Mike Gasson

Analysis of the genetic determinant for production of the peptide antibiotic nisin.

The structural gene for the precursor of the peptide antibiotic nisin was isolated and characterized. As with other lanthionine-containing antibiotics, nisin is synthesized as a pre-propeptide which undergoes post-translational modification to generate the mature antibiotic. The sequence data obtained agreed with those of precursor nisin genes isolated by other workers from different Lactococcus lactis strains. Analysis of regions flanking the precursor nisin gene revealed the presence of a downstream open reading frame that may be involved in maturation of the precursor molecule. Nucleotide sequences characteristic of an IS element were located upstream of the nisin determinant. This element, termed IS904, is present in multiple copies in the genome of L. lactis. The nisin determinant of L. lactis is a component of a large transmissible gene block that also encodes nisin resistance and sucrose-metabolizing genes. Gene probe experiments indicated that the nisin/sucrose gene block was located in the chromosome. Furthermore, the copy of IS904 identified adjacent to the precursor nisin gene lies at, or very close to, one end of this transmissible DNA segment and may play a role in mediating its transfer between strains.

Characterization and exploitation of conjugation in Lactococcus lactis

The sex factor in L. lactis MG1363 is a chromosomally-located element with an approximate 50kb size. The genetic study of this sex factor has previously been confined to the analysis of lactose plasmid cointegrates which often exhibit high transfer frequency and a constitutive cell aggregation phenotype. The cryptic nature of the sex factor limits its direct analysis. Accordingly, a tetracycline resistance gene (tet M) was integrated within the chromosomally-located sex factor and this allowed the demonstration of its independent capacity for high frequency transfer between a diverse group of lactococcal strains. Chromosomal genes were also shown to be transferred by the sex factor in a unidirectional polar manner reminiscent of an Escherichia coli Hfr strain. The sex factor was cloned from its chromosomal location and most of its sequence determined. Homology of proteins encoded by sex factor genes to known protein sequences revealed three of immediate interest. Tra D exhibits homology to the equivalent protein of the E. coli sex factor F as well as to the VirD4 protein of Agro-bacterium tumefaciens and it is likely to play a role in the transfer of DNA through the cell surface during conjugation. CluA is involved in the establishment of cell to cell contact and can induce a constitutive cell aggregation phenotype. A third protein exhibits strong homology to yeast mitochondrial intron maturases and the orf structure suggests that a type II intron is present in a sex factor gene. Sequence analysis also revealed the nature of sex factor integration and excision to be site-specific involving an identical 24 bp sequence on both the sex factor and the chromosome. The integration/ excision process resembled that of E. coli bacteriophage lambda.

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.

Controlled Release of Protein from Viable Lactococcus lactis Cells

ABSTRACT 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.

Structure and conformation of a novel genetically engineered polysaccharide P2

A new exocellular polysaccharide (P2) has been produced by the manipulation of a glycosyl transferase gene (aceP) involved in the biosynthesis of the polysaccharide acetan by the bacterium Acetobacter xylinum strain CKE5. The P2 polysaccharide has been studied by methylation analysis, reductive cleavage, and 1H and 13C NMR spectroscopy. The data are consistent with the structure predicted when the aceP gene is deactivated: [Molecular structure: see text]. The effect of cooling on proton NMR line width indicates a coil-helix transition in P2 at about 70 degrees C.

Generation of a novel polysaccharide by inactivation of the aceP gene from the acetan biosynthetic pathway in Acetobacter xylinum

The acetan biosynthetic pathway in Acetobacter xylinum is an ideal model system for engineering novel bacterial polysaccharides. To genetically manipulate this pathway, an Acetobacter strain (CKE5), more susceptible to gene-transfer methodologies, was developed. A new gene, aceP, involved in acetan biosynthesis was identified, sequenced and shown to have homology at the amino acid level with beta-D-glucosyl transferases from a number of different organisms. Disruption of aceP in strain CKE5 confirmed the function assigned above and was used to engineer a novel polysaccharide with a pentasaccharide repeat unit.

Genetic analysis of the acetan biosynthetic pathway in Acetobacter xylinum

We have identified, cloned and sequenced an 8422 base pair fragment of Acetobacter xylinum genomic DNA containing part of the acetan biosynthetic gene cluster. Computer analysis of the nucleotide sequence data generated revealed the presence of six open reading frames. Comparison of the translated sequences of putative genes to the amino acid sequences of genes from other organisms was used to assign functions to the aceA, aceC and manB genes. These genes were predicted to encode a UDP-glycosyl transferase, a GDP-mannosyl transferase and a phosphomannose isomerase/GDP-mannose pyrophosphorylase, respectively.

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.

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.

Characterisation of genetically modified nisin molecules by Fourier transform ion cyclotron resonance mass spectrometry

Modified nisin molecules, synthesised by strains of Lactococcus lactis with deliberately mutated nisA genes, have been characterised using Fourier transform ion cyclotron resonance mass spectrometry and electrospray ionisation. The predicted substitutions in the three nisin variants synthesised were first confirmed by precise measurement of the molecular mass (precision ±0.1 Da). Analysis of the lower intensity peaks in the mass spectra showed the presence of some minor components, notably hydrated molecules, in the first two samples. The third sample contained a major hydrated component that could be isolated in pure form by high-performance liquid chromatography. The engineered nisin molecules were further characterised by tandem mass spectrometry using the sustained off-resonance irradiation collisionally-activated decomposition technique. This yielded a number of sequence ions that were compared with those measured in a previous study of nisin A itself. The location of each substituent was deduced from the observed mass shifts of the sequence ions. This permitted definitive confirmation of the predicted substitutions. Out of several possible sites it was confirmed that position 33 contained the additional water molecule in the major hydrated form of one of the nisin variants.