Abstract
The transport of citrate in Lactococcus lactis biovar diacetylactis is mediated by the citrate permease P. This polypeptide is encoded by the citP gene carried by plasmid pCIT264. In this report, we characterize the citP transcript, identify a cluster of two genes cotranscribed with citP and describe their post-transcriptional regulation. The transcriptional promoter is located 1500 nucleotides upstream of the citP gene and the transcriptional terminator is positioned next to the 3′-end of this gene. The DNA sequence was determined of the region upstream of the citP gene, including the promoter. Two partially overlapping open reading frames, citQ and citR were identified, which could encode polypeptides of 3.9 and 13 kDa respectively. These two genes, together with citP, constitute the cit cluster. Moreover, an IS-like element located between the cit promoter and the citQ open reading frame was identified. This element includes an open reading frame ORF1, which could encode a 33 kDa polypeptide. A translational fusion between the citP and a cat reporter gene showed that translation of citR and citP is coupled, and regulated by CitR. The cit mRNA was subjected to specific cleavage after addition of rifampicin to the bacterial cultures. We propose that expression of the cit cluster is controlled at the post-transcriptional level by mRNA processing at a putative complex secondary structure and by translational repression mediated by CitR.
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References
Chen EJ, Seeburg PH (1985) Supercoil sequencing a fast method for sequencing DNA plasmid. DNA 4:165–170
David S, van der Rest ME, Driessen AJM, Simmons G, de Vos WM (1990) Nucleotide sequence and expression in Escherichia coli of the Lactococcus lactis citrate permease gene. J Bacteriol 172:5789–5794
Devereux J, Haeverli P, Smithies O (1984) A comprehensive set of programs for the Vax. Nucleic Acids Res 12:387–395
Diaz A, Pons ME, Lacks SA, López P (1992) Streptococcus pneumoniae DNA polymerase I lacks 3′-to-5′ exonuclease activity: localization of the 5′-to-3′ exonucleolytic domain. J Bacteriol 174:2014–2024
Dornan S, Collins MA (1990) High efficiency electroporation of L. lactis susp. lactis LM0230. (Letters) Appl Microbiol 11:62–64
Gamper M, Ganter B, Polito MA, Haas D (1992) RNA processing modulates the expression of the arcDABC operon in Pseudomonas aeruginosa. J Mol Biol 226:943–957
Gasson MJ (1983) Plasmid complements of Streptococcus lactis NCDO 712 and other streptococci after protoplast-induced curing. J Bacteriol 154:1–9
Hirschorn RR, Aller P, Yuan ZA, Gibson CW, Basenga R (1984) Cell-cycle specific cDNAs from mammalian cells temperature sensitive for growth. Proc Natl Acad Sci USA 81:6004–6008
Horinouchi S, Weisblum B (1982) Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance. J Bacteriol 150:815–825
Hugenholtz J (1993) Citrate metabolism in lactic acid bacteria. FEMS Microbiol Rev 12:165–178
Ishiguro N, Sato G (1985) Nucleotide sequence of the gene determining plasmid-mediated citrate utilization. J Bacteriol 164:977–982
Ishiguro N, Izawa H, Shinagawa M, Shimamoto T, Tsuchiya T (1992) Cloning and nucleotide sequence of the gene (citC) encoding a citrate carrier from several Salmonella serovars. J Biol Chem 267:9559–9564
Kempler GM, McKay LL (1981) Biochemistry and genetics of citrate utilization in Streptococcus lactis subsp. diacetylactis. J Dairy Sci 64:1527–1539
Lacks SA (1970) Mutants of Diplococcus pneumoniae that lack deoxyribonucleases and other activities possibly pertinent to genetic transformation. J Bacteriol 101:373–383
Lacks SA, Löpez P, Greenberg B, Espinosa M (1986) Identification and analysis of genes for tetracycline resistance and replication functions in the broad-host-range plasmid pLS1. J Mol Biol 192:753–765
Lesage P, Chiaruttini C, Graffe M, Dondon J, Milet M, Springer M (1992) Messenger RNA secondary structure and translational coupling in Escherichia coli operon encoding translation initiation factor IF3 and the ribosomal proteins, L35 and L20. J Mol Biol 228:366–385
Liu J, Barnell WO, Conway T (1992) The polycistronic mRNA of the Zygomonas mobilis glf-zwf-edd-glk operon is subjected to complex transcript processing. J Bacteriol 174:2824–2833
Löpez P, Diaz A, Espinosa M, Lacks SA (1989) Characterization of the polA gene of Streptococcus pneumoniae and comparison of the DNA polymerase I it encodes to homologous enzymes from E. coli and phage T7. J Biol Chem 264:4255–4263
Löpez de Felipe F, Corrates MA, Löpez P (1994) Comparative analysis of gene expression in Streptococcus lactis and Lactococcus lactis. FEMS Microbiol Lett 122:289–296
Ludwig W, Seewaldt E, Klipper-Balz R, Schleifer KH, Magrum L, Woese CR, Fox, GE, Stackebrandt E (1985) The phytogenetic position of Streptococcus and Enterococcus J Gen Microbiol 131:543–551
Magni C, Löpez de Felipe F, Sesma F, Löpez P, de Mendoza D (1994) Citrate transport in Lactococcus lactis subsp. lactis biovar diacetylactis. Expression of the citrate permease P. FEMS Microbiol Lett 118:75–82
Martinez S, Löpez P, Espinosa M, Lacks SA (1987) Complementation of B. subtilis polA mutants by DNA polymerase I from Streptococcus pneumoniae. Mol Gen Genet 210:203–210
Mayford M, Weisblum B (1989) Conformational alterations in the ermC transcript in vivo during induction. EMBO J 8:4307–4314
McCarthy JEG, Gualerzi C (1990) Translational control of prokaryotic gene expression. Trends Genet 6:78–85
Pons ME, Diaz A, Lacks, SA, Löpez P (1991) The polymerase domain of Streptococcus pneumoniae DNA polymerase I. High expression, purification and characterization. Eur J Biochem 201:147–155
Rosenberg M, Court D (1979) Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet 13:319–353
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Schurr T, Nadir E, Margalit H (1993) Identification and characterization of the Escherichia coli ribosomal binding sites by free energy computation. Nucleic Acids Res 21:4019–4023
Sesma F, Gardiol D, de Ruiz Holgado AP, de Mendoza D (1990) Cloning and expression of the citrate permease gene of Lactococcus lactis subsp. lactis biovar diacetylactis in Escherichia coli. Appl Environ Microbiol 56:2099–2103
Shaw WV (1975) Chloramphenicol acetyltransferase from chloramphenicol-resistance bacteria. Methods Enzymol 43:737–755
Simon D, Chopin A (1988) Construction of a vector plasmid family and its use for molecular cloning in Streptococcus lactis. Biochimie 70:559–566
Studier FW, Rosenberg AIL Dunn JJ (1989) Use of the T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185:60–89
Tinoco I, Borer PN, Dengler B, Levine MD (1973) Improved estimation of secondary structure in ribonucleic acids. Nature 246:40–41
Van de Guchte M, Kok J, Venema G (1992) Gene expression in Lactococcus lactis. FEMS Microbiol Rev 88:73–92
Van der Rest ME, Siewe RM, Abee T, Schwarz E, Oesterhelt D, Konings WN (1992) Nucleotide sequence and functional properties of a sodium-dependent citrate transport system from Klebsiella pneumoniae. J Biol Chem 267:8971–8976
Van Rooijen RJ, de Vos WM (1990) Molecular cloning, transcriptional analysis and nucleotide sequence of lacR, a gene encoding the repressor of the lactose phosphotransferase system of Lactococcus lactis. J Biol Chem 265:18499–18503
Yarchuk O, Jacques N, Guillerez J, Dreyfus M (1992) Interdependence of translation, transcription and mRNA degradation in the lacZ gene. J Mol Biol 226:581–596
Zuker M, Stiegler P (1981) Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res 9:133–148
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Communicated by B. J. Kilbey
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de Felipe, F.L., Magni, C., de Mendoza, D. et al. Citrate utilization gene cluster of the Lactococcus lactis biovar diacetylactis: organization and regulation of expression. Molec. Gen. Genet. 246, 590–599 (1995). https://doi.org/10.1007/BF00298965
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DOI: https://doi.org/10.1007/BF00298965