Lactococcus lactis is of great importance for the nutrition of hundreds of millions of people worldwide. This paper describes the genome sequence of Lactococcus lactis subsp. cremoris MG1363, the lactococcal strain most intensively studied throughout the world. The 2,529,478-bp genome contains 81 pseudogenes and encodes 2,436 proteins. Of the 530 unique proteins, 47 belong to the COG (clusters of orthologous groups) functional category "carbohydrate metabolism and transport," by far the largest category of novel proteins in comparison with L. lactis subsp. lactis IL1403. Nearly one-fifth of the 71 insertion elements are concentrated in a specific 56-kb region. This integration hot-spot region carries genes that are typically associated with lactococcal plasmids and a repeat sequence specifically found on plasmids and in the "lateral gene transfer hot spot" in the genome of Streptococcus thermophilus. Although the parent of L. lactis MG1363 was used to demonstrate lysogeny in Lactococcus, L. lactis MG1363 carries four remnant/satellite phages and two apparently complete prophages. The availability of the L. lactis MG1363 genome sequence will reinforce its status as the prototype among lactic acid bacteria through facilitation of further applied and fundamental research.Lactococcus lactis, a mesophilic fermentative bacterium producing lactic acid from sugar (hexose) fermentation, is an important industrial microorganism with extensive and diverse uses in food fermentation. Strains of L. lactis are used as defined mixtures or in undefined combinations with other lactic acid bacteria (LAB) in the production of fermented milk products. The organism has adapted to growth in milk under stringent human selection for better performance with respect to taste, flavor, and texture of dairy products, and this process continues today (57,98,99). In 1985, the "dairy streptococci" were reclassified into two L. lactis subspecies, Lactococcus lactis subsp. lactis (previously Streptococcus lactis) and Lactococcus lactis subsp. cremoris (previously Streptococcus cremoris), to distinguish them from the streptococci sensu stricto, which contain a number of notorious human pathogens (82, 83).The strain used in this study, L. lactis subsp. cremoris MG1363, is the international prototype for LAB genetics, and the knowledge gained from fundamental research on this strain has been exploited for a wide variety of biotechnological applications. The large and unstable complement of plasmid DNA of the parent strain, L. lactis NCDO712, was eliminated by employing UV treatment and protoplast-curing strategies in the early 1980s (41). The resultant plasmid-free strain, L. lactis MG1363, is robust and genetically amenable, which has facilitated the analysis of introduced lactococcal and heterologous DNA. Sophisticated systems have been developed for the expression of proteins and peptides in this strain, and it has been used as a cell factory for a wide variety of heterologous products (e.g., antimicrobials, including bacteriocins [50], bacteriop...
To analyse nod gene expression in Rhizobium leguminosarum, a broad host‐range lacZ protein fusion vector was constructed. Two protein fusions, nodC‐lacZ and nodD‐lacZ, were used to measure the regulation of expression of the promoters of the nodA,B,C and the nodD transcripts by measuring the induced levels of β‐galactosidase activity in R. leguminosarum. In the absence of plant root exudate the nodD‐lacZ hybrid was expressed but the nodC‐lacZ hybrid was not. The expression of the nodD‐lacZ hybrid was repressed in R. leguminosarum strains containing an intact cloned nodD gene indicating that the nodD gene is autoregulatory. The induction of the nodC‐lacZ hybrid required both the nodD gene and a component present in plant root exudate. Therefore the nodD gene acts both as a repressor and as an activator of gene expression. The nodD gene is adjacent to nodA and transcribed divergently from nodA,B,C with only ∼300 nucleotides between the coding regions of nodA and nodD. Within this intergenic region is a unique BclI site and, using nodC‐lacZ or nodD‐lacZ translational fusions with this BclI site as an end point, no induction of nodC‐lacZ or nodD‐lacZ was observed. Therefore the promoters of nodD and nodA,B,C overlap at least at this region, and the regulation of these overlapping promoters appears to be controlled by the nodD protein which becomes an activator only in the presence of a component from plant exudate.
The involvement of nicotinamide adenine nucleotides (NAD ؉ , NADH) in the regulation of glycolysis in Lactococcus lactis was investigated by using 13 C and 31 P NMR to monitor in vivo the kinetics of the pools of NAD ؉ , NADH, ATP, inorganic phosphate (P i ), glycolytic intermediates, and end products derived from a pulse of glucose. Nicotinic acid specifically labeled on carbon 5 was synthesized and used in the growth medium as a precursor of pyridine nucleotides to allow for in vivo detection of 13 C-labeled NAD ؉ and NADH. The capacity of L. lactis MG1363 to regenerate NAD ؉ was manipulated either by turning on NADH oxidase activity or by knocking out the gene encoding lactate dehydrogenase (LDH). An LDH ؊ deficient strain was constructed by double crossover. Upon supply of glucose, NAD ؉ was constant and maximal (ϳ5 mM) in the parent strain (MG1363) but decreased abruptly in the LDH ؊ strain both under aerobic and anaerobic conditions. NADH in MG1363 was always below the detection limit as long as glucose was available. The rate of glucose consumption under anaerobic conditions was 7-fold lower in the LDH ؊ strain and NADH reached high levels (2.5 mM), reflecting severe limitation in regenerating NAD ؉ . However, under aerobic conditions the glycolytic flux was nearly as high as in MG1363 despite the accumulation of NADH up to 1.5 mM. Glyceraldehyde-3-phosphate dehydrogenase was able to support a high flux even in the presence of NADH concentrations much higher than those of the parent strain. We interpret the data as showing that the glycolytic flux in wild type L. lactis is not primarily controlled at the level of glyceraldehyde-3-phosphate dehydrogenase by NADH. The ATP/ADP/P i content could play an important role.Lactococcus lactis plays an essential role in the manufacture of a wide range of dairy products. The relative simplicity of L. lactis metabolism that converts sugars via the glycolytic pathway to pyruvate, generating energy mainly through substrate level phosphorylation, makes it an attractive model organism to test metabolic engineering strategies. Moreover, the large number of genetic tools available for L. lactis (1) and the recent release of the complete genome sequence are additional incentives to study the physiology of this organism in great depth (2).Despite numerous studies, a satisfactory answer to the question, What controls the glycolytic flux in L. lactis? has not been put forward. During homolactic fermentation, regulation of the carbon flux has been associated with high levels of fructose 1,6-bisphosphate (FBP), 1 which activates lactate dehydrogenase (LDH; EC 1.1.1.27) and pyruvate kinase (PK; EC 2.7.1.40), directing the flux toward the production of lactate (3). A metabolic shift from homolactic (lactate production) to mixed acid fermentation (ethanol, acetate, and formate production) was observed in glucose-limited chemostat cultures (4). A deviation from homolactic fermentation was also reported under aerobic conditions (5) or during the metabolism of galactose (6). The format...
The DNA sequence of ∼3.5 kb of the nodulation (nod) region of the Rhizobium leguminosarum symbiotic plasmid pRL1JI was determined. Three open reading frames were identified; genes corresponding to these have been called nodD, nodE and nodF.nodD is adjacent to nodA and is transcribed in the opposite direction. The nodF and nodE genes are downstream of, and transcribed in the same direction as, nodD with 667 nucleotides between nodD and nodF and three nucleotides separating nodF and nodE. The induction of the nodFE operon requires the nodD gene product and a component present in plant root exudate. Regions of DNA sequence preceding nodF are similar to those preceding nodA; these sequences may be involved in the regulation of the expression of nodA and nodF. Analysis of nodD revealed an amino acid sequence similar to the predicted DNA‐binding domain of known DNA‐binding proteins. A protein comparison of the nodF protein showed it to be similar to the acyl‐carrier protein from Escherichia coli and barley, especially around the pantothenate‐binding region and on this basis it is thought that this protein may be involved in an acyl transfer reaction.
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