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September 22, 2019  |  

Mode of action and heterologous expression of the natural product antibiotic vancoresmycin.

Antibiotics that interfere with the bacterial cytoplasmic membrane have long-term potential for the treatment of infectious diseases as this mode of action is anticipated to result in low resistance frequency. Vancoresmycin is an understudied natural product antibiotic consisting of a terminal tetramic acid moiety fused to a linear, highly oxygenated, stereochemically complex polyketide chain. Vancoresmycin shows minimum inhibitory concentrations (MICs) from 0.125 to 2 µg/mL against a range of clinically relevant, antibiotic-resistant Gram-positive bacteria. Through a comprehensive mode-of-action study, utilizing Bacillus subtilis reporter strains, DiSC3(5) depolarization assays, and fluorescence microscopy, we have shown that vancoresmycin selectively targets the cytoplasmic membrane of Gram-positive bacteria via a non-pore-forming, concentration-dependent depolarization mechanism. Whole genome sequencing of the producing strain allowed identification of the 141 kbp gene cluster encoding for vancoresmycin biosynthesis and a preliminary model for its biosynthesis. The size and complex structure of vancoresmycin could confound attempts to generate synthetic analogues. To overcome this problem and facilitate future studies, we identified, cloned, and expressed the 141 kbp biosynthetic gene cluster in Streptomyces coelicolor M1152. Elucidation of the mode-of-action of vancoresmycin, together with the heterologous expression system, will greatly facilitate further studies of this and related molecules.

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September 22, 2019  |  

Complete genome sequence of Lactobacillus pentosus SLC13, isolated from mustard pickles, a potential probiotic strain with antimicrobial activity against foodborne pathogenic microorganisms.

Lactobacillus pentosus SLC13 is a high exopolysaccharide (EPS)-producing strain with broad-spectrum antimicrobial activity and the ability to grow in simulated gastrointestinal conditions. SLC13 was isolated from mustard pickles in Taiwan for potential probiotic applications. To better understand the molecular base for its antimicrobial activity and high EPS production, entire genome of SLC13 was determined by PacBio SMRT sequencing.L. pentosus SLC13 contains a genome with a 3,520,510-bp chromosome and a 62,498-bp plasmid. GC content of the complete genome was 46.5% and that of plasmid pSLC13 was 41.3%. Sequences were annotated at the RAST prokaryotic genome annotation server, and the results showed that the genome contained 3172 coding sequences and 82 RNA genes. Seventy-six protein-coding sequences were identified on the plasmid pSLC13. A plantaricin gene cluster, which is responsible for bacteriosins biosynthesis and could be associated with its broad-spectrum antimicrobial activity, was identified based on comparative genomic analysis. Two gene clusters involved in EPS production were also identified.This genomic sequence might contribute to a future application of this strain as probiotic in productive livestock potentially inhibiting competing and pathogenic organisms.

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September 22, 2019  |  

Multi-omics Reveals the Lifestyle of the Acidophilic, Mineral-Oxidizing Model Species Leptospirillum ferriphilumT.

Leptospirillum ferriphilum plays a major role in acidic, metal-rich environments, where it represents one of the most prevalent iron oxidizers. These milieus include acid rock and mine drainage as well as biomining operations. Despite its perceived importance, no complete genome sequence of the type strain of this model species is available, limiting the possibilities to investigate the strategies and adaptations that Leptospirillum ferriphilum DSM 14647T (here referred to as Leptospirillum ferriphilumT) applies to survive and compete in its niche. This study presents a complete, circular genome of Leptospirillum ferriphilumT obtained by PacBio single-molecule real-time (SMRT) long-read sequencing for use as a high-quality reference. Analysis of the functionally annotated genome, mRNA transcripts, and protein concentrations revealed a previously undiscovered nitrogenase cluster for atmospheric nitrogen fixation and elucidated metabolic systems taking part in energy conservation, carbon fixation, pH homeostasis, heavy metal tolerance, the oxidative stress response, chemotaxis and motility, quorum sensing, and biofilm formation. Additionally, mRNA transcript counts and protein concentrations were compared between cells grown in continuous culture using ferrous iron as the substrate and those grown in bioleaching cultures containing chalcopyrite (CuFeS2). Adaptations of Leptospirillum ferriphilumT to growth on chalcopyrite included the possibly enhanced production of reducing power, reduced carbon dioxide fixation, as well as elevated levels of RNA transcripts and proteins involved in heavy metal resistance, with special emphasis on copper efflux systems. Finally, the expression and translation of genes responsible for chemotaxis and motility were enhanced.IMPORTANCELeptospirillum ferriphilum is one of the most important iron oxidizers in the context of acidic and metal-rich environments during moderately thermophilic biomining. A high-quality circular genome of Leptospirillum ferriphilumT coupled with functional omics data provides new insights into its metabolic properties, such as the novel identification of genes for atmospheric nitrogen fixation, and represents an essential step for further accurate proteomic and transcriptomic investigation of this acidophile model species in the future. Additionally, light is shed on adaptation strategies of Leptospirillum ferriphilumT for growth on the copper mineral chalcopyrite. These data can be applied to deepen our understanding and optimization of bioleaching and biooxidation, techniques that present sustainable and environmentally friendly alternatives to many traditional methods for metal extraction. Copyright © 2018 Christel et al.

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September 22, 2019  |  

Comparative genomics and transcriptomics analysis-guided metabolic engineering of Propionibacterium acidipropionici for improved propionic acid production.

Acid stress induced by the accumulation of organic acids during the fermentation of propionibacteria is a severe limitation in the microbial production of propionic acid (PA). To enhance the acid resistance of strains, the tolerance mechanisms of cells must first be understood. In this study, comparative genomic and transcriptomic analyses were conducted on wild-type and acid-tolerant Propionibacterium acidipropionici to reveal the microbial response of cells to acid stress during fermentation. Combined with the results of previous proteomic and metabolomic studies, several potential acid-resistance mechanisms of P. acidipropionici were analyzed. Energy metabolism and transporter activity of cells were regulated to maintain pH homeostasis by balancing transmembrane transport of protons and ions; redundant protons were eliminated by enhancing the metabolism of certain amino acids for a relatively stable intracellular microenvironment; and protective mechanism of macromolecules were also induced to repair damage to proteins and DNA by acids. Transcriptomic data indicated that the synthesis of acetate and lactate were undesirable in the acid-resistant mutant, the expression of which was 2.21-fold downregulated. In addition, metabolomic data suggested that the accumulation of lactic acid and acetic acid reduced the carbon flow to PA and led to a decrease in pH. On this basis, we propose a metabolic engineering strategy to regulate the synthesis of lactic acid and acetic acid that will reduce by-products significantly and increase the PA yield by 12.2% to 10.31?±?0.84?g/g DCW. Results of this study provide valuable guidance to understand the response of bacteria to acid stress and to construct microbial cell factories to produce organic acids by combining systems biology technologies with synthetic biology tools.© 2017 Wiley Periodicals, Inc.

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September 22, 2019  |  

Complete genome sequence of Bacillus velezensis 157 isolated from Eucommia ulmoides with pathogenic bacteria inhibiting and lignocellulolytic enzymes production by SSF.

Bacillus velezensis 157 was isolated from the bark of Eucommia ulmoides, and exhibited antagonistic activity against a broad spectrum of pathogenic bacteria and fungi. Moreover, B. velezensis 157 also showed various lignocellulolytic activities including cellulase, xylanase, a-amylase, and pectinase, which had the ability of using the agro-industrial waste (soybean meal, wheat bran, sugarcane bagasse, wheat straw, rice husk, maize flour and maize straw) under solid-state fermentation and obtained several industrially valuable enzymes. Soybean meal appeared to be the most efficient substrate for the single fermentation of B. velezensis 157. Highest yield of pectinase (19.15 ± 2.66 U g-1), cellulase (46.69 ± 1.19 U g-1) and amylase (2097.18 ± 15.28 U g-1) was achieved on untreated soybean meal. Highest yield of xylanase (22.35 ± 2.24 U g-1) was obtained on untreated wheat bran. Here, we report the complete genome sequence of the B. velezensis 157, composed of a circular 4,013,317 bp chromosome with 3789 coding genes and a G + C content of 46.41%, one circular 8439 bp plasmid and a G + C content of 40.32%. The genome contained a total of 8 candidate gene clusters (bacillaene, difficidin, macrolactin, butirosin, bacillibactin, bacilysin, fengycin and surfactin), and dedicates over 15.8% of the whole genome to synthesize secondary metabolite biosynthesis. In addition, the genes encoding enzymes involved in degradation of cellulose, xylan, lignin, starch, mannan, galactoside and arabinan were found in the B. velezensis 157 genome. Thus, the study of B. velezensis 157 broadened that B. velezensis can not only be used as biocontrol agents, but also has potentially a wide range of applications in lignocellulosic biomass conversion.

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September 22, 2019  |  

Identification of the streptothricin and tunicamycin biosynthetic gene clusters by genome mining in Streptomyces sp. strain fd1-xmd.

The genus Streptomyces have been highly regarded for their important source of natural products. Combined with the technology of genome sequencing and mining, we could identify the active ingredients from fermentation broth quickly. Here, we report on Streptomyces sp. strain fd1-xmd, which was isolated from a soil sample collected in Shanghai. Interestingly, the fermentation broth derived from this strain demonstrated broad-spectrum antimicrobial activity against gram-positive bacteria, gram-negative bacteria, and eukaryotes. To identify the antimicrobial substances and their biosynthetic gene clusters, we sequenced the fd1-xmd strain and obtained a genome 7,929,999 bp in length. The average GC content of the chromosome was 72.5 mol%. Knockout experiments demonstrated that out of eight biosynthetic gene clusters we could identify, two are responsible for the biosynthesis of the antibiotics streptothricin (ST) and tunicamycin (TM). The ST biosynthetic gene cluster from fd1-xmd was verified via successful heterologous expression in Streptomyces coelicolor M1146. ST production had a yield of up to 0.5 g/L after the optimization of culture conditions. This study describes a novel producer of ST and TM and outlines the complete process undertaken for Streptomyces sp. strain fd1-xmd genome mining.

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September 22, 2019  |  

Characterization of Lactobacillus amylolyticus L6 as potential probiotics based on genome sequence and corresponding phenotypes

The potential of newly isolated Lactobacillus amylolyticus L6 as probiotics was investigated based on the whole genome sequence and corresponding phenotypes. With Lactobacillus acidophilus NCFM as positive control, several established methods of evaluating potential probiotics were performed on L. amylolyticus L6. The results indicated that L. amylolyticus L6 retained higher viability in human gastrointestinal (GI) tract and it also had strong inhibitory effect on pathogenic bacteria. Meanwhile, the candidate probiotics exhibited similar adhesion level as that of L. acidophilus NCFM in vitro test. As for carbohydrate utilization profile, L. amylolyticus L6 had high ability of utilizing raffinose and stachyose which were known as flatulence factors in soybean products. And this strain could also utilize starch. Besides, the mechanisms of probiotic and metabolic properties for L. amylolyticus L6 were further illustrated with the identification of related genes through the analysis of genome sequence. Therefore, we proposed that L. amylolyticus L6 have the potential to be used as probiotics from phenotypes to genotypes. And it is the first time that the complete genome sequence of L. amylolyticus L6 and the potential of this strain to be used as probiotics were reported in this study.

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September 22, 2019  |  

Pathogen-specific binding soluble Down syndrome cell adhesion molecule (Dscam) regulates phagocytosis via membrane-bound Dscam in crab.

The Down syndrome cell adhesion molecule (Dscam) gene is an extraordinary example of diversity that can produce thousands of isoforms and has so far been found only in insects and crustaceans. Cumulative evidence indicates that Dscam may contribute to the mechanistic foundations of specific immune responses in insects. However, the mechanism and functions of Dscam in relation to pathogens and immunity remain largely unknown. In this study, we identified the genome organization and alternative Dscam exons from Chinese mitten crab, Eriocheir sinensis. These variants, designated EsDscam, potentially produce 30,600 isoforms due to three alternatively spliced immunoglobulin (Ig) domains and a transmembrane domain. EsDscam was significantly upregulated after bacterial challenge at both mRNA and protein levels. Moreover, bacterial specific EsDscam isoforms were found to bind specifically with the original bacteria to facilitate efficient clearance. Furthermore, bacteria-specific binding of soluble EsDscam via the complete Ig1-Ig4 domain significantly enhanced elimination of the original bacteria via phagocytosis by hemocytes; this function was abolished by partial Ig1-Ig4 domain truncation. Further studies showed that knockdown of membrane-bound EsDscam inhibited the ability of EsDscam with the same extracellular region to promote bacterial phagocytosis. Immunocytochemistry indicated colocalization of the soluble and membrane-bound forms of EsDscam at the hemocyte surface. Far-Western and coimmunoprecipitation assays demonstrated homotypic interactions between EsDscam isoforms. This study provides insights into a mechanism by which soluble Dscam regulates hemocyte phagocytosis via bacteria-specific binding and specific interactions with membrane-bound Dscam as a phagocytic receptor.

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September 22, 2019  |  

Complete genome analysis of Gluconacetobacter xylinus CGMCC 2955 for elucidating bacterial cellulose biosynthesis and metabolic regulation.

Complete genome sequence of Gluconacetobacter xylinus CGMCC 2955 for fine control of bacterial cellulose (BC) synthesis is presented here. The genome, at 3,563,314?bp, was found to contain 3,193 predicted genes without gaps. There are four BC synthase operons (bcs), among which only bcsI is structurally complete, comprising bcsA, bcsB, bcsC, and bcsD. Genes encoding key enzymes in glycolytic, pentose phosphate, and BC biosynthetic pathways and in the tricarboxylic acid cycle were identified. G. xylinus CGMCC 2955 has a complete glycolytic pathway because sequence data analysis revealed that this strain possesses a phosphofructokinase (pfk)-encoding gene, which is absent in most BC-producing strains. Furthermore, combined with our previous results, the data on metabolism of various carbon sources (monosaccharide, ethanol, and acetate) and their regulatory mechanism of action on BC production were explained. Regulation of BC synthase (Bcs) is another effective method for precise control of BC biosynthesis, and cyclic diguanylate (c-di-GMP) is the key activator of BcsA-BcsB subunit of Bcs. The quorum sensing (QS) system was found to positively regulate phosphodiesterase, which decomposed c-di-GMP. Thus, in this study, we demonstrated the presence of QS in G. xylinus CGMCC 2955 and proposed a possible regulatory mechanism of QS action on BC production.

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September 22, 2019  |  

Genome-wide comparison reveals a probiotic strain Lactococcus lactis WFLU12 isolated from the gastrointestinal tract of olive flounder (Paralichthys Olivaceus) harboring genes supporting probiotic action.

Our previous study has shown that dietary supplementation with Lactococcus lactis WFLU12 can enhance the growth of olive flounder and its resistance against streptococcal infection. The objective of the present study was to use comparative genomics tools to investigate genomic characteristics of strain WFLU12 and the presence of genes supporting its probiotic action using sequenced genomes of L. lactis strains. Dispensable and singleton genes of strain WFLU12 were found to be more enriched in genes associated with metabolism (e.g., energy production and conversion, and carbohydrate transport and metabolism) than pooled dispensable and singleton genes in other L. lactis strains, reflecting WFLU12 strain-specific ecosystem origin and its ability to metabolize different energy sources. Strain WFLU12 produced antimicrobial compounds that could inhibit several bacterial fish pathogens. It possessed the nisin gene cluster (nisZBTCIPRKFEG) and genes encoding lysozyme and colicin V. However, only three other strains (CV56, IO-1, and SO) harbor a complete nisin gene cluster. We also found that L. lactis WFLU12 possessed many other important functional genes involved in stress responses to the gastrointestinal tract environment, dietary energy extraction, and metabolism to support the probiotic action of this strain found in our previous study. This strongly indicates that not all L. lactis strains can be used as probiotics. This study highlights comparative genomics approaches as very useful and powerful tools to select probiotic candidates and predict their probiotic effects.

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September 22, 2019  |  

Genetic and molecular basis of the immune system in the brachiopod Lingula anatina.

The extension of comparative immunology to non-model systems, such as mollusks and annelids, has revealed an unexpected diversity in the complement of immune receptors and effectors among evolutionary lineages. However, several lophotrochozoan phyla remain unexplored mainly due to the lack of genomic resources. The increasing accessibility of high-throughput sequencing technologies offers unique opportunities for extending genome-wide studies to non-model systems. As a result, the genome-based study of the immune system in brachiopods allows a better understanding of the alternative survival strategies developed by these immunologically neglected phyla. Here we present a detailed overview of the molecular components of the immune system identified in the genome of the brachiopod Lingula anatina. Our findings reveal conserved intracellular signaling pathways as well as unique strategies for pathogen detection and killing in brachiopods. Copyright © 2017 Elsevier Ltd. All rights reserved.

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September 22, 2019  |  

In silico exploration of Red Sea Bacillus genomes for natural product biosynthetic gene clusters.

The increasing spectrum of multidrug-resistant bacteria is a major global public health concern, necessitating discovery of novel antimicrobial agents. Here, members of the genus Bacillus are investigated as a potentially attractive source of novel antibiotics due to their broad spectrum of antimicrobial activities. We specifically focus on a computational analysis of the distinctive biosynthetic potential of Bacillus paralicheniformis strains isolated from the Red Sea, an ecosystem exposed to adverse, highly saline and hot conditions.We report the complete circular and annotated genomes of two Red Sea strains, B. paralicheniformis Bac48 isolated from mangrove mud and B. paralicheniformis Bac84 isolated from microbial mat collected from Rabigh Harbor Lagoon in Saudi Arabia. Comparing the genomes of B. paralicheniformis Bac48 and B. paralicheniformis Bac84 with nine publicly available complete genomes of B. licheniformis and three genomes of B. paralicheniformis, revealed that all of the B. paralicheniformis strains in this study are more enriched in nonribosomal peptides (NRPs). We further report the first computationally identified trans-acyltransferase (trans-AT) nonribosomal peptide synthetase/polyketide synthase (PKS/ NRPS) cluster in strains of this species.B. paralicheniformis species have more genes associated with biosynthesis of antimicrobial bioactive compounds than other previously characterized species of B. licheniformis, which suggests that these species are better potential sources for novel antibiotics. Moreover, the genome of the Red Sea strain B. paralicheniformis Bac48 is more enriched in modular PKS genes compared to B. licheniformis strains and other B. paralicheniformis strains. This may be linked to adaptations that strains surviving in the Red Sea underwent to survive in the relatively hot and saline ecosystems.

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September 22, 2019  |  

Isolation and characterization of Bacillus sp. GFP-2, a novel Bacillus strain with antimicrobial activities, from Whitespotted bamboo shark intestine.

The abuse of antibiotics and following rapidly increasing of antibiotic-resistant pathogens is the serious threat to our society. Natural products from microorganism are regarded as the important substitution antimicrobial agents of antibiotics. We isolated a new strain, Bacillus sp. GFP-2, from the Chiloscyllium plagiosum (Whitespotted bamboo shark) intestine, which showed great inhibitory effects on the growth of both Gram-positive and Gram-negative bacteria. Additionally, the growth of salmon was effectively promoted when fed with inactivated strain GFP-2 as the inhibition agent of pathogenic bacteria. The genes encoding antimicrobial peptides like LCI, YFGAP and hGAPDH and gene clusters for secondary metabolites and bacteriocins, such as difficidin, bacillibactin, bacilysin, surfactin, butirosin, macrolactin, bacillaene, fengycin, lanthipeptides and LCI, were predicted in the genome of Bacillus sp. GFP-2, which might be expressed and contribute to the antimicrobial activities of this strain. The gene encoding ß-1,3-1,4-glucanase was successfully cloned from the genome and this protein was detected in the culture supernatant of Bacillus sp. GFP-2 by the antibody produced in rabbit immunized with the recombinant ß-1,3-1,4-glucanase, indicating that this strain could express ß-1,3-1,4-glucanase, which might partially contribute to its antimicrobial activities. This study can enhance a better understanding of the mechanism of antimicrobial activities in genus Bacillus and provide a useful material for the biotechnology study in antimicrobial agent development.

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September 22, 2019  |  

Genomic and probiotic characterization of SJP-SNU strain of Pichia kudriavzevii.

The yeast strain SJP-SNU was investigated as a probiotic and was characterized with respect to growth temperature, bile salt resistance, hydrogen sulfide reducing activity, intestinal survival ability and chicken embryo pathogenicity. In addition, we determined the complete genomic and mitochondrial sequences of SJP-SNU and conducted comparative genomics analyses. SJP-SNU grew rapidly at 37 °C and formed colonies on MacConkey agar containing bile salt. SJP-SNU reduced hydrogen sulfide produced by Salmonella serotype Enteritidis and, after being fed to 4-week-old chickens, could be isolated from cecal feces. SJP-SNU did not cause mortality in 10-day-old chicken embryos. From 13 initial contigs, 11 were finally assembled and represented 10 chromosomal sequences and 1 mitochondrial DNA sequence. Comparative genomic analyses revealed that SJP-SNU was a strain of Pichia kudriavzevii. Although SJP-SNU possesses pathogenicity-related genes, they showed very low amino acid sequence identities to those of Candida albicans. Furthermore, SJP-SNU possessed useful genes, such as phytases and cellulase. Thus, SJP-SNU is a useful yeast possessing the basic traits of a probiotic, and further studies to demonstrate its efficacy as a probiotic in the future may be warranted.

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September 22, 2019  |  

Mycobacterial biomaterials and resources for researchers.

There are many resources available to mycobacterial researchers, including culture collections around the world that distribute biomaterials to the general scientific community, genomic and clinical databases, and powerful bioinformatics tools. However, many of these resources may be unknown to the research community. This review article aims to summarize and publicize many of these resources, thus strengthening the quality and reproducibility of mycobacterial research by providing the scientific community access to authenticated and quality-controlled biomaterials and a wealth of information, analytical tools and research opportunities.

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