Mapping, sequence, and apparent lack of function of araJ, a gene of the Escherichia coli arabinose regulon

Abstract

We report the mapping, sequencing, and study of the physiological role of the fourth arabinose-inducible operon from Escherichia coli, araJ. It is located at 9 min on the chromosome and codes for a single 42-kDa protein that shows no significant homology to other known proteins. Destruction of the chromosomal araJ gene does not detectably affect either of the two arabinose transport systems, the ability of cells to grow The enteric bacterium Escherichia coli can utilize L-arabinose as a sole carbon source. In the presence of arabinose, expression from the three known ara operons is induced up to 300-fold. These are the araBAD operon (5, 6), which codes for proteins involved in the catabolism of arabinose, and the araE (27) and araFGH (2, 17, 31) operons, which code for the low-and high-affinity arabinose transport systems, respectively: In addition to the promoters of these three well-characterized operons, there exists a fourth promoter in E. coli that is induced by arabinose. This promoter was originally cloned by Kosiba and Schleif (18) and at that time was misidentified as the araFGH promoter. Subsequent work (11, 13) has identified the actual araFGH promoter, and the arabinose-inducible promoter cloned by Kosiba and Schleif (18) is now known as pJ. Recent work (11,18) has shown that arabinose induces substantial transcription from pJ in vivo and in vitro. In vivo, as well as in vitro (11,18), transcription from pJ is dependent upon the arabinosespecific transcriptional regulator protein AraC and the global catabolite gene activator protein CAP, as is transcription from the araBAD, araE, and araFGH operons (7,8,32).Since all known ara mutations are confined to the araBAD, araC, araE, and araFGH operons, we were interested to learn whether pJ drives an intact gene and whether its product plays any detectable role in vivo. In particular, we wished to determine whether the araJ product was a fourth component in the high-affinity arabinose transport system, since most analogous systems contain four protein components (1, 34).Here we report the mapping of the araJ gene to 9 min on the E. coli chromosome and the cloning and sequencing of the araJ gene and surrounding regions of the chromosome. We also show that insertion/deletion mutations in araJ have no detectable effect on the ability of the bacteria to grow on arabinose and to induce the araBAD operon. MATERIALS AND METHODSMedia, strains, and general methods. Media and general methods have been described (21,33 Kosiba and Schleif (18) containing the araJ promoter. Three positive clones were selected and shown by restriction mapping to contain overlapping copies of the same piece of genomic DNA; these three clones were sent to us for further analysis.One of the lambda clones was digested with PvuI, and the 1.9-and 2.5-kb fragments that contained pJ and flanking sequences were isolated. The fragments were filled out with DNA polymerase (Klenow fragment) and then ligated into 7765 JOURNAL OF BACTERIOLOGY, Dec. 1991, p. 7765-7771

Decreasing Accumulation of Acetate in a Rich Medium byEscherichia colion Introduction of Genes on a Multicopy Plasmid

“…Both the NagC and putative Mlc proteins may influence the acetate metabolism of the host strain in a similar manner. Although the ORF gene downstream of the araJ gene 28 ) did not have this property, making large colonies, there is a possibility that this gene is not transcribed (the 5 f -region has not been sequenced).…”

“…The NagC protein, the regulator of the nag operon encoding proteins involved in the use of N-acetylglucosamine, 27) the ORF (the sequence published is incomplete and the function is unknown) downstream of the araJ gene 28 ) in E. coli, and the XylR protein, the regulator of the xyl operon encoding proteins involved in the use of xylose, in Bacillus subtilis 29 ) were found to have high homologies with the putative Mlc protein (Fig. 5).…”

In vivo induction kinetics of the arabinose promoters in Escherichia coli

“…These facts suggest that specific contacts are made between the AraC protein and RNA polymerase at p BAD . Providing further support for this theory is the almost identical arrangement of the protein binding sites for araBAD, araE, and araJ, a weak, arabinose-inducible promoter whose gene product is nonessential (24). Surprisingly, the araFGH promoter, p FGH , possesses a radically different structure (Fig.…”

Evolutionary relationships between sugar kinases and transcriptional repressors in bacteria

“…One is encoded downstream of the araJ gene o f I:. coli, possibly as part of the ara regulon (Reeder & Schleif, 1991), whereas the other is distantly linked to the na<<t--I gene coding for a hyaluronidase of Clustridiztm perfriqms (Carnard et al, GenBank M81878, unpublished:. Table 2 presents the binary percentage identity and RDF2 comp:it-tcon scores for most of the sequenced protein members of the ROK family.…”