ß-Glucan is a major growth substrate for human gut bacteria related to Coprococcus eutactus.

A clone encoding carboxymethyl cellulase activity was isolated during functional screening of a human gut metagenomic library using Lactococcus lactis MG1363 as heterologous host. The insert carried a glycoside hydrolase family 9 (GH9) catalytic domain with sequence similarity to a gene from Coprococcus eutactus ART55/1. Genome surveys indicated a limited distribution of GH9 domains among dominant human colonic anaerobes. Genomes of C. eutactus-related strains harboured two GH9-encoding and four GH5-encoding genes, but the strains did not appear to degrade cellulose. Instead, they grew well on ß-glucans and one of the strains also grew on galactomannan, galactan, glucomannan and starch. Coprococcus comes and Coprococcus catus strains did not harbour GH9 genes and were not able to grow on ß-glucans. Gene expression and proteomic analysis of C. eutactus ART55/1 grown on cellobiose, ß-glucan and lichenan revealed similar changes in expression in comparison to glucose. On ß-glucan and lichenan only, one of the four GH5 genes was strongly upregulated. Growth on glucomannan led to a transcriptional response of many genes, in particular a strong upregulation of glycoside hydrolases involved in mannan degradation. Thus, ß-glucans are a major growth substrate for species related to C. eutactus, with glucomannan and galactans alternative substrates for some strains.

Molecular characterization and structural instability of the industrially important composite metabolic plasmid pLP712.

The widely used plasmid-free Lactococcus lactis strain MG1363 was derived from the industrial dairy starter strain NCDO712. This strain carries a 55.39 kb plasmid encoding genes for lactose catabolism and a serine proteinase involved in casein degradation. We report the DNA sequencing and annotation of pLP712, which revealed additional metabolic genes, including peptidase F, d-lactate dehydrogenase and α-keto acid dehydrogenase (E3 complex). Comparison of pLP712 with other large lactococcal lactose and/or proteinase plasmids from L. lactis subsp. cremoris SK11 (pSK11L, pSK11P) and the plant strain L. lactis NCDO1867 (pGdh442) revealed their close relationship. The plasmid appears to have evolved through a series of genetic events as a composite of pGdh442, pSK11L and pSK11P. We describe in detail a scenario by which the metabolic genes relevant to the growth of its host in a milk environment have been unified on one replicon, reflecting the evolution of L. lactis as it changed its biological niche from plants to dairy environments. The extensive structural instability of pLP712 allows easy isolation of derivative plasmids lacking genes for casein degradation and/or lactose catabolism. Plasmid pLP712 is transferable by transduction and conjugation, and both of these processes result in significant molecular rearrangements. We report the detailed molecular analysis of insertion sequence element-mediated genetic rearrangements within pLP712 and several different mechanisms, including homologous recombination and adjacent deletion. Analysis of the integration of the lactose operon into the chromosome highlights the fluidity of the MG1363 integration hotspot and the potential for frequent movement of genes between plasmids and chromosomes in Lactococcus.

CsiA is a bacterial cell wall synthesis inhibitor contributing to DNA translocation through the cell envelope.

Conjugation is a widely spread mechanism allowing bacteria to adapt and evolve by acquiring foreign DNA. The chromosome of Lactococcus lactis MG 1363 contains a 60 kb conjugative element called the sex factor capable of high-frequency DNA transfer. Yet, little is known about the proteins involved in this process. Comparative genomics revealed a close relationship between the sex factor and elements found in Gram-positive pathogenic cocci. Among the conserved gene products, CsiA is a large protein that contains a highly conserved domain (HCD) and a C-terminal cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) domain in its C-terminal moiety. Here, we show that CsiA is required for DNA transfer. Surprisingly, increased expression of CsiA affects cell viability and the cells become susceptible to lysis. Point mutagenesis of HCD reveals that this domain is responsible for the observed phenotypes. Growth studies and electron microscope observations suggest that CsiA is acting as a cell wall synthesis inhibitor. In vitro experiments reveal the capacity of CsiA to bind d-Ala-d-Ala analogues and to prevent the action of penicillin binding proteins. Our results strongly suggest that CsiA sequesters the peptidoglycan precursor and prevents the final stage of cell wall biosynthesis to enable the localized assembly of the DNA transfer machinery through the cell wall.

Controlled release of protein from viable Lactococcus lactis cells.

Overexpression of the lactococcal CsiA protein affects the cell wall integrity of growing cells and leads to leakage of intracellular material. This property was optimized and exploited for the targeted release of biologically active compounds into the extracellular environment, thereby providing a new delivery system for bacterial proteins and peptides. The effects of different levels of CsiA expression on the leakage of endogenous lactate dehydrogenase and nucleic acids were measured and related to the impact of CsiA expression on Lactococcus lactis cell viability and growth. A leakage phenotype was obtained from cells expressing both recombinant and nonrecombinant forms of CsiA. As proof of principle, we demonstrated that CsiA promotes the efficient release of the heterologous Listeria bacteriophage endolysin LM4 in its active form. Under optimized conditions, native and heterologous active-molecule release is possible without affecting cell viability. The ability of CsiA to release intracellular material by controlled lysis without the requirement for an external lytic agent provides a technology for the control of both the extent of lysis and its timing. Taken together, these results demonstrate the potential of this novel approach for applications including product recovery in industrial fermentations, food processing, and medical therapy.

Involvement of dehydroalanine and dehydrobutyrine in the addition of glutathione to nisin.

Nisin variants and fragments were reacted with glutathione, and the products of the reactions were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography/mass spectrometry (LC-MS). Reactions between glutathione and either [Ala5]nisin or [Ala33]nisin resulted in products with two glutathione molecules conjugated to one nisin variant molecule. Only one glutathione molecule was added to [Ala5,Ala33]nisin. Fragmentation of the nisin molecule resulted in nisin 1-12, nisin 1-20, and nisin 1-32 fragments. Each fragment retained two dehydro residues, which subsequently underwent reaction with glutathione. The data indicated that the dehydroalanine residues of nisin are sites of addition for glutathione. Such addition renders the nisin molecule inactive.

The Tra domain of the lactococcal CluA surface protein is a unique domain that contributes to sex factor DNA transfer.

CluA is a cell surface-presented protein that causes cell aggregation and is essential for a high-efficiency conjugation process in Lactococcus lactis. We know from previous work that in addition to promoting cell-to-cell contact, CluA is involved in sex factor DNA transfer. To define the CluA domains involved in aggregation and in transfer, we first performed random mutagenesis of the cluA gene using a modified mini-Tn7 element which generated five amino acid insertions located throughout the encoded protein. Thirty independent cluA insertion mutants expressing modified CluA proteins at the cell surface were isolated and characterized further. The level of aggregation of each mutant was determined. The cell binding capacity of CluA was affected strongly when the protein had a mutation in its N-terminal region, which defined an aggregation domain extending from amino acid 153 to amino acid 483. Of the cluA mutants that still exhibited aggregation, eight showed an attenuated ability to conjugate, and six mutations were located in a 300-amino-acid C-terminal region of the protein defining a transfer domain (Tra). This result was confirmed by a phenotypic analysis of an additional five mutants obtained using site-directed mutagenesis in which charged amino acids of the Tra domain were replaced by alanine residues. Two distinct functional domains of the CluA protein were defined in this work; the first domain is involved in cell binding specificity, and the Tra domain is probably involved in the formation of the DNA transport machinery. This is the first report of a protein involved in conjugation that actively contributes to DNA transfer and mediates contact between donor and recipient strains.

Controlled expression of CluA in Lactococcus lactis and its role in conjugation.

CluA is a 136 kDa surface-bound protein encoded by the chromosomally located sex factor of Lactococcus lactis MG1363 and is associated with cell aggregation linked to high-frequency transfer of the sex factor. To further investigate the involvement of CluA in these phenomena, the cluA gene was cloned on a plasmid, downstream from the lactococcal nisA promoter. In a sex-factor-negative MG1363 derivative, nisin-controlled CluA expression resulted in aggregation, despite the absence of the other genes of the sex factor. Therefore, CluA is the only sex factor component responsible for aggregation. The direct involvement of CluA in the establishment of cell-to-cell contact for aggregate formation was observed by electron microscopy using immunogold-labelled CluA antibodies. Inactivation of cluA in an MG1363 background led to a dramatic decrease in sex factor conjugation frequency compared to the parental strain. Increasing levels of CluA expressed in trans in the cluA-inactivated donor strain facilitated a gradual restoration of conjugation frequency, reaching that of the parental strain. In conclusion, CluA is essential for efficient sex factor transfer in conjugation of L. lactis.

Discovering lactic acid bacteria by genomics.

This review summarizes a collection of lactic acid bacteria that are now undergoing genomic sequencing and analysis. Summaries are presented on twenty different species, with each overview discussing the organisms fundamental and practical significance, environmental habitat, and its role in fermentation, bioprocessing, or probiotics. For those projects where genome sequence data were available by March 2002, summaries include a listing of key statistics and interesting genomic features. These efforts will revolutionize our molecular view of Gram-positive bacteria, as up to 15 genomes from the low GC content lactic acid bacteria are expected to be available in the public domain by the end of 2003. Our collective view of the lactic acid bacteria will be fundamentally changed as we rediscover the relationships and capabilities of these organisms through genomics.