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July 7, 2019

Natural product diversity associated with the nematode symbionts Photorhabdus and Xenorhabdus.

Xenorhabdus and Photorhabdus species dedicate a large amount of resources to the production of specialized metabolites derived from non-ribosomal peptide synthetase (NRPS) or polyketide synthase (PKS). Both bacteria undergo symbiosis with nematodes, which is followed by an insect pathogenic phase. So far, the molecular basis of this tripartite relationship and the exact roles that individual metabolites and metabolic pathways play have not been well understood. To close this gap, we have significantly expanded the database for comparative genomics studies in these bacteria. Clustering the genes encoded in the individual genomes into hierarchical orthologous groups reveals a high-resolution picture of functional evolution in this clade. It identifies groups of genes-many of which are involved in secondary metabolite production-that may account for the niche specificity of these bacteria. Photorhabdus and Xenorhabdus appear very similar at the DNA sequence level, which indicates their close evolutionary relationship. Yet, high-resolution mass spectrometry analyses reveal a huge chemical diversity in the two taxa. Molecular network reconstruction identified a large number of previously unidentified metabolite classes, including the xefoampeptides and tilivalline. Here, we apply genomic and metabolomic methods in a complementary manner to identify and elucidate additional classes of natural products. We also highlight the ability to rapidly and simultaneously identify potentially interesting bioactive products from NRPSs and PKSs, thereby augmenting the contribution of molecular biology techniques to the acceleration of natural product discovery.

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July 7, 2019

Revealing the saline adaptation strategies of the halophilic bacterium Halomonas beimenensis through high-throughput omics and transposon mutagenesis approaches.

Studies on the halotolerance of bacteria are attractive to the fermentation industry. However, a lack of sufficient genomic information has precluded an investigation of the halotolerance of Halomonas beimenensis. Here, we describe the molecular mechanisms of saline adaptation in H. beimenensis based on high-throughput omics and Tn5 transposon mutagenesis. The H. beimenensis genome is 4.05 Mbp and contains 3,807 genes, which were sequenced using short and long reads obtained via deep sequencing. Sixteen Tn5 mutants with a loss of halotolerance were identified. Orthologs of the mutated genes, such as nqrA, trkA, atpC, nadA, and gdhB, have significant biological functions in sodium efflux, potassium uptake, hydrogen ion transport for energy conversion, and compatible solute synthesis, which are known to control halotolerance. Other genes, such as spoT, prkA, mtnN, rsbV, lon, smpB, rfbC, rfbP, tatB, acrR1, and lacA, function in cellular signaling, quorum sensing, transcription/translation, and cell motility also shown critical functions for promoting a halotolerance. In addition, KCl application increased halotolerance and potassium-dependent cell motility in a high-salinity environment. Our results demonstrated that a combination of omics and mutagenesis could be used to facilitate the mechanistic exploitation of saline adaptation in H. beimenensis, which can be applied for biotechnological purposes.

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July 7, 2019

Determination of the genome and primary transcriptome of syngas fermenting Eubacterium limosum ATCC 8486.

Autotrophic conversion of CO2 to value-added biochemicals has received considerable attention as a sustainable route to replace fossil fuels. Particularly, anaerobic acetogenic bacteria are naturally capable of reducing CO2 or CO to various metabolites. To fully utilize their biosynthetic potential, an understanding of acetogenesis-related genes and their regulatory elements is required. Here, we completed the genome sequence of the syngas fermenting Eubacterium limosum ATCC 8486 and determined its transcription start sites (TSS). We constructed a 4.4?Mb long circular genome with a GC content of 47.2% and 4,090 protein encoding genes. To understand the transcriptional and translational regulation, the primary transcriptome was augmented, identifying 1,458 TSSs containing a high pyrimidine (T/C) and purine nucleotide (A/G) content at the -1 and +1 position, respectively, along with 1,253 5′-untranslated regions, and principal promoter elements such as -10 (TATAAT) and -35 (TTGACA), and Shine-Dalgarno motifs (GGAGR). Further analysis revealed 93 non-coding RNAs, including one for potential transcriptional regulation of the hydrogenase complex via interaction with molybdenum or tungsten cofactors, which in turn controls formate dehydrogenase activity of the initial step of Wood-Ljungdahl pathway. Our results provide comprehensive genomic information for strain engineering to enhance the syngas fermenting capacity of acetogenic bacteria.

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July 7, 2019

Tryptorubin A: A polycyclic peptide from a fungus-derived Streptomycete.

Fungus-growing ants engage in complex symbiotic relationships with their fungal crop, specialized fungal pathogens, and bacteria that provide chemical defenses. In an effort to understand the evolutionary origins of this multilateral system, we investigated bacteria isolated from fungi. One bacterial strain (Streptomyces sp. CLI2509) from the bracket fungus Hymenochaete rubiginosa, produced an unusual peptide, tryptorubin A, which contains heteroaromatic links between side chains that give it a rigid polycyclic globular structure. The three-dimensional structure was determined by NMR and MS, including a (13)C-(13)C COSY of isotopically enriched material, degradation, derivatives, and computer modeling. Whole genome sequencing identified a likely pair of biosynthetic genes responsible for tryptorubin A’s linear hexapeptide backbone. The genome also revealed the close relationship between CLI2509 and Streptomyces sp. SPB78, which was previously implicated in an insect-bacterium symbiosis.

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July 7, 2019

A novel cold active esterase from a deep sea sponge Stelletta normani metagenomic library

Esterases catalyze the hydrolysis of ester bonds in fatty acid esters with short-chain acyl groups. Due to the widespread applications of lipolytic enzymes in various industrial applications, there continues to be an interest in novel esterases with unique properties. Marine ecosystems have long been acknowledged as a significant reservoir of microbial biodiversity and in particular of bacterial enzymes with desirable characteristics for industrial use, such as for example cold adaptation and activity in the alkaline pH range. We employed a functional metagenomic approach to exploit the enzymatic potential of one particular marine ecosystem, namely the microbiome of the deep sea sponge Stelletta normani. Screening of a metagenomics library from this sponge resulted in the identification of a number of lipolytic active clones. One of these encoded a highly, cold-active esterase 7N9, and the recombinant esterase was subsequently heterologously expressed in Escherichia coli. The esterase was classified as a type IV lipolytic enzyme, belonging to the GDSAG subfamily of hormone sensitive lipases. Furthermore, the recombinant 7N9 esterase was biochemically characterized and was found to be most active at alkaline pH (8.0) and displays salt tolerance over a wide range of concentrations. In silico docking studies confirmed the enzyme’s activity toward short-chain fatty acids while also highlighting the specificity toward certain inhibitors. Furthermore, structural differences to a closely related mesophilic E40 esterase isolated from a marine sediment metagenomics library are discussed.

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July 7, 2019

The complete genome sequence of Streptomyces albolongus YIM 101047, the producer of novel bafilomycins and odoriferous sesquiterpenoids.

Streptomyces albolongus YIM 101047 produces novel bafilomycins and odoriferous sesquiterpenoids with cytotoxic and antimicrobial activities. Here, we report the complete genome sequence of S. albolongus YIM 101047, which consists of an 8,027,788bp linear chromosome. Forty-six putative biosynthetic gene clusters of secondary metabolites were found. The sesquiterpenoid gene cluster was on the left arm (0.09-0.10Mb), and the bafilomycin biosynthetic gene cluster was on the right arm (7.46-7.64Mb) of the chromosome. Twenty-two putative gene clusters with high or moderate similarity to important antibiotic biosynthetic gene clusters were found, including the antitumor agents bafilomycin, epothilone and hedamycin; the antibacterial/antifungal agents clavulanic acid, collismycin A, frontalamides, kanamycin, streptomycin and streptothricin; the protein phosphatase inhibitor RK-682; and the acute iron poisoning medication desferrioxamine B. The genome sequence reported here will enable us to study the biosynthetic mechanism of these important antibiotics and will facilitate the discovery of novel secondary metabolites with potential applications to human health. Copyright © 2017 Elsevier B.V. All rights reserved.

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July 7, 2019

Complete genome sequence of endophyte Bacillus flexus KLBMP 4941 reveals its plant growth promotion mechanism and genetic basis for salt tolerance.

Bacillus flexus KLBMP 4941 is a halotolerant endophyte isolated from the halophyte Limonium sinense. This strain can improve host seedling growth under salt stress conditions. We here report the complete genome information of endophyte KLBMP 4941. It has a circular chromosome and two plasmids for a total genome 4,104,242 bp in size with a G+C content of 38.09%. Genes related to plant growth promotion (PGP), such as those associated with nitrogen fixation, siderophore, spermidine, and acetoin synthesis were found in the KLBMP 4941 genome. Some genes responsible for high salinity tolerance, like genes associated with the Na(+)/H(+) antiporter, glycine betaine transporter, and betaine-aldehyde dehydrogenase were also found in the KLBMP 4941 genome. The genome analysis will provide better understanding of the mechanisms underlying the promotion of plant growth in strain KLBMP 4941 under salt stress conditions and its ability to adapt to coastal salt marsh habitats, and provide a basis for its further biotechnological applications in agriculture. Copyright © 2017 Elsevier B.V. All rights reserved.

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July 7, 2019

Aestuarium zhoushanense gen. nov., sp. nov., Isolated from the Tidal Flat.

A gram-stain-negative, aerobic, ovoid or short rod-shaped, and non-motile strain, designed G7T was isolated from a tidal flat sample collected from the coast of East Sea in Zhoushan, China. Strain G7T grew at 4-40 °C and pH 6.0-9.0 (optimum, 28 °C and pH 7.5) and with 0-7% (w/v) NaCl (optimum, 1%). The predominant respiratory quinone was Q-10 and the major fatty acids (>10%) identified were C18:1 ?7c, C16:0 and summed feature 3 (C16:1 ?7c and/or C16:1 ?6c). The polar lipids of strain G7T consisted of phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, and four unidentified lipids. The genomic DNA G+C content was 56.7 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain G7T formed a distinct lineage belonging to the Roseobacter clade of the family Rhodobacteraceae. On the basis of morphological, physiological, and chemotaxonomic characteristics, together with the results of phylogenetic analysis, strain G7T is described as a novel species in a new genus, for which the name Aestuarium zhoushanense gen. nov., sp. nov. (type strain G7T = MCCC 1K03229T = KCTC 52584T) is proposed.

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July 7, 2019

Biosynthesis of ilamycins featuring unusual building blocks and engineered production of enhanced anti-tuberculosis agents.

Tuberculosis remains one of the world’s deadliest communicable diseases, novel anti-tuberculosis agents are urgently needed due to severe drug resistance and the co-epidemic of tuberculosis/human immunodeficiency virus. Here, we show the isolation of six anti-mycobacterial ilamycin congeners (1-6) bearing rare L-3-nitro-tyrosine and L-2-amino-4-hexenoic acid structural units from the deep sea-derived Streptomyces atratus SCSIO ZH16. The biosynthesis of the rare L-3-nitrotyrosine and L-2-amino-4-hexenoic acid units as well as three pre-tailoring and two post-tailoring steps are probed in the ilamycin biosynthetic machinery through a series of gene inactivation, precursor chemical complementation, isotope-labeled precursor feeding experiments, as well as structural elucidation of three intermediates (6-8) from the respective mutants. Most impressively, ilamycins E1/E2, which are produced in high titers by a genetically engineered mutant strain, show very potent anti-tuberculosis activity with an minimum inhibitory concentration value ˜9.8?nM to Mycobacterium tuberculosis H37Rv constituting extremely potent and exciting anti-tuberculosis drug leads.Tuberculosis (TB) remains one of the world’s deadliest communicable diseases, novel anti-TB agents are urgently needed due to severe drug resistance and the co-epidemic of TB/HIV. Here, the authors show that anti-mycobacterial ilamycin congeners bearing unusual structural units possess extremely potent anti-tuberculosis activities.

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July 7, 2019

CTX-M-15-producing Shewanella sp. clinical isolate expressing OXA-535, a chromosome-encoded OXA-48 variant, putative progenitor of the plasmid-encoded OXA-436.

Shewanella spp. constitute a reservoir of antibiotic resistance determinants. In a bile sample, we have identified three Extended Spectrum ß-lactamase (ESBL)-producing bacteria (Escherichia coli, Klebsiella pneumoniae and Shewanella sp. JAB-1) isolated from a child suffering from cholangitis. Our objectives were to characterize the genome and the resistome of the first ESBL-producing isolate of the genus Shewanella and determine whether plasmidic exchange occurred between the three-bacterial species. Bacterial isolates were characterized using MALDI-TOF, standard biochemical tools and antimicrobial susceptibility testing. Shewanella sp JAB-1 and ESBL gene-carrying plasmids were characterized using PacBio and Illumina whole genome sequencing, respectively. The Shewanella sp JAB-1 chromosome-encoded OXA-48-variant was cloned and functionally characterized.Whole genome sequencing (WGS) of the Shewanella sp. clinical isolate JAB-1 revealed the presence of a 193-kb plasmid belonging to IncA/C incompatibility group and harboring two ESBL genes: blaCTX-M-15 and blaSHV-2ablaCTX-M-15 gene carrying plasmids belonging to IncY and IncR incompatibility groups were also found in the E. coli and K. pneumoniae isolates from the same patient, respectively. Comparison of the blaCTX-M-15 genetic environment indicated the independent origin of these plasmids and dismissed in vivo transfers. Furthermore, characterization of the resistome of Shewanella sp. JAB-1 revealed the presence of a chromosome-encoded blaOXA-535 gene, likely the progenitor of the plasmid-encoded blaOXA-436 gene, a novel blaOXA-48-like gene. Expression of blaOXA-535 in E. coli showed the carbapenem-hydrolyzing activity of OXA-535. The production of OXA-535 in Shewanella sp. JAB-1 could be evidenced using molecular and immuno-enzymatic tests, but not with biochemical tests that monitor carbapenem-hydrolysis. In this study, we have identified a CTX-M-15-producing Shewanella species that was responsible of an hepatobiliary infection and that is likely the progenitor of OXA-436, a novel plasmid-encoded OXA-48-like class D carbapenemases. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Diversity oriented biosynthesis via accelerated evolution of modular gene clusters.

Erythromycin, avermectin and rapamycin are clinically useful polyketide natural products produced on modular polyketide synthase multienzymes by an assembly-line process in which each module of enzymes in turn specifies attachment of a particular chemical unit. Although polyketide synthase encoding genes have been successfully engineered to produce novel analogues, the process can be relatively slow, inefficient, and frequently low-yielding. We now describe a method for rapidly recombining polyketide synthase gene clusters to replace, add or remove modules that, with high frequency, generates diverse and highly productive assembly lines. The method is exemplified in the rapamycin biosynthetic gene cluster where, in a single experiment, multiple strains were isolated producing new members of a rapamycin-related family of polyketides. The process mimics, but significantly accelerates, a plausible mechanism of natural evolution for modular polyketide synthases. Detailed sequence analysis of the recombinant genes provides unique insight into the design principles for constructing useful synthetic assembly-line multienzymes.

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July 7, 2019

Complete genome sequence analysis of Enterobacter sp. SA187, a plant multi-stress tolerance promoting endophytic bacterium

Enterobacter sp. SA187 is an endophytic bacterium that has been isolated from root nodules of the indigenous desert plant Indigofera argentea. SA187 could survive in the rhizosphere as well as in association with different plant species, and was able to provide abiotic stress tolerance to Arabidopsis thaliana. The genome sequence of SA187 was obtained by using Pacific BioScience (PacBio) single-molecule sequencing technology, with average coverage of 275X. The genome of SA187 consists of one single 4,429,597 bp chromosome, with an average 56% GC content and 4,347 predicted protein coding DNA sequences (CDS), 153 ncRNA, 7 rRNA, and 84 tRNA. Functional analysis of the SA187 genome revealed a large number of genes involved in uptake and exchange of nutrients, chemotaxis, mobilization and plant colonization. A high number of genes were also found to be involved in survival, defense against oxidative stress and production of antimicrobial compounds and toxins. Moreover, different metabolic pathways were identified that potentially contribute to plant growth promotion. The information encoded in the genome of SA187 reveals the characteristics of a dualistic lifestyle of a bacterium that can adapt to different environments and promote the growth of plants. This information provides a better understanding of the mechanisms involved in plant-microbe interaction and could be further exploited to develop SA187 as a biological agent to improve agricultural practices in marginal and arid lands.

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July 7, 2019

Molecular approaches for high throughput detection and quantification of genetically modified crops: A review.

As long as the genetically modified crops are gaining attention globally, their proper approval and commercialization need accurate and reliable diagnostic methods for the transgenic content. These diagnostic techniques are mainly divided into two major groups, i.e., identification of transgenic (1) DNA and (2) proteins from GMOs and their products. Conventional methods such as PCR (polymerase chain reaction) and enzyme-linked immunosorbent assay (ELISA) were routinely employed for DNA and protein based quantification respectively. Although, these Techniques (PCR and ELISA) are considered as significantly convenient and productive, but there is need for more advance technologies that allow for high throughput detection and the quantification of GM event as the production of more complex GMO is increasing day by day. Therefore, recent approaches like microarray, capillary gel electrophoresis, digital PCR and next generation sequencing are more promising due to their accuracy and precise detection of transgenic contents. The present article is a brief comparative study of all such detection techniques on the basis of their advent, feasibility, accuracy, and cost effectiveness. However, these emerging technologies have a lot to do with detection of a specific event, contamination of different events and determination of fusion as well as stacked gene protein are the critical issues to be addressed in future.

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July 7, 2019

Identification of YfiH and the catalase CatA as polyphenol oxidases of Aeromonas media and CatA as a regulator of pigmentation by Its peroxyl radical scavenging capacity.

Pyomelanin is the major constituent of pigment in melanogenic Aeromonas strains of bacteria. However, eumelanin, synthesized from tyrosine via L-DOPA and polyphenol oxidases (PPOs), may also be present in this genus since L-DOPA is frequently detected in culture fluids of several species. To address this question, we used a deletion mutant of Aeromonas media strain WS, in which pyomelanin synthesis is completely blocked under normal culture conditions. When tyrosine was supplied to the medium, we observed residual melanin accumulation, which we interpret as evidence for existence of the DOPA-melanin pathway. We traced enzymatic activity in this bacterium using native-polyacrylamide gel electrophoresis. Two PPOs: YfiH, a laccase-like protein, and CatA, a catalase, were identified. However, neither protein was critical for the residual pigmentation in pyomelanin-deficient mutant. We speculate that eumelanin synthesis may require other unknown enzymes. Deletion of yfiH did not affect pigmentation in A. media strain WS, while deletion of the CatA-encoding gene katE resulted in a reduction of melanin accumulation, but it started 9 h earlier than in the wild-type. Since catalases regulate reactive oxygen species levels during melanogenesis, we speculated that CatA affects pigmentation through its peroxyl radical scavenging capacity. Consistent with this, expression of the catalases Hpi or Hpii from Escherichia coli in the katE deletion strain of A. media strain WS restored pigmentation to the wild-type level. Hpi and Hpii also exhibited PPO activity, suggesting that catalase may represent a new class of PPOs.

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July 7, 2019

High-quality draft genome sequence of Streptomyces agglomeratus 5-1-8 with strong anti-MRSA ability, isolated from the frozen soil of Tibet in China

Streptomyces agglomeratus 5-1-8 with strong anti methicillin-resistant Staphylococcus aureus (MRSA) ability, isolated from the frozen soil of Tibet in China, has a strong ability to kill the multi-drugs-resistant MRSA. To identify the second-ary metabolism ability of this strain, we describe here the phenotypic characteristics of this strain, along with its high-quality draft genome sequence, its annotation, and analysis. The 7.1M draft genome encodes 6,284 putative open reading frames (ORFs), of which 4,416 ORFs were assigned with clusters of orthologous genes (COG) categories. Also, 65 tRNA genes and 24 rRNA operons were identified. The genome contains 12 gene clusters involved in antibiotics production and 1 gene cluster involved in anticancer-compounds production; 4 gene clusters belong to polyketides and nonribosomal peptides, 1 gene cluster belong to the butyrolactone, 4 gene clusters belong to the bacteriocin or lantipeptide, and 3 gene clusters belong to the others. This genome-sequence data will facilitate efforts to probe the potential of new antibiotics to kill multi-drugs-resistant MRSA.

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