Characterization of a Theta-Type Plasmid from Lactobacillus sakei: a Potential Basis for Low-Copy-Number Vectors in Lactobacilli

The complete nucleotide sequence of the 13-kb plasmid pRV500, isolated from Lactobacillus sakei RV332, was determined. Sequence analysis enabled the identification of genes coding for a putative type I restriction-modification system, two genes coding for putative recombinases of the integrase family, and a region likely involved in replication. The structural features of this region, comprising a putative ori segment containing 11- and 22-bp repeats and a repA gene coding for a putative initiator protein, indicated that pRV500 belongs to the pUCL287 subfamily of theta-type replicons. A 3.7-kb fragment encompassing this region was fused to an Escherichia coli replicon to produce the shuttle vector pRV566 and was observed to be functional in L. sakei for plasmid replication. The L. sakei replicon alone could not support replication in E. coli. Plasmid pRV500 and its derivative pRV566 were determined to be at very low copy numbers in L. sakei. pRV566 was maintained at a reasonable rate over 20 generations in several lactobacilli, such as Lactobacillus curvatus, Lactobacillus casei, and Lactobacillus plantarum, in addition to L. sakei, making it an interesting basis for developing vectors. Sequence relationships with other plasmids are described and discussed

Intestinal Bacterial Communities That Produce Active Estrogen-Like Compounds Enterodiol and Enterolactone in Humans

Lignans are dietary diphenolic compounds which require activation by intestinal bacteria to exert possible beneficial health effects. The intestinal ecosystem plays a crucial role in lignan metabolism, but the organisms involved are poorly described. To characterize the bacterial communities responsible for secoisolariciresinol (SECO) activation, i.e., the communities that produce the enterolignans enterodiol (ED) and enterolactone (EL), a study with 24 human subjects was undertaken. SECO activation was detected in all tested fecal samples. The intestinal bacteria involved in ED production were part of the dominant microbiota (6 × 10(8) CFU g(−1)), as revealed by most-probable-number enumerations. Conversely, organisms that catalyzed the formation of EL occurred at a mean concentration of approximately 3 × 10(5) CFU g(−1). Women tended to have higher concentrations of both ED- and EL-producing organisms than men. Significantly larger amounts of EL were produced by fecal dilutions from individuals with moderate to high concentrations of EL-producing bacteria. Two organisms able to demethylate and dehydroxylate SECO were isolated from human feces. Based on 16S rRNA gene sequence analyses, they were named Peptostreptococcus productus SECO-Mt75m3 and Eggerthella lenta SECO-Mt75m2. A new 16S rRNA-targeted oligonucleotide probe specific for P. productus and related species was designed and further used in fluorescent in situ hybridization experiments, along with five additional group-specific probes. Significantly higher proportions of P. productus and related species (P = 0.012), as well as bacteria belonging to the Atopobium group (P = 0.035), were typical of individuals with moderate to high concentrations of EL-producing communities

Worst-case scenarios for horizontal gene transfer from Lactococcus lactis carrying heterologous genes to

A misprint occurred in this article: The graphs from Figure 1 and Figure 2 should be exchanged with the graphs from Figure 3 and Figure 4, respectively. The corresponding legends are in the correct position and should not be moved

Worst-case scenarios for horizontal gene transfer from

Since genetically modified (GM) lactic acid bacteria (LAB) might be released in open environments for future nutritional and medical applications, the purpose of this study was to determine an upper limit for the horizontal gene transfer (HGT) in the digestive tract (DT) from Lactococcus lactis carrying heterologous genes (lux genes encoding a bacterial luciferase) to Enterococcus faecalis. Two enterococcal wide host-range conjugative model systems were used: (i) a system composed of a mobilizable plasmid containing the heterologous lux genes and a native conjugative helper plasmid; and (ii) a Tn916-lux transposon. Both systems were tested under the most transfer-prone conditions, i.e. germfree mice mono-associated with the recipient E. faecalis. No transfer was observed with the transposon system. Transfers of the mobilizable plasmid carrying heterologous genes were below 102 transconjugants per g of faeces for a single donor dose and reached between 103 and 104 transconjugants per g of faeces when continuous inoculation of the donor strain was used. Once established in mice, transconjugants persisted at low levels in the mouse DT

Intraspecies Genomic Diversity and Natural Population Structure of the Meat-Borne Lactic Acid Bacterium Lactobacillus sakei▿ †

Lactobacillus sakei is a food-borne bacterium naturally found in meat and fish products. A study was performed to examine the intraspecies diversity among 73 isolates sourced from laboratory collections in several different countries. Pulsed-field gel electrophoresis analysis demonstrated a 25% variation in genome size between isolates, ranging from 1,815 kb to 2,310 kb. The relatedness between isolates was then determined using a PCR-based method that detects the possession of 60 chromosomal genes belonging to the flexible gene pool. Ten different strain clusters were identified that had noticeable differences in their average genome size reflecting the natural population structure. The results show that many different genotypes may be isolated from similar types of meat products, suggesting a complex ecological habitat in which intraspecies diversity may be required for successful adaptation. Finally, proteomic analysis revealed a slight difference between the migration patterns of highly abundant GapA isoforms of the two prevailing L. sakei subspecies (sakei and carnosus). This analysis was used to affiliate the genotypic clusters with the corresponding subspecies. These findings reveal for the first time the extent of intraspecies genomic diversity in L. sakei. Consequently, identification of molecular subtypes may in the future prove valuable for a better understanding of microbial ecosystems in food products