Expression of the staphylococcal protein A gene in Bacillus subtilis by gene fusions utilizing the promoter from a Bacillus amyloliquefaciens alpha-amylase gene.

Gene fusions of DNA sequences encoding protein A from Staphylococcus aureus (spa) with expression elements from an alpha-amylase gene from Bacillus amyloliquefaciens (amyEBamP) directed the synthesis and efficient secretion of protein A in Bacillus subtilis. The fusions were established on multicopy pUB110-based plasmid vectors, in contrast to the intact spa gene, which could not be stably established on plasmids in B. subtilis. Some of the resulting B. subtilis strains secreted protein A at levels in excess of 1 g/liter, demonstrating that a foreign protein encoded by an engineered gene can be secreted by B. subtilis at levels comparable to endogenous exoproteins.     

Expression of a germination-specific amidase, SleB, of Bacilli in the forespore compartment of sporulating cells and its localization on the exterior side of the cortex in dormant spores

A germination-specific amidase of bacilli is a major spore-lytic enzyme that is synthesized with a putative signal sequence and hydrolyses spore cortex in situ. The sleB gene encoding this amidase in Bacillus subtilis and Bacillus cereus was expressed in the forespore compartment of sporulating cells under the control of sigmaG, as shown by Northern blot and primer extension analyses. The forespore-specific expression of B. subtilis sleB was further indicated by the forespore-specific accumulation of a SleB-green fluorescent protein fusion protein from which a putative secretion signal of SleB was deleted. Immunoelectron microscopy with anti-SleB antiserum and a colloidal gold-immunoglobulin G complex showed that the enzymes from both Bacillus species are located just inside the spore coat layer in the dormant spore, and in the dormant spore, the amidases appear exist in a mature form lacking a signal sequence. These results indicate that SleB is translocated across the forespore's inner membrane by a secretion signal peptide and is deposited in cortex layer synthesized between the forespore inner and outer membranes. The peripheral location of the spore-lytic enzymes in the dormant spore suggests that spore germination is initiated at the exterior of the cortex.     

Homology between endoglucanase Z of Erwinia chrysanthemi and endoglucanases of Bacillus subtilis and alkalophilic Bacillus

Nucleotide sequencing of the celZ gene encoding the extracellular endoglucanase Z of Erwinia chrysanthemi indicated the presence of an open reading frame encoding 428 amino acids. The mature protein appeared to be extended by a signal peptide of 43 amino acids; this sequence is unusually long and positively charged (+5). It was shown to function as a signal peptide by fusing it to a truncated phoA gene encoding Escherichia coli alkaline phosphatase. Comparison of the encoded sequence with those of the endoglucanases of Bacillus subtilis and alkalophilic Bacillus revealed the existence of a region of extensive homology occurring in all three proteins at about the same distance from the NH2-terminal end. These regions may be involved in substrate binding and/or catalytic sites.     

Cloning of the maltose phosphorylase gene from Bacillus sp. strain RK-1 and efficient production of the cloned gene and the trehalose phosphorylase gene from Bacillus stearothermophilus SK-1 in Bacillus subtilis

The maltose phosphorylase (MPase) gene of Bacillus sp. strain RK-1 was cloned by PCR with oligonucleotide primers designed on the basis of a partial N-terminal amino acid sequence of the purified enzyme. The MPase gene consisted of 2,655 bp encoding a theoretical protein with a Mr of 88,460, and had no secretion signal sequence, although most of the MPase activity was detected in the culture supernatant of RK-1. This cloned MPase gene and the trehalose phosphorylase (TPase) gene from Bacillus stearothermophilus SK-1 were efficiently expressed intracellularly under the control of the Bacillus amyloliquefaciens alpha-amylase promoter in Bacillus subtilis. The production yields were estimated to be more than 2 g of enzyme per liter of medium, about 250 times the production of the original strains, in a simple shake flask. About 60% of maltose was converted into trehalose by the simultaneous action of both enzymes produced in B. subtilis.     

Expression and secretion of heterologous proteases by Corynebacterium glutamicum.

Genes encoding the basic protease of Dichelobacter nodosus (bprV) and the subtilisin of Bacillus subtilis (aprE) were cloned and expressed in Corynebacterium glutamicum. In each case, enzymatically active protein was detected in the supernatants of liquid cultures. While the secretion of subtilisin was directed by its own signal peptide, the natural signal peptide of the bprV basic protease did not facilitate secretion. A hybrid aprE-bprV gene in which the promoter and signal peptide coding sequences of subtilisin replaced those of bprV could be expressed, and basic protease was secreted by C. glutamicum. Expression of these proteases in C. glutamicum provides an opportunity to compare protein secretion from this gram-positive host with that from other gram-positive and gram-negative bacteria.     

Cloning of aldB, which encodes alpha-acetolactate decarboxylase, an exoenzyme from Bacillus brevis.

A gene for alpha-acetolactate decarboxylase (ALDC) was cloned from Bacillus brevis in Escherichia coli and in Bacillus subtilis. The 1.3-kilobase-pair nucleotide sequence of the gene, aldB, encoding ALDC and its flanking regions was determined. An open reading frame of 285 amino acids included a typical N-terminal signal peptide of 24 or 27 amino acids. A B. subtilis strain harboring the aldB gene on a recombinant plasmid processed and secreted ALDC. In contrast, a similar enzyme from Enterobacter aerogenes is intracellular.     

In vivo genetic engineering: homologous recombination as a tool for plasmid construction

This paper describes a novel method for creating exact DNA fusions between any two points in a plasmid carried in Bacillus subtilis. It exploits the homologous in vivo recombination between directly repeated sequences that can be established by insertion of a synthetic oligodeoxyribonucleotide. The method was used to enhance the productivity in B. subtilis of a cloned alpha-amylase (Amy)-encoding gene originating from Bacillus stearothermophilus. Thus, an exact fusion between nucleotide sequences encoding the expression signals, including the signal peptide, of a Bacillus licheniformis Amy-encoding gene and the mature Amy of B. stearothermophilus, was created. The resulting hybrid translational product was processed correctly in B. subtilis during secretion, giving rise to an Amy identical to the mature Amy secreted by B. stearothermophilus.     

A system for the inducible secretion of proteins from Bacillus subtilis during logarithmic growth

Abstract A Bacillus subtilis-Escherichia coli shuttle vector was constructed containing the B. subtilis levansucrase gene promoter and region encoding its signal sequence.
A site for the restriction enzyme Nae I was included to facilitate precise translational fusions to the DNA encoding the levansucrase signal sequence. Fusions of TEM β-lactamase to this construct displayed sucrose-inducible expression and secretion of B. subtilis .     

外源木聚糖酶在枯草芽胞杆菌中信号肽筛选

[目的]本试验旨在筛选引导表达外源木聚糖酶基因高效分泌的信号肽,为枯草芽胞杆菌木聚糖酶高效分泌表达系统提供元件.[方法]构建信号肽筛选载体,载体是以含壮观霉素抗性基因的大肠-枯草穿梭载体为基本骨架,目标蛋白为耐碱性木聚糖酶,可在麦芽糖启动子Pglv诱导下表达.从枯草芽胞杆菌A1747基因组中扩增获得24个Sec途径信号肽,并将其全部链接到至筛选载体上,并在枯草芽胞杆菌WB700中实现表达分泌.重组菌在3%麦芽糖诱导下培养24h后用DNS法测定上清酶活.[结果]成功构建信号肽筛选载体pGPSX及24个表达载体,实现木聚糖酶表达分泌.且不同信号肽对于引导外源木聚糖酶分泌能力不同,其中YnfF信号肽引导分泌目标蛋白效率最高,上清酶活为37.2IU/mL.[结论]试验证明在枯草杆菌中对外源蛋白进行信号肽筛选是提高其分泌的有效途径,并获得了针对木聚糖酶高效分泌信号肽YnfF.     

Cloning, sequencing and expression of the gene encoding the extracellular neutral protease, vibriolysin, of Vibrio proteolyticus.

The gene (nprV), encoding the extracellular neutral protease, vibriolysin (NprV), of the Gram- marine microorganism, Vibrio proteolyticus, was isolated from a V. proteolyticus DNA library constructed in Escherichia coli. The recombinant E. coli produced a protease that co-migrated with purified neutral protease from V. proteolyticus on non-denaturing polyacrylamide gels, and that demonstrated enzymatic specificity towards the neutral protease substrate N-[3-(2-furyl)acryloyl]-L-alanylphenylalanine amide. The nucleotide (nt) sequence of the cloned nprV gene revealed an open reading frame encoding 609 amino acids (aa) including a putative signal peptide sequence followed by a long 'pro' sequence consisting of 172 aa. The N-terminal aa sequence of NprV purified from cultures of V. proteolyticus, identified the beginning of the mature protein within the aa sequence deduced from the nt sequence. Comparative analysis of mature NprV to the sequences of the neutral proteases from Bacillus thermoproteolyticus (thermolysin) and Bacillus stearothermophilus identified extensive regions of conserved aa homology, particularly with respect to active-site residues, zinc-binding residues, and calcium-binding sites. NprV was overproduced in Bacillus subtilis by placing the DNA encoding the 'pro' and mature enzyme downstream from a Bacillus promoter and signal sequence.     

Cloning, sequencing, and characterization of the genetic region relevant to biosynthesis of the lipopeptides iturin A and surfactin in Bacillus subtilis

Bacillus subtilis B3 was found to produce lipopeptides iturins and fengycin that have activity against several plant pathogens such as Fusarium graminearum, Rhizoctonia solani, Rhizoctonia cerealis, and Pyricularia grisea. A 3642-bp genomic region of B. subtilis B3 comprising srfDB3, aspB3, lpaB3, and yczEB3 genes that resulted in biosynthesis of surfactin in B. subtilis 168 was cloned, sequenced, and characterized. Among them, the srfDB3 gene encodes thioesterase, which is required for biosynthesis of surfactin in B. subtilis; the aspB3 gene encodes a putative aspartate aminotransferase-like protein; the lpaB3 encodes phosphopantetheinyl transferase, which shows high identity to the product of lpa-14 gene regulating the biosynthesis of iturin A and surfactin in B. subtilis RB14; the yczEB3 encodes a YczE-like protein with significant similarities in signal peptide and part of the ABC transport system. The genetic regions between the srfD gene and lpa gene from B. subtilis B3 and B. subtilis A13, which produces iturin A, contain an approximate 1-kb nucleotide fragment encoding an aspartate aminotransferase-like protein; however, the relevant regions from B. subtilis 168 and B. subtilis ATCC21332 producing surfactin comprise an approximately 4-kb nucleotide fragment encoding four unknown proteins. There is 73% identity between the Lpa family and the Sfp family, although both are highly conserved.     

Expression of the MuIFN alpha 7 gene in Bacillus subtilis using the levansucrase system

The mouse interferon alpha 7 gene, the signal sequence of which has been removed by oligonucleotide-directed mutagenesis, was introduced into a Bacillus subtilis secretion vector containing the promoter and the signal sequence of the B. subtilis levansucrase gene. Different B. subtilis strains were transformed with the fused levansucrase-interferon gene; their cell extracts and culture supernatants tested for antiviral activity and the IFN alpha 7 protein showed the presence of IFN alpha 7 only in the cell extracts. To promote IFN alpha 7 secretion, constructs were realized in order to restore the alpha helix conformation of the signal sequence of levansucrase and interferon protein junction. Our results suggest that factors other than the structure of the peptide around the cleavage site are involved in the secretion of IFN alpha 7 by B. subtilis.     

Construction and use of signal sequence selection vectors in Escherichia coli and Bacillus subtilis.

To study the diversity and efficiency of signal peptides for secreted proteins in gram-positive bacteria, two plasmid vectors were constructed which were used to probe for export signal-coding regions in Bacillus subtilis. The vectors contained genes coding for extracellular proteins (the alpha-amylase gene from Bacillus licheniformis and the beta-lactamase gene from Escherichia coli) which lacked a functional signal sequence. By shotgun cloning of restriction fragments from B. subtilis chromosomal DNA, a great variety of different export-coding regions were selected. These regions were functional both in B. subtilis and in E. coli. In a number of cases where protein export had been restored, intracellular precursor proteins of increased size could be detected, which upon translocation across the cellular membrane were processed to mature products. The high frequency with which export signal-coding regions were obtained suggests that, in addition to natural signal sequences, many randomly cloned sequences can function as export signal.     

Functional Role of Hepatitis C Virus Chimeric Glycoproteins in the Infectivity of Pseudotyped Virus

The putative envelope glycoproteins of hepatitis C virus (HCV) likely play an important role in the initiation of viral infection. Available information suggests that the genomic regions encoding the putative envelope glycoproteins, when expressed as recombinant proteins in mammalian cells, largely accumulate in the endoplasmic reticulum. In this study, genomic regions which include the putative ectodomain of the E1 (amino acids 174 to 359) and E2 (amino acids 371 to 742) glycoproteins were appended to the transmembrane domain and cytoplasmic tail of vesicular stomatitis virus (VSV) G protein. This provided a membrane anchor signal and the VSV incorporation signal at the carboxy termini of the E1 and E2 glycoproteins. The chimeric gene constructs exhibited expression of the recombinant proteins on the cell surface in a transient expression assay. When infected with a temperature-sensitive VSV mutant (ts045) and grown at the nonpermissive temperature (40.5°C), cells transiently expressing the E1 or E2 chimeric glycoprotein generated VSV/HCV pseudotyped virus. The resulting pseudotyped virus generated from E1 or E2 surprisingly exhibited the ability to infect mammalian cells and sera derived from chimpanzees immunized with the homologous HCV envelope glycoproteins neutralized pseudotyped virus infectivity. Results from this study suggested a potential functional role for both the E1 and E2 glycoproteins in the infectivity of VSV/HCV pseudotyped virus in mammalian cells. These observations further suggest the importance of using both viral glycoproteins in a candidate subunit vaccine and the potential for using a VSV/HCV pseudotyped virus to determine HCV neutralizing antibodies.     

Conversion of a secretory protein into a transmembrane protein results in its transport to the Golgi complex but not to the cell surface

We have carried out experiments designed to ask if it is possible to convert a secretory protein into an integral membrane protein by appending the membrane spanning domain of an integral membrane protein to its carboxy terminus. We first obtained expression of a cDNA clone encoding rat growth hormone (rGH) in eucaryotic cells, and found that this protein was secreted. We then constructed and expressed a hybrid gene encoding rGH fused to the membrane spanning and cytoplasmic domains of the vesicular stomatitis virus (VSV) glycoprotein (G). This fusion protein was anchored in microsomal membranes in the expected transmembrane configuration. The fusion protein was transported to the Golgi apparatus, and was esterified to palmitic acid, but it was not transported to the cell surface. We suggest that the sorting signal which allows rapid secretion of soluble rGH does not function when the protein is bound to the membrane.     

Secretion of a heterologous protein from Bacillus subtilis with the aid of protease signal sequences.

Secretion vectors based on the genes from Bacillus amyloliquefaciens P for alkaline protease (aprBamP) and neutral protease (nprBamP) were constructed. With both aprBamP and nprBamP, a unique restriction site was introduced 3' of the predicted signal coding region by using the technique of oligonucleotide-directed mutagenesis. The new sites enabled us to fuse a heterologous gene to the expression and secretion elements. We used the protein A gene (spa) from Staphylococcus aureus as a heterologous gene. Bacillus subtilis cells carrying the resulting apr-spa or npr-spa gene fusions synthesized the fusion protein. B. subtilis cells were also capable of removing the signal peptide from the fusion protein, as indicated by the appearance of processed protein A into the growth medium. In addition, these gene fusions allowed us to identify the signal processing site of both the APR-SPA and NPR-SPA proteins.     

Protein secretion in Bacillus subtilis as influenced by the combination of signal sequence and the following mature portion

Abstract We have constructed secretion vector plasmids that have the signal sequence of the Bacillus licheniformis penicillinase gene ( penP ) or the Bacillus stearothermophilus α-amylase gene ( amyT ). We have also constructed penP, amyT and hsa (human salivary α-amylase gene) cartridges. Each of these cartridges was cloned on secretion vectors in Bacillus subtilis , and enzyme production was examined. When amyT vector was used, nearly the same efficiency of enzyme secretion was observed for amyT and penP cartridges. When penP vector was used, enzyme secretion for amyT decreased to about 3% of that for penP cartridges. The eukaryotic gene hsa was hardly expressed in any secretion vectors in B. subtilis .     

Novel plasmid-based expression vectors for intra- and extracellular production of recombinant proteins in Bacillus subtilis

Two plasmid-based expression vectors have been constructed where one allows intracellular production of recombinant proteins while the second directs the proteins into the culture medium. Both vectors use the strong promoter preceding the groESL operon (codes for the essential heat shock proteins GroES and GroEL) of Bacillus subtilis fused to the lac operator allowing their induction by addition of ITPG. While the background level of expression of these expression cassettes is very low in the absence of the inducer, an induction factor of about 1300 was measured. When the genes htpG and pbpE (coding for a heat shock protein and a penicillin-binding protein, respectively) were fused to the groE promoter, the amount of recombinant protein produced after addition of IPTG represented 10 and 13%, respectively, of the total cellular protein. To obtain secretion of recombinant proteins, the coding region for the signal peptide of the amyQ gene encoding an alpha-amylase from Bacillus amyloliquefasciens was fused to the groE promoter. High-level secretion of amyQ alpha-amylase and cellulase A and B of Clostridium thermocellum was demonstrated.     

Nucleotide sequence of the Clostridium thermocellum laminarinase gene.

The sequence presented (1022 bp) shows the Clostridium thermocellum laminarinase gene (lam1) and its flanking regions. The gene lam1 comprises an open reading frame of 726 nt, encoding a 242-aa protein with predicted Mr 27661. The ORF startswith the translation initiation codon ATG. This ATG codon is preceded at a spacing of 7 bp by a potential ribosome binding site (GGAGGT). A putative signal peptide was identified (the potential cleavage site is between position 27-28 aa). The comparison of the primary protein sequence with other beta-1, 3-1, 4-glucanases showed extensive homology for Bacillus amyloliqefaciens and Bacillus subtilis glucanases (identity-46.7%; similarity-57.0%).     

Secretion of staphylococcal nuclease by Bacillus subtilis.

The staphylococcal nuclease (nuc) gene from Staphylococcus aureus has been cloned and expressed in Bacillus subtilis. The nuclease protein was expressed either from its own promoter and translation start signals, or from a combination of a B. subtilis promoter, ribosome binding site, and a signal peptide sequence. Greater than 80% of the active gene product was secreted into the medium, whereas, when a signal peptide sequence was absent, as little as 4% of the nuclease activity was found in the culture medium. Intracellular (or cell-bound) nuclease, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, was shown to have the molecular weight of the predicted precursor protein with the signal peptide. Levels of nuclease reached 50 mg per liter in the culture medium, depending on the growth medium and the strain used. These findings indicate the prospective use of nuclease as a model system for studying secretion of heterologous proteins in B. subtilis.