Integrating and conjugative elements (ICEs) are one of the three principal types of self-transmissible mobile genetic elements in bacteria. ICEs, like plasmids, transfer via conjugation; but unlike plasmids and similar to many phages, these elements integrate into and replicate along with the host chromosome. Members of the SXT/R391 family of ICEs have been isolated from several species of gram-negative bacteria, including Vibrio cholerae, the cause of cholera, where they have been important vectors for disseminating genes conferring resistance to antibiotics. Here we developed a plasmid-based system to capture and isolate SXT/R391 ICEs for sequencing. Comparative analyses of the genomes of 13 SXT/R391 ICEs derived from diverse hosts and locations revealed that they contain 52 perfectly syntenic and nearly identical core genes that serve as a scaffold capable of mobilizing an array of variable DNA. Furthermore, selection pressure to maintain ICE mobility appears to have restricted insertions of variable DNA into intergenic sites that do not interrupt core functions. The variable genes confer diverse element-specific phenotypes, such as resistance to antibiotics. Functional analysis of a set of deletion mutants revealed that less than half of the conserved core genes are required for ICE mobility; the functions of most of the dispensable core genes are unknown. Several lines of evidence suggest that there has been extensive recombination between SXT/R391 ICEs, resulting in re-assortment of their respective variable gene content. Furthermore, our analyses suggest that there may be a network of phylogenetic relationships among sequences found in all types of mobile genetic elements. Integrative and conjugative elements (ICEs) are a class of mobile genetic elements that are key mediators of horizontal gene flow in bacteria. These elements integrate into the host chromosome, yet are able to excise and transfer via conjugation. Our understanding of ICE evolution is rudimentary. Here, we developed a method to capture ICEs on plasmids, thus facilitating their sequencing. Comparative analyses of the DNA sequences of ICEs from the same family revealed that they have an identical genetic structure consisting of syntenous, highly conserved core genes that are interrupted by clusters of diverse variable genes. Unexpectedly, many genes in the core backbone proved non-essential for ICE transfer. Comparisons of the variable gene content in the ICEs analyzed revealed that these elements are mosaics whose genomes have been shaped by inter–ICE recombination. Finally, our work suggests that ICEs contribute to a larger gene pool that connects all types of mobile elements.

Blood is arguably the most important bodily fluid and its analysis provides crucial health status information. A first routine measure to narrow down diagnosis in clinical practice is the differential blood count, determining the frequency of all major blood cells. What is lacking to advance initial blood diagnostics is an unbiased and quick functional assessment of blood that can narrow down the diagnosis and generate specific hypotheses. To address this need, we introduce the continuous, cell-by-cell morpho-rheological (MORE) analysis of diluted whole blood, without labeling, enrichment or separation, at rates of 1000 cells/sec. In a drop of blood we can identify all major blood cells and characterize their pathological changes in several disease conditions in vitro and in patient samples. This approach takes previous results of mechanical studies on specifically isolated blood cells to the level of application directly in blood and adds a functional dimension to conventional blood analysis.

Autotransporters are secreted bacterial proteins exhibiting diverse virulence functions. Various autotransporters have been identified amongEscherichia coliassociated with intestinal or extraintestinal infections; however, the specific distribution of autotransporter sequences among a diversity ofE. colistrains has not been investigated. We have validated the use of a multiplex PCR assay to screen for the presence of autotransporter sequences. Herein, we determined the presence of 13 autotransporter sequences and five allelic variants of antigen 43 (Ag43) among 491E. coliisolates from human urinary tract infections, diarrheagenicE. coli, and avian pathogenicE. coli(APEC) andE. colireference strains belonging to the ECOR collection. Clinical isolates were also classified into established phylogenetic groups. The results indicated that Ag43 alleles were significantly associated with clinical isolates (93%) compared to commensal isolates (56%) and thatagn43K12was the most common and widely distributed allele.agn43allelic variants were also phylogenetically distributed. Sequences encodingespC,espP, andsepAandagn43allelesEDL933andRS218were significantly associated with diarrheagenicE. colistrains compared to other groups.tshwas highly associated with APEC strains, whereassatwas absent from APEC.vat,sat, andpicwere associated with urinary tract isolates and were identified predominantly in isolates belonging to either group B2 or D of the phylogenetic groups based on the ECOR strain collection. Overall, the results indicate that specific autotransporter sequences are associated with the source and/or phylogenetic background of strains and suggest that, in some cases, autotransporter gene profiles may be useful for comparative analysis ofE. colistrains from clinical, food, and environmental sources.

Integrating conjugative elements (ICEs) are a class of bacterial mobile genetic elements that disseminate via conjugation and then integrate into the host cell genome. The SXT/R391 family of ICEs consists of more than 30 different elements that all share the same integration site in the host chromosome but often encode distinct properties. These elements contribute to the spread of antibiotic resistance genes in several gram-negative bacteria including Vibrio cholerae, the agent of cholera. Here, using comparative analyses of the genomes of several SXT/R391 ICEs, we found evidence that the genomes of these elements have been shaped by inter–ICE recombination. We developed a high throughput semi-quantitative method to explore the genetic determinants involved in hybrid ICE formation. Recombinant ICE formation proved to be relatively frequent, and to depend on host (recA) and ICE (s065 and s066) loci, which can independently and potentially cooperatively mediate hybrid ICE formation. s065 and s066, which are found in all SXT/R391 ICEs, are orthologues of the bacteriophage λ Red recombination genes bet and exo, and the s065/s066 recombination system is the first Red-like recombination pathway to be described in a conjugative element. Neither ICE excision nor conjugative transfer proved to be essential for generation of hybrid ICEs. Instead conjugation facilitates the segregation of hybrids and could provide a means to select for functional recombinant ICEs containing novel combinations of genes conferring resistance to antibiotics. Thus, ICEs promote their own diversity and can yield novel mobile elements capable of disseminating new combinations of antibiotic resistance genes. Integrating and conjugative elements (ICEs) are a class of mobile elements found in diverse bacteria. ICEs of the SXT/R391 family have enabled the dissemination of genes conferring resistance to antibiotics among several important pathogens, including Vibrio cholerae, the agent of cholera. Here, using comparative analyses of the genomes of several SXT/R391 ICEs, we found that these elements are mosaics that have been shaped by inter–ICE recombination. We developed a plate-based method for semi-quantitative analyses of the genetic requirements for hybrid ICE formation. We discovered that hybrids form at relatively high frequencies and that both host and ICE genes can function independently and potentially cooperatively to mediate hybrid formation. The ICE–encoded recombination genes, which are found in all SXT/R391 ICEs, are related to genes that mediate recombination in bacteriophages, but have not been described previously in conjugative elements. Conjugative ICE transfer was not required for hybrid ICE formation but facilitates the segregation of hybrids. Thus, ICEs promote their own diversity and the generation of recombinant ICEs can yield novel mobile elements capable of disseminating new combinations of antibiotic resistance genes.

RNA–protein interactions are the crucial basis for many steps of bacterial gene expression, including post‐transcriptional control by small regulatory RNAs (sRNAs). In stark contrast to recent progress in the analysis of Gram‐negative bacteria, knowledge about RNA–protein complexes in Gram‐positive species remains scarce. Here, we used the Grad‐seq approach to draft a comprehensive landscape of such complexes in Streptococcus pneumoniae , in total determining the sedimentation profiles of ~ 88% of the transcripts and ~ 62% of the proteins of this important human pathogen. Analysis of in‐gradient distributions and subsequent tag‐based protein capture identified interactions of the exoribonuclease Cbf1/YhaM with sRNAs that control bacterial competence for DNA uptake. Unexpectedly, the nucleolytic activity of Cbf1 stabilizes these sRNAs, thereby promoting their function as repressors of competence. Overall, these results provide the first RNA/protein complexome resource of a Gram‐positive species and illustrate how this can be utilized to identify new molecular factors with functions in RNA‐based regulation of virulence‐relevant pathways.

Streptococcus pneumoniae (pneumococcus) is the primary cause of bacterial meningitis. Pneumococcal bacteria penetrates the blood-brain barrier (BBB), but the bacterial factors that enable this process are not known. Here, we determined that expression of pneumococcal pilus-1, which includes the pilus adhesin RrgA, promotes bacterial penetration through the BBB in a mouse model. S. pneumoniae that colonized the respiratory epithelium and grew in the bloodstream were chains of variable lengths; however, the pneumococci that entered the brain were division-competent, spherical, single cocci that expressed adhesive RrgA–containing pili. The cell division protein DivIVA, which is required for an ovoid shape, was localized at the poles and septum of pneumococcal chains of ovoid, nonseparated bacteria, but was absent in spherical, single cocci. In the bloodstream, a small percentage of pneumococci appeared as piliated, RrgA-expressing, DivIVA-negative single cocci, suggesting that only a minority of S. pneumoniae are poised to cross the BBB. Together, our data indicate that small bacterial cell size, which is signified by the absence of DivIVA, and the presence of an adhesive RrgA-containing pilus-1 mediate pneumococcal passage from the bloodstream through the BBB into the brain to cause lethal meningitis.