Evidence that residues −15 to −46 of the haemolysin secretion signal are involved in early steps in secretion, leading to recognition of the translocator

We previously identified three well-dispersed mutations, E⁹⁷⁸-K, F⁹⁸⁹-L and D¹⁰⁰⁹-R within the haemolysin A signal region, located at positions –46, –35 and –15, with respect to the C-terminus, respectively. Each mutation reduces the efficiency of secretion two- to threefold leaving 30–45% of the wild-type activity. We have constructed by in vitro manipulations double mutants of HlyA carrying all combinations of these mutations and a triple mutant carrying all three mutations. The effects on secretion were determined and the results, including residual levels of secretion with the triple mutant of only 0.6%, compared with the wild type, indicated that these residues may interact to form a single function in the wild-type signal. To test this further, we developed a secretion competition assay in order to classify signal mutations. We demonstrated that a CIZ-HlyA fusion protein, containing the C-terminal 81 kDa of HlyA fused to virtually the whole LacZ protein, strongly inhibits the secretion of the wild-type HlyA co-expressed In the same cell. The properties of the fusion indicate that it blocks the translocator. The three mutations singly and in combinations were recombined in vitro into the 3′-end of the hybrid gene. In every case, the presence of a mutation in the secretion signal of the hybrid protein alleviated the inhibition of secretion of the co-expressed HiyA. All the mutations are therefore essentially recessive and we propose that they all affect an early function, probably recognition of the translocator, rather than a subsequent step involved in translocation or final release of the toxin to the medium. This would indicate that residues involved in recognition for steps     

Genome sequence of the probiotic strain Bifidobacterium animalis subsp. lactis CNCM I-2494

Bifidobacterium animalis subsp. lactis CNCM I-2494 is part of a commercialized fermented dairy product with documented health benefits revealed by multiple randomized placebo-controlled clinical trials. Here we report the complete genome sequence of this strain, which has a circular genome of 1,943,113 bp with 1,660 open reading frames and 4 ribosomal operons.     

Random and directed mutagenesis to elucidate the functional importance of helix II and F-989 in the C-terminal secretion signal of Escherichia coli hemolysin.

The HlyA secretion signal sequence of approximately 46 residues is predicted to contain helix I and an amphipathic helix II separated by a short loop including the conserved Phe residue, F-989. All nine substitutions of Phe-989 drastically reduce secretion of HlyA. Directed mutagenesis identified a functional hot spot, EISK, in helix II. However, genetic analysis did not provide strong support for a functional helix II; rather, the results emphasized that individual residues, for example, E-978 and F-989, are essential irrespective of a specific secondary structure.     

Lactobacillus paracasei Comparative Genomics: Towards Species Pan-Genome Definition and Exploitation of Diversity

Lactobacillus paracasei is a member of the normal human and animal gut microbiota and is used extensively in the food industry in starter cultures for dairy products or as probiotics. With the development of low-cost, high-throughput sequencing techniques it has become feasible to sequence many different strains of one species and to determine its “pan-genome”. We have sequenced the genomes of 34 different L. paracasei strains, and performed a comparative genomics analysis. We analysed genome synteny and content, focussing on the pan-genome, core genome and variable genome. Each genome was shown to contain around 2800–3100 protein-coding genes, and comparative analysis identified over 4200 ortholog groups that comprise the pan-genome of this species, of which about 1800 ortholog groups make up the conserved core. Several factors previously associated with host-microbe interactions such as pili, cell-envelope proteinase, hydrolases p40 and p75 or the capacity to produce short branched-chain fatty acids (bkd operon) are part of the L. paracasei core genome present in all analysed strains. The variome consists mainly of hypothetical proteins, phages, plasmids, transposon/conjugative elements, and known functions such as sugar metabolism, cell-surface proteins, transporters, CRISPR-associated proteins, and EPS biosynthesis proteins. An enormous variety and variability of sugar utilization gene cassettes were identified, with each strain harbouring between 25–53 cassettes, reflecting the high adaptability of L. paracasei to different niches. A phylogenomic tree was constructed based on total genome contents, and together with an analysis of horizontal gene transfer events we conclude that evolution of these L. paracasei strains is complex and not always related to niche adaptation. The results of this genome content comparison was used, together with high-throughput growth experiments on various carbohydrates, to perform gene-trait matching analysis, in order to link the distribution pattern of a specific phenotype to the presence/absence of specific sets of genes.     

Multilocus sequence typing of Lactobacillus casei reveals a clonal population structure with low levels of homologous recombination

Robust genotyping methods for Lactobacillus casei are needed for strain tracking and collection management, as well as for population biology research. A collection of 52 strains initially labeled L. casei or Lactobacillus paracasei was first subjected to rplB gene sequencing together with reference strains of Lactobacillus zeae, Lactobacillus rhamnosus, and other species. Phylogenetic analysis showed that all 52 strains belonged to a single compact L. casei-L. paracasei sequence cluster, together with strain CIP107868 (= ATCC 334) but clearly distinct from L. rhamnosus and from a cluster with L. zeae and CIP103137(T) (= ATCC 393(T)). The strains were genotyped using amplified fragment length polymorphism, multilocus sequence typing based on internal portions of the seven housekeeping genes fusA, ileS, lepA, leuS, pyrG, recA, and recG, and tandem repeat variation (multilocus variable-number tandem repeats analysis [MLVA] using nine loci). Very high concordance was found between the three methods. Although amounts of nucleotide variation were low for the seven genes (pi ranging from 0.0038 to 0.0109), 3 to 12 alleles were distinguished, resulting in 31 sequence types. One sequence type (ST1) was frequent (17 strains), but most others were represented by a single strain. Attempts to subtype ST1 strains by MLVA, ribotyping, clustered regularly interspaced short palindromic repeat characterization, and single nucleotide repeat variation were unsuccessful. We found clear evidence for homologous recombination during the diversification of L. casei clones, including a putative intragenic import of DNA into one strain. Nucleotides were estimated to change four times more frequently by recombination than by mutation. However, statistical congruence between individual gene trees was retained, indicating that recombination is not frequent enough to disrupt the phylogenetic signal. The developed multilocus sequence typing scheme should be useful for future studies of L. casei strain diversity and evolution.     

Multilocus Sequence Typing of Lactobacillus casei Reveals a Clonal Population Structure with Low Levels of Homologous Recombination

Robust genotyping methods for Lactobacillus casei are needed for strain tracking and collection management, as well as for population biology research. A collection of 52 strains initially labeled L. casei or Lactobacillus paracasei was first subjected to rplB gene sequencing together with reference strains of Lactobacillus zeae, Lactobacillus rhamnosus, and other species. Phylogenetic analysis showed that all 52 strains belonged to a single compact L. casei-L. paracasei sequence cluster, together with strain CIP107868 (= ATCC 334) but clearly distinct from L. rhamnosus and from a cluster with L. zeae and CIP103137^T (= ATCC 393^T). The strains were genotyped using amplified fragment length polymorphism, multilocus sequence typing based on internal portions of the seven housekeeping genes fusA, ileS, lepA, leuS, pyrG, recA, and recG, and tandem repeat variation (multilocus variable-number tandem repeats analysis [MLVA] using nine loci). Very high concordance was found between the three methods. Although amounts of nucleotide variation were low for the seven genes (π ranging from 0.0038 to 0.0109), 3 to 12 alleles were distinguished, resulting in 31 sequence types. One sequence type (ST1) was frequent (17 strains), but most others were represented by a single strain. Attempts to subtype ST1 strains by MLVA, ribotyping, clustered regularly interspaced short palindromic repeat characterization, and single nucleotide repeat variation were unsuccessful. We found clear evidence for homologous recombination during the diversification of L. casei clones, including a putative intragenic import of DNA into one strain. Nucleotides were estimated to change four times more frequently by recombination than by mutation. However, statistical congruence between individual gene trees was retained, indicating that recombination is not frequent enough to disrupt the phylogenetic signal. The developed multilocus sequence typing scheme should be useful for future studies of L. casei strain diversity and evolution.     

Identification of Restriction-Modification Systems of Bifidobacterium animalis subsp. lactis CNCM I-2494 by SMRT Sequencing and Associated Methylome Analysis

Bifidobacterium animalis subsp. lactis CNCM I-2494 is a component of a commercialized fermented dairy product for which beneficial effects on health has been studied by clinical and preclinical trials. To date little is known about the molecular mechanisms that could explain the beneficial effects that bifidobacteria impart to the host. Restriction-modification (R-M) systems have been identified as key obstacles in the genetic accessibility of bifidobacteria, and circumventing these is a prerequisite to attaining a fundamental understanding of bifidobacterial attributes, including the genes that are responsible for health-promoting properties of this clinically and industrially important group of bacteria. The complete genome sequence of B. animalis subsp. lactis CNCM I-2494 is predicted to harbour the genetic determinants for two type II R-M systems, designated BanLI and BanLII. In order to investigate the functionality and specificity of these two putative R-M systems in B. animalis subsp. lactis CNCM I-2494, we employed PacBio SMRT sequencing with associated methylome analysis. In addition, the contribution of the identified R-M systems to the genetic accessibility of this strain was assessed.