Extended −10 Promoter in ISAba-1 Upstream of bla_OXA-23 from Acinetobacter baumannii

Molecular studies showed that carbapenem-resistant Acinetobacter baumannii strains isolated from patients in hospitals in Cape Town contain bla_OXA-23 (7). Concordant with the findings of others (4, 11), PCR assays showed that of the 43 strains investigated, bla_OXA-23 was consistently associated with ISAba-1. One strain, designated RAM (8), was selected to identify the transcription promoter located upstream of bla_OXA-23.

Total RNA was extracted from strain RAM using the hot acid phenol method (1), and primer extension studies were carried out (9). The primer extension products were analyzed in conjunction with products of sequencing reactions performed on the corresponding DNA. The extension product mapped to a T 63 nucleotides upstream of the bla_OXA-23 start codon (Fig. 1). The hexamers TTAGAA (−35) and TTATTT (−10) are upstream of this start site. The sequence (TGACA) immediately upstream of the −10 box generates an extended −10 hexamer that shows similarity to sequences recognized by the σ^S subunit of RNA polymerase (5, 6, 12), chief regulator of the general stress response in Escherichia coli (5, 12, 13). Further, a putative distal UP element half site (GTATTTGTTT) and a CAP site, thought to play a role in σ^S selectivity (3, 12, 13), are upstream of the −10 box. It is noteworthy that RAM contains at least two other ISAba-1-linked resistance genes (sul2 and ampC). Primer extension studies indicated that the extended promoter also drives transcription of sul2 (2). Albeit in a different strain of A. baumannii, this promoter was identified upstream of ampC (9) and it is assumed to be transcriptionally active with respect to ampC in strain RAM. The copy number and implied mobility of ISAba-1 in Acinetobacter spp. (8), combined with its repertoire of regulatory sequences, suggest that this element may play a significant role in controlling the expression of a variety of genes in Acinetobacter, even within a single strain of this organism.

FIG. 1.

Nucleotide sequence upstream of bla_OXA-23 indicating transcription start sites (vertical arrows) of bla_OXA-23 in A. baumannii (RAM) (↓) and E. coli(pRK001) (↑). Major transcription start sites are in bold. The promoters are boxed and double underlined. The sequence (underlined) upstream of TTATTT creates an extended −10 promoter. The bla_OXA-23 start codon is boxed and shaded, and the left inverted repeat of ISAba-1 is in italics. The CAP site and UP element half site are underlined with dashed and dotted lines, respectively.

As transcription signals used in Acinetobacter are different from their counterparts in E. coli (10), primer extension studies were carried out to study the expression of bla_OXA-23 in E. coli. The functional bla_OXA-23 gene was amplified from RAM; cloned into pGEM-T EASY, generating pRK001; and expressed in E. coli JM109. Using RNA from E. coli(pRK001), five transcription start sites were identified (Fig. 1), corresponding to two major and three minor peaks. One of the major peaks mapped to a T which equates to the transcription start site identified upstream of bla_OXA-23 in strain RAM; presumably, the transcript in E. coli(pRK001) also emanates from the extended −10 promoter. A different ISAba-1 promoter is present upstream of the second major start site (Fig. 1). These data indicate that although A. baumannii and E. coli possess related transcription machinery, at least one of the ISAba-1-located promoters is more active in E. coli than in A. baumannii.