An extracellular halophilic protease SptA from a halophilic archaeon Natrinema sp. J7: gene cloning, expression and characterization

A gene encoding an extracellular protease, sptA, was cloned from the halophilic archaeon Natrinema sp. J7. It encoded a polypeptide of 565 amino acids containing a putative 49-amino acid signal peptide, a 103-amino acid propeptide, as well as a mature region and C-terminal extension, with a high proportion of acidic amino acid residues. The sptA gene was expressed in Haloferax volcanii WFD11, and the recombinant enzyme could be secreted into the medium as an active mature form. The N-terminal amino acid sequencing and MALDI-TOF mass spectrometry analysis of the purified SptA protease indicated that the 152-amino acid prepropeptide was cleaved and the C-terminal extension was not processed after secretion. The SptA protease was optimally active at 50°C in 2.5 M NaCl at pH 8.0. The NaCl removed enzyme retained 20% of its activity, and 60% of the activity could be restored by reintroducing 2.5 M NaCl into the NaCl removed enzyme. When the twin-arginine motif in the signal peptide of SptA protease was replaced with a twin-lysine motif, the enzyme was not exported from Hfx. volcanii WFD11, suggesting that the SptA protease was a Tat-dependent substrate.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Halolysin SptA有助于嗜盐古菌Natrinema sp.J7-2长期生存

【背景】嗜盐古菌可以在盐沉积物中存活长达几百万年,是著名的长寿菌。许多嗜盐古菌分泌胞外蛋白酶,大多数分泌的胞外蛋白酶被称为Halolysin,具有以下特征：属于枯草杆菌蛋白酶类蛋白酶;在胞内折叠后经Tat途径高效分泌至胞外;可自加工形成成熟酶;尤其在天然宿主中大多数Halolysin在对数生长后期表达并在稳定期达到最高水平。目前Halolysin的酶学性质、加工成熟及分泌机制已被广泛研究,然而其生理功能的研究较少。Halolysin SptA是嗜盐古菌Natrinema sp.J7-2的主要胞外蛋白酶,前期研究发现多个顺式调控元件协同调节SptA的生长期依赖性表达,使SptA参与J7-2菌株不同生长期之间的转变,而且在衰亡期之后SptA有助于J7-2菌株继续生存。【目的】研究Halolysin SptA对Natrinema sp.J7-2长期生存的作用。【方法】将J7-2菌株和突变体ΔsptA1分别在寡营养、无外源营养物质（液体）及营养丰富（固体）条件下长期培养,通过比较二者的生长、生存和SptA的表达分泌情况进一步探讨SptA的作用。【结果】J7-2菌株在寡营养条件下产生更多SptA,培养后期（33 d） J7-2菌株活细胞数显著高于ΔsptA1。在无外源营养物质情况下长期温育,J7-2菌株和ΔsptA1经历多次细胞分裂和细胞死亡,在延长温育期间（73—200 d）存活的J7-2菌株细胞数量均显著多于存活的ΔsptA1细胞数量。在营养丰富的固体平板上培养的后期（160 d）,由于营养物质消耗,J7-2菌株通过SptA吸收和利用来源于死细胞蛋白的降解产物,帮助其群体长期生存。【结论】SptA介导的细胞死亡和死细胞蛋白降解,促进J7-2菌株利用来源于死细胞的营养物质,从而有助于菌株群体在营养缺乏条件下长期存活。本研究提供了关于Halolysin生理作用的新见解。     

Autocatalytic maturation of the Tat-dependent halophilic subtilase Nep produced by the archaeon Natrialba magadii

Halolysins are subtilisin-like extracellular proteases produced by haloarchaea that possess unique protein domains and are salt dependent for structural integrity and functionality. In contrast to bacterial subtilases, the maturation mechanism of halolysins has not been addressed. The halolysin Nep is secreted by the alkaliphilic haloarchaeon Natrialba magadii, and the recombinant active enzyme has been synthesized in Haloferax volcanii. Nep contains an N-terminal signal peptide with the typical Tat consensus motif (GRRSVL), an N-terminal propeptide, the protease domain, and a C-terminal domain. In this study, we used Nep as a model protease to examine the secretion and maturation of halolysins by using genetic and biochemical approaches. Mutant variants of Nep were constructed by site-directed mutagenesis and expressed in H. volcanii, which were then analyzed by protease activity and Western blotting. The Tat dependence of Nep secretion was demonstrated in Nep RR/KK variants containing double lysine (KK) in place of the twin arginines (RR), in which Nep remained cell associated and the extracellular activity was undetectable. High-molecular-mass Nep polypeptides without protease activity were detected as cell associated and extracellularly in the Nep S/A variant, in which the catalytic serine 352 had been changed by alanine, indicating that Nep protease activity was needed for precursor processing and activation. Nep NSN 1-2 containing a modification in two potential cleavage sites for signal peptidase I (ASA) was not efficiently processed and activated. This study examined for the first time the secretion and maturation of a Tat-dependent halophilic subtilase.     

Identification and characterization of SNJ2, the first temperate pleolipovirus integrating into the genome of the SNJ1‐lysogenic archaeal strain

Proviral regions have been identified in the genomes of many haloarchaea, but only a few archaeal halophilic temperate viruses have been studied. Here, we report a new virus, SNJ2, originating from archaeal strain Natrinema sp. J7‐1. We demonstrate that this temperate virus coexists with SNJ1 virus and is dependent on SNJ1 for efficient production. Here, we show that SNJ1 is an icosahedral membrane‐containing virus, whereas SNJ2 is a pleomorphic one. Instead of producing progeny virions and forming plaques, SNJ2 integrates into the host tRNA^Met gene. The virion contains a discontinuous, circular, double‐stranded DNA genome of 16 992 bp, in which both nicks and single‐stranded regions are present preceded by a ‘GCCCA’ motif. Among 25 putative SNJ2 open reading frames (ORFs), five of them form a cluster of conserved ORFs homologous to archaeal pleolipoviruses isolated from hypersaline environments. Two structural protein encoding genes in the conserved cluster were verified in SNJ2. Furthermore, SNJ2‐like proviruses containing the conserved gene cluster were identified in the chromosomes of archaea belonging to 10 different genera. Comparison of SNJ2 and these proviruses suggests that they employ a similar integration strategy into a tRNA gene.     

Adaptation of protein secretion to extremely high-salt conditions by extensive use of the twin-arginine translocation pathway

Halophilic archaea thrive in environments with salt concentrations approaching saturation. However, little is known about the way in which these organisms stabilize their secreted proteins in such 'hostile' conditions. Here, we present data suggesting that the utilization of protein translocation pathways for protein secretion by the Halobacteriaceae differs significantly from that of non-haloarchaea, and most probably represents an adaptation to the high-salt environment. Although most proteins are secreted via the general secretion (Sec) machinery, the twin-arginine translocation (Tat) pathway is mainly used for the secretion of redox proteins and is distinct from the Sec pathway, in that it allows cytoplasmic folding of secreted proteins. tatfind (developed in this study) was used for systematic whole-genome analysis of Halobacterium sp. NRC-1 and several other prokaryotes to identify putative Tat substrates. Our analyses revealed that the vast majority of haloarchaeal secreted proteins were predicted substrates of the Tat pathway. Strikingly, most of these putative Tat substrates were non-redox proteins, the homologues of which in non-haloarchaea were identified as putative Sec substrates. We confirmed experimentally that the secretion of one such putative Tat substrate depended on the twin-arginine motif in its signal sequence. This extensive utilization of the Tat pathway in haloarchaea suggests an evolutionary adaptation to high-salt conditions by allowing cytoplasmic folding of secreted proteins before their secretion.     

A novel archaeal DNA repair factor that acts with the UvrABC system to repair mitomycin C‐induced DNA damage in a PCNA‐dependent manner

The sliding clamp proliferating cell nuclear antigen (PCNA) plays a vital role in a number of DNA repair pathways in eukaryotes and archaea by acting as a stable platform onto which other essential protein factors assemble. Many of these proteins interact with PCNA via a short peptide sequence known as a PIP (PCNA interacting protein) motif. Here we describe the identification and functional analysis of a novel PCNA interacting protein NreA that is conserved in the archaea and that has a PIP motif at its C‐terminus. Using the genetically tractable euryarchaeon Haloferax volcanii as a model system, we show that the NreA protein is not required for cell viability but that loss of NreA (or replacement of the wild‐type protein with a truncated version lacking the C‐terminal PIP motif) results in an increased sensitivity to the DNA damaging agent mitomycin C (MMC) that correlates with delayed repair of MMC‐induced chromosomal DNA damage monitored by pulsed‐field gel electrophoresis. Genetic epistasis analysis in Hfx. volcanii suggests that NreA works together with the UvrABC proteins in repairing DNA damage resulting from exposure to MMC. The wide distribution of NreA family members implies an important role for the protein in DNA damage repair in all archaeal lineages.     

Gentisate 1,2-dioxygenase from Haloferax sp. D1227

Gentisate 1,2-dioxygenase from the extreme halophile Haloferax sp. D1227 (Hf. D1227) was purified using a three-step procedure. The enzyme was found to be a homotetramer of 42 000 ± 1000 Da subunits, with a native molecular weight of 174 000 ± 6000 Da. The optimal salt concentration, temperature, and pH for enzyme activity were 2 M KCl or NaCl, 45°C, and pH 7.2, respectively. The gene encoding Hf. D1227 gentisate 1,2-dioxygenase was cloned, sequenced, and expressed in Haloferax volcanii. The deduced amino acid sequence exhibited a 9.2% excess acidic over basic amino acids typical of halophilic enzymes. Four novel histidine clusters and a possible extradiol dioxygenase fingerprint region were identified. Received: November 19, 1997 / Accepted: May 12, 1998     

Genetic and biochemical analysis of the twin-arginine translocation pathway in halophilic archaea

The twin-arginine translocation (Tat) pathway is present in a wide variety of prokaryotes and is capable of exporting partially or fully folded proteins from the cytoplasm. Although diverse classes of proteins are transported via the Tat pathway, in most organisms it facilitates the secretion of a relatively small number of substrates compared to the Sec pathway. However, computational evidence suggests that haloarchaea route nearly all secreted proteins to the Tat pathway. We have expanded previous computational analyses of the haloarchaeal Tat pathway and initiated in vivo characterization of the Tat machinery in a model haloarchaeon, Haloferax volcanii. Consistent with the predicted usage of the this pathway in the haloarchaea, we determined that three of the four identified tat genes in Haloferax volcanii are essential for viability when grown aerobically in complex medium. This represents the first report of an organism that requires the Tat pathway for viability when grown under such conditions. Deletion of the nonessential gene had no effect on the secretion of a verified substrate of the Tat pathway. The two TatA paralogs TatAo and TatAt were detected in both the membrane and cytoplasm and could be copurified from the latter fraction. Using size exclusion chromatography to further characterize cytoplasmic and membrane TatA proteins, we find these proteins present in high-molecular-weight complexes in both cellular fractions.     

Escherichia coli “TatExpress” strains super‐secrete human growth hormone into the bacterial periplasm by the Tat pathway

Numerous high‐value proteins are secreted into the Escherichia coli periplasm by the General Secretory (Sec) pathway, but Sec‐based production chassis cannot handle many potential target proteins. The Tat pathway offers a promising alternative because it transports fully folded proteins; however, yields have been too low for commercial use. To facilitate Tat export, we have engineered the TatExpress series of super‐secreting strains by introducing the strong inducible bacterial promoter, ptac , upstream of the chromosomal tatABCD operon, to drive its expression in E. coli strains commonly used by industry (e.g., W3110 and BL21). This modification significantly improves the Tat‐dependent secretion of human growth hormone (hGH) into the bacterial periplasm, to the extent that secreted hGH is the dominant periplasmic protein after only 1 hr induction. TatExpress strains accumulate in excess of 30 mg L^?1 periplasmic recombinant hGH, even in shake flask cultures. A second target protein, an scFv, is also shown to be exported at much higher rates in TatExpress strains.

     

Production of <Emphasis Type="Italic">Chryseobacterium proteolyticum</Emphasis> protein-glutaminase using the twin-arginine translocation pathway in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

The protein glutaminase (PG) secreted by the Gram-negative bacterium Chryseobacterium proteolyticum can deamidate glutaminyl residues in several substrate proteins, including insoluble wheat glutens. This enzyme therefore has potential application in the food industry. We assessed the possibility to produce PG containing a pro-domain in Corynebacterium glutamicum which we have successfully used for production of several kinds of proteins at industrial-scale. When it was targeted to the general protein secretion pathway (Sec) via its own signal sequence, the protein glutaminase was not secreted in this strain. In contrast, we showed that pro-PG could be efficiently produced using the recently discovered twin-arginine translocation (Tat) pathway when the typical Sec-dependent signal peptide was replaced by a Tat-dependent signal sequence from various bacteria. The accumulation of pro-PG in C. glutamicum ATCC13869 reached 183 mg/l, and the pro-PG was converted to an active form as the native one by SAM-P45, a subtilisin-like serine protease derived from Streptomyces albogriseolus. The successful secretion of PG via this approach confirms that the Tat pathway of C. glutamicum is an efficient alternative for the industrial-scale production of proteins that are not efficiently secreted by other systems.     

Biofilm formation by haloarchaea

A fluorescence‐based live‐cell adhesion assay was used to examine biofilm formation by 20 different haloarchaea, including species of Halobacterium, Haloferax and Halorubrum, as well as novel natural isolates from an Antarctic salt lake. Thirteen of the 20 tested strains significantly adhered (P‐value < 0.05) to a plastic surface. Examination of adherent cell layers on glass surfaces by differential interference contrast, fluorescence and confocal microscopy showed two types of biofilm structures. Carpet‐like, multi‐layered biofilms containing micro‐ and macrocolonies (up to 50 μm in height) were formed by strains of Halobacterium salinarum and the Antarctic isolate t‐ADL strain DL24. The second type of biofilm, characterized by large aggregates of cells adhering to surfaces, was formed by Haloferax volcanii DSM 3757^T and Halorubrum lacusprofundi DL28. Staining of the biofilms formed by the strongly adhesive haloarchaeal strains revealed the presence of extracellular polymers, such as eDNA and glycoconjugates, substances previously shown to stabilize bacterial biofilms. For Hbt. salinarum DSM 3754^T and Hfx. volcanii DSM 3757^T, cells adhered within 1 day of culture and remained viable for at least 2 months in mature biofilms. Adherent cells of Hbt. salinarum DSM 3754^T showed several types of cellular appendages that could be involved in the initial attachment. Our results show that biofilm formation occurs in a surprisingly wide variety of haloarchaeal species.     

Haloferax volcanii archaeosortase is required for motility,mating, and C‐terminal processing of the S‐layer glycoprotein

Cell surfaces are decorated by a variety of proteins that facilitate interactions with their environments and support cell stability. These secreted proteins are anchored to the cell by mechanisms that are diverse, and, in archaea, poorly understood. Recently published in silico data suggest that in some species a subset of secreted euryarchaeal proteins, which includes the S‐layer glycoprotein, is processed and covalently linked to the cell membrane by enzymes referred to as archaeosortases. In silico work led to the proposal that an independent, sortase‐like system for proteolysis‐coupled, carboxy‐terminal lipid modification exists in bacteria (exosortase) and archaea (archaeosortase). Here, we provide the first in vivo characterization of an archaeosortase in the haloarchaeal model organism Haloferax volcanii. Deletion of the artA gene (HVO_0915) resulted in multiple biological phenotypes: (a) poor growth, especially under low‐salt conditions, (b) alterations in cell shape and the S‐layer, (c) impaired motility, suppressors of which still exhibit poor growth, and (d) impaired conjugation. We studied one of the ArtA substrates, the S‐layer glycoprotein, using detailed proteomic analysis. While the carboxy‐terminal region of S‐layer glycoproteins, consisting of a putative threonine‐rich O‐glycosylated region followed by a hydrophobic transmembrane helix, has been notoriously resistant to any proteomic peptide identification, we were able to identify two overlapping peptides from the transmembrane domain present in the ΔartA strain but not in the wild‐type strain. This clearly shows that ArtA is involved in carboxy‐terminal post‐translational processing of the S‐layer glycoprotein. As it is known from previous studies that a lipid is covalently attached to the carboxy‐terminal region of the S‐layer glycoprotein, our data strongly support the conclusion that archaeosortase functions analogously to sortase, mediating proteolysis‐coupled, covalent cell surface attachment.     

Tantalizing evidence for caspase‐like protein expression and activity in the cellular stress response of Archaea

An enigmatic feature of microbial evolution is the emergence of programmed cell death (PCD), a genetically controlled form of cell suicide triggered by environmental stimuli. Archaea, the second major prokaryotic domain of life, have been notably absent from the PCD inheritance discussion, due to a lack of genetic homologues. Using the model haloarchaeon Haloferax volcanii, we document extremely high caspase‐specific activity and expression of immunoreactive proteins to human caspase 8 antisera, both of which were induced by salt stress and death and were abolished by in vivo addition of a broad‐spectrum caspase inhibitor. Caspase inhibition severely impaired cell growth under low and high salt stress, demonstrating a critical role in the cellular stress response. In silico analysis of the H. volcanii proteome identified a subset of 18 potential target proteins containing a signature tetrapeptide caspase cleavage motif (IETD), some with putative roles in allosteric regulation, signal transduction, osmotic stress and cell communication. Detection of similarly high activity and expression in other haloarchaea (Halorubrum and Haloarcula) and in diverse members of Euryarchaeota (the methanogen Methanosarcina acetivorans and the hyperthermophile Pyrococcus furiosus) and Crenarchaeota (the acidophile Sulfolobus solfataricus) argue for a broad representation within the archaeal domain. By playing a role in normal cell function, caspase‐like proteases in Archaea appear to have co‐evolved with other metabolic pathways, broadening their biological roles beyond apoptosis and cell death.     

Secretome analysis of Anabaena sp. PCC 7120 and the involvement of the TolC‐homologue HgdD in protein secretion

Secretion of proteins is a central strategy of bacteria to influence and respond to their environment. Until now, there has been very few discoveries regarding the cyanobacterial secrotome or the secretion machineries involved. For a mutant of the outer membrane channel TolC‐homologue HgdD of Anabaena sp. PCC 7120, a filamentous and heterocyst‐forming cyanobacterium, an altered secretome profile was reported. To define the role of HgdD in protein secretion, we have developed a method to isolate extracellular proteins of Anabaena sp. PCC 7120 wild type and an hgdD loss‐of‐function mutant. We identified 51 proteins of which the majority is predicted to have an extracellular secretion signal, while few seem to be localized in the periplasmic space. Eight proteins were exclusively identified in the secretome of wild‐type cells, which coincides with the distribution of type I secretion signal. We selected three candidates and generated hemagglutinin‐tagged fusion proteins which could be exclusively detected in the extracellular protein fraction. However, these proteins are not secreted in the hgdD‐mutant background, where they are rapidly degraded. This confirms a direct function of HgdD in protein secretion and points to the existence of a quality control mechanism at least for proteins secreted in an HgdD‐dependent pathway.     

Mapping the twin‐arginine protein translocation network of Bacillus subtilis

Bacteria employ twin‐arginine translocation (Tat) pathways for the transport of folded proteins to extracytoplasmic destinations. In recent years, most studies on bacterial Tat pathways addressed the membrane‐bound TatA(B)C subunits of the Tat translocase, and the specific interactions between this translocase and its substrate proteins. In contrast, relatively few studies investigated possible coactors in the TatA(B)C‐dependent protein translocation process. The present studies were aimed at identifying interaction partners of the Tat pathway of Bacillus subtilis, which is a paradigm for studies on protein secretion by Gram‐positive bacteria. Specifically, 36 interaction partners of the TatA and TatC subunits were identified by rigorous application of the yeast two‐hybrid (Y2H) approach. Our Y2H analyses revealed that the three TatA isoforms of B. subtilis can form homo‐ and heterodimers. Subsequently, the secretion of the Tat substrates YwbN and PhoD was tested in mutant strains lacking genes for the TatAC interaction partners identified in our genome‐wide Y2H screens. Our results show that the cell wall‐bound protease WprA is important for YwbN secretion, and that the HemAT and CsbC proteins are required for PhoD secretion under phosphate starvation conditions. Taken together, our findings imply that the Bacillus Tat pathway is embedded in an intricate protein–protein interaction network.     

The characterization and secretion pattern of the lysosomal trehalases ofDictyostelium discoideum

The enzyme trehalase II ofDictyostelium discoideum is efficiently secreted into the matrix of sori along with seven known lysosomal enzymes. The vegetative form of the enzyme, trehalase I, is particulate but the enzyme is secreted prior to cell aggregation or when cells are starved in phosphate buffer under standard secretion conditions. The secreted enzyme possesses properties common to lysosomal enzymes. Polyclonal and monoclonal antibodies raised against purified lysosomalN-acetylglucosaminidase precipitate the enzyme. The enzyme is released efficiently and about 62% of the initial cellular enzyme becomes extracellular. The secretion of trehalase is slightly sensitive to cycloheximide and completely blocked by sodium azide. Secretion is enhanced in the presence of disaccharides such as sucrose, lactose, and trehalose. Electrophoretograms of intracellular and secreted enzyme reveal no major processing of the enzyme during secretion. The pI of the trehalases has been estimated to be less than 2.5.     

Targeting and post‐translational processing of human α1‐antichymotrypsin in BY‐2 tobacco cultured cells†

The post‐translational processing of human α₁‐antichymotrypsin (AACT) in Bright Yellow‐2 (BY‐2) tobacco cells was assessed in relation to the cellular compartment targeted for accumulation. As determined by pulse‐chase labelling experiments and immunofluorescence microscopy, AACT sent to the vacuole or the endoplasmic reticulum (ER) was found mainly in the culture medium, similar to a secreted form targeted to the apoplast. Unexpectedly, AACT expressed in the cytosol was found in the nucleus under a stable, non‐glycosylated form, in contrast with secreted variants undergoing multiple post‐translational modifications during their transit through the secretory pathway. All secreted forms of AACT were N‐glycosylated, with the presence of complex glycans as observed naturally on human AACT. Proteolytic trimming was also observed for all secreted variants, both during their intracellular transit and after their secretion in the culture medium. Overall, the targeting of human AACT to different compartments of BY‐2 tobacco cells led to the production of two protein products: (i) a stable, non‐glycosylated protein accumulated in the nucleus; and (ii) a heterogeneous mixture of secreted variants resulting from post‐translational N‐glycosylation and proteolytic processing. Overall, these data suggest that AACT is sensitive to resident proteases in the ER, the Golgi and/or the apoplast, and that the production of intact AACT in the plant secretory pathway will require innovative approaches to protect its structural integrity in vivo. Studies are now needed to assess the activity of the different AACT variants, and to identify the molecular determinants for the nuclear localization of AACT expressed in the cytosol.