Environmental stress responses in Lactococcus lactis

Bacteria can encounter a variety of physical conditions during their life. Bacterial cells are able to survive these (often adverse) conditions by the induction of specific or general protection mechanisms. The lactic acid bacterium Lactococcus lactis is widely used for the production of cheese. Before and during this process as well as in its natural habitats, it is subjected to several stressful conditions. Such conditions include oxidation, heating and cooling, acid, high osmolarity/dehydration and starvation. In many environments combinations of these parameters occur. Understanding the stress response behaviour of L. lactis is important to optimize its application in industrial fermentations and is of fundamental interest as L. lactis is a non-differentiating Gram-positive bacterium. The stress response mechanisms of L. lactis have drawn increasing attention in recent years. The presence in L. lactis of a number of the conserved systems (e.g. the heat shock proteins) has been confirmed. Some of the regulatory mechanisms responding to an environmental stress condition are related to those found in other Gram-positive bacteria. Other stress response systems are conserved at the protein level but are under control of mechanisms unique for L. lactis. In a number of cases exposure to a single type of stress provides resistance to other adverse conditions. The unravelling of the underlying regulatory systems gives insight into the development of such cross resistance. Taken together, L. lactis has a unique set of stress response mechanisms, most of which have been identified on the basis of homology with proteins known from other bacteria. A number of the regulatory elements may provide attractive tools for the development of food grade inducible gene expression systems. Here an overview of the growth limits of L. lactis and the molecular characterization of its stress resistance mechanisms is presented.

Salt-inducible promoter derivable from a lactic acid bacterium, and its use in a lactic acid bacterium for production of a desired protein

The invention provides a salt-inducible promoter present in SEQ ID NO: 10 and derivable from a lactic acid bacterium in isolation from the coding sequence normally controlled by said promoter in a wild-type lactic acid bacterium, with modifications and important parts thereof. Also provided are a recombinant vector and a transformed lactic acid bacterium comprising such promoter, and the production of a desired protein by such transformed bacterium, whereby the gene encoding said desired protein or a precursor thereof is expressed under control of such promoter. The desired protein can be secreted by the bacterium due to the presence of a signal sequence. The action of the salt-inducible promoter is enhanced at a pH of about 4-4.5 and/or by the presence of glutamic acid. Such process can be used in a fermentation process, in which the desired protein is a lytic protein causing lysis of the cells and release of the cell content. Or the desired protein can be an enzyme involved in flavour formation, e.g. in producing dressings, spreads, sausages and sour dough, or a protein functioning in a cheese production process, such as chymosin or a precursor thereof, or an enzyme involved in cheese flavour formation

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.