Bovicin HJ50-Like Lantibiotics,a Novel Subgroup of Lantibiotics Featured by an Indispensable Disulfide Bridge

Lantibiotics are ribosomally-synthesized and posttranslationally modified peptides with potent antimicrobial activities. Discovery of novel lantibiotics has been greatly accelerated with the soaring release of genomic information of microorganisms. As a unique class II lantibiotic, bovicin HJ50 is produced by Streptococcus bovis HJ50 and contains one rare disulfide bridge. By using its precursor BovA as a drive sequence, 16 BovA-like peptides were revealed in a wide variety of species. From them, three representative novel lan loci from Clostridium perfringens D str. JGS1721, Bacillus cereus As 1.348 and B. thuringiensis As 1.013 were identified by PCR screening. The corresponding mature lantibiotics designated perecin, cerecin and thuricin were obtained and structurally elucidated to be bovicin HJ50-like lantibiotics especially by containing a conserved disulfide bridge. The disulfide bridge was substantiated to be essential for the function of bovicin HJ50-like lantibiotics as its disruption eliminated their antimicrobial activities. Further analysis indicated that the disulfide bridge played a crucial role in maintaining the hydrophobicity of bovicin HJ50, which might facilitate it to exert antimicrobial function. This study unveiled a novel subgroup of disulfide-containing lantibiotics from bacteria of different niches and further demonstrated the indispensable role of disulfide bridge in these novel bovicin HJ50-like lantibiotics.     

Dissecting the catalytic and substrate binding activity of a class II lanthipeptide synthetase BovM

LanM proteins are the synthetases of the class II lanthipeptides, which are responsible for lanthionine or methyllanthionine formation in lanthipeptides. LanMs are bifunctional enzymes with N-terminal dehydratase and C-terminal cyclase domains. However, the catalytic and especially the substrate binding function of LanM are not fully investigated. In this study, we analyzed the function of conserved residues of BovM, which is the synthetase of lanthipeptide bovicin HJ50, with alanine substitution method. Mass spectrometry (MS) and surface plasmon resonance (SPR) analyses showed six hydrophilic residues (e.g. Asp247) were involved in the dehydration activity of BovM and four hydrophobic residues (e.g. Ile254) were responsible for the substrate binding of BovM. In addition, a conserved Asp155 was proposed to be general base in the elimination of phosphates during the dehydration reactions. This research of BovM shed a light on the catalytic and substrate binding mechanism of LanM proteins.     

Characterization of a bacteriocin, Thermophilin 1277, produced by Streptococcus thermophilus SBT1277

AIMS: To assess the inhibitory activity and the influence of culture condition on the growth and bacteriocin, Thermophilin 1277, production by Streptococcus thermophilus SBT1277. METHODS AND RESULTS: Thermophilin 1277, which was produced by S. thermophilus SBT1277, showed an antimicrobial activity against several lactic acid bacteria and food spoilage bacteria including Clostridium butylicum, C. sprogenes and Bacillus cereus. Thermophilin 1277 was inactivated by proteinase K. Heating treatment did not affect the antimicrobial activity. The partially purified Thermophilin 1277 had an apparent molecular mass of 3.7 kDa. N-terminal sequence analysis revealed 15 amino acid residues that correspond with amino acid sequence of the lantibiotics bovicin HJ50 produced by Streptococcus bovis HJ50. The effects of culture condition for the bacteriocin production by S. thermophilus SBT1277 were studied. During the batch fermentation, Thermophilin 1277 was produced in M17 broth, but no bacteriocin production occurred in the sucrose-tryptone (ST) broth. Bacteriocin production was detected in pH controlled ST broth at pH values of 5.5-6.5. CONCLUSIONS: Thermophilin 1277 production from S. thermophilus strain depended on the culture conditions. Some characters and N-terminal amino acid sequence of Thermophilin 1277 differed from bacteriocins produced by S. thermophilus reported previously. SIGNIFICANCE AND IMPACT OF THE STUDY: Streptococcus thermophilus SBT1277 or its bacteriocin which has a wide inhibitory spectrum has a potential use as a biopreservative in dairy products.     

Gene cluster for biosynthesis of thermophilin 1277--a lantibiotic produced by Streptococcus thermophilus SBT1277, and heterologous expression of TepI, a novel immunity peptide

Aims: To identify genes cluster for thermophilin 1277 produced by Streptococcus thermophilus SBT1277. Methods and Results: To identify genes for thermophilin 1277 production, the chromosomal DNA region surrounding the structural gene, tepA, was sequenced using a primer‐walking method. The thermophilin 1277 biosynthesis gene locus (tep) is a 9·9‐kb region, which consists of at least ten open reading frames (ORFs) in the following order: tepAMTFEGKRI and ORF4. Homology analysis showed high similarity to genes involved in bovicin HJ50 production by Streptococcus bovis HJ50. tepI encodes a novel, small, positively charged hydrophobic peptide of 52 amino acids, which contains a putative transmembrane segment. By heterologous expression in Lactococcus lactis ssp. cremoris MG1363, the TepI‐expressing strain exhibited at least 1·3 times higher resistance to thermophilin 1277. Conclusions: Thermophilin 1277 biosynthesis genes were encoded by a 9·9‐kbp region containing at least ten ORFs. TepI is a novel immunity peptide, which protected Strep. thermophilus SBT1277 against thermophilin 1277 in addition to TepFEG, a putative ABC transporter. Significance and Impact of the Study: This is the first report regarding a lantibiotic gene cluster produced by Strep. thermophilus strain.     

N-terminal basic amino acids are not required for translocation and processing of preproparathyroid hormone

An N-terminal deletion mutant of preproparathyroid hormone that contains a single basic amino acid, lysine, in the N-terminal domain of the signal peptide is translocated across the endoplasmic reticulum membrane similarly to intact preproparathyroid hormone. To examine the function of charged residues preceeding the hydrophobic core, the lysine was replaced by an uncharged (methionine) or negatively charged (glutamic acid) amino acid. The translocational activity of the mutant signal peptides was assayed in a reticulocyte lysate system containing chicken oviduct microsomal membranes. Altering the net charge of the N-terminal domain did not abolish signal sequence activity, although the efficiency of translocation was decreased for the mutant with a glutamic acid substitution. Posttranslational, ribosome independent, translocation was observed for all the mutants tested, with the same dependence on N-terminal charge but with much lower efficiency than cotranslational translocation. These studies show that the presence of basic amino acids in the N-terminal domain of a eukaryotic signal sequence is not required for its activity.     

BATMAS30: amino acid substitution matrix for alignment of bacterial transporters

Aligned amino acid sequences of three functionally independent samples of transmembrane (TM) transport proteins have been analyzed. The concept of TM-kernel is proposed as the most probable transmembrane region of a sequence. The average amino acid composition of TM-kernels differs from the published amino acid composition of transmembrane segments. TM-kernels contain more alanines, glycines, and less polar, charged, and aromatic residues in contrast to non-TM-proteins. There are also differences between TM-kernels of bacterial and eukaryotic proteins. We have constructed amino acid substitution matrices for bacterial TM-kernels, named the BATMAS (BActerial Transmembrane MAtrix of Substitutions) series. In TM-kernels, polar and charged residues, as well as proline and tyrosine, are highly conserved, whereas there are more substitutions within the group of hydrophobic residues, in contrast to non-TM-proteins that have fewer, relatively more conserved, hydrophobic residues. These results demonstrate that alignment of transmembrane proteins should be based on at least two amino acid substitution matrices, one for loops (e.g., the BLOSUM series) and one for TM-segments (the BATMAS series), and the choice of the TM-matrix should be different for eukaryotic and bacterial proteins.     

Cloning of cDNA encoding the membrane-bound form of bovine beta 1,4-galactosyltransferase

UDPgalactose: N-acetyl-D-glucosamine 4-beta-D-galactosyltransferase (EC 2.4.1.38) (GalT) is a Golgi-membrane-bound enzyme that participates in the biosynthesis of the oligosaccharide structures of glycoproteins and glycolipids. Synthetic DNA oligomers representing segments of the published partial cDNA sequence for bovine GalT were used as molecular probes to isolate from bovine-liver cDNA libraries overlapping cDNA clones that span 1728 nucleotides and potentially code for the entire polypeptide chain of bovine galactosyltransferase. The cDNA sequence for bovine GalT reveals a 1206-base-pair open reading frame that codes for 402 amino acids, including a presumptive N-terminal membrane anchoring domain of 20 hydrophobic amino acids. The colinearity between the cDNA sequence and 29 non-overlapping amino acid residues which were positively identified by N-terminal sequencing of two polypeptides isolated from the soluble form of the enzyme was consistent with the translation frame and confirmed the authenticity of the cDNA clones. The finding of an N-terminal hydrophobic segment which serves as the membrane anchor and signal sequence suggests that the C-terminal region of the GalT polypeptide is oriented within the lumen of the Golgi membranes. This conclusion is in agreement with previous biochemical studies which indicated that the 51-kDa and 42-kDa soluble forms of the enzyme which encompass the C-terminal 324 and 297 amino acid residues of the entire GalT polypeptide, respectively, include the catalytic site.     

The amino acid sequence of a gonococcal growth inhibitor from Staphylococcus haemolyticus.

A gonococcal inhibitor produced by Staphylococcus haemolyticus was separated into three components by reverse-phase h.p.l.c. The amino acid composition analysis of each of the three components indicated extensive similarities. N-Terminal sequence analysis of all three components allowed the identification of the first 27-30 residues of each. The complete primary structure of each component was determined from the sequence analysis of trypic peptides and peptides generated by mild acid hydrolysis. Each component is composed of 44 amino acid residues, with evidence suggesting the presence of an N-terminal formylmethionine residue in each. The components I, II and III have respectively 33, 29 and 33 identical amino acid residues in their sequences, which represents 75%, 65.9% and 75% homology. These components contain a high proportion of hydrophobic amino acids, and their hydrophobicity profiles are closely related. Also, each of the three components contains a positively charged residue (lysine) as the third residue, followed by a core of hydrophobic residues. These results suggest that the three components are possible signal sequences of one or more secreted or membrane-associated proteins.     

Involvement of Val 315 located in the C-terminal region of thermolysin in its expression in Escherichia coli and its thermal stability

Thermolysin is a thermophilic and halophilic zinc metalloproteinase that consists of β-rich N-terminal (residues 1–157) and α-rich C-terminal (residues 158–316) domains. Expression of thermolysin variants truncated from the C-terminus was examined in E. coli culture. The C-terminal Lys316 residue was not significant in the expression, but Val315 was critical. Variants in which Val315 was substituted with fourteen amino acids were prepared. The variants substituted with hydrophobic amino acids such as Leu and Ile were almost the same as wild-type thermolysin (WT) in the expression amount, α-helix content, and stability. Variants with charged (Asp, Glu, Lys, and Arg), bulky (Trp), or small (Gly) amino acids were lower in these characteristics than WT. All variants exhibited considerably high activities (50–100% of WT) in hydrolyzing protein and peptide substrates. The expression amount, helix content, and stability of variants showed good correlation with hydropathy indexes of the amino acids substituted for Val315. Crystallographic study of thermolysin has indicated that V315 is a member of the C-terminal hydrophobic cluster. The results obtained in the present study indicate that stabilization of the cluster increases thermolysin stability and that the variants with higher stability are expressed more in the culture. Although thermolysin activity was not severely affected by the variation at position 315, the stability and specificity were modified significantly, suggesting the long-range interaction between the C-terminal region and active site.     

Bovicin HC5 inhibits wasteful amino acid degradation by mixed ruminal bacteria in vitro

Streptococcus bovis HC5 produces a broad spectrum lantibiotic (bovicin HC5) that inhibits pure cultures of hyper ammonia-producing bacteria (HAB). Experiments were preformed to see if: (1) S. bovis HC5 cells could inhibit the deamination of amino acids by mixed ruminal bacteria taken directly from a cow, (2) semi-purified bovicin was as effective as S. bovis HC5 cells, and 3) semi-purified and the feed additive monensin were affecting the same types of ammonia-producing ruminal bacteria. Because purified and semi-purified bovicin HC5 was as effective as S. bovis HC5 cells, it appeared that bovicin HC5 was penetrating the cell membranes of HAB before it could be degraded by peptidases and proteinases. Mixed ruminal bacteria that were successively transferred and enriched nine times with trypticase did not become significantly more resistant to either bovicin HC5 (50 AU mL⁻¹) or monensin (5 μM), and amplified rDNA restriction analysis indicated that bovicin HC5 and monensin appeared to be selecting against the same types of bacteria.     

The DNA replication inhibitor microcin B17 is a forty-three-amino-acid protein containing sixty percent glycine

Microcin B17 is a low-molecular-weight protein that inhibits DNA replication in a number of enteric bacteria. It is produced by bacterial strains which harbor a 70-kilobase plasmid called pMccB17. Four plasmid genes (named mcbABCD) are required for its production. The product of the mcbA gene was identified by labelling minicells. The mcbA gene product was slightly larger when a mutation in any of the other three production genes was present. This indicates that these genes are involved in processing the primary mcbA product to yield the active molecule. The mcbA gene product predicted from the nucleotide sequence has 69 amino acids including 28 glycine residues. Microcin B17 was extracted from the cells by boiling in 100 mM acetic acid, 1 mM EDTA, and purified to homogeneity in a single step by high-performance liquid chromatography through a C18 column. The N-terminal amino acid sequence and amino acid composition demonstrated that mcbA is the structural gene for microcin B17. The active molecule is a processed product lacking the first 26 N-terminal residues. The 43 remaining residues include 26 glycines. While microcin B17 is an exported protein, the cleaved N-terminal peptide does not have the characteristic properties of a "signal sequence", which suggests that it is secreted by a mechanism different from that used by most secreted proteins of E. coli.     

Sensor Domain of Histidine Kinase KinB of Pseudomonas: A HELIX-SWAPPED DIMER*

The overproduction of polysaccharide alginate is responsible for the formation of mucus in the lungs of cystic fibrosis patients. Histidine kinase KinB of the KinB-AlgB two-component system in Pseudomonas aeruginosa acts as a negative regulator of alginate biosynthesis. The modular architecture of KinB is similar to other histidine kinases. However, its periplasmic signal sensor domain is unique and is found only in the Pseudomonas genus. Here, we present the first crystal structures of the KinB sensor domain. The domain is a dimer in solution, and in the crystal it shows an atypical dimer of a helix-swapped four-helix bundle. A positively charged cavity is formed on the dimer interface and involves several strictly conserved residues, including Arg-60. A phosphate anion is bound asymmetrically in one of the structures. In silico docking identified several monophosphorylated sugars, including β-d-fructose 6-phosphate and β-d-mannose 6-phosphate, a precursor and an intermediate of alginate synthesis, respectively, as potential KinB ligands. Ligand binding was confirmed experimentally. Conformational transition from a symmetric to an asymmetric structure and decreasing dimer stability caused by ligand binding may be a part of the signal transduction mechanism of the KinB-AlgB two-component system.     

The topogenic contribution of uncharged amino acids on signal sequence orientation in the endoplasmic reticulum

Signal sequences for insertion of proteins into the endoplasmic reticulum induce translocation of either the C- or the N-terminal sequence across the membrane. The end that is translocated is primarily determined by the flanking charges and the hydrophobic domain of the signal. To characterize the hydrophobic contribution to topogenesis, we have challenged the translocation machinery in vivo in transfected COS cells with model proteins differing exclusively in the apolar segment of the signal. Homo-oligomers of hydrophobic amino acids as different in size and shape as Val(19), Trp(19), and Tyr(22) generated functional signal sequences with similar topologies in the membrane. The longer a homo-oligomeric sequence of a given residue, the more N-terminal translocation was obtained. To determine the topogenic contribution of all uncharged amino acids in the context of a hydrophobic signal sequence, two residues in a generic oligoleucine signal were exchanged for all uncharged amino acids. The resulting scale resembles a hydrophobicity scale with the more hydrophobic residues promoting N-terminal translocation. In addition, the helix breakers glycine and proline showed a position-dependent effect, which raises the possibility of a conformational contribution to topogenesis.     

Studies on influenza haemagglutinin fusion peptide mutants generated by reverse genetics

Influenza haemagglutinin (HA) is responsible for fusing viral and endosomal membranes during virus entry. In this process, conformational changes in the HA relocate the HA(2) N-terminal 'fusion peptide' to interact with the target membrane. The highly conserved HA fusion peptide shares composition and sequence features with functionally analogous regions of other viral fusion proteins, including the presence and distribution of glycines and large side-chain hydrophobic residues. HAs with mutations in the fusion peptide were expressed using vaccinia virus recombinants to examine the requirement for fusion of specific hydrophobic residues and the significance of glycine spacing. Mutant HAs were also incorporated into infectious influenza viruses for analysis of their effects on infectivity and replication. In most cases alanine, but not glycine substitutions for the large hydrophobic residues, yielded fusion-competent HAs and infectious viruses, suggesting that the conserved spacing of glycines may be structurally significant. When viruses containing alanine substitutions for large hydrophobic residues were passaged, pseudoreversion to valine was observed, indicating a preference for large hydrophobic residues at specific positions. Viruses were also obtained with serine, leucine or phenylalanine as the N-terminal residue, but these replicated to significantly lower levels than wild-type virus with glycine at this position.     

Identification and characterization of a mitochondrial targeting signal in rat cytochrome P450 2E1 (CYP2E1)

Cytochrome P450 2E1 (CYP2E1) lacking the hydrophobic NH(2)-terminal hydrophobic transmembrane domain is specifically targeted to mitochondria, where it is processed to a soluble and catalytically active form (Delta2E1) with a mass of about 40 kDa. Small amounts of Delta2E1 were also observed in mitochondria isolated from rat liver, indicating that this form of CYP2E1 is also present in vivo. In the present study the mitochondrial targeting signal was identified and characterized by the use of several NH(2)-terminally truncated and mutated forms of CYP2E1 that were expressed in the mouse H2.35 hepatoma cell line. Two potential mitochondrial targeting sequences were identified in the NH(2) terminus of CYP2E1. Deletion of the first potential mitochondrial targeting sequence located between amino acids 50 and 65, as in Delta(2-64)2E1, still resulted in mitochondrial targeting and processing, but when, in addition to the first, the second potential mitochondrial targeting sequence located between amino acids 74 and 95 was also deleted, as in Delta(2-95)2E1, the mitochondrial targeting was abolished. Mutation of the four positively charged Arg and Lys residues present in this sequence to neutral Ala residues resulted in the abrogation of mitochondrial targeting. Deletion of a hydrophobic stretch of amino acids between residues 76 and 83 also abolished mitochondrial targeting and import. Once imported in the mitochondria, these constructs were further processed to the mature protein Delta2E1. It is concluded that mitochondrial targeting of CYP2E1 is mediated through a sequence located between residues 74 and 95 and that positively charged residues as well as a hydrophobic stretch present in the beginning of this sequence are essential for this process.     

The primary structure of a procaryotic glycoprotein. Cloning and sequencing of the cell surface glycoprotein gene of halobacteria

The hexagonally patterned surface layer of halobacteria consists of a true glycoprotein. This procaryotic glycoprotein has recently been shown to exhibit novel features with respect to saccharide structure and saccharide biosynthesis. The primary structure and the location of glycosylation sites were determined by cloning and sequencing of the glycoprotein gene of Halobacterium halobium. According to the predicted amino acid sequence, the glycoprotein is synthesized with a N-terminal leader sequence of 34 amino acid residues reminiscent of eucaryotic and procaryotic signal peptides. A hydrophobic stretch of 21 amino acid residues at the C terminus probably serves as a transmembrane domain. 14 threonine residues are clustered adjacent to this membrane anchor and linked to these threonines are all the disaccharides of the cell surface glycoprotein. 12 N-glycosylation sites are distributed over the polypeptide chain.     

Molecular cloning of a light-inducible gene (lipA) encoding a novel pilin from Arthrobacter photogonimos

Development of pili on cells of Arthrobacter photogonimos is induced by photo-oxidative conditions. The nucleotide sequence was determined of a light-inducible gene (lipA) that encodes the precursor of a light-inducible pilin (designated LIP), a polypeptide of 212 amino acids. The N-terminal leader peptide includes a typical signal sequence with a consensus cleavage site for signal peptidase I after residue 28, which should generate N-terminal arginine. However, the next amino acid, alanine, is the N-terminal residue of the mature protein. The abundance of charged amino acids (27% of total), a calculated pI of 9.98, and recovery of mostly monomers when cells were washed with 1 M NaCl suggest that electrostatic interactions play a dominant role in association of LIP, a novel mechanism for assembly of pili.     

Identification of N-terminal Residues of Sonic Hedgehog Important for Palmitoylation by Hedgehog Acyltransferase

Sonic Hedgehog (Shh) is a secreted morphogen that regulates embryonic development. After removal of the signal peptide, Shh is processed to the mature, active form through autocleavage and a series of lipid modifications, including the attachment of palmitate. Covalent attachment of palmitate to the N-terminal cysteine of Shh is catalyzed by Hedgehog acyltransferase (Hhat) and is critical for proper signaling. The sequences within Shh that are responsible for palmitoylation by Hhat are not known. Here we show that the first six amino acids of mature Shh (CGPGRG) are sufficient for Hhat-mediated palmitoylation. Alanine scanning mutagenesis was used to determine the role of each amino acid and the positional sequence requirement in a cell-based Shh palmitoylation assay. Mutation of residues in the GPGR sequence to Ala had no effect on palmitoylation, provided that a positively charged residue was present within the first seven residues. The N-terminal position exhibited a strong but not exclusive requirement for Cys. Constructs with an N-terminal Ala were not palmitoylated. However, an N-terminal Ser served as a substrate for Hhat, but not the Drosophila melanogaster ortholog Rasp, highlighting a critical difference between the mammalian and fly enzymes. These findings define residues and regions within Shh that are necessary for its recognition as a substrate for Hhat-mediated palmitoylation. Finally, we report the results of a bioinformatics screen to identify other potential Hhat substrates encoded in the human genome.