Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1983 Apr;154(1):10–16. doi: 10.1128/jb.154.1.10-16.1983

Molecular cloning and characterization of the genes (pbpA and rodA) responsible for the rod shape of Escherichia coli K-12: analysis of gene expression with transposon Tn5 mutagenesis and protein synthesis directed by constructed plasmids.

S Asoh, H Matsuzawa, M Matsuhashi, T Ohta
PMCID: PMC217424  PMID: 6300030

Abstract

Two cell shape-determining genes of Escherichia coli K-12, pbpA, the structural gene for penicillin-binding protein 2, and rodA, whose protein is unknown, were subcloned into plasmid vectors from the transducing phage lambda MAd lip24, which carries the lip-leuS region of the E. coli chromosome. Plasmids with restriction enzyme-created deletions or transposon Tn5 insertions were isolated, and studies of genetic complementation of these plasmids with chromosomal mutations were carried out. Thus, a physical and genetic map of the rodA-pbpA region was established. The genes rodA and pbpA lie side by side within a 4.4-kilobase-pair region. The size of the rodA gene has been shown to be between 0.86 and 1.6 kilobase pairs; such DNA would encode a protein with a molecular weight between 32,000 and 59,000. Since Tn5 mutagenesis of the rodA gene did not affect the expression of the pbpA gene and vice versa, the genes rodA and pbpA seem to have independent promoters. Analysis of the proteins synthesized from the constructed plasmids in maxicells revealed that the plasmid carrying the pbpA gene encoded penicillin-binding protein 2 and amplification of the protein occurred. The product of the rodA gene was not identified.

Full text

PDF
10

Images in this article

13Image
on p.13
15Image
on p.15

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bachmann B. J., Low K. B. Linkage map of Escherichia coli K-12, edition 6. Microbiol Rev. 1980 Mar;44(1):1–56. doi: 10.1128/mr.44.1.1-56.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berg D. E., Weiss A., Crossland L. Polarity of Tn5 insertion mutations in Escherichia coli. J Bacteriol. 1980 May;142(2):439–446. doi: 10.1128/jb.142.2.439-446.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  4. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Clewell D. B., Helinski D. R. Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an opern circular DNA form. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1159–1166. doi: 10.1073/pnas.62.4.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Colman A., Byers M. J., Primrose S. B., Lyons A. Rapid purification of plasmid DNAs by hydroxyapatite chromatography. Eur J Biochem. 1978 Nov 2;91(1):303–310. doi: 10.1111/j.1432-1033.1978.tb20966.x. [DOI] [PubMed] [Google Scholar]
  7. Csonka L. N., Clark A. J. Construction of an Hfr strain useful for transferring recA mutations between Escherichia coli strains. J Bacteriol. 1980 Jul;143(1):529–530. doi: 10.1128/jb.143.1.529-530.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dagert M., Ehrlich S. D. Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene. 1979 May;6(1):23–28. doi: 10.1016/0378-1119(79)90082-9. [DOI] [PubMed] [Google Scholar]
  9. Felton J., Michaelis S., Wright A. Mutations in two unlinked genes are required to produce asparagine auxotrophy in Escherichia coli. J Bacteriol. 1980 Apr;142(1):221–228. doi: 10.1128/jb.142.1.221-228.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jorgensen R. A., Rothstein S. J., Reznikoff W. S. A restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance. Mol Gen Genet. 1979;177(1):65–72. doi: 10.1007/BF00267254. [DOI] [PubMed] [Google Scholar]
  11. Matsuzawa H., Hayakawa K., Sato T., Imahori K. Characterization and genetic analysis of a mutant of Escherichia coli K-12 with rounded morphology. J Bacteriol. 1973 Jul;115(1):436–442. doi: 10.1128/jb.115.1.436-442.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Morris C. F., Hama-Inaba H., Mace D., Sinha N. K., Alberts B. Purification of the gene 43, 44, 45, and 62 proteins of the bacteriophage T4 DNA replication apparatus. J Biol Chem. 1979 Jul 25;254(14):6787–6796. [PubMed] [Google Scholar]
  13. Sancar A., Hack A. M., Rupp W. D. Simple method for identification of plasmid-coded proteins. J Bacteriol. 1979 Jan;137(1):692–693. doi: 10.1128/jb.137.1.692-693.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Schrenk W. J., Weisberg R. A. A simple method for making new transducing lines of coliphage lambda. Mol Gen Genet. 1975;137(2):101–107. doi: 10.1007/BF00341676. [DOI] [PubMed] [Google Scholar]
  15. Spratt B. G., Boyd A., Stoker N. Defective and plaque-forming lambda transducing bacteriophage carrying penicillin-binding protein-cell shape genes: genetic and physical mapping and identification of gene products from the lip-dacA-rodA-pbpA-leuS region of the Escherichia coli chromosome. J Bacteriol. 1980 Aug;143(2):569–581. doi: 10.1128/jb.143.2.569-581.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Spratt B. G. Distinct penicillin binding proteins involved in the division, elongation, and shape of Escherichia coli K12. Proc Natl Acad Sci U S A. 1975 Aug;72(8):2999–3003. doi: 10.1073/pnas.72.8.2999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Spratt B. G. Temperature-sensitive cell division mutants of Escherichia coli with thermolabile penicillin-binding proteins. J Bacteriol. 1977 Jul;131(1):293–305. doi: 10.1128/jb.131.1.293-305.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Stüber D., Bujard H. Organization of transcriptional signals in plasmids pBR322 and pACYC184. Proc Natl Acad Sci U S A. 1981 Jan;78(1):167–171. doi: 10.1073/pnas.78.1.167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Tamaki S., Matsuzawa H., Matsuhashi M. Cluster of mrdA and mrdB genes responsible for the rod shape and mecillinam sensitivity of Escherichia coli. J Bacteriol. 1980 Jan;141(1):52–57. doi: 10.1128/jb.141.1.52-57.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Tamaki S., Nakajima S., Matsuhashi M. Thermosensitive mutation in Escherichia coli simultaneously causing defects in penicillin-binding protein-1Bs and in enzyme activity for peptidoglycan synthesis in vitro. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5472–5476. doi: 10.1073/pnas.74.12.5472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Weiss B., Jacquemin-Sablon A., Live T. R., Fareed G. C., Richardson C. C. Enzymatic breakage and joining of deoxyribonucleic acid. VI. Further purification and properties of polynucleotide ligase from Escherichia coli infected with bacteriophage T4. J Biol Chem. 1968 Sep 10;243(17):4543–4555. [PubMed] [Google Scholar]
  22. Yamamoto K. R., Alberts B. M., Benzinger R., Lawhorne L., Treiber G. Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large-scale virus purification. Virology. 1970 Mar;40(3):734–744. doi: 10.1016/0042-6822(70)90218-7. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES