James R. Walker

Cloning of the Escherichia coli dnaZX region and identification of its products

SummaryThe Escherichia coli DNA replication genes dnaZ and dnaX have previously been localized very near each other at 10.4 to 10.5 min on the chromosome map. These genes were cloned from a dnaZ+X+ plasmid of the Clarke and Carbon collection by identifying complementing fragments and both were located on a 2.1 kilobase pair (kb) fragment. The organization of the Z and X genes was investigated by Tn5 mutagenesis of a Z+Y+ plasmid. Insertions which abolished Z or X complementing activity were mapped by restriction enzyme analysis within the 2.1 kb fragment. With the exception of one atypical insertion, all the insertions inactivated both Z and X complementation.The protein products of the dnaZ-dnaX region were labelled in minicells containing dnaZ+X+ and dnaZX:: Tn5 plasmids. The 2.1 kb ZX region (which has a maximum coding capacity of 77,000 daltons of protein in a single reading frame) directed the synthesis of two proteins, one of 75,000 daltons, designated dnaX, and another of 56,500 daltons, designated dnaZ. Tn5 insertion into the ZX region interrupted the synthesis of these proteins; the detection of truncated fragments of dnaX determined the direction of transcription. In vitro, using a coupled transcription-translation system dependent on plasmid DNA, synthesis of the 75,000 dalton dnaX protein was demonstrated, but there was no detectable synthesis of the smaller dnaZ protein. Probably, therefore, the 75,000 dalton dnaX protein is cleaved in vivo to generate the dnaZ protein. It is possible that the 75,000 dalton product is the τ subunit of DNA polymerase III because they migrated similarly in electrophoresis.

A Replication-Inhibited Unsegregated Nucleoid at Mid-Cell Blocks Z-Ring Formation and Cell Division Independently of SOS and the SlmA Nucleoid Occlusion Protein in Escherichia coli

Chromosome replication and cell division of Escherichia coli are coordinated with growth such that wild-type cells divide once and only once after each replication cycle. To investigate the nature of this coordination, the effects of inhibiting replication on Z-ring formation and cell division were tested in both synchronized and exponentially growing cells with only one replicating chromosome. When replication elongation was blocked by hydroxyurea or nalidixic acid, arrested cells contained one partially replicated, compact nucleoid located mid-cell. Cell division was strongly inhibited at or before the level of Z-ring formation. DNA cross-linking by mitomycin C delayed segregation, and the accumulation of about two chromosome equivalents at mid-cell also blocked Z-ring formation and cell division. Z-ring inhibition occurred independently of SOS, SlmA-mediated nucleoid occlusion, and MinCDE proteins and did not result from a decreased FtsZ protein concentration. We propose that the presence of a compact, incompletely replicated nucleoid or unsegregated chromosome masses at the normal mid-cell division site inhibits Z-ring formation and that the SOS system, SlmA, and MinC are not required for this inhibition.

Clostridium taeniosporum spore ribbon‐like appendage structure, composition and genes

Clostridium taeniosporum spores have about 12 large, flat, ribbon‐like appendages attached through a common trunk at one spore pole to a previously unknown surface layer outside the coat that is proposed to be called the ‘encasement’. Appendages are about 4.5 μm long, 0.5 μm wide and 30 nm thick and taper at the attachment end into a semicircle that is twisted relative to the flat ribbon. Individual fibrils, about 45 nm in length with spherical heads and long thin tails, form a hair‐like nap, visible along the appendage edge. Four appendage proteins have been detected: a paralogous pair of 29 kDa (designated P29a and P29b), a glycoprotein of about 37 kDa (designated GP85) and an orthologue of the Bacillus spore morphogenetic protein SpoVM. The P29 proteins consist of duplicated regions and each region includes a domain of unknown function 11. The GP85 glycoprotein contains a collagen‐like region. The genes for P29a and b, GP85 and possibly related proteins are closely linked on two small chromosome fragments. Putative σK‐dependent promoters upstream of the P29a and b genes indicate that they likely are expressed late in the mother cell, consistent with their deposition into the layer external to the coat.

An e. coli DNA fragment 118 base pairs in length provides dnaY+ complementing activity

The dnaY gene of E. coli, thought to be involved in the polymerization phase of DNA replication, was localized on a fragment 118 base pairs in length. This fragment, cloned in two different vectors and tested in a dnaY (Ts) recA host, has dnaY + complementing activity. The nucleotide sequence of the 118 base pairs and flanking bases was determined. The dnaY complementing activity was inactivated by transposon insertion and by localized chemical mutagenesis. Three independent insertions of Tn5 into the 118 base pair region eliminated dnaY activity. Eight single-base-change mutations that resulted in loss of dnaY activity also were located within the 118 base pair region. Analysis of the nucleotide sequence reveals a potential promoter but reveals no open reading frames likely to be translated into polypeptides. However, an RNA transcript of the dnaY region is synthesized in vivo. Perhaps the active product of dnaY is a small RNA or perhaps the dnaY region functions as a site.

Growth of phages λ, ØX174, and M13 requires the dnaZ (previously dnaH) gene product of Escherichia coli

A functional dnaZ (previously designated dnaH) product, known to be involved in DNA polymerization, is required for phage λ, OX174, and M13, but not T4 or T7, growth.

Suppression of Temperature-Sensitive Chromosome Replication of an Escherichia coli dnaX(Ts) Mutant by Reduction of Initiation Efficiency

Temperature sensitivity of DNA polymerization and growth of a dnaX(Ts) mutant is suppressible at 39 to 40 degrees C by mutations in the initiator gene, dnaA. These suppressor mutations concomitantly cause initiation inhibition at 20 degrees C and have been designated Cs,Sx to indicate both phenotypic characteristics of cold-sensitive initiation and suppression of dnaX(Ts). One dnaA(Cs,Sx) mutant, A213D, has reduced affinity for ATP, and two mutants, R432L and T435K, have eliminated detectable DnaA box binding in vitro. Two models have explained dnaA(Cs,Sx) suppression of dnaX, which codes for both the tau and gamma subunits of DNA polymerase III. The initiation deficiency model assumes that reducing initiation efficiency allows survival of the dnaX(Ts) mutant at the somewhat intermediate temperature of 39 to 40 degrees C by reducing chromosome content per cell, thus allowing partially active DNA polymerase III to complete replication of enough chromosomes for the organism to survive. The stabilization model is based on the idea that DnaA interacts, directly or indirectly, with polymerization factors during replication. We present five lines of evidence consistent with the initiation deficiency model. First, a dnaA(Cs,Sx) mutation reduced initiation frequency and chromosome content (measured by flow cytometry) and origin/terminus ratios (measured by real-time PCR) in both wild-type and dnaX(Ts) strains growing at 39 and 34 degrees C. These effects were shown to result specifically from the Cs,Sx mutations, because the dnaX(Ts) mutant is not defective in initiation. Second, reduction of the number of origins and chromosome content per cell was common to all three known suppressor mutations. Third, growing the dnaA(Cs,Sx) dnaX(Ts) strain on glycerol-containing medium reduced its chromosome content to one per cell and eliminated suppression at 39 degrees C, as would be expected if the combination of poor carbon source, the Cs,Sx mutation, the Ts mutation, and the 39 degrees C incubation reduced replication to the point that growth (and, therefore, suppression) was not possible. However, suppression was possible on glycerol medium at 38 degrees C. Fourth, the dnaX(Ts) mutation can be suppressed also by introduction of oriC mutations, which reduced initiation efficiency and chromosome number per cell, and the degree of suppression was proportional to the level of initiation defect. Fifth, introducing a dnaA(Cos) allele, which causes overinitiation, into the dnaX(Ts) mutant exacerbated its temperature sensitivity.

The Escherichia coli argU10(Ts) Phenotype Is Caused by a Reduction in the Cellular Level of the argU tRNA for the Rare Codons AGA and AGG

The Escherichia coli argU10(Ts) mutation in the argU gene, encoding the minor tRNA(Arg) species for the rare codons AGA and AGG, causes pleiotropic defects, including growth inhibition at high temperatures, as well as the Pin phenotype at 30 degrees C. In the present study, we first showed that the codon selectivity and the arginine-accepting activity of the argU tRNA are both essential for complementing the temperature-sensitive growth, indicating that this defect is caused at the level of translation. An in vitro analysis of the effects of the argU10(Ts) mutation on tRNA functions revealed that the affinity with elongation factor Tu-GTP of the argU10(Ts) mutant tRNA is impaired at 30 and 43 degrees C, and this defect is more serious at the higher temperature. The arginine acceptance is also impaired significantly but to similar extents at the two temperatures. An in vivo analysis of aminoacylation levels showed that 30% of the argU10(Ts) tRNA molecules in the mutant cells are actually deacylated at 30 degrees C, while most of the argU tRNA molecules in the wild-type cells are aminoacylated. Furthermore, the cellular level of this mutant tRNA is one-tenth that of the wild-type argU tRNA. At 43 degrees C, the cellular level of the argU10(Ts) tRNA is further reduced to a trace amount, while neither the cellular abundance nor the aminoacylation level of the wild-type argU tRNA changes. We concluded that the phenotypic properties of the argU10(Ts) mutant result from these reduced intracellular levels of the tRNA, which are probably caused by the defective interactions with elongation factor Tu and arginyl-tRNA synthetase.

Interaction of host and viral regulatory mechanisms: Effect of the lon cell division defect on regulation of repression by bacteriophage lambda

Abstract The Escherichia coli lon cell division mutant, which is lysogenized with reduced frequency by λ and other lambdoid phages, produced only one-half the λ repressor activity found in lon+ cells after λ infection. A similar reduction of repressor level was observed in λcro-infected lon cells, compared to λcro-infected lon+ cells, indicating that the lon effect on repressor activity is not the result of an altered interaction with the λcro+ gene product. λ late gene functions were not efficiently shut off in λ+-infected lon cells, as demonstrated by elevated endolysin levels and phage yields. Although lon cells are lysogenized inefficiently, lon lysogens can be isolated and are stable. Mono-lysogens of lon+ and lon strains contained similar repressor levels. Thus, the lon defect results in reduced repressor activity during the establishment of repression, but not during maintenance of lysogeny. λ mutants (λtp) have been selected for the ability to form turbid plaques on lon hosts. After infection by λtp, repressor levels in both lon+ and lon cells were twofold higher than the levels in λ+-infected cells. Established lon+ and lon mono-lysogens of the λtp mutants contained repressor activity levels similar to those in λ+ mono-lysogens.

Defective initiation in an Escherichia coli dnaA(Cs,Sx) mutant.

Mutations in the Escherichia coli gene for initiation of DNA replication, dnaA, which suppress the polymerization defect and growth inhibition caused by temperature-sensitive (Ts) mutations in the polymerization gene, dnaX, are called Cs,Sx. We show here that these mutations, on their own, also cause defects in initiation, including inhibition of initiation at both low (20 degrees C) and high (44 degrees C) temperatures and asynchronous initiation at both the permissive (34 degrees C) and suppression (39 degrees C) temperatures. These findings suggests a relationship between partially defective initiation and suppression of the polymerization defect, both of which occur at 39 degrees C.

Cell division of the Escherichia coli lon-mutant

SummaryEscherichia coli lon-cells were subjected to treatments which produced a decrease in the DNA/mass ratio of the cell. Thymine starvation, a shift-up from minimal medium to rich medium, and exposure to BUdR each caused greater inhibition of cell division in lon-cells than in lon+cells. DNA metabolism was found to be the same in both lon+and lon-cells during these treatments. The results are consistent with the hypothesis that the lon-defect leads to inhibition of cell division under conditions which produce a decreased DNA/mass ratio.