Identification of bacteria harboring transglutaminase gene and its cloning and expression in Escherichia coli

Abstract

The bacterial strain C1112T was isolated from seafood processing wastewater collected from a treatment pond of the seafood factory in Songkhla Province, Thailand. It was Gram-negative, rod shape, non-spore former and produced transglutaminase (TGase). Phylogenetic analysis based on concatenated sequences from the 16S rRNA gene and five housekeeping genes, fusA, lepA, leuS, gyrB and ileS, respectively showed that the strain C1112T belonged to the genus Providencia, and shared 91.75% similarity with P. stuartii DSM 4539T. DNA-DNA hybridization between the strain C1112T and P. stuartii KCTC 2568T was 48.1% relatedness. Moreover, some results from biochemical properties indicated that the strain C1112T was distinguished from the phylogenetically closest relatives. The major fatty acids of strain C1112T were C16:0, iso-C15:0, C14:0 and C17:0 cyclo and the DNA G+C content was 41 mol%. Base on the genotypic and phenotypic considerations, it was classified as a novel species of the genus Providencia for which the name Providencia thailandensis sp. nov. was proposed. The type strain is C1112T (= KCTC 23281T =NBRC 106720T). A novel strain of Enterobacter, C2361T, a Gram-negative, non-spore-forming rod-shaped and facultative anaerobic bacterium with capability to produce the TGase, was isolated from seafood processing wastewater collected from a treatment pond of the seafood factory in Songkhla Province, Thailand. Phylogenetic analysis and phenotypic characteristics, including chemotaxonomic characteristics, showed that the strain was a member of the genus Enterobacter. The 16S rRNA gene sequence similarities between the strain C2361T and Enterobacter cloacae subsp. cloacae ATCC 13047T and Enterobacter cloacae subsp. dissolvens LMG 2683T were 97.5 and 97.5%, respectively. The major fatty acids were C16:0, C17:0cyclo and C14:0. The DNA G+C content was 53.0 mol%. On the basis of the polyphasic evidences gathered in this study, it was classified as a novel species of the genus Enterobacter for which the name Enterobacter siamensis sp. nov. was proposed. The type strain is C2361T (= KCTC 23282T = NBRC 107138T). TGase genes of P. thailandensis and E. siamensis were amplified by specific and degenerate primers using 2 Taq Master Mix (Vivantis) and Takara Ex Taq as DNA polymerase in PCR reactions. 1,100-bp and 1,200-bp were amplified products from E. siamensis genomic DNA, using F1R1 and F2R1 primer with 2 Taq Master. In addition, 1100-bp amplified product from P. thailandensis genomic DNA was obtained by using tgFtgR2 primer with Takara Ex Taq DNA polymerase and ligated with pSSBm97 and expressed into B. megaterium YYBm1. However, TGase activity could not detect from the expressed proteins. Due to P. thailandensis and E. siamensis are novel species, therefore no information regarding their TGase genes could be acquired from the database. This makes it quite difficult to design primers for TGase gene amplification from these two bacteria. Previously, TGase was produced by the genus Streptomyces isolated from soil. In this study, Streptomyces-like strains were isolated from soil and identified using 16S rRNA gene. The stain AH6 showed 99 % similarity with that of Streptomyces thermocarboxydus and named as Streptomyces sp. AH6. The TGase gene of Streptomyces sp. AH6 was constructed by fusion of TGase precursor and pET22b plasmid. The recombinant DNAs were transformed into Escherichia coli BL21 (DE3). The TGase ORF of this gene encoding 410 amino acids. Although the amino acid sequence of theTGase precursor was 100% homologous to that of S. mobaraensis NBRC 13476, the nucleotide sequence was different at 6 nucleotide proteins, including the nucleotide 231, 234, 237, 240, 246 and 249. The pET22b-TG-His6 was secreted as a soluble inclusion body in E. coli and the activity of partially purified enzyme was 3.2 U/ml. The optimal pH and temperature of this enzyme was 6.0 and 40˚C, respectively. The enzyme was inhibited by Cu2+ and Fe2.