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September 22, 2019

Identification of the biosynthetic pathway for the antibiotic bicyclomycin.

Diketopiperazines (DKPs) make up a large group of natural products with diverse structures and biological activities. Bicyclomycin is a broad-spectrum DKP antibiotic with unique structure and function: it contains a highly oxidized bicyclic [4.2.2] ring and is the only known selective inhibitor of the bacterial transcription termination factor, Rho. Here, we identify the biosynthetic gene cluster for bicyclomycin containing six iron-dependent oxidases. We demonstrate that the DKP core is made by a tRNA-dependent cyclodipeptide synthase, and hydroxylations on two unactivated sp(3) carbons are performed by two mononuclear iron, a-ketoglutarate-dependent hydroxylases. Using bioinformatics, we also identify a homologous gene cluster prevalent in a human pathogen Pseudomonas aeruginosa. We detect bicyclomycin by overexpressing this gene cluster and establish P. aeruginosa as a new producer of bicyclomycin. Our work uncovers the biosynthetic pathway for bicyclomycin and sheds light on the intriguing oxidation chemistry that converts a simple DKP into a powerful antibiotic.

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September 22, 2019

Revisiting the contribution of gene duplication of blaOXA-23 in carbapenem-resistant Acinetobacter baumannii.

Gene duplication has been discovered for many antimicrobial resistance genes in bacterial genomes and has been considered a source of elevated antimicrobial resistance.1 The gene blaOXA-23is a major determinant in the emergence of carbapenem-resistant Acinetobacter baumannii (CRAB).2–4 We have previously reported the widespread duplication of blaOXA-23by surveying 113 clinical CRAB isolates in China.5 However, in these isolates the blaOXA-23 copy number did not correlate well with the MIC of imipenem. A similar phenomenon was also reported recently by Yoon et al.6 One reasonable explanation is that, in addition to gene duplica- tions, other mechanisms might also impact on the MIC, such as the presence of specific outer membrane proteins and/ortheover-expression of resistance–nodulation–division (RND)-type efflux pumps.7 Often, these mechanisms might vary in their performance when in different genomic contexts. Instead of making comparisons between clinical isolates, in this study we cultured A. baumannii under treatment with carbapenem, thus avoiding any interference induced in different genomic contexts. If an increase in the blaOXA-23 copy number or MIC were to occur within the same strain, the contribution of gene duplication to carbapenem resistance would be acknowledged.

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September 22, 2019

Extensively drug-resistant Escherichia coli sequence type 1642 carrying an IncX3 plasmid containing the blaKPC-2 gene associated with transposon Tn4401a.

Extensively drug-resistant (XDR) Enterobacteriaceae carrying the bla(KPC) gene have emerged as a major global therapeutic concern. The purpose of this study was to analyze the complete sequences of plasmids from KPC-2 carbapenemase-producing XDR Escherichia coli sequence type (ST) 1642 isolates.We performed antimicrobial susceptibility testing, PCR, multilocus sequence typing (MLST), and whole-genome sequencing to characterize the plasmid-mediated KPC-2-producing E. coli clinical isolates.The isolates were resistant to most available antibiotics, including meropenem, ampicillin, ceftriaxone, gentamicin, and ciprofloxacin, but susceptible to tigecycline and colistin. The isolates were identified as the rare ST1642 by MLST. The isolates carried four plasmids: the first 69-kb conjugative IncX3 plasmid harbors bla(KPC-2) within a truncated Tn4401a transposon and bla(SHV-11) with duplicated conjugative elements. The second 142-kb plasmid with a multireplicon consisting of IncQ, IncFIA, and IncIB carries bla(TEM-1b) and two class 1 integrons. This plasmid also harbors a wide variety of additional antimicrobial resistance genes including aadA5, dfrA17, mph(A), sul1, tet(B), aac(3′)-IId, strA, strB, and sul2.The complete sequence analysis of plasmids from an XDR E. coli strain related to persistent infection showed the coexistence of a bla(KPC-2)-carrying IncX3-type plasmid and a class 1 integron-harboring multireplicon, suggesting its potential to cause outbreaks. Of additional clinical significance, the rare ST1642, identified in a cat, could constitute the source of human infection.

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September 22, 2019

Identification of a biosynthetic gene cluster for the polyene macrolactam sceliphrolactam in a Streptomyces strain isolated from mangrove sediment.

Streptomyces are a genus of Actinobacteria capable of producing structurally diverse natural products. Here we report the isolation and characterization of a biosynthetically talented Streptomyces (Streptomyces sp. SD85) from tropical mangrove sediments. Whole-genome sequencing revealed that Streptomyces sp. SD85 harbors at least 52 biosynthetic gene clusters (BGCs), which constitute 21.2% of the 8.6-Mb genome. When cultivated under lab conditions, Streptomyces sp. SD85 produces sceliphrolactam, a 26-membered polyene macrolactam with unknown biosynthetic origin. Genome mining yielded a putative sceliphrolactam BGC (sce) that encodes a type I modular polyketide synthase (PKS) system, several ß-amino acid starter biosynthetic enzymes, transporters, and transcriptional regulators. Using the CRISPR/Cas9-based gene knockout method, we demonstrated that the sce BGC is essential for sceliphrolactam biosynthesis. Unexpectedly, the PKS system encoded by sce is short of one module required for assembling the 26-membered macrolactam skeleton according to the collinearity rule. With experimental data disfavoring the involvement of a trans-PKS module, the biosynthesis of sceliphrolactam seems to be best rationalized by invoking a mechanism whereby the PKS system employs an iterative module to catalyze two successive chain extensions with different outcomes. The potential violation of the collinearity rule makes the mechanism distinct from those of other polyene macrolactams.

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September 22, 2019

Packaging of Dinoroseobacter shibae DNA into gene transfer agent particles is not random.

Gene transfer agents (GTAs) are phage-like particles which contain a fragment of genomic DNA of the bacterial or archaeal producer and deliver this to a recipient cell. GTA gene clusters are present in the genomes of almost all marine Rhodobacteraceae (Roseobacters) and might be important contributors to horizontal gene transfer in the world’s oceans. For all organisms studied so far, no obvious evidence of sequence specificity or other nonrandom process responsible for packaging genomic DNA into GTAs has been found. Here, we show that knock-out of an autoinducer synthase gene of Dinoroseobacter shibae resulted in overproduction and release of functional GTA particles (DsGTA). Next-generation sequencing of the 4.2-kb DNA fragments isolated from DsGTAs revealed that packaging was not random. DNA from low-GC conjugative plasmids but not from high-GC chromids was excluded from packaging. Seven chromosomal regions were strongly overrepresented in DNA isolated from DsGTA. These packaging peaks lacked identifiable conserved sequence motifs that might represent recognition sites for the GTA terminase complex. Low-GC regions of the chromosome, including the origin and terminus of replication, were underrepresented in DNA isolated from DsGTAs. DNA methylation reduced packaging frequency while the level of gene expression had no influence. Chromosomal regions found to be over- and underrepresented in DsGTA-DNA were regularly spaced. We propose that a “headful” type of packaging is initiated at the sites of coverage peaks and, after linearization of the chromosomal DNA, proceeds in both directions from the initiation site. GC-content, DNA-modifications, and chromatin structure might influence at which sides GTA packaging can be initiated.© The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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September 22, 2019

Comparative genome and phenotypic analysis of three Clostridioides difficile strains isolated from a single patient provide insight into multiple infection of C. difficile.

Clostridioides difficile infections (CDI) have emerged over the past decade causing symptoms that range from mild, antibiotic-associated diarrhea (AAD) to life-threatening toxic megacolon. In this study, we describe a multiple and isochronal (mixed) CDI caused by the isolates DSM 27638, DSM 27639 and DSM 27640 that already initially showed different morphotypes on solid media.The three isolates belonging to the ribotypes (RT) 012 (DSM 27639) and 027 (DSM 27638 and DSM 27640) were phenotypically characterized and high quality closed genome sequences were generated. The genomes were compared with seven reference strains including three strains of the RT 027, two of the RT 017, and one of the RT 078 as well as a multi-resistant RT 012 strain. The analysis of horizontal gene transfer events revealed gene acquisition incidents that sort the strains within the time line of the spread of their RTs within Germany. We could show as well that horizontal gene transfer between the members of different RTs occurred within this multiple infection. In addition, acquisition and exchange of virulence-related features including antibiotic resistance genes were observed. Analysis of the two genomes assigned to RT 027 revealed three single nucleotide polymorphisms (SNPs) and apparently a regional genome modification within the flagellar switch that regulates the fli operon.Our findings show that (i) evolutionary events based on horizontal gene transfer occur within an ongoing CDI and contribute to the adaptation of the species by the introduction of new genes into the genomes, (ii) within a multiple infection of a single patient the exchange of genetic material was responsible for a much higher genome variation than the observed SNPs.

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September 22, 2019

The non-specific adenine DNA methyltransferase M.EcoGII.

We describe the cloning, expression and characterization of the first truly non-specific adenine DNA methyltransferase, M.EcoGII. It is encoded in the genome of the pathogenic strain Escherichia coli O104:H4 C227-11, where it appears to reside on a cryptic prophage, but is not expressed. However, when the gene encoding M.EcoGII is expressed in vivo – using a high copy pRRS plasmid vector and a methylation-deficient E. coli host-extensive in vivo adenine methylation activity is revealed. M.EcoGII methylates adenine residues in any DNA sequence context and this activity extends to dA and rA bases in either strand of a DNA:RNA-hybrid oligonucleotide duplex and to rA bases in RNAs prepared by in vitro transcription. Using oligonucleotide and bacteriophage M13mp18 virion DNA substrates, we find that M.EcoGII also methylates single-stranded DNA in vitro and that this activity is only slightly less robust than that observed using equivalent double-stranded DNAs. In vitro assays, using purified recombinant M.EcoGII enzyme, demonstrate that up to 99% of dA bases in duplex DNA substrates can be methylated thereby rendering them insensitive to cleavage by multiple restriction endonucleases. These properties suggest that the enzyme could also be used for high resolution mapping of protein binding sites in DNA and RNA substrates.© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

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September 22, 2019

Comparative genomics and transcriptome analysis of Lactobacillus rhamnosus ATCC 11443 and the mutant strain SCT-10-10-60 with enhanced L-lactic acid production capacity.

Mechanisms for high L-lactic acid production remain unclear in many bacteria. Lactobacillus rhamnosus SCT-10-10-60 was previously obtained from L. rhamnosus ATCC 11443 via mutagenesis and showed improved L-lactic acid production. In this study, the genomes of strains SCT-10-10-60 and ATCC 11443 were sequenced. Both genomes are a circular chromosome, 2.99 Mb in length with a GC content of approximately 46.8%. Eight split genes were identified in strain SCT-10-10-60, including two LytR family transcriptional regulators, two Rex redox-sensing transcriptional repressors, and four ABC transporters. In total, 60 significantly up-regulated genes (log2fold-change?=?2) and 39 significantly down-regulated genes (log2fold-change?=?-?2) were identified by a transcriptome comparison between strains SCT-10-10-60 and ATCC 11443. KEGG pathway enrichment analysis revealed that “pyruvate metabolism” was significantly different (P?

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September 22, 2019

Genomic diversity in the endosymbiotic bacterium Rhizobium leguminosarum.

Rhizobium leguminosarum bv. viciae is a soil a-proteobacterium that establishes a diazotrophic symbiosis with different legumes of the Fabeae tribe. The number of genome sequences from rhizobial strains available in public databases is constantly increasing, although complete, fully annotated genome structures from rhizobial genomes are scarce. In this work, we report and analyse the complete genome of R. leguminosarum bv. viciae UPM791. Whole genome sequencing can provide new insights into the genetic features contributing to symbiotically relevant processes such as bacterial adaptation to the rhizosphere, mechanisms for efficient competition with other bacteria, and the ability to establish a complex signalling dialogue with legumes, to enter the root without triggering plant defenses, and, ultimately, to fix nitrogen within the host. Comparison of the complete genome sequences of two strains of R. leguminosarum bv. viciae, 3841 and UPM791, highlights the existence of different symbiotic plasmids and a common core chromosome. Specific genomic traits, such as plasmid content or a distinctive regulation, define differential physiological capabilities of these endosymbionts. Among them, strain UPM791 presents unique adaptations for recycling the hydrogen generated in the nitrogen fixation process.

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September 22, 2019

Multi-heme cytochromes provide a pathway for survival in energy-limited environments.

Bacterial reduction of oxidized sulfur species (OSS) is critical for energy production in anaerobic marine subsurfaces. In organic-poor sediments, H2 has been considered as a major energy source for bacterial respiration. We identified outer-membrane cytochromes (OMCs) that are broadly conserved in sediment OSS-respiring bacteria and enable cells to directly use electrons from insoluble minerals via extracellular electron transport. Biochemical, transcriptomic, and microscopic analyses revealed that the identified OMCs were highly expressed on the surface of cells and nanofilaments in response to electron donor limitation. This electron uptake mechanism provides sufficient but minimum energy to drive the reduction of sulfate and other OSS. These results suggest a widespread mechanism for survival of OSS-respiring bacteria via electron uptake from solid minerals in energy-poor marine sediments.

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September 22, 2019

Characterization of the SN35N strain-specific exopolysaccharide encoded in the whole circular genome of a plant-derived Lactobacillus plantarum.

Lactobacillus plantarum SN35N, which has been previously isolated from pear, secretes exopolysaccharide (EPS). The aim of the present study is to characterize the EPS chemically and to find the EPS-biosynthesizing gene cluster. The present study demonstrates that the strain produces an acidic EPS carrying phosphate residue, which is composed of glucose, galactose, and mannose at a molecular ratio of 15.0?:?5.7?:?1.0. We also show that acidic EPS strongly inhibits the catalytic activity of hyaluronidase (EC 3.2.1.35), promoting an inflammatory reaction. In the present study, we also determined the complete genome sequence of the SN35N strain, demonstrating that the genome is a circular DNA with 3267626?bp, and the number of predicted coding genes is 3146, with a GC content of 44.51%. In addition, the strain harbors four plasmids, designated pSN35N-1, -2, -3, and -4. Although four EPS-biosynthesizing genes, designated lpe1, lpe2, lpe3, and lpe4, are present in the SN35N chromosomal DNA, another EPS gene cluster, lpe5, is located in the pSN35N-3 plasmid, composed of 35425?bp. EPS low-producing mutants, which were obtained by treating SN35N cells with novobiocin, lost the lpe5 gene cluster in the plasmid-curing experiment, suggesting that the gene cluster for the biosynthesis of acidic EPS is present in the plasmid. The present study shows the chemical characterization of the acidic EPS and its inhibitory effect to the hyaluronidase.

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September 22, 2019

Re-classification of Clavibacter michiganensis subspecies on the basis of whole-genome and multi-locus sequence analyses.

Although the genus Clavibacter was originally proposed to accommodate all phytopathogenic coryneform bacteria containing B2? diaminobutyrate in the peptidoglycan, reclassification of all but one species into other genera has resulted in the current monospecific status of the genus. The single species in the genus, Clavibacter michiganensis, has multiple subspecies, which are all highly host-specific plant pathogens. Whole genome analysis based on average nucleotide identity and digital DNA-DNA hybridization as well as multi-locus sequence analysis (MLSA) of seven housekeeping genes support raising each of the C. michiganensis subspecies to species status. On the basis of whole genome and MLSA data, we propose the establishment of two new species and three new combinations: Clavibacter capsici sp. nov., comb. nov. and Clavibacter tessellarius sp. nov., comb. nov., and Clavibacter insidiosus comb. nov., Clavibacter nebraskensis comb. nov. and Clavibacter sepedonicus comb. nov.

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September 22, 2019

Whole-genome-sequencing characterization of bloodstream infection-causing hypervirulent Klebsiella pneumoniae of capsular serotype K2 and ST374.

Hypervirulent K. pneumoniae variants (hvKP) have been increasingly reported worldwide, causing metastasis of severe infections such as liver abscesses and bacteremia. The capsular serotype K2 hvKP strains show diverse multi-locus sequence types (MLSTs), but with limited genetics and virulence information. In this study, we report a hypermucoviscous K. pneumoniae strain, RJF293, isolated from a human bloodstream sample in a Chinese hospital. It caused a metastatic infection and fatal septic shock in a critical patient. The microbiological features and genetic background were investigated with multiple approaches. The Strain RJF293 was determined to be multilocis sequence type (ST) 374 and serotype K2, displayed a median lethal dose (LD50) of 1.5 × 102 CFU in BALB/c mice and was as virulent as the ST23 K1 serotype hvKP strain NTUH-K2044 in a mouse lethality assay. Whole genome sequencing revealed that the RJF293 genome codes for 32 putative virulence factors and exhibits a unique presence/absence pattern in comparison to the other 105 completely sequenced K. pneumoniae genomes. Whole genome SNP-based phylogenetic analysis revealed that strain RJF293 formed a single clade, distant from those containing either ST66 or ST86 hvKP. Compared to the other sequenced hvKP chromosomes, RJF293 contains several strain-variable regions, including one prophage, one ICEKp1 family integrative and conjugative element and six large genomic islands. The sequencing of the first complete genome of an ST374 K2 hvKP clinical strain should reinforce our understanding of the epidemiology and virulence mechanisms of this bloodstream infection-causing hvKP with clinical significance.

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September 22, 2019

Translating genomics into practice for real-time surveillance and response to carbapenemase-producing Enterobacteriaceae: evidence from a complex multi-institutional KPC outbreak.

Until recently, Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae were rarely identified in Australia. Following an increase in the number of incident cases across the state of Victoria, we undertook a real-time combined genomic and epidemiological investigation. The scope of this study included identifying risk factors and routes of transmission, and investigating the utility of genomics to enhance traditional field epidemiology for informing management of established widespread outbreaks.All KPC-producing Enterobacteriaceae isolates referred to the state reference laboratory from 2012 onwards were included. Whole-genome sequencing was performed in parallel with a detailed descriptive epidemiological investigation of each case, using Illumina sequencing on each isolate. This was complemented with PacBio long-read sequencing on selected isolates to establish high-quality reference sequences and interrogate characteristics of KPC-encoding plasmids.Initial investigations indicated that the outbreak was widespread, with 86 KPC-producing Enterobacteriaceae isolates (K. pneumoniae 92%) identified from 35 different locations across metropolitan and rural Victoria between 2012 and 2015. Initial combined analyses of the epidemiological and genomic data resolved the outbreak into distinct nosocomial transmission networks, and identified healthcare facilities at the epicentre of KPC transmission. New cases were assigned to transmission networks in real-time, allowing focussed infection control efforts. PacBio sequencing confirmed a secondary transmission network arising from inter-species plasmid transmission. Insights from Bayesian transmission inference and analyses of within-host diversity informed the development of state-wide public health and infection control guidelines, including interventions such as an intensive approach to screening contacts following new case detection to minimise unrecognised colonisation.A real-time combined epidemiological and genomic investigation proved critical to identifying and defining multiple transmission networks of KPC Enterobacteriaceae, while data from either investigation alone were inconclusive. The investigation was fundamental to informing infection control measures in real-time and the development of state-wide public health guidelines on carbapenemase-producing Enterobacteriaceae surveillance and management.

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September 22, 2019

Adaptive strategies of Bacillus thuringiensis isolated from acid mine drainage site in Sabah, Malaysia.

The adaptive process in bacteria is driven by specific genetic elements which regulate phenotypic characteristics such as tolerance to high metal ion concentrations and the secretion of protective biofilms. Extreme environments such as those associated with heavy metal pollution and extremes of acidity offer opportunities to study the adaptive mechanisms of microorganisms. This study focused on the genome analysis of Bacillus thuringiensis (Bt MCMY1), a gram positive rod shaped bacterium isolated from an acid mine drainage site in Sabah, Malaysia by using a combination of Single Molecule Real Time DNA Sequencing, Scanning Electron Microscopy (SEM) and Fourier Transform Infrared Spectroscopy (FTIR). The genome size of Bt MCMY1 was determined to be 5,458,152 bases which was encoded on a single chromosome. Analysis of the genome revealed genes associated with resistance to Copper, Mercury, Arsenic, Cobalt, Zinc, Cadmium and Aluminum. Evidence from SEM and FTIR indicated that the bacterial colonies form distinct films which bear the signature of polyhydroxyalkanoates (PHA) and this finding was supported by the genome data indicating the presence of a genetic pathway associated with the biosynthesis of PHAs. This is the first report of a Bacillus sp. isolated from an acid mine drainage site in Sabah, Malaysia and the genome sequence will provide insights into the manner in which B. thuringiensis adapts to acid mine drainage.

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