Diversity of Nonribosomal Peptide Synthetase Genes in the AnticancerProducing Actinomycetes Isolated from Marine Sediment in Indonesia

Marine actinomycetes is a group of bacteria that is highly potential in producing novel bioactive compound. It has unique characteristics and is different from other terrestrial ones. Extreme environmental condition is suspected to lead marine actinomycetes produce different types of bioactive compound found previously. The aim of this study was to explore the presence and diversity of NRPS genes in 14 anticancer-producing actinomycetes isolated from marine sediment in Indonesia. PCR amplification and restriction fragment analysis of NRPS genes with HaeIII from 14 marine actinomycetes were done to assess the diversity of NRPS genes. Genome mining of one species of marine actinomycetes (strain GMY01) also was employed towards this goal. The result showed that NRPS gene sequence diversity in 14 marine actinomycetes could be divided into 4 groups based on NRPS gene restriction patterns. Analysis of 16S rRNA gene sequences of representatives from each group showed that all isolates belong to genus of Streptomyces. Genome mining result showed that strain GMY01 harboring 10 different NRPS gene clusters that encode secondary metabolites, as pure NRPS or hybrid between NRPS and other compounds. These results indicated that marine actinomycetes having a high potential to be developed as source of anticancer drugs development

Whole-genome analyses reveal genetic instability of Acetobacter pasteurianus

Acetobacter species have been used for brewing traditional vinegar and are known to have genetic instability. To clarify the mutability, Acetobacter pasteurianus NBRC 3283, which forms a multi-phenotype cell complex, was subjected to genome DNA sequencing. The genome analysis revealed that there are more than 280 transposons and five genes with hyper-mutable tandem repeats as common features in the genome consisting of a 2.9-Mb chromosome and six plasmids. There were three single nucleotide mutations and five transposon insertions in 32 isolates from the cell complex. The A. pasteurianus hyper-mutability was applied for breeding a temperature-resistant strain grown at an unviable high-temperature (42°C). The genomic DNA sequence of a heritable mutant showing temperature resistance was analyzed by mutation mapping, illustrating that a 92-kb deletion and three single nucleotide mutations occurred in the genome during the adaptation. Alpha-proteobacteria including A. pasteurianus consists of many intracellular symbionts and parasites, and their genomes show increased evolution rates and intensive genome reduction. However, A. pasteurianus is assumed to be a free-living bacterium, it may have the potentiality to evolve to fit in natural niches of seasonal fruits and flowers with other organisms, such as yeasts and lactic acid bacteria

SECONDARY BIOACTIVE METABOLITE GENE CLUSTERS IDENTIFICATION OF ANTICANDIDA-PRODUCING Streptomyces Sp. GMR22 ISOLATED FROM WANAGAMA FOREST AS REVEALED BY GENOME MINING APPROACH

Streptomyces are a group of Gram-positive bacteria belonging to the Actinobacteria class, which are among the most important bacteria for producing secondary bioactive metabolites such as antibiotics, chemotherapeutics, insecticides and other high-value chemicals. Genome mining of gene clusters that encode the biosynthetic pathways for these metabolites has become a key methodology for novel compound discovery. Recently, we have isolated the Streptomyces sp. GMR22 from Cajuput rhizospheric soil at Wanagama Forest, Indonesia. GMR22 produced secondary metabolite that inhibited Candida albicans with IC50 of 62,5 μg/mL. The objective of this work was to reveal the novel secondary metabolites from GMR22 by genome mining approach. The antiSMASH 3.0 was used to predict gene clusters that encode the biosynthetic pathways of secondary metabolites in the genome of GMR22, and their core chemical structures. The pylogenomic analysis showed that GMR22 was closely related to Streptomyces bingchenggensis BCW1, as well as to the large genome size (9.5-12.7Mbp) groups of Streptomyces. AntiSMASH 3.0 analysis revealed that the genome of Streptomyces sp. GMR22 harbored at least 63 gene clusters that encode the biosynthetic pathways of secondary metabolites. It was the highest number of gene clusters had been observed among the members of Streptomyces groups, with PKS was predicted as the major groups of the identified gene cluster products. The results suggested that GMR22 could be a strong potential candidate of secondary bioactive metabolites source

In Silico Analysis of PKS and NRPS Gene Clusters in Arisostatin- and Kosinostatin-Producers and Description of Micromonospora okii sp. nov.

Micromonospora sp. TP-A0316 and Micromonospora sp. TP-A0468 are producers of arisostatin and kosinostatin, respectively. Micromonospora sp. TP-A0316 showed a 16S rRNA gene sequence similarity of 100% to Micromonosporaoryzae CP2R9-1T whereas Micromonospora sp. TP-A0468 showed a 99.3% similarity to Micromonospora haikouensis 232617T. A phylogenetic analysis based on gyrB sequences suggested that Micromonospora sp. TP-A0316 is closely related to Micromonospora oryzae whereas Micromonospora TP-A0468 is an independent genomospecies. As Micromonospora sp. TP-A0468 showed some phenotypic differences to its closely related species, it was classified as a novel species, for which the name Micromonospora okii sp. nov. is proposed. The type strain is TP-A0468T (= NBRC 110461T). Micromonospora sp. TP-A0316 and M. okii TP-A0468T were both found to harbor 15 gene clusters for secondary metabolites such as polyketides and nonribosomal peptides in their genomes. Arisostatin-biosynthetic gene cluster (BGC) of Micromonospora sp. TP-A0316 closely resembled tetrocarcin A-BGC of Micromonospora chalcea NRRL 11289. A large type-I polyketide synthase gene cluster was present in each genome of Micromonospora sp. TP-A0316 and M. okii TP-A0468T. It was an ortholog of quinolidomicin-BGC of M. chalcea AK-AN57 and widely distributed in the genus Micromonospora

Biosynthesis of Akaeolide and Lorneic Acids and Annotation of Type I Polyketide Synthase Gene Clusters in the Genome of Streptomyces sp. NPS554

The incorporation pattern of biosynthetic precursors into two structurally unique polyketides, akaeolide and lorneic acid A, was elucidated by feeding experiments with 13C-labeled precursors. In addition, the draft genome sequence of the producer, Streptomyces sp. NPS554, was performed and the biosynthetic gene clusters for these polyketides were identified. The putative gene clusters contain all the polyketide synthase (PKS) domains necessary for assembly of the carbon skeletons. Combined with the 13C-labeling results, gene function prediction enabled us to propose biosynthetic pathways involving unusual carbon-carbon bond formation reactions. Genome analysis also indicated the presence of at least ten orphan type I PKS gene clusters that might be responsible for the production of new polyketides

Draft genome sequence of Streptomyces hyaluromycini MB-PO13T, a hyaluromycin producer

Abstract Streptomyces hyaluromycini MB-PO13T (=NBRC 110483T = DSM 100105T) is type strain of the species, which produces a hyaluronidase inhibitor, hyaluromycin. Here, we report the draft genome sequence of this strain together with features of the organism and generation, annotation and analysis of the genome sequence. The 11.5 Mb genome of Streptomyces hyaluromycini MB-PO13T encoded 10,098 putative ORFs, of which 5317 were assigned with COG categories. The genome harbored at least six type I PKS clusters, three type II PKS gene clusters, two type III PKS gene clusters, six NRPS gene clusters, and one hybrid PKS/NRPS gene cluster. The type II PKS gene cluster including 2-amino-3-hydroxycyclopent-2-enone synthetic genes was identified to be responsible for hyaluromycin synthesis. We propose the biosynthetic pathway based on bioinformatic analysis

In Vitro and In Vivo Antibacterial Activities of L-084, a Novel Oral Carbapenem, against Causative Organisms of Respiratory Tract Infections

L-084 (a prodrug of LJC 11,036 [L-036]) is a new oral carbapenem. Here we compared the in vitro and in vivo antibacterial activities of L-036 with those of imipenem, faropenem, ceditoren-pivoxil, cefdinir, amoxicillin, and levofloxacin. The MICs at which 90% of the isolates were inhibited of L-036 against methicillin-susceptible staphylococci, Streptococcus pneumoniae including penicillin-resistant organisms, Escherichia coli, Klebsiella pneumoniae, Haemophilus influenzae including ampicillin-resistant organisms, Legionella pneumophila, and Moraxella catarrhalis were equal to or less than 1 μg/ml. In pharmacokinetics studies of L-084 in lungs of mice, the maximum concentration in serum, half-life, and area under the concentration-time curve of this drug were 9.09 μg/g of tissue, 6.18 h, and 31.0 μg · h/ml, respectively. In murine respiratory infection models of penicillin-susceptible and -resistant S. pneumoniae and H. influenzae, the efficacies of L-084 were better than those of reference drugs. Our results indicate that the in vitro high potency and good distribution in the lungs might be the underlying mechanisms of its efficacy in the murine model of pneumonia

Complete genome sequence of natural rubber-degrading, gram-negative bacterium, Rhizobacter gummiphilus strain NS21T

Gram-negative natural rubber-degrader, Rhizobacter gummiphilus NS21T, which was isolated from soil in the botanical garden in Japan, is a newly proposed species of genus of Rhizobacter. It has been reported that the latA1 gene is involved in the natural rubber degradation in this strain. To gain novel insights into natural rubber degradation pathway, the complete genome sequence of this strain was determined. The genome of strain NS21T consists of 6,398,096 bp of circular chromosome (GenBank accession number CP015118.1) with G + C content of 69.72%. The genome contains 5687 protein-coding and 68 RNA genes. Among the predicted genes, 4810 genes were categorized as functional COGs. Homology search revealed that existence of latA1 homologous gene (latA2) in this genome. Quantitative reverse-transcription-PCR and deletion analyses indicated that natural rubber degradation of this strain requires latA2 as well as latA1. Keywords: Natural rubber, Rubber oxygenase, Gram-negative natural-rubber degrading bacteri