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Asset Tag: Microbiology

September 22, 2019

Characterization of LE3 and LE4, the only lytic phages known to infect the spirochete Leptospira.

Leptospira is a phylogenetically unique group of bacteria, and includes the causative agents of leptospirosis, the most globally prevalent zoonosis. Bacteriophages in Leptospira are largely unexplored. To date, a genomic sequence is available for only one temperate leptophage called LE1. Here, we sequenced and analysed the first genomes of the lytic phages LE3 and LE4 that can infect the saprophyte Leptospira biflexa using the lipopolysaccharide O-antigen as receptor. Bioinformatics analysis showed that the 48-kb LE3 and LE4 genomes are similar and contain 62% genes whose function cannot be predicted. Mass spectrometry led to the identification of 21 and 23 phage proteins in LE3 and LE4, respectively. However we did not identify significant similarities with other phage genomes. A search for prophages close to LE4 in the Leptospira genomes allowed for the identification of a related plasmid in L. interrogans and a prophage-like region in the draft genome of a clinical isolate of L. mayottensis. Long-read whole genome sequencing of the L. mayottensis revealed that the genome contained a LE4 phage-like circular plasmid. Further isolation and genomic comparison of leptophages should reveal their role in the genetic evolution of Leptospira.

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September 22, 2019

Linking genotype and phenotype in an economically viable propionic acid biosynthesis process

Propionic acid (PA) is used as a food preservative and increasingly, as a precursor for the synthesis of monomers. PA is produced mainly through hydrocarboxylation of ethylene, also known as the `oxo-process’; however, Propionibacterium species are promising biological PA producers natively producing PA as their main fermentation product. However, for fermentation to be competitive, a PA yield of at least 0.6 g/g is required.

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September 22, 2019

Citrobacter freundii fitness during bloodstream infection.

Sepsis resulting from microbial colonization of the bloodstream is a serious health concern associated with high mortality rates. The objective of this study was to define the physiologic requirements of Citrobacter freundii in the bloodstream as a model for bacteremia caused by opportunistic Gram-negative pathogens. A genetic screen in a murine host identified 177 genes that contributed significantly to fitness, the majority of which were broadly classified as having metabolic or cellular maintenance functions. Among the pathways examined, the Tat protein secretion system conferred the single largest fitness contribution during competition infections and a putative Tat-secreted protein, SufI, was also identified as a fitness factor. Additional work was focused on identifying relevant metabolic pathways for bacteria in the bloodstream environment. Mutations that eliminated the use of glucose or mannitol as carbon sources in vitro resulted in loss of fitness in the murine model and similar results were obtained upon disruption of the cysteine biosynthetic pathway. Finally, the conservation of identified fitness factors was compared within a cohort of Citrobacter bloodstream isolates and between Citrobacter and Serratia marcescens, the results of which suggest the presence of conserved strategies for bacterial survival and replication in the bloodstream environment.

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September 22, 2019

Biology and genome of a newly discovered sibling species of Caenorhabditis elegans.

A ‘sibling’ species of the model organism Caenorhabditis elegans has long been sought for use in comparative analyses that would enable deep evolutionary interpretations of biological phenomena. Here, we describe the first sibling species of C. elegans, C. inopinata n. sp., isolated from fig syconia in Okinawa, Japan. We investigate the morphology, developmental processes and behaviour of C. inopinata, which differ significantly from those of C. elegans. The 123-Mb C. inopinata genome was sequenced and assembled into six nuclear chromosomes, allowing delineation of Caenorhabditis genome evolution and revealing unique characteristics, such as highly expanded transposable elements that might have contributed to the genome evolution of C. inopinata. In addition, C. inopinata exhibits massive gene losses in chemoreceptor gene families, which could be correlated with its limited habitat area. We have developed genetic and molecular techniques for C. inopinata; thus C. inopinata provides an exciting new platform for comparative evolutionary studies.

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September 22, 2019

Whole genome sequencing, de novo assembly and phenotypic profiling for the new budding yeast species Saccharomyces jurei.

Saccharomyces sensu stricto complex consist of yeast species, which are not only important in the fermentation industry but are also model systems for genomic and ecological analysis. Here, we present the complete genome assemblies of Saccharomyces jurei, a newly discovered Saccharomyces sensu stricto species from high altitude oaks. Phylogenetic and phenotypic analysis revealed that S. jurei is more closely related to S. mikatae, than S. cerevisiae, and S. paradoxus The karyotype of S. jurei presents two reciprocal chromosomal translocations between chromosome VI/VII and I/XIII when compared to the S. cerevisiae genome. Interestingly, while the rearrangement I/XIII is unique to S. jurei, the other is in common with S. mikatae strain IFO1815, suggesting shared evolutionary history of this species after the split between S. cerevisiae and S. mikatae The number of Ty elements differed in the new species, with a higher number of Ty elements present in S. jurei than in S. cerevisiae Phenotypically, the S. jurei strain NCYC 3962 has relatively higher fitness than the other strain NCYC 3947T under most of the environmental stress conditions tested and showed remarkably increased fitness in higher concentration of acetic acid compared to the other sensu stricto species. Both strains were found to be better adapted to lower temperatures compared to S. cerevisiae. Copyright © 2018 Naseeb et al.

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September 22, 2019

Novel enterobacter lineage as leading cause of nosocomial outbreak involving carbapenemase-producing strains.

We investigated unusual carbapenemase-producing Enterobacter cloacae complex isolates (n = 8) in the novel sequence type (ST) 873, which caused nosocomial infections in 2 hospitals in France. Whole-genome sequence typing showed the 1-year persistence of the epidemic strain, which harbored a blaVIM-4 ST1-IncHI2 plasmid, in 1 health institution and 2 closely related strains harboring blaCTX-M-15 in the other. These isolates formed a new subgroup in the E. hormaechei metacluster, according to their hsp60 sequences and phylogenomic analysis. The average nucleotide identities, specific biochemical properties, and pangenomic and functional investigations of isolates suggested isolates of a novel species that had acquired genes associated with adhesion and mobility. The emergence of this novel Enterobacter phylogenetic lineage within hospitals should be closely monitored because of its ability to persist and spread.

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September 22, 2019

Identification of natural product compounds as quorum sensing inhibitors in Pseudomonas fluorescens P07 through virtual screening.

Pseudomonas fluorescens, a Gram-negative psychrotrophic bacteria, is the main microorganism causing spoilage of chilled raw milk and aquatic products. Quorum sensing (QS) widely exists in bacteria to monitor their population densities and regulate numerous physiological activities, such as the secretion of siderophores, swarming motility and biofilm formation. Thus, searching for quorum sensing inhibitors (QSIs) may be another promising way to control the deterioration of food caused by P. fluorescens. Here, we screened a traditional Chinese medicine (TCM) database to discover potential QSIs with lesser toxicity. The gene sequences of LuxI- and LuxR-type proteins of P. fluorescens P07 were obtained through whole-genome sequencing. In addition, the protein structures built by homology modelling were used as targets to screen for QSIs. Twenty-one compounds with a dock score greater than 6 were purchased and tested by biosensor strains (Chromobacterium violaceum CV026 and Agrobacterium tumefaciens A136). The results showed that 10 of the compounds were determined as hits (hit rate: 66.67%). Benzyl alcohol, rhodinyl formate and houttuynine were effective QSIs. The impact of the most active compound (benzyl alcohol) on the phenotypes of P. fluorescens P07, including swimming and swarming motility, production of extracellular enzymes and siderophores, N-acylhomoserine lactone (AHLs) content and biofilm formation were determined. The inhibitory mechanism of benzyl alcohol on the QS system of P. fluorescens P07 is further discussed. This study reveals the feasibility of searching for novel QSIs through virtual screening. Copyright © 2018 Elsevier Ltd. All rights reserved.

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September 22, 2019

Catching up with Lyme disease antigenic variation computationally.

The spirochetes that cause Lyme disease have an elaborate antigenic variation system that produces millions of variants, thus evading the immune response. Verhey et al. have applied next-generation sequencing and computational analysis to gain new insights into how these bacteria keep ‘one step ahead’ of elimination by the host. Copyright © 2018 Elsevier Ltd. All rights reserved.

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September 22, 2019

Wheat microbiome bacteria can reduce virulence of a plant pathogenic fungus by altering histone acetylation.

Interactions between bacteria and fungi have great environmental, medical, and agricultural importance, but the molecular mechanisms are largely unknown. Here, we study the interactions between the bacterium Pseudomonas piscium, from the wheat head microbiome, and the plant pathogenic fungus Fusarium graminearum. We show that a compound secreted by the bacteria (phenazine-1-carboxamide) directly affects the activity of fungal protein FgGcn5, a histone acetyltransferase of the SAGA complex. This leads to deregulation of histone acetylation at H2BK11, H3K14, H3K18, and H3K27 in F. graminearum, as well as suppression of fungal growth, virulence, and mycotoxin biosynthesis. Therefore, an antagonistic bacterium can inhibit growth and virulence of a plant pathogenic fungus by manipulating fungal histone modification.

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September 22, 2019

The complete methylome of an entomopathogenic bacterium reveals the existence of loci with unmethylated adenines.

DNA methylation can serve to control diverse phenomena in eukaryotes and prokaryotes, including gene regulation leading to cell differentiation. In bacteria, DNA methylomes (i.e., methylation state of each base of the whole genome) have been described for several species, but methylome profile variation during the lifecycle has rarely been studied, and only in a few model organisms. Moreover, major phenotypic changes have been reported in several bacterial strains with a deregulated methyltransferase, but the corresponding methylome has rarely been described. Here we report the first methylome description of an entomopathogenic bacterium, Photorhabdus luminescens. Eight motifs displaying a high rate of methylation (>94%) were identified. The methylome was strikingly stable over course of growth, but also in a subpopulation responsible for a critical step in the bacterium’s lifecycle: successful survival and proliferation in insects. The rare unmethylated GATC motifs were preferentially located in putative promoter regions, and most of them were methylated after Dam methyltransferase overexpression, suggesting that DNA methylation is involved in gene regulation. Our findings bring key insight into bacterial methylomes and encourage further research to decipher the role of loci protected from DNA methylation in gene regulation.

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September 22, 2019

Whole-genome sequencing and comparative analysis of two plant-associated strains of Rhodopseudomonas palustris (PS3 and YSC3).

Rhodopseudomonas palustris strains PS3 and YSC3 are purple non-sulfur phototrophic bacteria isolated from Taiwanese paddy soils. PS3 has beneficial effects on plant growth and enhances the uptake efficiency of applied fertilizer nutrients. In contrast, YSC3 has no significant effect on plant growth. The genomic structures of PS3 and YSC3 are similar; each contains one circular chromosome that is 5,269,926 or 5,371,816?bp in size, with 4,799 or 4,907 protein-coding genes, respectively. In this study, a large class of genes involved in chemotaxis and motility was identified in both strains, and genes associated with plant growth promotion, such as nitrogen fixation-, IAA synthesis- and ACC deamination-associated genes, were also identified. We noticed that the growth rate, the amount of biofilm formation, and the relative expression levels of several chemotaxis-associated genes were significantly higher for PS3 than for YSC3 upon treatment with root exudates. These results indicate that PS3 responds better to the presence of plant hosts, which may contribute to the successful interactions of PS3 with plant hosts. Moreover, these findings indicate that the existence of gene clusters associated with plant growth promotion is required but not sufficient for a bacterium to exhibit phenotypes associated with plant growth promotion.

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September 22, 2019

Genomic analysis of the insect-killing fungus Beauveria bassiana JEF-007 as a biopesticide.

Insect-killing fungi have high potential in pest management. A deeper insight into the fungal genes at the whole genome level is necessary to understand the inter-species or intra-species genetic diversity of fungal genes, and to select excellent isolates. In this work, we conducted a whole genome sequencing of Beauveria bassiana (Bb) JEF-007 and characterized pathogenesis-related features and compared with other isolates including Bb ARSEF2860. A large number of Bb JEF-007 genes showed high identity with Bb ARSEF2860, but some genes showed moderate or low identity. The two Bb isolates showed a significant difference in vegetative growth, antibiotic-susceptibility, and virulence against Tenebrio molitor larvae. When highly identical genes between the two Bb isolates were subjected to real-time PCR, their transcription levels were different, particularly in heat shock protein 30 (hsp30) gene which is related to conidial thermotolerance. In several B. bassiana isolates, chitinases and trypsin-like protease genes involved in pathogenesis were highly conserved, but other genes showed noticeable sequence variation within the same species. Given the transcriptional and genetic diversity in B. bassiana, a selection of virulent isolates with industrial advantages is a pre-requisite, and this genetic approach could support the development of excellent biopesticides with intellectual property protection.

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September 22, 2019

Whole-genome resequencing and pan-transcriptome reconstruction highlight the impact of genomic structural Variation on secondary metabolite gene clusters in the grapevine Esca pathogen Phaeoacremonium minimum.

The Ascomycete fungus Phaeoacremonium minimum is one of the primary causal agents of Esca, a widespread and damaging grapevine trunk disease. Variation in virulence among Pm. minimum isolates has been reported, but the underlying genetic basis of the phenotypic variability remains unknown. The goal of this study was to characterize intraspecific genetic diversity and explore its potential impact on virulence functions associated with secondary metabolism, cellular transport, and cell wall decomposition. We generated a chromosome-scale genome assembly, using single molecule real-time sequencing, and resequenced the genomes and transcriptomes of multiple isolates to identify sequence and structural polymorphisms. Numerous insertion and deletion events were found for a total of about 1 Mbp in each isolate. Structural variation in this extremely gene dense genome frequently caused presence/absence polymorphisms of multiple adjacent genes, mostly belonging to biosynthetic clusters associated with secondary metabolism. Because of the observed intraspecific diversity in gene content due to structural variation we concluded that a transcriptome reference developed from a single isolate is insufficient to represent the virulence factor repertoire of the species. We therefore compiled a pan-transcriptome reference of Pm. minimum comprising a non-redundant set of 15,245 protein-coding sequences. Using naturally infected field samples expressing Esca symptoms, we demonstrated that mapping of meta-transcriptomics data on a multi-species reference that included the Pm. minimum pan-transcriptome allows the profiling of an expanded set of virulence factors, including variable genes associated with secondary metabolism and cellular transport.

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September 22, 2019

Characterization of a novel blaKLUC variant with reduced ß-lactam resistance from an IncA/C group plasmid in a clinical Klebsiella pneumoniae isolate.

Similar to other CTX-M family enzymes, KLUC is a recently identified and emerging determinant of cefotaxime resistance that has been recovered from at least three Enterobacteriaceae species, including Kluyvera cryocrescens, Escherichia coli, and Enterobacter cloacae. Whether this extended-spectrum ß-lactamase (ESBL) has been disseminated among commonly isolated Enterobacteriaceae is worthy of further investigation. In this study, we screened 739 nosocomial Enterobacteriaceae isolates (240 Klebsiella pneumoniae and 499 E. coli strains) and found that one K. pneumoniae and four E. coli isolates harbored the blaKLUC gene. Three blaKLUC determinants isolated from E. coli were entirely identical to a blaKLUC-3 gene previously recovered in the same hospital. PFGE of four blaKLUC-harboring E. coli strains showed that prevalence of these determinants was most likely mediated by horizontal gene transfer but not clonal dissemination. However, the variant isolated from K. pneumoniae belonged to a novel member of the KLUC enzyme group. This newly identified enzyme (KLUC-5) has an amino acid substitution compared with previously identified KLUC-1 (G18S) and KLUC-3 (G240D). Antimicrobial susceptibility tests showed that KLUC-5 significantly reduced resistance activity to almost all the selected antimicrobials compared to previously identified KLUC-3. Site-directed mutagenesis showed that blaKLUC-5-D240G and blaKLUC-5-S18G significantly enhanced the MIC against its best substrate. Conjugation and S1-PFGE indicated that blaKLUC-5 was located on a transferable plasmid, which was further decoded by single-molecule, real-time sequencing. Comparative genome analysis showed that its backbone exhibited genetic homology to the IncA/C incompatibility group plasmids. A transposable element, ISEcp1, was detected 256-bp upstream of the blaKLUC-5 gene; this location was inconsistent with the previously identified blaKLUC-1 but congruent with the variants recovered from E. coli in the same hospital. These data provide evidence of the increasingly emerging KLUC group of ESBLs in China.

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September 22, 2019

Phenotypic and genomic properties of Brachybacterium vulturis sp. nov. and Brachybacterium avium sp. nov.

Two strains, VM2412T and VR2415T, were isolated from the feces of an Andean condor (Vultur gryphus) living in Seoul Grand Park, Gyeonggi-do, South Korea. Cells of both strains were observed to be Gram-stain positive, non-motile, aerobic, catalase positive and oxidase negative. Growth was found to occur at 10-30°C, showing optimum growth at 30°C. The strains could tolerate up to 15% (w/v) NaCl concentration and grow at pH 6-9. The strains shared 99.3% 16S rRNA gene sequence similarity to each other but were identified as two distinct species based on 89.0-89.2% ANIb, 90.3% ANIm, 89.7% OrthoANI and 38.0% dDDH values calculated using whole genome sequences. Among species with validly published names, Brachybacterium ginsengisoli DCY80T shared high 16S rRNA gene sequence similarities with strains VM2412T (98.7%) and VR2415T (98.4%) and close genetic relatedness with strains VM2412T (83.3-83.5% ANIb, 87.0% ANIm, 84.3% OrthoANI and 27.8% dDDH) and VR2415T (82.8-83.2% ANIb, 86.7% ANIm, 83.9% OrthoANI and 27.2% dDDH). The major fatty acid of the two strains was identified as anteiso-C15:0 and the polar lipids consisted of phosphatidylglycerol, diphosphatidylglycerol, presumptively phosphatidylethanolamine and three unidentified glycolipids. Strain VR2415T also produced an unidentified phospholipid. The cell walls of the two strains contained meso-diaminopimelic acid as diagnostic diamino acid and the whole cell sugars were ribose, glucose, and galactose. The strains contained MK-7 as their predominant menaquinone. The genomes of strains VM2412T, VR2415T, and B. ginsengisoli DCY80T were sequenced in this study. The genomic G+C contents of strains VM2412T and VR2415T were determined to be 70.8 and 70.4 mol%, respectively. A genome-based phylogenetic tree constructed using an up-to-date bacterial core gene set (UBCG) showed that the strains formed a clade with members of the genus Brachybacterium, supporting their taxonomic classification into the genus Brachybacterium. Based on phenotypic and genotypic analyses in this study, strains VM2412T and VR2415T are considered to represent two novel species of the genus Brachybacterium and the names Brachybacterium vulturis sp. nov. and Brachybacterium avium sp. nov. are proposed for strains VM2412T (=KCTC 39996T = JCM 32142T) and VR2415T (=KCTC 39997T = JCM 32143T), respectively.

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