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July 7, 2019  |  

Stenotrophomonas comparative genomics reveals genes and functions that differentiate beneficial and pathogenic bacteria.

In recent years, the number of human infections caused by opportunistic pathogens has increased dramatically. Plant rhizospheres are one of the most typical natural reservoirs for these pathogens but they also represent a great source for beneficial microbes with potential for biotechnological applications. However, understanding the natural variation and possible differences between pathogens and beneficials is the main challenge in furthering these possibilities. The genus Stenotrophomonas contains representatives found to be associated with human and plant host.We used comparative genomics as well as transcriptomic and physiological approaches to detect significant borders between the Stenotrophomonas strains: the multi-drug resistant pathogenic S. maltophilia and the plant-associated strains S. maltophilia R551-3 and S. rhizophila DSM14405T (both are biocontrol agents). We found an overall high degree of sequence similarity between the genomes of all three strains. Despite the notable similarity in potential factors responsible for host invasion and antibiotic resistance, other factors including several crucial virulence factors and heat shock proteins were absent in the plant-associated DSM14405T. Instead, S. rhizophila DSM14405T possessed unique genes for the synthesis and transport of the plant-protective spermidine, plant cell-wall degrading enzymes, and high salinity tolerance. Moreover, the presence or absence of bacterial growth at 37°C was identified as a very simple method in differentiating between pathogenic and non-pathogenic isolates. DSM14405T is not able to grow at this human-relevant temperature, most likely in great part due to the absence of heat shock genes and perhaps also because of the up-regulation at increased temperatures of several genes involved in a suicide mechanism.While this study is important for understanding the mechanisms behind the emerging pattern of infectious diseases, it is, to our knowledge, the first of its kind to assess the risk of beneficial strains for biotechnological applications. We identified certain traits typical of pathogens such as growth at the human body temperature together with the production of heat shock proteins as opposed to a temperature-regulated suicide system that is harnessed by beneficials.

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July 7, 2019  |  

Association mapping, patterns of linkage disequilibrium and selection in the vicinity of the PHYTOCHROME C gene in pearl millet.

Linkage analysis confirmed the association in the region of PHYC in pearl millet. The comparison of genes found in this region suggests that PHYC is the best candidate. Major efforts are currently underway to dissect the phenotype-genotype relationship in plants and animals using existing populations. This method exploits historical recombinations accumulated in these populations. However, linkage disequilibrium sometimes extends over a relatively long distance, particularly in genomic regions containing polymorphisms that have been targets for selection. In this case, many genes in the region could be statistically associated with the trait shaped by the selected polymorphism. Statistical analyses could help in identifying the best candidate genes into such a region where an association is found. In a previous study, we proposed that a fragment of the PHYTOCHROME C gene (PHYC) is associated with flowering time and morphological variations in pearl millet. In the present study, we first performed linkage analyses using three pearl millet F2 families to confirm the presence of a QTL in the vicinity of PHYC. We then analyzed a wider genomic region of ~100 kb around PHYC to pinpoint the gene that best explains the association with the trait in this region. A panel of 90 pearl millet inbred lines was used to assess the association. We used a Markov chain Monte Carlo approach to compare 75 markers distributed along this 100-kb region. We found the best candidate markers on the PHYC gene. Signatures of selection in this region were assessed in an independent data set and pointed to the same gene. These results foster confidence in the likely role of PHYC in phenotypic variation and encourage the development of functional studies.

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July 7, 2019  |  

Methyltransferases acquired by lactococcal 936-type phage provide protection against restriction endonuclease activity

BACKGROUND:So-called 936-type phages are among the most frequently isolated phages in dairy facilities utilising Lactococcus lactis starter cultures. Despite extensive efforts to control phage proliferation and decades of research, these phages continue to negatively impact cheese production in terms of the final product quality and consequently, monetary return.RESULTS:Whole genome sequencing and in silico analysis of three 936-type phage genomes identified several putative (orphan) methyltransferase (MTase)-encoding genes located within the packaging and replication regions of the genome. Utilising SMRT sequencing, methylome analysis was performed on all three phages, allowing the identification of adenine modifications consistent with N-6 methyladenine sequence methylation, which in some cases could be attributed to these phage-encoded MTases. Heterologous gene expression revealed that M.Phi145I/M.Phi93I and M.Phi93DAM, encoded by genes located within the packaging module, provide protection against the restriction enzymes HphI and DpnII, respectively, representing the first functional MTases identified in members of 936-type phages.CONCLUSIONS:SMRT sequencing technology enabled the identification of the target motifs of MTases encoded by the genomes of three lytic 936-type phages and these MTases represent the first functional MTases identified in this species of phage. The presence of these MTase-encoding genes on 936-type phage genomes is assumed to represent an adaptive response to circumvent host encoded restriction-modification systems thereby increasing the fitness of the phages in a dynamic dairy environment.

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July 7, 2019  |  

The odd one out: Bacillus ACT bacteriophage CP-51 exhibits unusual properties compared to related Spounavirinae W.Ph. and Bastille.

The Bacillus ACT group includes three important pathogenic species of Bacillus: anthracis, cereus and thuringiensis. We characterized three virulent bacteriophages, Bastille, W.Ph. and CP-51, that infect various strains of these three species. We have determined the complete genome sequences of CP-51, W.Ph. and Bastille, and their physical genome structures. The CP-51 genome sequence could only be obtained using a combination of conventional and second and third next generation sequencing technologies – illustrating the problems associated with sequencing highly modified DNA. We present evidence that the generalized transduction facilitated by CP-51 is independent of a specific genome structure, but likely due to sporadic packaging errors of the terminase. There is clear correlation of the genetic and morphological features of these phages validating their placement in the Spounavirinae subfamily (SPO1-related phages) of the Myoviridae. This study also provides tools for the development of phage-based diagnostics/therapeutics for this group of pathogens. Copyright © 2014 Elsevier Inc. All rights reserved.

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July 7, 2019  |  

Diversification of bacterial genome content through distinct mechanisms over different timescales.

Bacterial populations often consist of multiple co-circulating lineages. Determining how such population structures arise requires understanding what drives bacterial diversification. Using 616 systematically sampled genomes, we show that Streptococcus pneumoniae lineages are typically characterized by combinations of infrequently transferred stable genomic islands: those moving primarily through transformation, along with integrative and conjugative elements and phage-related chromosomal islands. The only lineage containing extensive unique sequence corresponds to a set of atypical unencapsulated isolates that may represent a distinct species. However, prophage content is highly variable even within lineages, suggesting frequent horizontal transmission that would necessitate rapidly diversifying anti-phage mechanisms to prevent these viruses sweeping through populations. Correspondingly, two loci encoding Type I restriction-modification systems able to change their specificity over short timescales through intragenomic recombination are ubiquitous across the collection. Hence short-term pneumococcal variation is characterized by movement of phage and intragenomic rearrangements, with the slower transfer of stable loci distinguishing lineages.

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July 7, 2019  |  

Complete genome sequence of a multidrug-resistant Salmonella enterica serovar Typhimurium var. 5- strain isolated from chicken breast.

Salmonella enterica subsp. enterica serovar Typhimurium is a leading cause of salmonellosis. Here, we report a closed genome sequence, including sequences of 3 plasmids, of Salmonella serovar Typhimurium var. 5- CFSAN001921 (National Antimicrobial Resistance Monitoring System [NARMS] strain ID N30688), which was isolated from chicken breast meat and shows resistance to 10 different antimicrobials. Whole-genome and plasmid sequence analyses of this isolate will help enhance our understanding of this pathogenic multidrug-resistant serovar.

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July 7, 2019  |  

Mutation in the C-di-AMP cyclase dacA affects fitness and resistance of methicillin resistant Staphylococcus aureus.

Faster growing and more virulent strains of methicillin resistant Staphylococcus aureus (MRSA) are increasingly displacing highly resistant MRSA. Elevated fitness in these MRSA is often accompanied by decreased and heterogeneous levels of methicillin resistance; however, the mechanisms for this phenomenon are not yet fully understood. Whole genome sequencing was used to investigate the genetic basis of this apparent correlation, in an isogenic MRSA strain pair that differed in methicillin resistance levels and fitness, with respect to growth rate. Sequencing revealed only one single nucleotide polymorphism (SNP) in the diadenylate cyclase gene dacA in the faster growing but less resistant strain. Diadenylate cyclases were recently discovered to synthesize the new second messenger cyclic diadenosine monophosphate (c-di-AMP). Introduction of this mutation into the highly resistant but slower growing strain reduced resistance and increased its growth rate, suggesting a direct connection between the dacA mutation and the phenotypic differences of these strains. Quantification of cellular c-di-AMP revealed that the dacA mutation decreased c-di-AMP levels resulting in reduced autolysis, increased salt tolerance and a reduction in the basal expression of the cell wall stress stimulon. These results indicate that c-di-AMP affects cell envelope-related signalling in S. aureus. The influence of c-di-AMP on growth rate and methicillin resistance in MRSA indicate that altering c-di-AMP levels could be a mechanism by which MRSA strains can increase their fitness levels by reducing their methicillin resistance levels.

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July 7, 2019  |  

Comparing the genomes of Helicobacter pylori clinical strain UM032 and mice-adapted derivatives.

Helicobacter pylori is a Gram-negative bacterium that persistently infects the human stomach inducing chronic inflammation. The exact mechanisms of pathogenesis are still not completely understood. Although not a natural host for H. pylori, mouse infection models play an important role in establishing the immunology and pathogenicity of H. pylori. In this study, for the first time, the genome sequences of clinical H. pylori strain UM032 and mice-adapted derivatives, 298 and 299, were sequenced using the PacBio Single Molecule, Real-Time (SMRT) technology.Here, we described the single contig which was achieved for UM032 (1,599,441 bp), 298 (1,604,216 bp) and 299 (1,601,149 bp). Preliminary analysis suggested that methylation of H. pylori genome through its restriction modification system may be determinative of its host specificity and adaptation.Availability of these genomic sequences will aid in enhancing our current level of understanding the host specificity of H. pylori.

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July 7, 2019  |  

Direct sequencing of small genomes on the Pacific Biosciences RS without library preparation.

We have developed a sequencing method on the Pacific Biosciences RS sequencer (the PacBio) for small DNA molecules that avoids the need for a standard library preparation. To date this approach has been applied toward sequencing single-stranded and double-stranded viral genomes, bacterial plasmids, plasmid vector models for DNA-modification analysis, and linear DNA fragments covering an entire bacterial genome. Using direct sequencing it is possible to generate sequence data from as little as 1 ng of DNA, offering a significant advantage over current protocols which typically require 400-500 ng of sheared DNA for the library preparation.

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July 7, 2019  |  

Implementation and data analysis of Tn-seq, whole genome resequencing, and single-molecule real time sequencing for bacterial genetics.

Few discoveries have been more transformative to the biological sciences than the development of DNA sequencing technologies. The rapid advancement of sequencing and bioinformatics tools has revolutionized bacterial genetics, deepening our understanding of model and clinically relevant organisms. Although application of newer sequencing technologies to studies in bacterial genetics is increasing, the implementation of DNA sequencing technologies and development of the bioinformatics tools required for analyzing the large data sets generated remains a challenge for many. In this minireview, we have chosen to summarize three sequencing approaches that are particularly useful for bacterial genetics. We provide resources for scientists new to and interested in their application. Herein, we discuss the analysis of Tn-seq data to determine gene disruptions differentially represented in a mutant population, Illumina sequencing for identification of suppressor or other mutations, and we summarize single-molecule real time (SMRT) sequencing for de novo genome assembly and the use of the output data for detection of DNA base modifications. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 7, 2019  |  

Detection, isolation and characterization of Fusobacterium gastrosuis sp. nov. colonizing the stomach of pigs.

Nine strains of a novel Fusobacterium sp. were isolated from the stomach of 6-8 months old and adult pigs. The isolates were obligately anaerobic, although they endured 2h exposure to air. Phylogenetic analysis based on 16S rRNA and gyrase B genes demonstrated that the isolates showed high sequence similarity with Fusobacterium mortiferum, Fusobacterium ulcerans, Fusobacterium varium, Fusobacterium russii and Fusobacterium necrogenes, but formed a distinct lineage in the genus Fusobacterium. Comparative analysis of the genome of the type strain of this novel Fusobacterium sp. confirmed that it is different from other recognized Fusobacterium spp. DNA-DNA hybridization, fingerprinting and genomic %GC determination further supported the conclusion that the isolates belong to a new, distinct species. The isolates were also distinguishable from these and other Fusobacterium spp. by phenotypical characterization. The strains produced indole and exhibited proline arylamidase and glutamic acid decarboxylase activity. They did not hydrolyse esculin, did not exhibit pyroglutamic acid arylamidase, valine arylamidase, a-galactosidase, ß-galactosidase, ß-galactosidase-6-phosphate or a-glucosidase activity nor produced acid from cellobiose, glucose, lactose, mannitol, mannose, maltose, raffinose, saccharose, salicin or trehalose. The major fatty acids were C16:0 and C18:1?9c. The name Fusobacterium gastrosuis sp. nov. is proposed for the novel isolates with the type strain CDW1(T) (=DSM 101753(T)=LMG 29236(T)). We also demonstrated that Clostridium rectum and mortiferum Fusobacterium represent the same species, with nomenclatural priority for the latter. Copyright © 2016 Elsevier GmbH. All rights reserved.

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July 7, 2019  |  

Complete genome sequence of Pseudomonas brassicacearum strain L13-6-12, a biological control agent from the rhizosphere of potato

Pseudomonas brassicacearum strain L13-6-12 is a rhizosphere colonizer of potato, lettuce and sugar beet. Previous studies have shown that this motile, Gram-negative, non-sporulating bacterium is an effective biocontrol agent against different phytopathogens. Here, we announce and describe the complete genome sequence of P. brassicacearum L13-6-12 consisting of a single 6.7 Mb circular chromosome that consists of 5773 protein coding genes and 85 RNA-only encoding genes. Genome analysis revealed genes encoding specialized functions for pathogen suppression, thriving in the rhizosphere and interacting with eukaryotic organisms.

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