Cloning and expression of Klebsiella pneumoniae genes coding for citrate transport and fermentation.

Schwarz, Elisabeth; Oesterhelt, Dieter

doi:10.1002/j.1460-2075.1985.tb03823.x

Cited by 32 publications

(34 citation statements)

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“…Moreover, mutants deficient in malate uptake systems are available that do not grow or grow only poorly on malate, the other main substrate of the transporters of the 2HCT family (25,75). The genera Klebsiella and Salmonella are closely related to the genus Escherichia, and CitS and CitW of K. pneumoniae (58,126,143) and CitC of several Salmonella serovars (50) were successfully expressed in E. coli and characterized. However, CimH and MaeN of B. subtilis and MalP of S. bovis, more distantly related grampositive bacteria, could also be functionally expressed in E. coli (60,63,151).…”

Section: Cloning and Expressionmentioning

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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Section: Cloning and Expressionmentioning

confidence: 99%

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

Sobczak

Lolkema

2005

Microbiol Mol Biol Rev

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“…In this study, the role of the dp and the nature of the ions symported with citrate were analysed in membrane vesicles of E. coli DH1/pGR3. Plasmid pGR, is a derivative of plasmid pESl which is present in E. coli DHl/ CITl [8]. The citrate-transport gene on pGR3 is under the control of the promoters of the expression vector pIN3A [9].…”

Section: Resultsmentioning

confidence: 99%

“…E. coli DH1 recAl, endAl, gyrAg6, thi,,, S U~E~~, relAl, 2-1 was used as host for the plasmids pESl [8], which contains a 16-kb DNA fragment of K. pneumoniae DNA on which the gene for the citrate carrier is located, and pGR3 [l], a derivative of pESl containing a 1.7-kb fragment with the citrate determinant under control of the lipoprotein and lac promoter of the expression vector pIN3A [9].…”

Section: Bacterial Strains and Plasmidsmentioning

confidence: 99%

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Mechanism and energetics of a citrate‐transport system of Klebsiella pneumoniae

Vanderrest

Abee

Molenaar

et al. 1991

European Journal of Biochemistry

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The citrate-transport determinant of plasmid pESl from Klebsiella pneumoniue [Schwarz, E. & Oesterhelt, D. (1985) EMBO J . 4,1599 -16031 has been subcloned in Escherichiu coli DH1. Uptake of citrate in E. colimembrane vesicles via this uptake system is an electrogenic process, although the pH gradient is the main driving force for citrate uptake. The rate of citrate uptake, driven by artificially imposed ion-gradients, is high in the presence of an artificial ApH and low in the presence of an artificial A Y . Citrate transport does not depend on the presence of Na' or Mg2+ as has been observed for other citrate-transport systems. Citrate has three pKvalues: 3.14,4.77 and 5.40. Citrate forms a stable complex with Mg2+ with a stability constant of 3.2. Kinetic parameters and calculations of the different citrate (Cit) species at a given pH, indicate that the HCit2-is the species transported and that transport occurs in symport with three protons. This citrate-transport system is thus a unique example of a 3H+/solute symport system.In a previous paper we described the cloning and expression of a citrate-transport system from Klebsiellu pneumoniue in Escherichiu coli DH1 [l]. Unlike other species of the Enterobacteriaceae group, E. coli is unable to utilize citrate aerobically as the sole source of carbon and energy [2]. This property is one of the identification criteria of E. coli [3]. E. coli possesses all the enzymes necessary for citrate metabolism since citrate is a substrate in the tricarboxylic acid pathway. Therefore, the absence of a citrate-transport system seems to be the major cause of the inability to utilize citrate. Citrate-transport systems have been found and studied in several bacteria. Most transport systems for citrate are cation dependent [4-71. In this paper we elucidate the mechanism of a citrate-transport system from K. pneumoniue. This citratetransport system is a secondary transport system and transport is driven by the electrochemical gradient of protons across the membrane (dp) [l]. Citrate is a trivalent, negatively charged solute at neutral pH (pK1 = 3.14, pK2 = 4.77, pK3 = 5.40) which forms a stable complex with divalent cations. The apparent affinity of the transport system for total citrate was found to depend on the pH, while such a pH dependence was not observed if HCit2-is the solute transported by the citrate carrier of K. pneumoniae. This observation and the observed roles of the d Y and the ApH as driving forces for citrate uptake indicates that HCit' -is transported with at least three protons. MATERIALS AND METHODS Bacterial strains and plasmidsThe cit' gene was derived from genomic DNA of K. pneumoniae strain ATCC 13882. E. coli DH1 recAl, endAl, gyrAg6, thi,,, S U~E~~, relAl, 2-1 was used as host for the plasmids pESl [8], which contains a 16-kb DNA fragment of K. pneumoniae DNA on which the gene for the citrate carrier is located, and pGR3 [l], a derivative of pESl containing a 1.7-kb fragment with the citrate determinant under control of the lipoprotein and lac promot...

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“…

The citrate transport determinant of plasmid pESl from Klebsiella pneumoniue [Schwarz E. and D. Oesterhelt (1985) EMBO J . 4, 1599-16031 has been subcloned in Escherichia coli DH1.

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DNA sequence of a citrate carrier of Klebsiella pneumoniae

Rest

Schwarz

Oesterhelt

et al. 1990

European Journal of Biochemistry

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The citrate transport determinant of plasmid pESl from Klebsiella pneumoniue [Schwarz E. and D. Oesterhelt (1985) EMBO J . 4, 1599-16031 has been subcloned in Escherichia coli DH1. The DNA sequence of a 1723-base fragment that codes for the citrate carrier has been determined and the gene product has been characterized with the T7 promoter system. The DNA fragment contains an open reading frame of 1332 base pairs and codes for a protein of 444 amino acids. The hydropathy profile suggests that the protein is very hydrophobic and contains 12 membrane-spanning segments centered around a hydrophilic core. The gene for the citrate carrier has 66% similarity with a citrate carrier determinant from a naturally occurring plasmid responsible for secondary transport of citrate across the cytoplasmic membrane of E. coli.Citrate is abundant in nature since it is a natural constituent of all living cells. It is therefore not surprising that a large variety of bacterial species can utilize citrate as a carbon source. Special transport systems for citrate mediate the uptake of this solute into the cell. Most Enterobacteriaceae which are able to utilize citrate possess a citrate transport system under aerobic as well as under anaerobic conditions (for review see [l]). Unlike other species of this group, Escherichia coli is unable to utilize citrate aerobically as the sole source of carbon and energy [2]. This inability to grow on citrate is used in the identification of E. coli [3]. E. coli possesses all the enzymes necessary for citrate metabolism since citrate is a substrate in the tricarboxylic acid pathway. Therefore, the inability to transport citrate into the cell seems to be the major barrier for the utilization of citrate. Occasionally, however, natural Cit' E. coli strains can be found. This Cit' phenotype is usually attributed to a plasmid [4] or to a transposon [5]. The nucleotide sequences of two citi genes have been determined 16, 71. The sequences of these genes are 97% identical and are also virtually identical to a cit' gene from Citrobacter amulonaticus [8]. The 43 1-amino-acid polypeptide translated from these genes is classified as a hydrophobic membrane protein which mediates the transport of citrate across the cytoplasmic membrane via a proton-citrate symport system [9]. From a cosmid bank of Klebsiellapneumoniue DNA, three E. coli clones have been isolated which grow on citrate as the sole carbon and energy source [lo]. One of these clones, DHl/ CIT3 contains an inducible Na+-dependent anaerobic citrate Abbreviations. IPTG, isopropylthiogalactopyranoside; ORF, open reading frame; PQQ, 2,7,9,tricdrboxy-l H-pyrrolo-(2,3f)-quinoline-4,5-dione.Note. The novel nucleotidc sequence data published here have been deposited with the EMBL, Genbank and DDBJ sequence data banks and are available under the accession number X51479. The novel amino acid sequence data have been deposited with the EMBL, Genbank and DDBJ sequence data banks. transport system [ll]. DH1/CIT2 contains an inducible aerobic citrate transport syst...

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Cloning and expression of Klebsiella pneumoniae genes coding for citrate transport and fermentation.

Cited by 32 publications

References 19 publications

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

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

Mechanism and energetics of a citrate‐transport system of Klebsiella pneumoniae

DNA sequence of a citrate carrier of Klebsiella pneumoniae

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