Site-specific serine incorporation by Lif and Epr into positions 3 and 5 of the Staphylococcal peptidoglycan interpeptide bridge

The FemAB-like factors Lif and Epr confer resistance to glycylglycine endopeptidases lysostaphin and Ale-1, respectively, by incorporating serine residues into the staphylococcal peptidoglycan interpeptide bridges specifically at positions 3 and 5. This required the presence of FemA and/or FemB, in contrast to earlier postulations.     

Specificities of FemA and FemB for different glycine residues: FemB cannot substitute for FemA in staphylococcal peptidoglycan pentaglycine side chain formation.

The femAB operon codes for two nearly identical approximately 50-kDa proteins involved in the formation of the staphylococcal pentaglycine interpeptide bridge. Sequencing and analysis of the femA region of mutants isolated by chemical mutagenesis and selection for lysostaphin resistance revealed point mutations leading to the expression of truncated FemA proteins. These femA mutants, although still producing an intact FemB, exhibited a phenotype identical as that described for femAB double mutants. Thus, FemA seems to be essential for the addition of glycine residues 2 and 3 only, whereas FemB is involved in the attachment of exclusively glycine residues 4 and 5. Although FemB has 39% identity with FemA, it cannot substitute for FemA. The FemA and FemB proteins seem to be highly specific in regard to the position of the glycine residues that they attach.     

Studies on prolysostaphin processing and characterization of the lysostaphin immunity factor (Lif) of Staphylococcus simulans biovar staphylolyticus

Lysostaphin is an extracellular glycylglycine endopep-tidase produced by Staphylococcus simulans biovar staphylolyticus ATCC1362 that lyses staphylococcal cells by hydrolysing the polyglycine interpeptide bridges of the peptidoglycan. Renewed analysis of the sequence of the lysostaphin gene (Iss), and the sequencing of the amino-terminus of purified prolysostaphin and of mature lysostaphin revealed that lysostaphin is organized as a preproprotein of 493 amino acids (aa), with a signal peptide consisting of 36 aa, a propeptide of 211 aa from which 195 aa are organized in 15 tandem repeats of 13 aa length, and a mature protein of 246 aa. Prolysostaphin is processed in the culture supernatant of S. simulans biovar staphylolyticus by an extracellular cysteine protease. Although prolysostaphin was staphylolytically active, the mature lysostaphin was about 4.5-fold more active. The controlled expression in Staphylococcus carnosus of Iss and Iss with deletions in the prepropeptide region indicated that the tandem repeats of the propeptide are not necessary for protein export or activation of Lss, but keep Lss in a less active state. Intracellular expressed pro- and mature lysostaphin exert staphy-lolytic activity in cell-free extracts, but do not affect growth of the corresponding clones. We characterized a lysostaphin immunity factor gene (lif) which is located in the opposite direction to Iss. The expression of lif in S. carnosus led to an increase in the serine/glycine ratio of the interpeptide bridges of peptidoglycan from 2 to 35%, suggesting that lysostaphin immunity depends on serine incorporation into the interpeptide bridge. If, in addition to lif, Iss is co-expressed the serine/glycine ratio is further increased to 58%, suggesting that Lss selects for optimal serine incorporation. Lif shows similarity to FemA and FemB     

In vitro assembly of a complete, pentaglycine interpeptide bridge containing cell wall precursor (lipid II-Gly5) of Staphylococcus aureus

Staphylococcus aureus peptidoglycan is cross-linked via a characteristic pentaglycine interpeptide bridge. Genetic analysis had identified three peptidyltransferases, FemA, FemB and FemX, to catalyse the formation of the interpeptide bridge, using glycyl t-RNA as Gly donor. To analyse the pentaglycine bridge formation in vitro, we purified the potential substrates for FemA, FemB and FemX, UDP-MurNAc-pentapeptide, lipid I and lipid II and the staphylococcal t-RNA pool, as well as His-tagged Gly-tRNA-synthetase and His-tagged FemA, FemB and FemX. We found that FemX used lipid II exclusively as acceptor for the first Gly residue. Addition of Gly 2,3 and of Gly 4,5 was catalysed by FemA and FemB, respectively, and both enzymes were specific for lipid II-Gly1 and lipid II-Gly3 as acceptors. None of the FemABX enzymes required the presence of one or two of the other Fem proteins for activity; rather, bridge formation was delayed in the in vitro system when all three enzymes were present. The in vitro assembly system described here will enable detailed analysis of late, membrane-associated steps of S. aureus peptidoglycan biosynthesis.     

The Nonantibiotic Small Molecule Cyslabdan Enhances the Potency of β-Lactams against MRSA by Inhibiting Pentaglycine Interpeptide Bridge Synthesis

The nonantibiotic small molecule cyslabdan, a labdan-type diterpene produced by Streptomyces sp. K04-0144, markedly potentiated the activity of the β-lactam drug imipenem against methicillin-resistant Staphylococcus aureus (MRSA). To study the mechanism of action of cyslabdan, the proteins that bind to cyslabdan were investigated in an MRSA lysate, which led to the identification of FemA, which is involved in the synthesis of the pentaglycine interpeptide bridge of the peptidoglycan of MRSA. Furthermore, binding assay of cyslabdan to FemB and FemX with the function similar to FemA revealed that cyslabdan had an affinity for FemB but not FemX. In an enzyme-based assay, cyslabdan inhibited FemA activity, where as did not affected FemX and FemB activities. Nonglycyl and monoglycyl murein monomers were accumulated by cyslabdan in the peptidoglycan of MRSA cell walls. These findings indicated that cyslabdan primarily inhibits FemA, thereby suppressing pentaglycine interpeptide bridge synthesis. This protein is a key factor in the determination of β-lactam resistance in MRSA, and our findings provide a new strategy for combating MRSA.     

Identification of three additional femAB-like open reading frames in Staphylococcus aureus

Three new proteins, FmhA, FmhB and FmhC, with significant identities to FemA and FemB were identified in the Staphylococcus aureus (ATCC 55748) genome database. They were mapped to the SmaI-C, SmaI-H and SmaI-A fragments of the S. aureus 8325 chromosome, respectively. Whereas insertional inactivation of fmhA and fmhC had no effects on growth, antibiotic susceptibility, lysostaphin resistance, or peptidoglycan composition of the strains, fmhB could not be inactivated, strongly suggesting that fmhB may be an essential gene. As deduced from the functions of FemA and FemB which are involved in the synthesis of the peptidoglycan pentaglycine interpeptide, FmhB may be a candidate for the postulated FemX thought to add the first glycine to the nascent interpeptide.     

Cell wall monoglycine cross-bridges and methicillin hypersusceptibility in a femAB null mutant of methicillin-resistant Staphylococcus aureus.

The femAB operon is involved in the formation of the characteristic pentaglycine side chain of the staphylococcal peptidoglycan. Allele replacement of the femAB operon with the tetracycline resistance determinant tetK in a methicillin-resistant Staphylococcus aureus strain resulted in impaired growth, methicillin hypersusceptibility, and lysostaphin resistance. The usual pentaglycine cross-bridges were replaced by monoglycine bridges exclusively, and cross-linking of the peptidoglycan strands was drastically reduced. Complementation of the femAB null mutant by either femA or femAB resulted in the extension of the cross-bridges to a triglycine or a pentaglycine, respectively. This finding suggests that FemA is responsible for the formation of glycines 2 and 3, and FemB is responsible for formation of glycines 4 and 5, of the pentaglycine side chain of the peptidoglycan precursor. Moreover, it can be deduced that addition of the first glycine must occur by a femAB-independent mechanism.     

epr, which encodes glycylglycine endopeptidase resistance, is homologous to femAB and affects serine content of peptidoglycan cross bridges in Staphylococcus capitis and Staphylococcus aureus.

Staphylococcus capitis EPK1 produces a glycylglycine endopeptidase, ALE-1 (M. Sugai, T. Fujiwara, T. Akiyama, M. Ohara, H. Komatsuzawa, S. Inoue, and H. Suginaka, J. Bacteriol. 179:1193-1202, 1997), which hydrolyzes interpeptide pentaglycine chains of cell wall peptidoglycan of S. aureus. Characterizations of the enzyme activity and cloning of ale-1 revealed that ALE-1 is very similar to prolysostaphin produced by S. simulans bv. staphylolyticus. Strain EPK1 is resistant to lysis by ALE-1 and by lysostaphin. A gene that renders the cells resistant to glycylglycine endopeptidase (epr) was found 322 bp upstream of and in the opposite orientation to ale-1. The deduced amino acid sequence of epr showed similarities to FemA and FemB, which have been characterized as factors essential for methicillin resistance of S. aureus. Inactivation of either femA or femB causes decreased resistance to methicillin, increased resistance to lysostaphin, and decreased glycine content in the interpeptide chains of peptidoglycan. Therefore, femAB is suggested to be involved in the addition of glycine to pentapeptide peptidoglycan precursor. S. aureus with epr on a multicopy plasmid had phenotypes similar to those of femAB mutants except that it did not alter resistance level to methicillin. These results suggest that epr and femAB belong to the protein family involved in adding amino acids to the pentapeptide peptidoglycan precursor and that epr is involved in the addition of serine to the pentapeptide.     

Influence of Lif, the Lysostaphin Immunity Factor, on Acceptors of Surface Proteins and Cell Wall Sorting Efficiency in Staphylococcus carnosus

Proteins harboring a C-terminal cell wall sorting signal are covalently linked to pentaglycine acceptors within the staphylococcal peptidoglycan. This pentaglycine was modified when the lysostaphin immunity factor (Lif) of Staphylococcus simulans was expressed in Staphylococcus carnosus, likely by the exchange of two glycine residues for serine residues. A reporter protein was efficiently linked to the modified acceptor, indicating that the sorting reaction is not strictly dependent on the wild-type structures of the acceptors.     

X-ray crystal structure of Staphylococcus aureus FemA

The latter stages of peptidoglycan biosynthesis in Staphylococci involve the synthesis of a pentaglycine bridge on the epsilon amino group of the pentapeptide lysine side chain. Genetic and biochemical evidence suggest that sequential addition of these glycines is catalyzed by three homologous enzymes, FemX (FmhB), FemA, and FemB. The first protein structure from this family, Staphylococcus aureus FemA, has been solved at 2.1 A resolution by X-ray crystallography. The FemA structure reveals a unique organization of several known protein folds involved in peptide and tRNA binding. The surface of the protein also reveals an L-shaped channel suitable for a peptidoglycan substrate. Analysis of the structural features of this enzyme provides clues to the mechanism of action of S. aureus FemA.     

Random Mutagenesis Identifies Novel Genes Involved in the Secretion of Antimicrobial, Cell Wall-Lytic Enzymes by Lactococcus lactis

Lactococcus lactis is a gram-positive bacterium that is widely used in the food industry and is therefore desirable as a candidate for the production and secretion of recombinant proteins. Previously, we generated a L. lactis strain that expressed and secreted the antimicrobial cell wall-lytic enzyme lysostaphin. To identify lactococcal gene products that affect the production of lysostaphin, we isolated and characterized mutants generated by random transposon mutagenesis that had altered lysostaphin activity. Out of 35,000 mutants screened, only one with no lysostaphin activity was identified, and it was found to contain an insertion in the lysostaphin expression cassette. Ten mutants with higher lysostaphin activity contained insertions in only four different genes, which encode an uncharacterized putative transmembrane protein (llmg_0609) (three mutants), an enzyme catalyzing the first step in peptidoglycan biosynthesis (murA2) (five mutants), a putative regulator of peptidoglycan modification (trmA) (one mutant), and an uncharacterized enzyme possibly involved in ubiquinone biosynthesis (llmg_2148) (one mutant). These mutants were found to secrete larger amounts of lysostaphin than the control strain (MG1363[lss]), and the greatest increase in secretion was 9.8- to 16.1-fold, for the llmg_0609 mutants. The lysostaphin-oversecreting llmg_0609, murA2, and trmA mutants were also found to secrete larger amounts of another cell wall-lytic enzyme (the Listeria monocytogenes bacteriophage endolysin Ply511) than the control strain, indicating that the phenotype is not limited to lysostaphin.     

Engineering active lysostaphin variants that incorporate noncanonical amino acids and characterizing the effects of site-specific PEGylation

We describe a facile strategy to identify sites for the incorporation of noncanonical amino acids into lysostaphin—an enzyme that degrades the cell wall of Staphylococcus aureus—while retaining stapholytic activity. We used this strategy to generate active variants of lysostaphin incorporating para-azidophenylalanine. The incorporation of this “reactive handle” enabled the orthogonal site-specific modification of the enzyme variants with polyethylene glycol (PEG) using copper-free click cycloaddition. PEGylated lysostaphin variants could retain their stapholytic activity, with the extent of retention depending on the site of modification and the PEG molecular weight. The site-specific modification of lysostaphin could be useful not only for PEGylation to improve biocompatibility but also for the incorporation of the enzyme into hydrogels and other biomaterials and for studies of protein structure and dynamics. Moreover, the approach described herein could be readily applied to identify suitable sites for the incorporation of reactive handles into other proteins of interest.     

<Emphasis Type="Italic">Staphylococcus simulans</Emphasis> recombinant lysostaphin: Production,purification, and determination of antistaphylococcal activity

Staphylococcus simulans lysostaphin is an endopeptidase lysing staphylococcus cell walls by cleaving pentaglycine cross-bridges in their peptidoglycan. A synthetic gene encoding S. simulans lysostaphin was cloned in Escherichia coli cells, and producer strains were designed. The level of produced biologically active lysostaphin comprised 6-30% of total E. coli cell protein (depending on E. coli M15 or BL21 producer) under batch cultivation conditions. New methods were developed for purification of lysostaphin without affinity domains and for testing its enzymatic activity. As judged by PAGE, the purified recombinant lysostaphin is of >97% purity. The produced lysostaphin lysed cells of Staphylococcus aureus and Staphylococcus haemolyticus clinical isolates. In vitro activity and general biochemical properties of purified recombinant lysostaphin produced by M15 or BL21 E. coli strains were identical to those of recombinant lysostaphin supplied by SigmaAldrich (USA) and used as reference in other known studies. The prepared recombinant lysostaphin represents a potential product for development of enzymatic preparation for medicine and veterinary due to the simple purification scheme enabling production of the enzyme of high purity and antistaphylococcal activity.     

FemA, a host-mediated factor essential for methicillin resistance in Staphylococcus aureus: Molecular cloning and characterization

Summary The methicillin resistance determinant (mec) in Staphylococcus aureus resides on additional DNA not present in isogenic sensitive cells. However, besides mec, other chromosomally determined factors are essential for expression of methicillin resistance. We cloned and characterized a chromosomally determined gene which encodes a factor essential for the expression of methicillin resistance (femA) in S. aureus. femA mapped in chromosomal segment number 18, genetically very distant from the methicillin resistance determinant (mec). The product of femA was a protein of an apparent size of 48 kDa. FemA restored methicillin resistance in S. aureus that had become sensitive to methicillin by insertion of 22003 (femA::Tn551). Although FemA was needed for cell growth in the presence of -lactam antibiotics, it had no influence on the synthesis of the low affinity, additional penicillin-binding protein (PBP2) encoded by mec and known to be essential for cell wall synthesis in the presence of inhibitory concentrations of methicillin. Nucleotide sequence analysis, Northern RNA blotting and S1 nuclease RNA mapping suggested that femA was transcribed on a polycistronic mRNA. This mRNA contained the coding region for FemA (ORF433) and a second coding region (ORF419) producing a protein of 47 kDa. The nucleotide and amino acid sequence of FemA showed homologies with ORF419, suggesting that these genes arose by gene duplication. In addition we present evidence for a second chromosomal factor, femB, involved in expression of methicillin resistance which maps close to femA.     

Lif1 SUMOylation and its role in non-homologous end-joining

Non-homologous end-joining (NHEJ) repairs DNA double-strand breaks by tethering and ligating the two DNA ends. The mechanisms regulating NHEJ efficiency and interplay between its components are not fully understood. Here, we identify and characterize the SUMOylation of budding yeast Lif1 protein, which is required for the ligation step in NHEJ. We show that Lif1 SUMOylation occurs throughout the cell cycle and requires the Siz SUMO ligases. Single-strand DNA, but not double-strand DNA or the Lif1 binding partner Nej1, is inhibitory to Lif1 SUMOylation. We identify lysine 301 as the major conjugation site and demonstrate that its replacement with arginine completely abolishes Lif1 SUMOylation in vivo and in vitro. The lif1-K301R mutant cells exhibit increased levels of NHEJ repair compared with wild-type cells throughout the cell cycle. This is likely due to the inhibitory effect of Lif1 SUMOylation on both its self-association and newly observed single-strand DNA binding activity. Taken together, these findings suggest that SUMOylation of Lif1 represents a new regulatory mechanism that downregulates NHEJ in a cell cycle phase-independent manner.     

Effects of specific and prolonged expression of zebrafish growth factors,Fgf2 and Lif in primordial germ cells in vivo

Primordial germ cells (PGCs), specified early in development, proliferate and migrate to the developing gonad before sexual differentiation occurs in the embryo and eventually give rise to spermatogonia or oogonia. In this study, we discovered that nanos3 3′UTR, a common method used to label PGCs, not only directed PGC-specific expression of DsRed but also prolonged this expression up to 26 days post fertilization (dpf) when DsRed-nanos3 3′UTR hybrid mRNAs were introduced into 1- to 2-cell-stage embryos. As such, we employed this knowledge to express zebrafish leukemia inhibitory factor (Lif), basic fibroblast growth factor (Fgf2) and bone morphogenetic protein 4 (Bmp4) in the PGCs and evaluate their effects on PGC development in vivo for over a period of 3 weeks. The results show that expression of Fgf2 significantly increased PGC number at 14- and 21-dpf while Bmp4 resulted in severe ventralization and death of the embryos by 3 days. Expression of Lif resulted in a significant disruption of PGC migration. Mopholino knockdown experiments indicated that Lif illicited its effect on PGC migration through Lif receptor a (Lifra) but not Lifrb. The general approach described in this study could be used to achieve prolonged PGC-specific expression of other proteins to investigate their roles in germ cell and gonad development. The results also indicate that zebrafish PGCs have a mechanism to stabilize and prolong the expression of mRNA that carries nanos3 3′UTR. Understanding this mechanism may make it possible to achieve prolonged RNA expression in other cell types.     

A crystal molecular conformation of leucine-enkephalin related to the morphine molecule

Leucine-enkephalin (Try1-Gly2-Gly3-Phe4-Leu5) has been crystallized as a trihydrate from water solution. X-ray diffraction reveals a tightly folded molecular conformation with two fused beta III- (Gly2-Gly3) and beta I- (Gly3-Phe4) turns. The Tyr1 and Phe4 aromatic rings have a close orthogonal arrangement analogous to the tyramine and cyclohexenyl rings in morphine. This suggests that the conformation found in the trihydrate crystal structure could be required for recognition by mu-receptor sites.     

Inhibition of the Activity of Both Domains of Lysostaphin through Peptidoglycan Modification by the Lysostaphin Immunity Protein

Resistance to lysostaphin, a staphylolytic glycylglycine endopeptidase, is due to a FemABX-like immunity protein that inserts serines in place of some glycines in peptidoglycan cross bridges. These modifications inhibit both binding of the recombinant cell wall targeting domain and catalysis by the recombinant catalytic domain of lysostaphin.Lysostaphin is a glycylglycine endopeptidase produced by Staphylococcus simulans biovar staphylolyticus (18) that lyses susceptible staphylococci by hydrolyzing the polyglycine cross bridges in their cell wall peptidoglycans (3). The lysostaphin gene sequence was independently determined in 1987 by two groups (8, 13). BLAST analysis (1) of mature lysostaphin revealed two domains: an N-terminal catalytic domain (CAT), which is a member of the M23 family of zinc metalloendopeptidases, and a C-terminal cell wall targeting domain (CWT), which is a member of the SH3b domain family (Fig. ​(Fig.11 A).Open in a separate windowFIG. 1.(A) Schematic diagram of mature lysostaphin, the recombinant catalytic domain (rCAT) (lysostaphin residues 1 to 148), and the recombinant cell wall targeting domain (rCWT) (lysostaphin residues 149 to 246). The numbers represent the beginning and end of the domains, and the solid boxes indicate the N-terminal His₆ tag of the recombinant proteins. (B) SDS-PAGE analysis of rCAT and rCWT purified by a nickel affinity column. Mobilities of molecular mass standards are given on the left side of the gel.The lysostaphin endopeptidase resistance gene (epr or lif) encodes a FemABX-like immunity protein that is located adjacent to the lysostaphin gene on the plasmid pACK1 in S. simulans bv. staphylolyticus (4, 7, 20). Members of the FemABX family of proteins are nonribosomal peptidyl transferases that are involved in the addition of cross bridge amino acids during peptidoglycan subunit synthesis in the cytoplasm (15). In S. simulans bv. staphylolyticus, the lysostaphin immunity protein inserts serines in place of some glycines during peptidoglycan synthesis, which provides resistance to lysostaphin (4, 20).Originally it was suggested that the incorporation of serines in these peptidoglycan cross bridges gave increased resistance to lysostaphin because of the inability of the enzyme to hydrolyze glycyl-serine or seryl-glycine bonds (4, 14, 16). Others later reported that the CWT specifically binds to the polyglycine cross bridges in staphylococci (6) and the binding of CWT to producer-strain cells was less than that to susceptible cells (2). However, the ability of the enzyme or its targeting domain to bind to purified peptidoglycans from staphylococci containing the lysostaphin resistance gene has not been determined. Therefore, we determined if the modification to staphylococcal peptidoglycan cross bridges made by the lysostaphin immunity protein affected the activity of the binding domain, the catalytic domain, or both.     

FemA of Staphylococcus aureus: Isolation and immunodetection

Abstract FemA, a cytoplasmic protein necessary for the expression of methicillin resistance in Staphylococcus aureus and also involved in the biosynthesis of staphylococcal cell walls, was detected and quantified in several S. aureus strains under different growth conditions by Western immunoblot. Two types of antigens were used for the production of polyclonal antibodies against FemA: (i) a synthetic peptide comprising 14 amino acids of its C-terminal sequence; and (ii) FemA isolated by preparative gel electrophoresis and electroelution from an overproducing staphylococcal strain. Immunodetection revealed that all investigated strains, either methicillin-resistant or susceptible, expressed FemA during the exponential growth phase in varying amounts. In the stationary phase, the FemA content was diminished. Strains in which femA was inactivated by insertion of Tn557 into the control region of the fem AB operon still expressed about 10% of the protein compared to their parent strains. Tn55 I insertion in the middle of the fem B gene did not affect the FemA expression. In 40 methicillin-susceptible and 6 resistant clinical isolates of S. aureus , the FemA content or its affinity to the antibodies was reduced compared to laboratory parent strains. In susceptible strains, an additional protein of higher molecular weight, present in large quantities, was also able to bind the FemA antibodies. Such a protein was also present in methicillin-resistant isolates, although it was not as pronounced as in the susceptible strains.     

Characterization of a novel lipase and its specific foldase from Acinetobacter sp. XMZ-26

A novel operon containing a lipase gene (lip26) and its specific foldase gene (lif26) was discovered from Acinetobacter sp. XMZ-26 by creating and screening a gene library and then using genome walking. The amino acid sequence of Lip26 and Lif26 showed only 46.4% and 37.3% identity with the LipA and LipB (Lif) sequences from Acinetobacter sp. SY-01, respectively. The expressed recombinant Lip26 formed inactive inclusion bodies in Escherichia coli. However, the active Lip26 was refolded by the dilution refolding method with the assistance of purified recombinant Lif26, and the refolded Lip26 had a high specific activity. Lip26 hydrolyzed p-nitrophenyl (pNP) esters of fatty acids with C₂ to C₁₆ acyl chain lengths and had a substrate preference for pNP myristate. Maximal Lip26 activity was dependent on both the temperature (55 °C) and pH (9.0). In addition, Lip26 was capable of maintaining its activity in the presence of many detergents and organic solutions, and its activity was enhanced by the presence of Ca²⁺, Mn²⁺, and Ba²⁺. To directly obtain active Lip26, an E. coli strain was co-transformed with two expression plasmids containing the lip26 and lif26 genes. The co-expression of both proteins in vivo resulted in the expression of half of the recombinant Lip26 as a soluble protein with demonstrable lipase activity. A direct protein interaction between Lif26 and Lip26A was detected by both a pull-down assay and a yeast two-hybrid experiment.