| Abstract: | Bacteria are able to activate or switch off specific sets of genes as they face changing environmental conditions. This can be achieved through the activities of RNA polymerases (RNAP) containing alternate sigma factors and their cognate regulatory proteins. The sigma factor σ54 recognizes a conserved sequence motif centered at -24 and -12 nucleotides from the transcriptional start site (5''-TGGC-N8- TGCA/T-3'') and confers RNAP properties different from those of the major house-keeping form of RNAP such asσ70.
Xanthomonas campestris pv. campestris (XCC) is the Gram-negative bacterium causing black rot in crucifers, resulting in tremendous loss in agriculture. It produces great amounts of an exopolysaccharide, xanthan gum, which is used as viscosifying, emulsifying, plasticizer and stabilizing agent in food, cosmetics, agriculture and industry. The fully sequenced XCC genome has two homologues of the rpoN genes, rpoN1 and rpoN2. The two deduced RpoN sequences share only 41% of identity and both show different levels of homology to the RpoN proteins from other bacteria. Theσ54-dependent regulon has previously been studied in several groups of bacteria, but not in XCC. Preliminary physiological studies have demonstrated that the two rpoN genes are functionally independent and not interchangeable. In this study, I carried out searches on the recently published complete genome sequence of XCC in order to predict the possibleσ54-dependent genes. Molecular biological methods were then used to confirm whether the predicted σ54-dependent genes are indeed controlled by RpoN1 or RpoN2. The results revealed that glnA (encoding glutamine synthetase), nasA (encoding nitrate transporter), pilA1 (encoding pilin), and prpB (encoding carboxyphosphonoenolpyruvate phosphonomutase) are RpoN1-dependent, whereas flhF (encoding flagellar biosynthetic protein), flgG (encoding the distal rod protein of the flagellum basal body), flgB (encoding the proximal rod protein of the basal body), fliE (encoding MS-ring rod junction protein), and fliQ (encoding export component of flagellar protein) are RpoN2-dependent. Based on the RpoN-binding sequence in the upstream regions of the above genes and their homologs, the consensus RpoN1- and RpoN2-binding sites complied are 5''-TTGGC-AN7-TGCG/T- 3'' and 5''-TTTGGC-N8-TGCA-3'', respectively. In other words, the two Xcc RpoN proteins each has its specific recognition sequences. |
