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

A new colorimetric peptide nucleic acid-based assay for the specific detection of bacteria

Aim: Developments on synthetic molecules, such as peptide nucleic acid (PNA), make FISH procedures more robust for microbial identification. Fluorochromes use might hinder a broader implementation of PNA-FISH, but colorimetric applications are inexistent so far. Methods: A biotin-labeled eubacteria probe was used to develop a colorimetric PNA-in situ hybridization (ISH) assay. An enzymatic-conjugate, targeting biotin, was introduced. The procedure was optimized and evaluated regarding sensitivity, specificity and detection limit. Results: Results have shown strong ISH signals. The method was specific, but permeabilization problems were observed for Gram-positive bacteria. Detection limit was 5 × 107 CFU/ml, limiting current applications to pre-enriched samples. Conclusion: The PNA-ISH procedure described here is a simple alternative to other detection methods, and is also the base for the development of other PNA colorimetric systems