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Asset Tag: Industrial biotechnology

April 21, 2020

Acid stress response of Staphylococcus xylosus elicits changes in the proteome and cellular membrane.

Coagulase-negative Staphylococcus xylosus strains are used as starter organisms for sausage fermentation. As those strains have to cope with low pH-values during fermentation, the aim of this study was to identify the acid adaptation mechanisms of S. xylosus TMW 2.1523 previously isolated from salami.A comparative proteomic study between two different acid tolerant mutants was performed. Therefore, both S. xylosus mutants were grown pH-static under acid stress (pH 5·1) and reference conditions (pH 7·0). Proteomic data were supported by metabolite and cell membrane lipid analysis. Staphylococcus xylosus acid stress adaptation is mainly characterized by a metabolic change towards neutral metabolites, enhanced urease activity, reduced ATP consumption, an increase in membrane fluidity and changes in the membrane thickness.This study corroborates mechanisms as previously described for other Gram-positive bacteria. Additionally, the adjustment of membrane structure and composition in S. xylosus TMW 2.1523 play a prominent role in its acid adaptation.This study demonstrates for the first time changes in the membrane lipid composition due to acid stress adaptation in staphylococci. © 2019 The Society for Applied Microbiology.

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April 21, 2020

A physical and genetic map of Cannabis sativa identifies extensive rearrangements at the THC/CBD acid synthase loci.

Cannabis sativa is widely cultivated for medicinal, food, industrial, and recreational use, but much remains unknown regarding its genetics, including the molecular determinants of cannabinoid content. Here, we describe a combined physical and genetic map derived from a cross between the drug-type strain Purple Kush and the hemp variety “Finola.” The map reveals that cannabinoid biosynthesis genes are generally unlinked but that aromatic prenyltransferase (AP), which produces the substrate for THCA and CBDA synthases (THCAS and CBDAS), is tightly linked to a known marker for total cannabinoid content. We further identify the gene encoding CBCA synthase (CBCAS) and characterize its catalytic activity, providing insight into how cannabinoid diversity arises in cannabis. THCAS and CBDAS (which determine the drug vs. hemp chemotype) are contained within large (>250 kb) retrotransposon-rich regions that are highly nonhomologous between drug- and hemp-type alleles and are furthermore embedded within ~40 Mb of minimally recombining repetitive DNA. The chromosome structures are similar to those in grains such as wheat, with recombination focused in gene-rich, repeat-depleted regions near chromosome ends. The physical and genetic map should facilitate further dissection of genetic and molecular mechanisms in this commercially and medically important plant. © 2019 Laverty et al.; Published by Cold Spring Harbor Laboratory Press.

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April 21, 2020

Depiction of secondary metabolites and antifungal activity of Bacillus velezensis DTU001.

For a safe and sustainable environment, effective microbes as biocontrol agents are in high demand. We have isolated a new Bacillus velezensis strain DTU001, investigated its antifungal spectrum, sequenced its genome, and uncovered the production of lipopeptides in HPLC-HRMS analysis. To test the antifungal efficacy, extracts of B. velezensis DTU001 was tested against a range of twenty human or plant pathogenic fungi. We demonstrate that inhibitory potential of B. velezensis DTU001 against selected fungi is superior in comparison to single lipopeptide, either iturin or fengycin. The isolate showed analogous biofilm formation to other closely related Bacilli. To further support the biocontrol properties of the isolate, coculture with Candida albicans demonstrated that B. velezensis DTU001 exhibited excellent antiproliferation effect against C. albicans. In summary, the described isolate is a potential antifungal agent with a broad antifungal spectrum that might assist our aims to avoid hazardous pathogenic fungi and provide alternative to toxicity caused by chemicals.

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April 21, 2020

Mitochondrial genome of the entomophthoroid fungus Conidiobolus heterosporus provides insights into evolution of basal fungi.

Entomophthoroid fungi represent an ecologically important group of fungal pathogens on insects. Here, the whole mitogenome of Conidiobolus heterosporus, one of the entomophthoroid fungi, was described and compared to those early branching fungi with available mitogenomes. The 53,364-bp circular mitogenome of C. heterosporus contained two rRNA genes, 14 standard protein-coding genes, 26 tRNA genes, and three free-standing ORFs. Thirty introns interrupted nine mitochondrial genes. Phylogenetic analysis based on mitochondrion-encoded proteins revealed that C. heterosporus was most close to Zancudomyces culisetae in the Zoopagomycota of basal fungi. Comparison on mitogenomes of 23 basal fungi revealed great variabilities in terms of mitogenome conformation (circular or linear), genetic code (codes 1, 4, or 16), AT contents (53.3-85.5%), etc. These mitogenomes varied from 12.0 to 97.3 kb in sizes, mainly due to different numbers of genes and introns. They showed frequent DNA rearrangement events and a high variability of gene order, although high synteny and conserved gene order were also present between closely related species. By reporting the first mitogenome in Entomophthoromycotina and the second in Zoopagomycota, this study greatly enhanced our understanding on evolution of basal fungi.

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April 21, 2020

Identification and characterisation of anti – Pseudomonas aeruginosa proteins in mucus of the brown garden snail, Cornu aspersum.

Background: Novel antimicrobial treatments are urgently needed. Previous work has shown that the mucus of the brown garden snail (Cornu aspersum) has antimicrobial properties, in particular against type culture collection strains of Pseudomonas aeruginosa. We hypothesised that it would also be effective against clinical isolates of the bacterium and that investigation of fractions of the mucus would identify one or more proteins with anti-pseudomonal properties, which could be further characterised. Materials and methods: Mucus was extracted from snails collected from the wild. Antimicrobial activity against laboratory and clinical isolates of Ps. aeruginosa was determined in disc diffusion assays. Mucus was purified using size exclusion chromatography and fractions containing anti-pseudomonal activity identified. Mass spectroscopy and high performance liquid chromatography analysis of these fractions yielded partial peptide sequences. These were used to interrogate an RNA transcriptome generated from whole snails. Results: Mucus from C. aspersum inhibited growth of type collection strains and clinical isolates of Ps. aeruginosa. Four novel C. aspersum proteins were identified; at least three are likely to have antimicrobial properties. The most interesting is a 37.4 kDa protein whilst smaller proteins, one 17.5 kDa and one 18.6 kDa also appear to have activity against Ps. aeruginosa.Conclusions: The study has identified novel proteins with antimicrobial properties which could be used to develop treatments for use in human medicine.

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April 21, 2020

The complete mitochondrial genome sequences of Senna tora (Fabales: Fabaceae)

Cassia tora (Senna tora), known as an economically important plant, is medicinal in nature and belongs to the Fabaceae family. The complete mitochondrial genome sequences of S. tora were 566,589bp in length with a 45.23% GC content. A total of 63 genes were annotated including 36 protein-coding genes, 22 tRNA genes, and 5 rRNA genes. Phylogenetic tree based on the mitochondrial genome dem- onstrated that S. tora was most closely related to the Senna occidentalis and Caesalpinioideae subfamily and is definitely separated from the Faboideae subfamily.

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April 21, 2020

PacBio sequencing reveals bacterial community diversity in cheeses collected from different regions.

Cheese is a fermented dairy product that is popular for its unique flavor and nutritional value. Recent studies have shown that microorganisms in cheese play an important role in the fermentation process and determine the quality of the cheese. We collected 12 cheese samples from different regions and studied the composition of their bacterial communities using PacBio small-molecule real-time sequencing (Pacific Biosciences, Menlo Park, CA). Our data revealed 144 bacterial genera (including Lactobacillus, Streptococcus, Lactococcus, and Staphylococcus) and 217 bacterial species (including Lactococcus lactis, Streptococcus thermophilus, Staphylococcus equorum, and Streptococcus uberis). We investigated the flavor quality of the cheese samples using an electronic nose system and we found differences in flavor-quality indices among samples from different regions. We found a clustering tendency based on flavor quality using principal component analysis. We found correlations between lactic acid bacteria and the flavor quality of the cheese samples. Biodegradation and metabolism of xenobiotics, and lipid-metabolism-related pathways, were predicted to contribute to differences in cheese flavor using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt). This preliminary study explored the bacterial communities in cheeses collected from different regions and their potential genome functions from the perspective of flavor quality.Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

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April 21, 2020

Effect of sulfur-iron modified biochar on the available cadmium and bacterial community structure in contaminated soils.

Cadmium contamination in paddy soils has aroused increasing concern around the world, and biochar has many positive properties, such as large specific surface areas, micro porous structure for the heavy metal immobilization in soils. However there are few studies on sulfur-iron modified biochar as well as its microbiology effects. The purpose of this study was to evaluate the Cd immobilization effects of sulfur or sulfur-iron modified biochar and its related microbial community changes in Cd-contaminated soils. SEM-EDX analysis confirmed that sulfur and iron were loaded on the raw biochar successfully. Sulfur-modified biochar (S-BC) and sulfur-iron modified biochar (SF-BC) addition increased pH value and the content of soil organic matter, and also decreased DTPA-extractable Cd. There was a negative significant correlation between organic matter content and the available Cd (P?

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April 21, 2020

Isarubrolones Containing a Pyridooxazinium Unit from Streptomyces as Autophagy Activators.

Isarubrolones are bioactive polycyclic tropoloalkaloids from Streptomyces. Three new isarubrolones (2-4), together with the known isarubrolone C (1) and isatropolones A (5) and C (6, 3( R)-hydroxyisatropolone A), were identified from Streptomyces sp. CPCC 204095. The structures of these compounds were determined using a combination of mass spectrometry, 1D and 2D NMR spectroscopy, and ECD. Compounds 3 and 4 feature a pyridooxazinium unit, which is rarely seen in natural products. Compound 6 could conjugate with amino acids or amines to expand the structural diversity of isarubrolones with a pentacyclic or hexacyclic core. Importantly, 1 and 3-6 were found to induce complete autophagy.

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April 21, 2020

Identifying the Biosynthetic Gene Cluster for Triacsins with an N-Hydroxytriazene Moiety.

Triacsins are a family of natural products having in common an N-hydroxytriazene moiety not found in any other known secondary metabolites. Though many studies have examined the biological activity of triacsins in lipid metabolism, their biosynthesis has remained unknown. Here we report the identification of the triacsin biosynthetic gene cluster in Streptomyces aureofaciens ATCC 31442. Bioinformatic analysis of the gene cluster led to the discovery of the tacrolimus producer Streptomyces tsukubaensis NRRL 18488 as a new triacsin producer. In addition to targeted gene disruption to identify necessary genes for triacsin production, stable isotope feeding was performed in vivo to advance the understanding of N-hydroxytriazene biosynthesis. © 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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April 21, 2020

Functional genomics of the rapidly replicating bacterium Vibrio natriegens by CRISPRi.

The fast-growing Gram-negative bacterium Vibrio natriegens is an attractive microbial system for molecular biology and biotechnology due to its remarkably short generation time1,2 and metabolic prowess3,4. However, efforts to uncover and utilize the mechanisms underlying its rapid growth are hampered by the scarcity of functional genomic data. Here, we develop a pooled genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) screen to identify a minimal set of genes required for rapid wild-type growth. Targeting 4,565 (99.7%) of predicted protein-coding genes, our screen uncovered core genes comprising putative essential and growth-supporting genes that are enriched for respiratory pathways. We found that 96% of core genes were located on the larger chromosome 1, with growth-neutral duplicates of core genes located primarily on chromosome 2. Our screen also refines metabolic pathway annotations by distinguishing functional biosynthetic enzymes from those predicted on the basis of comparative genomics. Taken together, this work provides a broadly applicable platform for high-throughput functional genomics to accelerate biological studies and engineering of V. natriegens.

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April 21, 2020

Combining orthogonal CRISPR and CRISPRi systems for genome engineering and metabolic pathway modulation in Escherichia coli.

CRISPR utilizing Cas9 from Streptococcus pyogenes (SpCas9) and CRISPR interference (CRISPRi) employing catalytically inactive SpCas9 (SpdCas9) have gained popularity for Escherichia coli engineering. To integrate the SpdCas9-based CRISPRi module using CRISPR while avoiding mutual interference between SpCas9/SpdCas9 and their cognate single-guide RNA (sgRNA), this study aimed at exploring an alternative Cas nuclease orthogonal to SpCas9. We compared several Cas9 variants from different microorganisms such as Staphylococcus aureus (SaCas9) and Streptococcus thermophilius CRISPR1 (St1Cas9) as well as Cas12a derived from Francisella novicida (FnCas12a). At the commonly used E. coli model genes  LacZ, we found that SaCas9 and St1Cas9 induced DNA cleavage more effectively than FnCas12a. Both St1Cas9 and SaCas9 were orthogonal to SpCas9 and the induced DNA cleavage promoted the integration of heterologous DNA of up to 10?kb, at which size St1Cas9 was superior to SaCas9 in recombination frequency/accuracy. We harnessed the St1Cas9 system to integrate SpdCas9 and sgRNA arrays for constitutive knockdown of three genes, knock-in pyc and knockout adhE, without compromising the CRISPRi knockdown efficiency. The combination of orthogonal CRISPR/CRISPRi for metabolic engineering enhanced succinate production while inhibiting byproduct formation and may pave a new avenue to E. coli engineering. © 2019 Wiley Periodicals, Inc.

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April 21, 2020

Isolation, cloning and characterization of an azoreductase and the effect of salinity on its expression in a halophilic bacterium.

Understanding the molecular mechanisms of azo dye decolorization is important for the development of effective bioremediation for textile-colored wastewater. A halophilic bacterium Halomonas sp. strain GT was isolated, which could degrade the azo dye Acid Brilliant Scarlet GR at 10% NaCl. The complete genome sequence of this strain was obtained using the PacBio RS II platform. Genome annotation revealed that four proteins are related to decolorization of azo dyes, such as azoreductase, laccases, benzene 1,2-dioxygenase, and catechol 1,2-dioxygenase. The putative azoreductase gene of Halomonas sp. strain GT responsible for the decolorization of azo dye in high salt environment was isolated. Phylogenetic tree analysis showed that the azoG (azoreductase gene of Halomonas sp. strain GT) and its homologs constituted a new branch of the NADH depending azoreductases, with all the homologous sequence of the protein from halophilic bacteria. At high NaCl concentrations, azoreductase gene expression and azoreductase activity were restrained in Halomonas sp. strain GT, which resulted in low a decolorization rate. Copyright © 2018. Published by Elsevier B.V.

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April 21, 2020

Comparative Genome Characterization of a Petroleum-Degrading Bacillus subtilis Strain DM2.

The complete genome sequence of Bacillus subtilis strain DM2 isolated from petroleum-contaminated soil on the Tibetan Plateau was determined. The genome of strain DM2 consists of a circular chromosome of 4,238,631 bp for 4458 protein-coding genes and a plasmid of 84,240 bp coding for 103 genes. Thirty-four genomic islands coding for 330 proteins and 5 prophages are found in the genome. The DDH value shows that strain DM2 belongs to B. subtilis subsp. subtilis subspecies, but significant variations of the genome are also present. Comparative analysis showed that the genome of strain DM2 encodes some strain-specific proteins in comparison with B. subtilis subsp. subtilis str. 168, such as carboxymuconolactone decarboxylase family protein, gfo/Idh/MocA family oxidoreductases, GlsB/YeaQ/YmgE family stress response membrane protein, HlyC/CorC family transporters, LLM class flavin-dependent oxidoreductase, and LPXTG cell wall anchor domain-containing protein. Most of the common strain-specific proteins in DM2 and MJ01 strains, or proteins unique to DM2 strain, are involved in the pathways related to stress response, signaling, and hydrocarbon degradation. Furthermore, the strain DM2 genome contains 122 genes coding for developed two-component systems and 138 genes coding for ABC transporter systems. The prominent features of the strain DM2 genome reflect the evolutionary fitness of this strain to harsh conditions and hydrocarbon utilization.

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April 21, 2020

Identification of plasmid encoded osmoregulatory genes from halophilic bacteria isolated from the rhizosphere of halophytes.

Bacterial plasmids carry genes that code for additional traits such as osmoregulation, CO2 fixation, antibiotic and heavy metal resistance, root nodulation and nitrogen fixation. The main objective of the current study was to identify plasmid-conferring osmoregulatory genes in bacteria isolated from rhizospheric and non-rhizospheric soils of halophytes (Salsola stocksii and Atriplex amnicola). More than 55% of halophilic bacteria from the rhizosphere and 70% from non-rhizospheric soils were able to grow at 3?M salt concentrations. All the strains showed optimum growth at 1.5-3.0?M NaCl. Bacterial strains from the Salsola rhizosphere showed maximum (31%) plasmid elimination during curing experiments as compared to bacterial strains from the Atriplex rhizosphere and non-rhizospheric soils. Two plasmid cured strains Bacillus HL2HP6 and Oceanobacillus HL2RP7 lost their ability to grow in halophilic medium, but they grew well on LB medium. The plasmid cured strains also showed a change in sensitivity to specific antibiotics. These plasmids were isolated and transformed into E. coli strains and growth response of wild-type and transformed E. coli strains was compared at 1.5-4?M NaCl concentrations. Chromosomal DNA and plasmids from Bacillus filamentosus HL2HP6 were sequenced by using high throughput sequencing approach. Results of functional analysis of plasmid sequences showed different proteins and enzymes involved in osmoregulation of bacteria, such as trehalose, ectoine synthetase, porins, proline, alanine, inorganic ion transporters, dehydrogenases and peptidases. Our results suggested that plasmid conferring osmoregulatory genes play a vital role to maintain internal osmotic balance of bacterial cells and these genes can be used to develop salt tolerant transgenic crops.Copyright © 2019 Elsevier GmbH. All rights reserved.

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