Proteomics Analysis Of Aromatic Catabolic Pathways In Thermophilic Geobacillus Thermodenitrificans NG80-2and Complete Genome Sequence Of Aeromonas Veronii Strain B565

IIncreasing attention has been directed towards crude-oil pollution in recent years. Among the complex chemicals in crude oil, mono-aromatic hydrocarbons, mainly the BTEX group （benzene, toluene, ethyl benzene and xylene）, most soluble constituents, tend to be one of the dominant pollutants and serious threats in contaminated groundwater. Aromatic acids, metabolic intermediates such as benzoic acid, o-, m-,/>-toluic acids, frequently detected in the contaminated aquifer indicated a series of active microbial-mediated oxidation/reduction on the aromatic hydrocarbon components. Furthermore, mono-aromatics are produced in large amounts during the conversion of aromatic amino acids in nearly all living organisms, including phenylalanine, tryptophan and tyrosine. Next to glucosyl structures, aromatics are most widely distributed in nature, not simply resulting from lignin depolymerization of higher plants, but the release from the accidents and by-products in pharmaceutical, chemical and petroleum industries. Aromatic organic wastes are of great concern for their long-term persistence due to the high thermodynamic stability of the benzene moiety, and their acute carcinogenic, mutagenic, and teratogenic risks to public health. Therefore, considerable efforts have been devoted to understanding the fate of the aromatic hydrocarbons and their various metabolic pathways in the past two decades.Geobacillus thermodenitrificans NG80-2is a Gram-positive, facultative thermophilic, long-chain alkane-degrading bacterium isolated from an oil reservoir in China. Previous analysis of the NG80-2genome revealed the presence of four distinct aromatic catabolic gene clusters predicted for benzoate （via benzoyl-CoA, GTNG₁888-GTNG₁899）, phenylacetate （via phenylacetyl-CoA, GTNG₁919-GTNG₁930）,4-hydroxyphenylacetate （via3,4-dihydroxyphenylacetate, GTNG2973-GTNG₂993）,and anthranilate （via3-hydroxyanthranilate, GTNG₃150-GTNG₃164）,indicating the ability of NG80-2to utilize diverse aromatic compounds.In this study, these gene clusters and pathways for the degradation of benzoate （via benzoyl-CoA）, phenylacetate （via phenylacetyl-CoA）,4-hydroxyphenylacetate （via3,4-dihydroxyphenylacetate） and anthranilate （via3-hydroxyanthranilate） were confirmed using combined in silico analysis and proteomics approaches. It was found that synthesis of the enzymes responsible for the initial activation, ring oxidation and ring cleavage reactions were generally induced specifically by their respective substrates, while many of the enzymes catalyzing downstream reactions exhibited broader substrate specificities. Novel genes encoding benzoyl-CoA epoxidase and3,4-dihydroxyphenylacetate2,3-dioxygenase, were proposed. A paaX homologue in the benzoate gene cluster serves as a positive regulator of benzoate degradation. Downregulation of the glycolysis pathway, along with upregulation of the gluconeogenesis pathway were detected in association with the utilization of the aromatics. Moreover, the glyoxylate bypass was upregulated in the phenylacetate-cultured NG80-2cells as shown by the increased synthesis of isocitrate lyase （GTNG₅83） and malate synthase G （GTNG₁384）, indicating the utilization of acetyl-CoA as the sole carbon source, which is produced in excess （2copies of acetyl-CoA and1succinyl-CoA per phenylacetate）. This novel proteomics analysis confirmed the presence of multiple metabolic pathways for aromatic compounds in NG80-2, which is highly advantageous to the survival of this thermophilic bacterium under reservoir conditions.ⅡAeromonas veronii is a Gram-negative, rod-shaped bacterium commonly found in the environmental, clinical, and food samples. The pathogens in the genus Aeromonas can cause diseases ranging from wound infections and diarrhoea to septicaemia in immune compromised patients.A. veronii is also the causative agent of bacterial hemorrhagic septicemia in fish, which is a major economic problem in the fish-farming industry. A. veronii strain B565was isolated from aquaculture pond sediment in Tianjin, China. In a separate study, it was found to be able to produce chitinase which can be used to control fungal or Myxozoa-related diseases.In this study, the complete genome sequence of B565was presented here and compared with2published genome sequences of the pathogenic strains in Aeromonas genus, which are A.hydrophila ATCC7966and A.sa/monicida A449.B565encodes5 chitinase genes and some putative virulence factors, such as hemolysins, RTX protein, adhesion factor, flagella, and mannose-sensitive hemagglutinin （MSHA）.The result represents an independent step-wise acquisition of virulence factors of pathogenic strains in this genus:some virulence genes were inherited long time ago from their common ancestors, while most of the other weapons can be acquired quickly from other related species by lateral transfer.