A protein phosphorylation module patterns the Bacillus subtilis spore outer coat

Assembly of the Bacillus subtilis spore coat involves over 80 proteins which self-organize into a basal layer, a lamellar inner coat, a striated electrodense outer coat and a more external crust. CotB is an abundant component of the outer coat. The C-terminal moiety of CotB, SKR^B, formed by serine-rich repeats, is polyphosphorylated by the Ser/Thr kinase CotH. We show that another coat protein, CotG, with a central serine-repeat region, SKR^G, interacts with the C-terminal moiety of CotB and promotes its phosphorylation by CotH in vivo and in a heterologous system. CotG itself is phosphorylated by CotH but phosphorylation is enhanced in the absence of CotB. Spores of a strain producing an inactive form of CotH, like those formed by a cotG deletion mutant, lack the pattern of electrondense outer coat striations, but retain the crust. In contrast, deletion of the SKR^B region, has no major impact on outer coat structure. Thus, phosphorylation of CotG by CotH is a key factor establishing the structure of the outer coat. The presence of the cotB/cotH/cotG cluster in several species closely related to B. subtilis hints at the importance of this protein phosphorylation module in the morphogenesis of the spore surface layers.     

Surface-displayed VP28 on Bacillus subtilis spores induce protection against white spot syndrome virus in crayfish by oral administration

Aim: Surface‐displayed heterologous antigens on Bacillus subtilis spores can induce the vertebrate animals tested to generate local and systematic immune response through oral immunization. Here, the protection potential of the recombinant spores displaying the VP28 protein of white spot syndrome virus (WSSV) was investigated in the invertebrate crayfish (Cambarus clarkii). Methods and Results: The VP28 protein was successfully displayed on the surfaces of B. subtilis spores using CotB or CotC as a fusion partner. Crayfish were administrated orally by feeding the feed pellets coated with B. subtilis spores for 7 days and immediately followed by WSSV challenge. Oral administration of either spores expressing CotB‐VP28 or CotC‐VP28 resulted in significantly higher relative survival rates of 37·9 and 44·8% compared with the crayfish orally administrated with the spores nonexpressing VP28 (10·3% relative survival rate). When challenges were separately conducted at 7 and 21 days after oral administration, the relative survival rates increased to 46·4 and 50% at 7 days post‐oral administration, but decreased to 30 and 33·3% at 21 days after oral administration. Conclusion: These evidences indicate that the surface‐displayed VP28 on B. subtilis spore could induce protection of crayfish against WSSV via oral administration. Significance and Impact of the Study: This is the first report to use the spore surface display system to deliver orally a heterologous antigen in an aquatic invertebrate animal, crayfish. The results presented here suggest that the spore‐displayed VP28 might be suitable for an oral booster vaccine on prevention of WSSV infection in shrimp farming.     

Bacterial Surface Display of a Co-Factor Containing Enzyme, ω-Transaminase from Vibrio fluvialis Using the Bacillus subtilis Spore Display System

To improve the conventional bacterial surface display systems and to display a co-factor containing enzyme, ω-transaminase from Vibrio fluvialis, which needs pyridoxal phosphate (PLP) for efficient transamination, Bacillus subtilis spore display system with cotG, as an anchoring motif was used. Flow cytometry of the B. subtilis spore-expressing ω-transaminase proved its surface localization on the spore. The enzymatic activity of the spore expressing ω-transaminase was more than 30 times higher than that of the host spore. Protease treatment of the ω-transaminase displaying spores resulted in decreased transaminase activity, which is in keeping with the surface location of the fusion protein, CotG-ω-transaminase.     

Recent progress in Bacillus subtilis spore-surface display: concept,progress, and future

With the increased knowledge on spore structure and advances in biotechnology engineering, the newly developed spore-surface display system confers several inherent advantages over other microbial cell-surface display systems including enhanced stability and high safety. Bacillus subtilis is the most commonly used Bacillus species for spore-surface display. The expression of heterologous antigen or protein on the surface of B. subtilis spores has now been practiced for over a decade with noteworthy success. As an update and supplement to other previous reviews, we comprehensively summarize recent studies in the B. subtilis spore-surface display technique. We focus on its benefits as well as the critical factors affecting its display efficiency and offer suggestions for the future success of this field.

     

枯草芽孢杆菌表面展示技术用于黏膜疫苗的研究进展

枯草杆菌全名枯草芽孢杆菌(Bacillus subtilis),因其优秀的益生特性及芽孢良好的抗逆性而备受研究者青睐,由于芽孢的特殊结构及独特的生理特性,是酶和免疫原等外源蛋白的理想锚定点。采用枯草杆菌进行芽孢表面展示被认为是表达高活性和高稳定性的外源蛋白的方法之一。本文主要对枯草杆菌芽孢表面展示抗原蛋白以生产黏膜疫苗的策略和应用前景进行综述。     

Amino-terminal structure of spoOA protein and sequence homology with spoOF and spoOB proteins

Summary The previously reported nucleotide sequence of the spoOA coding region of Bacillus subtilis suggested that the protein is initiated with either of two possible initiation codons, ATG and GTG, 84 base pairs apart. To determine which codon is utilized as an initiator in B. subtilis, we constructed a fusion gene in which the promoter and NH₂-terminal region of the spoOA gene was connected to the chloramphenicol acetyltransferase gene (cat gene). After introduction of the plasmid carrying the spoOA-cat fusion gene into B. subtilis cells, the fusion protein was purified by affinity chromatography. The sequence of NH₂-terminal amino acids of the fusion protein was determined and the result established that the GTG codon is utilized as an initiator in B. subtilis.Comparison of the amino acid sequences revealed a marked homology between the spoOA (NH₂-terminal half) and spoOF proteins. A less striking but significant homology was also found between the spoOA (COOH-terminal half) and spoOB proteins. This suggests the presence of a common functional domain structure for these proteins that are supposed to play key regulatory roles in sporulation.     

Effect of depletion of FtsY on spore morphology and the protein composition of the spore coat layer in Bacillus subtilis

Bacillus subtilis FtsY is a homolog of the alpha-subunit of mammalian signal recognition particle (SRP) receptor, and is essential for protein translocation and vegetative cell growth. An FtsY conditional null mutant (strain ISR39) can express ftsY during the vegetative stage but not during spore formation. Spores of ISR39 have the same resistance to heat and chloroform as the wild-type, while their resistance to lysozyme is reduced. Electron microscopy showed that the outer coat of spores was incompletely assembled. The coat protein profile of the ftsY mutant spores was different from that of wild-type spores. The amounts of CotA, and CotE were reduced in spore coat proteins of ftsY mutant spores and the molecular mass of CotB was reduced. In addition, CotA, CotB, and CotE are present in normal form at T(8) of sporulation in ftsY mutant cells. These results suggest that FtsY has a pivotal role in assembling coat proteins onto the coat layer during spore morphogenesis.     

Production of glucoamylase in pyruvate decarboxylase deletion mutants of the yeast Kluyveromyces lactis

We have developed a novel Escherichia coli cell surface display system by employing PgsA as an anchoring motif. In our display system, C-terminal fusion to PgsA anchor protein from Bacillus subtilis was used. The enzymes selected for display were α-amylase (AmyA) from Streptococcus bovis 148 and lipase B (CALB) from Candida antarctica. The molecular mass values of AmyA and CALB are approximately 77 and 34 kDa, respectively. The enzymes were displayed on the surface as a fusion protein with a FLAG peptide tag at the C terminus. Both the PgsA-AmyA-FLAG and PgsA-CALB-FLAG fusion proteins were shown to be displayed by immunofluorescence labeling using anti-FLAG antibody. The displayed enzymes were active forms, and AmyA and CALB activities reached 990 U/g (dry cell weight) and 4.6 U/g (dry cell weight), respectively. AmyA-displaying E. coli cells grew utilizing cornstarch as the sole carbon source, while CALB-displaying E. coli cells catalyzed enantioselective transesterification, indicating that they are effective whole-cell biocatalysts. Since a target enzyme with a size of 77 kDa and an industrially useful lipase have been successfully displayed on the cell surface of E. coli for the first time, PgsA protein is probably a useful anchoring motif to display various enzymes.     

Surface display of recombinant proteins on Bacillus subtilis spores

We developed a novel surface display system based on the use of bacterial spores. A protein of the Bacillus subtilis spore coat, CotB, was found to be located on the spore surface and used as fusion partner to express the 459-amino-acid C-terminal fragment of the tetanus toxin (TTFC). Western, dot blot and fluorescent-activated cell sorting analyses were used to monitor TTFC surface expression on purified spores. We estimated that more than 1.5 x 10(3) TTFC molecules were exposed on the surface of each spore and recognized by TTFC-specific antibodies. The efficient surface presentation of the heterologous protein, together with the simple purification procedure and the high stability and safety record of B. subtilis spores, makes this spore-based display system a potentially powerful approach for surface expression of bioactive molecules.     

Oral vaccination with envelope protein VP28 against white spot syndrome virus in Procambarus clarkii using Bacillus subtilis as delivery vehicles

Aims: To achieve high‐level expression and secretion of active VP28 directed by a processing‐efficient signal peptide in Bacillus subtilis WB600 and exploit the possibility of obtaining an oral vaccine against white spot syndrome virus (WSSV) using vegetative cells or spores as delivery vehicles. Methods and Results: The polymerase chain reaction (PCR)‐amplified vp28 gene was inserted into a shuttle expression vector with a novel signal peptide sequence. After electro‐transformation, time‐courses for recombinant VP28 (rVP28) secretion level in B. subtilis WB600 were analysed. Crayfish were divided into three groups subsequently challenged by 7‐h immersion at different time points after vaccination. Subgroups including 20 inter‐moult crayfish with an average weight of 15 g in triplicate were vaccinated by feeding coated food pellets with vegetative cells or spores for 20 days. Vaccination trials showed that rVP28 by spore delivery induced a higher resistance than using vegetative cells. Challenged at 14 days postvaccination, the relative per cent survival (RPS) values of groups of rVP28‐bv and rVP28‐bs was 51·7% and 78·3%, respectively. Conclusions: The recombinant B. subtilis strain with the ability of high‐level secretion of rVP28 can evoke protection of crayfish against WSSV by oral delivery. Significance and Impact of the Study: Oral vaccination by the B. subtilis vehicle containing VP28 opens a new way for designing practical vaccines to control WSSV.     

Heterologous protein secretion in Lactococcus lactis is enhanced by the Bacillus subtilis chaperone-like protein PrsA

The Bacillus subtilis lipoprotein PrsA enhances the yield of several homologous and heterologous exported proteins in B. subtilis by being involved in the posttranslocational stage of the secretion process. In this work, we have studied the effect of B. subtilis PrsA on the secretion of Bacillus amyloliquefaciens α-amylase (AmyQ), a target protein for PrsA, and Bacillus licheniformis penicillinase (PenP) a nontarget protein for PrsA, in Lactococcus lactis. Two compatible plasmids were constructed and introduced into L. lactis strain NZ9000: one high copy plasmid, expressing the AmyQ gene (amyQ) or the PenP gene (penP), and one low copy plasmid, expressing the PrsA encoding gene (prsA). When amyQ and prsA were simultaneously expressed under the nisin-inducible promoter P _nisA , Western blotting experiments revealed a 15- to 20-fold increase in the total yield of AmyQ and a sixfold increase in secreted AmyQ activity, compared to a control strain lacking prsA. When expressed under the same induction conditions, PrsA had no effect on the secretion or total yield of PenP. These results show that the secretion yield of some heterologous proteins can be significantly increased in L. lactis when coproduced with the B. subtilis PrsA protein.     

Differences in pattern of a DNA protein complex isolated from vegetative cells and spores of Bacillus subtilis.

Summary A DNA protein complex has been isolated from vegetative cells and spores of Bacillus subtilis. Properties of the DNA protein complex prepared from vegetative cells were studied and SDS gel electrophoresis was employed to compare the different DNase-untreated and-treated DNA protein complexes. It is concluded that proteins are associated with the DNA and differences in protein pattern in polyacrylamide gels indicates the involvement of DNA-binding proteins in the regulation of spore formation.     

An outer membrane protein (OmpA) of Escherichia coli can be translocated across the cytoplasmic membrane of Bacillus subtllis

The translocation of secretory proteins derived from a Gram-positive (Staphylococcus hyicus prolipase) or a Gram-negative (Escherichia coli pre-OmpA protein) bacterium across the cytoplasmic membrane was studied in E. coli and Bacillus subtilis. in both microorganisms, the prolipase was found to be secreted across the plasma membrane when either the pre-prolipase signal peptide (38 amino acids in length) or the pre-OmpA signal peptide (21 amino acids in length) was used. Expression of the gene encoding the authentic pre-OmpA protein in B. subtilis resulted in the translocation of mature OmpA protein across the plasma membrane. Processing of the OmpA precursor in B. subtilis required the electrochemical potential and was sensitive to sodium azide, suggesting that the B. subtilis SecA homologue was involved in the translocation process. The mature OmpA protein, which was most likely present in an aggregated state, was fully accessible to proteases in protoplasted cells. Therefore, our results clearly demonstrate that an outer membrane protein can be secreted by B. subtilis, supporting the notion that the basic mechanism of protein translocation is highly conserved in Gram-positive and Gram-negative bacteria.     

Development of a <Emphasis Type="Italic">Bacillus subtilis</Emphasis> expression system using the improved P<Emphasis Type="Italic">glv</Emphasis> promoter

Background  

B. subtilis is an important organism in the biotechnological application. The efficient expression system is desirable in production of recombinant gene products in B. subtilis. Recently, we developed a new inducible expression system in B. subtilis, which directed by B. subtilis maltose utilization operon promoter P _glv . The system demonstrated high-level expression for target proteins in B. subtilis when induced by maltose. However, the system was markedly repressed by glucose. This limited the application of the system as a high-expression tool in biotechnology field. The aim of this study was to further improve the P _glv promoter system and enhance its expression strength.     

Alternate promoters direct stress-induced transcription of the Bacillus subtilis clpC operon

clpC ofBacillus subtilis is part of an operon containing six genes. Northern blot analysis suggested that all genes are co-transcribed and encode stress-inducible proteins. Two promoters (P_A and P_B) were mapped upstream of the first gene. P_A resembles promoters recognized by the vegetative RNA polymerase Eσ^A. The other promoter (P_B) was shown to be dependent on σ^B, the general stress σ factor in B. subtilis, suggesting that clpC, a potential chaperone, is expressed in a σ^B-dependent manner. This is the first evidence that σ^B in B, subtilis is involved in controlling the expression of a gene whose counterpart, clpB, is subject to regulation by σ³² in Escherichia coli, indicating a new function of σ^B-dependent general stress proteins. P_B deviated from the consensus sequence of σ^B promoters and was only slightly induced by starvation conditions. Nevertheless, strong induction by heat, ethanol, and salt stress occurred at the σ^B-dependent promoter, whereas the vegetative promoter was only weakly induced under these conditions. However, in a sigB mutant, the σ^A-like promoter became inducible by heat and ethanol stress, completely compensating for sigB deficiency. Only the downstream σ^A-like promoter was induced by certain stress conditions such as hydrogen peroxide or puromycin. These results suggest that novel stress-induction mechanisms are acting at a vegetative promoter. Involvement of additional elements in this mode of induction are discussed.     

Survival of Plasmid-Containing Bacillus subtilis Released into Mushroom Compost

Abstract The survival of a plasmid-containing Bacillus subtilis released into mushroom compost was investigated. The indigenous Bacillus population of mushroom compost exhibited an antibiotic-resistance profile that was distinguished by almost complete absence of chloramphenicol resistance. Bacillus subtilis containing the chloramphenicol-resistance plasmid pC194 was released into mushroom compost microcosms and populations were monitored at different incubation temperatures. The organism colonized both sterile and untreated compost at 37°C, and to a lesser extent at 50°C, but was eliminated after 30 d at 65°C. Although sporulation of the B. subtilis population occurred within compost, the population was maintained for up to 13 weeks at 50°C, largely as vegetative cells. Experiments in which the B. subtilis host strain, without plasmid, was released demonstrated that plasmid carriage had no effect on the ability of the bacterium to colonize and survive in compost. Furthermore, the size and composition of the indigenous bacterial population was unaffected by the presence of the introduced B. subtilis strain. Virtually no loss of plasmid pC194 from the B. subtilis population in compost was observed, and experiments at low growth rates in chemostats confirmed the stability of this host/vector system in the absence of positive selection pressure. Received: 9 July 1997; Accepted: 20 October 1997     

The roles of signal peptide and mature protein in RNase (barnase) export from Bacillus subtilis

Barnase, an extracellular RNAse from Bacillus amyloliquefaciens is secreted post-translationally from B. subtilis. The rate of secretion of barnase from B. subtilis was improved by replacement of the barnase signal peptide with a heterologous signal peptide. However, the barnase signal peptide exported Escherichia coli alkaline phosphatase faster than mature barnase. Heat shock of B. subtilis cells did not significantly alter the export of barnase using the barnase signal peptide. The slow rate of export of barnase from B. subtilis is due to both the signal peptide and the mature protein sequence rather than either alone.     

An improved system for the surface immobilisation of proteins on Bacillus thuringiensis vegetative cells and spores through a new spore cortex-lytic enzyme anchor

An improved surface-immobilisation system was engineered to target heterologous proteins onto vegetative cells and spores of Bacillus thuringiensis plasmid-free recipient strain BMB171. The sporulation-dependent spore cortex-lytic enzyme from B. thuringiensis YBT-1520, SceA, was expressed in vegetative cells and used as the surface anchoring motif. Green fluorescent protein (GFP) and a Bacillus endo-β-1,3-1,4-glucanase (BglS) were used as the fusion partners to test the binding efficiency and the functional activities of immobilised surface proteins. The surface localisation of the SceA-GFP fusion protein on vegetative cells and spores was confirmed by Western blot, immunofluorescence microscopy and flow cytometry. The GFP fluorescence intensity from both vegetative cells and spores was measured and compared to a previously characterised surface display system using a peptidoglycan hydrolase anchor (Mbg). Results demonstrated comparable efficiency of SceA- and Mbg-mediated immobilisation on vegetative cells but a more efficient immobilisation on spores using the SceA anchor, suggesting SceA has greater potential for spore-based applications. The SceA protein was then applied to target BglS onto vegetative cells and spores, and the surface immobilisation was verified by the substantial whole-cell enzymatic activity and enhanced whole-spore enzymatic activity compared to vegetative cells. A dually active B. thuringiensis vegetative cell and spore display system could prove especially valuable for the development of regenerable and heat-stable biocatalysts that function under adverse environmental conditions, for example, an effective feed additive for improved digestion and nutrient absorption by livestock.     

Surface display of lipolytic enzyme,Lipase A and Lipase B of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> on the <Emphasis Type="Italic">Bacillus subtilis</Emphasis> spore

For the enhancement of lipase stability in organic solvent containing reaction, live immobilization method, using Bacillus subtilis spore as a display vehicle was attempted. Bacillus subtilis coat protein cotE was used as an anchoring motif for the display of lipA and lipB of Bacillus subtilis. Using this motif, lipolytic enzyme Lipase A and Lipase B were functionally displayed on the surface of Bacillus subtilis spore. Purified spore displaying CotE-LipB fusion protein showed higher lipolytic activity compared to that of CotE-LipA fusion protein. The surface localization of Lipase B was verified with flow cytometry and protease accessibility experiment. Spore displayed lipase retained its activity against acetone and benzene which completely deactivated free soluble lipase in the same reaction condition.