Structural organization, nucleotide sequence, and regulation of the Haemophilus influenzae rec-1+ gene

Abstract

The Haemophilus influenzae rec-1+ protein plays a central role in DNA metabolism, participating in general homologous recombination, recombinational (postreplication) DNA repair, and prophage induction. Although many H. influenzae rec-1 mutants have been phenotypically characterized, little is known about the rec-1+ gene at the molecular level. In this study, we present the genetic organization of the rec-1+ locus, the DNA sequence of rec-1+, and studies of the transcriptional regulation of rec-1+ during cellular assault by DNA-damaging agents and during the induction of competence for genetic transformation. Although little is known about promoter structure in H. influenzae, we identified a potential rec-1+ promoter that is identical in 11 of 12 positions to the bacterial sigma 70-dependent promoter consensus sequence. Results from a primer extension analysis revealed that the start site of rec-1+ transcription is centered 6 nucleotides downstream of this promoter. We identified potential DNA binding sites in the rec-1+ gene for LexA, integration host factor, and cyclic AMP receptor protein. We obtained evidence that at least one of the proposed cyclic AMP receptor protein binding sites is active in modulating rec-1+ transcription. This finding makes rec-1+ control circuitry novel among recA+ homologs. Two H. influenzae DNA uptake sequences that may function as a transcription termination signal were identified in inverted orientations at the end of the rec-1+ coding sequence. In addition, we report the first use of the Escherichia coli lacZ operon fusion technique in H. influenzae to study the transcriptional control of rec-1+. Our results indicate that rec-1+ is transcriptionally induced about threefold during DNA-damaging events. Furthermore, we show that rec-1+ can substitute for recA+ in E. coli to modulate SOS induction of dinB1 expression. Surprisingly, although 5% of the H. influenzae genome is in the form of single-stranded DNA during competence for genetic transformation, an event that could be a potent SOS-inducing signal, we failed to detect significant changes in rec-1+ transcription during the induction of genetic competence.

The expression of the Acinetobacter calcoaceticus recA gene increases in response to DNA damage independently of RecA and of development of competence for natural transformation

“…It was found that in a wild-type A. calcoaceticzls background, the E. coli recA gene is induced twofold upon treatment with mitomycin C, which is comparable to our results. The authors' conclusion that this indicates that A. calcoaceticzls possesses a LexA-dependent SOS induction mechanism, comparable to the E. coli mechanism, has to be questioned on the basis of the RecAindependent induction, as we report in this study (and because (Zulty & Barcak, 1993) Multiple copies of the recA promoter region (introduced via pARA11) appear to titrate a repressor from the chromosomal recA promoter, This titration effect of the recA promoter is not recognizable in a comparison of AAC406 and AAC407. We interpret this latter observation, however, as a consequence of differences in mRNA stability (see below).…”

“…The typical SOS response of E. coli (Little & Mount, 1982), but e.g. also of B. subtilis (Cheo etal., 1991), P. stutperi (Vosman e t al., 2993) and H. injuenpae (Zulty & Barcak, 1993), is dependent on a functional RecA protein.…”

“…One of these strains, AAC406, exhibits a RecA-phenotype, due to disruption of the recA gene by the lacZ insertion (Fig. la) , 1993;Zulty & Barcak, 1993). When the p-galactosidase level of cultures of strains AAC406 and AAC407 was followed with time during growth in batch culture (Fig.…”

Induction of the SOS regulon of Haemophilus influenzae does not affect phase variation rates at tetranucleotide or dinucleotide repeats

“…In E. coli, only 5-10 % of the recA transcripts are recA-recX co-transcripts, because the majority of transcripts terminate at a palindromic repeat sequence that is located between these two genes (Pagès et al, 2003). Whilst we have not accurately determined the relative levels of the recA and recX transcripts, it is interesting to note that there are two inverted copies of the H. influenzae uptake sequence 19 bp 39 of the H. influenzae recA stop codon, which could form a stem-loop and thereby terminate transcription between recA and recX (Zulty & Barcak, 1993). Additionally, it was recently shown that the E. coli RecX competes with DinI (an E. coli SOS-regulated protein that is absent from the H. influenzae genome) to modulate RecA activity; RecX being a negative regulator and DinI positive (Lusetti et al, 2004).…”

“…Only three genes (lexA, recA and recN) were found to have an SOS box present in the promoter region in all the organisms analysed, indicating that, although the SOS box sequence and RecA/LexA controlling mechanism are conserved within this subclass, the number of genes included in the LexA regulon varies considerably such that individual species may differ in the way in which they survive and repair DNA damage. Zulty & Barcak (1993) showed that the recA gene of H. influenzae was induced approximately 3-fold in response to SOS-activating signals, but no detailed studies of the LexA regulon of H. influenzae have been performed. Some differences between the SOS regulons of H. influenzae and E. coli are apparent.…”