Studies on the alteration of chromosome copy number and cell division potential in a dnaA mutant of Escherichia coli.

The dnaA167 mutant of Escherichia coli, N167, maintains, on the average, two replicating chromosomes per cell at the permissive growth temperature of 30 degrees C and only one per cell at the higher permissive growth temperature of 38 degrees C. When the growth temperature of this mutant is changed from 30 degrees to 38 degrees C the cells rapidly readjust their chromosome copy number from two to one. I have examined the kinetics of this transition with reference to DNA replication and cell division. My results indicate that this mutant uncouples cell division from chromosome duplication to achieve the appropriate copy number, suggesting that the dnaA gene product may be involved in the coordination between these two cellular events.

Evidence that TolC is required for functioning of the Mar/AcrAB efflux pump of Escherichia coli.

A study examining the influence of TolC on AcrA, AcrR, and MarR1 mutants indicates that functional TolC is required for the operation of the AcrAB efflux system and for the expression of the Mar phenotype. That the effect of TolC on the AcrAB pump is not regulatory in nature is shown by studies measuring the influence of a tolC::Tn10 insertion mutation on the expression of an acrA::lacZ reporter fusion. These results are compatible with the hypothesis that TolC is a component of the AcrAB efflux complex.

Studies on the expression of outer membrane protein 2 in escherichia coli.

The relative level of protein 2 expressed in the outer membrane of strains of Escherichia coli K-12 lysogenized with bacteriophage PA-2 was found to be influenced by both the growth temperature and lc+ gene dosage. An increase in either of these parameters was accompanied by an increase in the level of protein 2 up to an apparent saturation level. Any increase in the amount of protein 2 was accompanied by a concomittant decrease in the amount of OmpF and OmpC porins. This inverse relationship led to the maintenance of an approximately constant protein mass per unit of peptidoglycan. Our results are discussed in light of recent genetic studies on the regulation of the OmpF and OmpC porins and can be explained through the competition of these three matrix proteins for a common export or insertion site.

Relationship between the OmpC and LamB proteins of Escherichia coli and its influence on the protein mass of the outer membrane.

Cultures of Escherichia coli K-12 which possessed varied amounts of the LamB protein were found to contain reduced amounts of the OmpC protein. This process was regulated in part at the level of transcription. However, additional controls were inferred from anomalously high levels of the OmpC protein present at low levels of the LamB protein. Both proteins were present at increased levels, and this led to an increase in the total outer membrane protein mass per cell. The absolute amount of outer membrane protein per cell was found not to be a constant as had been tacitly assumed.

Additive effect of tolC and rfa mutations on the hydrophobic barrier of the outer membrane of Escherichia coli K-12.

Studies using tolC mutant derivatives of deep rough (rfa) mutants indicate that tolC and rfa mutations have an additive effect with respect to their sensitivity to hydrophobic agents, suggesting that they are not acting through a mutual mechanism to alter the permeability of the outer membrane.

Isolation of an Lc-specific Escherichia coli bacteriophage.

We isolated an OmpF-specific bacteriophage whose host range mutant, SQ108h2, requires the presence of the Lc porin for its attachment and which can be used to screen or select for Lc-defective mutants among Escherichia coli K-12 strains lysogenic for the PA-2 converting phage.

Isolation and characterization of a putative multidrug resistance pump from Vibrio cholerae.

Multidrug-resistant strains of Vibrio cholerae (the causative agent of the diarrhoeal disease cholera) have recently been described. In an attempt to identify a homologue of the Escherichia coli TolC in V. cholerae, we isolated a DNA fragment (pVC) that enabled an E. coli tolC mutant to grow in the presence of 0.05% deoxycholate (DOC). However, other TolC defects were not complemented. Nucleotide sequence analysis of this fragment revealed the presence of two open reading frames (ORF1 and ORF2) separated by 9 bp and encoding 42.4 and 55.8 kDa proteins respectively. The translational products of these two ORFs correlated closely with the molecular weights of the predicted proteins. The deduced amino acid sequences of ORF1 and ORF2 showed a high degree of similarity with conserved regions of the E. coli efflux pump proteins, EmrA and EmrB. The presence of pVC2 within the E. coli efflux pump mutants defective in either the emrAB or the acrAB genes provided the mutants with resistance against several antibiotics. A V. cholerae isogenic mutant defective in ORF2 was constructed by gene replacement. Characterization of this mutant has shown it to be more sensitive to CCCP, PMA, PCP, nalidixic acid and DOC than the parent strain. These results suggest that ORF1 and ORF2 constitute an operon encoding two components of a putative multidrug resistance pump in V. cholerae. In addition, the presence of both structural and functional similarities between VceAB and EmrAB suggests that VceAB is a homologue of EmrAB.

Polyuridylic acid-directed phenylalanine incorporation in minicell extracts.

Cell-free extracts of miniature Escherichia coli cells deficient in deoxyribonucleic acid (DNA) and DNA-dependent ribonucleic acid polymerase have been shown to be capable of polyuridylic acid-directed [(14)C]phenylalanine incorporation.

The requirement of DNA synthesis for the induction of alkaline phosphatase by bromodeoxyuridine in a derivative of the HeLa cell line.

Non-lethal concentrations of bromodeoxyuridine induce a 2- to 5-fold increase in the specific activity of alkaline phosphatase in a HeLa subclone, S3G. Experiments employing 10-hour pulses of BRdU showed that 48 hours were required before induction commenced, and that maximal induction was attained by 96 hours. Under conditions in which DNA synthesis was prevented with hydroxyurea induction did not occur. Upon removal of hydroxyurea both DNA synthesis and induction were rapidly reestablished. Furthermore, experiments employing radiolabelled BRdU demonstrated that the kinetics of the induction process paralleled the incorporation of the analogue into cellular DNA. These results indicate that DNA synthesis, or some process intimately linked to DNA synthesis, is required for the induction of alkaline phosphatase, and suggest that the mode of the induction may be through the incorporation of the analogue into cellular DNA.

Temperature dependent release of beta-beta' subunits of DNA dependent RNA polymerase from the folded chromosome of a dnaAts mutant of Escherichia coli.

DNA-dependent RNA polymerase has been found to be preferentially released at 43 degrees C from the folded nucleoids of an E. coli dnaAts mutant when compared with the same nucleoids at 30 degrees C or with nucleoids of a dnaA+ strain at either 30 degrees or 43 degrees C. The polypeptides released are identical in molecular weight with those of the beta and beta' constituent polypeptides of the core enzyme of a known E. coli RNA polymerase. In addition, these polypeptides are precipitated by specific anti-RNA polymerase rabbit IgG. The implications of the interactions of RNA polymerase with the dnaA gene product are discussed.

Membrane association of conjugally transferred deoxyribonucleic acid in Escherichia coli minicells.

The conjugally acquired deoxyribonucleic acid (DNA) of small, anucleate cells ("minicells") of a mutant strain of Escherichia coli K-12 was found to be predominantly associated with the bacterial membrane. Evidence from X-irradiation studies in vivo shows that there is no decrease in DNA-membrane association under conditions which reduce the DNA to one-sixth its original size and suggests the possibility of multiple DNA-membrane association sites. Preliminary enzymatic studies indicate the involvement of protein, DNA, and lipids in the membrane association of the DNA.