EVOLUTION OF DNA UPTAKE IN HAEMOPHILUS AND NEISSERIA

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DESCRIPTION (Adapted from the Applicant's Abstract): This project centers on DNA uptake sequences (USS) in Haemophilus influenzae. It includes a mixture of modeling experiments, genomic analyses and experimental work. Modeling will attempt to answer the question whether the benefits expected from the acquisition of nucleotides from other bacteria are sufficient to justify the evolution of bacterial competence for DNA transformation. In a separate approach, a model will be developed to test whether a receptor for DNA transformation with a sequence bias will result in the accumulation of USS sequences in the genome. Modeling will also examine the distribution of USS around the genome and attempt to determine whether it is random or not. Genomic analyses will be performed with genome sequences of Actinobacillus actinomycetemcomitans, Neisseria gonorrhoeae and Neisseria meningitidis to determine whether the same patterns of organization of USS apply to these organisms as to H. influenzae. These analyses will distinguish between single and repeated copies of USS and their distribution within and between ORFs. The genomes will also be scanned to determine whether they contain genes that are unusually homologous across species barriers. Experimental work will test whether killing by DNA transformation is restricted to H. influenzae strain Rd in order to determine whether lethality is a common phenomenon. Mucus contains DNA and therefore analyses will determine whether DNA can be taken up from mucus or not and whether the mucus that H. influenzae normally lives in inhibits transformation or not. The specificity of the USS will be tested by synthesizing 50mer USS that differ by one bp at each of 27 positions from the consensus sequence and testing them for uptake and ability to inhibit DNA uptake. Larger DNA fragments that differ in sequence will also be obtained by PCR amplification and tested. The absolute necessity of an USS for DNA uptake will be tested using longer incubation times than formerly used. Attempts will be made to identify a USS-binding protein on the bacterial cell surface and intracellularly. If a protein can be identified, it will be sequenced. Attempts will also be made to determine whether the USS are protected in nucleoids.