Cloning and DNA sequence of a plasmid-determined citrate utilization system in Escherichia coli.

The citrate utilization determinant from a large 200-kilobase (kb) naturally occurring plasmid was previously cloned into the PstI site of plasmid vector pBR325 creating the Cit+ tetracycline resistance plasmid pWR61 (15 kb). Tn5 insertion mutagenesis analysis of plasmid pWR61 limited the segment responsible for citrate utilization to a 4.8-kb region bordered by EcoRI and PstI restriction nuclease sites. The 4.8-kb fragment was cloned into phage M13, and the DNA sequence was determined by the dideoxyribonucleotide method. Within this sequence was a 1,296-base-pair open reading frame with a preceding ribosomal binding site. The 431-amino-acid polypeptide that could be translated from this open reading frame would be highly hydrophobic. A second long open reading frame with the potential of encoding a 379-amino-acid polypeptide preceded the larger open reading frame. Portions of the 4.8-kb fragment were further subcloned with restriction endonucleases BglII and BamHI, reducing the minimum size needed for a citrate-positive phenotype to a 1.9-kb BamHI-BglII fragment (which includes the coding region for the 431-amino-acid polypeptide, but only the distal 2/3 of the reading frame for the 379-amino-acid polypeptide). Citrate utilization results from a citrate transport activity encoded by the plasmid. With the 4.8-kb fragment (as with larger fragments) the citrate transport activity was inducible by growth on citrate. On transfer from glucose, succinate, malate, or glycerol medium to citrate medium, the Cit+ Escherichia coli strains showed a delay of 36 to 48 h before growth.     

Location of plasmid-mediated citrate utilization determinant in R27 and incidence in other H incompatibility group plasmids.

Citrate utilization (Cit+) is encoded by a specific subgroup of incompatibility HI plasmids, viz., IncHI1 plasmids. Only one IncHI1 plasmid, pRG1271, which originated in a Mexican typhoid outbreak in 1972, did not specify Cit+. All other Cit+ plasmids hybridized to a Cit+ probe, a 2-kilobase BglII fragment derived from the Cit+ transposon Tn3411. The position of the Cit+ determinant was mapped to a 13.5-kilobase ApaI fragment within the prototype IncHI1 plasmid R27. No other functions have been mapped within this region. Citrate utilization mediated by IncHI1 was observed only after a prolonged lag period of approximately 150 h, and certain Escherichia coli strains, e.g., E. coli K-12 J53-1, were not able to utilize citrate specified by the H plasmids. Most E. coli strains harboring the multicopy Cit+ plasmid pOH2, a derivative of pBR322, required only 18 to 24 h to express the Cit+ phenotype, but E. coli J53-1 (pOH2) required at least 72 h for expression.     

Location of plasmid-mediated citrate utilization determinant in R27 and incidence in other H incompatibility group plasmids

Citrate utilization (Cit+) is encoded by a specific subgroup of incompatibility HI plasmids, viz., IncHI1 plasmids. Only one IncHI1 plasmid, pRG1271, which originated in a Mexican typhoid outbreak in 1972, did not specify Cit+. All other Cit+ plasmids hybridized to a Cit+ probe, a 2-kilobase BglII fragment derived from the Cit+ transposon Tn3411. The position of the Cit+ determinant was mapped to a 13.5-kilobase ApaI fragment within the prototype IncHI1 plasmid R27. No other functions have been mapped within this region. Citrate utilization mediated by IncHI1 was observed only after a prolonged lag period of approximately 150 h, and certain Escherichia coli strains, e.g., E. coli K-12 J53-1, were not able to utilize citrate specified by the H plasmids. Most E. coli strains harboring the multicopy Cit+ plasmid pOH2, a derivative of pBR322, required only 18 to 24 h to express the Cit+ phenotype, but E. coli J53-1 (pOH2) required at least 72 h for expression.     

Tricarboxylate transport in a Cit+ Escherichia coli: evidence for the role of an outer membrane protein

A strain of Escherichia coli of bovine origin able to use tricarboxylates as single carbon source is described. Tricarboxylate utilization (Cit+) and fluorocitrate sensitivity (FCs) could be transferred conjugatively to E. coli K12 and were not plasmid borne. No evidence was found for tct gene products of Salmonella typhimurium. A citrate-inducible outer membrane protein of 21-22 kilodaltons (kd) was found only in Cit+ strains. A protein (21-22kd) protein was also found in wild-type E. coli K12 and in fluorocitrate-resistant mutants of Cit+ strains, but it was present in a cryptic form no longer inducible by citrate. Fluorocitrate-resistant mutants of Cit+ strains were still able to transport citrate by a fluorocitrate-insensitive system. High levels of the 22-kd protein correlated with reduced growth induction times on citrate and with the ability to effectively transport citrate.     

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

Three Escherichia coli clones (DH1/Cit1, DH1/Cit2 and DH1/Cit3) capable of utilizing citrate as a sole carbon source were isolated from a cosmid bank of Klebsiella pneumoniae wild-type DNA. Two of these clones (DH1/Cit1 and DH1/Cit2) only grew aerobically on citrate minimal medium, the third clone (DH1/Cit3) could also be cultured under fermentative conditions. The aerobic as well as the anaerobic generation times of the three clones were from 4.5 to 7 h. Whereas clone DH1/Cit3 showed a pronounced lag phase on citrate when the cells were pre-grown in medium without citrate, clone DH1/Cit1 immediately started growth, while with clone DH1/Cit2 a short lag phase could be observed upon transfer to citrate minimal medium. Restriction analyses of the three plasmids showed that no common fragments had been cloned. The length of the inserts were 13 and 6 kb for the aerobic Cit+ clones and 27 kb (10 kb) for the anaerobic one. Cultures of the anaerobic Cit+ clone were analyzed by immunoblotting techniques and shown to contain oxaloacetate decarboxylase, which confers citrate utilization under anaerobic conditions to K. pneumoniae. Enzyme assays demonstrated the active state of this biotin-containing membrane protein. The specific activity in vesicle preparations from the E. coli clone was 30% of the wild-type K. pneumoniae vesicles. Citrate acts as an inducer of enzyme protein synthesis in the E. coli clone as it does in K. pneumoniae.     

Citrate utilization by Escherichia coli: plasmid- and chromosome-encoded systems

Citrate utilization plasmids have previously been identified in atypical Escherichia coli isolates. A different citrate-utilizing (Cit+) variant of E. coli K-12 arose as a consequence of two chromosomal mutations (B. G. Hall, J. Bacteriol. 151:269-273, 1982). The processes controlling the transport of citrate in both a Cit+ chromosomal mutant and a Cit+ plasmid system were studied. Both systems were found to be inducible in growth experiments. In transport assays with whole cells, citrate-grown cells accumulated [1,5-14C]citrate at two to three times the rate of uninduced cells. Only the Vmax was affected by induction, and the Km for whole cells remained at 67 microM citrate for the chromosomal strain and 120 microM citrate for the plasmid-conferred system. There was no detectable accumulation of radioactivity with [6-14C]citrate, because of rapid metabolism and the release of 14CO2. Energy-dependent citrate transport was found with membrane vesicles obtained from both the chromosome-conferred and the plasmid Cit+ systems. The vesicle systems were inhibited by valinomycin and carbonyl cyanide m-chloro-phenylhydrazone but not by nigericin and monensin. In contrast to whole cells, the vesicle systems were resistant to Hg2+ and showed identical kinetics with [1,5-14C]citrate and [6-14C]citrate. H+ appeared to be important for citrate transport in whole cells and membranes. Monovalent cations such as Na+ and K+, divalent cations such as Mg2+ and Mn2+, and anions such as PO4(3-), SO4(2-), and NO3- were not required. The two systems differed in inhibition by citrate analogs.     

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.     

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.     

Plasmids in Escherichia coli controlling citrate-utilizing ability.

The citrate-utilizing ability of 19 out of 22 citrate-positive Escherichia coli strains isolated from pig sewage was transferred via conjugation to E. coli K-12. The conjugal transfer of citrate-utilizing (Cit) abilities was thermosensitive and concurrent with transfer of drug resistance. Weakly citrate-positive colonies were readily obtained in conjugation experiments. Their Cit characters could be transmitted to the other E. coli strains at a similar frequency in the retransfer experiments, and the transconjugants obtained still showed same characteristic growth on Simmons citrate agar plates. The 19 thermosensitive plasmids conferring citrate utilization and drug resistance were Fi-, and 16 of these plasmids belonged to incompatibility group H1. However, occasionally two conjugative plasmids (pOH3122-1 and pOH3124-1) carrying only the citrate utilization were also obtained in the conjugation experiments, and they were Fi+ and compatible with 19 reference R plasmids. In the two citrate-positive E. coli strains, it was suggested that the conjugative Cit plasmid showing Fi+ character and the more thermosensitive H1 plasmid conferring both the Cit character and drug resistance coexisted in the strain. The characterization of citrate utilization plasmids derived from pig farm sewage is discussed.     

Plasmids in Escherichia coli controlling citrate-utilizing ability.

The citrate-utilizing ability of 19 out of 22 citrate-positive Escherichia coli strains isolated from pig sewage was transferred via conjugation to E. coli K-12. The conjugal transfer of citrate-utilizing (Cit) abilities was thermosensitive and concurrent with transfer of drug resistance. Weakly citrate-positive colonies were readily obtained in conjugation experiments. Their Cit characters could be transmitted to the other E. coli strains at a similar frequency in the retransfer experiments, and the transconjugants obtained still showed same characteristic growth on Simmons citrate agar plates. The 19 thermosensitive plasmids conferring citrate utilization and drug resistance were Fi-, and 16 of these plasmids belonged to incompatibility group H1. However, occasionally two conjugative plasmids (pOH3122-1 and pOH3124-1) carrying only the citrate utilization were also obtained in the conjugation experiments, and they were Fi+ and compatible with 19 reference R plasmids. In the two citrate-positive E. coli strains, it was suggested that the conjugative Cit plasmid showing Fi+ character and the more thermosensitive H1 plasmid conferring both the Cit character and drug resistance coexisted in the strain. The characterization of citrate utilization plasmids derived from pig farm sewage is discussed.     

Cloning and nucleotide sequence of the gene (citA) encoding a citrate carrier from Salmonella typhimurium.

A cryptic citrate transport gene (citA) from Salmonella typhimurium chromosome was cloned and its nucleotide sequence was determined. The cloned plasmid conferred citrate-utilizing ability on wild-type Escherichia coli, which cannot grow on citrate as the sole source of carbon. The resultant E. coli transformant was able to transport citrate. A 1,302-base-pair open reading frame with a preceding ribosomal binding site was found in the cloned DNA fragment. The 434-amino-acid protein that could be translated from this open reading frame is highly hydrophobic (69% nonpolar amino acid residues), consistent with the fact that the transport protein is an intrinsic membrane protein. The molecular weight of this protein was calculated to be 47,188. The gene sequence determined is highly homologous to those of Cit+ plasmid-mediated citrate transport gene, citA, from E. coli, the chromosomal citA gene from Citrobacter amalonaticus and the chromosomal cit+ gene from Klebsiella pneumoniae. The hydropathy profile of the deduced amino acid sequence suggests that this carrier has 12 hydrophobic segments, which may span the membrane lipid bilayer.     

Thermosensitive H1 plasmids determining citrate utilization.

Twelve thermosensitive H1 plasmids from strains of Salmonella typhi that had caused outbreaks of chloramphenicol-resistant typhoid fever in Vietnam, Thailand and India mediated citrate utilization (Cit+) in a prototrophic Escherichia coli K12 strain but not in the S. typhi strains from which they were derived. Four H1 plasmids from a similar outbreak in Mexico differed from the Far Eastern plasmids in not mediating citrate utlization but in mediating mercury resistance. H1 plasmids resembling the Far Eastern and the Mexican plasmids in regard to citrate utilization and mercury resistance were found in sewage in Britain. Citrate utilization was transferred to eight pathogenic strains of E. coli and to one strain each of Shigella flexneri and Shigella sonnei. Cultures of Cit+ bacteria grew more rapidly in citrate media at 28 degrees C than at 37 degrees C. Plasmid mutants that were more efficient at utilizing citrate were present in all such cultures--they grew equally well or better at 37 degrees C than at 28 degrees C. None of 222 strains of E. coli or Shigella that contained a variety of different plasmids were able to utilize citrate. This property was not transferred to the prototrophic E. coli K12 strain from Citrobacter (3 strains), Salmonella (39 strains), Proteus (44 strains), Klebsiella pneumoniae (33 strains) or Pseudomonas aeruginosa (44 strains).     

Molybdenum cofactor negative mutants of Escherichia coli use citrate anaerobically

Anaerobically, Escherichia coli cannot grow using either glycerol or citrate as sole carbon and energy source. However, it has been reported that a mixture of glycerol and citrate will support growth. We have found that wild-type strains of E. coli K-12 do not grow on glycerol plus citrate anaerobically. However, growth eventually occurs due to the frequent appearance of mutants. We found that such Cit+ mutants were defective in anaerobic respiration with nitrate or trimethylamine-N-oxide and were chlorate resistant (i.e. molybdenum cofactor deficient). Conversely, well characterized mutants in any of chlA, B, D, E, G and N were also able to use citrate anaerobically. No anaerobic growth differences between wild type and chl mutants were observed either with fermentable sugars or with glycerol plus fumarate or glycerol plus tartrate. Citrate lyase was induced anaerobically by citrate and repressed by glucose in both wild type strains and chl mutants. Furthermore, levels of citrate lyase, fumarate reductase, malate dehydrogenase, fumarase and alcohol dehydrogenase were similar in both types of strains under anaerobic conditions. It is conceivable that a functioning molybdenum cofactor prevents use of citrate by keeping citrate lyase in the inactive form.     

Nucleotide sequence of a citrate utilization gene from Citrobacter amalonaticus.

The chromosomal DNA fragment (BamHI-BglII fragment of 2,972 base pairs) conferring citrate utilization in Citrobacter amalonaticus ATCC 25405 was cloned and sequenced. Two genes (citA and citB) identified in the Cit+ determinant were found in a BamHI-BglII fragment from C. amalonaticus. Southern DNA-DNA hybridization experiments and the construction of Cit- mutants of C. amalonaticus showed that C. amalonaticus has a single copy of the cit gene on the chromosome.     

Distribution of citrate utilization plasmids in Salmonella strains of bovine origin in Japan.

Attempts to detect transferable citrate-utilizing (Cit) ability in enterobacterial strains were carried out by conjugation experiments. Of 318 strains of Salmonella typhimurium and 1 strain of Salmonella bredeney isolated from cattle in Japan from 1970 to 1979, 107 (33.5%) strains contained transferable Cit characters. Most of the strains transferred the Cit characters to recipient Escherichia coli more efficiently at 28 degrees C than at 37 degrees C, indicating that their transfer of the Cit character is thermosensitive. Transferred Cit characters were found in association with drug resistance markers such as ampicillin, chloramphenicol, kanamycin, streptomycin, sulfonamides, and tetracycline or with mercury resistance, but Cit plasmids conferring Cit ability alone were also obtained. Of 221 conjugative Cit plasmids tested for fertility inhibition (Fi), all but 2 were Fi- and exhibited thermosensitive transfer; 2 Cit plasmids showing the Fi+ character were also isolated from 2 S. typhimurium strains. No transferable Cit character was detected from strains of Proteus, Serratia, Klebsiella, Enterobacter, and Citrobacter spp. isolated from humans or cows in the present study. The utilization of tricarboxylic acids by strains with plasmid-borne Cit ability was examined, and two different patterns of utilization were found in the Cit+ E. coli transconjugants.     

Spontaneous deletion of citrate-utilizing ability promoted by insertion sequences.

The citrate utilization (Cit+) transposon Tn3411 was shown to be flanked by directly repeated sequences (IS3411L and IS3411R) by restriction enzyme analysis and electron microscope observation. Cit- deletion mutants were frequently found to be generated in pBR322::Tn3411 by intramolecular recombination between the two copies of IS3411. The flanking IS3411 elements of Tn3411 were shown to be functional insertion sequences by Tn3411-mediated direct and inverse transposition. Tn3411-mediated inverse transposition from pBR322::Tn3411 to the F-plasmid derivative pED100 occurred more efficiently than that of direct transposition of the Cit+ determinant. This was thought to be due to the differential transposability of IS3411L and IS3411R in the transposition process. The frequency of transposition of IS3411 marked with a chloramphenicol resistance determinant was much higher than IS3411-mediated cointegrate formation, suggesting that replicon fusions are not essential intermediates in the transposition process of Tn3411 or IS3411. Spontaneous deletions occurred with high frequency in recA hosts. The spontaneous deletion promoted by homologous recombination between two IS3411 elements in Tn3411 was examined with deletion mutants.     

Nucleotide sequence of the gene determining plasmid-mediated citrate utilization.

The citrate utilization determinant from transposon Tn3411 has been cloned and sequenced, and its polypeptide products have been characterized in minicell experiments. The nucleotide sequence was determined for a 2,047-base-pair BglII restriction endonuclease fragment that includes the citrate determinant. This region contains an open reading frame that would encode a 431-amino-acid very hydrophobic polypeptide and which is preceded by a reasonable ribosomal binding site. However, the single polypeptide found in minicell experiments had an apparent molecular weight of 35,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Comparison of DNA sequences required for the function of citrate utilization among different citrate utilization plasmids

The relatedness of DNA sequences encoding citrate utilization was examined by hybridization with a cloned DNA fragment from a citrate utilization (Cit) plasmid, pOH30221, and DNA of other Cit plasmids. This revealed that there are at least two groups of Cit plasmids: the Inc W Cit plasmids, which show homology with the probe, and the Inc H1 plasmids, which do not.     

Identification of citrate utilization transposon Tn3411 from a naturally occurring citrate utilization plasmid.

We have isolated a new transposon, Tn3411, encoding citrate-utilizing ability, from a naturally occurring citrate utilization (Cit) plasmid, pOH3001. Citrate transposon Tn3411 was transposed from pOH3001 to lambda b519 b515 cI857 S7 (abbreviated lambda bb) phage, and further from the resulting lambda bb:Tn3411 to a vector plasmid, pBR322, in recA-deficient strains. The Cit+ plasmids (pOH2 and pOH3) constructed by the integration of Tn3411 into pBR322 were examined by restriction endonuclease and heteroduplex analysis. The results obtained were as follows: (i) Tn3411 was 7.4 kilobases long and flanked by small inverted repeats, and it contained one more pair of inverted repeats at the opposite orientation in the internal region, thus making alternate repeats; and (ii) the Cit+ structure gene was located on the fragment (5.5 kilobases) between two SalI cleavage sites on Tn3411.