Minisatellite polymorphism as a tool to distinguish closely related Lactococcus lactis strains

Genome plasticity is considered as a means for bacteria to adapt to their environment. Plasticity in tandem repeat sequences on bacterial genomes has been recently exploited to trace the epidemiology of pathogens. Here, we examine the utility of minisatellite (i.e., a repeat unit of six nucleotides or more) typing in non-pathogenic food bacteria of the species Lactococcus lactis. Thirty-four minisatellites identified on the sequenced L. lactis ssp. lactis strain IL1403 genome were first analyzed in 10 closely related ssp. lactis strains, as determined by randomly amplified polymorphic DNA (RAPD). The selected tandem repeats varied in length, percent identity between repeats, and locations. We showed that: (i) the greatest polymorphism was in orfs encoding exported proteins or in intergenic regions; (ii) two thirds of minisatellites were little- or non-variable, despite as much as 90% identity between tandem repeats; and (iii) dendrograms based on either RAPD or minisatellite analyses were similar. Seven minisatellites identified in this study are potentially useful for lactococcal typing. We then asked whether tandem repeats in L. lactis were stable upon very long-term (up to two years) storage. Despite large rearrangements previously reported in derivative strains, just one of 10 minisatellites tested underwent an alteration, suggesting that tandem repeat rearrangements probably occur during active DNA replication. We conclude that multiple locus minisatellite analysis can be a valuable tool to follow lactococcal strain diversity.     

Genetic and transcriptional analysis of a novel plasmid-encoded copper resistance operon from Lactococcus lactis

A plasmid-borne copper resistance operon (lco) was identified from Lactococcus lactis subsp. lactis LL58-1. The lco operon consists of three structural genes lcoABC. The predicted products of lcoA and lcoB were homologous to chromosomally encoded prolipoprotein diacylglyceral transferases and two uncharacterized proteins respectively, and the product of lcoC is similar to several multicopper oxidases, which are generally plasmid-encoded. This genetic organization represents a new combination of genes for copper resistance in bacteria. The three genes are co-transcribed from a copper-inducible promoter, which is controlled by lcoRS encoding a response regulator and a kinase sensor. The five genes are flanked by two insertion sequences, almost identical to IS-LL6 from L. lactis. Transposon mutagenesis and subcloning analysis indicated that the three structural genes were all required for copper resistance. Copper assay results showed that the extracellular concentration of copper of L. lactis LM0230 containing the lco operon was significantly higher than that of the host strain when copper was added at concentrations from 2 to 3 mM. The results suggest that the lco operon conferred copper resistance by reducing the intracellular accumulation of copper ions in L. lactis.     

Lactococcus lactis phage operon coding for an endonuclease homologous to RuvC

The function of the Lactococcus lactis bacteriophage bIL66 middle time-expressed operon (M-operon), involved in sensitivity to the abortive infection mechanism AbiD1, was examined. Expression of the M-operon is detrimental to Escherichia coli cells, induces the SOS response and is lethal to recA and recBC E. coli mutants, which are both deficient in recombinational repair of chromosomal double-stranded breaks (DSBs). The use of an inducible expression system allowed us to demonstrate that the M-operon-encoded proteins generate a limited number of randomly distributed chromosomal DSBs that are substrates for ExoV-mediated DNA degradation. DSBs were also shown to occur upstream of the replication initiation point of unidirectionally theta-replicating plasmids. The characteristics of the DSBs lead us to propose that the endonucleolytic activity of the M-operon is not specific to DNA sequence, but rather to branched DNA structures. Genetic and physical analysis performed with different derivatives of the M-operon indicated that two orf s ( orf2 and orf3 ) are needed for nucleolytic activity. The orf3 product has amino acid homology with the E. coli RuvC Holliday junction resolvase. By site-specific mutagenesis, we have shown that one of the amino acid residues constituting the active centre of RuvC enzyme (Glu-66) and conserved in ORF3 (Glu-67) is essential for the nucleolytic activity of the M-operon gene product(s). We therefore propose that orf2 and orf3 of the M-operon code for a structure-specific endonuclease (M-nuclease), which might be essential for phage multiplication.     

Network analysis as a tool to understand social development in spider monkeys

The emerging field of network science has demonstrated that an individual's connectedness within their social network has cascading effects to other dimensions of life. Like humans, spider monkeys live in societies with high fission–fusion dynamics, and are remarkably social. Social network analysis (SNA) is a powerful tool for quantifying connections that may vary as a function of initiating or receiving social behaviors, which has been described as shifting social roles. In primatology, the SNA literature is dominated by work in catarrhines, and has yet to be applied to the study of development in a platyrrhine model. Here, SNA was utilized in combination with R-Index social role calculation to characterize social interaction patterns in juvenile and adult Colombian spider monkeys (Ateles fusciceps rufiventris). Connections were examined across five behaviors: embrace, face-embrace, grooming, agonism, and tail-wrapping from 186 hr of observation and four network metrics. Mann–Whitney U tests were utilized to determine differences between adult and juvenile social network patterns for each behavior. Face-embrace emerged as the behavior with different network patterns for adults and juveniles for every network metric. With regard to social role, juveniles were receivers, not initiators, for embrace, face-embrace, and grooming (ps < .05). Network and social role differences are discussed in light of social development and aspects of the different behaviors.     

Phage operon involved in sensitivity to the Lactococcus lactis abortive infection mechanism AbiD1.

Phage bIL66 is unable to grow on Lactococcus lactis cells harboring the abortive infection gene abiD1. Spontaneous phage mutants able to grow on AbiD1 cells were used to study phage-Abi interaction. A 1.33-kb DNA segment of a mutant phage allowed growth of AbiD1s phages in AbiD1 cells when present in trans. Sequence analysis of this segment revealed an operon composed of four open reading frames, designated orf1 to orf4. The operon is transcribed 10 min after infection from a promoter presenting an extended -10 consensus sequence but no -35 sequence. Analysis of four independent AbiD1r mutants revealed a different point mutation localized in orf1, implying that this open reading frame is needed for sensitivity to AbiD1. However, the sensitivity is partly suppressed when orf3 is expressed in trans on a high-copy-number plasmid, suggesting that AbiD1 acts by decreasing the concentration of an available orf3 product.     

The las enzymes control pyruvate metabolism in Lactococcus lactis during growth on maltose

The fermentation pattern of Lactococcus lactis with altered activities of the las enzymes was examined on maltose. The wild type converted 65% of the maltose to mixed acids. An increase in phosphofructokinase or lactate dehydrogenase expression shifted the fermentation towards homolactic fermentation, and with a high level of expression of the las operon the fermentation was homolactic.     

Construction of a food-grade host/vector system for Lactococcus lactis based on the lactose operon

Abstract A plasmid-based food-grade vector system was developed for Lactococcus lactis by exploiting the genes for lactose metabolism. L. lactis MGS267 is a plasmid-free strain containing the entire lactose operon as a chromosomal insertion. The lacF gene was deleted from this strain by a double cross-over homologous recombination event. The lacF -deficient strain produced a Lac⁻ phenotype on indicator agar. A cloned copy of the lacF gene expressed on a plasmid was capable of complementing the lacF -deficient strain resulting in a Lac⁺ phenotype. This stably maintained system fits the requirements of a self-selecting vector system and has the potential to be exploited in the food industry.     

Genome-scale metabolic model for Lactococcus lactis MG1363 and its application to the analysis of flavor formation

Lactococcus lactis subsp. cremoris MG1363 is a paradigm strain for lactococci used in industrial dairy fermentations. However, despite of its importance for process development, no genome-scale metabolic model has been reported thus far. Moreover, current models for other lactococci only focus on growth and sugar degradation. A metabolic model that includes nitrogen metabolism and flavor-forming pathways is instrumental for the understanding and designing new industrial applications of these lactic acid bacteria. A genome-scale, constraint-based model of the metabolism and transport in L. lactis MG1363, accounting for 518 genes, 754 reactions, and 650 metabolites, was developed and experimentally validated. Fifty-nine reactions are directly or indirectly involved in flavor formation. Flux Balance Analysis and Flux Variability Analysis were used to investigate flux distributions within the whole metabolic network. Anaerobic carbon-limited continuous cultures were used for estimating the energetic parameters. A thorough model-driven analysis showing a highly flexible nitrogen metabolism, e.g., branched-chain amino acid catabolism which coupled with the redox balance, is pivotal for the prediction of the formation of different flavor compounds. Furthermore, the model predicted the formation of volatile sulfur compounds as a result of the fermentation. These products were subsequently identified in the experimental fermentations carried out. Thus, the genome-scale metabolic model couples the carbon and nitrogen metabolism in L. lactis MG1363 with complete known catabolic pathways leading to flavor formation. The model provided valuable insights into the metabolic networks underlying flavor formation and has the potential to contribute to new developments in dairy industries and cheese-flavor research.     

Control analysis of the importance of phosphoglycerate enolase for metabolic fluxes in Lactococcus lactis subsp. lactis IL1403

The glycolytic enzyme phosphoglycerate enolase (PGE) catalyses the step from 2-phosphoglycerate to phosphoenolpyruvate in glycolysis. A control analysis of PGE on growth, glycolytic flux and product formation in Lactococcus lactis subsp. lactis IL1403 is presented. A library of strains with a modulated expression of PGE from 36 to 232% relative to wildtype level was constructed. Selected strains were studied with respect to growth, glycolytic flux and product formation in a chemically defined medium. On the basis of these data, flux control coefficients of PGE on the respective fluxes were calculated. At wildtype level, PGE was found to have no significant flux control on growth, glycolytic flux or product formation, but at 36% of PGE activity relative to wildtype, the flux control on the growth rate was estimated to be C(PGE)J(micro) approximately equal to 0.7, on the glycolytic flux C(PGE)J(g) approximately equal to 0.8, on lactate formation C(PGE)J(lactate) approximately equal to 1.3, on formate formation C(PGE)J(formate) approximately equal to 0.5 and on acetate formation C(PGE) J(acetate) approximately equal to 0.25. These flux control coefficients show that the metabolism of L. lactis subsp. lactis IL1403 becomes slightly more mixed acid at reduced PGE activities. Estimation of the relative turnover of PGE indicates that excess capacity of PGE in L. lactis IL1403 may be as low as twofold.