• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. Oct 1995; 177(19): 5554–5560.
PMCID: PMC177364

Functional analysis of the ffh-trmD region of the Escherichia coli chromosome by using reverse genetics.


We have analyzed the essentiality or contribution to growth of each of four genes in the Escherichia coli trmD operon (rpsP, 21K, trmD, and rplS) and of the flanking genes ffh and 16K by a reverse genetic method. Mutant alleles were constructed in vitro on plasmids and transferred by recombination to the corresponding lambda phage clone (lambda 439) and from the phage clone to the E. coli chromosome. An ability to obtain recombinants only in cells carrying a complementing plasmid indicated that the mutated gene was essential, while an ability to obtain recombinants in plasmid-free cells indicated nonessentiality. In this way, Ffh, the E. coli homolog to the 54-kDa protein of the signal recognition particle of mammalian cells, and ribosomal proteins S16 and L19 were shown to be essential for viability. A deletion of the second gene, 21K, of the trmD operon reduced the growth rate of the cells fivefold, indicating that the wild-type 21-kDa protein is important for viability. A deletion-insertion in the same gene resulted in the accumulation of an assembly intermediate of the 50S ribosomal subunit, as a result of polar effects on the expression of a downstream gene, rplS, which encodes ribosomal protein L19. This finding suggests that L19, previously not considered to be an assembly protein, contributes to the assembly of the 50S ribosomal subunits. Strains deleted for the trmD gene, the third gene of the operon, encoding the tRNA (m1G37)methyltransferase (or TrmD) showed a severalfold reduced growth rate. Since such a strain grew much slower than a strain lacking the tRNA(m(1)G37) methyltransferase activity because of a point mutation, the TrmD protein might have a second function in the cell. Finally, a 16-kDa protein encoded by the gene located downstream of, and convergently transcribed to, the trmD operon was found to be nonessential and not to contribute to growth.

Full Text

The Full Text of this article is available as a PDF (279K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Bachmann BJ. Linkage map of Escherichia coli K-12, edition 8. Microbiol Rev. 1990 Jun;54(2):130–197. [PMC free article] [PubMed]
  • Bernstein HD, Poritz MA, Strub K, Hoben PJ, Brenner S, Walter P. Model for signal sequence recognition from amino-acid sequence of 54K subunit of signal recognition particle. Nature. 1989 Aug 10;340(6233):482–486. [PubMed]
  • BERTANI G. Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol. 1951 Sep;62(3):293–300. [PMC free article] [PubMed]
  • Björk GR, Ericson JU, Gustafsson CE, Hagervall TG, Jönsson YH, Wikström PM. Transfer RNA modification. Annu Rev Biochem. 1987;56:263–287. [PubMed]
  • Björk GR, Wikström PM, Byström AS. Prevention of translational frameshifting by the modified nucleoside 1-methylguanosine. Science. 1989 May 26;244(4907):986–989. [PubMed]
  • Byström AS, Björk GR. Chromosomal location and cloning of the gene (trmD) responsible for the synthesis of tRNA (m1G) methyltransferase in Escherichia coli K-12. Mol Gen Genet. 1982;188(3):440–446. [PubMed]
  • Byström AS, Björk GR. The structural gene (trmD) for the tRNA(m1G)methyltransferase is part of a four polypeptide operon in Escherichia coli K-12. Mol Gen Genet. 1982;188(3):447–454. [PubMed]
  • Byström AS, Hjalmarsson KJ, Wikström PM, Björk GR. The nucleotide sequence of an Escherichia coli operon containing genes for the tRNA(m1G)methyltransferase, the ribosomal proteins S16 and L19 and a 21-K polypeptide. EMBO J. 1983;2(6):899–905. [PMC free article] [PubMed]
  • Byström AS, von Gabain A, Björk GR. Differentially expressed trmD ribosomal protein operon of Escherichia coli is transcribed as a single polycistronic mRNA species. J Mol Biol. 1989 Aug 20;208(4):575–586. [PubMed]
  • Chow WY, Berg DE. Tn5tac1, a derivative of transposon Tn5 that generates conditional mutations. Proc Natl Acad Sci U S A. 1988 Sep;85(17):6468–6472. [PMC free article] [PubMed]
  • Connolly T, Gilmore R. Formation of a functional ribosome-membrane junction during translocation requires the participation of a GTP-binding protein. J Cell Biol. 1986 Dec;103(6 Pt 1):2253–2261. [PMC free article] [PubMed]
  • Dabbs ER, Wittman HG. A strain of Escherichia coli which gives rise to mutations in a large number of ribosomal proteins. Mol Gen Genet. 1976 Dec 22;149(3):303–309. [PubMed]
  • de Boer HA, Comstock LJ, Vasser M. The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci U S A. 1983 Jan;80(1):21–25. [PMC free article] [PubMed]
  • Guthrie C, Nashimoto H, Nomura M. Structure and function of E. coli ribosomes. 8. Cold-sensitive mutants defective in ribosome assembly. Proc Natl Acad Sci U S A. 1969 Jun;63(2):384–391. [PMC free article] [PubMed]
  • Hagervall TG, Tuohy TM, Atkins JF, Björk GR. Deficiency of 1-methylguanosine in tRNA from Salmonella typhimurium induces frameshifting by quadruplet translocation. J Mol Biol. 1993 Aug 5;232(3):756–765. [PubMed]
  • Hasenbank R, Guthrie C, Stöffler G, Wittmann HG, Rosen L, Apirion D. Electrophoretic and immunological studies on ribosomal proteins of 100 Escherichia coli revertants from streptomycin dependence. Mol Gen Genet. 1973 Dec 14;127(1):1–18. [PubMed]
  • Held WA, Nomura M. Escherichia coli 30 S ribosomal proteins uniquely required for assembly. J Biol Chem. 1975 Apr 25;250(8):3179–3184. [PubMed]
  • Isono K, Cumberlidge AG, Isono S. Further temperature-sensitive mutants of Escherichia coli with altered ribosomal proteins. Mol Gen Genet. 1977 Apr 29;152(3):239–243. [PubMed]
  • Isono K, Krauss J, Hirota Y. Isolation and characterization of temperature-sensitive mutants of Escherichia coli with altered ribosomal proteins. Mol Gen Genet. 1976 Dec 22;149(3):297–302. [PubMed]
  • Kohara Y, Akiyama K, Isono K. The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library. Cell. 1987 Jul 31;50(3):495–508. [PubMed]
  • Krieg UC, Walter P, Johnson AE. Photocrosslinking of the signal sequence of nascent preprolactin to the 54-kilodalton polypeptide of the signal recognition particle. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8604–8608. [PMC free article] [PubMed]
  • Krishnan BR, Blakesley RW, Berg DE. Linear amplification DNA sequencing directly from single phage plaques and bacterial colonies. Nucleic Acids Res. 1991 Mar 11;19(5):1153–1153. [PMC free article] [PubMed]
  • Kulakauskas S, Wikström PM, Berg DE. Efficient introduction of cloned mutant alleles into the Escherichia coli chromosome. J Bacteriol. 1991 Apr;173(8):2633–2638. [PMC free article] [PubMed]
  • Kurzchalia TV, Wiedmann M, Girshovich AS, Bochkareva ES, Bielka H, Rapoport TA. The signal sequence of nascent preprolactin interacts with the 54K polypeptide of the signal recognition particle. Nature. 1986 Apr 17;320(6063):634–636. [PubMed]
  • Lanzer M, Bujard H. Promoters largely determine the efficiency of repressor action. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8973–8977. [PMC free article] [PubMed]
  • Lerner CG, Inouye M. Low copy number plasmids for regulated low-level expression of cloned genes in Escherichia coli with blue/white insert screening capability. Nucleic Acids Res. 1990 Aug 11;18(15):4631–4631. [PMC free article] [PubMed]
  • Médigue C, Viari A, Hénaut A, Danchin A. Escherichia coli molecular genetic map (1500 kbp): update II. Mol Microbiol. 1991 Nov;5(11):2629–2640. [PubMed]
  • Mullis KB, Faloona FA. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 1987;155:335–350. [PubMed]
  • Nierhaus KH. The assembly of prokaryotic ribosomes. Biochimie. 1991 Jun;73(6):739–755. [PubMed]
  • Nomura M, Gourse R, Baughman G. Regulation of the synthesis of ribosomes and ribosomal components. Annu Rev Biochem. 1984;53:75–117. [PubMed]
  • Persson BC, Gustafsson C, Berg DE, Björk GR. The gene for a tRNA modifying enzyme, m5U54-methyltransferase, is essential for viability in Escherichia coli. Proc Natl Acad Sci U S A. 1992 May 1;89(9):3995–3998. [PMC free article] [PubMed]
  • Phillips GJ, Silhavy TJ. The E. coli ffh gene is necessary for viability and efficient protein export. Nature. 1992 Oct 22;359(6397):744–746. [PubMed]
  • Poritz MA, Bernstein HD, Strub K, Zopf D, Wilhelm H, Walter P. An E. coli ribonucleoprotein containing 4.5S RNA resembles mammalian signal recognition particle. Science. 1990 Nov 23;250(4984):1111–1117. [PubMed]
  • Regalado A. Commotion over E. coli project. Science. 1995 Mar 31;267(5206):1899–1900. [PubMed]
  • Riley M. Functions of the gene products of Escherichia coli. Microbiol Rev. 1993 Dec;57(4):862–952. [PMC free article] [PubMed]
  • Römisch K, Webb J, Herz J, Prehn S, Frank R, Vingron M, Dobberstein B. Homology of 54K protein of signal-recognition particle, docking protein and two E. coli proteins with putative GTP-binding domains. Nature. 1989 Aug 10;340(6233):478–482. [PubMed]
  • Russell DR, Bennett GN. Construction and analysis of in vivo activity of E. coli promoter hybrids and promoter mutants that alter the -35 to -10 spacing. Gene. 1982 Dec;20(2):231–243. [PubMed]
  • Rydén-Aulin M, Shaoping Z, Kylsten P, Isaksson LA. Ribosome activity and modification of 16S RNA are influenced by deletion of ribosomal protein S20. Mol Microbiol. 1993 Mar;7(6):983–992. [PubMed]
  • Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350–1354. [PubMed]
  • Shapiro J, Machattie L, Eron L, Ihler G, Ippen K, Beckwith J. Isolation of pure lac operon DNA. Nature. 1969 Nov 22;224(5221):768–774. [PubMed]
  • Stöffler G, Noah M, Stöffler-Meilicke M, Dabbs ER. The localization of protein L19 on the surface of 50 S subunits of Escherichia coli aided by the use of mutants lacking protein L19. J Biol Chem. 1984 Apr 10;259(7):4521–4526. [PubMed]
  • Tai PC, Kessler DP, Ingraham J. Cold-sensitive mutations in Salmonella typhimurium which affect ribosome synthesis. J Bacteriol. 1969 Mar;97(3):1298–1304. [PMC free article] [PubMed]
  • Tsai MM, Wong RY, Hoang AT, Deonier RC. Transposition of Tn1000: in vivo properties. J Bacteriol. 1987 Dec;169(12):5556–5562. [PMC free article] [PubMed]
  • Vieira J, Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. [PubMed]
  • VOGEL HJ, BONNER DM. Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem. 1956 Jan;218(1):97–106. [PubMed]
  • Walter P, Blobel G. Purification of a membrane-associated protein complex required for protein translocation across the endoplasmic reticulum. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7112–7116. [PMC free article] [PubMed]
  • Wikström PM, Björk GR. Noncoordinate translation-level regulation of ribosomal and nonribosomal protein genes in the Escherichia coli trmD operon. J Bacteriol. 1988 Jul;170(7):3025–3031. [PMC free article] [PubMed]
  • Wikström PM, Björk GR. A regulatory element within a gene of a ribosomal protein operon of Escherichia coli negatively controls expression by decreasing the translational efficiency. Mol Gen Genet. 1989 Nov;219(3):381–389. [PubMed]
  • Wikström PM, Byström AS, Björk GR. Non-autogenous control of ribosomal protein synthesis from the trmD operon in Escherichia coli. J Mol Biol. 1988 Sep 5;203(1):141–152. [PubMed]
  • Zumstein L, Wang JC. Probing the structural domains and function in vivo of Escherichia coli DNA topoisomerase I by mutagenesis. J Mol Biol. 1986 Oct 5;191(3):333–340. [PubMed]

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


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...