Skip to main content
NCBI home page
Infection and Immunity logoLink to Infection and Immunity
. 1997 Dec;65(12):5017–5027. doi: 10.1128/iai.65.12.5017-5027.1997

Variable number of tandem repeats in clinical strains of Haemophilus influenzae.

A van Belkum 1, S Scherer 1, W van Leeuwen 1, D Willemse 1, L van Alphen 1, H Verbrugh 1
PMCID: PMC175724  PMID: 9393791

Abstract

An algorithm capable of identifying short repeat motifs was developed and used to screen the whole genome sequence available for Haemophilus influenzae, since some of these repeats have been shown to affect bacterial virulence. Various di- to hexanucleotide repeats were identified, confirming and extending previous findings on the existence of variable-number-of-tandem-repeat loci (VNTRs). Repeats with units of 7 or 8 nucleotides were not encountered. For all of the 3- to 6-nucleotide repeats in the H. influenzae chromosome, PCR tests capable of detecting allelic polymorphisms were designed. Fourteen of 18 of the potential VNTRs were indeed highly polymorphic when different strains were screened. Two of the potential VNTRs appeared to be short and homogeneous in length; another one may be specific for the H. influenzae Rd strain only. One of the primer sets generated fingerprint-type DNA banding patterns. The various repeat types differed with respect to intrinsic stability as well. It was noted for separate colonies derived from a single clinical specimen or strains passaged for several weeks on chocolate agar plates that the lengths of the VNTRs did not change. When several strains from different patients infected during an outbreak of lung disease were analyzed, increased but limited variation was encountered in all VNTR sites analyzed. One of the 5-nucleotide VNTRs proved to be hypervariable. This variability may reflect the molecular basis of a mechanism used by H. influenzae bacteria to successfully colonize and infect different human individuals.

Full Text

The Full Text of this article is available as a PDF (2.1 MB).

Selected References

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

  1. Asayama M., Kabasawa M., Takahashi I., Aida T., Shirai M. Highly repetitive sequences and characteristics of genomic DNA in unicellular cyanobacterial strains. FEMS Microbiol Lett. 1996 Apr 1;137(2-3):175–181. doi: 10.1111/j.1574-6968.1996.tb08102.x. [DOI] [PubMed] [Google Scholar]
  2. Boom R., Sol C. J., Salimans M. M., Jansen C. L., Wertheim-van Dillen P. M., van der Noordaa J. Rapid and simple method for purification of nucleic acids. J Clin Microbiol. 1990 Mar;28(3):495–503. doi: 10.1128/jcm.28.3.495-503.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bult C. J., White O., Olsen G. J., Zhou L., Fleischmann R. D., Sutton G. G., Blake J. A., FitzGerald L. M., Clayton R. A., Gocayne J. D. Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii. Science. 1996 Aug 23;273(5278):1058–1073. doi: 10.1126/science.273.5278.1058. [DOI] [PubMed] [Google Scholar]
  4. Caskey C. T., Pizzuti A., Fu Y. H., Fenwick R. G., Jr, Nelson D. L. Triplet repeat mutations in human disease. Science. 1992 May 8;256(5058):784–789. doi: 10.1126/science.1589758. [DOI] [PubMed] [Google Scholar]
  5. Cookson B. D., Aparicio P., Deplano A., Struelens M., Goering R., Marples R. Inter-centre comparison of pulsed-field gel electrophoresis for the typing of methicillin-resistant Staphylococcus aureus. J Med Microbiol. 1996 Mar;44(3):179–184. doi: 10.1099/00222615-44-3-179. [DOI] [PubMed] [Google Scholar]
  6. Elena S. F., Cooper V. S., Lenski R. E. Punctuated evolution caused by selection of rare beneficial mutations. Science. 1996 Jun 21;272(5269):1802–1804. doi: 10.1126/science.272.5269.1802. [DOI] [PubMed] [Google Scholar]
  7. Field D., Wills C. Long, polymorphic microsatellites in simple organisms. Proc Biol Sci. 1996 Feb 22;263(1367):209–215. doi: 10.1098/rspb.1996.0033. [DOI] [PubMed] [Google Scholar]
  8. Fleischmann R. D., Adams M. D., White O., Clayton R. A., Kirkness E. F., Kerlavage A. R., Bult C. J., Tomb J. F., Dougherty B. A., Merrick J. M. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science. 1995 Jul 28;269(5223):496–512. doi: 10.1126/science.7542800. [DOI] [PubMed] [Google Scholar]
  9. Fraser C. M., Gocayne J. D., White O., Adams M. D., Clayton R. A., Fleischmann R. D., Bult C. J., Kerlavage A. R., Sutton G., Kelley J. M. The minimal gene complement of Mycoplasma genitalium. Science. 1995 Oct 20;270(5235):397–403. doi: 10.1126/science.270.5235.397. [DOI] [PubMed] [Google Scholar]
  10. Frénay H. M., Bunschoten A. E., Schouls L. M., van Leeuwen W. J., Vandenbroucke-Grauls C. M., Verhoef J., Mooi F. R. Molecular typing of methicillin-resistant Staphylococcus aureus on the basis of protein A gene polymorphism. Eur J Clin Microbiol Infect Dis. 1996 Jan;15(1):60–64. doi: 10.1007/BF01586186. [DOI] [PubMed] [Google Scholar]
  11. Frénay H. M., Theelen J. P., Schouls L. M., Vandenbroucke-Grauls C. M., Verhoef J., van Leeuwen W. J., Mooi F. R. Discrimination of epidemic and nonepidemic methicillin-resistant Staphylococcus aureus strains on the basis of protein A gene polymorphism. J Clin Microbiol. 1994 Mar;32(3):846–847. doi: 10.1128/jcm.32.3.846-847.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Goh S. H., Byrne S. K., Zhang J. L., Chow A. W. Molecular typing of Staphylococcus aureus on the basis of coagulase gene polymorphisms. J Clin Microbiol. 1992 Jul;30(7):1642–1645. doi: 10.1128/jcm.30.7.1642-1645.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Heath D. G., An F. Y., Weaver K. E., Clewell D. B. Phase variation of Enterococcus faecalis pAD1 conjugation functions relates to changes in iteron sequence region. J Bacteriol. 1995 Oct;177(19):5453–5459. doi: 10.1128/jb.177.19.5453-5459.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. High N. J., Deadman M. E., Moxon E. R. The role of a repetitive DNA motif (5'-CAAT-3') in the variable expression of the Haemophilus influenzae lipopolysaccharide epitope alpha Gal(1-4)beta Gal. Mol Microbiol. 1993 Sep;9(6):1275–1282. doi: 10.1111/j.1365-2958.1993.tb01257.x. [DOI] [PubMed] [Google Scholar]
  15. Hood D. W., Deadman M. E., Jennings M. P., Bisercic M., Fleischmann R. D., Venter J. C., Moxon E. R. DNA repeats identify novel virulence genes in Haemophilus influenzae. Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):11121–11125. doi: 10.1073/pnas.93.20.11121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jeffreys A. J., Wilson V., Thein S. L., Weatherall D. J., Ponder B. A. DNA "fingerprints" and segregation analysis of multiple markers in human pedigrees. Am J Hum Genet. 1986 Jul;39(1):11–24. [PMC free article] [PubMed] [Google Scholar]
  17. Jordens J. Z., Leaves N. I., Anderson E. C., Slack M. P. Polymerase chain reaction-based strain characterization of noncapsulate Haemophilus influenzae. J Clin Microbiol. 1993 Nov;31(11):2981–2987. doi: 10.1128/jcm.31.11.2981-2987.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Karlin S., Mrázek J., Campbell A. M. Frequent oligonucleotides and peptides of the Haemophilus influenzae genome. Nucleic Acids Res. 1996 Nov 1;24(21):4263–4272. doi: 10.1093/nar/24.21.4263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Koeuth T., Versalovic J., Lupski J. R. Differential subsequence conservation of interspersed repetitive Streptococcus pneumoniae BOX elements in diverse bacteria. Genome Res. 1995 Nov;5(4):408–418. doi: 10.1101/gr.5.4.408. [DOI] [PubMed] [Google Scholar]
  20. Landgraf I. M., Vieira M. F. Biotypes and serotypes of Haemophilus influenzae from patients with meningitis in the city of São Paulo, Brazil. J Clin Microbiol. 1993 Mar;31(3):743–745. doi: 10.1128/jcm.31.3.743-745.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Levinson G., Gutman G. A. Slipped-strand mispairing: a major mechanism for DNA sequence evolution. Mol Biol Evol. 1987 May;4(3):203–221. doi: 10.1093/oxfordjournals.molbev.a040442. [DOI] [PubMed] [Google Scholar]
  22. Levy J., Verhaegen G., De Mol P., Couturier M., Dekegel D., Butzler J. P. Molecular characterization of resistance plasmids in epidemiologically unrelated strains of multiresistant Haemophilus influenzae. J Infect Dis. 1993 Jul;168(1):177–187. doi: 10.1093/infdis/168.1.177. [DOI] [PubMed] [Google Scholar]
  23. Madoff L. C., Michel J. L., Gong E. W., Kling D. E., Kasper D. L. Group B streptococci escape host immunity by deletion of tandem repeat elements of the alpha C protein. Proc Natl Acad Sci U S A. 1996 Apr 30;93(9):4131–4136. doi: 10.1073/pnas.93.9.4131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Martin B., Humbert O., Camara M., Guenzi E., Walker J., Mitchell T., Andrew P., Prudhomme M., Alloing G., Hakenbeck R. A highly conserved repeated DNA element located in the chromosome of Streptococcus pneumoniae. Nucleic Acids Res. 1992 Jul 11;20(13):3479–3483. doi: 10.1093/nar/20.13.3479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Moxon E. R., Rainey P. B., Nowak M. A., Lenski R. E. Adaptive evolution of highly mutable loci in pathogenic bacteria. Curr Biol. 1994 Jan 1;4(1):24–33. doi: 10.1016/s0960-9822(00)00005-1. [DOI] [PubMed] [Google Scholar]
  26. Peak I. R., Jennings M. P., Hood D. W., Bisercic M., Moxon E. R. Tetrameric repeat units associated with virulence factor phase variation in Haemophilus also occur in Neisseria spp. and Moraxella catarrhalis. FEMS Microbiol Lett. 1996 Mar 15;137(1):109–114. doi: 10.1111/j.1574-6968.1996.tb08091.x. [DOI] [PubMed] [Google Scholar]
  27. Peterson S. N., Bailey C. C., Jensen J. S., Borre M. B., King E. S., Bott K. F., Hutchison C. A., 3rd Characterization of repetitive DNA in the Mycoplasma genitalium genome: possible role in the generation of antigenic variation. Proc Natl Acad Sci U S A. 1995 Dec 5;92(25):11829–11833. doi: 10.1073/pnas.92.25.11829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Philipp W. J., Poulet S., Eiglmeier K., Pascopella L., Balasubramanian V., Heym B., Bergh S., Bloom B. R., Jacobs W. R., Jr, Cole S. T. An integrated map of the genome of the tubercle bacillus, Mycobacterium tuberculosis H37Rv, and comparison with Mycobacterium leprae. Proc Natl Acad Sci U S A. 1996 Apr 2;93(7):3132–3137. doi: 10.1073/pnas.93.7.3132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Roche R. J., Moxon E. R. Phenotypic variation in Haemophilus influenzae: the interrelationship of colony opacity, capsule and lipopolysaccharide. Microb Pathog. 1995 Feb;18(2):129–140. doi: 10.1016/s0882-4010(95)90117-5. [DOI] [PubMed] [Google Scholar]
  30. Rosche W. A., Jaworski A., Kang S., Kramer S. F., Larson J. E., Geidroc D. P., Wells R. D., Sinden R. R. Single-stranded DNA-binding protein enhances the stability of CTG triplet repeats in Escherichia coli. J Bacteriol. 1996 Aug;178(16):5042–5044. doi: 10.1128/jb.178.16.5042-5044.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Samuelson A., Freijd A., Jonasson J., Lindberg A. A. Turnover of nonencapsulated Haemophilus influenzae in the nasopharynges of otitis-prone children. J Clin Microbiol. 1995 Aug;33(8):2027–2031. doi: 10.1128/jcm.33.8.2027-2031.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Smith-Vaughan H. C., Sriprakash K. S., Mathews J. D., Kemp D. J. Long PCR-ribotyping of nontypeable Haemophilus influenzae. J Clin Microbiol. 1995 May;33(5):1192–1195. doi: 10.1128/jcm.33.5.1192-1195.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Stern A., Meyer T. F. Common mechanism controlling phase and antigenic variation in pathogenic neisseriae. Mol Microbiol. 1987 Jul;1(1):5–12. doi: 10.1111/j.1365-2958.1987.tb00520.x. [DOI] [PubMed] [Google Scholar]
  34. Sturm A. W., Mostert R., Rouing P. J., van Klingeren B., van Alphen L. Outbreak of multiresistant non-encapsulated Haemophilus influenzae infections in a pulmonary rehabilitation centre. Lancet. 1990 Jan 27;335(8683):214–216. doi: 10.1016/0140-6736(90)90291-c. [DOI] [PubMed] [Google Scholar]
  35. Weiser J. N., Love J. M., Moxon E. R. The molecular mechanism of phase variation of H. influenzae lipopolysaccharide. Cell. 1989 Nov 17;59(4):657–665. doi: 10.1016/0092-8674(89)90011-1. [DOI] [PubMed] [Google Scholar]
  36. Wiid I. J., Werely C., Beyers N., Donald P., van Helden P. D. Oligonucleotide (GTG)5 as a marker for Mycobacterium tuberculosis strain identification. J Clin Microbiol. 1994 May;32(5):1318–1321. doi: 10.1128/jcm.32.5.1318-1321.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. van Belkum A., Duim B., Regelink A., Möller L., Quint W., van Alphen L. Genomic DNA fingerprinting of clinical Haemophilus influenzae isolates by polymerase chain reaction amplification: comparison with major outer-membrane protein and restriction fragment length polymorphism analysis. J Med Microbiol. 1994 Jul;41(1):63–68. doi: 10.1099/00222615-41-1-63. [DOI] [PubMed] [Google Scholar]
  38. van Belkum A., Kluytmans J., van Leeuwen W., Bax R., Quint W., Peters E., Fluit A., Vandenbroucke-Grauls C., van den Brule A., Koeleman H. Multicenter evaluation of arbitrarily primed PCR for typing of Staphylococcus aureus strains. J Clin Microbiol. 1995 Jun;33(6):1537–1547. doi: 10.1128/jcm.33.6.1537-1547.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. van Belkum A., Sluijuter M., de Groot R., Verbrugh H., Hermans P. W. Novel BOX repeat PCR assay for high-resolution typing of Streptococcus pneumoniae strains. J Clin Microbiol. 1996 May;34(5):1176–1179. doi: 10.1128/jcm.34.5.1176-1179.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. van Ham S. M., van Alphen L., Mooi F. R., van Putten J. P. Phase variation of H. influenzae fimbriae: transcriptional control of two divergent genes through a variable combined promoter region. Cell. 1993 Jun 18;73(6):1187–1196. doi: 10.1016/0092-8674(93)90647-9. [DOI] [PubMed] [Google Scholar]
  41. van Ham S. M., van Alphen L., Mooi F. R., van Putten J. P. The fimbrial gene cluster of Haemophilus influenzae type b. Mol Microbiol. 1994 Aug;13(4):673–684. doi: 10.1111/j.1365-2958.1994.tb00461.x. [DOI] [PubMed] [Google Scholar]
  42. van Soolingen D., de Haas P. E., Hermans P. W., Groenen P. M., van Embden J. D. Comparison of various repetitive DNA elements as genetic markers for strain differentiation and epidemiology of Mycobacterium tuberculosis. J Clin Microbiol. 1993 Aug;31(8):1987–1995. doi: 10.1128/jcm.31.8.1987-1995.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES