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R C Gayda

Publications and source records attributed to R C Gayda.

13 recordsLinked to original sources
PubMed1992

Quantitative determination of FtsA at different growth rates in Escherichia coli using monoclonal antibodies.

FtsA is an essential cell division protein in Escherichia coli. Its synthesis in low amounts makes the investigation of its functions difficult. Partially purified FtsA protein was obtained by solubilizing cellular inclusion bodies after overexpression of the ftsA gene for the purpose of raising monoclonal antibodies. Mice were immunized with this FtsA protein fraction and their spleen cells were fused to Sp2/0-AG14 mouse myeloma cells. Hybrid cells were screened and two clones were positively identified as FtsA monoclonal antibody producers by enzyme-linked immunosorbent assay and Western blotting. A quantitative assay using these monoclonal antibodies indicated that the average number of FtsA molecules per cell to be between 50 and 200. However, the concentration of FtsA protein normalized to total cell protein was constant over a wide range of growth rates. This finding is in agreement with the hypothesized role of FtsA protein as a stoichiometric component of the septum.

Animals↗
PubMed1992

C-shaped cells caused by expression of an ftsA mutation in Escherichia coli.

A plasmid, pDLL4, was isolated from a Tn5tac1 mutagenesis experiment with plasmid pZAQ. When pDLL4 was transformed into wild-type rod-shaped cells, it caused cells in the population to become curved (C-shaped or convoluted). The Tn5tac1 transposon was integrated within the carboxyl end of the ftsA gene in pDLL4. This mutation was designated ftsAc. Subcloning ftsAc DNA into another plasmid vector verified that the curved-cell phenotype was caused by the expression of this altered gene. DNA sequence analysis of the ftsAc mutation revealed that the transposition event changed the DNA so that the last 28 amino acids of the FtsA protein were lost and 5 new amino acids were added. A radioactive peptide band corresponding to this truncated FtsAc protein was identified by a T7 promoter-T7 polymerase protein labeling system. Observations of thin sections of these curved cells with an electron microscope revealed aggregates of striated cylindrical structures traversing the cytoplasm. The ends of these aggregates appear to be at or near the cell membrane. The linear periodicity of the cylinders was approximately 11 nm, and the diameter of a cylinder was about 15 nm. Aggregates of as many as five cylinders were arrayed diagonally to the long axis of the curved cells, a finding that suggests that some type of internal organization may be causing the curved cell shape.

Amino Acid Sequence↗
PubMed1985

Regulatory region of the heat shock-inducible capR (lon) gene: DNA and protein sequences.

The CapR protein is an ATP hydrolysis-dependent protease as well as a DNA-stimulated ATPase and a nucleic acid-binding protein. The sequences of the 5' end of the capR (lon) gene DNA and N-terminal end of the CapR protein were determined. The sequence of DNA that specifies the N-terminal portion of the CapR protein was identified by comparing the amino acid sequence of the CapR protein with the sequence predicted from the DNA. The DNA and protein sequences established that the mature protein is not processed from a precursor form. No sequence corresponding to an SOS box was found in the 5' sequence of DNA. There were sequences that corresponded to a putative -35 and -10 region for RNA polymerase binding. The capR (lon) gene was recently identified as one of 17 heat shock genes in Escherichia coli that are positively regulated by the product of the htpR gene. A comparison of the 5' DNA region of the capR gene with that of several other heat shock genes revealed possible consensus sequences.

Amino Acid Sequence↗
PubMed1984

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

The DNA of the promoter region of omp T, including the putative start for the pro-Omp T protein (pro-protein a), has been sequenced. Previous studies showed that trypsin inhibitors prevent the processing of pro-Omp T to Omp T 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-Omp T and Omp T 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 per A (tpo, envZ) gene product is required for synthesis of Omp T protein (as well as several outer membrane and periplasmic proteins). The perA gene product could be activating translation of Omp T protein by disrupting the mRNA secondary structure that sequesters the SD sequence. Omp T protein synthesis is reduced at temperatures below 32 degrees C and this may also be related to the greater stability of the sequestered SD sequence of the mRNA at low temperature.

Bacterial Outer Membrane Proteins↗
PubMed1984

Regulation of cell division in Escherichia coli: SOS induction and cellular location of the sulA protein, a key to lon-associated filamentation and death.

Mutations in sulA (sfiA) block the filamentation and death of capR (lon) mutants that occur after treatments that either damage DNA or inhibit DNA replication and thereby induce the SOS response. Previous sulA-lacZ gene fusion studies showed that sulA is transcriptionally regulated by the SOS response system (lexA/recA). SulA protein has been hypothesized to be additionally regulated proteolytically through the capR (lon) protease, i.e., in lon mutants lacking a functional ATP-dependent protease there would be more SulA protein. A hypothesized function for SulA protein is an inhibitor of cell septation. To investigate aspects of this model, we attempted to construct lon, lon sulA, and lon sulB strains containing multicopy plasmids specifying the sulA+ gene. Multicopy sulA+ plasmids could not be established in lon strains because more SulA protein accumulates than in a lon+ strain. When the sulA gene was mutated by a mini Mu transposon the plasmid could be established in the lon strains. In contrast, sulA+ plasmids could be established in lon+, lon sulA, and lon sulB strains. The sulA+ plasmids caused lon sulA and lon sulB cells to exist as filaments without SOS induction and to be sensitive to UV light and nitrofurantoin. Evidence implicated higher basal levels of SulA protein in these lon plasmid sulA+ strains as the cause of filamentation. We confirmed that the SulA protein is an 18-kilodalton polypeptide and demonstrated that it was induced by treatment with nalidixic acid. The SulA protein was rapidly degraded in a lon+ strain, but was comparatively more stable in vivo in a lon sulB mutant. Furthermore, the SulA protein was localized to the membrane by several techniques.

Bacterial Proteins↗
PubMed1983

omp T: Escherichia coli K-12 structural gene for protein a (3b).

Chromosomal DNA from strain UT400, a previously described deletion mutant of Escherichia coli K-12 that lacks outer membrane protein a, failed to hybridize with plasmid DNA (pGGC110) containing the structural gene for protein a. We designate the genetic locus for protein a, located at approximately 12.5 min of the E. coli chromosome, ompT.

Bacterial Outer Membrane Proteins↗
PubMed1979

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

capR (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 non-mucoid 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 Kdal outer membrane protein a or were deficient in synthesis of 25 Kdal and 14.5 Kdal polypeptides specified by the 2 Mdal DNA fragments. 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.

Bacterial Proteins↗
PubMed1979

Neuroactive drugs inhibit trypsin and outer membrane protein processing in Escherichia coli K-12.

Previous studies demonstrated that a cloned 2-megadalton (MDal) fragment of Escherichia coli DNA contained the structural gene for major outer membrane protein a (also known as 3b or M2 (40 kDal). The present study demonstrates that M2 is synthesized from a 42-kDal precursor that also is present in the outer membrane. The conversion of the 42-kDal precursor to M2 is inhibited by a number of different local anesthetics (procaine, piperocaine, lidocaine, cocaine), by the neuroactive drug atropine, and by the classical trypsin inhibitors N alpha-tosyllysine chloromethyl ketone (TLCK) and benzamidine. Our kinetic studies demonstrate that the amidase action of pure trypsin is inhibited competitively by the local anesthetics tested (excluding lidocaine) as well as by atropine and neostigmine. A mechanism of action for local anesthetics as well as atropine in E. coli may to be inhibit trypsinlike proteases, in a competitive manner, in the region of the outer membrane. The mechanism of action of these compounds in regulating nerve conduction in man have certain features in common with the mechanism proposed in E. coli.

Anesthetics, Local↗
PubMed1978

Cloned DNA fragment specifying major outer membrane protein a in Escherichia coli K-12.

Plasmid pMC44 is a recombinant plasmid that contains a 2-megadalton EcoRI fragment of Escherichia coli K-12 DNA joined to the cloning vehicle, pSC101. The polypeptides specified by plasmid pMC44 were identified and compared with those specified by pSC101 to determine those that are unique to pMC44. Three polypeptides specified by plasmid pMC44 were localized in the cell envelope fraction of minicells: a Sarkosyl-insoluble outer membrane polypeptide (designated M2), specified by the cloned 2-megadalton DNA fragment, and two Sarkosyl-soluble membrane polypeptides specified by the cloning plasmid pSC101. Bacteria containing plasmid pMC44 synthesized quantities of M2 approximately equal to the most abundant E. coli K-12 outer membrane protein. Evidence is presented that outer membrane polypeptide M2, specified by the recombinant plasmid pMC44, is the normal E. coli outer membrane protein designated protein a by Lugtenberg and 3b by Schnaitman.

Bacterial Proteins↗
PubMed1976

Second-site mutations in capR (lon) strains of Escherichia coli K-12 that prevent radiation sensitivity and allow bacteriophage lambda to lysogenize.

capR (lon) mutants of Escherichia coli K-12 are mucoid and sensitive to ultraviolet (UV) and X-ray radiation as well as to nitrofurantoin. The mutants form filaments after exposure to these agents. capR mutants are also conditionally lethal since they die when plated on complex medium even without UV treatment; this phenomenon is designated "complex medium-induced killing". Furthermore, capR mutants are poorly lysogenized by bacteriophage lambda. Second-site revertants were isolated by plating on media containing nitrofurantoin. All 17 of the independent revertants studied were still mucoid but resistant to UV radiation. Sixteen of the 17 revertants contained a mutation, sulA, that cotransduced with pyrD (21 min). A second locus, sulB, was also found that cotransduced with leu (2 min). Studies with partial diploids (F'pyrD+ sulA+/pyrD36 sulA17 capR9 (lon) demonstrated that sulA+ is dominant to sulA; thus the indicated partial diploid is UV sensitive, whereas the haploid parent is UV resistant. Furthermore, two other phenotypic traits of capR (lon) mutants were reversed by the sul mutation:complex medium-induced killing and the inability of lambda phage to efficiently lysogenize capR strains. On the basis of these and other results, the following model is suggested to explain capR (lon) and sul gene interactions. capR (lon) is a regulator gene for the structural genes sulA+ and sulB+. Depression of both sul operons results in UV sensitivity and decreased ability of lambda to lysogenize, whereas inactivation of either sul+ protein by mutation to sul prevents these phenomena.

Chromosome Mapping↗