Although the composition of the gut microbiota and its symbiotic contribution to key host physiological functions are well established, little is known as yet about the bacterial factors that account for this symbiosis. We selected Lactobacillus casei as a model microorganism to proceed to genomewide identification of the functions required for a symbiont to establish colonization in the gut. As a result of our recent development of a transposonmutagenesis tool that overcomes the barrier that had prevented L. casei random mutagenesis, we developed a signature-tagged mutagenesis approach combining whole-genome reverse genetics using a set of tagged transposons and in vivo screening using the rabbit ligated ileal loop model. After sequencing transposon insertion sites in 9,250 random mutants, we assembled a library of 1,110 independent mutants, all disrupted in a different gene, that provides a representative view of the L. casei genome. By determining the relative quantity of each of the 1,110 mutants before and after the in vivo challenge, we identified a core of 47 L. casei genes necessary for its establishment in the gut. They are involved in housekeeping functions, metabolism (sugar, amino acids), cell wall biogenesis, and adaptation to environment. Hence we provide what is, to our knowledge, the first global functional genomics analysis of L. casei symbiosis.commensalism | Lactic acid bacteria T he pioneering studies that led to the characterization of the gut microbiota were reviewed in 2001 (1). These studies and recent investigations have revealed mutualistic functions (2), including a barrier effect against allogenic microbes (3), fermentation of complex sugars (4, 5), and maturation and homeostasis of the immune system (6). Recent metagenomic studies have revealed an extraordinary diversity of genes constituting the gut microbiome (7), opening the way to correlative studies linking microbiome diversity, homeostasis, and diseases (5,8,9).In parallel, some representative species, i.e., "model symbionts," now are being studied functionally (10). As it was done for pathogens, it is essential to develop the cellular microbiology of symbionts and particularly to identify the genes required for their establishment and persistence in the gut. Transcriptomic profiling identified up-regulated genes linked to metabolic functions, stress responses, and pili synthesis during early colonization (11-13). Comparative genomics among Lactobacilli identified strain-specific candidate genes for extended colonization: In Lactobacillus rhamnosus, persistence was attributed to an spaCBA locus encoding LPXTG-like pilins (14), and in Lactobacillus johnsonii it was attributed to specific glycosyltransferases, a phosphotransfer system, and a protease (15). Otherwise, a functional in vivo screening based on the expression of a genomic library of Bacteroides fragilis identified a locus encoding polysaccharide utilization as essential for stable colonization of murine colonic crypts (16). Alternatively, colonization of germ-free mic...

These results demonstrate that some ribosome-targeting antibiotics can induce the transfer of a given mobile genetic element, here Tn916, although it does not provide any resistance determinant for most of the triggering drugs. This implies that specific antibiotic therapies can have dramatic impacts on the dissemination of unexpected and unlinked resistance genes, with the clear risk of reducing our therapeutic potential for later treatments.

The random transposon mutagenesis system P junc -TpaseIS 1223 is composed of plasmids pVI129, expressing IS1223 transposase, and pVI110, a suicide transposon plasmid carrying the P junc sequence, the substrate of the IS1223 transposase. This system is particularly efficient in Lactobacillus casei, as more than 10,000 stable, random mutants were routinely obtained via electroporation. Lactic acid bacteria (LAB) are widely used in food fermentations, as well as for their probiotic properties. Lactobacilli delbrueckii subsp. bulgaricus and Lactobacillus casei have been shown to provide beneficial effects to the immune system (29, 35). However, due to the lack of reliable tools such as a random mutagenesis system to perform global reverse genetics, the overall mechanisms underlying their probiotic effects are poorly understood.Neither the Gram-positive transposition systems based on transposon delivery by a suicide or a thermosensitive vector (19,27,34,36) nor in vitro transposon mutagenesis using Tn5-based transposons (17) is adapted to all species of LAB, due to low transformation efficiencies or unwanted stability of the transposon delivery vector (23).IS3 sequences are surrounded by imperfect inverted repeats (IR). They carry two consecutive and partially overlapping open reading frames, orfA and orfB, which encode a transposase. IS3 sequences undergo a "cut-and-paste" transposition mechanism that occurs by generating a covalently closed circular transposition intermediate, which promotes transposase induction resulting from the generation of a strong promoter named P junc . The P junc promoter corresponds to abutted IRR (inverted repeat right) and IRL (inverted repeat left) sequences as a result of insertion sequence circularization and constitutes an efficient transposition substrate (13,14). Here, we report the construction of a novel in trans transposition procedure, named the P junc -TpaseIS 1223 system and dedicated to in vivo random mutagenesis in LAB, and its application for random mutagenesis in L. casei. It is based on IS1223, a member of the IS3 family from Lactobacillus johnsonii (39) that transposes efficiently in Lactobacillus delbrueckii subsp. bulgaricus (31,39). This system is composed of two plasmids: pVI129, carrying the IS1223 transposase gene, and pVI110, a suicide transposon plasmid carrying the P junc sequence, the substrate of the IS1223 transposase.Construction of the P junc -TpaseIS 1223 system and validation in Escherichia coli. Plasmid pVI116 was constructed as described in Fig. 1 and its legend to provide the transposase of IS1223 expressed under the control of the L. delbrueckii subsp. bulgaricus P hlbA promoter (9). Plasmid pVI115 was constructed from pVI162 (see Table 1 for details of construction) to provide the transposition substrate corresponding to an abutted IRR-IRL junction of IS1223 separated by 3 base pairs, named P junc (Fig. 1A). It replicates only in the TG1 RepA strain of Escherichia coli (18). Plasmid pVI116 (P hlbA -Tpase IS1223 ) and the control plasmids, pGB2 and ...

Lactic acid bacteria (LAB) are associated with various plant, animal, and human niches and are also present in many fermented foods and beverages. Thus, they are subjected to several stress conditions and have developed advanced response mechanisms to resist, adapt, and grow. This work aimed to identify the genes involved in some stress adaptation mechanisms in LAB. For this purpose, global reverse genetics was applied by screening a library of 1287 Lactobacillus paracasei transposon mutants for mild monofactorial stresses. This library was submitted independently to heat (52°C, 30 min), ethanol (170 g.L−1, 30 min), salt (NaCl 0.8 M, 24 h), acid (pH 4.5, 24 h), and oxidative (2 mM H2O2, 24 h) perturbations which trigger mild monofactorial stresses compatible with bacterial adaptation. Stress sensitivity of mutants was determined either by evaluating viability using propidium iodide (PI) staining, or by following growth inhibition through turbidity measurement. The screening for heat and ethanol stresses lead respectively to the identification of 63 and 27 genes/putative promoters whose disruption lead to an increased sensitivity. Among them, 14 genes or putative promoters were common for both stresses. For salt, acid and oxidative stresses, respectively 8, 6, and 9 genes or putative promoters were identified as essential for adaptation to these unfavorable conditions, with only three genes common to at least two stresses. Then, RT-qPCR was performed on selected stress response genes identified by mutant screenings in order to evaluate if their expression was modified in response to stresses in the parental strain. Eleven genes (membrane, transposase, chaperone, nucleotide and carbohydrate metabolism, and hypothetical protein genes) were upregulated during stress adaptation for at least two stresses. Seven genes, encoding membrane functions, were upregulated in response to a specific stress and thus could represent potential transcriptomic biomarkers. The results highlights that most of the genes identified by global reverse genetics are specifically required in response to one stress and that they are not differentially transcribed during stress in the parental strain. Most of these genes have not been characterized as stress response genes and provide new insights into the adaptation of lactic acid bacteria to their environment.

Olive brine represents a stressful environment due to the high NaCl concentration, presence of phenolic compounds known as antimicrobials, and low availability of nutrients. Thus, only a few strains of lactic acid bacteria (LAB) are adapted to grow in and ferment table olives. To identify the mechanisms by which these few strains are able to grow in olive brine, Lactobacillus pentosus C11, a particularly resistant strain isolated from naturally fermented table olives, was mutagenized by random transposition using the P junc -TpaseIS1223 system (H. Licandro-Seraut, S. Brinster, M. van de Guchte, H. Scornec, E. Maguin, P. Sansonetti, J. F. Cavin, and P. Serror, Appl. Environ. Microbiol. 78:5417-5423, 2012). A library of 6,000 mutants was generated and screened for adaptation and subsequent growth in a medium, named BSM (brine screening medium), which presents the stressful conditions encountered in olive brine. Five transposition mutants impaired in growth on BSM were identified. Transposition occurred in two open reading frames and in three transcription terminators affecting stability of transcripts. Thus, several essential genes for adaptation and growth of L. pentosus C11 in olive brine were identified.

No abstract

No abstract

The cell surface is the primary recognition site between the bacterium and the host. An operon of three genes, LSEI_0219 (cwaR), LSEI_0220 (cwaS), and LSEI_0221 (ldcA), has been previously identified as required for the establishment of Lactobacillus paracasei in the gut. The genes cwaR and cwaS encode a predicted two-component system (TCS) and ldcA a predicted D-alanyl-D-alanine carboxypeptidase which is a peptidoglycan (PG) biosynthesis enzyme. We explored the functionality and the physiological role of these three genes, particularly their impact on the bacterial cell wall architecture and on the bacterial adaptation to environmental perturbations in the gut. The functionality of CwaS/R proteins as a TCS has been demonstrated by biochemical analysis. It is involved in the transcriptional regulation of several genes of the PG biosynthesis. Analysis of the muropeptides of PG in mutants allowed us to re-annotate LSEI_0221 as a putative L,D-carboxypeptidase (LdcA). The absence of this protein coincided with a decrease of two surface antigens: LSEI_0020, corresponding to p40 or msp2 whose implication in the host epithelial homeostasis has been recently studied, and LSEI_2029 which has never been functionally characterized. The inactivation of each of these three genes induces susceptibility to antimicrobial peptides (hBD1, hBD2, and CCL20), which could be the main cause of the gut establishment deficiency. Thus, this operon is necessary for the presence of two surface antigens and for a suitable cell wall architecture.