The Unconventional Xer Recombination Machinery of Streptococci/Lactococci

Homologous recombination between circular sister chromosomes during DNA replication in bacteria can generate chromosome dimers that must be resolved into monomers prior to cell division. In Escherichia coli, dimer resolution is achieved by site-specific recombination, Xer recombination, involving two paralogous tyrosine recombinases, XerC and XerD, and a 28-bp recombination site (dif) located at the junction of the two replication arms. Xer recombination is tightly controlled by the septal protein FtsK. XerCD recombinases and FtsK are found on most sequenced eubacterial genomes, suggesting that the Xer recombination system as described in E. coli is highly conserved among prokaryotes. We show here that Streptococci and Lactococci carry an alternative Xer recombination machinery, organized in a single recombination module. This corresponds to an atypical 31-bp recombination site (difSL) associated with a dedicated tyrosine recombinase (XerS). In contrast to the E. coli Xer system, only a single recombinase is required to recombine difSL, suggesting a different mechanism in the recombination process. Despite this important difference, XerS can only perform efficient recombination when difSL sites are located on chromosome dimers. Moreover, the XerS/difSL recombination requires the streptococcal protein FtsKSL, probably without the need for direct protein-protein interaction, which we demonstrated to be located at the division septum of Lactococcus lactis. Acquisition of the XerS recombination module can be considered as a landmark of the separation of Streptococci/Lactococci from other firmicutes and support the view that Xer recombination is a conserved cellular function in bacteria, but that can be achieved by functional analogs

Genes but Not Genomes Reveal Bacterial Domestication of Lactococcus Lactis

BACKGROUND: The population structure and diversity of Lactococcus lactis subsp. lactis, a major industrial bacterium involved in milk fermentation, was determined at both gene and genome level. Seventy-six lactococcal isolates of various origins were studied by different genotyping methods and thirty-six strains displaying unique macrorestriction fingerprints were analyzed by a new multilocus sequence typing (MLST) scheme. This gene-based analysis was compared to genomic characteristics determined by pulsed-field gel electrophoresis (PFGE). METHODOLOGY/PRINCIPAL FINDINGS: The MLST analysis revealed that L. lactis subsp. lactis is essentially clonal with infrequent intra- and intergenic recombination; also, despite its taxonomical classification as a subspecies, it displays a genetic diversity as substantial as that within several other bacterial species. Genome-based analysis revealed a genome size variability of 20%, a value typical of bacteria inhabiting different ecological niches, and that suggests a large pan-genome for this subspecies. However, the genomic characteristics (macrorestriction pattern, genome or chromosome size, plasmid content) did not correlate to the MLST-based phylogeny, with strains from the same sequence type (ST) differing by up to 230 kb in genome size. CONCLUSION/SIGNIFICANCE: The gene-based phylogeny was not fully consistent with the traditional classification into dairy and non-dairy strains but supported a new classification based on ecological separation between "environmental" strains, the main contributors to the genetic diversity within the subspecies, and "domesticated" strains, subject to recent genetic bottlenecks. Comparison between gene- and genome-based analyses revealed little relationship between core and dispensable genome phylogenies, indicating that clonal diversification and phenotypic variability of the "domesticated" strains essentially arose through substantial genomic flux within the dispensable genome

Les multiples facettes de Lactococcus lactis: de la plasticité chromosomique à la diversité génomique

Depuis toujours, le concept d’espèce bactérienne a été sujet à polémique entre les microbiologistes (Rosselló-Mora & Amann, 2001). Ceci est dû en partie aux nombreux échanges génétiques (transfert horizontal), même entre individus très éloignés. Pour des raisons pratiques et aussi pour satisfaire nos esprits cartésiens soucieux « de mettre tout individu dans une case », les scientifiques ont regroupé les microorganismes en fonction de certains caractères qu’ils avaient en commun, caractères définis par des méthodes phénotypiques et moléculaires (Stackebrandt et al., 2002). Pourtant comment regrouper sous une même définition des organismes dont la diversité génétique et phénotypique est continuellement mise à jour ? Le développement du séquençage en masse des génomes couplé à des analyses de génomique comparative ont permis de montrer une grande variabilité, même entre souches d’une même espèce. Les premiers travaux effectués dans ce sens par Tettelin (Tettelin et al., 2005), portaient sur la séquence de 8 souches de Streptococcus agalactiae. Environ 20% des gènes de la souche de référence étaient absents d’au moins une des autres souches. Ainsi, une seule souche ne pouvait plus représenter la totalité d’une espèce. La notion de pan-génome était née. Ce dernier est composé du génome coeur, commun à toutes les souches de l’espèce, et du génome accessoire partagé par une ou plusieurs souches. Ce génome accessoire est souvent appelé génome adaptatif car il contient des gènes impliqués dans des styles de vie particuliers. Enfin, un peu de diversité arrivait dans ce monde trop uniforme de microorganismes ! Il n’y avait plus une souche représentative d’une espèce, mais des dizaines, centaines, milliers de souches pour satisfaire nos esprits. Pour décrire ce pan-génome, la première question qui se pose est d’évaluer l’étendue de la diversité. La diversité peut s’appréhender à différents niveaux : tout d’abord au niveau des réarrangements chromosomiques (translocations, inversions) qui peuvent se produire. Ces derniers ne modifient pas le contenu génétique, mais perturbent l’architecture du chromosome. Ensuite, les réarrangements introduisant des insertions ou des délétions vont modifier le contenu génétique et amènent une diversité génomique. Enfin le dernier niveau, mais qui va au-delà de la notion de pan-génome est la diversité phénotypique existant au sein de souches génétiquement très proches. Oserais-je parler de pan-phénotype ? C’est dans ce cadre que j’ai débuté mes travaux au sein du Laboratoire de Microbiologie et de Génétique Moléculaires du CNRS. Lactococcus lactis a été notre modèle d’étude pour plusieurs raisons : cette espèce est phylogénétiquement reliée à la famille des Streptococcaceae, mais contrairement aux autres streptocoques, elle ne renferme pas de souches pathogènes. Peu de travaux existaient à l’époque sur la diversité intra-spécifique de cette espèce puisque la majorité des études étaient focalisées sur les bactéries pathogènes. Ensuite, le fait que cette bactérie d’intérêt biotechnologique soit présente dans différents environnements et sujette à différents stress technologiques en faisait un très bon modèle d’étude de l’évolution de l’espèce en fonction des conditions environnementales. Ce manuscrit décrit l’ensemble de ces travaux, allant de la plasticité chromosomique de Lactococcus lactis à la diversité phénotypique en passant par la diversité génomique. L’étude de la diversité de cette espèce m’a amenée à m’intéresser aux questions d’adaptation de cette espèce à différents environnements, notamment le lait

Cre-loxP Recombination System for Large Genome Rearrangements in Lactococcus lactis

We have used a new genetic strategy based on the Cre-loxP recombination system to generate large chromosomal rearrangements in Lactococcus lactis. Two loxP sites were sequentially integrated in inverse order into the chromosome either at random locations by transposition or at fixed points by homologous recombination. The recombination between the two chromosomal loxP sites was highly efficient (approximately 1 × 10(−1)/cell) when the Cre recombinase was provided in trans, and parental- or inverted-type chromosomal structures were isolated after removal of the Cre recombinase. The usefulness of this approach was demonstrated by creating three large inversions of 500, 1,115, and 1,160 kb in size that modified the lactococcal genome organization to different extents. The Cre-loxP recombination system described can potentially be used for other gram-positive bacteria without further modification

Natural diversity of lactococci in γ-aminobutyric acid (GABA) production and genetic and phenotypic determinants

Abstract Background γ-aminobutyric acid (GABA) is a bioactive compound produced by lactic acid bacteria (LAB). The diversity of GABA production in the Lactococcus genus is poorly understood. Genotypic and phenotypic approaches were therefore combined in this study to shed light on this diversity. A comparative genomic study was performed on the GAD-system genes (gadR, gadC and gadB) involved in GABA production in 36 lactococci including L. lactis and L. cremoris species. In addition, 132 Lactococcus strains were screened for GABA production in culture medium supplemented with 34 mM L-glutamic acid with or without NaCl (0.3 M). Results Comparative analysis of the nucleotide sequence alignments revealed the same genetic organization of the GAD system in all strains except one, which has an insertion sequence element (IS981) into the P gadCB promoter. This analysis also highlighted several deletions including a 3-bp deletion specific to the cremoris species located in the P gadR promoter, and a second 39-bp deletion specific to L. cremoris strains with a cremoris phenotype. Phenotypic analysis revealed that GABA production varied widely, but it was higher in L. lactis species than in L. cremoris, with an exceptional GABA production of up to 14 and 24 mM in two L. lactis strains. Moreover, adding chloride increased GABA production in some L. cremoris and L. lactis strains by a factor of up to 16 and GAD activity correlated well with GABA production. Conclusions This genomic analysis unambiguously characterized the cremoris phenotype of L. cremoris species and modified GadB and GadR proteins explain why the corresponding strains do not produce GABA. Finally, we found that glutamate decarboxylase activity revealing GadB protein amount, varied widely between the strains and correlated well with GABA production both with and without chloride. As this protein level is associated to gene expression, the regulation of GAD gene expression was identified as a major contributor to this diversity

Precise Populations' Description in Dairy Ecosystems Using Digital Droplet PCR: The Case of L. lactis Group in Starters

Lactococcus lactis group (composed of the lactis and cremoris subspecies, recently reassigned as two distinct species) plays a major role in dairy fermentations. Usually present in starter cultures, the two species enable efficient acidification and improve the organoleptic qualities of the final product. Biovar diacetylactis strains produce diacetyl and acetoin, aromas from the citrate metabolization. As these populations have distinct genomic and phenotypic characteristics, the proportions of each other will affect the final product. Today, there is no quantitative test able to distinguish between the two species and the biovar in dairy ecosystems. In this study, we developed a specific, reliable, and accurate strategy to quantify these populations using, species-, and diacetylactis-specific fluorescent probes in digital droplet PCR assays (ddPCR). Species were distinguished based on three single nucleotide polymorphisms in the glutamate decarboxylase gadB gene, and the citD gene involved in citrate metabolism was used to target the biovar. Used in duplex or singleplex, these probes made it possible to measure the proportion of each population. At 59 • C, the probes showed target specificity and responded negatively to the non-target species usually found in dairy environments. Depending on the probe, limit of detection values in milk matrix ranged from 3.6 × 10 3 to 1.8 × 10 4 copies/ml. The test was applied to quantify sub-populations in the L. lactis group during milk fermentation with a commercial starter. The effect of temperature and pH on the balance of the different populations was pointed out. At the initial state, lactis and cremoris species represent, respectively, 75% and 28% of the total L. lactis group and biovar diacetylactis strains represent 21% of the lactis species strains. These ratios varied as a function of temperature (22 • C or 35 • C) and acidity (pH 4.5 or 4.3) with cremoris species promoted at 22 • C and pH4.5 compared to at 35 • C. The biovar diacetylactis strains were less sensitive to acid stress at 35 • C. This methodology proved to be useful for quantifying lactis and cremoris species and biovar diacetylactis, and could complete 16S metagenomics studies for the deeply description of L. lactis group in complex ecosystems

From genome to phenotype: An integrative approach to evaluate the biodiversity of Lactococcus lactis

Lactococcus lactis is one of the most extensively used lactic acid bacteria for the manufacture of dairy products. Exploring the biodiversity of L. lactis is extremely promising both to acquire new knowledge and for food and health-driven applications. L. lactis is divided into four subspecies: lactis, cremoris, hordniae and tructae, but only subsp. lactis and subsp. cremoris are of industrial interest. Due to its various biotopes, Lactococcus subsp. lactis is considered the most diverse. The diversity of L. lactis subsp. lactis has been assessed at genetic, genomic and phenotypic levels. Multi-Locus Sequence Type (MLST) analysis of strains from different origins revealed that the subsp. lactis can be classified in two groups: “domesticated” strains with low genetic diversity, and “environmental” strains that are the main contributors of the genetic diversity of the subsp. lactis. As expected, the phenotype investigation of L. lactis strains reported here revealed highly diverse carbohydrate metabolism, especially in plant- and gut-derived carbohydrates, diacetyl production and stress survival. The integration of genotypic and phenotypic studies could improve the relevance of screening culture collections for the selection of strains dedicated to specific functions and applications

New insights into Lactococcus lactis diacetyl- and acetoin-producing strains isolated from diverse origins

Lactococcus lactis subsp. lactis biovar diacetylactis strains are used in the dairy industry for generating acetoin and notably diacetyl which imparts a high level of buttery flavor notes. A collection of domesticated and environmental strains was screened for the production of diacetyl or acetoin (D/A), and citrate fermentation. Unexpectedly, both domesticated and environmental strains produced D/A. Domesticated strains belonging to the currently named "biovar diacetylactis" metabolized citrate and produced large amounts of D/A during early growth. They harbored the citP plasmid gene encoding citrate permease and a chromosomal region citM-citI-citCDEFXG involved in citrate metabolism. In these strains, citrate consumption was identified as the major determinant of aroma production. Environmental strains, specifically UCMA5716 and A12, produced as much D/A as the CitP strains, though at slightly lower rates. UCMA5716 was found to contain the citM-citI-citCDEFXG cluster but not the citP gene. A12 had neither. In these strains, production rate of D/A was linearly correlated with pyruvate synthesis rate. However, the correlation factor was strain-dependent, suggesting different modes of regulation for pyruvate rerouting towards fermentation end-products and flavors. This work highlights the genetic and metabolic differences between environmental and domesticated strains. The introduction of environmental strains into industrial processes could considerably increase the diversity of starters, enhancing the delivery of new technological properties. (C) 2012 Elsevier B.V. All rights reserved