Nucleotide sequence and expression in Escherichia coli of the Lactococcus lactis citrate permease gene

Abstract

The plasmid-encoded citrate determinant of the Lactococcus lactis subsp. lactis var. diacetylactis NCDO176 was cloned and functionally expressed in a Cit-Escherichia coli K-12 strain. From deletion derivative analysis, a 3.4-kilobase region was identified which encodes the ability to transport citrate. Analysis of proteins encoded by the cloned fragment in a T7 expression system revealed a 32,000-dalton protein band, which correlated with the ability of cells to transport citrate. Energy-dependent [1,5-'4Cjcitrate transport was found with membrane vesicles prepared from E. coli cells harboring the citrate permease-expressing plasmid. The gene encoding citrate transport activity, citP, was located on the cloned fragment by introducing a site-specific mutation that abolished citrate transport and resulted in a truncated form of the 32,000-dalton expression product. The nucleotide sequence for a 2.2-kilobase fragment that includes the citP gene contained an open reading frame of 1,325 base pairs coding for a very hydrophobic protein of 442 amino acids, which shows no sequence homology with known citrate carriers.As in members of the family Enterobacteriaceae (25), the ability to utilize citrate is a useful metabolic characteristic for identifying Lactococcus lactis species (6,34). The citrate-fermenting ability of these gram-negative bacteria appears to be linked to the presence of genetically unstable determinants such as plasmids (13,14,18,32,33,37,38) (12,36,41), the energetics of citrate uptake are not yet understood. Kempler and McKay (19) demonstrated that the ability to transport citrate was linked to a 7.9-kilobase (kb) plasmid that appears to be present in all citrate-fermenting L. lactis strains analyzed. A detailed physical map of one of these citrate plasmids, pCT176, has been reported (10).In the bacterial species described until now, the ability to grow on citrate is associated with cation-dependent transport systems. Na+-dependent citrate utilization is found in Enterobacter aerogenes (16,28) and Salmonella typhimurium, which also possess a K+-dependent transport system (1,18,40 Media and growth conditions. E. coli strains were grown in L-broth (24) with vigorous shaking at 37°C. When appropriate, the medium was supplemented with carbenicillin (100 ,ug/ml), kanamycin (20 p,g/ml), or tetracycline (12.5 ,ug/ml) or a combination of these antibiotics.Citrate-positive recombinants of E. coli DH1 were selected after overnight incubation on Simmons citrate agar plates (Difco Laboratories).Cloning of the citP gene. CsCl-ethidium bromide density gradient-purified plasmid DNA from L. lactis NCDO176 was prepared by the method of Maniatis et al. (24) with minor variations as described previously (6) and was digested to completion with EcoRI. The 7.9-kb linearized plasmid band of pCT176 was isolated, inserted into the unique EcoRI site of vector pBR328, and transformed to E. coli MC1061.

Energy transduction in lactic acid bacteria

“…The plasmid-encoded citrate transport gene (citP) of L. lactis subsp, diacetvlactis has been isolatcd and functionally expressed in E. coil [140]. The CitP protein is homologous to the citratc-H*-Na + symporter (CitS) of Kh'hsiella imeumoniae [141].…”

Cloning, nucleotide sequence and expression in Stveptomyces lividans and Escherichia coli of pabB from Lactococcus lactis subsp. lactis NCDO 496

“…Either of these alternatives could explain why expression of the L. lactis gene in both S. lividans and E. coli required a vector promoter. A similar apparent requirement for vector promoters was observed when a citrate permease gene from L. lactis was cloned and expressed in E. coli; the cloned sequence did not contain any regions resembling E. coli or L. lactis consensus promoter sequences for transcription initiation (David et al, 1990). Lack of expression due to absence of appropriate promoter sequences in cloned DNA from L. lactis subspecies lactis containing genes However, an operon containing all of the genes required for tryptophan biosynthesis was fully expressed in E. coli (Bardowski et al, 1992).…”

Regulation of expression of the Lactococcus lactis subsp. lactis biovar diacetylactis citrate transport system

“…Thus, the transport of citrate limits the rate of citrate metabolization and may modify the bioconversion yield of citrate into aroma compounds. This uptake is catalyzed by the citrate permease P (CitP) encoded by the plasmidic citP gene in L. diacetylactis [3] and Leuconostoc [13]. Here, we will review the actual knowledge conceming the citrate transport in LAB with special emphasis on the molecular basis of this process.…”