Complementation analysis of derepressed mutants of F-like plasmids of Escherichia coli

Complementation analysis of a number of conjugative transfer functions was performed in derepressed (drd) mutants of E. coli F-like plasmids. The major part of double plasmid complexes investigated has revealed the formation of complementation transfer inhibitor of Fin V-type, or less frequently--the formation of Fin U-type inhibitor. An additional complementation analysis of drd plasmids defective at Fin V region genes has demonstrated at least three genes (denoted A, B, C) in the structure of this region.     

Escherichia coli dnaK null mutants are inviable at high temperature.

DnaK, a major Escherichia coli heat shock protein, is homologous to major heat shock proteins (Hsp70s) of Drosophila melanogaster and humans. Null mutations of the dnaK gene, both insertions and a deletion, were constructed in vitro and substituted for dnaK+ in the E. coli genome by homologous recombination in a recB recC sbcB strain. Cells carrying these dnaK null mutations grew slowly at low temperatures (30 and 37 degrees C) and could not form colonies at a high temperature (42 degrees C); furthermore, they also formed long filaments at 42 degrees C. The shift of the mutants to a high temperature evidently resulted in a loss of cell viability rather than simply an inhibition of growth since cells that had been incubated at 42 degrees C for 2 h were no longer capable of forming colonies at 30 degrees C. The introduction of a plasmid carrying the dnaK+ gene into these mutants restored normal cell growth and cell division at 42 degrees C. These null mutants showed a high basal level of synthesis of heat shock proteins except for DnaK, which was completely absent. In addition, the synthesis of heat shock proteins after induction in these dnaK null mutants was prolonged compared with that in a dnaK+ strain. The well-characterized dnaK756 mutation causes similar phenotypes, suggesting that they are caused by a loss rather than an alteration of DnaK function. The filamentation observed when dnaK mutations were incubated at a high temperature was not suppressed by sulA or sulB mutations, which suppress SOS-induced filamentation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decreased conjugation efficiency by Fla-mutants of Escherichia coli K-12 bearing F-like plasmids.

Fla-, Pil-mutants of Escherichia coli K-12 were found to have decreased transfer efficiency of F-like resistance plasmids as compared to the parent strains. This was accompanied by decreased production of conjugation pili and decreased resistance level to some, but not all, of the antibiotics to which resistance was conferred. There was no reduction in pilus production or transfer efficiency in any of the mutants when the plasmid was F'gal. This host-mediated influence on conjugation pilus production is discussed with reference to a possible loss of cell envelope integrity which causes the simultaneous loss of all cellular appendage structures.     

deoP1 promoter and operator mutants in Escherichia coli: isolation and characterization

Plasmid DNA containing deoP1, one of the two major promoters of the deo operon, has been mutagenized using hydroxylamine, and promoter down-mutations and operator mutations were selected. The isolated mutants are all located within a 16 bp palindromic sequence containing the -10 region of deoP1. The results show that RNA polymerase and DeoR repressor compete for the same DNA target. The deoP1 promotor activity is dependent on a TG motif one base pair upstream of the -10 consensus sequence. The sequence of the deo operator site was further verified by use of a synthetic linker.     

Chromosome structure of Escherichia coli mutants temperature sensitive for deoxyribonucleic acid replication.

Folded chromosomes were isolated from Eschericia coli thermosensitive dnaA initiation mutants incubated at the nonpermissive temperature and were analyzed by neutral sucrose density gradient centrifugation. A chromosomal structure that sedimented at approximately 1,500S accumulated when the dnaA gene product was inactive. When the cells were returned to a permissive temperature, the folded chromosomes exhibited a decrease in sedimentation velocity to 1,300S but still retained their uniform structure. Very little deoxyribonucleic acid synthesis occurred during the period in which the chromosomes exhibited the reduction in sedimentation velocity. A dnaG elongation mutant showed no unique chromosome structure when the dnaG gene product was inactive.     

Factors influencing the copy number of F-like plasmids in Escherichia coli and Salmonella typhimurium.

The copy numbers of Flac, four F-like plasmids and pLT2 were estimated in two strains of Salmonella typhimurium and (for all except pLT2) one strain of Escherichia coli. For organisms grown in casamino acids minimal medium, the plasmids spanned a 7--8 fold range of copy number with ColB-K98 having the highest copy number in each strain and R124 the lowest. The copy number of ColB-K98 was substantially greater than 1 in each of the strains tested. There was no clear relation between the plasmid size and copy number, although the plasmids studied spanned only a narrow size range. The copy number of individual plasmids was slightly reduced or not affected at all by the presence of a second plasmid in the same strain. Derivatives harbouring each of the plasmids were grown in three different media to ascertain how plasmid copy number responds to changes in growth rate. For each plasmid, the copy number increased with decreasing growth rate. Extracts from each of the three strains harbouring ColB-K98 contained two distinct plasmid species. One appeared to be about twice as large as the other and both were absent from Col- segregants.     

Isolation and characterization of plasmids carrying a partially defective Escherichia coli replication origin

The replication origin (oriC) of the Escherichia coli chromosome has been cloned and the region essential for chromosomal replication has been delimited to 245 base pairs. In previous studies the ability of recombinants between oriC and ColE1-type vectors, to transform E. coli polA- strains was used to determine which nucleotides in oriC are essential for replication. In this paper we have used a different approach by isolating partial defective replication mutants of a minichromosome (pCM959) that contains oriC as the single replication origin. Our results demonstrate that many mutations are allowed within oriC that do not affect oriC function as measured by the ability to transform E. coli polA- strains. In the minimal oriC region we detected 8 mutations at positions that are conserved in the sequence of six bacterial origins. The implications of these results on previous work will be discussed. Our data also demonstrate that a mutation producing an oriC- phenotype may be suppressed by secondary mutations. An E. coli strain was found that facilitates the isolation of partially defective minichromosomes. The results with this strain indicate a specific function of the sequence surrounding the base pair at position 138.     

Nitrofurantoin-resistant mutants of Escherichia coli: isolation and mapping

Summary Mutants of Escherichia coli resistant to nitrofurantoin have been isolated. The mutations, designated nfnA and nfnB were introduced individually into a multiply auxotrophic E. coli F^– strain and mapped by conjugation and transduction. nfnA is located at 79.8 min and nfnB at 13.0 min on the E. coli chromosome.     

Replication of Fpoh+ plasmid in mafA mutants of Escherichia coli defective in plasmid maintenance.

A class of F' plasmids, designated Fpoh+, was previously shown to be able to replicate extra-chromosomally on Hfr strains by virtue of carrying the specific site or region poh+ (permissive on Hfr) of the E. coli chromosome (Hiraga, 1975, 1976a). These plasmids were now found to replicate on E. coli mafA mutants (mafA1 and mafA23) that cannot support vegetative replication of F and some other F-like plasmids. The derivatives of Fpoh+ that have lost the poh+ site, on the other hand, failed to replicate on mafA mutants. These mutants harboring Fpoh+ (but not Poh- derivatives thereof) exhibit abnormal cell division and form elongated cells, presumably due to competition between Fpoh+ and the host chromosome for some factor(s) essential for the initiation of DNA replication of the both replicons. It is tentatively concluded that the poh+ site is required for F' plasmids to replicate on mafA mutants as well as on Hfr strains. In view of the fact that the mechanism of inhibition of autonomous F DNA replication in mafA mutants and in Hfr strains are clearly different, the present data seem to provide strong support to the notion that the poh+ region contains the replication origin of the E. coli chromosome.     

OmpF assembly mutants of Escherichia coli K-12: isolation, characterization, and suppressor analysis.

This paper describes a novel genetic method used to isolate mutations that alter proper assembly of OmpF in the outer membrane. The thermolabile nature of assembly intermediates allowed selection of temperature-sensitive mutations within the ompF gene. A variant allele of ompF (ompF-Dex) was used because it provided a convenient selectable phenotype (Dex+). Assembly mutants were isolated in two steps. First, amber mutations were obtained that mapped in ompF-Dex. This resulted in a Dex- phenotype. Starting with these Dex- strains, Dex+ revertants were isolated. Mutants that displayed a temperature-sensitive Dex+ phenotype were further characterized. Three such mutants possessed a single substitution within ompF that reverted the nonsense codon to a sense codon which replaced W214 with either an E or Q and Y231 with a Q residue in the mature OmpF protein. All three mutant OmpF proteins showed an assembly defect. This defect led to a substantial reduction in the amount of stable OmpF trimers with the concomitant increase of a high-molecular-weight form of OmpF which migrated at the top of the gel. Suppressor mutations were sought that corrected the assembly defect of OmpF. These extragenic suppressor mutations were mapped at 45 min on the Escherichia coli chromosome. The suppressor mutations displayed no allele specificity and were recessive to the wild-type allele. In the presence of a suppressor, mutant stable trimers appeared in an almost normal manner. The appearance of stable trimers concurred with a substantial loss of the high-molecular-weight OmpF species. At this stage, it is not clear whether the high-molecular-weight species of OmpF is a normal assembly intermediate or a dead-end assembly product. The results presented in this study raise the intriguing possibility of a chaperone-like activity for the wild-type suppressor gene product.     

Isolation and characterization of thermosensitive Escherichia coli mutants defective in deoxyribonucleic acid replication

Thermosensitive deoxyribonucleic acid replication-defective mutants have been isolated by using an autoradiographic selection method. The mutants have been analyzed genetically and biochemically. Some of the mutants show thermosensitivity of in vitro deoxyribonucleic acid replication. These can be classified into three groups according to their behavior in in vitro complementation assays. This classification is congruent with that obtained by genetic mapping by using cotransduction frequencies with selected markers in P1 transduction analysis.     

Perturbed chromosomal replication in recA mutants of Escherichia coli.

When initiation of DNA replication is inhibited in wild-type Escherichia coli cells by rifampin or chloramphenicol, completion of ongoing rounds of replication (runout of replication) leads to cells containing two, four, or eight fully replicated chromosomes, as measured by flow cytometry. In recombination-deficient recA strains, a high frequency of cells with three, five, six, or seven fully replicated chromosomes was observed in addition to cells with two, four, or eight chromosomes. recA mutants affected only in the protease-stimulating function behaved like wild-type cells. Thus, in the absence of the recombinase function of RecA protein, the frequency of productive initiations was significantly reduced compared with that in its presence. DNA degradation during runout of replication in the presence of rifampin was about 15%. The DNA degradation necessary to account for the whole effect described above was in this range or even lower. However, a model involving selective and complete degradation of partially replicated chromosomes is considered unlikely. It is suggested that the lack of RecA protein causes initiations or newly formed replication forks to stall but remain reactivatable for a period of time by functional RecA protein.     

Identification and characterization of gyrB mutants of Escherichia coli that are defective in partitioning of mini-F plasmids.

hopA mutants, which have been suggested to be defective in mini-F plasmid partitioning (H. Niki, C. Ichinose, T. Ogura, H. Mori, M. Morita, M. Hasegawa, N. Kusukawa, and S. Hiraga, J. Bacteriol. 170:5272-5278, 1988), were found to carry mutations in the gyrB gene, coding for the B subunit of DNA gyrase. In gyrB(HopA) mutants, relaxation of the superhelicity of plasmids, increased IncG incompatibility, and increased SopB protein production were observed. It is suggested that altered expression of the sop genes, which is due to relaxation of the mini-F plasmid DNA, causes both defective partitioning of the mini-F plasmids and increased IncG incompatibility in gyrB(HopA) mutants.     

High-affinity arabinose transport mutants of Escherichia coli: isolation and gene location.

The gene araF, the product of which is the L-arabinose-binding protein--a component of the high-affinity L-arabinose transport system, was located on the Escherichia coli linkage map at 45 min. We established this location using bacteriophage P2 eductates and bacteriophage P1 cotransduction frequencies with the adjacent genetic loci, his (histidine biosynthesis) and mgl (methylgalactoside transport). In addition, we isolated a number of mutants that phenotypically exhibited altered high-affinity L-arabinose transport capacities. At least two of these mutations were located in the araF gene, as binding protein purified from these strains exhibited altered in vitro arabinose-binding properties.     

A temperature sensitive mutant of Escherichia coli which does not allow replication of RNA phage at a high temperature.

A conditional mutant, referred to as RepR43, was isolated from Escherichia coli W2252 by N-methyl-N'-nitro-N-nitroso-guanidine mutagenesis. Although RepR43 does not permit growth of RNA phage beta at the restrictive temperature, 43 degrees C, cell growth and synthesis of macromolecules such as RNA and protein continue at a somewhat reduced rate. Several lines of evidence indicate that a RepR43 function is indispensable for normal phage RNA replication. In addition, this function appears to be involved in the maintenance of the perpetuated phage genome. The addition of 10% sucrose to the medium at the restrictive temperature resulted in the production of the phage, suggesting that the mutant cell might have an altered membrane organization which interferes with normal viral replication.     

The isolation of fumB mutants of Escherichia coli

Escherichia coli strains lacking the terminus region of the chromosome (min 29-36) due to an IS10-promoted deletion did not grow well in rich medium; they also did not grow on fumarate minimal medium because fumAC (min 35.7) is deleted. Strains with secondary mutations that partially suppress the deletion phenotype displayed healthier growth on rich medium and grew on minimal fumarate medium. These suppressor mutants had an IS10 insertion just upstream of the fumB structural gene (min 93.4). A strain with a Tn10 insertion at this location was constructed and used to delete nonessential fumB; fumB deletion mutants grew well on both rich and minimal fumarate media.     

Regulation of tyrosine biosynthesis in Escherichia coli K-12: isolation and characterization of operator mutants

Mutant strains of Escherichia coli have been isolated in which the synthesis of two of the enzymes involved in tyrosine biosynthesis, 3-deoxy-d-arabinoheptulosonic acid-7 phosphate synthetase (tyr) and chorismate mutase T-prephenate dehydrogenase, is partially constitutive. The mutations involved are closely linked to aroF and tyrA, the structural genes of these enzymes. The gene in which the mutations occur has been designated aroK, and the gene sequence is aroK, aroF, tyrA. In aroK(+)/aroK diploids, the aroK allele only affects the structural genes in the cis position. The mutant allele aroK is not recessive to aroK(+) and aroK/aroK(+) strains exhibit the aroK phenotype of resistance to 4-aminophenylalanine. It is proposed that aroK is an operator locus for an aroF tyrA operon.