Igor Mierau

The proteolytic systems of lactic acid bacteria

Proteolysis in dairy lactic acid bacteria has been studied in great detail by genetic, biochemical and ultrastructural methods. From these studies the picture emerges that the proteolytic systems of lactococci and lactobacilli are remarkably similar in their components and mode of action. The proteolytic system consists of an extracellularly located serine-proteinase, transport systems specific for di-tripeptides and oligopeptides (> 3 residues), and a multitude of intracellular peptidases. This review describes the properties and regulation of individual components as well as studies that have led to identification of their cellular localization. Targeted mutational techniques developed in recent years have made it possible to investigate the role of individual and combinations of enzymes in vivo. Based on these results as well as in vitro studies of the enzymes and transporters, a model for the proteolytic pathway is proposed. The main features are: (i) proteinases have a broad specificity and are capable of releasing a large number of different oligopeptides, of which a large fraction falls in the range of 4 to 8 amino acid residues; (ii) oligopeptide transport is the main route for nitrogen entry into the cell; (iii) all peptidases are located intracellularly and concerted action of peptidases is required for complete degradation of accumulated peptides.

10 Years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis

Lactococcus lactis is a Gram-positive lactic acid bacterium that, in addition to its traditional use in food fermentations, is increasingly used in modern biotechnological applications. In the last 25 years great progress has been made in the development of genetic engineering tools and the molecular characterization of this species. A new versatile and tightly controlled gene expression system, based on the auto-regulation mechanism of the bacteriocin nisin, was developed 10 years ago—the NIsin Controlled gene Expression system, called NICE. This system has become one of the most successful and widely used tools for regulated gene expression in Gram-positive bacteria. The review describes, after a brief introduction of the host bacterium L. lactis, the fundaments, components and function of the NICE system. Furthermore, an extensive overview is provided of the different applications in lactococci and other Gram-positive bacteria: (1) over-expression of homologous and heterologous genes for functional studies and to obtain large quantities of specific gene products, (2) metabolic engineering, (3) expression of prokaryotic and eukaryotic membrane proteins, (4) protein secretion and anchoring in the cell envelope, (5) expression of genes with toxic products and analysis of essential genes and (6) large-scale applications. Finally, an overview is given of growth and induction conditions for lab-scale and industrial-scale applications.

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.

Bile Salt Hydrolase of Bifidobacterium longum—Biochemical and Genetic Characterization

ABSTRACT A bile salt hydrolase (BSH) was isolated from Bifidobacterium longum SBT2928, purified, and characterized. Furthermore, we describe for the first time cloning and analysis of the gene encoding BSH (bsh) in a member of the genusBifidobacterium. The enzyme has a native molecular weight of 125,000 to 130,000 and a subunit molecular weight of 35,024, as determined from the deduced amino acid sequence, indicating that the enzyme is a tetramer. The pH optimum of B. longum BSH is between 5 and 7, and the temperature optimum is 40°C. The enzyme is strongly inhibited by thiol enzyme inhibitors, indicating that a Cys residue is likely to be involved in the catalytic reaction. The BSH ofB. longum can hydrolyze all six major human bile salts and at least two animal bile salts. A slight preference for glycine-conjugated bile acids was detected based on both the specificity and the Km values. The nucleotide sequence of bsh was determined and used for homology studies, transcript analysis, and construction and analysis of various mutants. The levels of homology with BSH of other bacteria and with penicillin V acylase (PVA) of Bacillus sphaericus were high. On the basis of the similarity of BSH and PVA, whose crystal structure has been elucidated, BSH can be classified as an N-terminal nucleophile hydrolase with Cys as the N-terminal amino acid. This classification was confirmed by the fact that a Cys1Ala exchange by site-directed mutagenesis resulted in an inactive protein. Reverse transcription-PCR experiments revealed that bsh is part of an operon containing at least two genes, bsh andglnE (GlnE is glutamine synthetase adenylyltransferase). Two UV-induced BSH-negative mutants and one spontaneous BSH-negative mutant were isolated from B. longum SBT2928 cultures and characterized. These mutants had point mutations that inactivatedbsh by premature termination, frameshift, or amino acid exchange.

Increased Production of Folate by Metabolic Engineering of Lactococcus lactis

ABSTRACT The dairy starter bacterium Lactococcus lactis is able to synthesize folate and accumulates large amounts of folate, predominantly in the polyglutamyl form. Only small amounts of the produced folate are released in the extracellular medium. Five genes involved in folate biosynthesis were identified in a folate gene cluster in L. lactis MG1363: folA, folB, folKE, folP, and folC. The gene folKE encodes the biprotein 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase and GTP cyclohydrolase I. The overexpression of folKE in L. lactis was found to increase the extracellular folate production almost 10-fold, while the total folate production increased almost 3-fold. The controlled combined overexpression of folKE and folC, encoding polyglutamyl folate synthetase, increased the retention of folate in the cell. The cloning and overexpression of folA, encoding dihydrofolate reductase, decreased the folate production twofold, suggesting a feedback inhibition of reduced folates on folate biosynthesis.

Cholic Acid Is Accumulated Spontaneously, Driven by Membrane ΔpH, in Many Lactobacilli

Many lactobacilli from various origins were found to apparently lack cholic acid extrusion activity. Cholic acid was accumulated spontaneously, driven by the transmembrane proton gradient. Accumulation is a newly identified kind of interaction between intestinal microbes and unconjugated bile acids and is different from extrusion and modification, which have been described previously.

Controlled Modulation of Folate Polyglutamyl Tail Length by Metabolic Engineering of Lactococcus lactis

ABSTRACT The dairy starter bacterium Lactococcus lactis is able to synthesize folate and accumulates >90% of the produced folate intracellularly, predominantly in the polyglutamyl form. Approximately 10% of the produced folate is released into the environment. Overexpression of folC in L. lactis led to an increase in the length of the polyglutamyl tail from the predominant 4, 5, and 6 glutamate residues in wild-type cells to a maximum of 12 glutamate residues in the folate synthetase overproducer and resulted in a complete retention of folate in the cells. Overexpression of folKE, encoding the bifunctional protein 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase and GTP-cyclohydrolase I, resulted in reduction of the average polyglutamyl tail length, leading to enhanced excretion of folate. By simultaneous overexpression of folKE and folC, encoding the enzyme folate synthetase or polyglutamyl folate synthetase, the average polyglutamyl tail length was increased, again resulting in normal wild-type distribution of folate. The production of bioavailable monoglutamyl folate and almost complete release of folate from the bacterium was achieved by expressing the gene for γ-glutamyl hydrolase from human or rat origin. These engineering studies clearly establish the role of the polyglutamyl tail length in intracellular retention of the folate produced. Also, the potential application of engineered food microbes producing folates with different tail lengths is discussed.

High-Level Expression of Lactobacillus β-Galactosidases in Lactococcus lactis Using the Food-Grade, Nisin-Controlled Expression System NICE

In this work the overlapping genes (lacL and lacM) encoding heterodimeric beta-galactosidases from Lactobacillus reuteri , Lb. acidophilus , Lb. sakei , and Lb. plantarum were cloned into two different nisin-controlled expression (NICE) vectors and expressed using Lactococcus lactis NZ9000 and NZ3900 as hosts. The lacL gene, encoding the large subunit of the beta-galactosidases, was fused translationally downstream of the nisin-inducible promoter nisA. Chloramphenicol was employed as selection marker for the standard system using L. lactis NZ9000, whereas lactose utilization based on the complementation of the lacF gene was used as a dominant selection marker for the food-grade system employing L. lactis NZ3900. Comparison of the standard and the food-grade expression system, differing only in their selection markers, gave considerable differences in volumetric beta-galactosidase activity, ranging from 1.17 to 14 kU/L of fermentation broth, depending on both the origin of the lacLM genes and the selection marker used. The occurrence of codons less frequently used by L. lactis especially at the beginning of the lacL gene could be an explanation for the significant differences between the expression levels of lacLM from different origins, while plasmid stability might cause the difference obtained when employing the different selection markers.

Fate of peptides in peptidase mutants of Lactococcus lactis

The utilization of exogenous peptides was studied in mutants of Lactococcus lactis in which combinations of the peptidase genes pepNpepCpepOpepX and pepT were deleted. Multiple mutants lacking PepN, PepC, PepT plus PepX could not grow on peptides such as Leu–Gly–Gly, Gly–Phe–Leu, Leu–Gly–Pro, Ala–Pro–Leu and Gly–Leu–Gly–Leu, respectively, indicating that no other peptidases are present to release the essential amino acid Leu. In these mutants, peptides accumulate intracellularly, demonstrating that peptides are translocated as whole entities prior to degradation. The mutant lacking all five peptidases could still grow on Gly–Leu and Tyr–Gly–Gly–Phe–Leu, which confirmed the presence of a dipeptidase and led to the identification of an unknown PepO‐like endopeptidase. These studies have also shown that the general aminopeptidases PepN, PepC and PepT have overlapping but not identical specificities and differ in their overall activity towards individual peptides. In contrast, PepX has an unique specificity, because it is the only enzyme which can efficiently degrade Ala–Pro–Leu. The concerted action of peptidases in the breakdown of particular peptides revealed how these substrates are utilized as sources of nitrogen.

Control of Folate Production in Lactic Acid Bacteria by Using Metabolic Engineering

Folates are essential components in the human diet. They are involved as cofactor in many metabolic reactions, including the biosynthesis of nucleotides, the building blocks of DNA and RNA. Folates are produced in different (green) plants (folium (Latin) = leaf) and by some micro-organisms. Therefore, vegetables and dairy products are the main source of folates for humans. The daily recommended intake for an adult is 200 µg. For pregnant women a double dose is recommended, since folates are known to prevent neural-tube defect in newborns (1). Moreover, folates are reported to protect against some forms of cancer (2). A low folate level in the diet is associated with high homocysteine levels in the blood and, consequently with coronary diseases (3, 4).