Constitutive septal murein synthesis in Escherichia coli with impaired activity of the morphogenetic proteins RodA and penicillin-binding protein 2.

The pattern of peptidoglycan (murein) segregation in cells of Escherichia coli with impaired activity of the morphogenetic proteins penicillin-binding protein 2 and RodA has been investigated by the D-cysteine-biotin immunolabeling technique (M. A. de Pedro, J. C. Quintela, J.-V. H?ltje, and H. Schwarz, J. Bacteriol. 179:2823-2834, 1997). Inactivation of these proteins either by amdinocillin treatment or by mutations in the corresponding genes, pbpA and rodA, respectively, leads to the generation of round, osmotically stable cells. In normal rod-shaped cells, new murein precursors are incorporated all over the lateral wall in a diffuse manner, being mixed up homogeneously with preexisting material, except during septation, when strictly localized murein synthesis occurs. In contrast, in rounded cells, incorporation of new precursors is apparently a zonal process, localized at positions at which division had previously taken place. Consequently, there is no mixing of new and old murein. Old murein is preserved for long periods of time in large, well-defined areas. We propose that the observed patterns are the result of a failure to switch off septal murein synthesis at the end of septation events. Furthermore, the segregation results confirm that round cells of rodA mutants do divide in alternate, perpendicular planes as previously proposed (K. J. Begg and W. D. Donachie, J. Bacteriol. 180:2564-2567, 1998).     

Mutation in Escherichia coli K-12 Mediating Spherelike Envelopes and Changed Tolerance to Ultraviolet Irradiation and Some Antibiotics

In a mutation experiment with a rough, ampicillin-resistant strain of Escherichia coli K-12, two smooth ampicillin-sensitive mutants were isolated. One of the mutants (with the envA gene) was recently described. The second mutant (strain D23) with the envB gene which has been mapped to a position close to streptomycin resistance (strA) at 64 min is described. The envB gene gives rise to spherelike cells. Electron microscopy revealed an abnormal septum formation and a circular distribution of the nuclear material. Strain D23 (with envB) showed a changed resistance to several antibiotics as well as an increased tolerance to ultraviolet irradiation. The envB gene decreased the ampicillin resistance mediated by the ampA gene (at 82 min), but no effect was found on episomally mediated penicillin resistance.     

Defective and plaque-forming lambda transducing bacteriophage carrying penicillin-binding protein-cell shape genes: genetic and physical mapping and identification of gene products from the lip-dacA-rodA-pbpA-leuS region of the Escherichia coli chromosome

A series of defective lambda transducing phage carrying genes from the lip-leuS region of the Escherichia coli chromosome (min 14 on the current linkage map) has been isolated. The phage defined the gene order as lac---lip-dacA-rodA-pbpA-leuS---gal. These included the structural genes for penicillin-binding protein 2 (pbpA) and penicillin-binding protein 5 (dacA) as well as a previously unidentified cell shape gene that we have called rodA. rodA mutants were spherical and very similar to pbpA mutants but were distinguishable from them in that they had no defects in the activity of penicillin-binding protein 2. The separation into two groups of spherical mutants with mutations that mapped close to lip was confirmed by complementation analysis. The genes dacA, rodA, and pbpA lie within a 12-kilobase region, and represent a cluster of genes involved in cell shape determination and peptidoglycan synthesis. A restriction map of the lip-leuS region was established, and restriction fragments were cloned from defective transducing phage into appropriate lambda vectors to generate plaque-forming phage that carried genes from this region. Analysis of the proteins synthesized from lambda transducing phage in ultraviolet light-irradiated cells of E. coli resulted in the identification of the leuS, pbpA, dacA, and lip gene products, but the product of the rodA gene was not identified. The nine proteins that were synthesized from the lip-leuS region accounted for 57% of its coding capacity. Phage derivatives were constructed that allowed about 50-fold amplification of the levels of penicillin-binding proteins 2 and 5 in the cytoplasmic membrane.     

Extracellular polysaccharide is required for wild-type virulence of Pseudomonas solanacearum.

Several Pseudomonas solanacearum strains which produced no detectable extracellular polysaccharide (EPS) in planta had been reported to remain highly virulent when tested at high inoculum concentrations (P. Xu, M. Iwata, S. Leong, and L. Sequeira, J. Bacteriol. 172:3946-3951, 1990; P. Xu, S. Leong, and L. Sequeira, J. Bacteriol. 170:617-622, 1988). Two of these mutants, KD700 and KD710, have now been molecularly and genetically mapped to the EPSI gene cluster described by Denny and Baek (Mol. Plant-Microbe Interact. 4:198-206, 1991). When a range of inoculum concentrations was used, these two mutants and all other EPS-defective mutants tested were found to be reduced in virulence to eggplants and tobacco relative to the wild-type strain. Thus, EPS consistently is required for the wild-type level of virulence in P. solanacearum.     

Gene in the major cotransduction gap of the Escherichia coli K-12 linkage map required for the expression of chromosomal resistance to tetracycline and other antibiotics

In Escherichia coli K-12, amplifiable resistance to tetracycline, chloramphenicol, and other unrelated antibiotics was mediated by at least four spatially separated loci. Tetracycline-sensitive mutants were isolated by Tn5 insertional inactivation of an amplified multiply resistant strain. One of these, studied in detail, showed coordinate loss of expression of all other resistance phenotypes. The Tn5 element in this mutant mapped to 34 min on the E. coli K-12 linkage map. We have designated the locus marA (multiple antibiotic resistance). Tetracycline-sensitive mutants containing marA::Tn5 regained all resistance phenotypes at frequencies of 10(-8) to 10(-7) upon precise excision of Tn5. Moreover, a newly described tetracycline efflux system (A. M. George and S. B. Levy, J. Bacteriol. 155:531-540, 1983) was inactivated in tetracycline-sensitive mutants, but recovered in tetracycline-resistant revertants. In merodiploids, F-prime marA+ expressed partial or complete dominance over corresponding mutant chromosomal alleles. Dominance tests also established that a previously amplified host and a mutant marA allele were preconditions for the expression of phenotypic resistances.     

Ferric citrate transport in Escherichia coli requires outer membrane receptor protein fecA.

Mutants of Escherichia coli K-12 AB2847 and of E. coli K-12 AN92 were isolated which were unable to grow on ferric citrate as the sole iron source. Of 22 mutants, 6 lacked an outer membrane protein, designated FecA protein, which was expressed by growing cells in the presence of 1 mM citrate. Outer membranes showed an enhanced binding of radioactive iron, supplied as a citrate complex, depending on the amount of FecA protein. The FecA protein was the most resistant of the proteins involved in ferric irion iron translocation across the outer membrane (FhuA = TonA, FepA, Cir, or 83K proteins) to the action of pronase P. It is also shown that previously isolated fec mutants (G. C. Woodrow et al., J. Bacteriol. 133:1524-1526, 1978) which are cotransducible with argF all lack the FecA protein. They were termed fecA to distinguish them from the other ferric citrate transport mutants, now designated fecB, which mapped in the same gene region at 7 min but were not cotransducible with ArgF. E. coli W83-24 and Salmonella typhimurium, which are devoid of a citrate-dependent iron transport system, lacked the FecA protein. It is proposed that the FecA protein participates in the transport of ferric citrate.     

Inositol monophosphatase activity from the Escherichia coli suhB gene product.

The suhB gene is located at 55 min on the Escherichia coli chromosome and encodes a protein of 268 amino acids. Mutant alleles of suhB have been isolated as extragenic suppressors for the protein secretion mutation (secY24), the heat shock response mutation (rpoH15), and the DNA synthesis mutation (dnaB121) (K. Shiba, K. Ito, and T. Yura, J. Bacteriol. 160:696-701, 1984; R. Yano, H. Nagai, K. Shiba, and T. Yura, J. Bacteriol. 172:2124-2130, 1990; S. Chang, D. Ng, L. Baird, and C. Georgopoulos, J. Biol. Chem. 266:3654-3660, 1991). These mutant alleles of suhB cause cold-sensitive cell growth, indicating that the suhB gene is essential at low temperatures. Little work has been done, however, to elucidate the role of the product of suhB in a normal cell and the suppression mechanisms of the suhB mutations in the aforementioned mutants. The sequence similarity shared between the suhB gene product and mammalian inositol monophosphatase has prompted us to test the inositol monophosphatase activity of the suhB gene product. We report here that the purified SuhB protein showed inositol monophosphatase activity. The kinetic parameters of SuhB inositol monophosphatase (Km = 0.071 mM; Vmax = 12.3 mumol/min per mg) are similar to those of mammalian inositol monophosphatase. The ssyA3 and suhB2 mutations, which were isolated as extragenic suppressors for secY24 and rpoH15, respectively, had a DNA insertion at the 5' proximal region of the suhB gene, and the amount of SuhB protein within mutant cells decreased. The possible role of suhB in E. coli is discussed.     

Chromosomal location of the structural gene for glycerol kinase in Escherichia coli

Cozzarelli, N. R. (Harvard Medical School, Boston, Mass.), and E. C. C. Lin. Chromosomal location of the structural gene for glycerol kinase in Escherichia coli. J. Bacteriol. 91:1763-1766. 1966.-A glycerol kinase mutant site has been mapped by transduction and sexual conjugation. Three-factor crosses with the two procedures yielded the following gene order: arginine-1-methionine-1-glycerol kinase-isoleucine, valine-16. An additional 13 independent glycerol kinase mutant sites mapped in the same region. Since some of the mutants were able to produce a protein serologically indistinguishable from the wild-type enzyme, it is concluded that the region mapped represents the structural gene for the kinase.     

A New Fungal Metabolite and Root Growth-stimulating Activity of Its Methyl Ester

Escherichia coli rodA mutant AOS151 grows as round cells at 30 and 42°C (H. Matsuzawa, K. Hayakawa, T. Sato, and K. Imahori, J. Bacteriol., 115, 436–442 (1973)). The mutant was found to be resistant to mecillinam at both temperatures. lip⁺ transductants were prepared by Pl phage transduction via strain AOS151, the cotransduction frequency of round morphology (Rod^?) at 42°C with the lip gene being about 90%. At 42°C all 54 Rod^? transductants tested were resistant to mecillinam. At 30°C all but two of these Rod^? (at 42°C)-type transductants were rod-shaped, and all were sensitive to mecillinam; the two strains grew as ovoid cells. The original rodA mutant AOS151 probably involves an additional mutation(s), that expresses the round cell shape at lower temperature, whereas the rodA51 mutation alone seems to result in temperature-sensitive expression of round cell morphology and mecillinam resistance. rodA mutant cells cultured at either 30 or 42°C had wild-type penicillin-binding protein 2, judging from penicillin-binding activity, electrophoretic mobility, and thermosensitivity.     

Morphology of an Escherichia coli mutant with a temperature-dependent round cell shape.

Mutants of Escherichia coli capable of growing in the presence of 10 microgram of mecillinam per ml were selected after intensive mutagenesis. Of these mutants, 1.4% formed normal, rod-shaped cells at 30 degrees C but grew as spherical cells at 42 degrees C. The phenotype of one of these rod(Ts) mutants was 88% cotransducible with lip (14.3 min), and all lip+ rod(Ts) transductants of a lip recipient had the following characteristics: (i) growth was relatively sensitive to mecillinam at 30 degrees C but relatively resistant to mecillinam at 42 degrees C; (ii) penicillin-binding protein 2 was present in membranes of cells grown at 30 degrees C in reduced amounts and was undetectable in the membranes of cells grown at 42 degrees C. The mecillinam resistance, penicillin-binding protein 2 defect, and rod phenotypes all cotransduced with lip with high frequency. Thus the mutation [rodA(Ts)] is most likely in the gene for penicillin-binding protein 2 and causes the organism to grow as a sphere at 42 degrees C, although it grows with normal rodlike morphology at 30 degrees C. At 42 degrees C, cells of this strain were round with many wrinkles on their surfaces, as revealed by scanning electron microscopy. In these round cells, chromosomes were dispersed or distributed peripherally, in contrast to normal rod-shaped cells which had centrally located, more condensed chromosomes. The round cells divided asymmetrically on solid agar, and it seemed that the plane of each successive division was perpendicular to the preceding one. On temperature shift-down in liquid medium many cells with abnormal morphology appeared before normal rod-shaped cells developed. Few abnormal cells were seen when cells were placed on solid medium during temperature shift-down. These pleiotropic effects are presumably caused by one or more mutations in the rodA gene.     

Genetics of host-controlled restriction and modification of deoxyribonucleic acid in Escherichia coli

Lederberg, Seymour (Brown University, Providence, R.I.). Genetics of host-controlled restriction and modification of deoxyribonucleic acid in Escherichia coli. J. Bacteriol. 91:1029-1036. 1966.-The locus for the host specific restriction and modification of deoxyribonucleic acid in Escherichia coli has been mapped by matings between mutants for these characters in strains K-12, C600, and B. Linkage analysis and kinetics of marker transfer indicate that a single or closely linked multiple chromosomal site located about 4 min counterclockwise to leucine is responsible for these activities. Secondary factors which affect the quantitative level of restriction also were detected. Wild-type recombinants were isolated in crosses between rm(-) (restriction or modification, or both) mutants. The expression in zygotes of the restrictionless character of a rm(-) donor is masked by a separate, physiological impairment of restriction, which results from mating and is independent of the modification state of the donor. The relevance of the restriction character to mating incompatibilities in these and other bacterial strains is considered.     

Bacteriophage T4 gol site: sequence analysis and effects of the site on plasmid transformation.

The Escherichia coli lit gene product is required for the multiplication of bacteriophage T4 at temperatures below 34 degrees C. After infection of a lit mutant host, early gene product synthesis is normal, as is T4 DNA replication; however, the late gene products never appear, and early gene product synthesis eventually ceases. Consequently, at late times, there is no protein synthesis of any kind (W. Cooley, K. Sirotkin, R. Green, and L. Snyder, J. Bacteriol. 140:83-91, 1979; W. Champness and L. Snyder, J. Mol. Biol. 155:395-407, 1982), and no phage are produced. We have isolated T4 mutants which can multiply in lit mutant hosts. The responsible T4 mutations (called gol mutations) completely overcome the block to T4 gene expression (Cooley et al., J. Bacteriol. 140:83-91). We have proposed that gol mutations alter a cis-acting regulatory site on T4 DNA rather than a diffusible gene product and that the wild-type form of the gol site (gol+) somehow interferes with gene expression late in infection (Champness and Snyder, J. Mol. Biol. 155:395-409). In this communication, we report the sequence of the gol region of the T4 genome from five different gol mutants. The gol mutations are all single-base-pair transitions within 40 base pairs of DNA. Therefore, the gol site is at least 40 base pairs long. The sequence data confirm that the gol phenotype is not due to an altered protein. We also report that the gol+ site in plasmids prevents transformation of Lit- but not Lit+ E. coli. Thus, the gol site is at least partially active in the absence of the T4 genome.     

Characterization and Genetic Analysis of a Mutant of Escherichia coli K-12 with Rounded Morphology

A morphological mutant of Escherichia coli K-12 that grows as round cells at 30, 37, or 42 C in a variety of complex and synthetic media has been isolated and characterized. The gene concerned, designated rodA, has been shown to be on the chromosome between the purE and pyrC loci and to be located at about minute 15. The rodA gene has been found to be co-transducible with the lip gene at a frequency of 95%. The rodA mutant showed an increased resistance to ultraviolet irradiation and a changed sensitivity to drugs. The resistance to ultraviolet irradiation and mitomycin C appears to be co-transducible with the rodA gene.     

Mutants of Myxococcus xanthus dsp defective in fibril binding.

The dsp mutant of Myxococcus xanthus lacks extracellular fibrils and as a result is unable to undergo cohesion, group motility, or development (J. W. Arnold and L. J. Shimkets, J. Bacteriol. 170:5765-5770, 1983; J. W. Arnold and L. J. Shimkets, J. Bacteriol. 170:5771-5777, 1983; R. M. Behmlander and M. Dworkin, J. Bacteriol. 173:7810-7821, 1991; L. J. Shimkets, J. Bacteriol. 166:837-841, 1986; L. J. Shimkets, J. Bacteriol. 166:842-848, 1986). However, cohesion and development can be phenotypically restored by the addition of isolated fibrils (R. M. Behmlander, Ph.D. thesis, University of Minnesota, Minneapolis, 1994; B.-Y. Chang and M. Dworkin, J. Bacteriol. 176:7190-7196, 1994). As part of our attempts to examine the interaction of fibrils and cells of M. xanthus, we have isolated a series of secondary mutants of M. xanthus dsp in which cohesion, unlike that of the parent strain, could not be rescued by the addition of isolated fibrils. Cells of M. xanthus dsp were mutagenized either by ethyl methanesulfonate or by Tn5 insertions. Mutagenized cultures were enriched by selection of those cells that could not be rescued, i.e., that failed to cohere in the presence of isolated fibrils. Seven mutants of M. xanthus dsp, designated fbd mutants, were isolated from 6,983 colonies; these represent putative fibril receptor-minus mutants. The fbd mutants, like the parent dsp mutant, still lacked fibrils, but displayed a number of unexpected properties. They regained group motility and the ability to aggregate but not the ability to form mature fruiting bodies. In addition, they partially regained the ability to form myxospores. The fbd mutant was backcrossed into the dsp mutant by Mx4 transduction. Three independently isolated transconjugants showed essentially the same properties as the fbd mutants--loss of fibril rescue of cohesion, partial restoration of myxospore morphogenesis, and restoration of group motility. These results suggest that the physical presence of fibrils is not necessary for group motility, myxospore formation, or the early aggregative stage of development. We propose, however, that the perception of fibril binding is required for normal social behavior and development. The dsp fbd mutants (from here on referred to as fbd mutants) open the possibility of isolating and characterizing a putative fibril receptor gene.     

Cloning and sequencing of Escherichia coli murZ and purification of its product, a UDP-N-acetylglucosamine enolpyruvyl transferase.

The Escherichia coli gene murZ, encoding the enzyme UDP-N-acetylglucosamine enolpyruvyl transferase, has been cloned and sequenced. Identified by screening an E. coli genomic library for clones that conferred phosphomycin resistance, murZ encoded a 419-amino-acid polypeptide and was mapped to 69.3 min on the E. coli chromosome. MurZ protein was purified to near homogeneity and found to have the expected UDP-N-acetylglucosamine enolpyruvyl transferase activity. Sequence analysis of the predicted product revealed 44% identity to OrfR from Bacillus subtilis (K. Trach, J.W. Chapman, P. Piggot, D. LeCoq, and J.A. Hoch, J. Bacteriol. 170:4194-4208, 1988), suggesting that orfR may also encode a UDP-N-acetylglucosamine enolpyruvyl transferase enzyme. MurZ is also homologous to the aromatic amino acid biosynthetic enzyme enolpyruvyl shikimate phosphate synthase, the other enzyme known to catalyze an enolpyruvyl transfer.     

Escherichia coli mutants with defects in the biosynthesis of 5-methylaminomethyl-2-thio-uridine or 1-methylguanosine in their tRNA.

Two tRNA methyltransferase mutants, isolated as described in the accompanying paper (G.R. Bj?rk and K. Kjellin-Str?by, J. Bacteriol. 133:499-207, 1978), are biochemicaaly and genetically characterized. tRNA from mutant IB13 lacks 5-methylaminomethyl-2-thio-uridine in vivo due to a permanently nonfunctional methyltransferase. Thus tRNA from this mutant is a specific substrate for the corresponding tRNA methyltransferase in vitro. In spite of this defect in tRNA, such a mutant is viable. Mutant IB11 is conditionally defective in the biosynthesis of 1-methylguanosine in tRNA due to a temperature-sensitive tRNA (1-methyl-guanosine) methyltransferase. In mutant cells grown at a high temperature, the level of 1-methylguanosine in bulk tRNA is 20% of that of the wild type, demonstrating that in this mutant an 80% deficiency of 1-methylguanosine in tRNA is not lethal. Genetically these two distinct lesions, trmC2, causing 5=methylaminomethyl-2-thio-uridine deficiency, and trmD1, giving a temperature-sensitive tRNA (1-methylguanosine)methyltransferase, are both located between 50 and 61 min on the Escherichia coli chromosome.     

Precise mapping of the rnpB gene encoding the RNA component of RNase P in Escherichia coli K-12.

In Kohara's library derived from Escherichia coli K-12 W3110 (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987), multiple copies of chromosomal sequence are found at 68 and at 64 to 65 min (M. Umeda and E. Ohtsubo, J. Mol. Biol. 213:229-237, 1990). We have determined that the rnpB gene (previously mapped at 70 min [B. J. Bachmann, Microbiol. Rev. 54:130-197, 1990]) is located within these segments of repeated sequences as five separate copies, together with tdcA, B, C, and R (mapped at 68 min [Bachmann, 1990]) and six unidentified open reading frames. Since close linkage of rnpB and tdc is found in various strains of E. coli K-12, the rnpB gene should be mapped at 68 min rather than 70 min.     

Suppression of mutations conferring penicillin tolerance by interference with the stringent control mechanism of Escherichia coli.

Mutations in Escherichia coli previously reported (R. E. Harkness and E. E. Ishiguro, J. Bacteriol. 155:15-21, 1983; L. C. Shimmin, D. Vanderwel, R. E. Harkness, B. R. Currie, A. Galloway, and E. E. Ishiguro, J. Gen. Microbiol. 130:1315-1323, 1984) as conferring a temperature-dependent tolerance to lysis induced by inhibitors of peptidoglycan synthesis were suppressed by treatment with inhibitors of the stringent response or by introduction of a relA mutation. The relA+ derivatives of the mutants exhibited a stringent response at the nonpermissive temperature. The consequent inhibition of the autolytic enzyme system (W. Kusser and E. E. Ishiguro, J. Bacteriol. 164:861-865, 1985) was apparently responsible for the lysis-tolerant phenotypes of these mutants.     

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.     

Genetic and physiological analysis of an envB spherelike mutant of Escherichia coli K-12 and characterization of its transductants.

The envB1 mutation mediating a distorted cell morphology of Escherichia coliK-12 was cotransducible with strA, aroE, aspB, and argG. The mapping data is consistent with a gene location for envB around 62.5 min. In partial diploids envB1 was recessive to its wild-type allele. The original envB mutant contained a second mutation in a locus denoted sloB close to strA. The following gene order is suggested: sloB-strA-aroE-envB-aspB-argG. The sloB1 mutation caused a marked reduction in the growth rate of both envB and envB+ strains. Moreover, this mutation in the presence of envB1 appears to increase the ratio between deoxyribonucleic acid and protein in cells growing in rich medium. The phenotypic properties of envB1, sloB+ and envB+ transductants were characterized. Cells with envB1, sloB+ genotype were hypersensitive to several penicillins including the beta-lactam compound, amidino penicillin. Penicillin hypersensitivity could not be explained by increased outer membrane penetrability. The original envB mutant (envB1,SLOB1), as well as envB1, sloB1 or envB+, SLOB1 transductants were resistant to amidino penicillin. Resistance was explained by the slow growth rate mediated by the sloB1 mutation. The similarity between envB cells and wild-type cells treated with sublethal concentrations of amidino penicillin was emphasized.