Establishment Genes Present on pLS20 Family of Conjugative Plasmids Are Regulated in Two Different Ways

During conjugation, a conjugative DNA element is transferred from a donor to a recipient cell via a connecting channel. Conjugation has clinical relevance because it is the major route for spreading antibiotic resistance and virulence genes. The conjugation process can be divided into different steps. The initial steps carried out in the donor cell culminate in the transfer of a single DNA strand (ssDNA) of the conjugative element into the recipient cell. However, stable settlement of the conjugative element in the new host requires at least two additional events: conversion of the transferred ssDNA into double-stranded DNA and inhibition of the hosts’ defence mechanisms to prevent degradation of the transferred DNA. The genes involved in this late step are historically referred to as establishment genes. The defence mechanisms of the host must be inactivated rapidly and—importantly—transiently, because prolonged inactivation would make the cell vulnerable to the attack of other foreign DNA, such as those of phages. Therefore, expression of the establishment genes in the recipient cell has to be rapid but transient. Here, we studied regulation of the establishment genes present on the four clades of the pLS20 family of conjugative plasmids harboured by different Bacillus species. Evidence is presented that two fundamentally different mechanisms regulate the establishment genes present on these plasmids. Identification of the regulatory sequences were critical in revealing the establishment regulons. Remarkably, whereas the conjugation genes involved in the early steps of the conjugation process are conserved and are located in a single large operon, the establishment genes are highly variable and organised in multiple operons. We propose that the mosaical distribution of establishment genes in multiple operons is directly related to the variability of defence genes encoded by the host bacterial chromosomes.

3CAC: improving the classification of phages and plasmids from metagenomic assemblies using assembly graphs

Bacteriophages and plasmids usually coexist with their host bacteria in microbial communities and play important roles in microbial evolution. Accurately identifying sequence contigs as phages, plasmids, and bacterial chromosomes in mixed metagenomic assemblies is critical for further unraveling their functions. Many classification tools have been developed for identifying either phages or plasmids in metagenomic assemblies. However, only two classifiers, PPR-Meta and viralVerify, were proposed to simultaneously identify phages and plasmids in mixed metagenomic assemblies. Due to the very high fraction of chromosome contigs in the assemblies, both tools achieve high precision in the classification of chromosomes but perform poorly in classifying phages and plasmids. Short contigs in these assemblies are often wrongly classified or classified as uncertain. Here we present 3CAC, a new three-class classifier that improves the precision of phage and plasmid classifications. 3CAC starts with an initial three-class classification generated by existing classifiers and further improves the classification of short contigs and contigs with low confidence classification by using proximity in the assembly graph. Evaluation on simulated metagenomes and on real human gut microbiome samples showed that 3CAC outperformed PPR-Meta and viralVerify in both precision and recall, and increased F1-score by at least 10 percentage points.

The Xer activation factor of TLCΦ expands the possibilities for Xer recombination

ABSTRACTMany mobile elements take advantage of the highly-conserved chromosome dimer resolution system of bacteria, Xer. They participate in the transmission of antibiotic resistance and pathogenicity determinants. In particular, the toxin-linked cryptic satellite phage (TLCΦ) plays an essential role in the continuous emergence of new toxigenic clones of the Vibrio cholerae strain at the origin of the ongoing 7th cholera pandemic. The Xer machinery is composed of two chromosomally-encoded tyrosine recombinases, XerC and XerD. They resolve chromosome dimers by adding a crossover between sister copies of a specific 28 base pair site of bacterial chromosomes, dif. The activity of XerD depends on a direct contact with a cell division protein, FtsK, which spatially and temporally constrains the process. TLCΦ encodes for a XerD-activation factor (XafT), which drives the integration of the phage into the dif site of the primary chromosome of V. cholerae independently of FtsK. However, XerD does not bind to the attachment site (attP) of TLCΦ, which raised questions on the integration process. Here, we compared the integration efficiency of thousands of synthetic mini-TLCΦ plasmids harbouring different attP sites and assessed their stability in vivo. In addition, we compared the efficiency with which XafT and the XerD activation domain of FtsK drive recombination reactions in vitro. Taken together, our results suggest that XafT promotes the formation of synaptic complexes between canonical Xer recombination sites and imperfect sites.

Scanning mutagenesis of RNA-binding protein ProQ reveals a quality control role for the Lon protease

The FinO-domain protein ProQ belongs to a widespread family of RNA-binding proteins (RBPs) involved in gene regulation in bacterial chromosomes and mobile elements. Whilst the cellular RNA targets of ProQ have been established in diverse bacteria, the functionally crucial ProQ residues remain to be identified under physiological conditions. Following our discovery that ProQ deficiency alleviates growth suppression of Salmonella with succinate as the sole carbon source, an experimental evolution approach was devised to exploit this phenotype. By coupling mutational scanning with loss-of-function selection, we identified multiple ProQ residues in both the N-terminal FinO domain and the variable C-terminal region that are required for ProQ activity. Two C-terminal mutations abrogated ProQ function and mildly impaired binding of a model RNA target. By contrast, several mutations in the FinO domain rendered ProQ both functionally inactive and unable to interact with target RNA in vivo. Alteration of the FinO domain stimulated the rapid turnover of ProQ by Lon-mediated proteolysis, suggesting a quality control mechanism that prevents the accumulation of non-functional ProQ molecules. We extend this observation to Hfq, the other major sRNA chaperone of enteric bacteria. The Hfq Y55A mutant protein, defective in RNA-binding and oligomerization, proved to be labile and susceptible to degradation by Lon. Taken together, our findings connect the major AAA+ family protease Lon with RNA-dependent quality control of Hfq and ProQ, the two major sRNA chaperones of Gram-negative bacteria.

Whole-Genome Analysis Reveals That the Nucleoid Protein IHF Predominantly Binds to the Replication Origin oriC Specifically at the Time of Initiation

The structure and function of bacterial chromosomes are dynamically regulated by a wide variety of nucleoid-associated proteins (NAPs) and DNA superstructures, such as DNA supercoiling. In Escherichia coli, integration host factor (IHF), a NAP, binds to specific transcription promoters and regulatory DNA elements of DNA replication such as the replication origin oriC: binding to these elements depends on the cell cycle but underlying mechanisms are unknown. In this study, we combined GeF-seq (genome footprinting with high-throughput sequencing) with synchronization of the E. coli cell cycle to determine the genome-wide, cell cycle-dependent binding of IHF with base-pair resolution. The GeF-seq results in this study were qualified enough to analyze genomic IHF binding sites (e.g., oriC and the transcriptional promoters of ilvG and osmY) except some of the known sites. Unexpectedly, we found that before replication initiation, oriC was a predominant site for stable IHF binding, whereas all other loci exhibited reduced IHF binding. To reveal the specific mechanism of stable oriC–IHF binding, we inserted a truncated oriC sequence in the terC (replication terminus) locus of the genome. Before replication initiation, stable IHF binding was detected even at this additional oriC site, dependent on the specific DnaA-binding sequence DnaA box R1 within the site. DnaA oligomers formed on oriC might protect the oriC–IHF complex from IHF dissociation. After replication initiation, IHF rapidly dissociated from oriC, and IHF binding to other sites was sustained or stimulated. In addition, we identified a novel locus associated with cell cycle-dependent IHF binding. These findings provide mechanistic insight into IHF binding and dissociation in the genome.

Intracellular Localization of the Proteins Encoded by Some Type II Toxin-Antitoxin Systems in Escherichia coli

Bacterial toxin-antitoxin (TA) systems, which were discovered in 1985, have since been studied extensively. These studies have focused particularly on the distribution of these bacterial TA systems on either plasmids or on bacterial chromosomes, their functionality, their targets, their relation to virulence, and their mechanisms of action.

Multiscale Dynamic Structuring of Bacterial Chromosomes

Since the nucleoid was isolated from bacteria in the 1970s, two fundamental questions emerged and are still in the spotlight: how bacteria organize their chromosomes to fit inside the cell and how nucleoid organization enables essential biological processes. During the last decades, knowledge of bacterial chromosome organization has advanced considerably, and today, such chromosomes are considered to be highly organized and dynamic structures that are shaped by multiple factors in a multiscale manner. Here we review not only the classical well-known factors involved in chromosome organization but also novel components that have recently been shown to dynamically shape the 3D structuring of the bacterial genome. We focus on the different functional elements that control short-range organization and describe how they collaborate in the establishment of the higher-order folding and disposition of the chromosome. Recent advances have opened new avenues for a deeper understanding of the principles and mechanisms of chromosome organization in bacteria. Expected final online publication date for the Annual Review of Microbiology, Volume 75 is October 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

A Fork Trap in the Chromosomal Termination Area Is Highly Conserved across All Escherichia coli Phylogenetic Groups

Termination of DNA replication, the final stage of genome duplication, is surprisingly complex, and failures to bring DNA synthesis to an accurate conclusion can impact genome stability and cell viability. In Escherichia coli, termination takes place in a specialised termination area opposite the origin. A ‘replication fork trap’ is formed by unidirectional fork barriers via the binding of Tus protein to genomic ter sites. Such a fork trap system is found in some bacterial species, but it appears not to be a general feature of bacterial chromosomes. The biochemical properties of fork trap systems have been extensively characterised, but little is known about their precise physiological roles. In this study, we compare locations and distributions of ter terminator sites in E. coli genomes across all phylogenetic groups, including Shigella. Our analysis shows that all ter sites are highly conserved in E. coli, with slightly more variability in the Shigella genomes. Our sequence analysis of ter sites and Tus proteins shows that the fork trap is likely to be active in all strains investigated. In addition, our analysis shows that the dif chromosome dimer resolution site is consistently located between the innermost ter sites, even if rearrangements have changed the location of the innermost termination area. Our data further support the idea that the replication fork trap has an important physiological role that provides an evolutionary advantage.

hafeZ: Active prophage identification through read mapping

Bacteriophages that have integrated their genomes into bacterial chromosomes, termed prophages, are widespread across bacteria. Prophages are key components of bacterial genomes, with their integration often contributing novel, beneficial, characteristics to the infected host. Likewise, their induction—through the production and release of progeny virions into the surrounding environment—can have considerable ramifications on bacterial communities. Yet, not all prophages can excise following integration, due to genetic degradation by their host bacterium. Here, we present hafeZ, a tool able to identify 'active' prophages (i.e. those undergoing induction) within bacterial genomes through genomic read mapping. We demonstrate its use by applying hafeZ to publicly available sequencing data from bacterial genomes known to contain active prophages and show that hafeZ can accurately identify their presence and location in the host chromosomes. Availability and Implementation: hafeZ is implemented in Python 3.7 and freely available under an open-source GPL-3.0 license from https://github.com/Chrisjrt/hafeZ. Bugs and issues may be reported by submitting them via the hafeZ github issues page.

CTP and parS coordinate ParB partition complex dynamics and ParA-ATPase activation for ParABS-mediated DNA partitioning

ParABS partition systems, comprising the centromere-like DNA sequence parS, the parS-binding ParB-CTPase and the nucleoid-binding ParA-ATPase, ensure faithful segregation of bacterial chromosomes and low-copy-number plasmids. F-plasmid partition complexes containing ParBF and parSF move by generating and following a local concentration gradient of nucleoid-bound ParAF. However, the process through which ParBF activates ParAF-ATPase has not been defined. We studied CTP- and parSF-modulated ParAF-ParBF complex assembly, in which DNA-bound ParAF-ATP dimers are activated for ATP hydrolysis by interacting with two ParBF N-terminal domains. CTP or parSF enhances the ATPase rate without significantly accelerating ParAF-ParBF complex assembly. Together, parSF and CTP accelerate ParAF-ParBF assembly without further significant increase in ATPase rate. Magnetic-tweezers experiments showed that CTP promotes multiple ParBF loading onto parSF-containing DNA, generating condensed partition complex-like assemblies. We propose that ParBF in the partition complex adopts a conformation that enhances ParBF-ParBF and ParAF-ParBF interactions promoting efficient partitioning.