Sequence analysis of the mobilizable lactococcal plasmid pGdh442 encoding glutamate dehydrogenase activity

Abstract

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.,...

Molecular characterization and structural instability of the industrially important composite metabolic plasmid pLP712

“…NC_009435), featuring a single replication origin, the RepA replication initiation protein and the partitioning proteins ParA and ParB, thus confirming the prediction of Tanous et al (2007) that this is the reason for the plasmid incompatibility observed between these two plasmids. Located upstream of this region is a short CDS transcribed in the opposite direction, which has been annotated as repC on lactococcal plasmids pND324 (U44843.1) and pHP003 (AF247159.1) (Christensson et al, 2001), and is also present but not annotated on pIL105 (AF116286.2) (Anba et al, 1995).…”

“…S1), and putative D-Ldhs are only found in L. lactis plasmids pSK11P and pGdh442. Siezen et al (2005) (Tanous et al, 2007). Hence, there is no proof that the D-Ldh homologues found on pGdh442 and pSK11L are functional (Tanous et al, 2007;Siezen et al, 2005).…”

“…They could all be related to common ancestor plasmids and have undergone rearrangements and deletions, and exchanged DNA regions with other plasmids and the chromosome. More than 80 % of pLP712 DNA can be attributed to pGdh442 (NC_009435) (Tanous et al, 2007), pSK11P (DQ149245) (Siezen et al, 2005), which carries the genes necessary for the proteolysis of caseins, and pSK11L (DQ149244) (Siezen et al, 2005), which carries the lactose metabolism genes. In the following description, these plasmids and their relatives are referred to by the name of the published representative.…”

“…Siezen et al (2005) (Tanous et al, 2007). Hence, there is no proof that the D-Ldh homologues found on pGdh442 and pSK11L are functional (Tanous et al, 2007;Siezen et al, 2005). The pLP712_45 gene product has homology (Table S2) with an aketoacid dehydrogenase (E3 lipoamide dehydrogenase; Lpd), which is part of a family of multicomplex enzymes with many roles in metabolism that includes pyruvate dehydrogenase, a-ketoglutarate dehydrogenase, acetoin dehydrogenase, branched-chain a-ketoacid dehydrogenases (and the glycine cleavage system), depending on the bacterial species (Hein & Steinbüchel, 1994;Smith et al, 2002;Krüger et al, 1994).…”

Glutamate Dehydrogenases: Enzymology, Physiological Role and Biotechnological Relevance

“…Cheeses are rich in amino acids and peptides but the concentration of 2-OG is low. Supplementation of several types of cheeses with 2-OG led to a decrease in the levels of amino acids important for aroma development, which indicated that catabolism of these amino acids had been enhanced [137,138], and sensory analyses indicated that addition of 2-OG resulted in changes in creamy character, aroma intensity and fruity notes. These results clearly established that 2-OG availability is the limiting factor for the transamination reactions that convert aminoacids to aroma compounds and prompted scientist to look for sources of 2-OG.…”