GlcNAc Utilization System In Aeromonas Cavaie CB101 And Its Regulator Protein NagC Regulates Transcription Of ChiI And NagAl

L10 is a tansposon mutants of Aeromonas caviae CB101 and it can secret chitinase and chitobiase in LB media constantly. Tail-PCR and sequencing results showed that the insertion site was at 3' end ofnagA gene. We constructed cosmid genome library of CB101 and screened one clone, by southern blotting, which contained nagA gene using Tail-PCR fragment of L10 as probe. After sequencing part of insertion fragment by genome walking, we found that there were four genes nagE, nagB, nagA, and nagC for utilizing GlcNAc in CB101 and the only chitobiase gene was just in the cascade ofnagE gene, but we did not find nagD gene downstream nagC.GlcNAc is phosphorylated while transported by the specific pts transporter NagE and becomes GlcNAc-6-P. The nagA gene encodes N-acetylgucosamine-PO₄ deacetylase and nagB encodes gulcosamine-6- PO₄ deaminase which converts GlcNAc-6-PO₄ to fructose-6-PO₄. The nagC gene is regulator which controls transcription of nag cluster. Result of analyzing nagBAC sequence showed that these three genes had same transcription direction and there were only a few base pairs between each gap of these genes. So we presumed that nagBAC was co-transcript and NagC was the regulator of chitinase and chitobiase of CB101. The transposon insertion in nagA not only broke nagA gene but also blocked transcription of nagC so that chitinase and chitobiase could be secreted constantly.To prove our presumption, we constructed nagC deletion mutant CB101ΔnagC. Analyzing result of enzyme activity and protein component in LB media of CB101ΔnagC and L10 demonstrated that both enzyme activity and excellular protein were nearly the same. Also results of Realtime-PCR for studying transcription level of nag cluster, chiI and nagA1 of CB101ΔnagC identified that NagC protein regulated not only nag cluster but chiI and nagA1. For discover if nagBAC was co-transcript, we performed. RT-PCR and we got the good amplification using cDNA as template. That meant nagB, nagA and nagC were co-transcript from one promoter upstream nagB. What's more, we also analyzed AA seqtience and function domain of NagC protein. There were two domains Helix-turn-Helix domain and ROK(repressors, opening reading frames, and kinases) motif showing high similarity to NagC protein of E. coli K12 which was the classical ROK family regulator. So we expressed NagC protein in E. coli and proved that NagC protein could strongly bind chiI and nagA1 promoter region.In one word, NagC protein regulates transcription of nag operon, chiI and nagA1. This is the first time to report NagC protein can regulate transcription of chitinase gene.