Each amplified DNA fragment covered the region from the 18th to 172nd of the lipase gene and that from the 541st to 711th of the 16S rRNA

gene, respectively. The annealing temperature of the oligonucletotides for lipase gene is 55°C and that for 16S rRNA is 51°C. The thermal protocol was 95°C for selleck chemical 3  min and then 35 cycles of 95°C for 10  sec and the annealing temperature indicated above for  30 sec and 72°C for 30  sec. Fluorescence was measured at the end of the 72°C step during every cycle. As a control, a reaction mixture without reverse transcriptase was prepared using same protocol. The threshold for fluorescence was properly positioned according to the manufacture’s Selleck MG 132 protocol, and the number of cycles at which fluorescence reached the threshold line was determined. The relative transcriptional level of lipase gene was calculated according to the formula of the ΔΔCt method (26). In order

to comprehensively examine the effect of NaCl on production of extracellular proteins, we cultured two strains, wild-type strain (A. sobria 288 [asp+, amp+]) and two protease gene-deleted mutant strains (A. sobria 288 [asp−, amp−]), in NB (0.5) and NB (3.0) at 37°C for 24  hrs with shaking. We treated these culture supernatants with TCA, and collected and separated the precipitates yielded by SDS-PAGE as described in Materials and Methods. The results are shown in Figure 1. We applied samples of the wild-type strain, which we prepared by culturing in NB (0.5) and NB (3.0), to lanes 1 and 2, respectively. Compared with lane 2, the number of protein bands in lane 1 was small and their density low. We believe that both ASP and AMP were

produced in the wild type culture supernatant in NB (0.5) and that almost all proteins released into the culture supernatant were decomposed by these proteases. In contrast, we prepared the sample for lane 2 from the culture supernatant in NB (3.0). In NB (3.0), production of ASP and Nintedanib (BIBF 1120) AMP is repressed (8, 17). Therefore, many proteins in the culture supernatant were not attacked by these proteases. Thus, the number of bands was large and their densities high in lane 2. The above results show that the protease activity of the culture supernatant strongly influences the appearance of protein in it. It is important to eliminate the influence of proteases when analyzing exoproteins released into the milieu from bacteria. We therefore analyzed the exoproteins of a two protease gene-deleted mutant strain (A. sobria 288 [asp−, amp−]).