Citrate Permease Research Articles

Transcriptional activation of the citrate permease P gene of Lactococcus lactis biovar diacetylactis by an insertion sequence-like element present in plasmid pCIT264.

The lactococcal plasmid pCIT264 contains a cluster of three genes (citQ, citR and citP) involved in the transport of citrate in Lactococcus lactis biovar diacetylactis. The cit cluster contains a copy of a newly discovered insertion sequence (IS)-like element located between its promoter P1 and the first gene of the cluster. In this report, we show that this IS-like element can act as a mobile switch for the downstream genes, creating two new transcriptional promoters named P2 and P2'. The P2 promoter is recognized by the lactococcal RNA polymerase in vivo. This is a hybrid promoter composed of a -35 region reading outwards 12bp from the right end of the IS-like element, and a nucleotide sequence from the recipient plasmid, adjacent to the element, which provides an appropriately spaced -10 region. Transcription of the citQRP cluster from this promoter takes place during the exponential and stationary phases of growth in L. lactis. Promoter P2' is included in the IS-like element and is the only promoter responsible for expression of citP in E. coli. Thus, it appears that the introduction of this element into pCIT264 allows expression of the citQRP cluster in E. coli, and increases its levels of expression in L. lactis.

Transcriptional activation of the citrate permease P gene of

Transcriptional activation of the citrate permease P gene of

Citrate utilization gene cluster of the Lactococcus lactis biovar diacetylactis: organization and regulation of expression.

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.

Detection and characterization of lactose-utilizing Lactococcus spp. in natural ecosystems

The presence of lactose-utilizing Lactococcus species in nondairy environments was studied by using identification methods based on PCR amplification and (sub)species-specific probes derived from 16S rRNA sequences. Environmental isolates from samples taken on cattle farms and in the waste flow of a cheese production plant were first identified to the genus level, using a Lactococcus genus-specific probe. Isolates which showed a positive signal with this probe were further identified to the (sub)species level. Lactococcus lactis isolates were also characterized at the phenotypic level for the ability to hydrolyze arginine, to ferment citrate, and to produce proteases and bacteriocins. With specific PCR amplifications, the presence of sequences related to citP, coding for citrate permease; prtP, coding for protease; and nisA or nisZ, the structural genes for production of nisin A or nisin Z, respectively, was verified. By these methods, it was possible to isolate lactococci from various environmental sources, such as soil, effluent water, and the skin of cattle. The strains of L. lactis isolated differed in a number of properties, such as the ability to hydrolyze arginine or the absence of citP-related sequences, from those found in industrial starter cultures. The results indicate that the majority of the industrially produced lactococci do not survive outside the dairy environment, although natural niches are available. However, from those niches strains with the potential to be developed into novel starter cultures may be isolated.

Citrate transport in Lactococcus lactis biovar diacetylactis: Expression of the plasmid-borne citrate permease P

The Lactococcus lactis subsp. lactis biovar diacetylactis citrate permease P is encoded by the citP gene borne by an 8.3-kb Cit+ plasmid (F. Sesma, Gardiol, D., de Ruiz Holgado, A.P. and de Mendoza, D., Appl. Environ. Microbiol. 56 (1990) 2099–2103). By subcloning different DNA fragments from the Cit+ plasmid it was found that citP is the only plasmidic gene required for the transport of citrate. Northern blot analysis revealed that citP is transcribed in two mRNA species with lengths of 2.9 and 1.9 kb. The transcriptional pattern, the total amount of citP mRNAs and the transport of citrate were unaffected by the presence of citrate in the growth media. Our results support the conclusion that transcription of citP as well as citrate transport are not induced by citrate in Lactococcus lactis subsp. lactis biovar diacetylactis.

Citrate transport inLactococcus lactisbiovar diacetylactis: Expression of the plasmid-borne citrate permease P

The Lactococcus lactis subsp. lactis biovar diacetylactis citrate permease P is encoded by the citP gene borne by an 8.3-kb Cit+ plasmid (F. Sesma, Gardiol, D., de Ruiz Holgado, A.P. and de Mendoza, D., Appl. Environ. Microbiol. 56 (1990) 2099–2103). By subcloning different DNA fragments from the Cit+ plasmid it was found that citP is the only plasmidic gene required for the transport of citrate. Northern blot analysis revealed that citP is transcribed in two mRNA species with lengths of 2.9 and 1.9 kb. The transcriptional pattern, the total amount of citP mRNAs and the transport of citrate were unaffected by the presence of citrate in the growth media. Our results support the conclusion that transcription of citP as well as citrate transport are not induced by citrate in Lactococcus lactis subsp. lactis biovar diacetylactis.

Nucleotide Sequence and Expression in Escherichia coli of the Lactococcus lactis Citrate Permease Gene

Nucleotide Sequence and Expression in <i>Escherichia coli</i> of the <i>Lactococcus lactis</i> Citrate Permease Gene

Nucleotide Sequence and Expression in Escherichia coli of the Lactococcus lactis Citrate Permease Gene

Nucleotide Sequence and Expression in Escherichia coli of the Lactococcus lactis Citrate Permease Gene

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

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 [1,5-14C]citrate 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.

Cloning of the citrate permease gene of Lactococcus lactis subsp. lactis biovar diacetylactis and expression in Escherichia coli

The citrate plasmid (Cit+ plasmid) from Lactococcus lactis subsp. lactis biovar diacetylactis was cloned into the EcoRI site of plasmid pUC18. This recombinant plasmid enabled Escherichia coli K-12 to transport and utilize citrate as a source of energy, indicating expression of the citrate permease from L. lactis biovar diacetylactis. The citrate permease was under the control of the lac promoter of pUC18. Genetic expression of the Cit+ plasmid in maxicells revealed that the plasmid encoded two polypeptides of 47 and 32 kilodaltons, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Strategies for genetic modification of Lactococci

Abstract Lactococci are used for the manufacture of fermented dairy products. Many technological functions are coded on plasmids: lactose and citrate utilization, a cell wall associated protease, bacteriocin and slime production and phage insensitivity phenomena. Natural and artificial methods of gene transfer are available: transduction, conjugation, protoplast fusion, transformation, transfection and electroporation. Plasmid vectors for homologous and heterologous gene cloning, transfer and expression have been developed. Cloned und expressed genes include: phospho‐ß‐galactosidase, sucrose‐6‐P hydrolase, citrate permease, alpha‐acetolactate decarboxylase, bacteriophage resistance, bacteriocin production and immunity, bacteriophage lysin, cell wall protease, ß‐galactosidase from Escherichia coli, bovine prochymosin and egg white lysozyme. The application of genetically modified Lactococcus constructs needs careful evaluation regarding biological safety and containment issues.

Biochemistry and Genetics of Citrate Utilization in Streptococcus lactis ssp. diacetylactis

A review of current literature regards Streptococcus lactis ssp. diacetylactis and its ability to utilize citrate to produce the flavor compound diacetyl. Plating media to isolate organisms capable of citrate utilization are reviewed, as are biochemical mechanisms of citrate utilization and diacetyl synthesis. In addition, historical aspects of the organism are discussed with particular emphasis on reports of instability of citrate utilization and its relevance to taxonomic classification of S. lactis ssp. diacetylactis. Finally, the relationship between plasmid deoxyribonucleic acid and citrate metabolism and future applications of genetic research are discussed. Because citrate permease activity appeared to be linked to a 5.5 megadalton (Mdal) plasmid in all strains of S. lactis ssp. diacetylactis examined, studies determined the relationship between the 5.5 Mdal plasmids. By restriction endonuclease digestion, evidence is provided that the 5.5 Mdal plasmids in all strains examined are identical.

Studies on the induction and turnover of citrate-oxidizing capacity in Klebsiella aerogenes using chemostat culture.

Steady-state chemostat cultures of Klebsiella aerogenes growing on a carbon-limited medium were challenged with pulse of carbon sources, and the resultant changes in dissolved oxygen tension were shown to reflect changes in respiration rate. These changes were used to study the kinetics of induction of citrate-oxidizing capacity, which is most probably limited by citrate permease, and the return to the preinduced state. Previously unexposed cells showed a lag phase, the duration of which decreased with increasing growth rate, with a minimum of 10 min, followed by an induction phase of linear increase of citrate oxidation rate which continued as long as citrate was present. The rate of increase in activity, which can be equated to the rate of induction of citrate permease, was independent of citrate concentration but increased with growth rate. Previously exposed cells showed no lag and some residual activity before further induction. The kinetics of return to the preinduced state were unusual in that activity was short-lived with a half-life of 16 to 23 min while the lag took over 8 h to re-establish. The rate of decay of activity decreased with increasing growth rate.

Failure in the Production of Citrate Permease by Streptococcus diacetilactis

Summary Five strains of Streptococcus diacetilactis contained variants unable to form acetoin and gas from citrate. The variants differed from parent-type bacteria by not producing citrate permease, required for transporting citrate through the cell permeability barrier. Production of citritase by the acetoinless mutants distinguished them from cultures of Streptococcus lactis and Streptococcus cremoris .