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July 7, 2019

Synergistic interaction between phage therapy and antibiotics clears Pseudomonas aeruginosa infection in endocarditis and reduces virulence.

Increasing antibiotic resistance warrants therapeutic alternatives. Here we investigated the efficacy of bacteriophage-therapy (phage) alone or combined with antibiotics against experimental endocarditis (EE) due to Pseudomonas aeruginosa, an archetype of difficult-to-treat infection.In vitro fibrin clots and rats with aortic EE were treated with an antipseudomonas phage cocktail alone or combined with ciprofloxacin. Phage pharmacology, therapeutic efficacy, and resistance were determined.In vitro, single-dose phage therapy killed 7 log colony-forming units (CFUs)/g of fibrin clots in 6 hours. Phage-resistant mutants regrew after 24 hours but were prevented by combination with ciprofloxacin (2.5 × minimum inhibitory concentration). In vivo, single-dose phage therapy killed 2.5 log CFUs/g of vegetations in 6 hours (P < .001 vs untreated controls) and was comparable with ciprofloxacin monotherapy. Moreover, phage/ciprofloxacin combinations were highly synergistic, killing >6 log CFUs/g of vegetations in 6 hours and successfully treating 64% (n = 7/11) of rats. Phage-resistant mutants emerged in vitro but not in vivo, most likely because resistant mutations affected bacterial surface determinants important for infectivity (eg, the pilT and galU genes involved in pilus motility and LPS formation).Single-dose phage therapy was active against P. aeruginosa EE and highly synergistic with ciprofloxacin. Phage-resistant mutants had impaired infectivity. Phage-therapy alone or combined with antibiotics merits further clinical consideration.

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July 7, 2019

Metatranscriptomics supports the mechanism for biocathode electroautotrophy by “Candidatus Tenderia electrophaga.”

Biocathodes provide a stable electron source to drive reduction reactions in electrotrophic microbial electrochemical systems. Electroautotrophic biocathode communities may be more robust than monocultures in environmentally relevant settings, but some members are not easily cultivated outside the electrode environment. We previously used metagenomics and metaproteomics to propose a pathway for coupling extracellular electron transfer (EET) to carbon fixation in “Candidatus Tenderia electrophaga,” an uncultivated but dominant member of an electroautotrophic biocathode community. Here we validate and refine this proposed pathway using metatranscriptomics of replicate aerobic biocathodes poised at the growth potential level of 310 mV and the suboptimal 470 mV (versus the standard hydrogen electrode). At both potentials, transcripts were more abundant from “Ca. Tenderia electrophaga” than from any other constituent, and its relative activity was positively correlated with current. Several genes encoding key components of the proposed “Ca. Tenderia electrophaga” EET pathway were more highly expressed at 470 mV, consistent with a need for cells to acquire more electrons to obtain the same amount of energy as at 310 mV. These included cyc2, encoding a homolog of a protein known to be involved in iron oxidation. Mean expression of all CO2 fixation-related genes is 0.27 log2-fold higher at 310 mV, indicating that reduced energy availability at 470 mV decreased CO2 fixation. Our results substantiate the claim that “Ca. Tenderia electrophaga” is the key electroautotroph, which will help guide further development of this community for microbial electrosynthesis. IMPORTANCE Bacteria that directly use electrodes as metabolic electron donors (biocathodes) have been proposed for applications ranging from microbial electrosynthesis to advanced bioelectronics for cellular communication with machines. However, just as we understand very little about oxidation of analogous natural insoluble electron donors, such as iron oxide, the organisms and extracellular electron transfer (EET) pathways underlying the electrode-cell direct electron transfer processes are almost completely unknown. Biocathodes are a stable biofilm cultivation platform to interrogate both the rate and mechanism of EET using electrochemistry and to study the electroautotrophic organisms that catalyze these reactions. Here we provide new evidence supporting the hypothesis that the uncultured bacterium “Candidatus Tenderia electrophaga” directly couples extracellular electron transfer to CO2 fixation. Our results provide insight into developing biocathode technology, such as microbial electrosynthesis, as well as advancing our understanding of chemolithoautotrophy.

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July 7, 2019

Whole genome sequencing and analysis of Campylobacter coli YH502 from retail chicken reveals a plasmid-borne type VI secretion system.

Campylobacter is a major cause of foodborne illnesses worldwide. Campylobacter infections, commonly caused by ingestion of undercooked poultry and meat products, can lead to gastroenteritis and chronic reactive arthritis in humans. Whole genome sequencing (WGS) is a powerful technology that provides comprehensive genetic information about bacteria and is increasingly being applied to study foodborne pathogens: e.g., evolution, epidemiology/outbreak investigation, and detection. Herein we report the complete genome sequence of Campylobacter coli strain YH502 isolated from retail chicken in the United States. WGS, de novo assembly, and annotation of the genome revealed a chromosome of 1,718,974 bp and a mega-plasmid (pCOS502) of 125,964 bp. GC content of the genome was 31.2% with 1931 coding sequences and 53 non-coding RNAs. Multiple virulence factors including a plasmid-borne type VI secretion system and antimicrobial resistance genes (beta-lactams, fluoroquinolones, and aminoglycoside) were found. The presence of T6SS in a mobile genetic element (plasmid) suggests plausible horizontal transfer of these virulence genes to other organisms. The C. coli YH502 genome also harbors CRISPR sequences and associated proteins. Phylogenetic analysis based on average nucleotide identity and single nucleotide polymorphisms identified closely related C. coli genomes available in the NCBI database. Taken together, the analyzed genomic data of this potentially virulent strain of C. coli will facilitate further understanding of this important foodborne pathogen most likely leading to better control strategies. The chromosome and plasmid sequences of C. coli YH502 have been deposited in GenBank under the accession numbers CP018900.1 and CP018901.1, respectively.

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July 7, 2019

Insight into potential probiotic markers predicted in Lactobacillus pentosus MP-10 genome sequence.

Lactobacillus pentosus MP-10 is a potential probiotic lactic acid bacterium originally isolated from naturally fermented Aloreña green table olives. The entire genome sequence was annotated to in silico analyze the molecular mechanisms involved in the adaptation of L. pentosus MP-10 to the human gastrointestinal tract (GIT), such as carbohydrate metabolism (related with prebiotic utilization) and the proteins involved in bacteria-host interactions. We predicted an arsenal of genes coding for carbohydrate-modifying enzymes to modify oligo- and polysaccharides, such as glycoside hydrolases, glycoside transferases, and isomerases, and other enzymes involved in complex carbohydrate metabolism especially starch, raffinose, and levan. These enzymes represent key indicators of the bacteria’s adaptation to the GIT environment, since they involve the metabolism and assimilation of complex carbohydrates not digested by human enzymes. We also detected key probiotic ligands (surface proteins, excreted or secreted proteins) involved in the adhesion to host cells such as adhesion to mucus, epithelial cells or extracellular matrix, and plasma components; also, moonlighting proteins or multifunctional proteins were found that could be involved in adhesion to epithelial cells and/or extracellular matrix proteins and also affect host immunomodulation. In silico analysis of the genome sequence of L. pentosus MP-10 is an important initial step to screen for genes encoding for proteins that may provide probiotic features, and thus provides one new routes for screening and studying this potentially probiotic bacterium.

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July 7, 2019

Sequencing a piece of history: complete genome sequence of the original Escherichia coli strain.

In 1885, Theodor Escherich first described the Bacillus coli commune, which was subsequently renamed Escherichia coli. We report the complete genome sequence of this original strain (NCTC 86). The 5?144?392?bp circular chromosome encodes the genes for 4805 proteins, which include antigens, virulence factors, antimicrobial-resistance factors and secretion systems, of a commensal organism from the pre-antibiotic era. It is located in the E. coli A subgroup and is closely related to E. coli K-12 MG1655. E. coli strain NCTC 86 and the non-pathogenic K-12, C, B and HS strains share a common backbone that is largely co-linear. The exception is a large 2?803?932?bp inversion that spans the replication terminus from gmhB to clpB. Comparison with E. coli K-12 reveals 41 regions of difference (577?351?bp) distributed across the chromosome. For example, and contrary to current dogma, E. coli NCTC 86 includes a nine gene sil locus that encodes a silver-resistance efflux pump acquired before the current widespread use of silver nanoparticles as an antibacterial agent, possibly resulting from the widespread use of silver utensils and currency in Germany in the 1800s. In summary, phylogenetic comparisons with other E. coli strains confirmed that the original strain isolated by Escherich is most closely related to the non-pathogenic commensal strains. It is more distant from the root than the pathogenic organisms E. coli 042 and O157?:?H7; therefore, it is not an ancestral state for the species.

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July 7, 2019

Gas fermentation: cellular engineering possibilities and scale up.

Low carbon fuels and chemicals can be sourced from renewable materials such as biomass or from industrial and municipal waste streams. Gasification of these materials allows all of the carbon to become available for product generation, a clear advantage over partial biomass conversion into fermentable sugars. Gasification results into a synthesis stream (syngas) containing carbon monoxide (CO), carbon dioxide (CO2), hydrogen (H2) and nitrogen (N2). Autotrophy-the ability to fix carbon such as CO2 is present in all domains of life but photosynthesis alone is not keeping up with anthropogenic CO2 output. One strategy is to curtail the gaseous atmospheric release by developing waste and syngas conversion technologies. Historically microorganisms have contributed to major, albeit slow, atmospheric composition changes. The current status and future potential of anaerobic gas-fermenting bacteria with special focus on acetogens are the focus of this review.

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July 7, 2019

Natural competence rates are variable among Xylella fastidiosa strains and homologous recombination occurs in vitro between subspecies fastidiosa and multiplex.

Xylella fastidiosa, an etiological agent of emerging crop diseases around the world, is naturally competent for the uptake of DNA from the environment that is incorporated into its genome by homologous recombination. Homologous recombination between subspecies of X. fastidiosa was inferred by in silico studies and was hypothesized to cause disease emergence. However, no experimental data are available on the degree to which X. fastidiosa strains are capable of competence and whether recombination can be experimentally demonstrated between subspecies. Here, using X. fastidiosa strains from different subspecies, natural competence in 11 of 13 strains was confirmed with plasmids containing antibiotic markers flanked by homologous regions and, in three of five strains, with dead bacterial cells used as source of donor DNA. Recombination frequency differed among strains and was correlated to growth rate and twitching motility. Moreover, intersubspecific recombination occurred readily between strains of subsp. fastidiosa and multiplex, as demonstrated by movement of antibiotic resistance and green fluorescent protein from donor to recipient cells and confirmed by DNA sequencing of the flanking arms of recombinant strains. Results demonstrate that natural competence is widespread among X. fastidiosa strains and could have an impact in pathogen adaptation and disease development.

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July 7, 2019

Critical points for an accurate human genome analysis.

Next-generation sequencing is radically changing how DNA diagnostic laboratories operate. What started as a single-gene profession is now developing into gene panel sequencing and whole-exome and whole-genome sequencing (WES/WGS) analyses. With further advances in sequencing technology and concomitant price reductions, WGS will soon become the standard and be routinely offered. Here, we focus on the critical steps involved in performing WGS, with a particular emphasis on points where WGS differs from WES, the important variables that should be taken into account, and the quality control measures that can be taken to monitor the process. The points discussed here, combined with recent publications on guidelines for reporting variants, will facilitate the routine implementation of WGS into a diagnostic setting.© 2017 Wiley Periodicals, Inc.

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July 7, 2019

Generation of a collection of mutant tomato lines using pooled CRISPR libraries.

The high efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)-mediated mutagenesis in plants enables the development of high-throughput mutagenesis strategies. By transforming pooled CRISPR libraries into tomato (Solanum lycopersicum), collections of mutant lines were generated with minimal transformation attempts and in a relatively short period of time. Identification of the targeted gene(s) was easily determined by sequencing the incorporated guide RNA(s) in the primary transgenic events. From a single transformation with a CRISPR library targeting the immunity-associated leucine-rich repeat subfamily XII genes, heritable mutations were recovered in 15 of the 54 genes targeted. To increase throughput, a second CRISPR library was made containing three guide RNAs per construct to target 18 putative transporter genes. This resulted in stable mutations in 15 of the 18 targeted genes, with some primary transgenic plants having as many as five mutated genes. Furthermore, the redundancy in this collection of plants allowed for the association of aberrant T0 phenotypes with the underlying targeted genes. Plants with mutations in a homolog of an Arabidopsis (Arabidopsis thaliana) boron efflux transporter displayed boron deficiency phenotypes. The strategy described here provides a technically simple yet high-throughput approach for generating a collection of lines with targeted mutations and should be applicable to any plant transformation system.© 2017 American Society of Plant Biologists. All Rights Reserved.

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July 7, 2019

Rifamorpholines A-E, potential antibiotics from locust-associated actinobacteria Amycolatopsis sp. Hca4.

Cultivation of locust associated rare actinobacteria, Amycolatopsis sp. HCa4, has provided five unusual macrolactams rifamorpholines A-E. Their structures were determined by interpretation of spectroscopic and crystallographic data. Rifamorpholines A-E possess an unprecedented 5/6/6/6 ring chromophore, representing a new subclass of rifamycin antibiotics. The biosynthetic pathway for compounds 1-5 involves a key 1,6-cyclization for the formation of the morpholine ring. Compounds 2 and 4 showed potent activities against methicillin-resistant Staphylococcus aureus (MRSA) with MICs of 4.0 and 8.0 µM, respectively.

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July 7, 2019

Complete genome analysis of Thermus parvatiensis and comparative genomics of Thermus spp. provide insights into genetic variability and evolution of natural competence as strategic survival attributes.

Thermophilic environments represent an interesting niche. Among thermophiles, the genus Thermus is among the most studied genera. In this study, we have sequenced the genome of Thermus parvatiensis strain RL, a thermophile isolated from Himalayan hot water springs (temperature >96°C) using PacBio RSII SMRT technique. The small genome (2.01 Mbp) comprises a chromosome (1.87 Mbp) and a plasmid (143 Kbp), designated in this study as pTP143. Annotation revealed a high number of repair genes, a squeezed genome but containing highly plastic plasmid with transposases, integrases, mobile elements and hypothetical proteins (44%). We performed a comparative genomic study of the group Thermus with an aim of analysing the phylogenetic relatedness as well as niche specific attributes prevalent among the group. We compared the reference genome RL with 16 Thermus genomes to assess their phylogenetic relationships based on 16S rRNA gene sequences, average nucleotide identity (ANI), conserved marker genes (31 and 400), pan genome and tetranucleotide frequency. The core genome of the analyzed genomes contained 1,177 core genes and many singleton genes were detected in individual genomes, reflecting a conserved core but adaptive pan repertoire. We demonstrated the presence of metagenomic islands (chromosome:5, plasmid:5) by recruiting raw metagenomic data (from the same niche) against the genomic replicons of T. parvatiensis. We also dissected the CRISPR loci wide all genomes and found widespread presence of this system across Thermus genomes. Additionally, we performed a comparative analysis of competence loci wide Thermus genomes and found evidence for recent horizontal acquisition of the locus and continued dispersal among members reflecting that natural competence is a beneficial survival trait among Thermus members and its acquisition depicts unending evolution in order to accomplish optimal fitness.

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July 7, 2019

Comparative genomic and regulatory analyses of natamycin production of Streptomyces lydicus A02.

Streptomyces lydicus A02 is used by industry because it has a higher natamycin-producing capacity than the reference strain S. natalensis ATCC 27448. We sequenced the complete genome of A02 using next-generation sequencing platforms, and to achieve better sequence coverage and genome assembly, we utilized single-molecule real-time (SMRT) sequencing. The assembled genome comprises a 9,307,519-bp linear chromosome with a GC content of 70.67%, and contained 8,888 predicted genes. Comparative genomics and natamycin biosynthetic gene cluster (BGC) analysis showed that BGC are highly conserved among evolutionarily diverse strains, and they also shared closer genome evolution compared with other Streptomyces species. Forty gene clusters were predicted to involve in the secondary metabolism of A02, and it was richly displayed in two-component signal transduction systems (TCS) in the genome, indicating a complex regulatory systems and high diversity of metabolites. Disruption of the phoP gene of the phoR-phoP TCS and nsdA gene confirmed phosphate sensitivity and global negative regulation of natamycin production. The genome sequence and analyses presented in this study provide an important molecular basis for research on natamycin production in Streptomyces, which could facilitate rational genome modification to improve the industrial use of A02.

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July 7, 2019

In silico analysis of protein toxin and bacteriocins from Lactobacillus paracasei SD1 genome and available online databases.

Lactobacillus paracasei SD1 is a potential probiotic strain due to its ability to survive several conditions in human dental cavities. To ascertain its safety for human use, we therefore performed a comprehensive bioinformatics analysis and characterization of the bacterial protein toxins produced by this strain. We report the complete genome of Lactobacillus paracasei SD1 and its comparison to other Lactobacillus genomes. Additionally, we identify and analyze its protein toxins and antimicrobial proteins using reliable online database resources and establish its phylogenetic relationship with other bacterial genomes. Our investigation suggests that this strain is safe for human use and contains several bacteriocins that confer health benefits to the host. An in silico analysis of protein-protein interactions between the target bacteriocins and the microbial proteins gtfB and luxS of Streptococcus mutans was performed and is discussed here.

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July 7, 2019

Large scale and significant expression from pseudogenes in Sodalis glossinidius – a facultative bacterial endosymbiont

The majority of bacterial genomes have high coding efficiencies, but there are some genomes of intracellular bacteria that have low gene density. The genome of the endosymbiont Sodalis glossinidius contains almost 50% pseudogenes containing mutations that putatively silence them at the genomic level. We have applied multiple omic strategies, combining: Illumina and Pacific Biosciences Single-Molecule Real Time DNA-sequencing and annotation; stranded RNA-sequencing; and proteome analysis to better understand the transcriptional and translational landscape of Sodalis pseudogenes, and potential mechanisms for their control. Between 53% and 74% of the Sodalis transcriptome remains active in cell-free culture. Mean sense transcription from Coding Domain Sequences (CDS) is four-times greater than that from pseudogenes. Comparative genomic analysis of six Illumina-sequenced Sodalis isolates from different host Glossina species shows pseudogenes make up ~40% of the 2,729 genes in the core genome, suggesting are stable and/or Sodalis is a recent introduction across the Glossina genus as a facultative symbiont. These data further shed light on the importance of transcriptional and translational control in deciphering host-microbe interactions, and demonstrate that pseudogenes are more complex than a simple degrading DNA sequence. The combination of genomics, transcriptomics and proteomics give a multidimensional perspective for studying prokaryotic genomes with a view to elucidating evolutionary adaptation to novel environmental niches.

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July 7, 2019

Comparative genome analysis of the Flavobacteriales bacterium strain UJ101, isolated from the gut of Atergatis reticulatus.

Here we report the comparative genomic analysis of strain UJ101 with 15 strains from the family Flavobacteriaceae, using the CGExplorer program. Flavobacteriales bacterium strain UJ101 was isolated from a xanthid crab, Atergatis reticulatus, from the East Sea near Korea. The complete genome of strain UJ101 is a 3,074,209 bp, single, circular chromosome with 30.74% GC content. While the UJ101 genome contains a number of annotated genes for many metabolic pathways, such as the Embden-Meyerhof pathway, the pentose phosphate pathway, the tricarboxylic acid (TCA) cycle, and the glyoxylate cycle, genes for the Entner-Douddoroff pathway are not found in the UJ101 genome. Overall, carbon fixation processes were absent but nitrate reduction and denitrification pathways were conserved. The UJ101 genome was compared to genomes from other marine animals (three invertebrate strains and 5 fish strains) and other marine animal- derived genera. Notable results by genome comparisons showed that UJ101 is capable of denitrification and nitrate reduction, and that biotin-thiamine pathway participation varies among marine bacteria; fish-dwelling bacteria, freeliving bacteria, invertebrate-dwelling bacteria, and strain UJ101. Pan-genome analysis of the 16 strains in this study included 7,220 non-redundant genes that covered 62% of the pan-genome. A core-genome of 994 genes was present and consisted of 8% of the genes from the pan-genome. Strain UJ101 is a symbiotic hetero-organotroph isolated from xanthid crab, and is a metabolic generalist with nitrate-reducing abilities but without the ability to synthesize biotin. There is a general tendency of UJ101 and some fish pathogens to prefer thiamine-dependent glycolysis to gluconeogenesis. Biotin and thiamine auxotrophy or prototrophy may be used as important markers in microbial community studies.

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