The gene coding for 3-deoxy-manno-octulosonic acid transferase and the rfaQ gene are transcribed from divergently arranged promoters in Escherichia coli

Abstract

The gene kd&4 in Escherichia coli codes for 3-deoxy-D-manno-octulosonic acid transferase, the enzyme responsible for attachment of the two 3-deoxy-D-manno-octulosonic acid residues that constitute the link between lipid A and the core oligosaccharide of the lipopolysaccharide. Cloning and subsequent sequencing of the region upstream of kdt4 revealed an open reading frame identified as the first gene (fiaQ) in an ia gene cluster. The kdt4 and raQ transcripts were identified, and the 5' ends of the transcripts were mapped by primer extension. Two main, divergently arranged promoters were found. These promoters generated transcripts with 5' ends separated by 289 bases. That the two divergent transcripts from the identified promoters represent the kdt4 and rfaQ transcripts was confirmed by fusing different parts of the intergenic region between the promoterless lacZ and phoA genes in promoter-screening plasmid pCB267.The anionic sugar 3-deoxy-D-manno-octulosonic acid (KDO) is an essential component of the lipopolysaccharide (LPS) in Escherichia coli and probably in most other gramnegative bacteria as well (21). As the proximal part of the inner core oligosaccharide, it links the polysaccharide portion of LPS to the membrane-anchored lipid A. Two molecules of KDO are sequentially transferred to lipid A by a KDO transferase with the activated sugar CMP-KDO as a substrate (6). The gene coding for the (KDO)2 transferase (kdtA) has been cloned, sequenced, and shown to code for a 43-kDa polypeptide (9). The incorporation in extracts of E. coli of the two stereochemically distinct KDO residues is stimulated about 100-fold when kdtA is expressed from a high-copy-number plasmid. It is thus strongly believed that kdtA encodes both the first and the second KDO Bacterial strains and growth conditions. Cultures were grown in Luria broth, consisting of 10 g of NaCl, 5 g of yeast extract, and 10 g of tryptone per liter, or in minimal medium A (2). Required amino acids were added to minimal medium A at 40 mg/liter, and carbon sources were used at 0.2%

Regulatory Architecture of the Iron-Regulated fepD-ybdA Bidirectional Promoter Region in Escherichia coli

“…It is predicted to have the potential of forming a stem-loop structure which (with some transcripts) may effectively sequester the lacZ ribosome binding site, leading to poor translation initiation (data not shown). Several other studies using this same and related vectors have reported low levels of enzyme activity (3,12,19,53). However, despite the low ␤-galactosidase reporter activity in these constructs, sequence modifications to the potential control elements led to changes in these activity levels that were consistent with changes in the actual transcript levels measured by primer extension.…”

RfaH and the ops element, components of a novel system controlling bacterial transcription elongation

“…Arakawa et al (1995) identified a weak homology between the JUMPstart sequence and a 54specific promoter sequence and suggested that it acts as a promoter. This is unlikely as the ops element is unable to direct transcription without an upstream promoter (Nieto et al, 1996) and, where transcript initiation sites of RfaHdependent operons have been mapped, the ops element lies in a non-translated leader sequence upstream of the first gene (Clementz, 1992;Leeds and Welch, 1996;Stevens et al, 1997).…”

GadE (YhiE): a novel activator involved in the response to acid environment in Escherichia coli

“…To determine the impact of GadE in such pathways, we analysed the DNA-binding properties of GadE to the promoter region of several gene targets whose products are involved in each pathway. The regulatory region of some of them has been described in detail previously: gadA (Castanie-Cornet De Biase et al, 1999), gadX (Tramonti et al, 2002), hdeAB (Arnqvist et al, 1994;Tucker et al, 2003), gltBD (Wiese et al, 1997), osmC (Bouvier et al, 1998), rcsA (Stout, 1996), rfaQGPSBIJYZK (Clementz, 1992) and ompC (Norioka et al, 1986). We characterized here the promoter region of hdeD.…”