Optimization Of Electroporation Conditions For Arthrobacter And Studies On Function Of Trehalose-6-phosphate Phosphatase Gene From Arthrobacter Sp.A3

Strains of Arthrobacter are among the most frequently isolated, indigenous, aerobic bacteria found in soils. Members of the genus are known to biodegrade organic compounds in the environment and have the ability to withstand various environmental stresses, such as cold, desiccation, starvation, ionizing radiation, and osmotic stress. The presence of genes for trehalose synthesis in Arthrobacter might be important for this organism's resistance to environmental stresses. The major pathway for trehalose biosynthesis in most organisms involves two enzymes, trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP). TPP has received great interest because of its abundant biological functions and potential importance for resistance to environmental stresses. Although TPP has been studied in thermophiles and mesophiles, few of them were characterized in cold-adapted microorganisms. A prerequisite for genetic manipulations of these specific genes in Arthrobacter is a high efficiency transformation system that allows for gene cloning, overexpression, and knock-out, as well as for studying trehalose biosynthetic pathway. Thus, in this study, a constitutive gene expression vector pART2 was selected to optimize the electroporation conditions for Arthrobacter sp. A3. Furthermore, the biochemical properties of TPP were studied in this psychrotrophic bacterium. The main results of the present study are as follows:(1) A simple, rapid, and highly efficient electroporation method which was suitable for Arthrobacter species was established through a systematic examination of the factors involved in the entire electroporation process. Of the parameters assayed, the addition of penicillin to cells during the early log phase of growth and the presence of 0.5 M sorbitol in the electroporation and recovery media produced the greatest increases in transformation efficiency and consistency of results. Using optimum conditions, we generally achieved an efficiency of 6.8 x 10'transformants per microgram of pART2 for Arthrobacter sp. A3. To our knowledge, this level of electro-competence is the highest ever reported for the Arthrobacter genus. The proposed method allows a transformation trial to be accomplished in 10 minutes. This protocol was also successfully applied to other five Arthrobacter species. This method constitutes a useful tool for the genetic manipulations of Arthrobacter, and will facilitate research of this economically important group of organism.(2) The tpp gene encoding trehalose-6-phosphate phosphatase from Arthrobacter sp. A3 was obtained by thermal asymmetric interlaced PCR (TAIL-PCR). The gene contained a 801-bp open reading frame encoding 266 amino acids. The result of amino acid sequence analysis by bioinformatics showed that there was no signal peptide and transmembrane domain, which indicated that the TPP of Arthrobacter sp. A3 was an intracellular enzyme. BLASTP result revealed that TPP belonged to the HAD superfamily.(3) The tpp gene was expressed heterogenously in Escherichia coli, and the recombinant enzyme was purified to apparent homogeneity with a calculated molecular weight of 27.9 kDa.(4) The properties of recombinant TPP were characterized in detail. This enzyme showed an absolute requirement for Mg²⁺or Co²⁺. The Arthrobacter sp. A3 TPP showed a broad pH optimum range (from pH 5.0 to 7.5), whereas other trehalose-6-phosphate phosphatase characterised do not possess this feature. The temperature optimum was about 30℃, which was also the point of the highest Kcat/Km value (catalytic efficiency) for the recombinant TPP enzyme. The enzyme was stable at low temperatures, as it could maintain 75%of the maximal activity at 4℃. On the other hand, the enzyme was generally heat-labile, losing 70%of its activity when subjected to heat treatment at 50℃for 6 min. The highest Kcat/Km value was 37.5 mM-1·s-1, which was much higher than values published for mesphilic E.coli TPP. The lowest Km value was at 20℃, however, even at 4℃, it possessed the lowest Km value compared to the counterparts from the other reported microorganisms. These characteristics may suggest that this TPP is a novel cold-adapted enzyme.(5) The tpp gene was expressed homologously in Arthrobacter sp. A3. Overexpression of tpp in Arthrobacter sp. A3 could increase the cell viability slightly at 0℃. The enhancement of viability of tpp-overproducing strain at 0℃, together with the low optimal temperature and the high-catalytic efficiency of TPP at low temperature may indicate that this TPP plays an important role in resistance of Arthrobacter sp. A3 to cold.This is the first report of the characterization of TPP from psychrotrophic Arthrobacter genus. The research of this paper would provide more knowledge to the functions of TPP, and provide basic information for cold-adapted enzymes, and also provide foundation for further understanding the survival strategies of Arthrobacter at low temperatures.