Alfred J. Haandrikman

Proteolytic enzymes of lactic acid bacteria

The proteolytic system of lactic acid bacteria is essential for their growth in milk and contributes significantly to flavour development in fermented milk products where these microorganisms are used as starter cultures. The proteolytic system is composed of proteinases which initially cleave the milk protein to peptides, peptidases which cleave the peptides thus formed into smaller peptides and amino acids and transport systems which are involved in the cellular uptake of small peptides and amino acids. An overview of the literature on the research to date performed on the proteolytic enzymes of lactic acid bacteria is presented. The review highlights the different types of lactococcal and non-lactococcal proteinases as well as the approach to molecular cloning of the lactococcal proteinase genes and the molecular control of the regulation of proteinase production. A wide spectrum of peptidases have been identified in lactic acid bacteria. The research on the characterisation, substrate specificity and localisation of endopeptidases, aminopeptidases, dipeptidases, tripeptidases and proline specific peptidases is presented in addition to the illuminating research which has been performed on the transport systems for peptides and amino acids in lactic acid bacteria.

CHITINASE-B FROM SERRATIA-MARCESCENS-BJL200 IS EXPORTED TO THE PERIPLASM WITHOUT PROCESSING

A gene encoding a chitinase from Serratia marcescens BJL200 was cloned and expressed in Escherichia coli and S. marcescens. Nucleotide sequencing revealed an open reading frame encoding a 55.5 kDa protein of 499 amino acids without a typical signal peptide for export. The cellular localization of the chitinase was studied, using two types of cell fractionation methods of immunocytochemical techniques. These analyses showed that the chitinase is located in the cytoplasm in E. coli, whereas it is exported to the periplasm in S. marcescens. Analysis of chitinase isolated from periplasmic fractions of S. marcescens carrying the cloned gene showed that export of the enzyme is not accompanied by processing at the N-terminus. The chitinase did not show any of the characteristics that have been proposed to direct the export of other non-processed extracellular proteins such as the E. coli haemolysin and might therefore be secreted via a hitherto unknown mechanism.

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.

EXPRESSION OF A CHITINASE GENE FROM SERRATIA-MARCESCENS IN LACTOCOCCUS-LACTIS AND LACTOBACILLUS-PLANTARUM

A chitinase gene from the Gram-negative bacterium Serratia marcescens BJL200 was cloned in Lactococcus lactis subsp. lactis MG1363 and in the silage inoculum strain Lactobacillus plantarum E19b. The chitinase gene was expressed as an active enzyme at a low level in Lactococcus lactis, when cloned in the same transcriptional orientation as the gene specifying the replication protein of the vector pIL253. Using the expression vectors pMG36e and pGKV259 with lactococcal promoter fragments p32 and p59, the expression in L. lactis was increased nine- and 27-fold, respectively. An additional twofold increase was obtained after cloning the gene under the control of p59 in the high-copy number replicon pIL253. In Lactobacillus plantarum, chitinase activity was expressed from p32, and the activity was at the same level as under p32 control in L. lactis.

Nonnisin Bacteriocins in Lactococci: Biochemistry, Genetics, and Mode of Action

I. Summary In recent years we have seen a rapid increase in our knowledge of the structure, genetics, and mode of action of a number of bacteriocins that are produced by certain strains of lactococci. A number of the new bacteriocins have been purified to homogeneity, allowing their amino acid sequence analysis. The genetic information for both the production of and immunity against a number of lactococcins has been analyzed at the nucleotide level and has revealed that these bacteriocins are processed at their amino-terminal end. In addition to the bacteriocin structural and immunity genes, two genes have been identified that are essential for lactococcin production and, on the basis of protein homology studies, the products of these genes tentatively form a dedicated secretion system for lactococcin. The effect of purified lactococcin A on whole lactococcal cells and vesicles indicates that the bacteriocin increases the permeability of the cytoplasmic membrane of sensitive lactococci in a voltage-independent way. The specificity of lactococcin A for lactococci seems to stem from the fact that the bacteriocin recognizes a Lactococcus- specific membrane receptor protein.

Heterologous Processing and Export of the Bacteriocins Pediocin PA-1 and Lactococcin A in Lactococcus Lactis : A Study with Leader Exchange

The bacteriocins pediocin PA-1 and lactococcin A are synthesized as precursors carrying N-terminal extensions with a conserved cleavage site preceded by two glycine residues in positions -2 and -1. Each bacteriocin is translocated through the cytoplasmic membrane by an integral membrane protein of the ABC cassette superfamily which, in the case of pediocin PA-1, has been shown to possess peptidase activity responsible for proteolytic cleavage of the pre-bacteriocin. In each case, another integral membrane protein is essential for bacteriocin production. In this study, a two-step PCR approach was used to permutate the leaders of pediocin PA-1 and lactococcin A. Wild-type and chimeric pre-bacteriocins were assayed for maturation by the processing/export machinery of pediocin PA-1 and lactococcin A. The results show that pediocin PA-1 can be efficiently exported by the lactococcin machinery whether it carries the lactococcin or the pediocin leader. It can also compete with wild-type lactococcin A for the lactococcin machinery. Pediocin PA-1 carrying the lactococcin A leader or lactococcin A carrying that of pediocin PA-1 was poorly secreted when complemented with the pediocin PA-1 machinery, showing that the pediocin machinery is more specific for its bacteriocin substrate. Wild-type pre-pediocin and chimeric pre-pediocin were shown to be processed by the lactococcin machinery at or near the double-glycine cleavage site. These results show the potential of the lactococcin LcnC/LcnD machinery as a maturation system for peptides carrying double-glycine-type amino-terminal leaders.