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July 7, 2019

Rifamorpholines A-E, potential antibiotics from locust-associated actinobacteria Amycolatopsis sp. Hca4.

Cultivation of locust associated rare actinobacteria, Amycolatopsis sp. HCa4, has provided five unusual macrolactams rifamorpholines A-E. Their structures were determined by interpretation of spectroscopic and crystallographic data. Rifamorpholines A-E possess an unprecedented 5/6/6/6 ring chromophore, representing a new subclass of rifamycin antibiotics. The biosynthetic pathway for compounds 1-5 involves a key 1,6-cyclization for the formation of the morpholine ring. Compounds 2 and 4 showed potent activities against methicillin-resistant Staphylococcus aureus (MRSA) with MICs of 4.0 and 8.0 µM, respectively.

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July 7, 2019

Adaptation of genetically monomorphic bacteria: evolution of copper resistance through multiple horizontal gene transfers of complex and versatile mobile genetic elements.

Copper-based antimicrobial compounds are widely used to control plant bacterial pathogens. Pathogens have adapted in response to this selective pressure. Xanthomonas citri pv. citri, a major citrus pathogen causing Asiatic citrus canker, was first reported to carry plasmid-encoded copper resistance in Argentina. This phenotype was conferred by the copLAB gene system. The emergence of resistant strains has since been reported in Réunion and Martinique. Using microsatellite-based genotyping and copLAB PCR, we demonstrated that the genetic structure of the copper-resistant strains from these three regions was made up of two distant clusters and varied for the detection of copLAB amplicons. In order to investigate this pattern more closely, we sequenced six copper-resistant X. citri pv. citri strains from Argentina, Martinique and Réunion, together with reference copper-resistant Xanthomonas and Stenotrophomonas strains using long-read sequencing technology. Genes involved in copper resistance were found to be strain dependent with the novel identification in X. citri pv. citri of copABCD and a cus heavy metal efflux resistance-nodulation-division system. The genes providing the adaptive trait were part of a mobile genetic element similar to Tn3-like transposons and included in a conjugative plasmid. This indicates the system’s great versatility. The mining of all available bacterial genomes suggested that, within the bacterial community, the spread of copper resistance associated with mobile elements and their plasmid environments was primarily restricted to the Xanthomonadaceae family.© 2017 John Wiley & Sons Ltd.

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July 7, 2019

Comparative genomic and regulatory analyses of natamycin production of Streptomyces lydicus A02.

Streptomyces lydicus A02 is used by industry because it has a higher natamycin-producing capacity than the reference strain S. natalensis ATCC 27448. We sequenced the complete genome of A02 using next-generation sequencing platforms, and to achieve better sequence coverage and genome assembly, we utilized single-molecule real-time (SMRT) sequencing. The assembled genome comprises a 9,307,519-bp linear chromosome with a GC content of 70.67%, and contained 8,888 predicted genes. Comparative genomics and natamycin biosynthetic gene cluster (BGC) analysis showed that BGC are highly conserved among evolutionarily diverse strains, and they also shared closer genome evolution compared with other Streptomyces species. Forty gene clusters were predicted to involve in the secondary metabolism of A02, and it was richly displayed in two-component signal transduction systems (TCS) in the genome, indicating a complex regulatory systems and high diversity of metabolites. Disruption of the phoP gene of the phoR-phoP TCS and nsdA gene confirmed phosphate sensitivity and global negative regulation of natamycin production. The genome sequence and analyses presented in this study provide an important molecular basis for research on natamycin production in Streptomyces, which could facilitate rational genome modification to improve the industrial use of A02.

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July 7, 2019

Complete genome sequence of Leuconostoc garlicum KCCM 43211 producing exopolysaccharide.

Leuconostoc garlicum KCCM 43211 isolated from traditional Korean fermented food is an intensive producer of exopolysaccharide (EPS). Here we report the first complete genome sequence of L. garlicum KCCM 43211. The genome sequence displayed that this strain contains genes involved in production of EPS possibly composed of glucose monomers. An uncharacterized EPS from the L. garlicum KCCM 43211 strains was also produced during fermentation in the sucrose medium. The MALDI-TOF results displayed the typical mass spectrometry pattern of dextran. This uncharacterized EPS may have use in commercial prebiotics, food additives, and medical purposes. The complete genome sequence of L. garlicum KCCM 43211 will provide valuable information for strain engineering based on the genetic information. Copyright © 2017 Elsevier B.V. All rights reserved.

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July 7, 2019

Genomic analysis of Clavibacter michiganensis reveals insight into virulence strategies and genetic diversity of a gram-positive bacterial pathogen.

Clavibacter michiganensis subsp. michiganensis is a gram-positive bacterial pathogen that proliferates in the xylem vessels of tomato, causing bacterial canker disease. In this study, we sequenced and assembled genomes of 11 C. michiganensis subsp. michiganensis strains isolated from infected tomato fields in California as well as five Clavibacter strains that colonize tomato endophytically but are not pathogenic in this host. The analysis of the C. michiganensis subsp. michiganensis genomes supported the monophyletic nature of this pathogen but revealed genetic diversity among strains, consistent with multiple introduction events. Two tomato endophytes that clustered phylogenetically with C. michiganensis strains capable of infecting wheat and pepper and were also able to cause disease in these plants. Plasmid profiles of the California strains were variable and supported the essential role of the pCM1-like plasmid and the CelA cellulase in virulence, whereas the absence of the pCM2-like plasmid in some pathogenic C. michiganensis subsp. michiganensis strains revealed it is not essential. A large number of secreted C. michiganensis subsp. michiganensis proteins were carbohydrate-active enzymes (CAZymes). Glycome profiling revealed that C. michiganensis subsp. michiganensis but not endophytic Clavibacter strains is able to extensively alter tomato cell-wall composition. Two secreted CAZymes found in all C. michiganensis subsp. michiganensis strains, CelA and PelA1, enhanced pathogenicity on tomato. Collectively, these results provide a deeper understanding of C. michiganensis subsp. michiganensis diversity and virulence strategies.

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July 7, 2019

Improved PKS gene expression with strong endogenous promoter resulted in geldanamycin yield increase.

The type I polyketide geldanamycin is a potent anti-tumor reagent. Its biosynthesis includes three steps: the biosynthesis of precursors, such as 3-amino-5-hydroxybenzoic acid (AHBA), the polyketide synthase (PKS) chain extension, and the post-PKS modifications. According to the genomic and transcriptomic analysis, the PKS chain extension was deduced to be the rate-limiting step for geldanamycin production in Streptomyces hygroscopicus XM201. In order to improve the expression of PKS genes, a strong endogenous promoter 5063p was obtained based on the transcriptomic analysis and XylE enzymatic assay. By replacing the native PKS promoter gdmA1p with 5063p, the expression of the PKS genes during geldanamycin fermentation was increased by 4-141-folds, and the geldanamycin yield was increased by 39%. Interestingly, AHBA feeding experiment showed that the supply of AHBA in turn become a new rate-limiting factor for geldanamycin production. Further combined overexpression of the 6-gene AHBA biosynthetic cassette and PKS genes increased the yield of geldanamycin by 88%, from 773?mg?L(-1) of the wild-type to 1450?mg?L(-1) in the derived strain. Our results suggested that improved expression of all PKS genes in a particular biosynthetic gene cluster is important for the yield increase of the corresponding polyketide natural product.© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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July 7, 2019

Insights into the red algae and eukaryotic evolution from the genome of Porphyra umbilicalis (Bangiophyceae, Rhodophyta).

Porphyra umbilicalis (laver) belongs to an ancient group of red algae (Bangiophyceae), is harvested for human food, and thrives in the harsh conditions of the upper intertidal zone. Here we present the 87.7-Mbp haploid Porphyra genome (65.8% G + C content, 13,125 gene loci) and elucidate traits that inform our understanding of the biology of red algae as one of the few multicellular eukaryotic lineages. Novel features of the Porphyra genome shared by other red algae relate to the cytoskeleton, calcium signaling, the cell cycle, and stress-tolerance mechanisms including photoprotection. Cytoskeletal motor proteins in Porphyra are restricted to a small set of kinesins that appear to be the only universal cytoskeletal motors within the red algae. Dynein motors are absent, and most red algae, including Porphyra, lack myosin. This surprisingly minimal cytoskeleton offers a potential explanation for why red algal cells and multicellular structures are more limited in size than in most multicellular lineages. Additional discoveries further relating to the stress tolerance of bangiophytes include ancestral enzymes for sulfation of the hydrophilic galactan-rich cell wall, evidence for mannan synthesis that originated before the divergence of green and red algae, and a high capacity for nutrient uptake. Our analyses provide a comprehensive understanding of the red algae, which are both commercially important and have played a major role in the evolution of other algal groups through secondary endosymbioses.

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July 7, 2019

Complete genome sequence of the sand-sediment actinobacterium Nocardioides dokdonensis FR1436(T).

Nocardioides dokdonensis, belonging to the class Actinobacteria, was first isolated from sand sediment of a beach in Dokdo, Korea, in 2005. In this study, we determined the genome sequence of FR1436, the type strain of N. dokdonensis, and analyzed its gene contents. The genome sequence is the second complete one in the genus Nocardioides after that of Nocardioides sp. JS614. It is composed of a 4,376,707-bp chromosome with a G + C content of 72.26%. From the genome sequence, 4,104 CDSs, three rRNA operons, 51 tRNAs, and one tmRNA were predicted, and 71.38% of the genes were assigned putative functions. Through the sequence analysis, dozens of genes involved in steroid metabolism, especially its degradation, were detected. Most of the identified genes were located in large gene clusters, which showed high similarities with the gene clusters in Pimelobacter simplex VKM Ac-2033D. Genomic features of N. dokdonensis associated with steroid catabolism indicate that it could be used for research and application of steroids in science and industry.

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July 7, 2019

Whole genome sequence of two Rathayibacter toxicus strains reveals a tunicamycin biosynthetic cluster similar to Streptomyces chartreusis.

Rathayibacter toxicus is a forage grass associated Gram-positive bacterium of major concern to food safety and agriculture. This species is listed by USDA-APHIS as a plant pathogen select agent because it produces a tunicamycin-like toxin that is lethal to livestock and may be vectored by nematode species native to the U.S. The complete genomes of two strains of R. toxicus, including the type strain FH-79, were sequenced and analyzed in comparison with all available, complete R. toxicus genomes. Genome sizes ranged from 2,343,780 to 2,394,755 nucleotides, with 2079 to 2137 predicted open reading frames; all four strains showed remarkable synteny over nearly the entire genome, with only a small transposed region. A cluster of genes with similarity to the tunicamycin biosynthetic cluster from Streptomyces chartreusis was identified. The tunicamycin gene cluster (TGC) in R. toxicus contained 14 genes in two transcriptional units, with all of the functional elements for tunicamycin biosynthesis present. The TGC had a significantly lower GC content (52%) than the rest of the genome (61.5%), suggesting that the TGC may have originated from a horizontal transfer event. Further analysis indicated numerous remnants of other potential horizontal transfer events are present in the genome. In addition to the TGC, genes potentially associated with carotenoid and exopolysaccharide production, bacteriocins and secondary metabolites were identified. A CRISPR array is evident. There were relatively few plant-associated cell-wall hydrolyzing enzymes, but there were numerous secreted serine proteases that share sequence homology to the pathogenicity-associated protein Pat-1 of Clavibacter michiganensis. Overall, the genome provides clear insight into the possible mechanisms for toxin production in R. toxicus, providing a basis for future genetic approaches.

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July 7, 2019

Towards systems metabolic engineering in Pichia pastoris.

The methylotrophic yeast Pichia pastoris is firmly established as a host for the production of recombinant proteins, frequently outperforming other heterologous hosts. Already, a sizeable amount of systems biology knowledge has been acquired for this non-conventional yeast. By applying various omics-technologies, productivity features have been thoroughly analyzed and optimized via genetic engineering. However, challenging clonal variability, limited vector repertoire and insufficient genome annotation have hampered further developments. Yet, in the last few years a reinvigorated effort to establish P. pastoris as a host for both protein and metabolite production is visible. A variety of compounds from terpenoids to polyketides have been synthesized, often exceeding the productivity of other microbial systems. The clonal variability was systematically investigated and strategies formulated to circumvent untargeted events, thereby streamlining the screening procedure. Promoters with novel regulatory properties were discovered or engineered from existing ones. The genetic tractability was increased via the transfer of popular manipulation and assembly techniques, as well as the creation of new ones. A second generation of sequencing projects culminated in the creation of the second best functionally annotated yeast genome. In combination with landmark physiological insights and increased output of omics-data, a good basis for the creation of refined genome-scale metabolic models was created. The first application of model-based metabolic engineering in P. pastoris showcased the potential of this approach. Recent efforts to establish yeast peroxisomes for compartmentalized metabolite synthesis appear to fit ideally with the well-studied high capacity peroxisomal machinery of P. pastoris. Here, these recent developments are collected and reviewed with the aim of supporting the establishment of systems metabolic engineering in P. pastoris. Copyright © 2017. Published by Elsevier Inc.

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July 7, 2019

Restriction-modification mediated barriers to exogenous DNA uptake and incorporation employed by Prevotella intermedia.

Prevotella intermedia, a major periodontal pathogen, is increasingly implicated in human respiratory tract and cystic fibrosis lung infections. Nevertheless, the specific mechanisms employed by this pathogen remain only partially characterized and poorly understood, largely due to its total lack of genetic accessibility. Here, using Single Molecule, Real-Time (SMRT) genome and methylome sequencing, bisulfite sequencing, in addition to cloning and restriction analysis, we define the specific genetic barriers to exogenous DNA present in two of the most widespread laboratory strains, P. intermedia ATCC 25611 and P. intermedia Strain 17. We identified and characterized multiple restriction-modification (R-M) systems, some of which are considerably divergent between the two strains. We propose that these R-M systems are the root cause of the P. intermedia transformation barrier. Additionally, we note the presence of conserved Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) systems in both strains, which could provide a further barrier to exogenous DNA uptake and incorporation. This work will provide a valuable resource during the development of a genetic system for P. intermedia, which will be required for fundamental investigation of this organism’s physiology, metabolism, and pathogenesis in human disease.

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July 7, 2019

Complete genome sequence of Streptomyces sp. TN58, a producer of acyl alpha-L-rhamnopyranosides.

Streptomyces sp. TN58, isolated from a Tunisian soil sample, produces several natural products, including acyl alpha-l-rhamnopyranosides. It possesses a 7.6-Mb linear chromosome. This is, to our knowledge, the first genome sequence of a microorganism known to produce acyl alpha-l-rhamnopyranosides, and it will be helpful to study the biosynthesis of these specialized metabolites. Copyright © 2017 Najah et al.

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July 7, 2019

Genome characterization of two bile-isolated Vibrio fluvialis strains: an insight into pathogenicity and bile salt adaption.

Vibrio fluvialis is recognized as an emerging pathogen. However, not much is known about the mechanism of its pathogenesis, and its adaptation to a special niche such as the gall bladder. Here we describe two V. fluvialis strains that cause acute cholecystitis. It is noteworthy that both strains were susceptible to all antibiotics tested, which is in contrast to previous studies, suggesting substantial genetic diversity among V. fluvialis isolates. In agreement with their survival and growth in the gall bladder, the genomes of strains 12605 and 3663 contain a considerable number of genes that confer resistance to bile, including toxR, omp U, tolC, cmeABC, rlpB, yrbK, rpoS, damX and gltK. Furthermore, integrative and conjugative elements (ICEs), virulence factors and prophage regions were also detected in strains 12605 and 3663, reflecting their flexibility in recombination during the evolution of pathogenicity. Comparative analysis of nine available genomes of V. fluvialis revealed a core genome consisting of 3,147 genes. Our results highlight the association of V. fluvialis with a rare disease profile and shed light on the evolution of pathogenesis and niche adaptation of V. fluvialis.

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July 7, 2019

The complete genome sequence of Streptomyces autolyticus CGMCC 0516, the producer of geldanamycin, autolytimycin, reblastatin and elaiophylin.

Streptomyces autolyticus CGMCC 0516 produces the anti-tumor benzoquinone ansamycins geldanamycin, autolytimycin, and reblastatin and the 16-membered macrodiolide elaiophylin. Here, we report the complete genome sequence of S. autolyticus CGMCC 0516, which consists of a 10,029,028bp linear chromosome and seven circular plasmids. Fifty-seven putative biosynthetic gene clusters for secondary metabolites were found. The geldanamycin, autolytimycin, and reblastatin biosynthetic gene clusters were located on the left arm (2.06-2.15Mb) of the chromosome, and the elaiophylin gene cluster was located on the right arm (9.45-9.53Mb). Twenty-one putative gene clusters with high or moderate similarity to important antibiotic biosynthetic gene clusters were found, including the antitumor agents echoside, bafilomycin, hygrocin, and toxoflavin; the antibacterial/antifungal agents nigericin, skyllamycin, kanamycin, naphthomycin, eco-02301, and bottromycin A2; the immunosuppressants meridamycin and brasilicardin A; the anti-inflammatory agent cyclooctatin; 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

Draft genome sequence of the plant pathogen Streptomyces sp. strain 11-1-2.

Streptomyces sp. strain 11-1-2 is a Gram-positive filamentous bacterium that was isolated from a common scab lesion on a potato tuber. The strain is highly pathogenic to plants but does not produce the virulence-associated Streptomyces phytotoxin thaxtomin A. Here, we report the draft genome sequence of Streptomyces sp. 11-1-2. Copyright © 2017 Bown and Bignell.

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