Cell division control in Escherichia coli K-12: some properties of the ftsZ84 mutation and suppression of this mutation by the product of a newly identified gene.

The Fts proteins play an important role in the control of cell division in Escherichia coli. These proteins, which possibly form a functional complex, are encoded by genes that form an operon. In this study, we examined the properties of the temperature-sensitive mutation ftsZ84 harbored by low- or high-copy-number plasmids. Cells of strain AB1157, which had the ftsZ84 mutation, did not form colonies on salt-free L agar at 30 degrees C. When a low-copy-number plasmid containing the ftsZ84 mutation was present in these mutant cells, colony formation was restored on this medium at 30 degrees C, suggesting that FtsZ84 is probably less active than the wild-type protein and is therefore limiting in its capacity to trigger cell divisions. On the other hand, when the ftsZ84 mutation was harbored by the high-copy-number plasmid pBR325, colony formation was prevented on salt-free L agar plates whether the recipients were ftsZ84 mutant or parental cells, suggesting that, at high levels, FtsZ84 acts as a division inhibitor. The fact that colony formation was also prevented at 42 degrees C indicates that the FtsZ84 protein is not inactivated at the nonpermissive temperature. The possibility that FtsZ84 is a more efficient division inhibitor than the wild-type FtsZ is discussed. Evidence is also presented showing that a gene adjacent to mutT codes for a product that, under certain conditions, suppresses the ftsZ84 mutation.     

Cloning and characterization of Escherichia coli K-12 regulator gene tyrR.

The Escherichia coli K-12 regulator gene tyrR was cloned into the multicopy plasmid pBR322 from a lambda(Tn10)tyrR+ specialized transducing phage. Further subcloning localized the gene within a 2.1-kilobase region. Analysis of plasmid-coded proteins showed that the tyrR gene codes for a 63,000-dalton polypeptide.     

A class of gyrB mutants, substantially unaffected in DNA topology, suppresses the Escherichia coli K12 ftsZ84 mutation.

Previous work in our laboratory suggested that DNA topology could be implicated in the regulation of the division gene ftsZ. To settle this question, we have selected and characterized mutants in the gyrB gene able to phenotypically suppress the defects of the ftsZ84 mutation. No strict correlation was found between the degree of plasmid DNA relaxation and the level of suppression of the thermosensitivity of the ftsZ84 strain. Interestingly, the class of mutants that shows maximal suppression is substantially unaffected in DNA topology. In addition, the amount of ftsZ-specific mRNA in this class of mutants is comparable to that present in the ftsZ84 strain. These results hint that the ability of these gyrB mutants to correct the effects of the ftsZ84 mutation is largely unrelated to the function of the GyrB (as a part of DNA gyrase) in the control of DNA superhelicity and suggest hitherto unsuspected interaction between the ftsZ and gyrB gene products.     

New locus for exopolysaccharide overproduction in Escherichia coli K-12.

A new locus for exopolysaccharide overproduction in Escherichia coli K-12 was mapped by insertion mutagenesis. A 66% linkage to serA, which is located at 62 min on the E. coli K-12 linkage map, was shown by P1 transduction. The polysaccharide produced by the mutant was isolated and was shown to be similar to colanic acid.     

Isolation and characterization of a new temperature-sensitive polynucleotide phosphorylase mutation in Escherichia coli K-12

Polynucleotide phosphorylase (PNPase) has been studied in detail since its discovery in 1955 [1]. In an attempt to determine what role, if any, it has in mRNA decay in Escherichia coli, we have isolated and characterized a temperature-sensitive mutation, pnp-200, in the pnp gene. In vitro phosphorolysis, polymerization and exchange activities of the partially purified Pnp-200 enzyme are all reduced to 30-40% of wild-type activity at 50 degrees C compared to 32 degrees C. The pnp-200 mutation alone does not affect cell growth or mRNA stability. A triple mutant strain containing pnp-200 in combination with other temperature-sensitive mutations in genes known to affect mRNA metabolism (rnb-500 and ams-1) is conditionally lethal and shows increased mRNA stability after shift to the non-permissive temperature.     

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.     

Identification and characterization of a cyanate permease in Escherichia coli K-12.

Escherichia coli contains an inducible enzyme, cyanase, that catalyzes the decomposition of cyanate into ammonia and bicarbonate. The gene encoding cyanase, cynS, was cloned and found to be on a DNA fragment that contained the lac operon. Characterization of a plasmid encoding cyanase indicated that a 26-kilodalton (kDa) protein of unknown function was also induced by cyanate (Y-C. Sung, D. Parsell, P.M. Anderson, and J.A. Fuchs, J. Bacteriol. 169:2639-2642, 1987). The gene encoding the 26-kDa protein was located between cynS and its promoter, indicating the existence of a cyn operon. The 26-kDa protein was identified as a cyanate permease that transports exogenous cyanate by active transport. E. coli was shown to contain a cyanate transport system that is energy dependent and saturable by cyanate.     

ftsZ is an essential cell division gene in Escherichia coli.

The ftsZ gene is thought to be an essential cell division gene in Escherichia coli. We constructed a null allele of ftsZ in a strain carrying additional copies of ftsZ on a plasmid with a temperature-sensitive replication defect. This strain was temperature sensitive for cell division and viability, confirming that ftsZ is an essential cell division gene. Further analysis revealed that after a shift to the nonpermissive temperature, cell division ceased when the level of FtsZ started to decrease, indicating that septation is very sensitive to the level of FtsZ. Subsequent studies showed that nucleoid segregation was normal while FtsZ was decreasing and that ftsZ expression was not autoregulated. The null allele could not be complemented by lambda 16-2, even though this bacteriophage can complement the thermosensitive ftsZ84 mutation and carries 6 kb of DNA upstream of the ftsZ gene.     

Genetic and biochemical characterization of the Escherichia coli K-12 fhuB mutation.

The fhuB region of Escherichia coli K-12 was subcloned from pLC4-44 into pP lac to obtain pCPN1. Deletions of this recombinant plasmid were made, and a 1.4-kilobase PstI fragment was further subcloned into the vector plasmid pKK177-2 to obtain pCPN12. The response of tonA and tonB strains and fhuB strains containing the plasmids to 15 hydroxamate siderophores were assayed. Results showed that tonA strains were deficient only in the utilization of ferrichrome-type siderophores, whereas fhuB strains were deficient in the utilization of all hydroxamate-type siderophores. The response of the plasmid-containing fhuB strains to the siderophores showed that the fhuB gene resides on a 1.4-kilobase PstI fragment of DNA. The proteins synthesized by these plasmids were examined in maxicells of strain CSR603. Plasmid pCPN1 expressed five proteins of molecular weights 78,000, 40,000, 30,000, 24,000, and 13,700. By the use of deletions of pCPN1, the approximate order of the genes for these proteins was determined. Plasmid pCPN12 expressed no proteins other than the beta-lactamase proteins in maxicell strain CSR603. However, in maxicell strain BN660, a lon mutant, it expressed a 20,000-molecular-weight protein. Inner membrane vesicles made from tonB and fhuB strains were able to transport [55Fe]ferrichrome and [55Fe]rhodotorulate at rates similar to those obtained in vesicles from tonB+ and fhuB+ strains.     

In vivo cell division gene product interactions in Escherichia coli K-12.

Overexpression of plasmid-coded PBP 3 was analyzed in strains harboring ftsA, ftsH, pbpB (ftsI), ftsQ, ftsZ, or recA441 (Tif) mutations. Higher cellular levels of PBP 3, the pbpB gene product, could not restore septum formation of ftsA, ftsQ, ftsZ, and recA (Tif) mutants at 42 degrees C. However, filamentation in strains harboring pbpB and ftsH mutations was fully suppressed by PBP 3 overexpression. Additional observations indicated that the Y16 (ftsH) strain, not transformed with the PBP 3-overproducing plasmid, had no detectable PBP 3 in envelopes after incubation at the restrictive temperature. These results suggest that suppression of filamentation of fts strains overexpressing wild-type cell division proteins after the shift to the restrictive temperature can be a useful strategy to demonstrate in vivo interactions of cell division gene products.     

Molecular cloning of the fdhF gene of Escherichia coli K-12

Abstract The fdhF gene of Escherichia coli , coding for at least one component of benzyl viologen-linked formate dehydrogenase (FDH-BV) activity, was isolated on a ColE1- fdhF hybrid plasmid from the Clarke and Carbon colony bank.
Endonuclease restriction maps of this plasmid and its pBR322-subcloned derivative, pLW06, were constructed. Various hybrid plasmids were further obtained by deletion of endonuclease-cleaved fragments from pLW06 DNA. Their complementation pattern was analyzed after introduction into different fdhF mutant strains. The fdhF gene was shown to be located on a 5.5 kb Bam HI- Pvu II-DNA fragment, which restored FDH-BV activity to the wild-type level.     

Isolation and characterization of a light-sensitive mutant of Escherichia coli K-12 with a mutation in a gene that is required for the biosynthesis of ubiquinone.

Cells with a novel mutation that is lethal when the cells are exposed to visible light were isolated from Escherichia coli K-12. The mutation was mapped at 63 min on the linkage map of the E. coli chromosome, and the gene, designated visB, was cloned and sequenced. From its map position and the evidence that the gene product VisB exhibits homology with flavin monooxygenase of Pseudomonas fluorescens, the visB gene was deduced to be identical to the ubiH gene, which is a gene required for the biosynthesis of ubiquinone and is thought to be similar to the gene for flavin monooxygenase. The photosensitive phenotype appears to be due to the accumulation of the substrate for the reaction catalyzed by the visB (ubiH) gene product because other mutations that block earlier steps in the biosynthesis of ubiquinone can reverse the photosensitivity. The accumulated intermediates may produce active species of oxygen in the mutant bacteria upon illumination by visible light, and these active oxygen species may cause the death of the cells by a mechanism similar to that associated with mutations in visA (hemH).     

Identification of a mutation affecting an alanine-alpha-ketoisovalerate transaminase activity in Escherichia coli K-12.

Summary A mutation affecting alanine--ketoisovalerate transaminase activity has been shown to be cotransducibe with the ilv gene cluster. The transaminase deficiency results in conditional isoleucine auxotrophy in the presence of alanine.     

Identification and characterization of a new Escherichia coli gene that is a dosage-dependent suppressor of a dnaK deletion mutation.

We report the isolation and characterization of a previously unidentified Escherichia coli gene that suppresses the temperature-sensitive growth and filamentation of a dnaK deletion mutant strain. Introduction of a multicopy plasmid carrying this wild-type gene into a dnaK deletion mutant strain rescued the temperature-sensitive growth of the dnaK deletion mutant strain at 40.5 degrees C and the filamentation, fully at 37 degrees C and partially at 40.5 degrees C. However, the inability of dnaK mutant cells to support bacteriophage lambda growth was not suppressed. This gene was also able to suppress the temperature-sensitive growth of a grpE280 mutant strain at 41 degrees C. Filamentation of the grpE280 mutant strain was suppressed at 37 degrees C but not at 41 degrees C. The dnaK suppressor gene, designated dksA, maps near the mrcB gene (3.7 min on the E. coli chromosome). DNA sequence analysis and in vivo experiments showed that dksA encodes a 17,500-Mr polypeptide. Gene disruption experiments indicated that dksA is not an essential gene.     

New cysE-pyrE-linked rfa mutation in Escherichia coli K-12 that results in a heptoseless lipopolysaccharide.

A new novobiocin-supersensitive mutant of Escherichia coli K-12 has been characterized biochemically and genetically. Lipopolysaccharide prepared from this mutant strain is truncated and contains 2-keto-3-deoxyoctulosonic acid as its only core sugar. This new core-defective mutation, designated rfa-2, results in increased sensitivity to several hydrophobic and some hydrophilic agents. Genetic analysis of the rfa mutant indicated that the rfa-2 locus is located at 81 min on the chromosome. The order of the genes in this region based on transduction analysis is xyl cysE rfa-2 rfaD70 pyrE. P1 transduction analyses indicate that the rfa-2 marker is nonallelic with the recently described cysE-pyrE-linked rfaD70 locus. Plasmids carrying the wild-type rfaD70+ allele failed to abolish the rfa-2 phenotypes. Further, the rfaD gene product, ADP-L-glycero-D-mannoheptose-6-epimerase, was detected in crude extracts of a rfa-2 mutant strain, CL609, and was absent in the rfaD70 mutant. The wild-type rfa-2 allele codes either for a specific heptose biosynthetic enzyme (different from the rfaD gene product) or an enzymatic activity required for the addition of heptose to the lipid A-2-keto-3-deoxyoctulosonic acid acceptor.     

Identification of two new hemagglutinins of Escherichia coli, N-acetyl-D-glucosamine-specific fimbriae and a blood group M-specific agglutinin, by cloning the corresponding genes in Escherichia coli K-12.

Genes encoding the Escherichia coli IH11165 hemagglutinins with specificity for terminal N-acetyl-D-glucosamine and blood group M antigen, respectively, were cloned by a cosmid cloning procedure. A 22-kilobase-pair subclone expressed both hemagglutination specificities in the nonhemagglutinating E. coli HB101 recipient strain. Derivatives obtained by insertion and deletion mutagenesis expressed either one of the two hemagglutination specificities. Both agglutinins were purified; the agglutinin recognizing terminal N-acetyl-D-glucosamine was associated with a new type of fimbria (G fimbria) with an apparent subunit molecular mass of 19.5 kilodaltons, whereas the blood group M agglutinin (M agglutinin) was nonfimbrial and had an apparent subunit mass of 21 kilodaltons.