Coli Genetic Map Research Articles

Identification and characterization of the tktB gene encoding a second transketolase in Escherichia coli K-12

We isolated a transposon Tn10 insertion mutant of Escherichia coli K-12 which could not grow on MacConkey plates containing D-ribose. Characterization of the mutant revealed that the level of the transketolase activity was reduced to one-third of that of the wild type. The mutation was mapped at 63.5 min on the E. coli genetic map, in which the transketolase gene (tkt) had been mapped. A multicopy suppressor gene which complemented the tkt mutation was cloned on a 7.8-kb PstI fragment. The cloned gene was located at 53 min on the chromosome. Subcloning and sequencing of a 2.7-kb fragment containing the suppressor gene identified an open reading frame encoding a polypeptide of 667 amino acids with a calculated molecular weight of 72,973. Overexpression of the protein and determination of its N-terminal amino acid sequence defined unambiguously the translational start site of the gene. The deduced amino acid sequence showed similarity to sequences of transketolases from Saccharomyces cerevisiae and Rhodobacter sphaeroides. In addition, the level of the transketolase activity increased in strains carrying the gene in multicopy. Therefore, the gene encoding this transketolase was designated tktB and the gene formerly called tkt was renamed tktA. Analysis of the phenotypes of the strains containing tktA, tktB, or tktA tktB mutations indicated that tktA and tktB were responsible for major and minor activities, respectively, of transketolase in E. coli.

Cloning and characterization of ftsN, an essential cell division gene in Escherichia coli isolated as a multicopy suppressor of ftsA12(Ts).

A new cell division gene, ftsN, was identified in Escherichia coli as a multicopy suppressor of the ftsA12(Ts) mutation. Remarkably, multicopy ftsN suppressed ftsI23(Ts) and to a lesser extent ftsQ1(Ts); however, no suppression of the ftsZ84(Ts) mutation was observed. The suppression of ftsA12(Ts), ftsI23(Ts), and ftsQ1(Ts) suggests that FtsN may interact with these gene products during cell division. The ftsN gene was located at 88.5 min on the E. coli genetic map just downstream of the cytR gene. ftsN was essential for cell division, since expression of a conditional null allele led to filamentation and cell death. DNA sequence analysis of the ftsN gene revealed an open reading frame of 319 codons which would encode a protein of 35,725 Da. The predicted gene product had a hydrophobic sequence near its amino terminus similar to the noncleavable signal sequences found in several other Fts proteins. The presumed extracellular domain was unusual in that it was rich in glutamine residues. A 36-kDa protein that was localized to the membrane fraction was detected in minicells containing plasmids with the ftsN gene, confirming that FtsN was a membrane protein.

Isolation and characterization of Escherichia coli strains containing new gene fusions (soi::lacZ) inducible by superoxide radicals

Gene fusions in Escherichia coli that showed increased beta-galactosidase expression in response to treatment with a superoxide radical (O2-) generator, methyl viologen (MV), were obtained. These fusions were constructed by using a Mud(Ap lac) phage to insert the lactose structural genes randomly into the E. coli chromosome. Ampicillin-resistant colonies were screened for increased expression of beta-galactosidase on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) plates containing MV at 1.25 micrograms/ml. Other O2- generators, menadione and plumbagin, also induced beta-galactosidase activity in these fusion strains. The induction by these drugs occurred only under aerobic conditions. Hyperoxygenation also elicited an induction of the fusions. On the other hand, no significant induction was observed with hydrogen peroxide and cumene hydroperoxide. The induction of these fusions by MV was not dependent on the peroxide stress control mediated by the oxyR gene or on the recA-dependent SOS system. These fusions were named soi (superoxide inducible)::lacZ. The induction of beta-galactosidase was significantly reduced by introducing a soxS::Tn10 locus into the fusion strains, indicating that the soi genes are members of the soxRS regulon. Five of the fusions were located in 6 to 26 min of the E. coli genetic map, while three fusions were located in 26 to 36 min, indicating that these fusions are not related to genes already known to be inducible by O2- under the control of soxRS. At least five mutants containing the soi::lacZ fusion were more sensitive to MV and menadione than the wild-type strain, suggesting that the products of these soi genes play an important role in protection against oxidative stress.

Escherichia coli thioesterase I, molecular cloning and sequencing of the structural gene and identification as a periplasmic enzyme

The structural gene for Escherichia coli thioesterase I (called tesA) has been cloned by use of sequence data obtained from the purified protein. The tesA gene was mapped at 530 kilobase pair of the E. coli physical map (minute 11.6 of E. coli genetic map). The DNA sequence of the tesA gene was obtained and the deduced protein sequence showed that thioesterase I consists of 182 amino acids and has a molecular mass of 20.5 kDa. Comparison of the DNA and protein sequence data suggested that a leader sequence of 26 amino acid residues is cleaved from the primary translation product, and this processing was confirmed by NH2-terminal sequencing of the primary translation product synthesized in vitro. These data predicted that thioesterase I (long believed to be a cytoplasmic protein) is exported to the cell periplasm, a prediction supported by release of the enzyme from cells upon osmotic shock. The TesA protein sequence does not exhibit any significant overall sequence similarity with other known proteins, although the sequence does contain two small sequence elements found in several other thioesterases. One of these elements is a sequence similar to the serine esterase active sites found in serine proteases and four other thioesterases. A serine residue within this TesA element was shown to be covalently labeled with [3H] diisopropyl fluorophosphate, a potent inhibitor of TesA activity. The second sequence element is a histidine-containing sequence found close to the carboxyl terminus that is also found in the carboxyl termini of the four known active serine thioesterases. The physiological role of thioesterase I is unclear. A strain carrying a null mutation of the tesA gene was constructed and found to have no growth phenotype. Moreover, a strain carrying a plasmid that gave massive overproduction of TesA (approximately 100-fold higher than that of the wild type) also grew normally. In addition a strain containing double null mutations in both tesA and tesB (the structural gene for E. coli thioesterase II) also failed to display any growth phenotype. Analysis of the fatty acid compositions of phospholipid, lipid A, and lipoprotein of the above strains showed no significant changes from a wild type strain.

Expression of theEscherichia coli ftsZgene: trials and tribulations of gene fusion studies

The ftsZ gene of Escherichia coli, which codes for an essential cell division protein, is subjected to multiple regulation, as shown in part with studies using an ftsZ::lacZ operon fusion located on phage lambda JFL100. Using this same fusion, we sought to isolate regulatory mutants overexpressing ftsZ by selecting mutants able to grow on lactose. One Lac+ mutant was obtained which overexpressed the ftsZ::lacZ fusion 70-fold. The mutation responsible for the overexpression lies in a new gene, cot, located near 56 min on the E. coli genetic map. The cot mutation probably affects the transcription of a chromosomal open reading frame, ORF1, lying downstream of the bioA gene and adjacent to the ftzZ::lacZ fusion of the lambda JFL100 prophage integrated at att lambda. Using an ftsZ84(Ts) strain, in which there was a double selection for overexpression of both ftsZ::lacZ and ftsZ+, no Lac+Tr mutants were obtained from 3.6 x 10(10) bacteria; the introduction of a mutL allele, increasing spontaneous base substitution mutation rates 75-fold, did not permit us to isolate such a mutant. We conclude that Lac+ ftsZ-constitutive mutations cannot be obtained in lambda JFL100 lysogens by a single base substitution.

DNA sequence of the murE-murD region of Bacillus subtilis 168

The sequence of a 4.4 kbp region of DNA from Bacillus subtilis 168, lying between sporulation genes spoVD and spoVE, has been determined as part of the B. subtilis genomic sequencing programme. The region contains three genes with high sequence similarity to the murE, mraY and murD genes of Escherichia coli. The products of these genes are likely to catalyse various steps in the formation of the precursors for peptidoglycan synthesis in B. subtilis. The regions at 133 degrees on the standard genetic map of the B. subtilis chromosome, and in the 2 min region of the E. coli genetic map, are now shown to contain a large cluster of functionally related genes. Although the linear order of the genes in the cluster is conserved, three genes that are present in the E. coli chromosome, and which are likely to be essential for peptidoglycan synthesis in both organisms, are absent from this region of the B. subtilis chromosome. In general, the B. subtilis cluster differs from that of E. coli in having more extensive intergenic regions, with less potential for translational coupling.

Function of the Escherichia coli nucleoid protein, H-NS: molecular analysis of a subset of proteins whose expression is enhanced in a hns deletion mutant.

The expression of numerous Escherichia coli cellular proteins was previously demonstrated to be greatly enhanced in a hns deletion background, relative to the levels in wild-type cells. In this study, a subset of such proteins, expression of which is affected by H-NS, was partially purified, and the genes coding for some of the proteins were identified and characterized. Two of the proteins thus characterized, 19K and 17K, were found to be encoded by previously predicted genes that are located adjacent to, and downstream of, the trpABCDE operon (27.6 min on the E. coli genetic map). The genes coding for the other two proteins, 10K-L and 10K-S, are located at 77.5 min on the genetic map. Their nucleotide sequences were determined and revealed that they may constitute an operon. To characterize the putative promoters for these genes, a set of promoter-lacZ transcriptional fusion genes was constructed on the E. coli chromosome. The results of such promoter-probe analyses indicated that H-NS represses the expression of these genes at the transcriptional level. Furthermore, H-NS appeared to exhibit relatively strong affinity for the putative promoter sequences in vitro. These results are compatible with the hypothesis that H-NS functions as a transcriptional repressor.

A genetic study of apramycin‐resistant mutants of Escherichia coli

The apramycin-resistant mutants of E. coli were isolated spontaneously. Mutations, aprA and aprB, conferring resistance to apramycin were located at 72 min on the E. coli genetic map where most of ribosomal genes were mapped. One of the mutants, carrying the aprB mutation, has an altered translational fidelity expressed as severe restriction of amber suppressor activity in vivo. The chromosome location and altered translational fidelity indicate that the apramycin-resistant phenotype could be a consequence of an alteration of the ribosomal structure.

The genes encoding the two carboxyltransferase subunits of Escherichia coli acetyl-CoA carboxylase.

We report characterization of the component proteins and molecular cloning of the genes encoding the two subunits of the carboxyltransferase component of the Escherichia coli acetyl-CoA carboxylase. Peptide mapping of the purified enzyme component indicates that the carboxyltransferase component is a complex of two nonidentical subunits, a 35-kDa alpha subunit and a 33-kDa beta subunit. The alpha subunit gene encodes a protein of 319 residues and is located immediately downstream of the polC gene (min 4.3 of the E. coli genetic map). The deduced amino acid composition, molecular mass, and amino acid sequence match those determined for the purified alpha subunit. Six sequenced internal peptides also match the deduced sequence. The amino-terminal sequence of the beta subunit was found within a previously identified open reading frame of unknown function called dedB and usg (min 50 of the E. coli genetic map) which encodes a protein of 304 residues. Comparative peptide mapping also indicates that the dedB/usg gene encodes the beta subunit. Moreover, the deduced molecular mass and amino acid composition of the dedB/usg-encoded protein closely match those determined for the beta subunit. The deduced amino acid sequences of alpha and beta subunits show marked sequence similarities to the COOH-terminal half and the NH2-terminal halves, respectively, of the rat propionyl-CoA carboxylase, a biotin-dependent carboxylase that catalyzes a similar carboxyltransferase reaction reaction. Several conserved regions which may function as CoA-binding sites are noted.

A novel sensor-regulator protein that belongs to the homologous family of signal-transduction proteins involved in adaptive responses in Escherichia coli.

Expression of the Escherichia coli outer membrane porins, OmpC and OmpF, is regulated in response to changes in the medium osmolarity through the functions of the regulatory factors, EnvZ and OmpR. A 3.0 kilobase pair DNA fragment cloned from E. coli is able phenotypically to suppress the defect in ompC and ompF expression caused by an envZ deletion mutation, provided that a certain gene located in this fragment is expressed on a high copy-number plasmid. Nucleotide sequencing revealed that the putative gene encodes a protein of 102,452 Da. The deduced amino acid sequence of the protein shows a high degree of homology to those of both EnvZ and OmpR, i.e. it contains both a 'sensory kinase domain' and a 'response regulator domain' in its primary amino acid sequence. The protein identified in this study is probably a novel member of the homologous family of proteins involved in bacterial adaptive responses. Hence, the gene encoding this novel sensor-regulator protein was designated as barA (bacterial adaptive responses) and mapped at 60 min on the E. coli genetic map. The BarA protein in isolated membranes was demonstrated in vitro to undergo phosphorylation in the presence of ATP.

Isolation and characterization of the Escherichia coli msbB gene, a multicopy suppressor of null mutations in the high-temperature requirement gene htrB

Previous work established that the htrB gene of Escherichia coli is required for growth in rich media at temperatures above 32.5 degrees C but not at lower temperatures. In an effort to determine the functional role of the htrB gene product, we have isolated a multicopy suppressor of htrB, called msbB. The msbB gene has been mapped to 40.5 min on the E. coli genetic map, in a 12- to 15-kb gap of the genomic library made by Kohara et al. (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987). Mapping data show that the order of genes in the region is eda-edd-zwf-pykA-msbB. The msbB gene codes for a protein of 37,410 Da whose amino acid sequence is similar to that of HtrB and, like HtrB, the protein is very basic in nature. The similarity of the HtrB and MsbB proteins could indicate that they play functionally similar roles. Mutational analysis of msbB shows that the gene is not essential for E. coli growth; however, the htrB msbB double mutant exhibits a unique morphological phenotype at 30 degrees C not seen with either of the single mutants. Analysis of both msbB and htrB mutants shows that these bacteria are resistant to four times more deoxycholate than wild-type bacteria but not to other hydrophobic substances. The addition of quaternary ammonium compounds rescues the temperature-sensitive phenotype of htrB bacteria, and this rescue is abolished by the simultaneous addition of Mg2+ or Ca2+. These results suggest that MsbB and HtrB play an important role in outer membrane structure and/or function.

Molecular cloning and sequence of the thdF gene, which is involved in thiophene and furan oxidation by Escherichia coli

Our previous work resulted in the isolation of mutant strains of Escherichia coli K-12 which were able to oxidize furans and thiophenes as a result of mutations in several novel genes. Some of the genes involved in thiophene oxidation were cloned into the multicopy vector pUC19. The plasmid pKA10 carries a 3.8-kb chromosomal fragment which encodes a previously undiscovered gene involved in thiophene oxidation. Three proteins with approximate molecular sizes of 48, 30, and 26 kDa were overproduced by cells carrying pKA10. Maxicell experiments and DNA sequence analysis indicated that the 48- and 26-kDa proteins are encoded by pKA10, whereas the 30-kDa protein is apparently chromosomally derived. A cassette specifying kanamycin resistance was inserted into various sites on pKA10. An insertion which abolished the 48-kDa protein also abolished thiophene oxidation. Chromosomal integration of pKA10::Kan allowed us to locate the chromosomal insert of pKA10 at 84 min on the E. coli genetic map by transduction. Since no previously identified genes involved in thiophene metabolism are located in this region, we designated the gene for the 48-kDa protein as thdF. Sequencing of the 3.8-kb insert revealed an overlap of several hundred bases with the regulatory and structural regions of the tnaA gene, which is also located at 84 min. The 26-kDa protein is probably truncated tnaA protein. An open reading frame corresponding to the 48-kDa thdF protein was located next to the tnaA gene, which encodes tryptophanase, but was transcribed in the opposite sense.

The Escherichia coli htrP gene product is essential for bacterial growth at high temperatures: mapping, cloning, sequencing, and transcriptional regulation of htrP

We identified and characterized a new Escherichia coli gene, htrP. The htrP gene was identified because its insertional inactivation by the Tn10 transposon results in the inability of E. coli to form colonies at temperatures above 37 degrees C and a slow growth phenotype at 30 degrees C. The htrP gene was cloned and mapped to 66.3 min on the E. coli genetic map, 4 kbp clockwise from the tolC gene. The htrP gene was sequenced and shown to code for an acidic, 27,471-Da polypeptide and to be transcribed counterclockwise with respect to the genetic map. The predicted HtrP protein has two potential transmembrane segments and shares an identity of 64.4% over a length of 210 amino acids with the LuxH protein. Despite the fact that the htrP gene is essential for E. coli growth exclusively at high temperatures, the levels of htrP-specific transcripts decrease with increasing temperature.

Relationships between phosphatidylcholine content, chitin synthesis, growth, and morphology of Aspergillus nidulans choC

The phosphatidylcholine (PC) content of Aspergillus nidulans choC was varied by growing the auxotroph in medium containing various concentrations of choline chloride. Direct linear correlations were observed between PC content and in vivo chitin synthase activity, between in vivo chitin synthase activity and mean hyphal extension rate, and between mean hyphal extension rate and hyphal growth unit length; hyphal growth unit length is a measure of hyphal branching. Further, there was a correlation between PC content and colony radial growth rate. Thus, membrane composition is an important determinant of both hyphal (and colony) extension rate and mycelial morphology.

Escherichia coli thymidine kinase: nucleotide sequence of the gene and relationships to other thymidine kinases

The thymidine kinase (TK)-encoding gene ( tdk) of Escherichia coli is located at min 27 of the E. coli genetic map. Sequence analysis of this region revealed an open reading frame of 205 codons. Identification of this region as the E. coli tdk gene was confirmed by its similarity to other TK-encoding genes. The E. coli amino acid (aa) sequence showed significant similarity to the corresponding TK polypeptides of vertebrates and large DNA viruses, but showed no similarity to known herpesvirus TK enzymes. Mapping of highly conserved positions among all sequences indicates the importance of these residues for catalytic activity and may facilitate further functional studies. Using a distance matrix method, the evolutionary relationships among the TK aa sequences of poxviruses, eukaryotes and prokaryotes were analyzed and a potential phylogenetic tree was established.

A novel L-serine deaminase activity in Escherichia coli K-12

We demonstrate here that Escherichia coli K-12 synthesizes two different L-serine deaminases (L-SD) catalyzing the nonoxidative deamination of L-serine to pyruvate, one coded for by the previously described sdaA gene and a second, hitherto undescribed enzyme which we call L-SD2. A strain carrying a null mutation in sdaA made no detectable L-SD in minimal medium, but had activity in Luria broth. We describe a mutation, sdaX, which affects the regulation of L-SD2 and permits its expression in minimal medium, and an insertion mutation, sdaB, which abolishes L-SD2 activity completely. Both mutations lie near 60.5 min on the E. coli genetic map. The two L-SD enzymes have similar enzyme parameters, and both require posttranslational activation.

Mutations permitting the anaerobic growth ofEscherichia colion trehalose

Wild-type strains of Escherichia coli K-12 do not grow anaerobically on trehalose or galactose. We isolated two operon fusion mutants of E. coli which gained the ability to grow on trehalose anaerobically (tan). The tanA-lac mutation was located at 41 min on the E. coli genetic map and also abolished growth on glucuronic acid both aerobically and anaerobically. The tanB-lac mutation was mapped to 68 min and permitted anaerobic growth on galactose as well as trehalose. The tanB-lac fusion was induced anaerobically whereas tanA-lac showed more or less constitutive beta-galactosidase expression.

Mutations permitting the anaerobic growth of Escherichia coli on trehalose

Wild-type strains of Escherichia coli K-12 do not grow anaerobically on trehalose or galactose. We isolated two operon fusion mutants of E. coli which gained the ability to grow on trehalose anaerobically (tan). The tanA-lac mutation was located at 41 min on the E. coli genetic map and also abolished growth on glucuronic acid both aerobically and anaerobically. The tanB-lac mutation was mapped to 68 min and permitted anaerobic growth on galactose as well as trehalose. The tanB-lac fusion was induced anaerobically whereas tanA-lac showed more or less constitutive beta-galactosidase expression.

Analysis of an Escherichia coli dnaB temperature-sensitive insertion mutation and its cold-sensitive extragenic suppressor

An Escherichia coli mutant, ts121, was isolated following random insertional mutagenesis using phage lambda Mu transposition. The mutant phenotype includes inability to form colonies at temperatures above 38 degrees C and inability to propagate phage lambda at all temperatures. A lambda i434 cI- (ts121)+ transducing phage was isolated on the basis of its ability to form plaques on ts121 mutant bacteria. Using this transducing phage, it was shown through complementation and protein analyses, that the ts121 mutation is located in the dnaB gene. The exact insertion event was identified by polymerase chain reaction amplification of the DNA sequences containing the insertion junction. The mutational insertion event in ts121 was mapped precisely between base pairs 1514 and 1515 of the dnaB gene. This result predicts that the mutant dnaB protein has lost its six terminal amino acids. The reading frame shifts into Mu-specific DNA sequences resulting in an additional 20 amino acid residues. The E. coli wild type dnaB protein participates in host replication and interacts with lambda P protein to initiate phage lambda DNA replication. Our results demonstrate that the extreme carboxyl end of the dnaB protein is required for productive interaction with the lambda P replication protein at all temperatures, and is important for dnaB function at temperatures above 38 degrees C. Cold-sensitive extragenic suppressors of the ts121 mutation were isolated on the basis of their ability to restore colony formation at 42 degrees C. One of these extragenic suppressors was mapped at 54 min on the E. coli genetic map and localized to the suhB gene, whose product may affect the expression of a number of genes at the translational level.

Isolation and characterization of the Escherichia coli htrB gene, whose product is essential for bacterial viability above 33 degrees C in rich media

We have identified and studied the htrB gene of Escherichia coli. Insertional inactivation of the htrB gene leads to bacterial death at temperatures above 33 degrees C. The mutant bacterial phenotype at nonpermissive temperatures includes an arrest of cell division followed by the formation of bulges or filaments. The htrB+ gene has been cloned by complementation and shown to reside at 23.4 min on the E. coli genetic map, the relative order of the neighboring loci being mboA-htrB-pyrC. The htrB gene is transcribed in a counterclockwise fashion, relative to the E. coli genetic map, and its product has been identified as a membrane-associated protein of 35,000 Da. Growth experiments in minimal media indicate that the HtrB function becomes dispensable at low growth rates.