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52700.9489Characterization of the bagremycin biosynthetic gene cluster in Streptomyces sp. Tü 4128. Bagremycin A and bagremycin B isolated from Streptomyces sp. Tü 4128 have activities against Gram-positive bacteria, fungi and also have a weak antitumor activity, which make them have great potential for development of novel antibiotics. Here, we report a draft genome 8,424,112 bp in length of S. sp. Tü 4128 by Illumina Hiseq2000, and identify the bagremycins biosynthetic gene cluster (BGC) by bioinformatics analysis. The putative bagremycins BGC includes 16 open reading frames (ORFs) with the functions of biosynthesis, resistance and regulation. Disruptions of relative genes and HPLC analysis of bagremycins production demonstrated that not all the genes within the BGC are responsible for the biosynthesis of bagremycins. In addition, the biosynthetic pathways of bagremycins are proposed for deeper inquiries into their intriguing biosynthetic mechanism.201930526412
37610.9481Construction of a reporter plasmid for screening in vivo promoter activity in Francisella tularensis. Francisella tularensis is a facultative intracellular bacterium that survives and multiplies inside macrophages. Here we constructed a new promoter probe plasmid denoted pKK214 by introduction of a promoter-less chloramphenicol acetyltransferase (cat) gene into the shuttle vector pKK202. A promoter library was created in F. tularensis strain LVS by cloning random chromosomal DNA fragments into pKK214. Approximately 15% of the recombinant bacteria showed chloramphenicol resistance in vitro. The promoter library was also used to infect macrophages in the presence of chloramphenicol and after two cycles of infection the library contained essentially only chloramphenicol resistance clones which shows that pKK214 can be used to monitor F. tularensis genes that are expressed during infection.200111728719
53420.9461Plasmid shuttle vector with two insertionally inactivable markers for coryneform bacteria. A new shuttle vector pCEM500 replicating in Escherichia coli and in Brevibacterium flavum was constructed. It carries two antibiotic resistance determinants (Kmr/Gmr from plasmid pSa of Gram-negative bacteria and Smr/Spr from plasmid pCG4 of Corynebacterium glutamicum) which are efficiently expressed in both hosts and can be inactivated by insertion of DNA fragments into the unique restriction endonuclease sites located within them. This vector was found to be stably maintained in B. flavum and can be used for transfer of the cloned genes into this amino-acid-producing coryneform bacterium.19902148164
37130.9457Single amino acid substitutions in the enzyme acetolactate synthase confer resistance to the herbicide sulfometuron methyl. Sulfometuron methyl, a sulfonylurea herbicide, blocks growth of bacteria, yeast, and higher plants by inhibition of acetolactate synthase (EC 4.1.3.18), the first common enzyme in the biosynthesis of branched-chain amino acids. Spontaneous mutations that confer increased resistance to the herbicide were obtained in cloned genes for acetolactate synthase from Escherichia coli and Saccharomyces cerevisiae. The DNA sequence of a bacterial mutant gene and a yeast mutant gene revealed single nucleotide differences from their respective wild-type genes. The mutations result in single amino acid substitutions in the structurally homologous aminoterminal regions of the two proteins, but at different positions. The bacterial mutation results in reduced levels of acetolactate synthase activity, reduced sensitivity to sulfometuron methyl, and unaltered resistance to feedback inhibition by valine. The yeast mutation results in unaltered levels of acetolactate synthase activity, greatly reduced sensitivity to sulfometuron methyl, and slightly reduced sensitivity to valine.198616593715
904240.9455Resistance of Francisella novicida to fosmidomycin associated with mutations in the glycerol-3-phosphate transporter. The methylerythritol phosphate (MEP) pathway is essential in most prokaryotes and some lower eukaryotes but absent from human cells, and is a validated target for antimicrobial drug development. The formation of MEP is catalyzed by 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR). MEP pathway genes have been identified in many category A and B biothreat agents, including Francisella tularensis, which causes the zoonosis tularemia. Fosmidomycin (Fos) inhibits purified Francisella DXR. This compound also inhibits the growth of F. tularensis NIH B38, F. novicida and F. tularensis subsp. holarctica LVS bacteria. Related compounds such as FR900098 and the lipophilic prodrug of FR900098 (compound 1) have been developed to improve the bioavailability of these DXR inhibitors. In performing disk-inhibition assays with these compounds, we observed breakthrough colonies of F. novicida in the presence of Fos, suggesting spontaneous development of Fos resistance (Fos(R)). Fos(R) bacteria had decreased sensitivity to both Fos and FR900098. The two most likely targets for the development of mutants would be the DXR enzyme itself or the glycerol-3-phosphate transporter (GlpT) that allows entry of Fos into the bacteria. Sensitivity of Fos(R)F. novicida bacteria to compound 1 was not abated suggesting that spontaneous resistance is not due to mutation of DXR. We thus predicted that the glpT transporter may be mutated leading to this resistant phenotype. Supporting this, transposon insertion mutants at the glpT locus were also found to be resistant to Fos. DNA sequencing of four different spontaneous Fos(R) colonies demonstrated a variety of deletions in the glpT coding region. The overall frequency of Fos(R) mutations in F. novicida was determined to be 6.3 × 10(-8). Thus we conclude that one mechanism of resistance of F. novicida to Fos is caused by mutations in GlpT. This is the first description of spontaneous mutations in Francisella leading to Fos(R).201222905031
53250.9449Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Disruption-deletion cassettes are powerful tools used to study gene function in many organisms, including Saccharomyces cerevisiae. Perhaps the most widely useful of these are the heterologous dominant drug resistance cassettes, which use antibiotic resistance genes from bacteria and fungi as selectable markers. We have created three new dominant drug resistance cassettes by replacing the kanamycin resistance (kan(r)) open reading frame from the kanMX3 and kanMX4 disruption-deletion cassettes (Wach et al., 1994) with open reading frames conferring resistance to the antibiotics hygromycin B (hph), nourseothricin (nat) and bialaphos (pat). The new cassettes, pAG25 (natMX4), pAG29 (patMX4), pAG31 (patMX3), pAG32 (hphMX4), pAG34 (hphMX3) and pAG35 (natMX3), are cloned into pFA6, and so are in all other respects identical to pFA6-kanMX3 and pFA6-kanMX4. Most tools and techniques used with the kanMX plasmids can also be used with the hph, nat and patMX containing plasmids. These new heterologous dominant drug resistance cassettes have unique antibiotic resistance phenotypes and do not affect growth when inserted into the ho locus. These attributes make the cassettes ideally suited for creating S. cerevisiae strains with multiple mutations within a single strain.199910514571
33160.9449MmpS4 promotes glycopeptidolipids biosynthesis and export in Mycobacterium smegmatis. The MmpS family (mycobacterial membrane protein small) includes over 100 small membrane proteins specific to the genus Mycobacterium that have not yet been studied experimentally. The genes encoding MmpS proteins are often associated with mmpL genes, which are homologous to the RND (resistance nodulation cell division) genes of Gram-negative bacteria that encode proteins functioning as multidrug efflux system. We showed by molecular genetics and biochemical analysis that MmpS4 in Mycobacterium smegmatis is required for the production and export of large amounts of cell surface glycolipids, but is dispensable for biosynthesis per se. A new specific and sensitive method utilizing single-chain antibodies against the surface-exposed glycolipids was developed to confirm that MmpS4 was dispensable for transport to the surface. Orthologous complementation demonstrated that the MmpS4 proteins are exchangeable, thus not specific to a defined lipid species. MmpS4 function requires the formation of a protein complex at the pole of the bacillus, which requires the extracytosolic C-terminal domain of MmpS4. We suggest that MmpS proteins facilitate lipid biosynthesis by acting as a scaffold for coupled biosynthesis and transport machinery.201021062372
80370.9449Nucleotide sequences and genetic analysis of hydrogen oxidation (hox) genes in Azotobacter vinelandii. Azotobacter vinelandii contains a heterodimeric, membrane-bound [NiFe]hydrogenase capable of catalyzing the reversible oxidation of H2. The beta and alpha subunits of the enzyme are encoded by the structural genes hoxK and hoxG, respectively, which appear to form part of an operon that contains at least one further potential gene (open reading frame 3 [ORF3]). In this study, determination of the nucleotide sequence of a region of 2,344 bp downstream of ORF3 revealed four additional closely spaced or overlapping ORFs. These ORFs, ORF4 through ORF7, potentially encode polypeptides with predicted masses of 22.8, 11.4, 16.3, and 31 kDa, respectively. Mutagenesis of the chromosome of A. vinelandii in the area sequenced was carried out by introduction of antibiotic resistance gene cassettes. Disruption of hoxK and hoxG by a kanamycin resistance gene abolished whole-cell hydrogenase activity coupled to O2 and led to loss of the hydrogenase alpha subunit. Insertional mutagenesis of ORF3 through ORF7 with a promoterless lacZ-Kmr cassette established that the region is transcriptionally active and involved in H2 oxidation. We propose to call ORF3 through ORF7 hoxZ, hoxM, hoxL, hoxO, and hoxQ, respectively. The predicted hox gene products resemble those encoded by genes from hydrogenase-related operons in other bacteria, including Escherichia coli and Alcaligenes eutrophus.19921624446
37580.9441A mariner transposon vector adapted for mutagenesis in oral streptococci. This article describes the construction and characterization of a mariner-based transposon vector designed for use in oral streptococci, but with a potential use in other Gram-positive bacteria. The new transposon vector, termed pMN100, contains the temperature-sensitive origin of replication repATs-pWV01, a selectable kanamycin resistance gene, a Himar1 transposase gene regulated by a xylose-inducible promoter, and an erythromycin resistance gene flanked by himar inverted repeats. The pMN100 plasmid was transformed into Streptococcus mutans UA159 and transposon mutagenesis was performed via a protocol established to perform high numbers of separate transpositions despite a low frequency of transposition. The distribution of transposon inserts in 30 randomly picked mutants suggested that mariner transposon mutagenesis is unbiased in S. mutans. A generated transposon mutant library containing 5000 mutants was used in a screen to identify genes involved in the production of sucrose-dependent extracellular matrix components. Mutants with transposon inserts in genes encoding glycosyltransferases and the competence-related secretory locus were predominantly found in this screen.201424753509
53090.9441Location of the genes for anthranilate synthase in Streptomyces venezuelae ISP5230: genetic mapping after integration of the cloned genes. The anthranilate synthase (trpEG) genes in Streptomyces venezuelae ISP5230 were located by allowing a segregationally unstable plasmid carrying cloned S. venezuelae trpEG DNA and a thiostrepton resistance (tsr) marker to integrate into the chromosome. The integrated tsr was mapped by conjugation and transduction to a location close to tyr-2, between arg-6 and trpA13. A genomic DNA fragment containing trpC from S. venezuelae ISP5230 was cloned by complementation of a trpC mutation in Streptomyces lividans. Evidence from restriction enzyme analysis of the cloned DNA fragments, from Southern hybridization using the cloned trp DNA as probes, and from cotransduction frequencies, placed trpEG at a distance of 12-45 kb from the trpCBA cluster. The overall arrangement of tryptophan biosynthesis genes in the S. venezuelae chromosome differs from that in other bacteria examined so far.19938515229
531100.9440p-Aminobenzoic acid and chloramphenicol biosynthesis in Streptomyces venezuelae: gene sets for a key enzyme, 4-amino-4-deoxychorismate synthase. Amplification of sequences from Streptomyces venezuelae ISP5230 genomic DNA using PCR with primers based on conserved prokaryotic pabB sequences gave two main products. One matched pabAB, a locus previously identified in S. venezuelae. The second closely resembled the conserved pabB sequence consensus and hybridized with a 3.8 kb NcoI fragment of S. venezuelae ISP5230 genomic DNA. Cloning and sequence analysis of the 3.8 kb fragment detected three ORFs, and their deduced amino acid sequences were used in BLAST searches of the GenBank database. The ORF1 product was similar to PabB in other bacteria and to the PabB domain encoded by S. venezuelae pabAB. The ORF2 product resembled PabA of other bacteria. ORF3 was incomplete; its deduced partial amino acid sequence placed it in the MocR group of GntR-type transcriptional regulators. Introducing vectors containing the 3.8 kb NcoI fragment of S. venezuelae DNA into pabA and pabB mutants of Escherichia coli, or into the Streptomyces lividans pab mutant JG10, enhanced sulfanilamide resistance in the host strains. The increased resistance was attributed to expression of the pair of discrete translationally coupled p-aminobenzoic acid biosynthesis genes (designated pabB/pabA) cloned in the 3.8 kb fragment. These represent a second set of genes encoding 4-amino-4-deoxychorismate synthase in S. venezuelae ISP5230. In contrast to the fused pabAB set previously isolated from this species, they do not participate in chloramphenicol biosynthesis, but like pabAB they can be disrupted without affecting growth on minimal medium. The gene disruption results suggest that S. venezuelae may have a third set of genes encoding PABA synthase.200111495989
349110.9440Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. A collection of Tn5-derived minitransposons has been constructed that simplifies substantially the generation of insertion mutants, in vivo fusions with reporter genes, and the introduction of foreign DNA fragments into the chromosome of a variety of gram-negative bacteria, including the enteric bacteria and typical soil bacteria like Pseudomonas species. The minitransposons consist of genes specifying resistance to kanamycin, chloramphenicol, streptomycin-spectinomycin, and tetracycline as selection markers and a unique NotI cloning site flanked by 19-base-pair terminal repeat sequences of Tn5. Further derivatives also contain lacZ, phoA, luxAB, or xylE genes devoid of their native promoters located next to the terminal repeats in an orientation that affords the generation of gene-operon fusions. The transposons are located on a R6K-based suicide delivery plasmid that provides the IS50R transposase tnp gene in cis but external to the mobile element and whose conjugal transfer to recipients is mediated by RP4 mobilization functions in the donor.19902172217
3766120.9438Identification of Genome-Wide Mutations in Ciprofloxacin-Resistant F. tularensis LVS Using Whole Genome Tiling Arrays and Next Generation Sequencing. Francisella tularensis is classified as a Class A bioterrorism agent by the U.S. government due to its high virulence and the ease with which it can be spread as an aerosol. It is a facultative intracellular pathogen and the causative agent of tularemia. Ciprofloxacin (Cipro) is a broad spectrum antibiotic effective against Gram-positive and Gram-negative bacteria. Increased Cipro resistance in pathogenic microbes is of serious concern when considering options for medical treatment of bacterial infections. Identification of genes and loci that are associated with Ciprofloxacin resistance will help advance the understanding of resistance mechanisms and may, in the future, provide better treatment options for patients. It may also provide information for development of assays that can rapidly identify Cipro-resistant isolates of this pathogen. In this study, we selected a large number of F. tularensis live vaccine strain (LVS) isolates that survived in progressively higher Ciprofloxacin concentrations, screened the isolates using a whole genome F. tularensis LVS tiling microarray and Illumina sequencing, and identified both known and novel mutations associated with resistance. Genes containing mutations encode DNA gyrase subunit A, a hypothetical protein, an asparagine synthase, a sugar transamine/perosamine synthetase and others. Structural modeling performed on these proteins provides insights into the potential function of these proteins and how they might contribute to Cipro resistance mechanisms.201627668749
609130.9437A metazoan ortholog of SpoT hydrolyzes ppGpp and functions in starvation responses. In nutrient-starved bacteria, RelA and SpoT proteins have key roles in reducing cell growth and overcoming stresses. Here we identify functional SpoT orthologs in metazoa (named Mesh1, encoded by HDDC3 in human and Q9VAM9 in Drosophila melanogaster) and reveal their structures and functions. Like the bacterial enzyme, Mesh1 proteins contain an active site for ppGpp hydrolysis and a conserved His-Asp-box motif for Mn(2+) binding. Consistent with these structural data, Mesh1 efficiently catalyzes hydrolysis of guanosine 3',5'-diphosphate (ppGpp) both in vitro and in vivo. Mesh1 also suppresses SpoT-deficient lethality and RelA-induced delayed cell growth in bacteria. Notably, deletion of Mesh1 (Q9VAM9) in Drosophila induces retarded body growth and impaired starvation resistance. Microarray analyses reveal that the amino acid-starved Mesh1 null mutant has highly downregulated DNA and protein synthesis-related genes and upregulated stress-responsible genes. These data suggest that metazoan SpoT orthologs have an evolutionarily conserved function in starvation responses.201020818390
533140.9437Construction of broad-host-range cosmid cloning vectors: identification of genes necessary for growth of Methylobacterium organophilum on methanol. Four new cloning vectors have been constructed from the broad-host-range cloning vector pRK290. These vectors, pLA2901, pLA2905, pLA2910, and pLA2917, confer resistance to kanamycin and tetracycline. The latter two are cosmid derivatives of pLA2901. The new vectors can be mobilized into, and are stably maintained in, a variety of gram-negative bacteria. A Sau3A genomic bank of Methylobacterium organophilum strain xx DNA has been constructed in pLA2917, and complementation analysis, with a variety of mutants unable to grow on methanol, revealed at least five separate regions necessary for growth on methanol. Complementation analysis and Tn5 mutagenesis data suggest that at least three genes are responsible for expression of active methanol dehydrogenase.19852982796
204150.9437RNA modification enzymes encoded by the gid operon: Implications in biology and virulence of bacteria. Ribonucleic acid (RNA) molecules consist of numerous chemically modified nucleosides that are highly conserved in eukarya, archeae, and bacteria, while others are unique to each domain of life. In bacteria, hundreds of RNA modification enzymes have been identified and implicated in biological pathways associated with many cell processes. The glucose-inhibited division (gid) operon encodes genes for two RNA modification enzymes named GidA and GidB. Studies have shown GidA is essential for the proper biosynthesis of 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U) of bacterial transfer RNA (tRNA) with GidB responsible for the methylation of the 16S ribosomal RNA (rRNA). Furthermore, deletion of gidA and gidB has shown to alter numerous bacterial properties like virulence, stress response, morphology, growth, antibiotic susceptibility, and others. In this review, we discuss the present knowledge of the RNA modification enzymes GidA and GidB, and their potential role in the biology and virulence of bacteria.201526427881
344160.9436Identification of genes in Rhizobium leguminosarum bv. trifolii whose products are homologues to a family of ATP-binding proteins. The specific interaction between rhizobia and their hosts requires many genes that influence both early and late steps in symbiosis. Three new genes, designated prsD, prsE (protein secretion) and orf3, were identified adjacent to the exo133 mutation in a cosmid carrying the genomic DNA of Rhizobium leguminosarum bv. trifolii TA1. The prsDE genes share significant homology to the genes encoding ABC transporter proteins PrtDE from Erwinia chrysanthemi and AprDE from Pseudomonas aeruginosa which export the proteases in these bacteria. PrsD shows at least five potential transmembrane hydrophobic regions and a large hydrophilic domain containing an ATP/GTP binding cassette. PrsE has only one potential transmembrane hydrophobic domain in the N-terminal part and is proposed to function as an accessory factor in the transport system. ORF3, like PrtF and AprF, has a typical N-terminal signal sequence but has no homology to these proteins. The insertion of a kanamycin resistance cassette into the prsD gene of the R. leguminosarum bv. trifolii TA1 wild-type strain created a mutant which produced a normal amount of exopolysaccharide but was not effective in the nodulation of clover plants.19979141701
369170.9436A gene fusion system using the aminoglycoside 3'-phosphotransferase gene of the kanamycin-resistance transposon Tn903: use in the yeast Kluyveromyces lactis and Saccharomyces cerevisiae. The aminoglycoside 3'-phosphotransferase type I (APHI)-coding gene of the bacterial transposon Tn903 confers resistance to kanamycin on bacteria and resistance to geneticin (G418) on many eukaryotes. We developed an APHI fusion system that can be used in the study of gene expression in these organisms, particularly in yeasts. The first 19 codons of the KmR (APHI) gene can be deleted, and replaced by other genes in a continuous reading frame, without loss of APH activity. Examples of vector constructions are given which are adapted to the yeast Kluyveromyces lactis transformation system. Their derivatives containing the 2 mu origin of replication can also be used in Saccharomyces cerevisiae.19882853096
529180.9436Crystal structure of the transcriptional repressor PagR of Bacillus anthracis. PagR is a transcriptional repressor in Bacillus anthracis that controls the chromosomal S-layer genes eag and sap, and downregulates the protective antigen pagA gene by direct binding to their promoter regions. The PagR protein sequence is similar to those of members of the ArsR repressor family involved in the repression of arsenate-resistance genes in numerous bacteria. The crystal structure of PagR was solved using multi-wavelength anomalous diffraction (MAD) techniques and was refined with 1.8 A resolution diffraction data. The PagR molecules form dimers, as observed in all SmtB/ArsR repressor family proteins. In the crystal lattice four PagR dimers pack together to form an inactive octamer. Model-building studies suggest that the dimer binds to a DNA duplex with a bend of around 4 degrees.201019926656
7190.9435An EDS1 heterodimer signalling surface enforces timely reprogramming of immunity genes in Arabidopsis. Plant intracellular NLR receptors recognise pathogen interference to trigger immunity but how NLRs signal is not known. Enhanced disease susceptibility1 (EDS1) heterodimers are recruited by Toll-interleukin1-receptor domain NLRs (TNLs) to transcriptionally mobilise resistance pathways. By interrogating the Arabidopsis EDS1 ɑ-helical EP-domain we identify positively charged residues lining a cavity that are essential for TNL immunity signalling, beyond heterodimer formation. Mutating a single, conserved surface arginine (R493) disables TNL immunity to an oomycete pathogen and to bacteria producing the virulence factor, coronatine. Plants expressing a weakly active EDS1(R493A) variant have delayed transcriptional reprogramming, with severe consequences for resistance and countering bacterial coronatine repression of early immunity genes. The same EP-domain surface is utilised by a non-TNL receptor RPS2 for bacterial immunity, indicating that the EDS1 EP-domain signals in resistance conferred by different NLR receptor types. These data provide a unique structural insight to early downstream signalling in NLR receptor immunity.201930770836