The following proposal for a Medical Research Council of Canada Studentship has been approved.
Consequently we now have a 'Studentship Voucher' which will entitle the student who undertakes this project to a MRC Studentship, providing up to five years of support.
More information about MRC Studentships is provided in MRC's Grants and Awards Guide.
Summary:
Our ability to investigate the DNA sequence specificity of the Haemophilus influenzae DNA uptake pathway is enhanced by the recent release of the complete genome sequence 5, and the analysis of uptake signal sequence (USS) repeats in the genome 9. However the extensive USS database is not matched by information about the DNA binding and uptake machinery, nor about the significance of sequence-specific DNA uptake in the human host. The holder of this Studentship will investigate the molecular basis of the sequence bias, and will characterize the effect of the USS on uptake of human and H. influenzae DNAs under conditions approximating those in respiratory mucosa.
Background
Although competent cells of gram-positive bacteria will bind and take up all double-stranded DNAs equally well, competent H. influenzae cells efficiently bind only DNAs from their own or a closely related species. H. influenzae DNA is favoured not because of its methylation state or other modification, but because it is highly enriched for a short sequence preferentially bound by competent cells. This uptake signal sequence (the USS) was initially identified by its presence on cloned DNA fragments, and its role in DNA binding confirmed by footprinting experiments and more DNA consensus analysis 4 7. These experiments identified the 9bp sequence AAGTGCGGT as the core USS, and implicated AT-rich flanking sequences in the binding. Experiments in which fragments with a known density of USSs competed with total genomic DNA for uptake by competent cells indicated that the H. influenzae genome contained 500 or more copies of the USS 6; about 8 copies would be expected if the sequence occurred randomly in the genome..
Because the biological role of DNA uptake has been thought to be the production of new genotypes by homologous recombination, the uptake specificity has been considered as an adaptation ensuring that competent cells did not waste efforts taking up DNA fragments from unrelated genomes. An unexpected finding was that the genomes of some other Haemophilus species, and of some species of the related genera Pasteurella and Actinobacillus, whose sequences had otherwise diverged dramatically, behaved in uptake-competition experiments as if they too had at least several hundred copies of the USS 2 3.
H. O. Smith has now analyzed the newly-available H. influenzae genome sequence for copies of the USS 5 9. He found 1465 perfect matches to the core sequence, and an additional 764 matches differing at single positions from the 9bp core sequence. Alignment of these revealed strong flanking-sequence consensus especially on one side, and agreeing well with the positions previously identified by footprinting 4.
There is no direct evidence that the USS affects any process other than DNA binding by competent cells. It has not been experimentally possible to determine whether processing or recombination of incoming DNA is influenced by the presence of a USS on the fragment. Nor is there direct evidence for any role of the USS in non-competent cells. Analysis of a functionally similar though unrelated uptake sequence in Neisseria gonorrhoeae found that these USS frequently occurred in inverted-repeat pairs downstream of open reading frames, where they might function as rho-independent transcriptional terminators 8. Only about 10% of the H. influenzae USSs occur in inverted-repeat orientation at the termini of genes; most USS occur singly, and most (65%) are within open reading frames, so any role involving stem-loop formation is likely to be secondary. Other hypothesized roles (suggested by functions of short repeated sequences in E. coli) are equally unconvincing: REP sequences must be palindromes, and Chi sequences are themselves over represented.
Experiments:
Do all parts of the USS consensus contribute to the DNA-binding specificity? One difficulty in interpreting past measurements is that the DNAs used were often undefined. For example, Albritton reported that cells will take up equivalent amounts of DNA from any source, but more time is required if the DNA is heterologous. It is not clear whether this is because the uptake system will slowly take DNAs that have no USS-like sequences, or if it must wait for the rare randomly-occurring USS-like sequences present in non-Haemophilus DNAs.
Although the genome analysis doesn't tell us directly about uptake specificity, the correspondence between the abundant sequence and what's known about the sequence bias of DNA binding gives us confidence to make predictions about the DNA binding specificity from the consensus. For example, the rightmost AT-rich region has not been directly implicated in DNA binding, but its strong conservation would suggest that it does play a role. This must be tested, because if the flanking sequences do not act in DNA binding, they must be conserved because of a binding-independent function. Consequently, one experiment will be to compare binding and uptake of core USSs with either perfect or imperfect flanking sequences.
A natural fragment containing a perfect extended USS will be identified in the genome sequence, amplified by PCR, and cloned. An imperfect derivative will be made by mutating the rightmost AT-segment to introduce several GCs. This will allow comparison of fragments differing only in the specific positions. The amount of each fragment taken up will be determined as a function of time allowed and fragment concentration, and then fragments will be tested in competition with each other.
Is the uptake bias totally independent of DNA methylation? The original experiments found that cloned H. influenzae fragments containing a USS were preferentially taken up. However, H. influenzae has two well-characterized restriction-modification systems, in addition to a number of uncharacterized DNA methylases identified in the genome sequence, and effects of methylation on uptake have not been ruled out. We will compare DNA binding/uptake between a directly PCR-amplified fragment and one purified from a H. influenzae-grown plasmid.
Experimental simulation of transformation in respiratory mucosa: H. influenzae cells are thought to normally grow in microcolonies in respiratory mucus. We do not know the extent to which DNA is transferred between cells in such conditions, although Stuy found very little exchange within mixed colonies 10, nor whether the large amount of DNA in mucus can be taken up by cells within microcolonies. When colonies on agar are resuspended in medium containing DNA, transformation frequencies are about 10-5 1 To simulate competence development and DNA uptake by microcolonies in respiratory mucus, artificial microcolonies of mixed genotypes will be overlaid with agar containing DNA with a different marker, or heterologous DNA.
Analysis of the genes and proteins responsible for sequence-specific DNA uptake: This is a very important goal, but not easily attained. No mutations directly affecting DNA binding or the first stage of uptake have been identified, although several genes are known that regulate of competence, and several more that are required for post-uptake processing. We have no immediate plans to seek these genes, but we hope they will be found soon. [Whether this project includes a search for this gene or genes will depend on the student's interests and on what is being undertaken by the few other labs interested in this problem.]
References:
1. Williams, P. M., L. A. Bannister and R. J. Redfield. 1994. The Haemophilus influenzae sxy-1 mutation is in a newly identified gene essential for competence. J. Bacteriol. 176:6789-6794.
2. Albritton, W. L. 1982. Infections due to Haemophilus species other than H. influenzae. Ann. Rev. Microbiol. 36:199-216.
3. Albritton, W. L., J. K. Setlow, M. Thomas, F. Sottnek and A. G. Steigerwalt. 1984. Heterospecific transformation in the genus Haemophilus. Molecular & General Genetics 193:358-63.
4. Danner, D. B., R. A. Deich, K. L. Sisco and H. O. Smith. 1980. An eleven-base-pair sequence determines the specificity of DNA uptake in Haemophilus transformation. Gene 11:311-8.
5. Fleischmann, R. D. a. 3. o. 1995. Whole-genome random sequencing and
assembly of Haemophilus influenzae Rd. Science 269:496-512.
The Institute for Genomic Research (TIGR)
6. Goodgal, S. H. 1982. DNA uptake in Haemophilus transformation. [Review]. Annual Review of Genetics 16:169-92.
7. Goodgal, S. H. and M. A. Mitchell. 1990. Sequence and uptake specificity of cloned sonicated fragments of Haemophilus influenzae DNA. Journal of Bacteriology 172:5924-8.
8. Goodman, S. D. and J. J. Scocca. 1988. Identification and arrangement of the DNA sequence recognized in specific transformation of Neisseria gonorrhoeae. Proceedings of the National Academy of Sciences of the United States of America 85:6982-6.
9. Smith, H. O., J.-F. Tomb, B. A. Dougherty, R. D. Fleischmann and J. C. Venter. 1995. Frequency and distribution of DNA uptake signal sequences in the Haemophilus influenzae Rd genome. Science 269:538-540.
10. Stuy, J. H. 1985. Transfer of genetic information within a colony of Haemophilus influenzae. Journal of Bacteriology 162:1-4.
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