Team:NWU-CHINA/Sense
AlkB2 Introduction
Alkane is a class of highly saturated hydrocarbons in the form of linear, cycloalkane, linear paraffins and etc. And with the relative molecular mass increasing, its solubility reduces. Researches have shown that there are two ways bacteria ingests alkane. The short-chain alkanes are highly water-soluble and can be directly ingested, while for long-chain alkane bacteria, surfactants produced by bacteria can increase solubility of long-chain alkane, which will help bacteria ingests alkane. For example. Pseudomonas aeruginosa can produce rhamnolipid to increase its ingest of n-hexadecane. Alkane oxidase catalyzes is the first step in the metabolic pathway of alkane catabolism, which is the key enzyme for alkane metabolism. The OCT plasmid of the P. putida GPol strain contains a number of genes encoding an alkane degrading enzyme [37]. The ikB gene encodes the protein A1kB as the first enzyme of the alkane metabolic pathway. A1kB is a single oxygenase with heme, and its catalytic action requires the synergistic effect of two soluble electron-transport proteins (A1kG and A1kT). Ferredoxin reductase transfers electrons from NADH to A1kG oxygenated protein through cofactor FAD. A1kG also transfers electrons to A1kB. A1kB electrons are transferred to oxygen molecules to complete the alkylation of alkanes.
GntR Introduction
By sensing different environment, microorganism can utilize various of regulatory mechanisms to control the expression of genes strictly, making themselves able to adapt to the environment. In general, the signal factors are always intermediate product during metabolism, perceived by the bacteria through a variety of HTH transcription factor. Then bacteria will do correspond reaction. Alkane Degradation Bacteria senses the presence of alkanes, induces the initiation of the regulatory mechanisms that express the relevant degrading enzymes. In the bacteria, the GntR family is one of the largest family of HTH-related transcription factors. The GntR family represents a class of proteins that are very diverse in terms of structure, function, and biochemistry. They are widely spread in microorganism. GntR protein may be directly involved in the regulation of many primary metabolic processes, response to changes of metabolite concentration rapidly, and then regulate the downstream gene transcription and expression, so that cells can quickly and sensitively respond to changes of environmental conditions. This can ensure normal and stable cell metabolism. Coding gene of GntR protein in the Pseudomonas aeruginosa is next to a1kB gene closely, which is directly involved in alkane oxidase A1kB transcription.
Relationship between GntR and AlkB2
As research knows, Liu constructed gene knocked out bacteria, included AlkB1 knocked out strain, MutAlkB1; AlkB1 and GntR knocked out strain, MutAlkB1/GntR; AlkB2, GntR knocked out strain, MutAlkB2/GntR and GntR knocked out strain, MutGntR. Subsequently, Liu analyzed the growth and degradation of MutAlkB2/GntR, MutAlkB1 and wild type strains on alkane culture (Figure 5.13D-F).We found that A1kB1 is responsible for the degradation of C-16 alkanes, while A1kB2 plays a major role in the degradation of medium-long paraffins and has the function of long-chain alkane degradation. It can be seen from the figure that MutAlkB1 grows slowly in C-14 and C-16 media, MutAlkB1/GntR is the fastest. When a1kB1 is absent, the growth is somewhat slowed down, indicating that A1kB1 does play a role in the degradation of these two alkanes. When the GntR and a1kB1 genes were knocked out simultaneously, the growth and degradation of alkanes were accelerated and the stagnation period was shortened. This is because the inhibition of a1kB2 transcription was relieved by GntR absence, and the expression of A1kB2 was improved obviously. Then we hypothesized that GntR can negatively regulate the transcription of the A1kB2 gene.
Results
We cloned AlB2 promoter and GntR protein coding gene by PCR. Our agarose gel electrophoresis results shows we got gene successfully.
Reference:
[1] Huan Liu.Molecular Mechanism of Degradation of Alkanes by Pseudomonas aeruginosa SJTD 1[D].Shanghai Jiao Tong University:Shanghai Jiao Tong University, 2014.
[2] Baptist, J. N.,Gholson, R. K.,Coon, M. J. Hydrocarbon oxidation by a bacterial enzyme system: I.Products of octane oxidation[J]. Biochim. Biophys. Acta, 1963, 69 40-47.
