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

Abstract

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.

FIG. 1.

Nucleotide conservation outside DUS in three species of Neisseria. The DUS of N. gonorrhoeae FA1090, N. lactamica ST640, and N. menigitidis Z2491, MC58, FAM18, and 8013 all show conservation of nucleotides T in position −1 and A in position −2. Values on the y axis are bits, as defined in reference , where the maximum sequence conservation per site is log₂ 4, or 2 bits for DNA/RNA.

FIG. 2.

Elongated DUS improves efficacy of transformation. The graphs show transformation frequencies of various DNA substrates on three different strains of Neisseria meningitidis and one strain of Neisseria gonorrhoeae. Transformation frequencies were calculated by dividing the number of erythromycin-resistant CFU by the total number of CFU. Values are the averages of at least three independent experiments and standard deviations are indicated by bars. Significantly different values (by Student's t test) from p10-DUS are labeled as follows: ** P < 0.01; *, P < 0.05; #, P < 0.1.

FIG. 3.

Higher DNA uptake of p12-DUS relative to p10-DUS and p0-DUS in N. meningitidis M1080. The graph shows DNA uptake as a percentage of maximum DNA uptake in strain M1080 and the noncompetent negative control, M1080ΔpilQ. Values are means plus standard deviations (n = 3). **, significantly different (P < 0.01) from maximum uptake achieved with p12-DUS in strain M1080.

FIG. 4.

Plasmids pSingle, pDR, and pIR transform two strains of N. meningitidis equally efficiently. The graphs show transformation frequencies (10⁻⁷) of four different DNA substrates containing none (p0), a single (pSingle), a direct repeat (pDR), and an inverted repeat (pIR) of DUS on two different strains of Neisseria meningitidis. Values are the averages of at least four independent experiments, and standard deviations are indicated by the bars.