Identification of four genes responsible for antimicrobial resistance of MEL-B against S. aureus.

There is increasing interest in the antimicrobial activity of mannosylerythritol lipids-B (MEL-B) against Gram-positive bacteria such as Staphylococcus aureus (S. aureus). However, the specific molecules involved in MEL-B's antimicrobial action against S. aureus have not been identified. This study utilized the Nebraska transposon mutant library (NTML), which contains 1920 mutants, each lacking three-quarters of the genes found in S. aureus. The NTML was screened to identify mutants resistant to MEL-B. Four mutants (Accession Number: SAUSA300_0904, SAUSA300_0752, SAUSA300_0387, and SAUSA300_2311) largely unaffected by incubation with MEL-B, indicating MEL-B resistance. Despite the strong binding of MEL-B to these mutants, the four molecules encoded by the deleted genes (yjbI, clpP, pbuX, or brpS) in each mutant were not directly recognized by MEL-B. Given that these molecules are not localized on the outer surface of S. aureus and that the antibacterial activity of MEL-B against S. aureus is facilitated by the effective transfer of two antibacterial fatty acids (caprylic acid and myristoleic acid) to S. aureus via ME, the deletion of each of the four molecules may alter the peptidoglycan structure, potentially inhibiting the effective transfer of these antimicrobial fatty acids into S. aureus.

l-Alanine Exporter AlaE Functions as One of the d-Alanine Exporters in Escherichia coli.

d-amino acids have recently been found to be present in the extracellular milieu at millimolar levels and are therefore assumed to play a physiological function. However, the pathway (or potential pathways) by which these d-amino acids are secreted remains unknown. Recently, Escherichia coli has been found to possess one or more energy-dependent d-alanine export systems. To gain insight into these systems, we developed a novel screening system in which cells expressing a putative d-alanine exporter could support the growth of d-alanine auxotrophs in the presence of l-alanyl-l-alanine. In the initial screening, five d-alanine exporter candidates, AlaE, YmcD, YciC, YraM, and YidH, were identified. Transport assays of radiolabeled d-alanine in cells expressing these candidates indicated that YciC and AlaE resulted in lower intracellular levels of d-alanine. Further detailed transport assays of AlaE in intact cells showed that it exports d-alanine in an expression-dependent manner. In addition, the growth constraints on cells in the presence of 90 mM d-alanine were mitigated by the overexpression of AlaE, implying that AlaE could export free d-alanine in addition to l-alanine under conditions in which intracellular d/l-alanine levels are raised. This study also shows, for the first time, that YciC could function as a d-alanine exporter in intact cells.

Initial Step of Selenite Reduction via Thioredoxin for Bacterial Selenoprotein Biosynthesis.

Many organisms reductively assimilate selenite to synthesize selenoprotein. Although the thioredoxin system, consisting of thioredoxin 1 (TrxA) and thioredoxin reductase with NADPH, can reduce selenite and is considered to facilitate selenite assimilation, the detailed mechanism remains obscure. Here, we show that selenite was reduced by the thioredoxin system from Pseudomonas stutzeri only in the presence of the TrxA (PsTrxA), and this system was specific to selenite among the oxyanions examined. Mutational analysis revealed that Cys33 and Cys36 residues in PsTrxA are important for selenite reduction. Free thiol-labeling assays suggested that Cys33 is more reactive than Cys36. Mass spectrometry analysis suggested that PsTrxA reduces selenite via PsTrxA-SeO intermediate formation. Furthermore, an in vivo formate dehydrogenase activity assay in Escherichia coli with a gene disruption suggested that TrxA is important for selenoprotein biosynthesis. The introduction of PsTrxA complemented the effects of TrxA disruption in E. coli cells, only when PsTrxA contained Cys33 and Cys36. Based on these results, we proposed the early steps of the link between selenite and selenoprotein biosynthesis via the formation of TrxA-selenium complexes.

Characterization of a novel class of glyoxylate reductase belonging to the ß-hydroxyacid dehydrogenase family in Acetobacter aceti.

Enzymes related to ß-hydroxyacid dehydrogenases/3-hydroxyisobutyrate dehydrogenases are ubiquitous, but most of them have not been characterized. An uncharacterized protein with moderate sequence similarities to Gluconobacter oxydans succinic semialdehyde reductase and plant glyoxylate reductases/succinic semialdehyde reductases was found in the genome of Acetobacter aceti JCM20276. The corresponding gene was cloned and expressed in Escherichia coli. The gene product was purified and identified as a glyoxylate reductase that exclusively catalyzed the NAD(P)H-dependent reduction of glyoxylate to glycolate. The strict substrate specificity of this enzyme to glyoxylate, the diverged sequence motifs for its binding sites with cofactors and substrates, and its phylogenetic relationship to homologous enzymes suggested that this enzyme represents a novel class of enzymes in the ß-hydroxyacid dehydrogenase family. This study may provide an important clue to clarify the metabolism of glyoxylate in bacteria. Abbreviations: GR: glyoxylate reductase; GRHPR: glyoxylate reductase/hydroxypyruvate reductase; HIBADH: 3-hydroxyisobutyrate dehydrogenase; SSA: succinic semialdehyde; SSAR: succinic semialdehyde reductase.

Selenite uptake by outer membrane porin ExtI and its involvement in the subcellular localization of rhodanese-like lipoprotein ExtH in Geobacter sulfurreducens.

Selenite reduction is a key step in the biogeochemical cycle of selenium-an essential trace element for life. A variety of bacteria can transform selenite into elemental selenium nanoparticles on the cell surface via anaerobic respiration or detoxification processes. However, the proteins associated with the uptake of selenite for these processes are poorly understood. In this study, we investigated the role of an outer membrane porin-like protein, ExtI, in selenite permeation in Geobacter sulfurreducens. We demonstrated that selenite uptake and selenium nanoparticle formation were impaired in an extI-deficient strain. A putative rhodanese-like lipoprotein is encoded by an extH gene located immediately upstream of extI in the genome. We showed that ExtH is translocated into inner and outer membranes and that extI deficiency exclusively affects the localization of ExtH in the outer membrane. Coelution of ExtI and ExtH during gel filtration analysis of the outer membrane fraction of wild-type cells suggests a direct protein-protein interaction between them. Taken together, these results lead us to propose a physiological role for ExtI as a selenite channel associated with ExtH in the outer membrane.

Characterization of a Novel Porin-Like Protein, ExtI, from Geobacter sulfurreducens and Its Implication in the Reduction of Selenite and Tellurite.

The extI gene in Geobacter sulfurreducens encodes a putative outer membrane channel porin, which resides within a cluster of extHIJKLMNOPQS genes. This cluster is highly conserved across the Geobacteraceae and includes multiple putative c-type cytochromes. In silico analyses of the ExtI sequence, together with Western blot analysis and proteinase protection assays, showed that it is an outer membrane protein. The expression level of ExtI did not respond to changes in osmolality and phosphate starvation. An extI-deficient mutant did not show any significant impact on fumarate or Fe(III) citrate reduction or sensitivity to ß-lactam antibiotics, as compared with those of the wild-type strain. However, extI deficiency resulted in a decreased ability to reduce selenite and tellurite. Heme staining analysis revealed that extI deficiency affects certain heme-containing proteins in the outer and inner membranes, which may cause a decrease in the ability to reduce selenite and tellurite. Based on these observations, we discuss possible roles for ExtI in selenite and tellurite reduction in G. sulfurreducens.

Selenophosphate synthetase 1 is an essential protein with roles in regulation of redox homoeostasis in mammals.

Selenophosphate synthetase (SPS) was initially detected in bacteria and was shown to synthesize selenophosphate, the active selenium donor. However, mammals have two SPS paralogues, which are designated SPS1 and SPS2. Although it is known that SPS2 catalyses the synthesis of selenophosphate, the function of SPS1 remains largely unclear. To examine the role of SPS1 in mammals, we generated a Sps1-knockout mouse and found that systemic SPS1 deficiency led to embryos that were clearly underdeveloped by embryonic day (E)8.5 and virtually resorbed by E14.5. The knockout of Sps1 in the liver preserved viability, but significantly affected the expression of a large number of mRNAs involved in cancer, embryonic development and the glutathione system. Particularly notable was the extreme deficiency of glutaredoxin 1 (GLRX1) and glutathione transferase Omega 1 (GSTO1). To assess these phenotypes at the cellular level, we targeted the removal of SPS1 in F9 cells, a mouse embryonal carcinoma (EC) cell line, which affected the glutathione system proteins and accordingly led to the accumulation of hydrogen peroxide in the cell. Furthermore, we found that several malignant characteristics of SPS1-deficient F9 cells were reversed, suggesting that SPS1 played a role in supporting and/or sustaining cancer. In addition, the overexpression of mouse or human GLRX1 led to a reversal of observed increases in reactive oxygen species (ROS) in the F9 SPS1/GLRX1-deficient cells and resulted in levels that were similar to those in F9 SPS1-sufficient cells. The results suggested that SPS1 is an essential mammalian enzyme with roles in regulating redox homoeostasis and controlling cell growth.

A non-radioactive assay for selenophosphate synthetase activity using recombinant pyruvate pyrophosphate dikinase from Thermus thermophilus HB8.

Biosynthesis of selenocysteine-containing proteins requires monoselenophosphate, a selenium-donor intermediate generated by selenophosphate synthetase (Sephs). A non-radioactive assay was developed as an alternative to the standard [8-(14)C] AMP-quantifying assay. The product, AMP, was measured using a recombinant pyruvate pyrophosphate dikinase from Thermus thermophilus HB8. The KM and kcat for Sephs2-Sec60Cys were determined to be 26 µM and 0.352 min(-1), respectively.

Deficiency of the 15-kDa selenoprotein led to cytoskeleton remodeling and non-apoptotic membrane blebbing through a RhoA/ROCK pathway.

The 15-kDa selenoprotein (Sep15) has been implicated in etiology of some types of cancer. Herein, inducible RNAi cell lines were established and cell morphology and motility were analyzed. The majority of Sep15-deficient cells (>95%) formed membrane blebs in a dynamic manner. Blebbing cells transformed cell morphology from a normal flat spindle shape to a spherical morphology. In blebbing cells, actin fibers moved to the cell periphery, covering and obscuring visualization of α-tubulin. Bleb formation was suppressed by the inhibitors of Rho-associated protein kinase (ROCK), RhoA or myosin light chain (MLC), restoring blebbing cells to wild-type morphology. RhoA activation and phosphorylation of myosin phosphatase target subunit 1 was induced by Sep15 knockdown. Sep15-deficient cells were non-apoptotic, and displayed a distinct relative localization of F-actin and α-tubulin from typical apoptotic blebbing cells. Our data suggest that Sep15 in Chang liver cells regulates the pathway that antagonizes RhoA/ROCK/MLC-dependent non-apoptotic bleb formation.

High error rates in selenocysteine insertion in mammalian cells treated with the antibiotic doxycycline, chloramphenicol, or geneticin.

Antibiotics target bacteria by interfering with essential processes such as translation, but their effects on translation in mammalian cells are less well characterized. We found that doxycycline, chloramphenicol, and Geneticin (G418) interfered with insertion of selenocysteine (Sec), which is encoded by the stop codon, UGA, into selenoproteins in murine EMT6 cells. Treatment of EMT6 cells with these antibiotics reduced enzymatic activities and Sec insertion into thioredoxin reductase 1 (TR1) and glutathione peroxidase 1 (GPx1). However, these proteins were differentially affected due to varying errors in Sec insertion at UGA. In the presence of doxycycline, chloramphenicol, or G418, the Sec-containing form of TR1 decreased, whereas the arginine-containing and truncated forms of this protein increased. We also detected antibiotic-specific misinsertion of cysteine and tryptophan. Furthermore, misinsertion of arginine in place of Sec was commonly observed in GPx1 and glutathione peroxidase 4. TR1 was the most affected and GPx1 was the least affected by these translation errors. These observations were consistent with the differential use of two Sec tRNA isoforms and their distinct roles in supporting accuracy of Sec insertion into selenoproteins. The data reveal widespread errors in inserting Sec into proteins and in dysregulation of selenoprotein expression and function upon antibiotic treatment.

Mammalian Trit1 is a tRNA([Ser]Sec)-isopentenyl transferase required for full selenoprotein expression.

Selenoproteins are proteins carrying the rare amino acid Sec (selenocysteine). Full expression of selenoproteins requires modification of tRNA([Ser]Sec), including N(6)-isopentenylation of base A(37). We show that Trit1 is a dimethylallyl:tRNA([Ser]Sec) transferase. Knockdown of Trit1 reduces expression of selenoproteins. Incubation of in vitro transcribed tRNA[Ser]Sec with recombinant Trit1 transfers [(14)C]dimethylallyl pyrophosphate to tRNA([Ser]Sec). 37A>G tRNA([Ser]Sec) is resistant to isopentenylation by Trit1.

Identification of Genes Associated with Resistance to Persulcatusin, a Tick Defensin from Ixodes persulcatus.

Antimicrobial peptides (AMPs) are present in a wide range of plants, animals, and microorganisms. Since AMPs are characterized by their effectiveness against emergent antibiotic-resistant bacteria, they are attracting attention as next-generation antimicrobial compounds that could solve the problem of drug-resistant bacteria. Persulcatusin (IP), an antibacterial peptide derived from the hard tick Ixodes persulcatus, shows high antibacterial activity against various Gram- positive bacteria as well as multidrug-resistant bacteria. However, reports on the antibacterial action and resistance mechanisms of IP are scarce. In this study, we spontaneously generated mutants showing increased a minimum inhibitory concentration (MIC) of IP and analyzed their cross-resistance to other AMPs and antibiotics. We also used fluorescent probes to investigate the target of IP activity by evaluating IP-induced damage to the bacterial cytoplasmic membrane. Our findings suggest that the antimicrobial activity of IP on bacterial cytoplasmic membranes occurs via a mechanism of action different from that of known AMPs. Furthermore, we screened for mutants with high susceptibility to IP using a transposon mutant library and identified 16 genes involved in IP resistance. Our results indicate that IP, like other AMPs, depolarizes the bacterial cytoplasmic membrane, but it may also alter membrane structure and inhibit cell-wall synthesis.

Thioredoxin reductase 1 deficiency enhances selenite toxicity in cancer cells via a thioredoxin-independent mechanism.

Selenium is an essential trace element in mammals, but is toxic at high levels. It is best known for its cancer prevention activity, but cancer cells are more sensitive to selenite toxicity than normal cells. Since selenite treatment leads to oxidative stress, and the Trx (thioredoxin) system is a major antioxidative system, we examined the interplay between TR1 (Trx reductase 1) and Trx1 deficiencies and selenite toxicity in DT cells, a malignant mouse cell line, and the corresponding parental NIH 3T3 cells. TR1-deficient cells were far more sensitive to selenite toxicity than Trx1-deficient or control cells. In contrast, this effect was not seen in cells treated with hydrogen peroxide, suggesting that the increased sensitivity of TR1 deficiency to selenite was not due to oxidative stress caused by this compound. Further analyses revealed that only TR1-deficient cells manifested strongly enhanced production and secretion of glutathione, which was associated with increased sensitivity of the cells to selenite. The results suggest a new role for TR1 in cancer that is independent of Trx reduction and compensated for by the glutathione system. The results also suggest that the enhanced selenite toxicity of cancer cells and simultaneous inhibition of TR1 can provide a new avenue for cancer therapy.

Thioredoxin reductase 1 protects against chemically induced hepatocarcinogenesis via control of cellular redox homeostasis.

Thioredoxin reductase 1 (TR1) controls the redox state of protein thiols in mammalian cells and has been shown to have roles in both preventing and promoting cancer. To define the role of this selenoenzyme in hepatocellular carcinoma development, we examined tumor incidence in the liver of mice with tissue-specific knockout of mouse TR1 subjected to the liver carcinogen, diethylnitrosamine (DEN). TR1-deficient livers manifested ~90% tumor incidence compared with ~16% in control livers. The TR1-dependent effect was observed independent of sex, and, in control mice, tumorigenesis did not affect the expression of TR1. On the other hand, we observed upregulation of another selenoenzyme, glutathione peroxidase 2 (GPx2), and components of the glutathione (GSH) system, including those that generate reduced GSH. Overall, this study shows that TR1 protects against chemically induced hepatocarcinogenesis via the control of the cellular redox state, whereas its role in promoting this type of cancer is minimal.

Characterization of Staphylococcus aureus Isolates from Bovine Mastitis and Bulk Tank Milk: First Isolation of Methicillin-Susceptible Staphylococcus aureus in Japan.

Staphylococcus aureus is one of the most important pathogens in humans as well as in livestock. Particularly, bovine mastitis caused by S. aureus is a serious issue in dairy farms due to disease recurrence. Here, cases of S. aureus-mediated intramammary infection occurring in the Miyagi Prefecture in Japan were monitored from May 2015 to August 2019; a total of 59 strains (49 from bovine milk and 10 from bulk milk) were obtained from 15 dairy farms and analyzed via sequence-based typing methods and antibiotic susceptibility tests. Two pairs of isolates were determined as recurrence cases from the same cows in distinct farms. The sequence type (ST), spa type, and coa type of each pair were the same: one pair showed ST705, t529, and VIb and the other showed ST352, t267, and VIc. In addition, the possession of toxin genes analyzed of each pair was exactly the same. Furthermore, seven oxacillin-sensitive clonal complex 398 isolates were obtained from a single farm. This is the first confirmed case of a Methicillin-Sensitive SA (MSSA) ST398 strain isolated from mastitis-containing cows in Japan. Our findings suggest that nationwide surveillance of the distribution of ST398 strains in dairy farms is important for managing human and animal health.

Group II truncated haemoglobin YjbI prevents reactive oxygen species-induced protein aggregation in Bacillus subtilis.

Oxidative stress-mediated formation of protein hydroperoxides can induce irreversible fragmentation of the peptide backbone and accumulation of cross-linked protein aggregates, leading to cellular toxicity, dysfunction, and death. However, how bacteria protect themselves from damages caused by protein hydroperoxidation is unknown. Here, we show that YjbI, a group II truncated haemoglobin from Bacillus subtilis, prevents oxidative aggregation of cell-surface proteins by its protein hydroperoxide peroxidase-like activity, which removes hydroperoxide groups from oxidised proteins. Disruption of the yjbI gene in B. subtilis lowered biofilm water repellence, which associated with the cross-linked aggregation of the biofilm matrix protein TasA. YjbI was localised to the cell surface or the biofilm matrix, and the sensitivity of planktonically grown cells to generators of reactive oxygen species was significantly increased upon yjbI disruption, suggesting that YjbI pleiotropically protects labile cell-surface proteins from oxidative damage. YjbI removed hydroperoxide residues from the model oxidised protein substrate bovine serum albumin and biofilm component TasA, preventing oxidative aggregation in vitro. Furthermore, the replacement of Tyr63 near the haem of YjbI with phenylalanine resulted in the loss of its protein peroxidase-like activity, and the mutant gene failed to rescue biofilm water repellency and resistance to oxidative stress induced by hypochlorous acid in the yjbI-deficient strain. These findings provide new insights into the role of truncated haemoglobin and the importance of hydroperoxide removal from proteins in the survival of aerobic bacteria.

Inducible L-alanine exporter encoded by the novel gene ygaW (alaE) in Escherichia coli.

We previously isolated a mutant hypersensitive to L-alanyl-L-alanine from a non-L-alanine-metabolizing Escherichia coli strain and found that it lacked an inducible l-alanine export system. Consequently, this mutant showed a significant accumulation of intracellular L-alanine and a reduction in the L-alanine export rate compared to the parent strain. When the mutant was used as a host to clone a gene(s) that complements the dipeptide-hypersensitive phenotype, two uncharacterized genes, ygaW and ytfF, and two characterized genes, yddG and yeaS, were identified. Overexpression of each gene in the mutant resulted in a decrease in the intracellular l-alanine level and enhancement of the L-alanine export rate in the presence of the dipeptide, suggesting that their products function as exporters of L-alanine. Since ygaW exhibited the most striking impact on both the intra- and the extracellular L-alanine levels among the four genes identified, we disrupted the ygaW gene in the non-L-alanine-metabolizing strain. The resulting isogenic mutant showed the same intra- and extracellular L-alanine levels as observed in the dipeptide-hypersensitive mutant obtained by chemical mutagenesis. When each gene was overexpressed in the wild-type strain, which does not intrinsically excrete alanine, only the ygaW gene conferred on the cells the ability to excrete alanine. In addition, expression of the ygaW gene was induced in the presence of the dipeptide. On the basis of these results, we concluded that YgaW is likely to be the physiologically most relevant exporter for L-alanine in E. coli and proposed that the gene be redesignated alaE for alanine export.

Complete Genome Sequence of Pseudomonas stutzeri Strain F2a, Isolated from Seleniferous Soil.

Pseudomonas stutzeri is a potential candidate for bioremediation of selenium-contaminated grounds and waters. Here, we report the complete genome sequence of a novel strain, F2a, which was isolated from a seleniferous area of Punjab, India. The genome sequence provides insight into the potential selenium oxyanion-reducing activity of this strain.

Genetic analysis of tellurate reduction reveals the selenate/tellurate reductase genes ynfEF and the transcriptional regulation of moeA by NsrR in Escherichia coli.

Several bacteria can reduce tellurate into the less toxic elemental tellurium, but the genes responsible for this process have not yet been identified. In this study, we screened the Keio collection of single-gene knockouts of Escherichia coli responsible for decreased tellurate reduction and found that deletions of 29 genes, including those for molybdenum cofactor (Moco) biosynthesis, iron-sulphur biosynthesis, and the twin-arginine translocation pathway resulted in decreased tellurate reduction. Among the gene knockouts, deletions of nsrR, moeA, yjbB, ynbA, ydaS and yidH affected tellurate reduction more severely than those of other genes. Based on our findings, we determined that the ynfEF genes, which code for the components of the selenate reductase YnfEFGH, are responsible for tellurate reduction. Assays of several molybdoenzymes in the knockouts suggested that nsrR, yjbB, ynbA, ydaS and yidH are essential for the activities of molybdoenzymes in E. coli. Furthermore, we found that the nitric oxide sensor NsrR positively regulated the transcription of the Moco biosynthesis gene moeA. These findings provided new insights into the complexity and regulation of Moco biosynthesis in E. coli.

Complete Genome Sequence of an Acetic Acid Bacterium, Acetobacter aceti JCM20276.

Acetobacter aceti is used in industry to produce vinegar by converting ethanol into acetic acid. We determined the complete genome sequence of A aceti JCM20276, which is composed of one chromosome and four plasmids. This study may contribute to a better understanding of the genes necessary for acetic acid production.