Kees Leenhouts

A general system for generating unlabelled gene replacements in bacterial chromosomes

Abstract A general system is described that facilitates gene replacements such that the recombinant strains are not labelled with antibiotic resistance genes. The method is based on the conditional replication of derivatives of the lactococcal plasmid pWV01, which lacks the repA gene encoding the replication initiation protein. Replacement vectors can be constructed in and isolated from gram-positive and gram-negative helper strains that provide RepA in trans. Cointegrate formation of the integration vectors with the chromosome of the target strain is selected by antibiotic resistance. Resolution of the cointegrate structure is identified in the second step of the procedure by the loss of the lacZ reporter gene present in the delivery vector. The second recombination event results either in gene replacement or in restoration of the original copy of the gene. As no antibiotic resistance marker is present in the genome of the mutant the system can be used to introduce multiple mutations in one strain. A feasibility study was performed using Lactococcus lactis and Bacillus subtilis as model organisms. The results indicate that the method should be applicable to any non-essential gene in numerous bacterial species.

A chloride‐inducible acid resistance mechanism in Lactococcus lactis and its regulation

Previously, a promoter was identified in Lactococcus lactis that is specifically induced by chloride. Here, we describe the nucleotide sequence and functional analysis of two genes transcribed from this promoter, gadC and gadB. GadC is homologous to putative glutamate‐γ‐aminobutyrate antiporters of Escherichia coli and Shigella flexneri and contains 12 putative membrane‐spanning domains. GadB shows similarity to glutamate decarboxylases. A L. lactis gadB mutant and a strain that is unable to express both gadB and gadC was more sensitive to low pH than the wild type when NaCl and glutamate were present. Expression of gadCB in L. lactis in the presence of chloride was increased when the culture pH was allowed to decrease to low levels by omitting buffer from the medium, while glutamate also stimulated gadCB expression. Apparently, these genes encode a glutamate‐dependent acid resistance mechanism of L. lactis that is optimally active under conditions in which it is needed to maintain viability. Immediately upstream of the chloride‐dependent gadCB promoter Pgad, a third gene encodes a protein (GadR) that is homologous to the activator Rgg from Streptococcus gordonii. gadR expression is chloride and glutamate independent. A gadR mutant did not produce the 3 kb gadCB mRNA that is found in wild‐type cells in the presence of NaCl, indicating that GadR is an activator of the gadCB operon.

Cell Wall Attachment of a Widely Distributed Peptidoglycan Binding Domain Is Hindered by Cell Wall Constituents

The C-terminal region (cA) of the major autolysin AcmA of Lactococcus lactis contains three highly similar repeated regions of 45 amino acid residues (LysM domains), which are separated by nonhomologous sequences. The cA domain could be deleted without destroying the cell wall-hydrolyzing activity of the enzyme in vitro. This AcmA derivative was capable neither of binding to lactococcal cells nor of lysing these cells while separation of the producer cells was incomplete. The cA domain and a chimeric protein consisting of cA fused to the C terminus of MSA2, a malaria parasite surface antigen, bound to lactococcal cells specifically via cA. The fusion protein also bound to many other Gram-positive bacteria. By chemical treatment of purified cell walls of L. lactis and Bacillus subtilis, peptidoglycan was identified as the cell wall component interacting with cA. Immunofluorescence studies showed that binding is on specific locations on the surface of L. lactis, Enterococcus faecalis, Streptococcus thermophilus, B. subtilis, Lactobacillus sake, and Lactobacillus casei cells. Based on these studies, we propose that LysM-type repeats bind to peptidoglycan and that binding is hindered by other cell wall constituents, resulting in localized binding of AcmA. Lipoteichoic acid is a candidate hindering component. For L. lactis SK110, it is shown that lipoteichoic acids are not uniformly distributed over the cell surface and are mainly present at sites where no MSA2cA binding is observed.

Nucleotide sequence and characterization of the broad-host-range lactococcal plasmid pWVO1.

The nucleotide sequence of the Lactococcus lactis broad-host-range plasmid pWVO1, replicating in both gram-positive and gram-negative bacteria, was determined. This analysis revealed four open reading frames (ORFs). ORF A appeared to encode a trans-acting 26.8-kDa protein (RepA), necessary for replication. The ORF C product was assumed to play a regulatory role in replication. Both RepA and the ORF C product showed substantial sequence similarity with the Rep proteins of the streptococcal plasmid pLS1. In addition, the plus origin of replication was identified on the basis of strong similarity with the plus origin of pLS1. Derivatives of pWVO1 produced single-stranded (ss) DNA in Bacillus subtilis and L. lactis, suggesting that this plasmid uses the rolling-circle mode of replication. In B. subtilis, but not in L. lactis, the addition of rifampicin resulted in increased levels of ssDNA, indicating that in the former organism the host-encoded RNA polymerase is involved in the conversion of the ssDNA to double-stranded plasmid DNA (dsDNA). Apparently, in L. lactis the conversion of ss to ds pWVO1 DNA occurs by a mechanism which does not require the host RNA polymerase.

Novel Surface Display System for Proteins on Non-Genetically Modified Gram-Positive Bacteria

ABSTRACT A novel display system is described that allows highly efficient immobilization of heterologous proteins on bacterial surfaces in applications for which the use of genetically modified bacteria is less desirable. This system is based on nonliving and non-genetically modified gram-positive bacterial cells, designated gram-positive enhancer matrix (GEM) particles, which are used as substrates to bind externally added heterologous proteins by means of a high-affinity binding domain. This binding domain, the protein anchor (PA), was derived from the Lactococcus lactis peptidoglycan hydrolase AcmA. GEM particles were typically prepared from the innocuous bacterium L. lactis, and various parameters for the optimal preparation of GEM particles and binding of PA fusion proteins were determined. The versatility and flexibility of the display and delivery technology were demonstrated by investigating enzyme immobilization and nasal vaccine applications.

NisT, the Transporter of the Lantibiotic Nisin, Can Transport Fully Modified, Dehydrated, and Unmodified Prenisin and Fusions of the Leader Peptide with Non-lantibiotic Peptides

Lantibiotics are lanthionine-containing peptide antibiotics. Nisin, encoded by nisA, is a pentacyclic lantibiotic produced by some Lactococcus lactis strains. Its thioether rings are posttranslationally introduced by a membrane-bound enzyme complex. This complex is composed of three enzymes: NisB, which dehydrates serines and threonines; NisC, which couples these dehydrated residues to cysteines, thus forming thioether rings; and the transporter NisT. We followed the activity of various combinations of the nisin enzymes by measuring export of secreted peptides using antibodies against the leader peptide and mass spectroscopy for detection. L. lactis expressing the nisABTC genes efficiently produced fully posttranslationally modified prenisin. Strikingly, L. lactis expressing the nisBT genes could produce dehydrated prenisin without thioether rings and a dehydrated form of a non-lantibiotic peptide. In the absence of the biosynthetic NisBC enzymes, the NisT transporter was capable of excreting unmodified prenisin and fusions of the leader peptide with non-lantibiotic peptides. Our data show that NisT specifies a broad spectrum (poly)peptide transporter that can function either in conjunction with or independently from the biosynthetic genes. NisT secretes both unmodified and partially or fully posttranslationally modified forms of prenisin and non-lantibiotic peptides. These results open the way for efficient production of a wide range of peptides with increased stability or novel bioactivities.

Anchoring of proteins to lactic acid bacteria

The anchoring of proteins to the cell surface of lactic acid bacteria (LAB) using genetic techniques is an exciting and emerging research area that holds great promise for a wide variety of biotechnological applications. This paper reviews five different types of anchoring domains that have been explored for their efficiency in attaching hybrid proteins to the cell membrane or cell wall of LAB. The most exploited anchoring regions are those with the LPXTG box that bind the proteins in a covalent way to the cell wall. In recent years, two new modes of cell wall protein anchoring have been studied and these may provide new approaches in surface display. The important progress that is being made with cell surface display of chimaeric proteins in the areas of vaccine development and enzyme- or whole-cell immobilisation is highlighted.

Construction of a food-grade multiple-copy integration system for Lactococcus lactis

Abstract A food-grade vector system was developed that allows stable integration of multiple plasmid copies in the chromosome of Lactococcus lactis. The vector consists of the plus origin of replication (Ori+) of the lactococcal plasmid pWV01, the sucrose genes of the lactic acid bacterium Pediococcus pentosaceus PPE1.0 as selectable marker, a multiple-cloning site, and a lactococcal DNA fragment of a well-characterized chromosomal region. The system includes two L. lactis strains, LL108 and LL302, which produce the pWV01 RepA protein essential for replication of the Ori+ vectors. These helper strains allow the construction and isolation of the replicating form of the integration plasmids from a homologous background. Single-cross-over integration of the plasmids in L. lactis MG1363 resulted in amplifications to a level of approximately 20 copies/chromosome after selection of the transformants on medium containing sucrose as the only fermentable sugar. The amplifications were stable under selective growth conditions. In glucose-containing medium a limited loss of integrated plasmid copies was detected at a rate of (7.5–15) × 10−2 copies per generation. One strain, MG124, was isolated that had retained 11 integrated copies after a period of 120 generations of non-selective growth. These results show that the single-cross-over integration system described here represents a simple procedure for the engineering of stable food-grade strains carrying multiple copies of a gene of interest.

Immunogenicity of a malaria parasite antigen displayed by Lactococcus lactis in oral immunisations

Abstract A putative protective protein from Plasmodium falciparum merozoites, MSA2, was expressed in two different ways on the cell surface of the Gram-positive food-grade bacterium, Lactococcus lactis. The first display format exploits an LPXTG-type anchoring motif of the lactococcal proteinase PrtP to covalently anchor MSA2 to the genetically modified producer cells. In a second display format, MSA2 was fused to the peptidoglycan-binding domain (Protein Anchor) of the lactococcal cell wall hydrolase AcmA and was non-covalently rebound to the surface of non-genetically modified, non-living high-binder L. lactis cells, termed Gram-positive enhancer matrix (GEM) particles. The L. lactis recombinants carrying covalently bound MSA2 were used to immunise rabbits through nasal and oral routes. The highest levels of IgG antibodies reacting with near-native MSA2 on merozoites was elicited by oral administration. Intestinal antibodies to MSA2 were produced only after oral immunisation. MSA2-specific Th-cell activation could be demonstrated. Based on these results, the immunogenicity in oral immunisations of MSA2, bound non-covalently to non-genetically modified L. lactis GEM particles, was compared with MSA2 that was bound covalently to genetically modified L. lactis. These two forms elicited similar titres of serum antibodies. The results illustrate the potential of using non-genetically modified L. lactis as a safe vaccine delivery vehicle to elicit systemic antibodies, thereby avoiding the dissemination of recombinant DNA into the environment.

Mode of action of LciA, the lactococcin A immunity protein

Monoclonal antibodies were raised against a fusion between the Escherichia coli maltose‐binding protein and LciA, the immunity protein that protects Lactococcus lactis against the effects of the bacteriocin lactococcin A. One of the antibodies directed against the LciA moiety of the fusion protein was used to locate the immunity protein in the L. lactis producer cell. LciA was present in the cytosolic. the membrane‐associated, and the membrane fractions in roughly equal amounts, irrespective of the production by the cells of lactococcin A.