Complementation of the Lactococcus lactis Secretion Machinery with Bacillus subtilis SecDF Improves Secretion of Staphylococcal Nuclease

Unlike Bacillus subtilis and Escherichia coli, the gram-positive lactic acid bacterium Lactococcus lactis does not possess the SecDF protein, a component of the secretion (Sec) machinery involved in late secretion stages and required for the high-capacity protein secretion in B. subtilis. In this study, we complemented the L. lactis Sec machinery with SecDF from B. subtilis and evaluated the effect on the secretion of two forms of staphylococcal nuclease, NucB and NucT, which are efficiently and poorly secreted, respectively. The B. subtilis SecDF-encoding gene was tested in L. lactis at different levels. Increased quantities of the precursor and mature forms were observed only at low levels of SecDF and at high NucT production levels. This SecDF secretion enhancement was observed at the optimal growth temperature (30°C) and was even greater at 15°C. Furthermore, the introduction of B. subtilis SecDF into L. lactis was shown to have a positive effect on a secreted form of Brucella abortus L7/L12 antigen.     

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.     

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.     

Secretion and processing of staphylococcal nuclease by Bacillus subtilis.

We have studied the secretion and processing of Staphylococcus aureus nuclease in Bacillus subtilis. We show that the initial species of nuclease found in the cell supernatants during short-term radioactive labeling (pulse-chase) had a molecular weight of approximately 18,800 and comigrated in a sodium dodecyl sulfate-polyacrylamide gel with staphylococcal nuclease B. This nuclease B form was processed to the mature nuclease A extracellularly by a phenylmethylsulfonyl fluoride-sensitive protease. The nuclease B-processing site is a consensus signal peptidase site, and the processing of nuclease B was coupled to secretion as judged by pulse-chase experiments. The nuclease A was shown by microsequencing of the N terminus to be 2 amino acid residues shorter than the nuclease A described for S. aureus Foggi. The nuclease B form was still the first species found in the culture supernatant after removal of the N-terminal 26 amino acids of the native 60-amino-acid signal peptide. However, removal of the N-terminal 72 amino acids abolishes secretion of any nuclease form and leads to the intracellular accumulation of nuclease.     

Heterologous gene expression in Lactococcus lactis subsp. lactis: synthesis, secretion, and processing of the Bacillus subtilis neutral protease

The Bacillus subtilis nprE gene lacking its own promoter sequence was inserted in the lactococcal expression vector pMG36e. Upon introduction of the recombinant plasmid into Lactococcus lactis subsp. lactis strain MG1363, neutral protease activity could be visualized by the appearance of large clearing zones around colonies grown on milk agar plates. By measuring the activities of the neutral protease and the intracellular enzyme lactate dehydrogenase in culture supernatants and cell fractions, it was demonstrated that the neutral protease was actively secreted into the growth medium. This was corroborated by using the Western blot (immunoblot) technique, which showed the presence of the mature form of the neutral protease in the culture supernatant. On the basis of these results, it is concluded that the B. subtilis neutral protease gene was expressed in L. lactis and that the gene product was secreted into the growth medium and was apparently correctly processed to produced a biologically active protein. The secretion of this particular enzyme may be helpful in achieving accelerated cheese ripening.     

Controlled intra- or extracellular production of staphylococcal nuclease and ovine omega interferon in Lactococcus lactis

A system for controlled targeting of heterologous protein was developed in the food-grade bacterium Lactococcus lactis. It is composed of the L. lactis strain NZ9000 and of two broad host range expression vectors pCYT:Nuc and pSEC:Nuc for, respectively, cytoplasmic and secreted staphylococcal nuclease (Nuc) nisin-inducible production. The level of intracellular production of Nuc measured with pCYT:Nuc (3 mg x l(-1)) is significantly lower than the one obtained with pSEC:Nuc ( approximately 20 mg x l(-1)). The secretion efficiency (SE) of Nuc is estimated to be approximately 70%, corresponding to approximately 15 mg of secreted Nuc x l(-1). Furthermore, we established that Nuc production continued in L. lactis 10 h after a 1-h nisin-pulse induction. This system was then used for intra- and extracellular production of a protein of therapeutical interest in L. lactis, the ovine interferon-omega (IFN-omega). The SE and the quantity of secreted active IFN-omega were evaluated respectively to be approximately 70% and approximately 1 mg x l(-1) ( approximately two-fold higher than the cytoplasmic form).     

Heterologous gene expression in Lactococcus lactis subsp. lactis: synthesis, secretion, and processing of the Bacillus subtilis neutral protease.

The Bacillus subtilis nprE gene lacking its own promoter sequence was inserted in the lactococcal expression vector pMG36e. Upon introduction of the recombinant plasmid into Lactococcus lactis subsp. lactis strain MG1363, neutral protease activity could be visualized by the appearance of large clearing zones around colonies grown on milk agar plates. By measuring the activities of the neutral protease and the intracellular enzyme lactate dehydrogenase in culture supernatants and cell fractions, it was demonstrated that the neutral protease was actively secreted into the growth medium. This was corroborated by using the Western blot (immunoblot) technique, which showed the presence of the mature form of the neutral protease in the culture supernatant. On the basis of these results, it is concluded that the B. subtilis neutral protease gene was expressed in L. lactis and that the gene product was secreted into the growth medium and was apparently correctly processed to produced a biologically active protein. The secretion of this particular enzyme may be helpful in achieving accelerated cheese ripening.     

Oral delivery of staphylococcal nuclease by Lactococcus lactis prevents type 1 diabetes mellitus in NOD mice

Applied Microbiology and Biotechnology - Type 1 diabetes mellitus (T1DM) is an autoimmune disease characterized by the self-destruction of insulin-producing β cells. Recently, studies have...     

Protein traffic for secretion and related machinery of Bacillus subtilis

Gram-positive sporulating Bacillus subtilis secretes high levels of protein. Its complete genome sequence, published in 1997, encodes 4,106 proteins. Bioinformatic searches have predicted that about half of all B. subtilis proteins are related to the cell membrane through export to the extracellular medium, insertion, and attachment. Key features of the B. subtilis protein secretion machinery are the absence of an Escherichia coli SecB homolog and the presence of an SRP (signal recognition particle) that is structurally rather similar to human SRP. In addition, B. subtilis contains five type I signal peptidases (SipS, T, U, V, and W). Our in vitro assay system indicated that co-operation between the SRP-protein targeting system to the cell membrane and the Sec protein translocation machinery across the cytoplasmic membrane constitutes the major protein secretion pathway in B. subtilis. Furthermore, the function of the SRP-Sec pathway in protein localization to the cell membrane and spore was analyzed.     

Sequence of a Lactococcus lactis DNA fragment homologous to the recF gene of Bacillus subtilis

The recF gene of Lactococcus lactis ATCC 7962 is located 3 kb downstream from the lacZ gene and is transcribed in the opposite orientation. The recF gene is immediately preceded by a 121-codon ORF, and both recF and orf121 may be transcribed from the same promoter. The deduced RecF amino-acid sequence shows high homology to that of the Bacillus subtilis and Streptococcus pyogenes RecF proteins     

Metabolic engineering and synthetic biology employing Lactococcus lactis and Bacillus subtilis cell factories

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Complementation studies with human ClpP in Bacillus subtilis

ATP-dependent intracellular proteolysis is essential for all living organisms. ClpP, the proteolytic subunit of the ATP-dependent Clp proteases, shares 56% protein identity between B. subtilis and man. The aim of this study was to verify, whether human ClpP (HClpP) is able to substitute the bacterial pendant, BClpP, irrespectively of the huge evolutionary distance. For this reason hclpP was expressed from the natural B. subtilis promoters at the original chromosomal site. Growth at 37 °C as well as sporulation in the presence of hclpP depict an intermediate phenotype between wild type and clpP mutant suggesting a partial functional substitution of BClpP by HClpP. Northern as well as Western blot analyses show a similar induction pattern of both, bclpP and hclpP during heat stress on the mRNA as well as on the protein levels. Co-immunoprecipitation experiments imply specific interaction of HClpP with bacterial ClpC, ClpX and ClpE during control as well as heat stress conditions. Radioactive pulse-chase labeling and immunoprecipitation revealed that a ClpXP substrate, the short-living regulatory protein MgsR, is degraded by HClpP, although with an extremely slower rate in comparison to BClpP. The occurrence of an exceptional thickened cell wall of a clpP mutant can be almost fully reversed by the complementation with HClpP. The utilization of the HClpP expressing strain as a test system for new biological or synthetic active substances targeting BClpP is discussed.     

Extracellular Expression of a Functional Recombinant Ganoderma lucidium Immunomodulatory Protein by Bacillus subtilis and Lactococcus lactis

Bacillus subtilis and Lactococcus lactis are ideal hosts for the production of extracellular heterologous proteins of major commercial importance. A recombinant gene for the novel Ganoderma lucidium immunomodulatory protein LZ-8, recombinant LZ-8, was designed encoding the same amino acid sequence but using the preferred codons for both strains and was synthesized by overlapping extension PCR. Using the signal peptide (SP) from subtilisin YaB (SP_YaB), recombinant LZ-8 was expressed extracellularly in Bacillus subtilis and Lactococcus lactis. In the absence of SP_YaB, recombinant LZ-8 was expressed extracellularly in B. subtilis, but not in L. lactis. The three expressed recombinant LZ-8s showed different capacities for modulating the production of Th1 and Th2 cytokines by peripheral blood mononuclear cells and of tumor necrosis factor alpha by a macrophage cell line.     

Bacillus subtilis extracellular nuclease production associated with the spoOH sporulation locus

A number of nuclease-deficient mutants of Bacillus subtilis were isolated and found to be concurrently asporogenous. The nuclease-deficient phenotype appeared to be associated with the spoOH sporulation locus. Spontaneously occurring sporogenous revertants concomitantly recovered the ability to produce extracellular nuclease activity. The position of the ncl mutation was determined using transformation and PBS1 transduction and found to map in the same site as spoOH. The map order of ncl and markers in the vicinity was determined to be purA-cysA-ncl(spoOH)-strA.     

The peptidyl-prolyl isomerase motif is lacking in PmpA,the PrsA-like protein involved in the secretion machinery of Lactococcus lactis

The prsA-like gene from Lactococcus lactis encoding its single homologue to PrsA, an essential protein triggering the folding of secreted proteins in Bacillus subtilis, was characterized. This gene, annotated pmpA, encodes a lipoprotein of 309 residues whose expression is increased 7- to 10-fold when the source of nitrogen is limited. A slight increase in the expression of the PrsA-like protein (PLP) in L. lactis removed the degradation products previously observed with the Staphylococcus hyicus lipase used as a model secreted protein. This shows that PmpA either triggers the folding of the secreted lipase or activates its degradation by the cell surface protease HtrA. Unlike the case for B. subtilis, the inactivation of the gene encoding PmpA reduced only slightly the growth rate of L. lactis in standard conditions. However, it almost stopped its growth when the lipase was overexpressed in the presence of salt in the medium. Like PrsA of B. subtilis and PrtM of L. lactis, the L. lactis PmpA protein could thus have a foldase activity that facilitates protein secretion. These proteins belong to the third family of peptidyl-prolyl cis/trans-isomerases (PPIases) for which parvulin is the prototype. Almost all PLP from gram-positive bacteria contain a domain with the PPIase signature. An exception to this situation was found only in Streptococcaceae, the family to which L. lactis belongs. PLP from Streptococcus pneumoniae and Enterococcus faecalis possess this signature, but those of L. lactis, Streptococcus pyogenes, and Streptococcus mutans do not. However, secondary structure predictions suggest that the folding of PLP is conserved over the entire length of the proteins, including the unconserved signature region. The activity associated with the expression of PmpA in L. lactis and these genomic data show that either the PPIase motif is not necessary for PPIase activity or, more likely, PmpA foldase activity does not necessarily require PPIase activity.     

Sec-mediated secretion of bacteriocin enterocin P by Lactococcus lactis

Most lactic acid bacterium bacteriocins utilize specific leader peptides and dedicated machineries for secretion. In contrast, the enterococcal bacteriocin enterocin P (EntP) contains a typical signal peptide that directs its secretion when heterologously expressed in Lactococcus lactis. Signal peptide mutations and the SecA inhibitor azide blocked secretion. These observations demonstrate that EntP is secreted by the Sec translocase.     

Function of Lactococcus lactis nisin immunity genes nisI and nisFEG after coordinated expression in the surrogate host Bacillus subtilis

Nisin-producing Lactococcus lactis strains show a high degree of resistance to the action of nisin, which is based upon expression of the self-protection (immunity) genes nisI, nisF, nisE, and nisG. Different combinations of nisin immunity genes were integrated into the chromosome of a nisin-sensitive Bacillus subtilis host strain under the control of an inducible promoter. For the recipient strain, the highest level of acquired nisin tolerance was achieved after coordinated expression of all four nisin immunity genes. But either the lipoprotein NisI or the ABC transporter-homologous system NisFEG, respectively, were also able to protect the Bacillus host cells. The acquired immunity was specific to nisin and provided no tolerance to subtilin, a closely related lantibiotic. Quantitative in vivo peptide release assays demonstrated that NisFEG diminished the quantity of cell-associated nisin, providing evidence that one role of NisFEG is to transport nisin from the membrane into the extracellular space. NisI solubilized from B. subtilis membrane vesicles and recombinant hexahistidine-tagged NisI from Escherichia coli interacted specifically with nisin and not with subtilin. This suggests a function of NisI as a nisin-intercepting protein.     

Complementation and characterization of chemotaxis mutants of Bacillus subtilis.

A set of chemotaxis mutants of Bacillus subtilis was complemented by using SP beta c2 transducing bacteriophage either containing cloned segments of DNA or derived from abnormal excision of SP beta c2 dl2::Tn917 inserted into the chemotaxis region. Representative mutants were characterized in capillary assays for chemotaxis toward four amino acids and mannitol and in tethered-cell experiments for addition and removal of two attractants and two repellents. Twenty complementation groups were identified, in addition to the cheR previously characterized. All were found to be defective in chemotaxis toward all chemoeffectors. They were assigned the names cheA through cheU. The large number of general chemotaxis genes in B. subtilis, in contrast to the six in Escherichia coli, suggests fundamental differences in the mechanism of chemotaxis in the two species.