New functional identity for the DNA uptake sequence in transformation and its presence in transcriptional terminators.

The frequently occurring DNA uptake sequence (DUS), recognized as a 10-bp repeat, is required for efficient genetic transformation in the human pathogens Neisseria meningitidis and Neisseria gonorrhoeae. Genome scanning for DUS occurrences in three different species of Neisseria demonstrated that 76% of the nearly 2,000 neisserial DUS were found to have two semiconserved base pairs extending from the 5' end of DUS to constitute a 12-mer repeat. Plasmids containing sequential variants of the neisserial DUS were tested for their ability to transform N. meningitidis and N. gonorrhoeae, and the 12-mer was found to outperform the 10-mer DUS in transformation efficiency. Assessment of meningococcal uptake of DNA confirmed the enhanced performance of the 12-mer compared to the 10-mer DUS. An inverted repeat DUS was not more efficient in transformation than DNA species containing a single or direct repeat DUS. Genome-wide analysis revealed that half of the nearly 1,500 12-mer DUS are arranged as inverted repeats predicted to be involved in rho-independent transcriptional termination or attenuation. The distribution of the uptake signal sequence required for transformation in the Pasteurellaceae was also biased towards transcriptional terminators, although to a lesser extent. In addition to assessing the intergenic location of DUS, we propose that the 10-mer identity of DUS should be extended and recognized as a 12-mer DUS. The dual role of DUS in transformation and as a structural component on RNA affecting transcription makes this a relevant model system for assessing significant roles of repeat sequences in biology.

The VanY(D) DD-carboxypeptidase of Enterococcus faecium BM4339 is a penicillin-binding protein.

VanD-type Enterococcus faecium BM4339 is constitutively resistant to vancomycin and to low levels of teicoplanin. This strain produces peptidoglycan precursors terminating in D-lactate but, unlike VanA- and VanB-type strains, E. faecium BM4339 has a mutated ddl ligase gene and cannot synthesize D-Ala-D-Ala. Consequently, although it possesses vanX(D) and vanY(D) genes, it should not require an active VanX-type DD-dipeptidase or a VanY-type DD-carboxypeptidase for resistance. The vanY(D) gene contains the signatures of a penicillin-binding protein (PBP) and is believed to encode a penicillin-sensitive DD-carboxypeptidase. The enzyme activity was found to be membrane-bound and inhibited by low concentrations of benzylpenicillin in membrane preparations and in intact bacteria, indicating that the active site was present on the outside surface of the membrane. The 38 kDa protein was revealed as a PBP present in more copies per cell than conventional PBPs and all the protein was accessible to benzylpenicillin added externally, confirming the localization of the active site. A glycopeptide-susceptible strain of E. faecium lacked this PBP, and the membrane-bound DD-carboxypeptidase activity was less than 5% of that of E. faecium BM4339. Although the active site of VanY(D) was external to the membrane, UDP-MurNAc-tetrapeptide was produced internally, probably from UDP-MurNAc-pentadepsipeptide. The presence of benzylpenicillin at low concentrations in the growth medium substantially reduced the amount of tetrapeptide produced, indicating that inhibition of VanY(D) by benzylpenicillin influenced production of peptidoglycan precursors internally. A model to explain these contrasting observations is proposed.