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

Felipe, Félix López de; Magni, Christian; Mendoza, Diego de; López, Paloma

doi:10.1007/bf00298965

Cited by 45 publications

(78 citation statements)

References 39 publications

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“…Total RNA concentration was determined by UV spectrophotometry and by gel quantification with Gel Doc 1000 (Bio-Rad). Primer extension analysis was performed as previously described (13). The primer used for detection of the start sites of the cit operon and citI were pMM264 and RI264U, respectively (Table 1).…”

Section: Methodsmentioning

confidence: 99%

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Acid-Inducible Transcription of the Operon Encoding the Citrate Lyase Complex of Lactococcus lactis Biovar diacetylactis CRL264

et al. 2004

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Although Lactococcus is one of the most extensively studied lactic acid bacteria and is the paradigm for biochemical studies of citrate metabolism, little information is available on the regulation of the citrate lyase complex. In order to fill this gap, we characterized the genes encoding the subunits of the citrate lyase of Lactococcus lactis CRL264, which are located on an 11.4-kb chromosomal DNA region. Nucleotide sequence analysis revealed a cluster of eight genes in a new type of genetic organization. The citM-citCDEFXG operon (cit operon) is transcribed as a single polycistronic mRNA of 8.6 kb. This operon carries a gene encoding a malic enzyme (CitM, a putative oxaloacetate decarboxylase), the structural genes coding for the citrate lyase subunits (citD, citE, and citF), and the accessory genes required for the synthesis of an active citrate lyase complex (citC, citX, and citG). We have found that the cit operon is induced by natural acidification of the medium during cell growth or by a shift to media buffered at acidic pHs. Between the citM and citC genes is a divergent open reading frame whose expression was also increased at acidic pH, which was designated citI. This inducible response to acid stress takes place at the transcriptional level and correlates with increased activity of citrate lyase. It is suggested that coordinated induction of the citrate transporter, CitP, and citrate lyase by acid stress provides a mechanism to make the cells (more) resistant to the inhibitory effects of the fermentation product (lactate) that accumulates under these conditions. Many bacteria can utilize citrate under fermentative conditions. The citrate pathway has been extensively studied in enterobacteria (4, 26). In all known citrate fermentation pathways, after its uptake into the cell, citrate is split into acetate and oxaloacetate by the enzyme citrate lyase. In Klebsiella pneumoniae the citrate-specific fermentation genes form a cluster of two divergent operons (4, 26). This cluster includes the genes citDEF, encoding the citrate lyase subunits ␥, ␤, and ␣, and the citS gene, encoding a citrate H 2Ϫ /Na 1ϩ proton motive force-dissipating transporter. Associated with this cluster, K. pneumoniae contains the oadGAB genes, encoding the biotin oxaloacetate decarboxylase, which allows growth with citrate as the sole carbon and energy source (Fig. 1). In Escherichia coli, the cit cluster includes the genes encoding citrate lyase and the CitT citrate/succinate antiporter (22) (Fig.

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Section: Methodsmentioning

confidence: 99%

“…Transfer of nucleic acids to nitrocellulose membranes and hybridization with radioactive probes were performed as previously described (13). The single-stranded probes used were synthesized as follows.…”

Section: Methodsmentioning

confidence: 99%

Acid-Inducible Transcription of the Operon Encoding the Citrate Lyase Complex of Lactococcus lactis Biovar diacetylactis CRL264

et al. 2004

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“…A transcriptional regulator (citR) and the citrate/lactate exchanger (citP) are downstream of the citDEFXG genes, coding for the citrate lyase subunits and accessory proteins, while the oxaloacetate decarboxylase gene (citM) is upstream (Table 1) (12,13,91). In L. lactis, the pathway is encoded in two separate operons; the transcriptional regulator is encoded together with the transporter on an 8-kb plasmid, while the remaining metabolic enzymes are encoded on the chromosome (35,84,88,149). The only 2HCT transporter found in a Clostridium species, CitN in Clostridium perfringens, is also clustered with the citrate lyase genes and a malic enzyme homolog, suggesting its involvement in citrate degradation ( Table 1).…”

Section: Physiological Functionmentioning

confidence: 99%

The 2-Hydroxycarboxylate Transporter Family: Physiology, Structure, and Mechanism

Sobczak

Lolkema

2005

Microbiol Mol Biol Rev

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SUMMARY The 2-hydroxycarboxylate transporter family is a family of secondary transporters found exclusively in the bacterial kingdom. They function in the metabolism of the di- and tricarboxylates malate and citrate, mostly in fermentative pathways involving decarboxylation of malate or oxaloacetate. These pathways are found in the class Bacillales of the low-CG gram-positive bacteria and in the gamma subdivision of the Proteobacteria. The pathways have evolved into a remarkable diversity in terms of the combinations of enzymes and transporters that built the pathways and of energy conservation mechanisms. The transporter family includes H+ and Na+ symporters and precursor/product exchangers. The proteins consist of a bundle of 11 transmembrane helices formed from two homologous domains containing five transmembrane segments each, plus one additional segment at the N terminus. The two domains have opposite orientations in the membrane and contain a pore-loop or reentrant loop structure between the fourth and fifth transmembrane segments. The two pore-loops enter the membrane from opposite sides and are believed to be part of the translocation site. The binding site is located asymmetrically in the membrane, close to the interface of membrane and cytoplasm. The binding site in the translocation pore is believed to be alternatively exposed to the internal and external media. The proposed structure of the 2HCT transporters is different from any known structure of a membrane protein and represents a new structural class of secondary transporters.

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“…This gene is carried by a 22-kb plasmid. It is included in an operon together with five genes coding for the citrate lyase multienzymatic complex (citCDEFG) (15) and two open reading frames (ORFs), named citM and citR, coding for a putative malic enzyme and a polypeptide with homology to a Lactococcus lactis cit regulator (12).…”

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“…For primer extension and endonuclease S1 mapping, RNA from L. paramesenteroides was isolated as previously described for L. lactis (12) except that cell lysis was performed by the addition of lysozyme at 30 g/ml. For Northern blot hybridization, RNA was isolated with a Ribolyser and Recovery kit from Hybaid as specified by the supplier.…”

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Transcriptional Control of the Citrate-Inducible citMCDEFGRP Operon, Encoding Genes Involved in Citrate Fermentation in Leuconostoc paramesenteroides

et al. 2000

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In this study we describe the expression pattern of the Leuconostoc paramesenteroides citMCDEFGRP operon in response to the addition of citrate to the growth medium. An 8.8-kb polycistronic transcript, which includes the citMCDEFGRP genes, was identified; its synthesis was dramatically induced upon addition of citrate to the growth medium. We also found that expression of the cit operon is subjected to posttranscriptional regulation, since processing sites included in four complex secondary structures (I, II, III, and IV) were identified by Northern blot analysis and mapped by primer extension. Upstream of the citMCDEFGRP operon a divergent open reading frame, whose expression was also increased by citrate, was identified by DNA sequencing and designated citI. The start and end sites of transcription of the cit operon and citI gene were mapped. The start sites are separated by a stretch of 188 bp with a very high A؉T content of 77% and are preceded by transcriptional promoters. The end sites of the transcripts are located next to the 3 end of two secondary structures characteristic of -independent transcriptional terminators. The effect of the citI gene on expression of the cit operon was studied in Escherichia coli. The presence of the citI gene in cis and in trans resulted in increased activity of the cit promoter. These data provide the first evidence that citrate fermentation in Leuconostoc is regulated at the transcriptional level by a transcriptional activator rather than by a repressor.Citrate metabolism is carried out by only a few strains of lactic acid bacteria. This metabolic ability is invariable linked to endogenous plasmids that contain the gene encoding the transporter responsible for citrate uptake from the medium. Citrate transporters (CitPs) have been found in strains belonging to the genera Lactococcus and Leuconostoc, bacteria in which the mechanism of citrate fermentation has been studied in detail (1, 14-16). The first step in the breakdown of citrate inside the cell involves its conversion to acetate and oxalacetate by citrate lyase, a three-subunit enzyme (2). In the next step, oxalacetate is decarboxylated by oxalacetate decarboxylase, yielding pyruvate and carbon dioxide (for a review, see reference 9). The pathway generates a proton motive force (PMF) by a secondary mechanism (10). Electrogenic exchange of divalent citrate and monovalent lactate, catalyzed by CitP, efficiently generates a membrane potential, inside negative (17). Moreover a pH gradient (inside alkaline) is formed by the consumption of scalar protons in the decarboxylation of oxalacetate (10). Together, the membrane potential and pH gradient constitute the PMF, which seems to contribute significantly to the growth advantage observed during cometabolism of citrate and glucose in both Lactococcus and Leuconostoc (14,17).Like all the known citrate lyases, the Leuconostoc enzyme forms a functional complex of three proteins: a ␥ subunit (acyl carrier protein [ACP]), a ␤ subunit (citryl-S-ACP lyase), and an ␣ subunit (citrate...

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

Cited by 45 publications

References 39 publications

Acid-Inducible Transcription of the Operon Encoding the Citrate Lyase Complex of Lactococcus lactis Biovar diacetylactis CRL264

Acid-Inducible Transcription of the Operon Encoding the Citrate Lyase Complex of Lactococcus lactis Biovar diacetylactis CRL264

The 2-Hydroxycarboxylate Transporter Family: Physiology, Structure, and Mechanism

Transcriptional Control of the Citrate-Inducible citMCDEFGRP Operon, Encoding Genes Involved in Citrate Fermentation in Leuconostoc paramesenteroides

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