RESUMO
Heterologous gene expression in bifidobacteria requires weak, strong, and inducible promoters depending on the objectives of different expression studies. Weak promoters in Escherichia coli can also be desirable for stable heterologous gene cloning. Here, we developed a reporter system using the Bifidobacterium longum α-galactosidase gene and investigated the activity and inducibility of seven bifidobacterial promoters in B. longum and their activities in E. coli. These studies revealed diverse promoter activities. Three promoters were highly active in B. longum, but only slightly active in E. coli. Among these, two phosphoketolase gene (xfp) promoters exhibited strong activity in B. longum cells grown on glucose. In contrast, the promoter activity of the fructose transporter operon (fruEKFG) was strongly induced by carbohydrates other than glucose, including fructose, xylose, and ribose. These promoters will allow strong or highly inducible expression in bifidobacteria and stable gene cloning in E. coli. In contrast to the functions of these promoters, the promoter of sucrose-utilization operon cscBA showed very high activity in E. coli but low activity in B. longum. Other three promoters were functional in both B. longum and E. coli. In particular, two sucrose phosphorylase gene (scrP) promoters showed inducible activity by sucrose and raffinose in B. longum, indicating their applicability for regulated expression studies. The diverse promoter functions revealed in this study will contribute to enabling the regulated expression of heterologous genes in bifidobacteria research.
Assuntos
Bifidobacterium/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Genes Reporter/genética , Regiões Promotoras Genéticas/genética , alfa-Galactosidase/genética , Bifidobacterium/efeitos dos fármacos , Carboidratos/farmacologia , Escherichia coli/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Óperon/efeitos dos fármacos , Óperon/genética , Regulação para Cima/efeitos dos fármacosRESUMO
Chlamydomonas acidophila KT-1 and Chlamydomonas acidophila DVB238 exhibit a strong heavy metal tolerance, but C. acidophila DVB238 can accumulate a much higher amount of Cadmium (Cd) than C. acidophila KT-1. Phytochelatins (PCs) are known to play an important role in the detoxification of several toxic heavy metals, but the relationship between PCs and Cd accumulation is not clear. PC metabolism and Cd accumulation were investigated by using three Chlamydomonas strains including Chlamydomonas reinhardtii C-9 as a standard alga. The results showed that the PC content did not correlate closely with the level of Cd accumulation, maintenance of a high GSH level seeming to be more important for Cd accumulation. The ultrastructure of C. acidophila KT-1 was extremely disrupted by a great increase in starch granules, which resulted in a moribund state, but hyper-accumulator C. acidophila DVB238 did not exhibit an increase in starch granules in its cells, in spite of Cd accumulation in its chloroplasts, cytosol and vacuoles. These results indicated that C. acidophila DVB238 probably has a developed detoxification system preventing such as destruction of the cells due to Cd toxicity.