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      Comparative Genomic and Functional Analysis of 100 Lactobacillus rhamnosus Strains and Their Comparison with Strain GG

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          Lactobacillus rhamnosus is a lactic acid bacterium that is found in a large variety of ecological habitats, including artisanal and industrial dairy products, the oral cavity, intestinal tract or vagina. To gain insights into the genetic complexity and ecological versatility of the species L. rhamnosus, we examined the genomes and phenotypes of 100 L. rhamnosus strains isolated from diverse sources. The genomes of 100 L. rhamnosus strains were mapped onto the L. rhamnosus GG reference genome. These strains were phenotypically characterized for a wide range of metabolic, antagonistic, signalling and functional properties. Phylogenomic analysis showed multiple groupings of the species that could partly be associated with their ecological niches. We identified 17 highly variable regions that encode functions related to lifestyle, i.e. carbohydrate transport and metabolism, production of mucus-binding pili, bile salt resistance, prophages and CRISPR adaptive immunity. Integration of the phenotypic and genomic data revealed that some L. rhamnosus strains possibly resided in multiple niches, illustrating the dynamics of bacterial habitats. The present study showed two distinctive geno-phenotypes in the L. rhamnosus species. The geno-phenotype A suggests an adaptation to stable nutrient-rich niches, i.e. milk-derivative products, reflected by the alteration or loss of biological functions associated with antimicrobial activity spectrum, stress resistance, adaptability and fitness to a distinctive range of habitats. In contrast, the geno-phenotype B displays adequate traits to a variable environment, such as the intestinal tract, in terms of nutrient resources, bacterial population density and host effects.

          Author Summary

          Some bacterial species are specialists and adapted to a single niche, while others are generalists and able to grow in various environmental conditions. Lactobacillus rhamnosus is a generalist and its members can often be found in different human cavities but also in various artisanal and industrial dairy products. To gain insights into the genetic complexity and ecological versatility of this species, we collected 100 L. rhamnosus strains from different niches. Genomic and functional analysis of these revealed a dichotomy within the species that reflected its adaptation to particular niches. The variable regions identified in the L. rhamnosus genome encode lifestyle traits that allowed us to demonstrate that some L. rhamnosus isolates possibly resided in multiple habitats. Our work brings valuable data on the ecological dynamics and adaptability of the species and provides a basis for a model explaining the ecology of L. rhamnosus in an anthropocentric perspective. Finally, we observed that a set of pheno-genomic markers, i.e. CRISPR oligotyping or carbohydrate metabolism, would be sufficient and among the best ways to differentiate the L. rhamnosus strains, providing a general approach to select the highest diversity in these and other bacterial species.

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          Genome analysis of multiple pathogenic isolates of Streptococcus agalactiae: implications for the microbial "pan-genome".

          The development of efficient and inexpensive genome sequencing methods has revolutionized the study of human bacterial pathogens and improved vaccine design. Unfortunately, the sequence of a single genome does not reflect how genetic variability drives pathogenesis within a bacterial species and also limits genome-wide screens for vaccine candidates or for antimicrobial targets. We have generated the genomic sequence of six strains representing the five major disease-causing serotypes of Streptococcus agalactiae, the main cause of neonatal infection in humans. Analysis of these genomes and those available in databases showed that the S. agalactiae species can be described by a pan-genome consisting of a core genome shared by all isolates, accounting for approximately 80% of any single genome, plus a dispensable genome consisting of partially shared and strain-specific genes. Mathematical extrapolation of the data suggests that the gene reservoir available for inclusion in the S. agalactiae pan-genome is vast and that unique genes will continue to be identified even after sequencing hundreds of genomes.
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            CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA.

            Horizontal gene transfer (HGT) in bacteria and archaea occurs through phage transduction, transformation, or conjugation, and the latter is particularly important for the spread of antibiotic resistance. Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci confer sequence-directed immunity against phages. A clinical isolate of Staphylococcus epidermidis harbors a CRISPR spacer that matches the nickase gene present in nearly all staphylococcal conjugative plasmids. Here we show that CRISPR interference prevents conjugation and plasmid transformation in S. epidermidis. Insertion of a self-splicing intron into nickase blocks interference despite the reconstitution of the target sequence in the spliced mRNA, which indicates that the interference machinery targets DNA directly. We conclude that CRISPR loci counteract multiple routes of HGT and can limit the spread of antibiotic resistance in pathogenic bacteria.
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              Phage response to CRISPR-encoded resistance in Streptococcus thermophilus.

              Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated genes are linked to a mechanism of acquired resistance against bacteriophages. Bacteria can integrate short stretches of phage-derived sequences (spacers) within CRISPR loci to become phage resistant. In this study, we further characterized the efficiency of CRISPR1 as a phage resistance mechanism in Streptococcus thermophilus. First, we show that CRISPR1 is distinct from previously known phage defense systems and is effective against the two main groups of S. thermophilus phages. Analyses of 30 bacteriophage-insensitive mutants of S. thermophilus indicate that the addition of one new spacer in CRISPR1 is the most frequent outcome of a phage challenge and that the iterative addition of spacers increases the overall phage resistance of the host. The added new spacers have a size of between 29 to 31 nucleotides, with 30 being by far the most frequent. Comparative analysis of 39 newly acquired spacers with the complete genomic sequences of the wild-type phages 2972, 858, and DT1 demonstrated that the newly added spacer must be identical to a region (named proto-spacer) in the phage genome to confer a phage resistance phenotype. Moreover, we found a CRISPR1-specific sequence (NNAGAAW) located downstream of the proto-spacer region that is important for the phage resistance phenotype. Finally, we show through the analyses of 20 mutant phages that virulent phages are rapidly evolving through single nucleotide mutations as well as deletions, in response to CRISPR1.

                Author and article information

                Role: Editor
                PLoS Genet
                PLoS Genet
                PLoS Genetics
                Public Library of Science (San Francisco, USA )
                August 2013
                August 2013
                15 August 2013
                : 9
                : 8
                [1 ]Department of Veterinary Biosciences, University of Helsinki, Helsinki, Finland
                [2 ]Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands
                [3 ]Infection Biology Program, Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, Helsinki, Finland
                [4 ]Department of Agri-Food and Environmental System Management, University of Catania, Catania, Italy
                [5 ]Institute of Biotechnology, University of Helsinki, Helsinki, Finland
                [6 ]Functional Foods Forum, University of Turku, Turku, Finland
                MicroTrek Incorporated, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: FPD AR RK AP WMdV. Performed the experiments: FPD AR RK TEP HMJ MM CLR LP PL JRi CC TL RS JRe. Analyzed the data: FPD AR RK WMdV. Contributed reagents/materials/analysis tools: RK LP PL IvO CC CLR SJJB. Wrote the paper: FPD AR RK WMdV.


                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Pages: 15
                This study was supported by the grant ERC 250172 - Microbes Inside from the European Research Council, the Center of Excellence in Microbial Food Safety Research (CoE-MiFoSa), Academy of Finland (grant 141140) and the University of Helsinki. FPD was also financially supported by a postdoctoral research fellowship from the Academy of Finland (grant 252123). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Research Article
                Microbial Evolution



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