Alvin Markovitz

A new in vivo termination function for DNA polymerase I of Escherichia coli K12

Escherichia coli deleted for the tus gene are viable. Thus there must be at least one other mechanism for terminating chromosome synthesis. The tus deletion strain yielded a small fraction of cells that overproduce DNA, as determined by flow cytometry after run‐out chromosome replication in the presence of rifampicin and cephalexin. A plasmid, paraBAD tus+, prevented the excess DNA replication only when arabinose was added to the medium to induce the synthesis of the Tus protein. Transduction studies were done to test whether or not additional chromosomal deletions could enhance the excess chromosome replication in the tus deletion strain. A strain containing a second deletion in metE udp overproduced DNA at a high level during run‐out replication. Further studies demonstrated that a spontaneous unknown mutation had occurred during the transduction. This mutation was mapped and sequenced. It is polA(G544D). Transduction of polA(G544D) alone into the tus deletion strain produced the high DNA overproduction phenotype. The polA(G544D) and six other polA alleles were then tested in wild‐type and in tus deletion backgrounds. The two alleles with low levels of 5′→3′ exonuclease (exo) overproduced DNA while those with either high or normal exo overproduce much less DNA in run‐out assays in the wild‐type background. In contrast, all seven mutant polA alleles caused the high DNA overproduction phenotype in a tus deletion background. To explain these results we propose a new in vivo function for wild‐type DNA polymerase I in chromosome termination at site(s) not yet identified.

Outer membrane protein a and other polypeptides regulate capsular polysaccharide synthesis in E. coli K-12

SummarycapR (lon) mutants of Escherichia coli K-12 are mucoid on minimal agar because they produce large quantities of capsular polysaccharide. When such mutants are transformed to tetracycline resistance by plasmid pMC44, a hybrid plasmid that contains a 2 megadalton (Mdal) endonuclease EcoR1 fragment of E. coli K-12 DNA joined to the cloning vehicle-pSC101, capsular polysaccharide synthesis is inhibited and the transformed colonies exhibit a nonmucoid phenotype. Re-cloning of the 2 Mdal EcoR1 fragment onto plasmid pHA105, a min-colE1 plasmid, yielded plasmid pFM100 which also inhibited capsular polysaccharide synthesis in the capR mutants. A comparison of the polypeptides specified by both plasmids pFM100 and pMC44 in minicells demonstrated that seven polypeptide bands were specified by the 2 Mdal DNA, one of which was previously demonstrated to be outer membrane protein a; also known as 3b or M2 (40 kilodaltons, Kdal). Plasmid mutants no longer repressing capsular polysaccharide synthesis were either unable to specify the 40 K dal outer membrane protein a or were deficient in synthesis of 25 K dal and 14.5 K dal polypeptides specified by the 2 Mdal DNA fragment. Studies with a minicell-producing strain that also contained a capR mutation indicated that the capR gene product regulated processing of at least one normal protein, the precursor of outer membrane protein a.

Molecular mechanism for dominance of a mutant allele of an ATP-dependent protease.

Abstract We have demonstrated that the lon + ( cap R + ) ATP-hydrolysis-dependent protease is inhibited by the addition of an enzymatically inactive mutant form of the protein ( cap R9 protein). The enzymatic activity of the cap R + - cap R9 protein mixture is also more stable to heat than the cap R + protein alone indicating that hybrid molecules are formed. Independent measurements demonstrated that the lon + ATP-hydrolysis-dependent protease is a tetramer of identical 94 × 10 3 molecular weight monomers. The pattern of defective subunit ( cap R9) inhibition indicates only tetramers containing three cap R9 monomers and one cap R + monomer are inactive, i.e. two or more cap R + monomers are required for a tetramer to act as an ATP-hydrolysis-dependent protease. Two molecular mechanisms to explain the dominance of a mutant allele, termed anticomplementation, are considered.

Identification by deletion analysis of an inducible protein required for plasmid pSC101-mediated tetracycline resistance

Abstract Plasmids containing small deletions within a tetracycline (Tc) resistance gene(s) of plasmid pHA121 were isolated. Plasmid pHA121 was formed by ligating the EcoRI-digested Tc resistance plasmid pSC101 and similarly digested mini-ColE1 plasmid pHA105. The DNA deletion plasmids were constructed by digesting plasmid pHA121 DNA with the restriction endonucleases BamH1 and Sal1 and, in addition, λ exonuclease. Two plasmids, designated pJT131 and pJT133, had small deletions of approximately 0.64 to 0.8 kb and a comparison of the radioactive polypeptides synthesized in plasmid-containing minicells revealed that a 34-kdal polypeptide was not specified by either pJT131 or pJT133. We conclude that the 34-kdal polypeptide is required for the expression of Tc resistance and that its structural gene probably maps in the deleted region of pSC101 DNA.

Characterization of a mini ColE1 cloning vector.

Abstract Plasmid pHA105 (formerly pAC105), a mini ColE1 plasmid containing one restriction endonuclease EcoRI site, was further characterized using restriction endonuclease analysis thereby revealing its relationship to ColE1. The polypeptides specified by plasmid pHA105 in minicells are of low molecular weight making it a useful plasmid to define cloned polypeptides larger than 16,000 daltons and its use for that purpose was demonstrated. pHA105 was used to clone two different sized fragments of DNA containing the gal operon. pHA105 was also used to reclone a 2 Mdal fragment of DNA that, when expressed, represses the synthesis of capsular polysaccharide. The repression of polysaccharide synthesis was expressed when a plasmid containing one molecule each of pHA105 and the 2 Mdal fragment was prepared (pFM100). In contrast, a plasmid containing two copies of pHA105 and one of the 2 Mdal fragment (pHA138) did not repress polysaccharide synthesis. The results demonstrate that expression of a cloned fragment gene may be prevented in certain arrangements of the vector and cloned fragment. Plasmid pHA105 fails to exhibit relaxation after treatment with sodium dodecyl sulfate in contrast to ColE1 treated in the same way. pHA105 replicates as a dimer form while ColE1 usually does not. A hypothesis that a function of a DNA-protein complex is required for monomeric DNA circle formation is discussed.

Sequence of the regulatory region of ompT, the gene specifying major outer membrane protein a (3b) of Escherichia coli K-12: Implications for regulation and processing

SummaryThe DNA of the promoter region of ompT, including the putative start for the pro-OmpT protein (proprotein a), has been sequenced. Previous studies showed that trypsin inhibitors prevent the processing of pro-OmpT to OmpT protein which led to the prediction that the processing site would be a lysine or an arginine. The deduced amino acid sequence contains a lysine at amino acid 12 and an arginine at amino acid 17 from the N terminus. Chou-Fassman analysis would predict processing at the lysine (but not the arginine) to remove a 1389 dalton peptide, consistent with the fact that the estimated molecular masses of pro-OmpT and OmpT are 42 kd and 40 kd respectively. In addition, the predicted mRNA of the promoter region can form a stable secondary structure (-17.1 kcal) that sequesters the Shine-Dalgarno (SD) sequence as well as the initiator AUG codon. There is evidence that the perA (tpo, envZ) gene product is required for synthesis of OmpT protein (as well as several outer membrane and periplasmic proteins). The perA gene product could be activating translation of OmpT protein by disrupting the mRNA secondary structure that sequesters the SD sequence. OmpT protein synthesis is reduced at temperatures below 32°C and this may also be related to the greater stability of the sequestered SD sequence of the mRNA at low temperature.

Altered bacteriophage lambda expression in cell division mutants capR(lon) of Escherichia coli K-12.

SummaryLambda phage containing double amber mutations in the N gene (λc1857N7N53) can replicate and produce progeny phage in capR(lon) bacteria, but not in isogenic capR+ (lon+) bacteria, when two additional mutations conferring weak suppression (supE44 and str-109) are present. Since weak suppression was necessary, the capR mutation does not provide a bacterial substitute for the N gene. In an attempt to explain and to relate the phenomenon with the finding that λ+ forms clear plaques on capR lawns and lysogenizes capR strains poorly N gene-dependent transcription, translation and functional mRNA decay was measured using a λlac phage in which β-galactosidase (β gal) synthesis is dependent on the N+ gene of the phage (λplacW205). No measurable difference in initiation of λ leftward N-dependent transcription or translation was observed in a capR strain compared to an isogenic capR+ strain. A differencewas found in the rate of synthesis of N-dependent β-gal however, between λ-infected capR and capR+ bacteria. λplacW205-infected capR9 strains exhibited a 2 to 4 fold lower rate of β-gal synthesis. This decreased rate of β-gal synthesis was the result of the size heterogeneity of the capR cells when grown in complex medium. That is, complex medium-induced capR filaments synthesized lower amounts of N-dependent β-gal. The results are discussed in relation to the findings in other laboratories.

Agal region mutant that requires cAMP for growth on galactose in an adenyl cyclase negative (cyaΔ) background

SummaryStrains ofEscherichia coli K12 that contain a deletion of the adenyl cyclase gene (cyaΔ), required for the synthesis of cyclic adenosine-3′; 5′ monophosphate (cAMP), grow on galactose-containing minimal medium. A mutant was isolated that grows on this medium only if cAMP is added. The mutation (designatedgalP20) is linked to thegal operon region as determined by both generalized transduction with bacteriophage P1 and specialized transduction with bacteriophage λ. Studies withgalP20 cyaΔ strains as well asgalΔ (deletions of thegal operon)cyaΔ strains indicate that synthesis of the physiologically important transport mechanism for galactose (galactose permease) requires either cAMP or a function missing from both thegalΔ strains and thegalP20 strain.