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Phages, short for bacteriophages, are viruses that specifically infect bacteria and are the most abundant biological entities on earth found in every explored environment, from the deep sea to the Sahara Desert. Phages are abundant within the human biome and are gaining increasing recognition as potential modulators of the gut ecosystem. For example, they have been connected to gastrointestinal diseases and the treatment efficacy of Fecal Microbiota Transplant. The ability of phages to modulate the human gut microbiome has been attributed to the predation of bacteria or the promotion of bacterial survival by the transfer of genes that enhance bacterial fitness upon infection. In addition, phages have been shown to interact with the human immune system with variable outcomes. Despite the increasing evidence supporting the importance of phages in the gut ecosystem, the extent of their influence on the shape of the gut ecosystem is yet to be fully understood. Here, we discuss evidence for phage modulation of the gut microbiome, postulating that phages are pivotal contributors to the gut ecosystem dynamics. We therefore propose novel research questions to further elucidate the role(s) that they have within the human ecosystem and its impact on our health and well-being.

Despite significant frequencies of lateral gene transfer between species, higher taxonomic groups of bacteria show ecological and phenotypic cohesion. This suggests that barriers prevent panmictic dissemination of genes via lateral gene transfer. We have proposed that most bacterial genomes have a functional architecture imposed by Architecture IMparting Sequences (AIMS). AIMS are defined as 8 base pair sequences preferentially abundant on leading strands, whose abundance and strand-bias are positively correlated with proximity to the replication terminus. We determined that inversions whose endpoints lie within a single chromosome arm, which would reverse the polarity of AIMS in the inverted region, are both shorter and less frequent near the replication terminus. This distribution is consistent with the increased selection on AIMS function in this region, thus constraining DNA rearrangement. To test the hypothesis that AIMS also constrain DNA transfer between genomes, AIMS were identified in genomes while ignoring atypical, potentially laterally-transferred genes. The strand-bias of AIMS within recently acquired genes was negatively correlated with the distance of those genes from their genome's replication terminus. This suggests that selection for AIMS function prevents the acquisition of genes whose AIMS are not found predominantly in the permissive orientation. This constraint has led to the loss of at least 18% of genes acquired by transfer in the terminus-proximal region. We used completely sequenced genomes to produce a predictive road map of paths of expected horizontal gene transfer between species based on AIMS compatibility between donor and recipient genomes. These results support a model whereby organisms retain introgressed genes only if the benefits conferred by their encoded functions outweigh the detriments incurred by the presence of foreign DNA lacking genome-wide architectural information.

  According to the authors, every E. coli strain is endowed with unusual characters, at least in terms of its gene content. Even as we grapple to understand the breadth of present-day E. coli, they continue to evolve at a breathtaking rate. Since the first detection of E. coli 0157:

The bacterium Paenibacillus larvae is responsible for the devastating honey bee (Apis mellifera) disease American Foulbrood. Research into bacteriophages that infect P. larvae is growing rapidly due to increasing antibiotic resistance and restrictions on antibiotic use in beehives in some countries. In this study, we present the sequenced and annotated genomes of 26 novel P. larvae phages recently isolated in New Zealand, which brings the total number of sequenced and annotated P. larvae phages to 96. The 26 novel phages belong to the pre-existing Vegas or Harrison clusters. We performed a comprehensive genomic analysis of all 96 phage genomes, grouping them into five divergent clusters and two singletons. The majority of these phages are temperate, with the possible exception of three phages that may be lytic. All 96 of these phages encode an N-acteylmuramoyl-L-alanine amidase that serves as their lysin. The amidases are from two divergent clusters, both of which show a high degree of intra-cluster similarity. Six phages and a prophage contain the Plx1 P. larvae toxin gene, which we suggest may be mobilizable. This study expands our knowledge of P. larvae phages from around the world.

Maintenance of rod-shape in bacterial cells depends on the actin-like protein MreB. Deletion of mreB from Pseudomonas fluorescens SBW25 results in viable spherical cells of variable volume and reduced fitness. Using a combination of time-resolved microscopy and biochemical assay of peptidoglycan synthesis, we show that reduced fitness is a consequence of perturbed cell size homeostasis that arises primarily from differential growth of daughter cells. A 1000-generation selection experiment resulted in rapid restoration of fitness with derived cells retaining spherical shape. Mutations in the peptidoglycan synthesis protein Pbp1A were identified as the main route for evolutionary rescue with genetic reconstructions demonstrating causality. Compensatory pbp1A mutations that targeted transpeptidase activity enhanced homogeneity of cell wall synthesis on lateral surfaces and restored cell size homeostasis. Mechanistic explanations require enhanced understanding of why deletion of mreB causes heterogeneity in cell wall synthesis. We conclude by presenting two testable hypotheses, one of which posits that heterogeneity stems from non-functional cell wall synthesis machinery, while the second posits that the machinery is functional, albeit stalled. Overall, our data provide support for the second hypothesis and draw attention to the importance of balance between transpeptidase and glycosyltransferase functions of peptidoglycan building enzymes for cell shape determination.

There is concern about second and subsequent waves of COVID-19 caused by the SARS-CoV-2 coronavirus occurring in communities globally that had an initial disease wave. Metropolitan Houston, TX, with a population of 7 million, is experiencing a massive second disease wave that began in late May 2020. To understand SARS-CoV-2 molecular population genomic architecture and evolution and the relationship between virus genotypes and patient features, we sequenced the genomes of 5,085 SARS-CoV-2 strains from these two waves. Our report provides the first molecular characterization of SARS-CoV-2 strains causing two distinct COVID-19 disease waves. We sequenced the genomes of 5,085 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains causing two coronavirus disease 2019 (COVID-19) disease waves in metropolitan Houston, TX, an ethnically diverse region with 7 million residents. The genomes were from viruses recovered in the earliest recognized phase of the pandemic in Houston and from viruses recovered in an ongoing massive second wave of infections. The virus was originally introduced into Houston many times independently. Virtually all strains in the second wave have a Gly614 amino acid replacement in the spike protein, a polymorphism that has been linked to increased transmission and infectivity. Patients infected with the Gly614 variant strains had significantly higher virus loads in the nasopharynx on initial diagnosis. We found little evidence of a significant relationship between virus genotype and altered virulence, stressing the linkage between disease severity, underlying medical conditions, and host genetics. Some regions of the spike protein—the primary target of global vaccine efforts—are replete with amino acid replacements, perhaps indicating the action of selection. We exploited the genomic data to generate defined single amino acid replacements in the receptor binding domain of spike protein that, importantly, produced decreased recognition by the neutralizing monoclonal antibody CR3022. Our report represents the first analysis of the molecular architecture of SARS-CoV-2 in two infection waves in a major metropolitan region. The findings will help us to understand the origin, composition, and trajectory of future infection waves and the potential effect of the host immune response and therapeutic maneuvers on SARS-CoV-2 evolution. IMPORTANCE There is concern about second and subsequent waves of COVID-19 caused by the SARS-CoV-2 coronavirus occurring in communities globally that had an initial disease wave. Metropolitan Houston, TX, with a population of 7 million, is experiencing a massive second disease wave that began in late May 2020. To understand SARS-CoV-2 molecular population genomic architecture and evolution and the relationship between virus genotypes and patient features, we sequenced the genomes of 5,085 SARS-CoV-2 strains from these two waves. Our report provides the first molecular characterization of SARS-CoV-2 strains causing two distinct COVID-19 disease waves.

CALR mutation analysis is routinely used to diagnose BCR/ABL1-negative myeloproliferative neoplasms. The 2 most common CALR mutations are a 52-base pair (bp) deletion and a 5-bp insertion, which account for approximately 85% of cases. To evaluate our new microfluidic chip assay, we tested CALR mutant and wild-type specimens that were previously analyzed using conventional methods at a reference laboratory. Samples included EDTA-anticoagulated peripheral blood and bone marrow specimens, air dried bone marrow aspirate smears, and formalin-fixed, paraffin-embedded bone marrow sections. CALR exon 9 was PCR amplified using 2 previously published primer pairs and a third unique primer pair designed for our new assay. Amplicons were sized using microfluidic chip analysis. Concordance with the reference method was 100% (42/42). Intra-run and inter-run reproducibility were also 100% (3/3 and 3/3, respectively). The limit of detection was confirmed to be 6% mutant alleles. We determined that the microfluidic chip assay to detect CALR exon 9 mutations was acceptable for clinical use. Compared with the conventional method, the microfluidic analysis assay benefits from a streamlined workflow, faster turnaround, and a smaller instrument footprint.

The bacteriophage population is vast, dynamic, old, and genetically diverse. The genomics of phages that infect bacterial hosts in the phylum Actinobacteria show them to not only be diverse but also pervasively mosaic, and replete with genes of unknown function. To further explore this broad group of bacteriophages, we describe here the isolation and genomic characterization of 116 phages that infect Microbacterium spp. Most of the phages are lytic, and can be grouped into twelve clusters according to their overall relatedness; seven of the phages are singletons with no close relatives. Genome sizes vary from 17.3 kbp to 97.7 kbp, and their G+C% content ranges from 51.4% to 71.4%, compared to ~67% for their Microbacterium hosts. The phages were isolated on five different Microbacterium species, but typically do not efficiently infect strains beyond the one on which they were isolated. These Microbacterium phages contain many novel features, including very large viral genes (13.5 kbp) and unusual fusions of structural proteins, including a fusion of VIP2 toxin and a MuF-like protein into a single gene. These phages and their genetic components such as integration systems, recombineering tools, and phage-mediated delivery systems, will be useful resources for advancing Microbacterium genetics.

Bacterial chromosomes are immense polymers whose faithful replication and segregation are crucial to cell survival. The ability of proteins such as FtsK to move unidirectionally toward the replication terminus, and direct DNA translocation into the appropriate daughter cell during cell division, requires that bacterial genomes maintain an architecture for the orderly replication and segregation of chromosomes. We suggest that proteins that locate the replication terminus exploit strand-biased sequences that are overrepresented on one DNA strand, and that selection increases with decreased distance to the replication terminus. We report a generalized method for detecting these architecture imparting sequences (AIMS) and have identified AIMS in nearly all bacterial genomes. Their increased abundance on leading strands and decreased abundance on lagging strands toward replication termini are not the result of changes in mutational bias; rather, they reflect a gradient of long-term positive selection for AIMS. The maintenance of the pattern of AIMS across the genomes of related bacteria independent of their positions within individual genes suggests a well-conserved role in genome biology. The stable gradient of AIMS abundance from replication origin to terminus suggests that the replicore acts as a target of selection, where selection for chromosome architecture results in the maintenance of gene order and in the lack of high-frequency DNA inversion within replicores.

Introduction: Next-generation-sequencing (NGS) is increasingly used in clinical and research protocols for patients with cancer. NGS assays are routinely used in clinical laboratories to detect mutations bearing on cancer diagnosis, prognosis and personalized therapy. A typical assay may interrogate 50 or more gene targets that encompass many thousands of possible gene variants. Analysis of NGS data in cancer is a labor-intensive process that can become overwhelming to the molecular pathologist or research scientist. Although commercial tools for NGS data analysis and interpretation are available, they are often costly, lack key functionality or cannot be customized by the end user. Methods: To facilitate NGS data analysis in our clinical molecular diagnostics laboratory, we created a custom bioinformatics tool termed Houston Methodist Variant Viewer (HMVV). HMVV is a Java-based solution that integrates sequencing instrument output, bioinformatics analysis, storage resources and end user interface. Results: Compared to the predicate method used in our clinical laboratory, HMVV markedly simplifies the bioinformatics workflow for the molecular technologist and facilitates the variant review by the molecular pathologist. Importantly, HMVV reduces time spent researching the biological significance of the variants detected, standardizes the online resources used to perform the variant investigation and assists generation of the annotated report for the electronic medical record. HMVV also maintains a searchable variant database, including the variant annotations generated by the pathologist, which is useful for downstream quality improvement and research projects. Conclusions: HMVV is a clinical grade, low-cost, feature-rich, highly customizable platform that we have made available for continued development by the pathology informatics community.