No abstract

Several enzymes have evolved as sensors in signal transduction pathways to control gene expression, thereby allowing bacteria to adapt efficiently to environmental changes. We recently identified the master regulator of cysteine metabolism in Bacillus subtilis, CymR, which belongs to the poorly characterized Rrf2 family of regulators. We now report that the signal transduction mechanism controlling CymR activity in response to cysteine availability involves the formation of a stable complex with CysK, a key enzyme for cysteine biosynthesis. We carried out a comprehensive quantitative characterization of this regulator-enzyme interaction by surface plasmon resonance and analytical ultracentrifugation. We also showed that O-acetylserine plays a dual role as a substrate of CysK and as an effector modulating the CymR-CysK complex formation. The ability of B. subtilis CysK to bind to CymR appears to be correlated to the loss of its capacity to form a cysteine synthase complex with CysE. We propose an original model, supported by the determination of the intracellular concentrations of the different partners, by which CysK positively regulates CymR in sensing the bacterial cysteine pool.

A novel plasmid named pGdh442 had previously been isolated from a plant Lactococcus lactis strain. This plasmid encodes two interesting properties with applications in the dairy industry: a glutamate dehydrogenase activity that stimulates amino acid conversion to aroma compounds, and cadmium/zinc resistance that can be used as a selectable marker. Moreover, this plasmid can be transferred naturally to other strains, but appears to be incompatible with certain other lactococcal plasmids. During this study, the complete sequence of pGdh442 (68 319 bp) was determined and analysed. This plasmid contains 67 ORFs that include 20 IS elements that may have mediated transfer events between L. lactis and other genera living in the same biotope, such as Streptococcus, Pediococcus and Lactobacillus. Even though it is a low-copy-number plasmid, it is relatively stable due to a theta replication mode and the presence of two genes involved in its maintenance system. However, pGdh442 is incompatible with pSK08-derived protease/lactose plasmids because both possess the same replication and partition system. pGdh442 is not self-transmissible, but can be naturally transmitted via mobilization by conjugative elements carried by the chromosome or by other plasmids, such as the 712-type sex factor, which is widely distributed in L. lactis. In addition to several genes already found on other L. lactis plasmids, such as the oligopeptide transport and utilization genes, pGdh442 also carries several genes not yet identified in L. lactis. Finally, it does not carry genes that would trigger concern over its presence in human food. INTRODUCTIONLactococcus lactis is a lactic acid bacterium (LAB) widely used as a starter culture for the production of various fermented dairy products. Although this bacterium is commonly found in milk, it is also naturally present on plants and on parts of the bodies of cows (Nomura et al., 2006;Salama et al., 1995;Teuber, 1995). Typically, Lactococcus strains possess an abundance of plasmid DNA. Many large L. lactis plasmids are self-transmissible or mobilizable by conjugative elements carried by the chromosome or plasmids of the host strain (Gasson et al., 1995). Through their natural transfer, these plasmids increase the probability of the gene moving between bacteria, and enable strains to acquire a wide variety of new genetic traits. They thus endow their hosts with many traits which offer a selective advantage to colonize specific biotopes. For example, the majority of dairy strains have adapted to growth in milk by acquiring plasmids that encode lactose catabolism (De Vos & Gasson, 1989), casein degradation (Kok, 1990), citrate utilization (Kempler & McKay, 1979) and oligopeptide transport (Yu et al., 1996). In addition, strains isolated from plant and animal environments, which contain a wide variety of cytotoxic compounds, have developed appropriate protection mechanisms (Putman et al., 2000), such as multidrug resistance, nisin resistance and heavy metal resistance (Dougherty et al., 1998;Liu et al.,...