Abstract
Many haloarchaea produce extracellular subtilisin-like proteases (halolysins) during late log phase; however, the physiological function and regulatory mechanism of growth phase-dependent production of halolysins are unknown. Halolysin SptA, the major extracellular protease of Natrinema sp. J7-2, is capable of intracellular self-activation to affect haloarchaeal growth. Here, we report that deletion of sptA leads to loss of extracellular and intracellular protease activities against azocasein and/or suc-AAPF-pNA, as well as a change in growth-phase transition of the haloarchaeon. Our results suggest that SptA is important for strain J7-2 to enter the stationary and death phases. Deletion and mutational analyses of the 5′-flanking region of sptA revealed two partially overlapping, semi-palindromic sequences upstream of the TATA box act as positive and negative cis-regulatory elements, respectively, to mediate sptA expression in late log phase. Additionally, a negative cis-regulatory element covering WW motif and a distant enhancer contribute to the modulation of sptA expression. Our results demonstrate that SptA functions both extracellularly and intracellularly, and that sptA expression relies on the cooperative action of multiple cis-regulatory elements, allowing SptA to exert its function properly at different growth stages in strain J7-2.
Highlights
Haloarchaea generally require 2–5.2 M NaCl for growth and thrive in hypersaline environments such as solar salterns, salt lakes, and salt deposits (Grant, 2004)
Many halolysinlike serine protease genes have been annotated in the genomes of Halobacterium salinarium and Natronococcus occultus, and the extracellular serine proteases of the two strains are produced during late log phase (Norberg and von Hofsten, 1969; Elsztein et al, 2001)
The results showed that the sptA core promoter contains some highly conserved, cis-acting elements of haloarchaeal promoters, including a transcription factor B (TFB) recognition element (BRE) motif, a TATA box recognized by the TATA-binding protein (TBP), and a WW motif that possibly interacts with TFB and RNA polymerase (RNAP) (Brenneis et al, 2007)
Summary
Haloarchaea generally require 2–5.2 M NaCl for growth and thrive in hypersaline environments such as solar salterns, salt lakes, and salt deposits (Grant, 2004). The production of Nep in Nab. magadii is repressed by ammonium (D’Alessandro et al, 2007). These findings show that the expression levels of halolysins and extracellular serine proteases in haloarchaea are strictly regulated. It has been suggested that the expression of nep is upregulated in response to regulatory factors (metabolites and/or regulatory molecules) present in high-density cultures of Nab. magadii (Paggi et al, 2010), and that the production of the extracellular serine protease in Ncc. occultus is induced in a quorum sensing-dependent manner by autoinducer molecules (Paggi et al, 2003). The fact that halolysin production occurs during late log phase suggests that halolysins are not essential for the exponential growth of haloarchaea. The physiological roles of halolysins in the growth and survival of haloarchaea remain to be elucidated
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