Efficient transformation system for Propionibacterium freudenreichii based on a novel vector.

A 3.6-kb endogenous plasmid was isolated from a Propionibacterium freudenreichii strain and sequenced completely. Based on homologies with plasmids from other bacteria, notably a plasmid from Mycobacterium, a region harboring putative replicative functions was defined. Outside this region two restriction enzyme recognition sites were used for insertion of an Escherichia coli-specific replicon and an erythromycin resistance gene for selection in Propionibacterium. Hybrid vectors obtained in this way replicated in both E. coli and P. freudenreichii. Whereas electroporation of P. freudenreichii with vector DNA isolated from an E. coli transformant yielded 10 to 30 colonies per microg of DNA, use of vector DNA reisolated from a Propionibacterium transformant dramatically increased the efficiency of transformation (> or =10(8) colonies per microg of DNA). It could be shown that restriction-modification was responsible for this effect. The high efficiency of the system described here permitted successful transformation of Propionibacterium with DNA ligation mixtures.

The S-layer protein of Lactobacillus acidophilus ATCC 4356: identification and characterisation of domains responsible for S-protein assembly and cell wall binding.

Lactobacillus acidophilus, like many other bacteria, harbors a surface layer consisting of a protein (S(A)-protein) of 43 kDa. S(A)-protein could be readily extracted and crystallized in vitro into large crystalline patches on lipid monolayers with a net negative charge but not on lipids with a net neutral charge. Reconstruction of the S-layer from crystals grown on dioleoylphosphatidylserine indicated an oblique lattice with unit cell dimensions (a=118 A; b=53 A, and gamma=102 degrees ) resembling those determined for the S-layer of Lactobacillus helveticus ATCC 12046. Sequence comparison of S(A)-protein with S-proteins from L. helveticus, Lactobacillus crispatus and the S-proteins encoded by the silent S-protein genes from L. acidophilus and L. crispatus suggested the presence of two domains, one comprising the N-terminal two-thirds (SAN), and another made up of the C-terminal one-third (SAC) of S(A)-protein. The sequence of the N-terminal domains is variable, while that of the C-terminal domain is highly conserved in the S-proteins of these organisms and contains a tandem repeat. Proteolytic digestion of S(A)-protein showed that SAN was protease-resistant, suggesting a compact structure. SAC was rapidly degraded by proteases and therefore probably has a more accessible structure. DNA sequences encoding SAN or Green Fluorescent Protein fused to SAC (GFP-SAC) were efficiently expressed in Escherichia coli. Purified SAN could crystallize into mono and multi-layered crystals with the same lattice parameters as those found for authentic S(A)-protein. A calculated S(A)-protein minus SAN density-difference map revealed the probable location, in projection, of the SAC domain, which is missing from the truncated SAN peptide. The GFP-SAC fusion product was shown to bind to the surface of L. acidophilus, L. helveticus and L. crispatus cells from which the S-layer had been removed, but not to non-stripped cells or to Lactobacillus casei.

Expression of cbsA encoding the collagen-binding S-protein of Lactobacillus crispatus JCM5810 in Lactobacillus casei ATCC 393(T).

The cbsA gene encoding the collagen-binding S-layer protein of Lactobacillus crispatus JCM5810 was expressed in L. casei ATCC 393(T). The S-protein was not retained on the surface of the recombinant bacteria but was secreted into the medium. By translational fusion of CbsA to the cell wall sorting signal of the proteinase, PrtP, of L. casei, CbsA was presented at the surface, rendering the transformants able to bind to immobilized collagens.

Characterization of the collagen-binding S-layer protein CbsA of Lactobacillus crispatus.

The cbsA gene of Lactobacillus crispatus strain JCM 5810, encoding a protein that mediates adhesiveness to collagens, was characterized and expressed in Escherichia coli. The cbsA open reading frame encoded a signal sequence of 30 amino acids and a mature polypeptide of 410 amino acids with typical features of a bacterial S-layer protein. The cbsA gene product was expressed as a His tag fusion protein, purified by affinity chromatography, and shown to bind solubilized as well as immobilized type I and IV collagens. Three other Lactobacillus S-layer proteins, SlpA, CbsB, and SlpnB, bound collagens only weakly, and sequence comparisons of CbsA with these S-layer proteins were used to select sites in cbsA where deletions and mutations were introduced. In addition, hybrid S-layer proteins that contained the N or the C terminus from CbsA, SlpA, or SlpnB as well as N- and C-terminally truncated peptides from CbsA were constructed by gene fusion. Analysis of these molecules revealed the major collagen-binding region within the N-terminal 287 residues and a weaker type I collagen-binding region in the C terminus of the CbsA molecule. The mutated or hybrid CbsA molecules and peptides that failed to polymerize into a periodic S-layer did not bind collagens, suggesting that the crystal structure with a regular array is optimal for expression of collagen binding by CbsA. Strain JCM 5810 was found to contain another S-layer gene termed cbsB that was 44% identical in sequence to cbsA. RNA analysis showed that cbsA, but not cbsB, was transcribed under laboratory conditions. S-layer-protein-expressing cells of strain JCM 5810 adhered to collagen-containing regions in the chicken colon, suggesting that CbsA-mediated collagen binding represents a true tissue adherence property of L. crispatus.

Engineering the microflora to vaccinate the mucosa: serum immunoglobulin G responses and activated draining cervical lymph nodes following mucosal application of tetanus toxin fragment C-expressing lactobacilli.

The delivery of antigens to mucosal-associated lymphoid tissues in paediatric and immunocompromised populations by safe, non-invasive vectors, such as commensal lactobacilli, represents a crucial improvement to prevailing vaccination options. In this report, we describe the oral and nasal immunization of mice with vaccines constructed through an original system for heterologous gene expression in Lactobacillus in which the 50 000-molecular weight (MW) fragment C of tetanus toxin (TTFC) is expressed either as an intracellular or a surface-exposed protein. Our data indicate that L. plantarum is more effective in this respect than L. casei and that, under the experimental conditions investigated, delivery of TTFC expressed as an intracellular antigen is more effective than cell-surface expression. Immunization of mice with live recombinant lactobacilli induced significant levels of circulating TTFC-specific immunoglobulin G (IgG) following nasal or oral delivery of vaccine strains. In addition, following nasal delivery, secretory immunoglobulin A (sIgA) was induced in bronchoalveolar lavage fluids, as were antigen-specific antibody-secreting cells and antigen-specific T-cell activation in draining lymph nodes, substantiating their potential for safe mucosal delivery of paediatric vaccines.

Live bacterial delivery systems for development of mucosal vaccines.

By expression of foreign antigens in attenuated strains derived from bacterial pathogens and in non-pathogenic commensal bacteria, recombinant vaccines are being developed that aim to stimulate mucosal immunity. Recent advances in the pathogenesis and molecular biology of these bacteria have allowed rational development of new and improved bacterial carriers and more effective gene expression systems. These advances have improved the performance and versatility of these delivery systems to induce mucosal immunity to recombinant antigens in animal models. Application of these (improved) technologies for development of human vaccines is still limited and awaits further exploration.

Transport of D-xylose in Lactobacillus pentosus, Lactobacillus casei, and Lactobacillus plantarum: evidence for a mechanism of facilitated diffusion via the phosphoenolpyruvate:mannose phosphotransferase system.

We have identified and characterized the D-xylose transport system of Lactobacillus pentosus. Uptake of D-xylose was not driven by the proton motive force generated by malolactic fermentation and required D-xylose metabolism. The kinetics of D-xylose transport were indicative of a low-affinity facilitated-diffusion system with an apparent K(m) of 8.5 mM and a V(max) of 23 nmol min(-1) mg of dry weight(-1). In two mutants of L. pentosus defective in the phosphoenolpyruvate:mannose phosphotransferase system, growth on D-xylose was absent due to the lack of D-xylose transport. However, transport of the pentose was not totally abolished in a third mutant, which could be complemented after expression of the L. curvatus manB gene encoding the cytoplasmic EIIB(Man) component of the EII(Man) complex. The EII(Man) complex is also involved in D-xylose transport in L. casei ATCC 393 and L. plantarum 80. These two species could transport and metabolize D-xylose after transformation with plasmids which expressed the D-xylose-catabolizing genes of L. pentosus, xylAB. L. casei and L. plantarum mutants resistant to 2-deoxy-D-glucose were defective in EII(Man) activity and were unable to transport D-xylose when transformed with plasmids containing the xylAB genes. Finally, transport of D-xylose was found to be the rate-limiting step in the growth of L. pentosus and of L. plantarum and L. casei ATCC 393 containing plasmids coding for the D-xylose-catabolic enzymes, since the doubling time of these bacteria on D-xylose was proportional to the level of EII(Man) activity.

Instruments for oral disease-intervention strategies: recombinant Lactobacillus casei expressing tetanus toxin fragment C for vaccination or myelin proteins for oral tolerance induction in multiple sclerosis.

Lactobacillus strains possess properties that make them attractive candidates as vehicles for oral administration of therapeutics. In this report we describe the construction and analysis of recombinant Lactobacillus casei applicable in oral vaccination against an infectious disease (tetanus) and in oral tolerance induction for intervention in an autoimmune disease, multiple sclerosis. Recombinant L. casei which express surface-anchored tetanus toxin fragment C (TTFC) were generated. Quantitative analysis by flow cytometry demonstrated a high level of cell wall-bound expression of TTFC and immunogenicity was demonstrated by parenteral immunization with whole cell extracts of the recombinants. A series of expression vectors was constructed to secrete human myelin basic protein (hMBP) or hMBP as a fusion protein with beta-glucuronidase from Escherichia coli. These heterologous products produced by L. casei were detected in the growth medium and parenteral immunization with this medium evoked antibodies against hMBP, confirming that secretion indeed had occurred. Based on the different localization of the heterologous proteins, lactobacilli expressing surface-anchored TTFC are ideally suited for the induction of antibody responses, whereas lactobacilli that secrete myelin proteins can be used for the induction of peripheral T-cell tolerance. In conclusion, the specific technology described here allows the construction of a wide array of safe live recombinant lactobacilli which may prove to be useful in oral intervention strategies for the prevention of infectious diseases or treatment of autoimmune diseases.

Molecular cloning and functional expression in lactobacillus plantarum 80 of xylT, encoding the D-xylose-H+ symporter of Lactobacillus brevis.

A 3-kb region, located downstream of the Lactobacillus brevis xylA gene (encoding D-xylose isomerase), was cloned in Escherichia coli TG1. The sequence revealed two open reading frames which could code for the D-xylulose kinase gene (xylB) and another gene (xylT) encoding a protein of 457 amino acids with significant similarity to the D-xylose-H+ symporters of E. coli, XylE (57%), and Bacillus megaterium, XylT (58%), to the D-xylose-Na+ symporter of Tetragenococcus halophila, XylE (57%), and to the L-arabinose-H+ symporter of E. coli, AraE (60%). The L. brevis xylABT genes showed an arrangement similar to that of the B. megaterium xylABT operon and the T. halophila xylABE operon. Southern hybridization performed with the Lactobacillus pentosus xylR gene (encoding the D-xylose repressor protein) as a probe revealed the existence of a xylR homologue in L. brevis which is not located with the xyABT locus. The existence of a functional XylR was further suggested by the presence of xylO sequences upstream of xylA and xylT and by the requirement of D-xylose for the induction of D-xylose isomerase, D-xylulose kinase, and D-xylose transport activities in L. brevis. When L. brevis was cultivated in a mixture of D-glucose and D-xylose, the D-xylose isomerase and D-xylulose kinase activities were reduced fourfold and the D-xylose transport activity was reduced by sixfold, suggesting catabolite repression by D-glucose of D-xylose assimilation. The xylT gene was functionally expressed in Lactobacillus plantarum 80, a strain which lacks proton motive force-linked D-xylose transport activity. The role of the XylT protein was confirmed by the accumulation of D-xylose in L. plantarum 80 cells, and this accumulation was dependent on the proton motive force generated by either malolactic fermentation or by the metabolism of D-glucose. The apparent affinity constant of XylT for D-xylose was approximately 215 microM, and the maximal initial velocity of transport was 35 nmol/min per mg (dry weight). Furthermore, of a number of sugars tested, only 6-deoxy-D-glucose inhibited the transport of D-xylose by XylT competitively, with a Ki of 220 microM.

Lactic acid bacteria as antigen delivery vehicles for oral immunization purposes.

In vaccination programmes in which large numbers of subjects are involved, the oral route of administration is more convenient as compared to the more frequently used parenteral route. This is particularly relevant when vaccines are to be applied in less industrialized countries. Lactic acid bacteria in general and strains of Lactobacillus in particular have a variety of properties which make them attractive candidates for oral vaccination purposes, e.g. GRAS status, adjuvant properties, mucosal adhesive properties and low intrinsic immunogenicity. An overview is given of current research aimed at unravelling the relationship between structure and properties of surface proteins of lactobacilli and in vivo colonization, in particular of species capable of adhering to epithelial cells in vitro. Secondly, the state of the art will be discussed with respect to antigen presentation by lactic acid bacteria. Finally, some preliminary immunological data of recombinant lactic acid bacterial strains expressing antigens from pathogens will be presented.

Functional expression in Lactobacillus plantarum of xylP encoding the isoprimeverose transporter of Lactobacillus pentosus.

The xylP gene of Lactobacillus pentosus, the first gene of the xylPQR operon, was recently found to be involved in isoprimeverose metabolism. By expression of xylP on a multicopy plasmid in Lactobacillus plantarum 80, a strain which lacks active isoprimeverose and D-xylose transport activities, it was shown that xylP encodes a transporter. Functional expression of the XylP transporter was shown by uptake of isoprimeverose in L. plantarum 80 cells, and this transport was driven by the proton motive force generated by malolactic fermentation. XylP was unable to catalyze transport of D-xylose.

Cloning, sequence analysis, and characterization of the genes involved in isoprimeverose metabolism in Lactobacillus pentosus.

Two genes, xylP and xylQ, from the xylose regulon of Lactobacillus pentosus were cloned and sequenced. Together with the repressor gene of the regulon, xylR, the xylPQ genes form an operon which is inducible by xylose and which is transcribed from a promoter located 145 bp upstream of xylP. A putative xylR binding site (xylO) and a cre-like element, mediating CcpA-dependent catabolite repression, were found in the promoter region. L. pentosus mutants in which both xylP and xylQ (LPE1) or only xylQ (LPE2) was inactivated retained the ability to ferment xylose but were impaired in their ability to ferment isoprimeverose (alpha-D-xylopyranosyl-(1,6)-D-glucopyranose). Disruption of xylQ resulted specifically in the loss of a membrane-associated alpha-xylosidase activity when LPE1 or LPE2 cells were grown on xylose. In the membrane fraction of wild-type bacteria, alpha-xylosidase could catalyze the hydrolysis of isoprimeverose and p-nitrophenyl-alpha-D-xylopyranoside with apparent Km and Vmax values of 0.2 mM and 446 nmol/min/mg of protein, and 1.3 mM and 54 nmol/min/mg of protein, respectively. The enzyme could also hydrolyze the alpha-xylosidic linkage in xyloglucan oligosaccharides, but neither methyl-alpha-D-xylopyranoside nor alpha-glucosides were substrates. Glucose repressed the synthesis of alpha-xylosidase fivefold, and 80% of this repression was released in an L. pentosus delta ccpA mutant. The alpha-xylosidase gene was also expressed in the absence of xylose when xylR was disrupted.

Regulation of expression of the Lactobacillus pentosus xylAB operon.

The xylose cluster of Lactobacillus pentosus consists of five genes, two of which, xylAB, form an operon and code for the enzymes involved in the catabolism of xylose, while a third encodes a regulatory protein, XylR. By introduction of a multicopy plasmid carrying the xyl operator and by disruption of the chromosomal xylR gene, it was shown that L. pentosus xylR encodes a repressor. Constitutive expression of xylAB in the xylR mutant is repressed by glucose, indicating that glucose repression does not require XylR. The xylR mutant displayed a prolonged lag phase compared to wild-type bacteria when bacteria were shifted from glucose to xylose medium. Differences in the growth rate in xylose medium at different stages of growth are not correlated with differences in levels of xylAB transcription in L. pentosus wild-type or xylR mutant bacteria but are positively correlated in Lactobacillus casei with a plasmid containing xylAB. Glucose repression was further investigated with a ccpA mutant. An 875-bp internal fragment of the ccpA gene of L. pentosus was isolated by PCR and used to construct a ccpA knockout mutant. Transcription analysis of L. pentosus xylA showed that CcpA is involved in glucose repression. CcpA was also shown to be involved in glucose repression of the alpha-amylase promoter of Lactobacillus amylovorus by demonstrating that glucose repression of the chloramphenicol acetyltransferase gene under control of the alpha-amylase promoter is strongly reduced in the L. pentosus ccpA mutant strain.

Molecular biology of S-layers.

In this chapter we report on the molecular biology of crystalline surface layers of different bacterial groups. The limited information indicates that there are many variations on a common theme. Sequence variety, antigenic diversity, gene expression, rearrangements, influence of environmental factors and applied aspects are addressed. There is considerable variety in the S-layer composition, which was elucidated by sequence analysis of the corresponding genes. In Corynebacterium glutamicum one major cell wall protein is responsible for the formation of a highly ordered, hexagonal array. In contrast, two abundant surface proteins from the S-layer of Bacillus anthracis. Each protein possesses three S-layer homology motifs and one protein could be a virulence factor. The antigenic diversity and ABC transporters are important features, which have been studied in methanogenic archaea. The expression of the S-layer components is controlled by three genes in the case of Thermus thermophilus. One has repressor activity on the S-layer gene promoter, the second codes for the S-layer protein. The rearrangement by reciprocal recombination was investigated in Campylobacter fetus. 7-8 S-layer proteins with a high degree of homology at the 5' and 3' ends were found. Environmental changes influence the surface properties of Bacillus stearothermophilus. Depending on oxygen supply, this species produces different S-layer proteins. Finally, the molecular bases for some applications are discussed. Recombinant S-layer fusion proteins have been designed for biotechnology.

Functions of S-layers.

Although S-layers are being increasingly identified on Bacteria and Archaea, it is enigmatic that in most cases S-layer function continues to elude us. In a few instances, S-layers have been shown to be virulence factors on pathogens (e.g. Campylobacter fetus ssp. fetus and Aeromonas salmonicida), protective against Bdellovibrio, a depository for surface-exposed enzymes (e.g. Bacillus stearothermophilus), shape-determining agents (e.g. Thermoproteus tenax) and nucleation factors for fine-grain mineral development (e.g. Synechococcus GL 24). Yet, for the vast majority of S-layered bacteria, the natural function of these crystalline arrays continues to be evasive. The following review up-dates the functional basis of S-layers and describes such diverse topics as the effect of S-layers on the Gram stain, bacteriophage adsorption in lactobacilli, phagocytosis by human polymorphonuclear leukocytes, the adhesion of a high-molecular-mass amylase, outer membrane porosity, and the secretion of extracellular enzymes of Thermoanaerobacterium. In addition, the functional aspect of calcium on the Caulobacter S-layer is explained.

The presence of two S-layer-protein-encoding genes is conserved among species related to Lactobacillus acidophilus.

Previously we have shown that the type strain of Lactobacillus acidophilus possesses two S-protein-encoding genes, one of which is silent, on a chromosomal segment of 6 kb. The S-protein-encoding gene in the expression site can be exchanged for the silent S-protein-encoding gene by inversion of this slp segment. In this study the presence of S-protein and corresponding S-protein-encoding genes of strains belonging to species that are closely related to L. acidophilus was determined. All strains investigated were identified by numerical comparison of highly standardized one-dimensional SDS-PAGE whole-cellular-protein patterns. Western blot and Southern blot methods were used to identify the presence of, and homology between, S-proteins and S-protein-encoding genes. From these analyses we conclude that strains of L. acidophilus, L. crispatus, L. amylovorus and L. gallinarum possess an S-layer and contain two slp genes. Strains of L. helveticus possess an S-layer but have only one intact slp gene. Strains of L. gasseri, L. johnsonii and L. delbrueckii subsp. bulgaricus have neither an S-layer nor S-protein-encoding genes hybridizing with probes derived from the L. acidophilus slpA or slpB region. The presence of a highly conserved 5' region in the slp genes of strains of L. acidophilus, L. crispatus, L. amylovorus and L. gallinarum suggests that S-layer variation is a common feature for strains of these species.

The Lactobacillus acidophilus S-layer protein gene expression site comprises two consensus promoter sequences, one of which directs transcription of stable mRNA.

S-proteins are proteins which form a regular structure (S-layer) on the outside of the cell walls of many bacteria. Two S-protein-encoding genes are located in opposite directions on a 6.0-kb segment of the chromosome of Lactobacillus acidophilus ATCC 4356 bacteria. Inversion of this chromosomal segment occurs through recombination between two regions with identical sequences, thereby interchanging the expressed and the silent genes. In this study, we show that the region involved in recombination also has a function in efficient S-protein production. Two promoter sequences are present in the S-protein gene expression site, although only the most downstream promoter (P-1) is used to direct mRNA synthesis. S-protein mRNA directed by this promoter has a half-life of 15 min. Its untranslated leader can form a stable secondary structure in which the 5' end is base paired, whereas the ribosome-binding site is exposed. Truncation of this leader sequence results in a reduction in protein production, as shown by reporter gene analysis of Lactobacillus casei. The results obtained indicate that the untranslated leader sequence of S-protein mRNA is involved in efficient S-protein production.

Expression, secretion and antigenic variation of bacterial S-layer proteins.

The function of the S-layer, a regularly arranged structure on the outside of numerous bacteria, appears to be different for bacteria living in different environments. Almost no similarity exists between the primary sequences of S-proteins, although their amino acid composition is comparable. S-protein production is directed by single or multiple promoters in front of the S-protein gene, yielding stable mRNAs. Most bacteria secrete S-proteins via the general secretory pathway (GSP). Translocation of S-protein across the outer membrane of Gram-negative bacteria sometimes occurs by S-protein-specific branches of the GSP. O-polysaccharide side-chains of the lipopolysaccharide component of the cell wall of Gram-negative bacteria appear to function as receptors for attachment of the S-layer. Silent S-protein genes have been found in Campylobacter fetus and Lactobacillus acidophilus. These silent genes are placed in the expression site in a fraction of the bacterial population via inversion of a chromosomal segment.

Interchange of the active and silent S-layer protein genes of Lactobacillus acidophilus by inversion of the chromosomal slp segment.

The most-dominant surface-exposed protein in many bacterial species is the S-protein. This protein crystallises into a regular monolayer on the outside surface of the bacteria: the S-layer. Lactobacillus acidophilus harbours two S-protein-encoding genes, slpA and slpB, only one of which (slpA) is expressed. In this study, we show by polymerase chain reaction (PCR) analysis that slpA and slpB are located on a 6 kb chromosomal segment, in opposite or orientations. In a small fraction of the bacterial population, this segment is inverted. The inversion leads to interchanging of the expressed and silent S-protein-encoding genes, and places the formerly silent gene behind the S-promoter which is located outside the inverted segment. A 26 bp sequence showing a high degree of similarity with the consensus sequence recognized by the Din family of invertases is present in the region where recombination occurs. Expression of the slpA gene seems to be favoured under laboratory growth conditions because 99.7% of the chromosomes of an L. acidophilus ATCC 4356 broth culture had the slpA gene present at the slp expression site.