Objective
The primary objective of the project was the characterisation of the Bacillus protein secretion apparatus to provide a knowledge base for optimised production of native and heterologous proteins. B. subtilis, the paradigm for Gram-positive bacteria, was used as the model organism.
Many genes specifying early (cytoplasmic), middle (translocase), and late releaseHfolding) components of the secretion apparatus have been identified and characterised. Several of the corresponding gene products were shown to be bottlenecks in the secretion of model proteins Strong evidence was obtained that the cell wall is also a bottleneck for the secretion of native and heterologous proteins. Good progres was made in the development of an in vitro protein translocation systems for B. subtilis. the new insights in the secretion process and the identified genes will be used in the follow-up project (CT960097) for the engineering/optimisation of the secretion apparatus.
MAJOR SCIANTIFIC BREAKTHROUGHS/INDUSTIRAL APPLICATIONS:
Four approache, labeled below as A, B, C, and D, were followed to achieve the objectives.
A. Identifiction and characterisation of components of the Bacillus secretion apparatus
I.Early components. The chaperone genes groEL/ES, dnaK/dnaJ/grpE wer placed under the control of regulatable promoters (02). No effects of DnaK/DnaJ/GrpE on protein secretion were observed (02): In contrast, overproduction of GroEL/ES stimulated the secretion of alpha-amylase (02). The potential role of the chaperone HtpG in protein secretion is being analysed (02). A secB-like gene is likely to be missing in bacilli (03). The ftsY gene encoding the putative receptor for the SRP-like targeting factor Ffh was found upstream of the ffh gene (03). In contrast to other known FtsY proteins, FtsY of B. subtilis appears to be a soluble cytosolic protein. The ftsY gene is esential for cell growth and, probably, protein secretion (03). Maximal expression of ftsY occurs at the end of the exponential growth phase, at the onset of increased secretion activity, suggesting that coregulation of secretion activity and synthesis of components of the secretion machinery may exist (03). Fragments of B. subtilis and Bacillus caldolyticus DNA were identified which stilulate the production and/or export of b-lactamase in E. coli through an, as yet, unidentified mechanism (01). A novel ABC transporter, ecs, was detected which increases both the expression and secretion of several secretory proteins (07), possibly through effects on expression of other components of the secretion pathway (01,05,07). The gene for an MPP-like processing protease is not required for protein secretion (01).
II.Translocase components. Genes encoding major components of the translocase complex, secA, secY and secE, are available (04,05). The secA gene of the thermophilic B. stearothermophilus was cloned (04). Efforts to clone a B. subtilis secG-like gene have not yet been successful, but the presence of this gene was shown by Southern hybridization, and its disruption resulted in impaired secretion of alpha-amylase (04). SecA of B. subtilis could be inhibited by azide (05). The nucleotide binding sites of SecA and the corresponding Mg2+-binding residues were identified (04,05). Low resolution images of SecA were obtained by electron microscopy (04). SecA has a two-domain structure; interactions between these domains are mediated by nucleotide binding (04). Interactions between SecA and SecY in intact B. subtilis werd shown by chemical cross-linking (04). Complementation of secA mutations in B. subtilis and other bacteria indicated that SecA interacts most effeciently with translocase components of the same host (05). The translocase of B. subtilis was shown to transport a heterologous precursor protein (OmpA) with its own signal peptide (05). Like the ftsY gene, the B. subtilis secA gene is maximally expressed at the end of exponential growth, at the onset of increased secretion activity (05). atthe end of exponential growth, at the onset of increased secretion activity (05).
III.Late functions. Type I signal peptidase (sip) genes were cloned from B. subtilis and several other bacilli, such as the thermophilic B. caldolyticus (01). From other projects, partly from within this consortium, two additional chromosomal B. subtilis sip genes (sipT and sipU), and two plasmid-encoded B. subtilis (natto) sipP genes, became available (01). None of these genes is essential for growth. SipS production is maximal during post-exponential growth and, like SecA (A.II) maximal synthesis seems to coincide with maximal secretion activity (01). SipS, SipT and SipU have a preference for different subsets of secretory proteins (01). The potential active site of SipS was identified (01). The gene for the lipoprotein-specific type II signal peptidase of B. subtilis was cloned (01). The FtsH protein, which appears to be an ATP-dependent protease with chaperone activity, is required for secretion in B. subtilis; ftsH mutants secrete strongly reduced amounts of protein, and no subtilisin (02). the lipoprotein PrsA, which shows homology to peptidyl-prolyl cis/trans isomerases, has been cloned from B. subtilis (07) and several other bacilli (02, 05, 07, 09). PrsA seems to catalyse correct folding of some secreted proteins and is required for their high-level production (06, 07, 08, 09). PrsA seems to catalyse correct folding of some secreted proteins and is required for their high-level production (06, 07, 08, 09). The product of the lgt gene is required for lipomodification of PrsA, which seems to be a prerequisite for full PrsA activity (07). Inducible variants of prsA were made, and the protein was overexpressed and purified (07). For several native reporter proteins it was shown that the post-translocational folding and release steps are rat-limiting for secretion in to the growth medium, with temperature and the presence of Ca2+ ions being critical parameters (06, 08, 09). Production and secretion of the human protein disulphide bond isomerase (PDI) did not improve the secretion of a heterologous protein that accumulates in the cell wall (01).
IV.Cell wall. To assess the role of the cell wall, an efficient system to analyse protein secretion in protoplasts was set up (07). In protoplasts, processing and release of translocated proteins was strongly reduced, and the stability of these proteins was no longer dependent on PrsA (07). The influence of the anionic polymer comporition of the wall on secretion was studied; cells with walls containing more teichuronic than teichoic acid showed the fastest processing and release kinetics of secreted proteins (08). Hybrid alpha-amylases with different pI-values were constructed to test the effects of charge of secretory proteins on cell wall passage (08,09). These structurally conserved changes normally resulted in reduced levels of secretion. This is probably due to reduced folding kinetics and increased susceptibility to as yet unidentified proteases (06, 08, 09). A major role of the wall in this process is probably maintaining the divalent metal ion concentration (06, 08), important for folding of secretory proteins (see A. III).
B. Development of in vitro systems
Three in vitro systems were developed: (i) an in vitro protein translocation system was established for B. subtilis by using membrane vesicles from strains with reduced levels of extracellular proteases which overproduce SecE and SecY of B. subtilis. The translocation activity of the vesicles was low, but could be strongly increased by including SecA and SecG of E. coli (A.II; 04). Further optimisation of this system awaits the cloning of the B. subtilis secG gene. Radiolabelled hexahistidine tagged variants of hybrid alpha-amylases (see A.IV) have been synthesized in vitro to study the influence of pI on protein translocation (04). (ii), The refolding kinetics of secreted proteins, such as levansucrase and B. subtilis alpha-amylase were analysed in vitro: pH, Ca2+/PO4ion concentration and temperature were important parameters for efficient refolding (06); GroEL and PrsA had no influence on refolding rates (04, 06, 07). (iii), An in vitro cell wall binding assay was developed; wild-type alpha-amylase (AmyL; pI 7) did not bind to the wall, whereas a mutant alpha-amylase with a pI of 10 was bound strongly.
C. Identification of secretion bottlenecks
Several bottlenecks have been identified. The (over-) production of E. coli SecB and B. subtilis Ffh had under some conditions a positive effect on the secretion of certain eukaryotic proteins via B. subtilis (03); overproduction of GroEL/ES stimulated the secretion of alpha-amylase (02); and processing of certain hybrid precursors was improved by overproduction of SipS (01). In contrast, processing of overproduced pre-alpha-amylase was improved by disruption of the sipS gene (01). The temporally regulated expression SecA (05), and SipS (01) are possible bottlenecks when (heterologous) secretory proteins are produced during the exponential phase. PrsA was limiting for the production of (heterologous( extracellular enzymes under certain conditions (03, 06, 07, 08, 09). Rapid folding after translocation across the membrane is essential for stability and release into the medium, in particular for the secretion of non-native proteins. Factors which promote folding, such as PrsA, divalent cations, and indirectly the cell wall, are therefore potential limiting factors (01, 0ì, 06, 07, 08, 09).
D. Development of strategies for the optimisation of secretion
Attempts to develop strategies for the improved production of secreted (heterologous) proteins will mainly be carried out in the follow-up project (CT60097). Some initial attempts were made. The use of (i), a strain lacking six extracellular proteases improved the secretion of human-a-amylase slightly (01); and (ii), overproduction of certain components of the secretion apparatus improved the secretion ot at least some heterologous proteins under certain conditions (see section C; 03, 07, 08, 09).
Fields of science (EuroSciVoc)
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
- natural sciences biological sciences microbiology bacteriology
- natural sciences physical sciences optics microscopy electron microscopy
- natural sciences biological sciences genetics mutation
- natural sciences biological sciences genetics nucleotides
- natural sciences biological sciences biochemistry biomolecules proteins enzymes
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