ABSTRACT:
Haemophilus influenzae Rd strains express specialized proteins that allow cells to bind and take up large pieces of DNA from the environment, a process called natural genetic competence. Natural competence, should not be confused with induced competence. (e.g., competent Escherichia coli cells used for cloning), which results from the chemical or electrical treatment of cells. Rd strains that contain the H. influenzae conjugative plasmid pRI234 or the H. influenzae bacteriophage N3 as a astable lysogen cannot be transformed with chromosomal DNA, phage DNA, or plasmid DNA. The gene designated as dunA (dun = DNA uptake negative), on the conjugative plasmid pRI234 and the gene (dunB) on the bacteriophage N3 that interfer with DNA-uptake by genetically competent H. influenzae Rd cells have been cloned, sequencedand characterized. DunA codes for a 16.3 kDa polypeptide (DUNA) of 144 amino acids. DunB codes for a 15.0 kDa protein (DUNB) composed of 135 amino acids. Comparisons of DunA and DunB to protein databases indicate that both DunA and DunB appear to be novel, low-molecular weight protein tyrosine phosphatases (LMW-PTPases). Following this lead, both genes were cloned into the expression vector pProEX-HTc (Life Technologies ) and expressed. The result recombinant proteins (DunA and DunB) were able to dephosphorylate the chemically synthesized tyrosine (Y) phosphopeptide DADE(pY)LIPQQG (Tyrosine Phosphatase Assay System, Promega Corporation), which confirms the comparison data.
Indication of competence in H. influenzae is accompanied by generation of membrane extensions called tranformosomes that facilitate DNA-uptake. Although cells containing either dunA or dunB appear to make transformosomes when subjected to a competence-inducing regime, analysis of the protein composition of these cells' membranes reveals that they are incomplete, lacking the same competence-specific 32kDa protein(s). It is possiblem that one or more of these proteins (if there are more than one) are part of the transformosome structure. Furthermore, a 32-dDa transformosome polypeptide which binds to both single-stranded and double-stranded DNA was isolated by Kahn and Smith (*). Such a protein might be a candidate for the as yet elusive competence-specific DNA-uptake receptor and its absence from the transformosome would prevent cells from taking up DNA, accounting for the DNA-uptake-negative phenotype of cells harboring dunA or dunB.
*Kahn, M. E., F. Barany, and H. O. Smith. 1983. Transformasomes: specialized membranous structures that protect DNA during Haemophilus transformation. Proc Natl Acad Sci U S A. 80(22):6027-31.
*Kahn, M. E., and H. O. Smtih. 1984.
Transformation
in Haemophilus: a problem in membrane biology. J
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