ABSTRACT
Bacillus subtilis is an ideal host for secretion and expression of foreign proteins. The promoter is one of the most important elements to facilitate the high-level production of recombinant protein. To expand the repertoire of strong promoters for biotechnological applications in Bacillus species, 14 highly transcribed genes based on transcriptome profiling of B. pumilus BA06 were selected and evaluated for their promoter strength in B. subtilis. Consequently, a strong promoter P2069 was obtained, which could drive the genes encoding alkaline protease (aprE) and green fluorescent protein (GFP) to express more efficiency by an increase of 3.65-fold and 18.40-fold in comparison with the control promoter (PaprE), respectively. Further, promoter engineering was applied to P2069, leading to a mutation promoter (P2069M) that could increase GFP expression by 3.67-fold over the wild-type promoter (P2069). Moreover, the IPTG-inducible expression systems were constructed using the lac operon based on the strong promoters of P2069 and P2069M, which could work well both in B. subtilis and B. pumilus. In this study, highly efficient expression system for Bacillus was constructed based on transcriptome data and promoter engineering, which provide not only a new option for recombinant expression in B. subtilis, but also novel genetic tool for B. pumilus.
ABSTRACT
BACKGROUND: Bacillus pumilus is a Gram-positive and endospore-forming bacterium broadly existing in a variety of environmental niches. Because it produces and secrets many industrially useful enzymes, a lot of studies have been done to understand the underlying mechanisms. Among them, scoC was originally identified as a pleiotropic transcription factor negatively regulating protease production and sporulation in B. subtilis. Nevertheless, its role in B. pumilus largely remains unknown. RESULTS: In this study we successfully disrupted scoC gene in B. pumilus BA06 and found increased total extracellular protease activity in scoC mutant strain. Surprisingly, we also found that scoC disruption reduced cell motility possibly by affecting flagella formation. To better understand the underlying mechanism, we performed transcriptome analysis with RNA sequencing. The result showed that more than one thousand genes were alternated at transcriptional level across multiple growth phases, and among them the largest number of differentially expressed genes (DEGs) were identified at the transition time point (12 h) between the exponential growth and the stationary growth phases. In accordance with the altered phenotype, many protease genes especially the aprE gene encoding alkaline protease were transcriptionally regulated. In contrast to the finding in B. subtilis, the aprN gene encoding neutral protease was transcriptionally downregulated in B. pumilus, implicating that scoC plays strain-specific roles. CONCLUSIONS: The pleiotropic transcription factor ScoC plays multiple roles in various cellular processes in B. pumilus, some of which were previously reported in B. subtilis. The supervising finding is the identification of ScoC as a positive regulator for flagella formation and bacterial motility. Our transcriptome data may provide hints to understand the underlying mechanism.
