Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1995 Jun;177(11):3235–3240. doi: 10.1128/jb.177.11.3235-3240.1995

DNA sequence and characterization of Haemophilus influenzae dprA+, a gene required for chromosomal but not plasmid DNA transformation.

S Karudapuram 1, X Zhao 1, G J Barcak 1
PMCID: PMC177016  PMID: 7768823

Abstract

Natural genetic transformation in Haemophilus influenzae involves DNA binding, uptake, translocation, and recombination. In this study, we cloned and sequenced a 3.8-kbp H. influenzae DNA segment capable of complementing in trans the transformation defect of an H. influenzae strain carrying the tfo-37 mutation. We used subcloning, deletion analysis, and in vivo protein labeling experiments to more precisely define the gene required for efficient DNA transformation on the cloned DNA. A novel gene, which we called dprA+, was shown to encode a 41.6-kDa polypeptide that was required for efficient chromosomal but not plasmid DNA transformation. Analysis of the deduced amino acid sequence of DprA suggested that it may be an inner membrane protein, which is consistent with its apparent role in DNA processing during transformation. Four other open reading frames (ORFs) on the cloned DNA segment were identified. Two ORFs were homologous to the phosphofructokinase A (pfkA) and alpha-isopropyl malate synthase (leuA) genes of Escherichia coli and Salmonella typhimurium, respectively. Homologs for the two other ORFs could not be identified.

Full Text

The Full Text of this article is available as a PDF (409.8 KB).

Selected References

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

  1. Barcak G. J., Chandler M. S., Redfield R. J., Tomb J. F. Genetic systems in Haemophilus influenzae. Methods Enzymol. 1991;204:321–342. doi: 10.1016/0076-6879(91)04016-h. [DOI] [PubMed] [Google Scholar]
  2. Barcak G. J., Tomb J. F., Laufer C. S., Smith H. O. Two Haemophilus influenzae Rd genes that complement the recA-like mutation rec-1. J Bacteriol. 1989 May;171(5):2451–2457. doi: 10.1128/jb.171.5.2451-2457.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chandler M. S. The gene encoding cAMP receptor protein is required for competence development in Haemophilus influenzae Rd. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1626–1630. doi: 10.1073/pnas.89.5.1626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Clifton S. W., McCarthy D., Roe B. A. Sequence of the rec-2 locus of Haemophilus influenzae: homologies to comE-ORF3 of Bacillus subtilis and msbA of Escherichia coli. Gene. 1994 Aug 19;146(1):95–100. doi: 10.1016/0378-1119(94)90840-0. [DOI] [PubMed] [Google Scholar]
  5. Dorocicz I. R., Williams P. M., Redfield R. J. The Haemophilus influenzae adenylate cyclase gene: cloning, sequence, and essential role in competence. J Bacteriol. 1993 Nov;175(22):7142–7149. doi: 10.1128/jb.175.22.7142-7149.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dreiseikelmann B. Translocation of DNA across bacterial membranes. Microbiol Rev. 1994 Sep;58(3):293–316. doi: 10.1128/mr.58.3.293-316.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. GOODGAL S. H., HERRIOTT R. M. Studies on transformations of Hemophilus influenzae. I. Competence. J Gen Physiol. 1961 Jul;44:1201–1227. doi: 10.1085/jgp.44.6.1201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gemmill R. M., Wessler S. R., Keller E. B., Calvo J. M. leu operon of Salmonella typhimurium is controlled by an attenuation mechanism. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4941–4945. doi: 10.1073/pnas.76.10.4941. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hellinga H. W., Evans P. R. Nucleotide sequence and high-level expression of the major Escherichia coli phosphofructokinase. Eur J Biochem. 1985 Jun 3;149(2):363–373. doi: 10.1111/j.1432-1033.1985.tb08934.x. [DOI] [PubMed] [Google Scholar]
  10. Herriott R. M., Meyer E. M., Vogt M. Defined nongrowth media for stage II development of competence in Haemophilus influenzae. J Bacteriol. 1970 Feb;101(2):517–524. doi: 10.1128/jb.101.2.517-524.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hui F. M., Zhou L., Morrison D. A. Competence for genetic transformation in Streptococcus pneumoniae: organization of a regulatory locus with homology to two lactococcin A secretion genes. Gene. 1995 Feb 3;153(1):25–31. doi: 10.1016/0378-1119(94)00841-f. [DOI] [PubMed] [Google Scholar]
  12. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  13. Larson T. G., Goodgal S. H. Donor DNA processing is blocked by a mutation in the com101A locus of Haemophilus influenzae. J Bacteriol. 1992 May;174(10):3392–3394. doi: 10.1128/jb.174.10.3392-3394.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Larson T. G., Goodgal S. H. Sequence and transcriptional regulation of com101A, a locus required for genetic transformation in Haemophilus influenzae. J Bacteriol. 1991 Aug;173(15):4683–4691. doi: 10.1128/jb.173.15.4683-4691.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Martinez E., Bartolomé B., de la Cruz F. pACYC184-derived cloning vectors containing the multiple cloning site and lacZ alpha reporter gene of pUC8/9 and pUC18/19 plasmids. Gene. 1988 Aug 15;68(1):159–162. doi: 10.1016/0378-1119(88)90608-7. [DOI] [PubMed] [Google Scholar]
  16. Meinnel T., Guillon J. M., Mechulam Y., Blanquet S. The Escherichia coli fmt gene, encoding methionyl-tRNA(fMet) formyltransferase, escapes metabolic control. J Bacteriol. 1993 Feb;175(4):993–1000. doi: 10.1128/jb.175.4.993-1000.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Notani N. K., Setlow J. K., McCarthy D., Clayton N. L. Transformation of Haemophilus influenzae by plasmid RSF0885. J Bacteriol. 1981 Dec;148(3):812–816. doi: 10.1128/jb.148.3.812-816.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. O'Neill M. C. Training back-propagation neural networks to define and detect DNA-binding sites. Nucleic Acids Res. 1991 Jan 25;19(2):313–318. doi: 10.1093/nar/19.2.313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Parsot C., Cossart P., Saint-Girons I., Cohen G. N. Nucleotide sequence of thrC and of the transcription termination region of the threonine operon in Escherichia coli K12. Nucleic Acids Res. 1983 Nov 11;11(21):7331–7345. doi: 10.1093/nar/11.21.7331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Perry K. L., Elledge S. J., Mitchell B. B., Marsh L., Walker G. C. umuDC and mucAB operons whose products are required for UV light- and chemical-induced mutagenesis: UmuD, MucA, and LexA proteins share homology. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4331–4335. doi: 10.1073/pnas.82.13.4331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pifer M. L. Plasmid establishment in competent Haemophilus influenzae occurs by illegitimate transformation. J Bacteriol. 1986 Nov;168(2):683–687. doi: 10.1128/jb.168.2.683-687.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Redfield R. J. sxy-1, a Haemophilus influenzae mutation causing greatly enhanced spontaneous competence. J Bacteriol. 1991 Sep;173(18):5612–5618. doi: 10.1128/jb.173.18.5612-5618.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ricca E., Calvo J. M. The nucleotide sequence of leuA from Salmonella typhimurium. Nucleic Acids Res. 1990 Mar 11;18(5):1290–1290. doi: 10.1093/nar/18.5.1290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sisco K. L., Smith H. O. Sequence-specific DNA uptake in Haemophilus transformation. Proc Natl Acad Sci U S A. 1979 Feb;76(2):972–976. doi: 10.1073/pnas.76.2.972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  27. Stuy J. H., Walter R. B. Effect of glycerol on plasmid transfer in genetically competent Haemophilus influenzae. Mol Gen Genet. 1986 May;203(2):296–299. doi: 10.1007/BF00333969. [DOI] [PubMed] [Google Scholar]
  28. Stuy J. H., Walter R. B. Homology-facilitated plasmid transfer in Haemophilus influenzae. Mol Gen Genet. 1986 May;203(2):288–295. doi: 10.1007/BF00333968. [DOI] [PubMed] [Google Scholar]
  29. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Tomb J. F., Barcak G. J., Chandler M. S., Redfield R. J., Smith H. O. Transposon mutagenesis, characterization, and cloning of transformation genes of Haemophilus influenzae Rd. J Bacteriol. 1989 Jul;171(7):3796–3802. doi: 10.1128/jb.171.7.3796-3802.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wilcox K. W., Smith H. O. Isolation and characterization of mutants of Haemophilus influenzae deficient in an adenosine 5'-triphosphate-dependent deoxyribonuclease activity. J Bacteriol. 1975 May;122(2):443–453. doi: 10.1128/jb.122.2.443-453.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Zulty J. J., Barcak G. J. Identification of a DNA transformation gene required for com101A+ expression and supertransformer phenotype in Haemophilus influenzae. Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3616–3620. doi: 10.1073/pnas.92.8.3616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Zulty J. J., Barcak G. J. Structural organization, nucleotide sequence, and regulation of the Haemophilus influenzae rec-1+ gene. J Bacteriol. 1993 Nov;175(22):7269–7281. doi: 10.1128/jb.175.22.7269-7281.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES