Activation of subtilin precursors by Bacillus subtilis extracellular serine proteases subtilisin (AprE), WprA,and Vpr

The maturation of the peptide antibiotic (lantibiotic) subtilin in Bacillus subtilis ATCC 6633 includes posttranslational modifications of the propeptide and proteolytic cleavage of the leader peptide. To identify subtilin processing activities, we used antimicrobial inactive subtilin precursors consisting of the leader peptide which was still attached to the fully matured propeptide. Two extracellular B. subtilis proteases were able to activate subtilin precursors, the commercially available serine protease prototype subtilisin (AprE) and WprA. The latter was isolated from B. subtilis WB600, a strain deficient in six extracellular proteases. Surprisingly, the aprE wprA double mutant of the ATCC 6633 strain was still able to produce active subtilin, however, with a reduced production rate. No subtilin processing was found within the culture supernatant of the WB800 strain, which is deficient in eight extracellular proteases. Vpr was identified as the third protease capable to process subtilin.     

Enhancement of the chemical and antimicrobial properties of subtilin by site-directed mutagenesis.

Subtilin and nisin are gene-encoded antibiotic peptides that are ribosomally synthesized by Bacillus subtilis and Lactococcus lactis, respectively. Gene-encoded antibiotics are unique in that their structures can be manipulated by mutagenesis of their structural genes. Although subtilin and nisin share considerable structural homology, subtilin has a greater tendency than nisin to undergo spontaneous inactivation. This inactivation is a accompanied by chemical modification of the dehydroalanine at position 5 (DHA5) with a kinetic first-order t1/2 of 0.8 days. It was hypothesized that the R group carboxyl of Glu4 in subtilin participates in the chemical modification of the adjacent DHA5. Noting that nisin has Ile at position 4, site-directed mutagenesis was used to change Glu4 of subtilin to Ile, in order to eliminate this carboxyl-group participation. The DHA5 of this mutant subtilin (E4I-subtilin) underwent modification with a t1/2 of 48 days, which is 57-fold slower than natural subtilin, and the rate of loss of biological activity dropped by a like amount. These results suggest that an intact DHA5 is critical for subtilin activity against bacterial spore outgrowth. A double mutant of subtilin, in which the DHA5 residue of E4I-subtilin was mutated to Ala was devoid of detectable inhibition against spore outgrowth. The specific activity of E4I-subtilin was 3-4-fold higher than natural subtilin, suggesting that an increase in the hydrophobicity of the N-terminal end of the molecule enhances activity. These are the first mutants of subtilin that have been reported, and E4I-subtilin is the first example of any lantibiotic whose properties have been improved by mutagenesis. In order to carry out the mutagenesis, a host-vector pair was constructed that permits a deletion replacement in which the natural subtilin gene is replaced by the mutant gene at the normal location in the chromosome. This maintains normal gene dosage and regulatory responses, as well as eliminates ambiguities caused by expression of the normal and mutant genes in the same cell.     

Engineering dehydrated amino acid residues in the antimicrobial peptide nisin.

The small antimicrobial peptide nisin, produced by Lactococcus lactis, contains the uncommon amino acid residues dehydroalanine and dehydrobutyrine and five thio ether bridges. Since these structures are posttranslationally formed from Ser, Thr, and Cys residues, it is feasible to study their role in nisin function and biosynthesis by protein engineering. Here we report the development of an expression system for mutated nisin Z (nisZ) genes, using nisin A producing L. lactis as a host. Replacement by site-directed mutagenesis of the Ser-5 codon in nisZ by a Thr codon, led to a mutant with a dehydrobutyrine instead of a dehydroalanine residue at position 5, as shown by NMR. Its antimicrobial activity was 2-10-fold lower relative to wild-type nisin Z, depending on the indicator strain used. In another mutagenesis study a double mutation was introduced in the nisZ gene by replacing the codons for Met-17 and Gly-18 by codons for Gln and Thr, respectively, as in the third lanthionine ring of the related antimicrobial peptide subtilin from Bacillus subtilis. This resulted in the simultaneous production of two mutant species, one containing a Thr residue and the other containing a dehydrobutyrine residue at position 18, both having different bacteriocidal properties.     

Engineering a disulfide bond and free thiols in the lantibiotic nisin Z.

The antimicrobial peptide nisin contains the uncommon amino acid residues lanthionine and methyl-lanthionine, which are post-translationally formed from Ser, Thr and Cys residues. To investigate the importance of these uncommon residues for nisin activity, a mutant was designed in which Thr13 was replaced by a Cys residue, which prevents the formation of the thioether bond of ring C. Instead, Cys13 couples with Cys19 via an intramolecular disulfide bridge, a bond that is very unusual in lantibiotics. NMR analysis of this mutant showed a structure very similar to that of wild-type nisin, except for the configuration of ring C. The modification was accompanied by a dramatic reduction in antimicrobial activity to less than 1% of wild-type activity, indicating that the lanthionine of ring C is very important for this activity. The nisin Z mutants S5C and M17C were also isolated and characterized; they are the first lantibiotics known that contain an additional Cys residue that is not involved in bridge formation but is present as a free thiol. Secretion of these peptides by the lactococcal producer cells, as well as their antimicrobial activity, was found to be strongly dependent on a reducing environment. Their ability to permeabilize lipid vesicles was not thiol-dependent. Labeling of M17C nisin Z with iodoacetamide abolished the thiol-dependence of the peptide. These results show that the presence of a reactive Cys residue in nisin has a strong effect on the antimicrobial properties of the peptide, which is probably the result of interaction of these residues with thiol groups on the outside of bacterial cells.     

Structure-activity relationships in the peptide antibiotic nisin: role of dehydroalanine 5.

A mutant of the peptide antibiotic nisin in which the dehydroalanine residue at position 5 has been replaced by an alanine has been produced and structurally characterized. It is shown to have activity very similar to that of wild-type nisin in inhibiting growth of Lactococcus lactis and Micrococcus luteus but is very much less active than nisin as an inhibitor of the outgrowth of spores of Bacillus subtilis. These observations, which parallel those of W. Liu and J. N. Hansen (Appl. Environ. Microbiol. 59:648-651, 1993) on the corresponding mutant of the related antibiotic subtilin, are discussed in terms of the mechanism(s) of action of these antibiotics.     

Entianin, a novel subtilin-like lantibiotic from Bacillus subtilis subsp. spizizenii DSM 15029T with high antimicrobial activity

Lantibiotics, such as nisin and subtilin, are lanthionine-containing peptides that exhibit antimicrobial as well as pheromone-like autoinducing activity. Autoinduction is specific for each lantibiotic, and reporter systems for nisin and subtilin autoinduction are available. In this report, we used the previously reported subtilin autoinduction bioassay in combination with mass spectrometric analyses to identify the novel subtilin-like lantibiotic entianin from Bacillus subtilis subsp. spizizenii DSM 15029(T). Linearization of entianin using Raney nickel-catalyzed reductive cleavage enabled, for the first time, the use of tandem mass spectrometry for the fast and efficient determination of an entire lantibiotic primary structure, including posttranslational modifications. The amino acid sequence determined was verified by DNA sequencing of the etnS structural gene, which confirmed that entianin differs from subtilin at 3 amino acid positions. In contrast to B. subtilis ATCC 6633, which produces only small amounts of unsuccinylated subtilin, B. subtilis DSM 15029(T) secretes considerable amounts of unsuccinylated entianin. Entianin was very active against several Gram-positive pathogens, such as Staphylococcus aureus and Enterococcus faecalis. The growth-inhibiting activity of succinylated entianin (S-entianin) was much lower than that of unsuccinylated entianin: a 40-fold higher concentration was required for inhibition. For succinylated subtilin (S-subtilin), a concentration 100-fold higher than that of unsuccinylated entianin was required to inhibit the growth of a B. subtilis test strain. This finding was in accordance with a strongly reduced sensing of cellular envelope stress provided by S-entianin relative to that of entianin. Remarkably, S-entianin and S-subtilin showed considerable autoinduction activity, clearly demonstrating that autoinduction and antibiotic activity underlie different molecular mechanisms.     

Engineering of a novel thioether bridge and role of modified residues in the lantibiotic Pep5.

Pep5 is a 34-amino-acid antimicrobial peptide, produced by Staphylococcus epidermidis 5, that contains the thioether amino acids lanthionine and methyllanthionine, which form three intramolecular ring structures. In addition, two didehydrobutyrines are present in the central part of the lantibiotic and an oxobutyryl residue is located at the N terminus. All rare amino acids are introduced by posttranslational modifications of a ribosomally made precursor peptide. To elucidate the function of the modified residues for the antimicrobial action of Pep5, mutant peptides, in which single modified residues had been eliminated, were produced by site-directed mutagenesis. All of these peptides showed a reduced antimicrobial activity. In addition, those peptides from which the ring structures had been deleted became susceptible to proteolytic digest. This demonstrates that the ring structures serve as stabilizers of conformations essential for activity, e.g., amphiphilicity, as well as for protecting Pep5 against proteases of the producing strains. In addition, residues that could serve as precursors of new modified amino acids in lantibiotics were introduced into the Pep5 precursor peptide. This way, a novel methyllanthionine and a didehydroalanine were inserted into the flexible central part of Pep5, demonstrating that biosynthesis of modified amino acids is feasible by protein engineering and use of the lantibiotic modification system.     

Use of alkaline phosphatase as a reporter polypeptide to study the role of the subtilin leader segment and the SpaT transporter in the posttranslational modifications and secretion of subtilin in Bacillus subtilis 168.

The subtilin leader segment of presubtilin was fused to alkaline phosphatase (AP), which was used as a reporter polypeptide to study the role of the subtilin leader segment in posttranslational modifications during the conversion of presubtilin to subtilin and in the translocation of presubtilin from the cytoplasm of Bacillus subtilis 168 to the extracellular medium. It was observed that the subtilin leader segment could be utilized by a wild-type transporter, but secretion was enhanced if the subtilin transporter was available. The subtilin leader was not cleaved away from the AP component of the precursor until the precursor had been transported to the cell wall, and none of the AP was released into the medium until after cleavage had occurred. The role of SpaT, which is an ABC transporter that has been implicated in subtilin secretion, was explored by making a large in-frame deletion from the central region of SpaT and observing the effect on translocation of the AP reporter. Instead of having an effect on translocation, the deletion disrupted proteolytic cleavage of the subtilin leader segment and release of the AP reporter into the medium. The AP that was secreted by means of the subtilin leader segment had not undergone any posttranslational modifications, as assessed by amino acid composition analysis and enzymatic activity analysis.     

Synthesis of novel unnatural amino acid as a building block and its incorporation into an antimicrobial peptide

Considering the biological mechanism and in vivo stability of antimicrobial peptides, we designed and synthesized novel unnatural amino acids with more positively charged and bulky side chain group than lysine residue. The unusual amino acids, which were synthesized by either solution phase or solid phase, were incorporated into an antimicrobial peptide. Its effect on the stability, activity, and the structure of the peptide was studied to evaluate the potential of these novel unnatural amino acids as a building block for antimicrobial peptides. The incorporation of this unusual amino acid increased the resistance of the peptide against serum protease more than three times without a decrease in the activity. Circular dichroism spectra of the peptides indicated that all novel unnatural amino acids must have lower helical forming propensities than lysine. Our results indicated that the unnatural amino acids synthesized in this study could be used not only as a novel building block for combinatorial libraries of antimicrobial peptides, but also for structure–activity relationship studies about antimicrobial peptides.     

Conversion of Bacillus subtilis 168 to a subtilin producer by competence transformation.

Subtilin is a ribosomally synthesized peptide antibiotic produced by Bacillus subtilis ATCC 6633. B. subtilis 168 was converted to a subtilin producer by competence transformation with chromosomal DNA from B. subtilis ATCC 6633. A chloramphenicol acetyltransferase gene was inserted next to the subtilin structural gene as a selectable marker. The genes that conferred subtilin production were derived from a 40-kb region of the B. subtilis ATCC 6633 chromosome that had flanking homologies to the B. subtilis 168 chromosome. The subtilin produced by the mutant was identical to natural subtilin in its biological activity, chromatographic behavior, amino acid composition, and N-terminal amino acid sequence.     

Characterization of modification enzyme NukM and engineering of a novel thioether bridge in lantibiotic nukacin ISK-1

The lantibiotic nukacin ISK-1 is an antimicrobial peptide containing unusual amino acids such as lanthionine and dehydrobutyrine. The nukacin ISK-1 prepeptide (NukA) undergoes posttranslational modifications, such as the dehydration and cyclization reactions required to form the unusual amino acids by the modification enzyme NukM. We have previously constructed a system for the introduction of unusual amino acids into NukA by coexpression of NukM in Escherichia coli. Using this system, we describe the substrate specificity of NukM by the coexpression of a series of NukA mutants. Our results revealed the following characteristics of NukM: (1) its dehydration activity is not coupled to its cyclization activity; (2) its dehydration activity is site-specific; (3) the length of the substrate is important for its dehydration activity. Furthermore, we succeeded in introducing a novel thioether bridge in NukA by replacing an unmodified Ser at position 27 with a Cys residue.     

Inactivation of vegetative cells,but not spores,of Bacillus anthracis,B. cereus,and B. subtilis on stainless steel surfaces coated with an antimicrobial silver- and zinc-containing zeolite formulation

Stainless steel surfaces coated with paints containing a silver- and zinc-containing zeolite (AgION antimicrobial) were assayed in comparison to uncoated stainless steel for antimicrobial activity against vegetative cells and spores of three Bacillus species, namely, B. anthracis Sterne, B. cereus T, and B. subtilis 168. Under the test conditions (25 degrees C and 80% relative humidity), the zeolite coating produced approximately 3 log(10) inactivation of vegetative cells within a 5- to 24-h period, but viability of spores of the three species was not significantly affected.     

Isolation and characterization of genetically engineered gallidermin and epidermin analogs.

Gallidermin (Gdm) and epidermin (Epi) are highly homologous tetracyclic polypeptide antibiotics that are ribosomally synthesized by a Staphylococcus gallinarum strain and a Staphylococcus epidermidis strain, respectively. These antibiotics are secreted into media and are distinguished by the presence of the unusual amino acids lanthionine, 3-methyllanthionine, didehydrobutyrine, and S-(2-aminovinyl)-D-cysteine, which are formed by posttranslational modification. To study the substrate specificities of the modifying enzymes and to obtain variants that exhibit altered or new biological activities, we changed certain amino acids by performing site-specific mutagenesis with the Gdm and Epi structural genes (gdmA and epiA, respectively). S. epidermidis Tü3298/EMS6, an epiA mutant of the Epi-producing strain, was used as the expression host. This mutant synthesized Epi, Gdm, or analogs of these antibiotics when the appropriate genes were introduced on a plasmid. No Epi or Gdm analogs were isolated from the supernatant when (i) hydroxyamino acids involved in thioether amino acid formation were replaced by nonhydroxyamino acids (S3N and S19A); (ii) C residues involved in thioether bridging were deleted (delta C21, C22 and delta C22); or (iii) a ring amino acid was replaced by an amino acid having a completely different character (G10E and Y20G). A strong decrease in production was observed when S residues involved in thioether amino acid formation were replaced by T residues (S16T and S19T). A number of conservative changes at positions 6, 12, and 14 on the Gdm backbone were tolerated and led to analogs that had altered biological properties, such as enhanced antimicrobial activity (L6V) or a remarkable resistance to proteolytic degradation (A12L and Dhb14P). The T14S substitution led to simultaneous production of two Gdm species formed by incomplete posttranslational modification (dehydration) of the S-14 residue. The fully modified Dhb14Dha analog exhibited antimicrobial activity similar to that of Gdm, whereas the Dhb14S analog was less active. Both peptides were more sensitive to tryptic cleavage than Gdm was.     

Analysis of genes involved in biosynthesis of the lantibiotic subtilin.

Lantibiotics are peptide-derived antibiotics with high antimicrobial activity against pathogenic gram-positive bacteria. They are ribosomally synthesized and posttranslationally modified (N. Schnell, K.-D. Entian, U. Schneider, F. Götz, H. Zähner, R. Kellner, and G. Jung, Nature [London] 333:276-278, 1988). The most important lantibiotics are subtilin and the food preservative nisin, which both have a very similar structure. By using a hybridization probe specific for the structural gene of subtilin, spaS, the DNA region adjacent to spaS was isolated from Bacillus subtilis. Sequence analysis of a 4.9-kb fragment revealed several open reading frames with the same orientation as spaS. Downstream of spaS, no reading frames were present on the isolated XbaI fragment. Upstream of spaS, three reading frames, spaB, spaC, and spaT, were identified which showed strong homology to genes identified near the structural gene of the lantibiotic epidermin. The SpaT protein derived from the spaT sequence was homologous to hemolysin B of Escherichia coli, which indicated its possible function in subtilin transport. Gene deletions within spaB and spaC revealed subtilin-negative mutants, whereas spaT gene disruption mutants still produced subtilin. Remarkably, the spaT mutant colonies revealed a clumpy surface morphology on solid media. After growth on liquid media, spaT mutant cells agglutinated in the mid-logarithmic growth phase, forming longitudinal 3- to 10-fold-enlarged cells which aggregated. Aggregate formation preceded subtilin production and cells lost their viability, possibly as a result of intracellular subtilin accumulation. Our results clearly proved that reading frames spaB and spaC are essential for subtilin biosynthesis whereas spaT mutants are probably deficient in subtilin transport.     

Structural and Mechanistic Insights into the Pseudomonas fluorescens 2-Nitrobenzoate 2-Nitroreductase NbaA

The bacterial 2-nitroreductase NbaA is the primary enzyme initiating the degradation of 2-nitrobenzoate (2-NBA), and its activity is controlled by posttranslational modifications. To date, the structure of NbaA remains to be elucidated. In this study, the crystal structure of a Cys194Ala NbaA mutant was determined to a 1.7-Å resolution. The substrate analog 2-NBA methyl ester was used to decipher the substrate binding site by inhibition of the wild-type NbaA protein. Tandem mass spectrometry showed that 2-NBA methyl ester produced a 2-NBA ester bond at the Tyr193 residue in the wild-type NbaA but not residues in the Tyr193Phe mutant. Moreover, covalent binding of the 2-NBA methyl ester to Tyr193 reduced the reactivity of the Cys194 residue on the peptide link. The Tyr193 hydroxyl group was shown to be essential for enzyme catalysis, as a Tyr193Phe mutant resulted in fast dissociation of flavin mononucleotide (FMN) from the protein with the reduced reactivity of Cys194. FMN binding to NbaA varied with solution NaCl concentration, which was related to the catalytic activity but not to cysteine reactivity. These observations suggest that the Cys194 reactivity is negatively affected by a posttranslational modification of the adjacent Tyr193 residue, which interacts with FMN and the substrate in the NbaA catalytic site.     

Analysis of genes involved in biosynthesis of the lantibiotic subtilin.

Lantibiotics are peptide-derived antibiotics with high antimicrobial activity against pathogenic gram-positive bacteria. They are ribosomally synthesized and posttranslationally modified (N. Schnell, K.-D. Entian, U. Schneider, F. G?tz, H. Z?hner, R. Kellner, and G. Jung, Nature [London] 333:276-278, 1988). The most important lantibiotics are subtilin and the food preservative nisin, which both have a very similar structure. By using a hybridization probe specific for the structural gene of subtilin, spaS, the DNA region adjacent to spaS was isolated from Bacillus subtilis. Sequence analysis of a 4.9-kb fragment revealed several open reading frames with the same orientation as spaS. Downstream of spaS, no reading frames were present on the isolated XbaI fragment. Upstream of spaS, three reading frames, spaB, spaC, and spaT, were identified which showed strong homology to genes identified near the structural gene of the lantibiotic epidermin. The SpaT protein derived from the spaT sequence was homologous to hemolysin B of Escherichia coli, which indicated its possible function in subtilin transport. Gene deletions within spaB and spaC revealed subtilin-negative mutants, whereas spaT gene disruption mutants still produced subtilin. Remarkably, the spaT mutant colonies revealed a clumpy surface morphology on solid media. After growth on liquid media, spaT mutant cells agglutinated in the mid-logarithmic growth phase, forming longitudinal 3- to 10-fold-enlarged cells which aggregated. Aggregate formation preceded subtilin production and cells lost their viability, possibly as a result of intracellular subtilin accumulation. Our results clearly proved that reading frames spaB and spaC are essential for subtilin biosynthesis whereas spaT mutants are probably deficient in subtilin transport.     

The inhibition of vegetative cell outgrowth and division from spores of Bacillus cereus T by hen egg albumen

Spores of Bacillus cereus T germinated and formed vegetative cells in Tryptone Soya broth (TSB), pH 9-0 and 7-4 at 30^oC. Spores germinated but did not form vegetative cells when suspended in hen egg white (pH 9-0) supplemented with L-alanine and inosine. Using a split image eyepiece, the volumes of germinating spores in egg white were seen to increase as a result of increases in both length and breadth. In TSB at the same pH, the major volume increase resulted from a progressive increase in cell length. Egg white supplemented with L-alanine and inosine (pH 7-6 30^oC) allowed limited outgrowth to occur but the vegetative cells differed in morphology to those in TSB. Fe(NH₄)₂(SO₄)2.6H₂O overcame the inhibition of outgrowth in egg white at pH 7–8 but not in egg white at pH 9-1. Solutions containing trace elements, growth factors and casamino acids could not replace iron in this respect. Sporulation occurred in egg white only when iron was present.     

Peptide antibiotic subtilin is synthesized via precursor proteins

Biogenesis of subtilin, an antimicrobial peptide produced by Bacillus subtilis ATCC 6633, was studied in growing cells. Pulse-chase labeling experiments with [35S]cysteine revealed the presence of precursor proteins of subtilin. The synthesis of both precursor proteins and subtilin was inhibited by inhibitors of protein and RNA synthesis. When the precursor proteins were incubated with crude extracts of the organism in vitro, they were converted to subtilin. Pepstatin and phenylmethylsulfonyl fluoride in combination inhibited this conversion.     

Tuning the biological properties of amphipathic alpha-helical antimicrobial peptides: rational use of minimal amino acid substitutions

In nature, alpha-helical antimicrobial peptides present the small and flexible residue glycine at positions 7 or 14 with a significant frequency. Based on the sequence of the non-proteinogenic alpha-helical model peptide P1(Aib7), with a potent, broad spectrum antimicrobial activity, six peptides were designed by effecting a single amino acid substitution to investigate how tuning the structural characteristics at position 7 could lead to optimization of selectivity without affecting antimicrobial activity against a broad panel of multidrug resistant bacterial and yeast indicator strains. The relationship between structural features (size/hydrophobicity of the side chain as well as conformation and flexibility) and biological activity, in terms of minimum inhibitory concentration, membrane permeabilization kinetics and lysis of red blood cells are discussed. On conversion of the peptide to proteinogenic residues, these principles allowed development of a potent antimicrobial peptide with a reduced cytotoxicity. However, while results suggest that both hydrophobicity of residue 7 and chain flexibility at this position can be modulated to improve selectivity, position 14 is less tolerant of substitutions.