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September 22, 2019

Conversion of methionine to cysteine in Lactobacillus paracasei depends on the highly mobile cysK-ctl-cysE gene cluster.

Milk and dairy products are rich in nutrients and are therefore habitats for various microbiomes. However, the composition of nutrients can be quite diverse, in particular among the sulfur containing amino acids. In milk, methionine is present in a 25-fold higher abundance than cysteine. Interestingly, a fraction of strains of the species L. paracasei – a flavor-enhancing adjunct culture species – can grow in medium with methionine as the sole sulfur source. In this study, we focus on genomic and evolutionary aspects of sulfur dependence in L. paracasei strains. From 24 selected L. paracasei strains, 16 strains can grow in medium with methionine as sole sulfur source. We sequenced these strains to perform gene-trait matching. We found that one gene cluster – consisting of a cysteine synthase, a cystathionine lyase, and a serine acetyltransferase – is present in all strains that grow in medium with methionine as sole sulfur source. In contrast, strains that depend on other sulfur sources do not have this gene cluster. We expanded the study and searched for this gene cluster in other species and detected it in the genomes of many bacteria species used in the food production. The comparison to these species showed that two different versions of the gene cluster exist in L. paracasei which were likely gained in two distinct events of horizontal gene transfer. Additionally, the comparison of 62 L. paracasei genomes and the two versions of the gene cluster revealed that this gene cluster is mobile within the species.


September 22, 2019

Complete genomic analysis of a kingdom crossing Klebsiella variicola isolate.

Bacterial isolate X39 was isolated from a community-acquired pneumonia patient in Beijing, China. A phylogenetic tree based on rpoB genes and average nucleotide identity data confirmed that isolate X39 belonged to Klebsiella variicola. The genome of K. variicola X39 contained one circular chromosome and nine plasmids. Comparative genomic analyses with other K. variicola isolates revealed that K. variicola X39 contained the most unique genes. Of these unique genes, many were prophages and transposases. Many virulence factors were shared between K. variicola X39 and Klebsiella pneumoniae F1. The pathogenicity of K. variicola X39 was compared with that of K. pneumoniae F1 in an abdominal infection model. The results indicated that K. variicola X39 was less virulent than typical clinical K. pneumoniae F1. The genome of K. variicola X39 also contained some genes involved in plant colonization, nitrogen fixation, and defense against oxidative stress. GFP-labeled K. variicola X39 could colonize maize as an endophytic bacterium. We concluded that K. variicola X39 was a kingdom-crossing strain.


September 22, 2019

Bacillus wiedmannii biovar thuringiensis: A specialized mosquitocidal pathogen with plasmids from diverse origins.

Bacillus cereus sensu lato also known as B. cereus group is composed of an ecologically diverse bacterial group with an increasing number of related species, some of which are medically or agriculturally important. Numerous e?orts have been undertaken to allow presumptive di?erentiation of B. cereus group species from one another. FCC41 is a Bacillus sp. strain toxic against mosquito species like Aedes aegypti, Aedes (Ochlerotatus) albifasciatus, Culex pipiens, Culex quinquefasciatus, and Culex apicinus, some of them responsible for the transmission of vector-borne diseases. Here, we report the complete genome sequence of FCC41 strain, which consists of one circular chromosome and eight circular plasmids ranging in size from 8 to 490?kb. This strain harbors six crystal protein genes, including cry24Ca, two cry4-like and two cry52-like, a cry41-like parasporin gene and multiple virulence factors. The phylogenetic analysis of the whole-genome sequence of this strain with molecular approaches places this strain into the Bacillus wiedmannii cluster. However, according with phenotypical characteristics such as the mosquitocidal activity due to the presence of Cry proteins found in the parasporal body and cry genes encoded in plasmids of different sizes, indicate that this strain could be renamed as B. wiedmannii biovar thuringiensis strain FCC41.


September 22, 2019

Type II restriction modification system in Ureaplasma parvum OMC-P162 strain.

Ureaplasma parvum serovar 3 strain, OMC-P162, was isolated from the human placenta of a preterm delivery at 26 weeks’ gestation. In this study, we sequenced the complete genome of OMC-P162 and compared it with other serovar 3 strains isolated from patients with different clinical conditions. Ten unique genes in OMC-P162, five of which encoded for hypothetical proteins, were identified. Of these, genes UPV_229 and UPV_230 formed an operon whose open reading frames were predicted to code for a DNA methyltransferase and a hypothetical protein, respectively. DNA modification analysis of the OMC-P162 genome identified N4-methylcytosine (m4C) and N6-methyladenine (m6A), but not 5-methylocytosine (m5C). UPV230 recombinant protein displayed endonuclease activity and recognized the CATG sequence, resulting in a blunt cut between A and T. This restriction enzyme activity was identical to that of the cultivated OMC-P162 strain, suggesting that this restriction enzyme was naturally expressed in OMC-P162. We designated this enzyme as UpaP162. Treatment of pT7Blue plasmid with recombinant protein UPV229 completely blocked UpaP162 restriction enzyme activity. These results suggest that the UPV_229 and UPV_230 genes act as a type II restriction-modification system in Ureaplasma OMC-P162.


September 22, 2019

Discovery of mcr-1-mediated colistin resistance in a highly virulent Escherichia coli lineage.

Resistance to last-line polymyxins mediated by the plasmid-borne mobile colistin resistance gene (mcr-1) represents a new threat to global human health. Here we present the complete genome sequence of an mcr-1-positive multidrug-resistant Escherichia coli strain (MS8345). We show that MS8345 belongs to serotype O2:K1:H4, has a large 241,164-bp IncHI2 plasmid that carries 15 other antibiotic resistance genes (including the extended-spectrum ß-lactamase blaCTX-M-1) and 3 putative multidrug efflux systems, and contains 14 chromosomally encoded antibiotic resistance genes. MS8345 also carries a large ColV-like virulence plasmid that has been associated with E. coli bacteremia. Whole-genome phylogeny revealed that MS8345 clusters within a discrete clade in the sequence type 95 (ST95) lineage, and MS8345 is very closely related to the highly virulent O45:K1:H4 clone associated with neonatal meningitis. Overall, the acquisition of a plasmid carrying resistance to colistin and multiple other antibiotics in this virulent E. coli lineage is concerning and might herald an era where the empirical treatment of ST95 infections becomes increasingly more difficult.IMPORTANCEEscherichia coli ST95 is a globally disseminated clone frequently associated with bloodstream infections and neonatal meningitis. However, the ST95 lineage is defined by low levels of drug resistance amongst clinical isolates, which normally provides for uncomplicated treatment options. Here, we provide the first detailed genomic analysis of an E. coli ST95 isolate that has both high virulence potential and resistance to multiple antibiotics. Using the genome, we predicted its virulence and antibiotic resistance mechanisms, which include resistance to last-line antibiotics mediated by the plasmid-borne mcr-1 gene. Finding an ST95 isolate resistant to nearly all antibiotics that also has a high virulence potential is of major clinical importance and underscores the need to monitor new and emerging trends in antibiotic resistance development in this important global lineage. Copyright © 2018 Forde et al.


September 22, 2019

An introduced crop plant is driving diversification of the virulent bacterial pathogen Erwinia tracheiphila.

Erwinia tracheiphila is the causal agent of bacterial wilt of cucurbits, an economically important phytopathogen affecting an economically important phytopathogen affecting few cultivated Cucurbitaceae few cultivated Cucurbitaceae host plant species in temperate eastern North America. However, essentially nothing is known about E. tracheiphila population structure or genetic diversity. To address this shortcoming, a representative collection of 88 E. tracheiphila isolates was gathered from throughout its geographic range, and their genomes were sequenced. Phylogenomic analysis revealed three genetic clusters with distinct hrpT3SS virulence gene repertoires, host plant association patterns, and geographic distributions. Low genetic heterogeneity within each cluster suggests a recent population bottleneck followed by population expansion. We showed that in the field and greenhouse, cucumber (Cucumis sativus), which was introduced to North America by early Spanish conquistadors, is the most susceptible host plant species and the only species susceptible to isolates from all three lineages. The establishment of large agricultural populations of highly susceptible C. sativus in temperate eastern North America may have facilitated the original emergence of E. tracheiphila into cucurbit agroecosystems, and this introduced plant species may now be acting as a highly susceptible reservoir host. Our findings have broad implications for agricultural sustainability by drawing attention to how worldwide crop plant movement, agricultural intensification, and locally unique environments may affect the emergence, evolution, and epidemic persistence of virulent microbial pathogens.IMPORTANCEErwinia tracheiphila is a virulent phytopathogen that infects two genera of cucurbit crop plants, Cucurbita spp. (pumpkin and squash) and Cucumis spp. (muskmelon and cucumber). One of the unusual ecological traits of this pathogen is that it is limited to temperate eastern North America. Here, we complete the first large-scale sequencing of an E. tracheiphila isolate collection. From phylogenomic, comparative genomic, and empirical analyses, we find that introduced Cucumis spp. crop plants are driving the diversification of E. tracheiphila into multiple lineages. Together, the results from this study show that locally unique biotic (plant population) and abiotic (climate) conditions can drive the evolutionary trajectories of locally endemic pathogens in unexpected ways. Copyright © 2018 Shapiro et al.


September 22, 2019

Comparative genomic analysis of Pseudomonas amygdali pv. lachrymans NM002: Insights into its potential virulence genes and putative invasion determinants.

Pseudomonas amygdali pv. lachrymans is currently of important plant pathogenic bacteria that causes cucumber angular leaf spot worldwide. The pathogen has been studied for its roles in pathogenicity and plant inheritance resistance. To further delineate traits critical to virulence, invasion and survival in the phyllosphere, we reported the first complete genome of P. amygdali pv. lachrymans NM002. Analysis of the whole genome in comparison with three closely-related representative pathovars of P. syringae identified the conservation of virulence genes, including flagella and chemotaxis, quorum-sensing systems, two-component systems, and lipopolysaccharide and antiphagocytosis. It also revealed differences of invasion determinants, such as type III effectors, phytotoxin (coronatine, syringomycin and phaseolotoxin) and cell wall-degrading enzyme, which may contribute to infectivity. The aim of this study was to derive genomic information that would reveal the probable molecular mechanisms underlying the virulence, infectivity and provide a better understanding of the pathogenesis of the P. syringae pathovars. Copyright © 2018. Published by Elsevier Inc.


September 22, 2019

Distribution of the pco gene cluster and associated genetic determinants among swine Escherichia coli from a controlled feeding trial.

Copper is used as an alternative to antibiotics for growth promotion and disease prevention. However, bacteria developed tolerance mechanisms for elevated copper concentrations, including those encoded by the pco operon in Gram-negative bacteria. Using cohorts of weaned piglets, this study showed that the supplementation of feed with copper concentrations as used in the field did not result in a significant short-term increase in the proportion of pco-positive fecal Escherichia coli. The pco and sil (silver resistance) operons were found concurrently in all screened isolates, and whole-genome sequencing showed that they were distributed among a diversity of unrelated E. coli strains. The presence of pco/sil in E. coli was not associated with elevated copper minimal inhibitory concentrations (MICs) under a variety of conditions. As found in previous studies, the pco/sil operons were part of a Tn7-like structure found both on the chromosome or on plasmids in the E. coli strains investigated. Transfer of a pco/sil IncHI2 plasmid from E. coli to Salmonellaenterica resulted in elevated copper MICs in the latter. Escherichia coli may represent a reservoir of pco/sil genes transferable to other organisms such as S. enterica, for which it may represent an advantage in the presence of copper. This, in turn, has the potential for co-selection of resistance to antibiotics.


September 22, 2019

The genomic basis of color pattern polymorphism in the Harlequin ladybird.

Many animal species comprise discrete phenotypic forms. A common example in natural populations of insects is the occurrence of different color patterns, which has motivated a rich body of ecological and genetic research [1-6]. The occurrence of dark, i.e., melanic, forms displaying discrete color patterns is found across multiple taxa, but the underlying genomic basis remains poorly characterized. In numerous ladybird species (Coccinellidae), the spatial arrangement of black and red patches on adult elytra varies wildly within species, forming strikingly different complex color patterns [7, 8]. In the harlequin ladybird, Harmonia axyridis, more than 200 distinct color forms have been described, which classic genetic studies suggest result from allelic variation at a single, unknown, locus [9, 10]. Here, we combined whole-genome sequencing, population-based genome-wide association studies, gene expression, and functional analyses to establish that the transcription factor Pannier controls melanic pattern polymorphism in H. axyridis. We show that pannier is necessary for the formation of melanic elements on the elytra. Allelic variation in pannier leads to protein expression in distinct domains on the elytra and thus determines the distinct color patterns in H. axyridis. Recombination between pannier alleles may be reduced by a highly divergent sequence of ~170 kb in the cis-regulatory regions of pannier, with a 50 kb inversion between color forms. This most likely helps maintain the distinct alleles found in natural populations. Thus, we propose that highly variable discrete color forms can arise in natural populations through cis-regulatory allelic variation of a single gene. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.


September 22, 2019

Characterization of the lytic bacteriophage phiEaP-8 effective against both Erwinia amylovora and Erwinia pyrifoliae causing severe diseases in apple and pear.

Bacteriophages, bacteria-infecting viruses, have been recently reconsidered as a biological control tool for preventing bacterial pathogens. Erwinia amylovora and E. pyrifoliae cause fire blight and black shoot blight disease in apple and pear, respectively. In this study, the bacteriophage phiEaP-8 was isolated from apple orchard soil and could efficiently and specifically kill both E. amylovora and E. pyrifoliae. This bacteriophage belongs to the Podoviridae family. Whole genome analysis revealed that phiEaP-8 carries a 75,929 bp genomic DNA with 78 coding sequences and 5 tRNA genes. Genome comparison showed that phiEaP-8 has only 85% identity to known bacteriophages at the DNA level. PhiEaP-8 retained lytic activity up to 50°C, within a pH range from 5 to 10, and under 365 nm UV light. Based on these characteristics, the bacteriophage phiEaP-8 is novel and carries potential to control both E. amylovora and E. pyrifoliae in apple and pear.


September 22, 2019

Endogenous rRNA sequence variation can regulate stress response gene expression and phenotype.

Prevailing dogma holds that ribosomes are uniform in composition and function. Here, we show that nutrient limitation-induced stress in E. coli changes the relative expression of rDNA operons to alter the rRNA composition within the actively translating ribosome pool. The most upregulated operon encodes the unique 16S rRNA, rrsH, distinguished by conserved sequence variation within the small ribosomal subunit. rrsH-bearing ribosomes affect the expression of functionally coherent gene sets and alter the levels of the RpoS sigma factor, the master regulator of the general stress response. These impacts are associated with phenotypic changes in antibiotic sensitivity, biofilm formation, and cell motility and are regulated by stress response proteins, RelA and RelE, as well as the metabolic enzyme and virulence-associated protein, AdhE. These findings establish that endogenously encoded, naturally occurring rRNA sequence variation can modulate ribosome function, central aspects of gene expression regulation, and cellular physiology. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.


September 22, 2019

Comparative genome analysis and evaluation of probiotic characteristics of Lactobacillus plantarum strain JDFM LP11.

In the current study, the probiotic potential of approximately 250 strains of lactic acid bacteria (LAB) isolated from piglet fecal samples were investigated; among them Lactobacillus plantarum strain JDFM LP11, which possesses significant probiotic potential, with enhanced acid/bile tolerance, attachment to porcine intestinal epithelial cells (IPEC-J2), and antimicrobial activity. The genetic characteristics of strain JDFM LP11 were explored by performing whole genome sequencing (WGS) using a PacBio system. The circular draft genome have a total length of 3,206,883 bp and a total of 3,021 coding sequences were identified. Phylogenetically, three genes, possibly related to survival and metabolic activity in the porcine host, were identified. These genes encode p60, lichenan permease IIC component, and protein TsgA, which are a putative endopeptidase, a component of the phosphotransferase system (PTS), and a major facilitator in the gut environment, respectively. Our findings suggest that understanding the functional and genetic characteristics of L. plantarum strain JDFM LP11, with its candidate genes for gut health, could provide new opportunities and insights into applications in the animal food and feed additive industries.


September 22, 2019

Complete genome sequence and characterization of linezolid-resistant Enterococcus faecalis clinical isolate KUB3006 carrying a cfr(B)-transposon on its chromosome and optrA-plasmid.

Linezolid (LZD) has become one of the most important antimicrobial agents for infections caused by gram-positive bacteria, including those caused by Enterococcus species. LZD-resistant (LR) genetic features include mutations in 23S rRNA/ribosomal proteins, a plasmid-borne 23S rRNA methyltransferase gene cfr, and ribosomal protection genes (optrA and poxtA). Recently, a cfr gene variant, cfr(B), was identified in a Tn6218-like transposon (Tn) in a Clostridioides difficile isolate. Here, we isolated an LR Enterococcus faecalis clinical isolate, KUB3006, from a urine specimen of a patient with urinary tract infection during hospitalization in 2017. Comparative and whole-genome analyses were performed to characterize the genetic features and overall antimicrobial resistance genes in E. faecalis isolate KUB3006. Complete genome sequencing of KUB3006 revealed that it carried cfr(B) on a chromosomal Tn6218-like element. Surprisingly, this Tn6218-like element was almost (99%) identical to that of C. difficile Ox3196, which was isolated from a human in the UK in 2012, and to that of Enterococcus faecium 5_Efcm_HA-NL, which was isolated from a human in the Netherlands in 2012. An additional oxazolidinone and phenicol resistance gene, optrA, was also identified on a plasmid. KUB3006 is sequence type (ST) 729, suggesting that it is a minor ST that has not been reported previously and is unlikely to be a high-risk E. faecalis lineage. In summary, LR E. faecalis KUB3006 possesses a notable Tn6218-like-borne cfr(B) and a plasmid-borne optrA. This finding raises further concerns regarding the potential declining effectiveness of LZD treatment in the future.


September 22, 2019

Genome sequence and metabolic analysis of a fluoranthene-degrading strain Pseudomonas aeruginosa DN1.

Pseudomonas aeruginosa DN1, isolated from petroleum-contaminated soil, showed excellent degradation ability toward diverse polycyclic aromatic hydrocarbons (PAHs). Many studies have been done to improve its degradation ability. However, the molecular mechanisms of PAHs degradation in DN1 strain are unclear. In this study, the whole genome of DN1 strain was sequenced and analyzed. Its genome contains 6,641,902 bp and encodes 6,684 putative open reading frames (ORFs), which has the largest genome in almost all the comparative Pseudomonas strains. Results of gene annotation showed that this strain harbored over 100 candidate genes involved in PAHs degradation, including those encoding 25 dioxygenases, four ring-hydroxylating dioxygenases, five ring-cleaving dioxygenases, and various catabolic enzymes, transcriptional regulators, and transporters in the degradation pathways. In addition, gene knockout experiments revealed that the disruption of some key PAHs degradation genes in DN1 strain, such as catA, pcaG, pcaH, and rhdA, did not completely inhibit fluoranthene degradation, even though their degradative rate reduced to some extent. Three intermediate metabolites, including 9-hydroxyfluorene, 1-acenaphthenone, and 1, 8-naphthalic anhydride, were identified as the dominating intermediates in presence of 50 µg/mL fluoranthene as the sole carbon source according to gas chromatography mass spectrometry analysis. Taken together, the genomic and metabolic analysis indicated that the fluoranthene degradation by DN1 strain was initiated by dioxygenation at the C-1, 2-, C-2, 3-, and C-7, 8- positions. These results provide new insights into the genomic plasticity and environmental adaptation of DN1 strain.


September 22, 2019

Characterization and genomic analyses of Pseudomonas aeruginosa podovirus TC6: establishment of genus Pa11virus.

Phages have attracted a renewed interest as alternative to chemical antibiotics. Although the number of phages is 10-fold higher than that of bacteria, the number of genomically characterized phages is far less than that of bacteria. In this study, phage TC6, a novel lytic virus of Pseudomonas aeruginosa, was isolated and characterized. TC6 consists of an icosahedral head with a diameter of approximately 54 nm and a short tail with a length of about 17 nm, which are characteristics of the family Podoviridae. TC6 can lyse 86 out of 233 clinically isolated P. aeruginosa strains, thus showing application potentials for phage therapy. The linear double-stranded genomic DNA of TC6 consisted of 49796 base pairs and was predicted to contain 71 protein-coding genes. A total of 11 TC6 structural proteins were identified by mass spectrometry. Comparative analysis revealed that the P. aeruginosa phages TC6, O4, PA11, and IME180 shared high similarity at DNA sequence and proteome levels, among which PA11 was the first phage discovered and published. Meanwhile, these phages contain 54 core genes and have very close phylogenetic relationships, which distinguish them from other known phage genera. We therefore proposed that these four phages can be classified as Pa11virus, comprising a new phage genus of Podoviridae that infects Pseudomonas spp. The results of this work promoted our understanding of phage biology, classification, and diversity.


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