poly-P) plays an important role in removing soluble phosphorus from wastewater. Strain JN459, isolated from a sewage system, was previously demonstrated to be Microlunatus phosphovorus. In this study, we analyzed the phosphorus-accumulating and phosphorus-releasing characteristics of strain JN459. Our analyses indicate that strain JN459 accumulates Poly-P under aerobic conditions but releases phosphorus under anaerobic conditions. To determine the mechanisms underlying Poly-P metabolism in strain JN459, we compared transcriptional profiles under aerobic and anaerobic conditions. Significant differences were detected in the expression levels of genes associated with Poly-P metabolism between aerobic and anaerobic conditions, including ppk (MLP_47700, MLP_50300 and MLP_05750), ppgk (MLP_05430 and MLP_26610), ppx (MLP_44770), pap (MLP_23310) and ppnk (MLP_17420). The high expression of polyphosphate glucokinase (MLP_05430) and polyphosphate/ATP-dependent NAD kinase (MLP_17420) indicated that both of them might be responsible for utilizing Poly-P as the energy resource for growth under anaerobic conditions. These findings enhance our understanding of phosphate metabolism in a major bacterial species involved in wastewater phosphorus reduction.

Deletion of the ß-Propeller Protein Gene Rv1057 Reduces ESAT-6 Secretion and Intracellular Growth of Mycobacterium tuberculosis.

Rv1057 is the only ß-propeller protein in Mycobacterium tuberculosis, but its biological function is still unclear. In this study, we generated a deletion mutant of Rv1057 (D1057) in the virulent M. tuberculosis strain H37Rv and examined the characteristics of the mutant in vitro and in macrophages. We found that deletion of Rv1057 reduces secretion of the major virulence factor ESAT-6 and ESAT-6 stops in the cell envelope fraction during secretion, although ESAT-6 levels were similar in lysates of the mutant and control strains. In infected macrophages, Rv1057 deletion significantly reduced the secretion levels of cytokines IL-1ß, IL-10, TNF-α, and INF-γ, but did not affect IL-4 and IL-8. D1057-infected macrophages also release less LDH and produce more nitric oxide (NO) than H37Rv- and D1057com (Rv1057 complemented strain of D1057com)-infected macrophages, indicating that D1057 has the decreased cytotoxicity compared to H37Rv or D1057com. In addition, the capacity of the Rv1057 deletion mutant to grow in macrophages was significantly lower than that of H37Rv and D1057com. Our findings support a role for Rv1057 in ESAT-6 secretion and in modulating the interactions between M. tuberculosis and macrophages.

Improvement of clavulanic acid production in Streptomyces clavuligerus F613-1 by using a claR-neo reporter strategy

Background: Streptomyces clavuligerus was the producer of clavulanic acid, claR, a pathway-specific transcriptional regulator in S. clavuligerus, positively regulates clavulanic acid biosynthesis. In this study, the promoter-less kanamycin resistance gene neo was fused with claR to obtain strain NEO from S. clavuligerus F613-1. The claR-neo fusion strain NEO was mutated using physical and chemical mutagens and then screened under high concentrations of kanamycin for high-yield producers of clavulanic acid. Results: The reporter gene neo was fused downstream of claR and used as an indicator for expression levels of claR in strain NEO. After three rounds of continuous treatment and screening, the high-yield clavulanic acid-producing strain M3-19 was obtained. In the shaking flask model, the clavulanic acid titer of M3-19 reached 4.33 g/L, which is an increase of 33% over the titer of 3.26 g/L for the starting strains S. clavuligerus F613-1 and NEO. Conclusions: Our results indicate that neo can be effectively used as a reporter for the expression of late-stage biosynthetic genes when screening for high-yield strains and that this approach has strong potential for improving Streptomyces strains of industrial value.

HAL2 overexpression induces iron acquisition in bdf1Δ cells and enhances their salt resistance.

The yeast Saccharomyces cerevisiae is capable of responding to various environmental stresses, such as salt stress. Such responses require a complex network and adjustment of the gene expression network. The goal of this study is to further understand the molecular mechanism of salt stress response in yeast, especially the molecular mechanism related to genes BDF1 and HAL2. The Bromodomain Factor 1 (Bdf1p) is a transcriptional regulator, which is part of the basal transcription factor TFIID. Cells lacking Bdf1p are salt sensitive with an abnormal mitochondrial function. We previously reported that the overexpression of HAL2 or deletion of HDA1 lowers the salt sensitivity of bdf1Δ. To better understand the mechanism behind the HAL2-related response to salt stress, we compared three global transcriptional profiles (bdf1Δ vs WT, bdf1Δ + HAL2 vs bdf1Δ, and bdf1Δhda1Δ vs bdf1Δ) in response to salt stress using DNA microarrays. Our results reveal that genes for iron acquisition and cellular and mitochondrial remodeling are induced by HAL2. Overexpression of HAL2 decreases the concentration of nitric oxide. Mitochondrial iron-sulfur cluster (ISC) assembly also decreases in bdf1Δ + HAL2. These changes are similar to the changes of transcriptional profiles induced by iron starvation. Taken together, our data suggest that mitochondrial functions and iron homeostasis play an important role in bdf1Δ-induced salt sensitivity and salt stress response in yeast.

The yeast BDF1 regulates endocytosis via LSP1 under salt stress.

Bromodomain-containing transcription factor, a kind of important regulating protein, can recognize and bind to acetylated histone. The homologous genes, BDF1 and BDF2, in Saccharomyces cerevisiae, respectively, encode a bromodomain-containing transcription factor. Previously study has demonstrated that both BDF1 and BDF2 participate in yeast salt stress response. Bdf1p deletion cells are sensitive to salt stress and this phenotype is suppressed by its homologue BDF2 in a dosage-dependent manner. In this study, we show that the histone deacetylase SIR2 over-expression enhanced dosage-dependent compensation of BDF2. SIR2 over-expression induced a global transcription change, and 1959 gene was down-regulated. We deleted some of the most significant down-regulated genes and did the spot assay. The results revealed that LSP1, an upstream component of endocytosis pathway, and CIN5, a transcription factor that mediates cellular resistance to stresses, can enhance salt resistance of bdf1∆. Further analysis demonstrated that under salt stress the endocytosis is over-activated in bdf1∆ but was recovered in bdf1∆ lsp1∆. To our best knowledge, this is the first report that the transcription factor Bdf1p regulates endocytosis under salt stress via LSP1, a major component of eisosomes that regulate the sites of endocytosis.

Reduced expression of PTPRD correlates with poor prognosis in gastric adenocarcinoma.

BACKGROUND: PTPRD, encoding protein tyrosine phosphatases receptor type D, is located at chromosome 9p23-24.1, a loci frequently lost in many types of tumors. Recently, PTPRD has been proposed to function as a tumor suppressor gene. The current study aimed to investigate PTPRD expression and its prognostic significance in primary gastric adenocarcinoma. METHODS AND RESULTS: Quantitative real time reverse transcription PCR (qRT-PCR) and western blotting were used to examine PTPRD expression in paired gastric tumourous and paracancerous tissues. Compared with the matched normal gastric mucosa tissues, both the mRNA (P = 0.0138) and protein (P = 0.0093) expression of PTPRD in fresh surgical specimens were significantly reduced. Clinicopathological and prognostic roles of PTPRD in gastric adenocarcinoma were investigated using immunohistochemistry with 513 paraffin-embedded gastric adenocarcinoma tissue blocks. Statistical analysis revealed that reduced PTPRD expression was significantly associated with T stage (P = 0.004), TNM stage (P<0.001) and tumor size (P = 0.003). Furthermore, Kaplan-Meier survival analysis revealed that low expression of PTPRD significantly correlated with poor survival of gastric cancer patients (P<0.001). Cox regression analysis confirmed PTPRD expression as independent predictor of the overall survival of gastric cancer patients. The MTT assay determined the effects of PTPRD on cell proliferation of MGC803 and GES1 cell lines. Restoring PTPRD expression in MGC803 cells significantly inhibited their growth rate. Silencing PTPRD expression by siRNA treatment in GES1 significantly enhanced cell proliferation compared with mock siRNA treatment. Methylation analysis of PTPRD promoter CpG island in 3 primary GC samples showed one case with partial methylation. CONCLUSIONS: These results indicated that PTPRD is a candidate tumour suppressor in gastric cancer. Thus, PTPRD may play an important role in gastric tumorigenesis and serve as a valuable prognostic marker of gastric adenocarcinoma.

Hal2p functions in Bdf1p-involved salt stress response in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae Bdf1p associates with the basal transcription complexes TFIID and acts as a transcriptional regulator. Lack of Bdf1p is salt sensitive and displays abnormal mitochondrial function. The nucleotidase Hal2p detoxifies the toxic compound 3' -phosphoadenosine-5'-phosphate (pAp), which blocks the biosynthesis of methionine. Hal2p is also a target of high concentration of Na(+). Here, we reported that HAL2 overexpression recovered the salt stress sensitivity of bdf1Δ. Further evidence demonstrated that HAL2 expression was regulated indirectly by Bdf1p. The salt stress response mechanisms mediated by Bdf1p and Hal2p were different. Unlike hal2Δ, high Na(+) or Li(+) stress did not cause pAp accumulation in bdf1Δ and methionine supplementation did not recover its salt sensitivity. HAL2 overexpression in bdf1Δ reduced ROS level and improved mitochondrial function, but not respiration. Further analyses suggested that autophagy was apparently defective in bdf1Δ, and autophagy stimulated by Hal2p may play an important role in recovering mitochondrial functions and Na(+) sensitivity of bdf1Δ. Our findings shed new light towards our understanding about the molecular mechanism of Bdf1p-involved salt stress response in budding yeast.