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An ideal bacterial phylogenetic tree accurately retraces evolutionary history and accurately incorporates mutational, recombination and other events on the appropriate branches. Current strain-level bacterial phylogenetic analysis based on large numbers of genomes lacks reliability and resolution, and is hard to be replicated, confirmed and reused, because of the highly divergent nature of microbial genomes. We present SNPs and Recombination Events Tree (SaRTree), a pipeline using six "living trees" modules that addresses problems arising from the high numbers and variable quality of bacterial genome sequences. It provides for reuse of the tree and offers a major step toward global standardization of phylogenetic analysis by generating deposit files including all steps involved in phylogenetic inference. The tree itself is a "living tree" that can be extended by addition of more sequences, or the deposit can be used to vary the programs or parameters used, to assess the effect of such changes. This approach will allow phylogeny papers to meet the traditional responsibility of providing data and analysis that can be repeated and critically evaluated by others. We used the Acinetobacter baumannii global clone I to illustrate use of SaRTree to optimize tree resolution. An Escherichia coli tree was built from 351 sequences selected from 11,162 genome sequences, with the others added back onto well-defined branches, to show how this facility can greatly improve the outcomes from genome sequencing. SaRTree is designed for prokaryote strain-level analysis but could be adapted for other usage. [ABSTRACT FROM AUTHOR]

Vibrio cholerae has caused seven cholera pandemics since 1817, imposing terror on much of the world, but bacterial strains are currently only available for the sixth and seventh pandemics. The El Tor biotype seventh pandemic began in 1961 in Indonesia, but did not originate directly from the classical biotype sixth-pandemic strain. Previous studies focused mainly on the spread of the seventh pandemic after 1970. Here, we analyze in unprecedented detail the origin, evolution, and transition to pandemicity of the seventh-pandemic strain. We used high-resolution comparative genomic analysis of strains collected from 1930 to 1964, covering the evolution from the first available El Tor biotype strain to the start of the seventh pandemic. We define six stages leading to the pandemic strain and reveal all key events. The seventh pandemic originated from a nonpathogenic strain in the Middle East, first observed in 1897. It subsequently underwent explosive diversification, including the spawning of the pandemic lineage. This rapid diversification suggests that, when first observed, the strain had only recently arrived in theMiddle East, possibly from the Asian homeland of cholera. The lineage migrated toMakassar, Indonesia, where it gained the important virulence-associated elements Vibrio seventh pandemic island I (VSP-I), VSP-II, and El Tor type cholera toxin prophage by 1954, and it then became pandemic in 1961 after only 12 additional mutations. Our data indicate that specific niches in the Middle East and Makassar were important in generating the pandemic strain by providing gene sources and the driving forces for genetic events. [ABSTRACT FROM AUTHOR]

Translocation of lipid-linked oligosaccharides is a common theme across prokaryotes and eukaryotes. For bacteria, such activity is used in cell wall construction, polysaccharide synthesis, and the relatively recently discovered protein glycosylation. To the best of our knowledge, the Gram-negative inner membrane flippase Wzx was the first protein identified as being involved in oligosaccharide translocation, and yet we still have only a limited understanding of this protein after 3 decades of research. At present, Wzx is known to be a multitransmembrane protein with enormous sequence diversity that flips oligosaccharide substrates with varied degrees of preference. In this review, we provide an overview of the major findings for this protein, with a particular focus on substrate preference. [ABSTRACT FROM AUTHOR]

Escherichia coli includes clonal groups of both commensal and pathogenic strains, with some of the latter causing serious infectious diseases. O antigen variation is current standard in defining strains for taxonomy and epidemiology, providing the basis for many serotyping schemes for Gram-negative bacteria. This review covers the diversity in E. coli O antigen structures and gene clusters, and the genetic basis for the structural diversity. Of the 187 formally defined O antigens, six (O31, O47, O67, O72, O94 and O122) have since been removed and three (O34, O89 and O144) strains do not produce any O antigen. Therefore, structures are presented for 176 of the 181 E. coli O antigens, some of which include subgroups. Most (93%) of these O antigens are synthesized via the Wzx/Wzy pathway, 11 via the ABC transporter pathway, with O20, O57 and O60 still uncharacterized due to failure to find their O antigen gene clusters. Biosynthetic pathways are given for 38 of the 49 sugars found in E. coli O antigens, and several pairs or groups of the E. coli antigens that have related structures show close relationships of the O antigen gene clusters within clades, thereby highlighting the genetic basis of the evolution of diversity., (© The Author(s) 2019. Published by Oxford University Press on behalf of FEMS.)

Cloning of whole polysaccharide biosynthesis gene clusters for expression in a common Escherichia coli tester strain has the major advantage of enabling direct functional comparisons between gene clusters that are normally found in different strains, where their expression is potentially under differential regulatory control. However, due to the large size of many of these gene clusters, classical cloning methods are highly inefficient, time-consuming, and/or labor-intensive. Here we describe a recently developed system, called the operon assembly protocol (OAP), in which yeast homologous recombination pathways are used to assemble overlapping PCR fragments onto a specially engineered yeast E. coli shuttle vector, resulting in full-length customizable gene cluster clones on single-copy plasmids. Multiple versions of the same gene cluster can also be assembled in parallel with genes deleted, replaced, or rearranged, allowing the function and/or specificity of individual genes to be examined. Since the vector can be easily modified to include other bacterial replicons, it can also be broadly applied to the functional analysis of a wide range of bacterial gene clusters and operons.

Translocation of lipid-linked oligosaccharides is a common theme across prokaryotes and eukaryotes. For bacteria, such activity is used in cell wall construction, polysaccharide synthesis, and the relatively recently discovered protein glycosylation. To the best of our knowledge, the Gram-negative inner membrane flippase Wzx was the first protein identified as being involved in oligosaccharide translocation, and yet we still have only a limited understanding of this protein after 3 decades of research. At present, Wzx is known to be a multitransmembrane protein with enormous sequence diversity that flips oligosaccharide substrates with varied degrees of preference. In this review, we provide an overview of the major findings for this protein, with a particular focus on substrate preference., (Copyright © 2017 American Society for Microbiology.)

The Wzx/Wzy O-antigen pathway involves synthesis of a repeat unit (O unit) consisting of 3 to 8 sugars on an inner-membrane-embedded lipid carrier. These O units are translocated across the membrane to its periplasmic face by Wzx, while retaining linkage to the carrier, and then polymerized by Wzy to O-antigen polymer, which WaaL ligase transfers to a lipopolysaccharide precursor to complete lipopolysaccharide synthesis, concomitantly releasing the lipid carrier. This lipid carrier is also used for peptidoglycan assembly, and sequestration is known to be toxic. Thus, O-unit synthesis must involve precise regulation to meet demand but avoid overproduction. Here we show that loss of WaaL reverses a known growth defect in a Salmonella mutant that otherwise accumulates O-unit intermediates and propose that WaaL is also involved in a novel feedback mechanism to regulate O-unit synthesis, based on the availability of O units on the periplasmic face of the membrane.