Richard van Kranenburg

Complete genome sequence of Lactobacillus plantarum WCFS1

The 3,308,274-bp sequence of the chromosome of Lactobacillus plantarum strain WCFS1, a single colony isolate of strain NCIMB8826 that was originally isolated from human saliva, has been determined, and contains 3,052 predicted protein-encoding genes. Putative biological functions could be assigned to 2,120 (70%) of the predicted proteins. Consistent with the classification of L. plantarum as a facultative heterofermentative lactic acid bacterium, the genome encodes all enzymes required for the glycolysis and phosphoketolase pathways, all of which appear to belong to the class of potentially highly expressed genes in this organism, as was evident from the codon-adaptation index of individual genes. Moreover, L. plantarum encodes a large pyruvate-dissipating potential, leading to various end-products of fermentation. L. plantarum is a species that is encountered in many different environmental niches, and this flexible and adaptive behavior is reflected by the relatively large number of regulatory and transport functions, including 25 complete PTS sugar transport systems. Moreover, the chromosome encodes >200 extracellular proteins, many of which are predicted to be bound to the cell envelope. A large proportion of the genes encoding sugar transport and utilization, as well as genes encoding extracellular functions, appear to be clustered in a 600-kb region near the origin of replication. Many of these genes display deviation of nucleotide composition, consistent with a foreign origin. These findings suggest that these genes, which provide an important part of the interaction of L. plantarum with its environment, form a lifestyle adaptation region in the chromosome.

Molecular characterization of the plasmid-encoded eps gene cluster essential for exopolysaccharide biosynthesis in Lactococcus lactis

Lactococcus lactis strain NIZO B40 produces an extracellular phosphopolysaccharide containing galactose, glucose, and rhamnose. A 40 kb plasmid encoding exopolysaccharide production was isolated through conjugal transfer of total plasmid DNA from strain NIZO B40 to the plasmid‐free L. lactis model strain MG1614 and subsequent plasmid curing. A 12 kb region containing 14 genes with the order epsRXABCDEFGHIJKL was identified downstream of an iso‐IS982 element. The predicted gene products of epsABCDEFGHIJK show sequence homologies with gene products involved in exopolysaccharide, capsular polysaccharide, lipopolysaccharide, or teichoic acid biosynthesis of other bacteria. Transcriptional analysis of the eps gene cluster revealed that the gene cluster is transcribed as a single 12 kb mRNA. The transcription start site of the promoter was mapped upstream of the first gene epsR. The involvement of epsD in exopolysaccharide (EPS) biosynthesis was demonstrated through a single gene disruption rendering an exopolysaccharide‐deficient phenotype. Heterologous expression of epsD in Escherichia coli showed that its gene product is a glucosyltransferase linking the first sugar of the repeating unit to the lipid carrier.

Flavour formation from amino acids by lactic acid bacteria: predictions from genome sequence analysis

Flavour development in dairy fermentations is the result of a series of chemical and biochemical processes during ripening. Starter lactic acid bacteria provide the enzymes involved in the formation of specific flavours. Amino acids, and in particular methionine, the aromatic and the branched-chain amino acids, are major precursors for volatile aroma compounds. The recent sequencing of complete genomes of several lactic acid bacteria (i.e. Lactococcus lactis, Lactobacillus plantarum, Streptococcus thermophilus) is beginning to provide insight into the full complement of proteins that may be involved in flavour-forming reactions, and hence the potential for formation of specific flavour compounds. Examples are given how bioinformatics tools can be used to search in genomes for essential components, such as proteinases, peptidases, aminotransferases, enzymes for biosynthesis of amino acids, and transport systems for peptides and amino acids.

Complete Sequences of Four Plasmids of Lactococcus lactis subsp. cremoris SK11 Reveal Extensive Adaptation to the Dairy Environment

ABSTRACT Lactococcus lactis strains are known to carry plasmids encoding industrially important traits. L. lactis subsp. cremoris SK11 is widely used by the dairy industry in cheese making. Its complete plasmid complement was sequenced and found to contain the plasmids pSK11A (10,372 bp), pSK11B (13,332 bp), pSK11L (47,165 bp), and pSK11P (75,814 bp). Six highly homologous repB-containing replicons were found, all belonging to the family of lactococcal theta-type replicons. Twenty-three complete insertion sequence elements segment the plasmids into numerous modules, many of which can be identified as functional units or containing functionally related genes. Plasmid-encoded functions previously known to reside on L. lactis SK11 plasmids were now mapped in detail, e.g., lactose utilization (lacR-lacABCDFEGX), the proteolytic system (prtM-prtP, pepO, pepF), and the oligopeptide permease system (oppDFBCA). Newly identified plasmid-encoded functions could facilitate the uptake of various cations, while the pabA and pabB genes could be essential for folate biosynthesis. A competitive advantage could be obtained by using the putative flavin adenine dinucleotide-dependent d-lactate dehydrogenase and oxalate:formate antiporter for enhanced ATP synthesis, while the activity of the predicted α-acetolactate decarboxylase may contribute to the formation of an additional electron sink. Various stress response proteins are plasmid encoded, which could enhance strain robustness. A substantial number of these “adaptation” genes have not been described before on L. lactis plasmids. Moreover, several genes were identified for the first time in L. lactis, possibly reflecting horizontal gene transfer.

Sugar catabolism and its impact on the biosynthesis and engineering of exopolysaccharide production in lactic acid bacteria

Over the last years, the production of exopolysaccharides (EPS) by lactic acid bacteria (LAB) has been extensively studied. These EPS play an important role in the rheology and texture of fermented food products. Significant progress in the understanding of EPS biosynthetic pathways, genetics, kinetic models, and physics has been made. This knowledge can now be applied to rationally design metabolic engineering studies to modify EPS production and composition. This mini review will discuss the potential engineering strategies of sugar catabolism for the production of EPS by lactic acid bacteria (LAB).

Regulation of the metC-cysK Operon, Involved in Sulfur Metabolism in Lactococcus lactis

Sulfur metabolism in gram-positive bacteria is poorly characterized. Information on the molecular mechanisms of regulation of genes involved in sulfur metabolism is limited, and no regulator genes have been identified. Here we describe the regulation of the lactococcal metC-cysK operon, encoding a cystathionine beta-lyase (metC) and cysteine synthase (cysK). Its expression was shown to be negatively affected by high concentrations of cysteine, methionine, and glutathione in the culture medium, while sulfur limitation resulted in a high level of expression. Other sulfur sources tested showed no significant effect on metC-cysK gene expression. In addition we found that O-acetyl-l-serine, the substrate of cysteine synthase, was an inducer of the metC-cysK operon. Using a random mutagenesis approach, we identified two genes, cmbR and cmbT, involved in regulation of metC-cysK expression. The cmbT gene is predicted to encode a transport protein, but its precise role in regulation remains unclear. Disruption of cmbT resulted in a two- to threefold reduction of metC-cysK transcription. A 5.7-kb region containing the cmbR gene was cloned and sequenced. The encoded CmbR protein is homologous to the LysR family of regulator proteins and is an activator of the metC-cysK operon. In analogy to CysB from Escherichia coli, we propose that CmbR requires acetylserine to be able to bind the activation sites and subsequently activate transcription of the metC-cysK operon.

Lactococcal aminotransferases AraT and BcaT are key enzymes for the formation of aroma compounds from amino acids in cheese

Amino acid catabolism plays a major role in cheese aroma development. Previously, we showed that the lactococcal aminotransferases AraT and BcaT initiate the conversion of aromatic amino acids, branched-chain amino acids and methionine to aroma compounds. In this study, we evaluated the importance of these two enzymes in the formation of aroma compounds in a cheese model by using single araT and bcaT mutants and a double araT/bcaT mutant. We confirmed that addition of α-ketoglutarate, a co-substrate of aminotransferases, stimulates the conversion of amino acids to aroma compounds in cheese. The results demonstrated that AraT and BcaT are essential for conversion of aromatic and branched-chain amino acids to aroma compounds by Lactococcus lactis in the cheese model and that they also play a major role in the formation of volatile sulphur compounds from methionine. However, another pathway or another aminotransferase appears also to be weakly involved in the formation of these sulphur compounds.

Next Generation Prokaryotic Engineering: The CRISPR-Cas Toolkit

The increasing demand for environmentally friendly production processes of green chemicals and fuels has stimulated research in microbial metabolic engineering. CRISPR-Cas-based tools for genome editing and expression control have enabled fast, easy, and accurate strain development for established production platform organisms, such as Escherichia coli and Saccharomyces cerevisiae. However, the growing interest in alternative production hosts, for which genome editing options are generally limited, requires further developing such engineering tools. In this review, we discuss established and emerging CRISPR-Cas-based tools for genome editing and transcription control of model and non-model prokaryotes, and we analyse the possibilities for further improvement and expansion of these tools for next generation prokaryotic engineering.

Identification and Genetic Characterization of a Novel Proteinase, PrtR, from the Human Isolate Lactobacillus rhamnosus BGT10

ABSTRACT A novel proteinase, PrtR, produced by the human vaginal isolate Lactobacillus rhamnosus strain BGT10 was identified and genetically characterized. The prtR gene and flanking regions were cloned and sequenced. The deduced amino acid sequence of PrtR shares characteristics that are common for other cell envelope proteinases (CEPs) characterized to date, but in contrast to the other cell surface subtilisin-like serine proteinases, it has a smaller and somewhat different B domain and lacks the helix domain, and the anchor domain has a rare sorting signal sequence. Furthermore, PrtR lacks the insert domain, which otherwise is situated inside the catalytic serine protease domain of all CEPs, and has a different cell wall spacer (W) domain similar to that of the cell surface antigen I and II polypeptides expressed by oral and vaginal streptococci. Moreover, the PrtR W domain exhibits significant sequence homology to the consensus sequence that has been shown to be the hallmark of human intestinal mucin protein. According to its αS1- and β-casein cleavage efficacy, PrtR is an efficient proteinase at pH 6.5 and is distributed throughout all L. rhamnosus strains tested. Proteinase extracts of the BGT10 strain obtained with Ca2+-free buffer at pH 6.5 were proteolytically active. The prtR promoter-like sequence was determined, and the minimal promoter region was defined by use of prtR-gusA operon fusions. The prtR expression is Casitone dependent, emphasizing that nitrogen depletion elevates its transcription. This is in correlation with the catalytic activity of the PrtR proteinase.

Impact of 4 Lactobacillus plantarum capsular polysaccharide clusters on surface glycan composition and host cell signaling.

BackgroundBacterial cell surface-associated polysaccharides are involved in the interactions of bacteria with their environment and play an important role in the communication between pathogenic bacteria and their host organisms. Cell surface polysaccharides of probiotic species are far less well described. Therefore, improved knowledge on these molecules is potentially of great importance to understand the strain-specific and proposed beneficial modes of probiotic action.ResultsThe Lactobacillus plantarum WCFS1 genome encodes 4 clusters of genes that are associated with surface polysaccharide production. Two of these clusters appear to encode all functions required for capsular polysaccharide formation (cps2A-J and cps4A-J), while the remaining clusters are predicted to lack genes encoding chain-length control functions and a priming glycosyl-transferase (cps1A-I and cps3A-J). We constructed L. plantarum WCFS1 gene deletion mutants that lack individual (Δcps1A-I, Δcps2A-J, Δcps3A-J and Δcps4A-J) or combinations of cps clusters (Δcps1A-3J and Δcps1A-3I, Δcps4A-J) and assessed the genome wide impact of these mutations by transcriptome analysis. The cps cluster deletions influenced the expression of variable gene sets in the individual cps cluster mutants, but also considerable numbers of up- and down-regulated genes were shared between mutants in cps cluster 1 and 2, as well as between mutant in cps clusters 3 and 4. Additionally, the composition of overall cell surface polysaccharide fractions was altered in each mutant strain, implying that despite the apparent incompleteness of cps1A-I and cps3A-J, all clusters are active and functional in L. plantarum. The Δcps1A-I strain produced surface polysaccharides in equal amounts as compared to the wild-type strain, while the polysaccharides were characterized by a reduced molar mass and the lack of rhamnose. The mutants that lacked functional copies of cps2A-J, cps3A-J or cps4A-J produced decreased levels of surface polysaccharides, whereas the molar mass and the composition of polysaccharides was not affected by these cluster mutations. In the quadruple mutant, the amount of surface polysaccharides was strongly reduced. The impact of the cps cluster mutations on toll-like receptor (TLR)-mediated human nuclear factor (NF)-κB activation in host cells was evaluated using a TLR2 reporter cell line. In comparison to a L. plantarum wild-type derivative, TLR2 activation remained unaffected by the Δcps1A-I and Δcps3A-J mutants but appeared slightly increased after stimulation with the Δcps2A-J and Δcps4A-J mutants, while the Δcps1A-3J and Δcps1A-3J, Δcps4A-J mutants elicited the strongest responses and clearly displayed enhanced TLR2 signaling.ConclusionsOur study reveals that modulation of surface glycan characteristics in L. plantarum highlights the role of these molecules in shielding of cell envelope embedded host receptor ligands. Although the apparently complete cps clusters (cps2A-J and cps4A-J) contributed individually to this shielding, the removal of all cps clusters led to the strongest signaling enhancement. Our findings provide new insights into cell surface glycan biosynthesis in L. plantarum, which bears relevance in the context of host-cell signaling by probiotic bacteria.