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Biomedical subjects

R H Baltz

Publications and source records attributed to R H Baltz.

At least 19 recordsLinked to original sources
PubMed2001

Cloning and analysis of the spinosad biosynthetic gene cluster of Saccharopolyspora spinosa.

BACKGROUND: Spinosad is a mixture of novel macrolide secondary metabolites produced by Saccharopolyspora spinosa. It is used in agriculture as a potent insect control agent with exceptional safety to non-target organisms. The cloning of the spinosyn biosynthetic gene cluster provides the starting materials for the molecular genetic manipulation of spinosad yields, and for the production of novel derivatives containing alterations in the polyketide core or in the attached sugars. RESULTS: We cloned the spinosad biosynthetic genes by molecular probing, complementation of blocked mutants, and cosmid walking, and sequenced an 80 kb region. We carried out gene disruptions of some of the genes and analyzed the mutants for product formation and for the bioconversion of intermediates in the spinosyn pathway. The spinosyn gene cluster contains five large open reading frames that encode a multifunctional, multi-subunit type I polyketide synthase (PKS). The PKS cluster is flanked on one side by genes involved in the biosynthesis of the amino sugar forosamine, in O-methylations of rhamnose, in sugar attachment to the polyketide, and in polyketide cross-bridging. Genes involved in the early common steps in the biosynthesis of forosamine and rhamnose, and genes dedicated to rhamnose biosynthesis, were not located in the 80 kb cluster. CONCLUSIONS: Most of the S. spinosa genes involved in spinosyn biosynthesis are found in one 74 kb cluster, though it does not contain all of the genes required for the essential deoxysugars. Characterization of the clustered genes suggests that the spinosyns are synthesized largely by mechanisms similar to those used to assemble complex macrolides in other actinomycetes. However, there are several unusual genes in the spinosyn cluster that could encode enzymes that generate the most striking structural feature of these compounds, a tetracyclic polyketide aglycone nucleus.

Amino Acid Sequence↗
PubMed2001

Genetic methods and strategies for secondary metabolite yield improvement in actinomycetes.

The foundation for any strain improvement program is efficient random chemically-induced mutagenesis coupled with highly reproducible fermentation and product assays. The broad spectrum of spontaneous mutations can be leveraged in some cases by direct selection of mutants with desired traits. Transposons containing outward-reading promoter activity might be used to enhance yields by inducing promoter fusions, disrupting negative regulatory elements, or disrupting genes involved in competing pathways. Transposons might also be used to identify and clone positive regulatory genes. As knowledge of the key elements in the fermentation process and secondary metabolite biosynthesis grows, gene cloning and targeted gene duplication becomes an important tool. Duplication of genes involved in rate limiting steps can be achieved to improve product yields by inserting the desired gene(s) into neutral sites in the chromosome by homologous recombination or by site-specific integration. The probabilities and frequencies of success of the molecular genetic approaches should increase with an increasing knowledge of key factors influencing product yields. This knowledge can be broadened dramatically by a combination of structural and functional genomics, gene disruption analysis and metabolic modeling. Protoplast fusion can be used to recombine beneficial traits from any of the other approaches.

Actinomycetales↗
PubMed2001

Genes for the biosynthesis of spinosyns: applications for yield improvement in Saccharopolyspora spinosa.

Spinosyns A and D are the active ingredients in an insect control agent produced by fermentation of Saccharopolyspora spinosa. Spinosyns are macrolides with a 21-carbon, tetracyclic lactone backbone to which the deoxysugars forosamine and tri-O-methylrhamnose are attached. The spinosyn biosynthesis genes, except for the rhamnose genes, are located in a cluster that spans 74 kb of the S. spinosa genome. DNA sequence analysis, targeted gene disruptions and bioconversion studies identified five large genes encoding type I polyketide synthase subunits, and 14 genes involved in sugar biosynthesis, sugar attachment to the polyketide or cross-bridging of the polyketide. Four rhamnose biosynthetic genes, two of which are also necessary for forosamine biosynthesis, are located outside the spinosyn gene cluster. Duplication of the spinosyn genes linked to the polyketide synthase genes stimulated the final step in the biosynthesis--the conversion of the forosamine-less pseudoaglycones to endproducts. Duplication of genes involved in the early steps of deoxysugar biosynthesis increased spinosyn yield significantly.

Anti-Bacterial Agents↗
PubMed2001

Genome of the bacterium Streptococcus pneumoniae strain R6.

Streptococcus pneumoniae is among the most significant causes of bacterial disease in humans. Here we report the 2,038,615-bp genomic sequence of the gram-positive bacterium S. pneumoniae R6. Because the R6 strain is avirulent and, more importantly, because it is readily transformed with DNA from homologous species and many heterologous species, it is the principal platform for investigation of the biology of this important pathogen. It is also used as a primary vehicle for genomics-based development of antibiotics for gram-positive bacteria. In our analysis of the genome, we identified a large number of new uncharacterized genes predicted to encode proteins that either reside on the surface of the cell or are secreted. Among those proteins there may be new targets for vaccine and antibiotic development.

Bacterial Proteins↗
PubMed2000

A cluster of genes for the biosynthesis of spinosyns, novel macrolide insect control agents produced by Saccharopolyspora spinosa.

Spinosyns A and D are the active ingredients in a family of insect control agents produced by fermentation of Saccharopolyspora spinosa. Spinosyns are 21-carbon tetracyclic lactones to which are attached two deoxysugars. Most of the genes involved in spinosyn biosynthesis are clustered in an 74 kb region of the S. spinosa genome. This region has been characterized by DNA sequence analysis and by targeted gene disruptions. The spinosyn biosynthetic gene cluster contains five large genes encoding a type I polyketide synthase, and 14 genes involved in modification of the macrolactone, or in the synthesis, modification and attachment of the deoxysugars. Four genes required for rhamnose biosynthesis (two of which are also required for forosamine biosynthesis) are not present in the cluster. A pathway for the biosynthesis of spinosyns is proposed.

Animals↗
PubMed1998

Genetic manipulation of antibiotic-producing Streptomyces.

The genetic manipulation of Streptomyces species has been facilitated by the development of versatile cloning vectors, robust gene transfer systems and transposable elements. These molecular genetic tools have been used to construct antibiotic-producing strains with improved properties and recombinants for the production of hybrid glycopeptide and macrolide antibiotics, novel anthelminthic agents and novel cytotoxic anthracyclines.

Anthelmintics↗
PubMed1998

DNA sequence sampling of the Streptococcus pneumoniae genome to identify novel targets for antibiotic development.

We initiated a survey of the Streptococcus pneumoniae genome by DNA sequence sampling. More than 9,500 random DNA sequences of approximately 500 bases average length were determined. Partial sequences sufficient to identify approximately 95% of the aminoacyl tRNA synthetase genes and ribosomal protein (rps) genes were found by comparing the database of partial sequences to known sequences from other organisms. Many genes involved in DNA replication, repair, and mutagenesis are present in S. pneumoniae. Genes for the major subunits of RNA polymerase are also present, as are genes for two alternative sigma factors, rpoD and rpoN. Many genes necessary for amino acid or cofactor biosynthesis and aerobic energy metabolism in other bacteria appear to be absent from the S. pneumoniae genome. A number of genes involved in cell wall biosynthesis and septation were identified, including six homologs to different penicillin binding proteins. Interestingly, four genes involved in the addition of D-alanine to lipoteicoic acid in other gram positive bacteria were found, even though the lipoteicoic acid in S. pneumoniae has not been shown to contain D-alanine. The S. pneumoniae genome contains a number of chaperonin genes similar to those found in other bacteria, but apparently does not contain genes involved in the type III secretion commonly observed in gram negative pathogens. The G+C content of S. pneumoniae genomic DNA is approximately 43 mole percent and the size of the genome is approximately 2.0 Mb as determined by pulsed-field gel electrophoresis. Many of the genes identified by sequence sampling have been physically mapped to the 19 different SmaI fragments derived from the S. pneumoniae genome. The database of random genome sequence tags (GSTs) provides the starting material for determining the complete genome sequence, gene disruption analysis, and comparative genomics to identify novel targets for antibiotic development.

Amino Acyl-tRNA Synthetases↗
PubMed1998

Molecular cloning and physical mapping of the daptomycin gene cluster from Streptomyces roseosporus.

The daptomycin biosynthetic gene cluster of Streptomyces roseosporus was analyzed by Tn5099 mutagenesis, molecular cloning, partial DNA sequencing, and insertional mutagenesis with cloned segments of DNA. The daptomycin biosynthetic gene cluster spans at least 50 kb and is located about 400 to 500 kb from one end of the approximately 7,100-kb linear chromosome. We identified two peptide synthetase coding regions interrupted by a 10- to 20-kb region that may encode other functions in lipopeptide biosynthesis.

Bacterial Proteins↗
PubMed1997

Production of hybrid glycopeptide antibiotics in vitro and in Streptomyces toyocaensis.

BACKGROUND: The glycopeptide antibiotics vancomycin and teicoplanin are currently the last line of defence against some microorganisms that are resistant to many drugs. The emergence of vancomycin-resistant and teicoplanin-resistant enterococci underscores the need for more potent antibiotics. The glycosylation patterns of glycopeptides and chemical modifications of the glycosyl moieties have been shown to greatly influence their antibiotic activity, and certain combinations have resulted in highly active new compounds. To explore further the production of more potent glycopeptide antibiotics, we assessed whether glycosyltransferases could be used to produce hybrid compounds that contain various combinations of sugars and peptide cores. RESULTS: We cloned five glycosyltransferase genes from Amycolatopsis orientalis strains that produce vancomycin or a related glycopeptide, A82846. The gtfB and gtfE' genes from A. orientalis strains expressed in Escherichia coli produced glucosyltransferase activities that added glucose or xylose to the vancomycin heptapeptide. The GtfE' protein added glucose efficiently to two other heptapeptides related to teicoplanin to produce hybrid glycopeptide antibiotics. The cloned gtfE' gene, driven by the strong constitutive promoter ermEp*, was introduced into Streptomyces toyocaensis, which produces the antibiotic A47934, a heptapeptide related to teicoplanin; recombinant organisms produced glucosyl A47934, a hybrid glycopeptide antibiotic. CONCLUSIONS: Cloned glycosyltransferases from glycopeptide antibiotic producers can be used to produce novel hybrid antibiotics, both in vitro and in vivo. Because similar enzymes have differing degrees of substrate specificity, it is advantageous to characterize the substrate specificity with enzymes expressed in E. coli prior to constructing recombinant actinomycetes for production.

Actinobacteria↗
PubMed1997

Applications of transposition mutagenesis in antibiotic producing streptomycetes.

Several transposons have been developed from the streptomycete insertion sequence IS493. They have broad host specificity in Streptomyces species and insert relatively randomly into a consensus target sequence of gNCaNTgNNy. Collectively, they have specialized features that facilitate the following: cloning of DNA flanking insertions; physical mapping of insertions; construction of highly stable mutants; and efficient construction of mutant libraries. All of the transposons can be introduced into streptomycetes by conjugation from E. coli, and can be delivered by curing the temperature sensitive delivery plasmid. Tn5099 was used to physically map genes involved in daptomycin and red pigment production in Streptomyces roseosporus, and to clone daptomycin biosynthetic genes. Tn5099 was also used in Streptomyces fradiae to identify and clone a neutral genomic site for the insertion of a second copy of the tylF gene. Recombinants containing two copies of the tylF gene carried out the normally rate limiting conversion of macrocin to tylosin very efficiently, thus causing substantial increases in tylosin yield.

Anti-Bacterial Agents↗
PubMed1997

Use of rpsL for dominance selection and gene replacement in Streptomyces roseosporus.

We developed a gene replacement system using the rpsL gene of Streptomyces roseosporus and demonstrated its utility by constructing a deletion in the S. roseosporus glnA gene. A 1.3-kb BamHI fragment that hybridized to the Mycobacterium smegmatis rpsL gene was subcloned from an S. roseosporus cosmid library and sequenced. Plasmid pRHB514 containing the rpsL gene conferred streptomycin sensitivity (Sm(S)) to the Sm(r) S. roseosporus TH149. The temperature-sensitive plasmid pRHB543 containing rpsL and the S. roseosporus glnA gene disrupted with a hygromycin resistance (Hm(r)) gene was introduced into S. roseosporus TH149, and recombinants containing single and double crossovers were obtained after a temperature increase. Southern hybridization analysis revealed that single crossovers occurred in the glnA or rpsL genes and that double crossovers resulted in replacement of the chromosomal glnA gene with the disrupted glnA. Glutamine synthetase activity was undetectable in the recombinant containing the disrupted glnA gene.

Amino Acid Sequence↗
PubMed1996

Transposition mutagenesis in Streptomyces fradiae: identification of a neutral site for the stable insertion of DNA by transposon exchange.

We explored transposition in Streptomyces fradiae (Sf) as a means to insert a second copy of the tylF gene to improve tylosin (Ty) production. Transposons Tn5096 and Tn5099 transposed relatively randomly in Sf, and many of the insertions caused no deleterious effects on Ty production yields. Tn5098, a derivative of Tn5096 containing tylF and tylJ genes, recombined into the chromosome into the tyl gene cluster and transposition was not observed. However, following the tagging of a neutral site (NS) by Tn5099 transposition, tylF was effectively inserted into the NS by homologous recombination (transposon exchange). Recombinants obtained by transposon exchange produced higher yields of Ty.

Anti-Bacterial Agents↗
PubMed1996

Molecular genetic methods for improving secondary-metabolite production in actinomycetes.

The practical applications of genetic engineering to improve secondary-metabolite production in actinomycetes are potentially numerous, but have been limited by: (1) restriction barriers, which can hinder the introduction of DNA into many actinomycetes; (2) self-replicating plasmid-cloning vectors, which generally inhibit secondary-metabolite production; and (3) recombinants containing heterologous DNA, which may be subject to additional regulatory hurdles. Recently, intergeneric conjugation has been used to circumvent host restriction, and integration of cloned DNA into neutral genomic sites prevents product inhibition by self-replicating plasmids, and has enabled construction of recombinant strains lacking heterologous DNA sequences. The rpsL system permits direct selection for gene replacements and gene insertions that can facilitate this process.

Actinomycetales↗
PubMed1996

Gene transfer and transposition mutagenesis in Streptomyces roseosporus: mapping of insertions that influence daptomycin or pigment production.

Streptomyces reseosporus, the producer of the cyclic lipopeptide antibiotic daptomycin, was shown to be a suitable host for molecular genetic manipulation. S. roseosporus does not appear to express significant restriction barriers based upon bacteriophage plaque formation studies. Plasmid DNA can be introduced into S. roseosporus by bacteriophage-FP43-mediated transduction and by conjugation from Escherichia coli. The streptomycete transposons Tn5096 and Tn5099, derived from IS493, transpose in S. roseosporus, and Tn5099-induced transposition mutants altered in the production of daptomycin, red pigment or black pigment were identified, and mapped to Dral and Asnl fragments. Three auxotrophic mutations (argB1, ade-1 and metB1) were identified among 100 individual Tn5096 insertions. Alignment and physical mapping of several Tn5099 insertions in Dral-E and Asnl-B fragments was facilitated by the presence of Dral and Asnl cleavage sites in Tn5099.

Bacteriophages↗
PubMed1996

Mutants of Streptomyces roseosporus that express enhanced recombination within partially homologous genes.

Streptomyces roseosporus mutants that express enhanced recombination between partially homologous (homeologous) sequences were isolated by selection for recombination between the bacteriophage phi C31 derivative KC570 containing the Streptomyces coelicolor glucose kinase (glk) gene and the S. roseosporus chromosome. The frequencies of homeologous recombination in the ehr mutants were determined by measuring the chromosomal insertion frequencies of plasmids containing S. coelicolor glnA or whiG genes. S. roseosporus ehr mutants showed 10(2)- to 10(4)-fold increases in homeologous recombination relative to Ehr+ strains, but no increase in homologous recombination. Southern hybridization analysis revealed single unique sites for the insertion of each of the plasmids, and the crossovers occurred in frame and in proper translational register, yielding functional chimeric glnA and whiG genes.

Amino Acid Sequence↗
PubMed1994

Hypertransposing derivatives of the streptomycete insertion sequence IS493.

Transposons derived from the Streptomyces lividans insertion sequence IS493 are useful for the genetic analysis and manipulation of a number of Streptomyces spp. Tn5099-10, an IS493 derivative that contains a spontaneous deletion terminating in the left inverted repeat (IR-L), transposed at a 1000-fold higher frequency in Streptomyces griseofuscus, and at a tenfold higher frequency in Streptomyces fradiae, than the IS493 derivatives, Tn5096 and Tn5099. The IR-L from Tn5099-10 was used to construct a cassette which hypertransposes from plasmids containing the transposon genes, ORFA and ORFB, outside of the inverted repeats. The target sequences of two Tn5099-10 insertions conformed to the consensus target sequence of the other IS493 derivatives, gNCaNTgNNy (where lower-case letters indicate that other nt have been observed at this position and N is any nt). Transposition mutant libraries of S. griseofuscus and S. fradiae can be easily prepared in broth culture by using the hypertransposing elements and a temperature-sensitive delivery plasmid.

Base Sequence↗
PubMed1994

Conjugal transfer of cosmid DNA from Escherichia coli to Saccharopolyspora spinosa: effects of chromosomal insertions on macrolide A83543 production.

Cosmid pOJ436, containing large inserts of Saccharopolyspora spinosa (Ss) DNA, was transferred by conjugation from Escherichia coli to Ss an integrated into the chromosome, apparently by homologous recombination, at high frequencies (10(-5) to 10(-4) per recipient). Transfer was mediated by the plasmid RP4 (RK2) transfer functions in E. coli, and the RK2 oriT function located on pOJ436 [Bierman et al., Gene 116 (1992) 43-49]. pOJ436 lacking Ss DNA, or containing a small insert (approx. 2 kb) of Ss DNA, conjugated from E. coli and integrated at either of two bacteriophage phi C31 attB sites at low frequency (approx. 10(-7) per recipient). Exconjugants containing homologous inserts or inserts at the phi C31 attB sites were stable in the absence of antibiotic selection, and most produced control levels of tetracyclic macrolide A83543 factors. Some exconjugants contained similar kinds of large deletions and were defective in macrolide production.

Anti-Bacterial Agents↗