Characterization of tRNA-dependent peptide bond formation by MurM in the synthesis of Streptococcus pneumoniae peptidoglycan

MurM is an aminoacyl ligase that adds l-serine or l-alanine as the first amino acid of a dipeptide branch to the stem peptide lysine of the pneumococcal peptidoglycan. MurM activity is essential for clinical pneumococcal penicillin resistance. Analysis of peptidoglycan from the highly penicillin-resistant Streptococcus pneumoniae strain 159 revealed that in vivo and in vitro, in the presence of the appropriate acyl-tRNA, MurM(159) alanylated the peptidoglycan epsilon-amino group of the stem peptide lysine in preference to its serylation. However, in contrast, identical analyses of the penicillin-susceptible strain Pn16 revealed that MurM(Pn16) activity supported serylation more than alanylation both in vivo and in vitro. Interestingly, both MurM(Pn16) acylation activities were far lower than the alanylation activity of MurM(159). The resulting differing stem peptide structures of 159 and Pn16 were caused by the profoundly greater catalytic efficiency of MurM(159) compared with MurM(Pn16) bought about by sequence variation between these enzymes and, to a lesser extent, differences in the in vivo tRNA(Ala):tRNA(Ser) ratio in 159 and Pn16. Kinetic analysis revealed that MurM(159) acted during the lipid-linked stages of peptidoglycan synthesis, that the d-alanyl-d-alanine of the stem peptide and the lipid II N-acetylglucosaminyl group were not essential for substrate recognition, that epsilon-carboxylation of the lysine of the stem peptide was not tolerated, and that lipid II-alanine was a substrate, suggesting an evolutionary link to staphylococcal homologues of MurM such as FemA. Kinetic analysis also revealed that MurM recognized the acceptor stem and/or the TPsiC loop stem of the tRNA(Ala). It is anticipated that definition of the minimal structural features of MurM substrates will allow development of novel resistance inhibitors that will restore the efficacy of beta-lactams for treatment of pneumococcal infection.     

Functional analysis of Streptococcus pneumoniae MurM reveals the region responsible for its specificity in the synthesis of branched cell wall peptides

The recently identified murMN operon of Streptococcus pneumoniae encodes enzymes involved in the synthesis of branched structured muropeptides of the pneumococcal cell wall peptidoglycan. Its inactivation was shown to cause production of a peptidoglycan composed exclusively of linear muropeptides and a virtually complete loss of resistance in penicillin-resistant strains. The studies described in this communication follow up these observations in several directions. The substrate of the MurM-catalyzed reaction (addition of alanine or serine) was identified as the lipid-linked N-acetylglucosamine-muramyl pentapeptide. Different murM alleles from several penicillin-resistant S. pneumoniae strains, each with a characteristic branched peptide pattern, were cloned into pLS578, a pneumococcal plasmid capable of replicating in S. pneumoniae, and transformed into the penicillin-susceptible laboratory strain R36A. All transformants remained penicillin-susceptible; however, their cell wall composition changed in directions corresponding to the muropeptide pattern of the strain from which the murM allele was derived. This suggests that the muropeptide composition of the pneumococcal cell walls is determined by the particular murM allele carried by the cells. A 30-amino acid long sequence within the MurM protein was shown to be the main determinant of the specificity of the reaction (addition of alanine versus serine).     

Lipid II-independent trans Editing of Mischarged tRNAs by the Penicillin Resistance Factor MurM

Streptococcus pneumoniae is a causative agent of nosocomial infections such as pneumonia, meningitis, and septicemia. Penicillin resistance in S. pneumoniae depends in part upon MurM, an aminoacyl-tRNA ligase that attaches l-serine or l-alanine to the stem peptide lysine of Lipid II in cell wall peptidoglycan. To investigate the exact substrates the translation machinery provides MurM, quality control by alanyl-tRNA synthetase (AlaRS) was investigated. AlaRS mischarged serine and glycine to tRNA^Ala, as observed in other bacteria, and also transferred alanine, serine, and glycine to tRNA^Phe. S. pneumoniae tRNA^Phe has an unusual U4:C69 mismatch in its acceptor stem that prevents editing by phenylalanyl-tRNA synthetase (PheRS), leading to the accumulation of misaminoacylated tRNAs that could serve as substrates for translation or for MurM. Although the peptidoglycan layer of S. pneumoniae tolerates a combination of both branched and linear muropeptides, deletion of MurM results in a reversion to penicillin sensitivity in strains that were previously resistant. However, because MurM is not required for cell viability, the reason for its functional conservation across all strains of S. pneumoniae has remained elusive. We now show that MurM can directly function in translation quality control by acting as a broad specificity lipid-independent trans editing factor that deacylates tRNA. This activity of MurM does not require the presence of its second substrate, Lipid II, and can functionally substitute for the activity of widely conserved editing domain homologues of AlaRS, termed AlaXPs proteins, which are themselves absent from S. pneumoniae.     

Characterization of the murMN operon involved in the synthesis of branched peptidoglycan peptides in Streptococcus pneumoniae

The murMN operon, recently identified in the genome of Streptococcus pneumoniae, encodes for enzymes involved in the synthesis of branched structured muropeptides in the pneumococcal peptidoglycan; inactivation of murMN causes production of a peptidoglycan composed exclusively of linear muropeptides and a virtually complete loss of resistance in penicillin-resistant strains (Filipe, S. R., and Tomasz, A. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 4891-4896). The experiments described in this paper follow up these observations. Primer extension analysis was used to identify the putative promoter region of the murMN operon in penicillin-susceptible and -resistant strains. Selective inactivation of the murN gene in the penicillin-resistant strain Pen6 caused production of an unusual peptidoglycan that contained only single amino acid residues in the muropeptide branches, indicating that the product of murN was involved with the addition of the second amino acid and the product of murM was involved with the addition of the first amino acid (alanine or serine) to the peptidoglycan cross-bridge. Allelic replacement of the mosaic murM gene of strain Pen6 with murM of the penicillin-susceptible laboratory strain caused enrichment of the peptidoglycan in linear muropeptides. The findings suggest that the genetic determinant primarily controlling the synthesis of branched muropeptides in the pneumococcal peptidoglycan is murM.     

Evolution of penicillin resistance in Streptococcus pneumoniae; the role of Streptococcus mitis in the formation of a low affinity PBP2B in S. pneumoniae

Penicillin-resistant strains of Streptococcus pneumoniae possess forms of penicillin-binding proteins (PBPs) that have a low affinity for penicillin compared to those from penicillin-sensitive strains. PBP genes from penicillin-resistant isolates are very variable and have a mosaic structure composed of blocks of nucleotides that are similar to those found in PBP genes from penicillin-sensitive isolates and blocks that differ by up to 21%. These chromosomally encoded mosaic genes have presumably arisen following transformation and homologous recombination with PBP genes from a number of closely related species. This study shows that PBP2B genes from many penicillin-resistant isolates of S. pneumoniae contain blocks of nucleotides originating from Streptococcus mitis. In several instances it would appear that this material alone is sufficient to produce a low affinity PBP2B. In other examples PBP2B genes possess blocks of nucleotides from S. mitis and at least one additional unidentified species. Mosaic structure was aiso found in the PBP2B genes of penicillin-sensitive isolates of S. mitis or S. pneumoniae. These mosaics did not confer penicillin resistance but nevertheless reveal something of the extent to which localized recombination occurs in these naturally transformable streptococci.     

Extensive re-modelling of the transpeptidase domain of penicillin-binding protein 2B of a penicillin-resistant South African isolate of Streptococcus pneumoniae

Clinical isolates of Streptococcus pneumoniae that have greatly increased levels of resistance to penicillin (greater than 1000-fold) have been reported from South Africa during the last ten years. Penicillin resistance in these strains is entirely due to the development of penicillin-binding proteins (PBPs) with decreased affinity for penicillin. We have cloned and sequenced the coding region for the transpeptidase domain of penicillin-binding protein 2B from three penicillin-sensitive strains of S. pneumoniae and from a penicillin-resistant South African strain. The amino acid sequences of the transpeptidase domains of PBP2B of the three penicillin-sensitive strains were identical and there were only between one and four differences in the nucleotide sequences of their coding regions. The corresponding region of the PBP2B gene from the penicillin-resistant strain differed by 74 nucleotide substitutions which resulted in 17 alterations in the amino acid sequence of PBP2B. The most remarkable alteration that has occurred during the development of the 'penicillin-resistant' form of PBP2B is the substitution of seven consecutive residues in a region that is predicted to form a loop at the bottom of the penicillin-binding site.     

Engineering subtilisin and its substrates for efficient ligation of peptide bonds in aqueous solution

Protein engineering techniques were used to construct a derivative of the serine protease subtilisin that ligates peptides efficiently in water. The subtilisin double mutant in which the catalytic Ser221 was converted to Cys (S221C) and Pro225 converted to Ala (P225A) has 10-fold higher peptide ligase activity and at least 100-fold lower amidase activity than the singly mutated thiolsubtilisin (S221C) that was previously shown to have some peptide ligase activity [Nakatsuka, T., Sasaki, T., & Kaiser, E.T. (1987) J. Am. Chem. Soc. 109, 3808-3810]. A 1.5-A X-ray crystal structure of an oxidized derivative of the double mutant (S221C/P225A) supports the protein design strategy in showing that the P225A mutation partly relieves the steric crowding expected from the S221C substitution, thus accounting for its improved catalytic efficiency. Stable and synthetically reasonable alkyl ester peptide substrates were prepared that rapidly acylate the S221C/P225A enzyme, and aminolysis of the resulting thioacyl-enzyme intermediate by various peptides is strongly preferred over hydrolysis. The efficiency of aminolysis is relatively insensitive to the sequence of the first two residues in the acyl acceptor peptide whose alpha-amino group attacks the thioacyl-enzyme. To obtain greater flexibility in the choice of coupling sites, a set of three additional peptide ligases were engineered by introducing mutations into the parent ligase (S221C/P225A) that were previously shown to change the specificity of subtilisin for the residue nearest the acyl bond (the P1 residue). The specificity properties of the parent ligase and derivatives of it paralleled those of wild type and corresponding specificity variants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aslfm, the D-aspartate ligase responsible for the addition of D-aspartic acid onto the peptidoglycan precursor of Enterococcus faecium

D-aspartate ligase has remained the last unidentified peptide bond-forming enzyme in the peptidoglycan assembly pathway of Gram-positive bacteria. Here we show that a two-gene cluster of Enterococcus faecium encodes aspartate racemase (Racfm) and ligase (Aslfm) for incorporation of D-Asp into the side chain of the peptidoglycan precursor. Aslfm was identified as a new member of the ATP-grasp protein superfamily, which includes a diverse set of enzymes catalyzing ATP-dependent carboxylate-amine ligation reactions. Aslfm specifically ligated the beta-carboxylate of D-Asp to the epsilon-amino group of L-Lys in the nucleotide precursor UDP-N-acetylmuramyl-pentapeptide. D-iso-asparagine was not a substrate of Aslfm, indicating that the presence of this amino acid in the peptidoglycan of E. faecium results from amidation of the alpha-carboxyl of D-Asp after its addition to the precursor. Heterospecific expression of the genes encoding Racfm and Aslfm in Enterococcus faecalis led to production of stem peptides substituted by D-Asp instead of L-Ala2, providing evidence for the in vivo specificity and function of these enzymes. Strikingly, sequencing of the cross-bridges revealed that substitution of L-Ala2 by D-Asp is tolerated by the d,d-transpeptidase activity of the penicillin-binding proteins both in the acceptor and in the donor substrates. The Aslfm ligase appears as an attractive target for the development of narrow spectrum antibiotics active against multiresistant E. faecium.     

Biochemical characterization and physiological properties of Escherichia coli UDP-N-acetylmuramate:L-alanyl-gamma-D-glutamyl-meso-diaminopimelate ligase

The UDP-N-acetylmuramate:L-alanyl-gamma-D-glutamyl-meso-diaminopimelate ligase (murein peptide ligase [Mpl]) is known to be a recycling enzyme allowing reincorporation into peptidoglycan (murein) of the tripeptide L-alanyl-gamma-D-glutamyl-meso-diaminopimelate released during the maturation and constant remodeling of this bacterial cell wall polymer that occur during cell growth and division. Mpl adds this peptide to UDP-N-acetylmuramic acid, thereby providing an economical additional source of UDP-MurNAc-tripeptide available for de novo peptidoglycan biosynthesis. The Mpl enzyme from Escherichia coli was purified to homogeneity as a His-tagged form, and its kinetic properties and parameters were determined. Mpl was found to accept tri-, tetra-, and pentapeptides as substrates in vitro with similar efficiencies, but it accepted the dipeptide L-Ala-D-Glu and L-Ala very poorly. Replacement of meso-diaminopimelic acid by L-Lys resulted in a significant decrease in the catalytic efficacy. The effects of disruption of the E. coli mpl gene and/or the ldcA gene encoding the LD-carboxypeptidase on peptidoglycan metabolism were investigated. The differences in the pools of UDP-MurNAc peptides and of free peptides between the wild-type and mutant strains demonstrated that the recycling activity of Mpl is not restricted to the tripeptide and that tetra- and pentapeptides are also directly reused by this process in vivo. The relatively broad substrate specificity of the Mpl ligase indicates that it is an interesting potential target for antibacterial compounds.     

Biochemical characterisation of the chlamydial MurF ligase, and possible sequence of the chlamydial peptidoglycan pentapeptide stem

Chlamydiaceae are obligate intracellular bacteria that do not synthesise detectable peptidoglycan although they possess an almost complete arsenal of genes encoding peptidoglycan biosynthetic activities. In this paper, the murF gene from Chlamydia trachomatis was shown to be capable of complementing a conditional Escherichia coli mutant impaired in UDP-MurNAc-tripeptide:D-Ala-D-Ala ligase activity. Recombinant MurF from C. trachomatis was overproduced and purified from E. coli. It exhibited ATP-dependent UDP-MurNAc-X-γ-D-Glu-meso-A(2)pm:D-Ala-D-Ala ligase activity in vitro. No significant difference of kinetic parameters was seen when X was L-Ala, L-Ser or Gly. The L-Lys-containing UDP-MurNAc-tripeptide was a poorer substrate as compared to the meso-A(2)pm-containing one. Based on the respective substrate specificities of the chlamydial MurC, MurE, MurF and Ddl enzymes, a sequence L-Ala/L-Ser/Gly-γ-D-Glu-meso-A(2)pm-D-Ala-D-Ala is expected for the chlamydial pentapeptide stem, with Gly at position 1 being less likely.     

Inhibition of tRNA-dependent ligase MurM from Streptococcus pneumoniae by phosphonate and sulfonamide inhibitors

Ligase MurM catalyses the addition of Ala from alanyl-tRNA^Ala, or Ser from seryl-tRNA^Ser, to lipid intermediate II in peptidoglycan biosynthesis in Streptococcus pneumoniae, and is a determinant of high-level penicillin resistance. Phosphorus-based transition state analogues were designed as inhibitors of the MurM-catalysed reaction. Phosphonamide analogues mimicking the attack of a lysine nucleophile upon Ala-tRNA^Ala showed no inhibition of MurM, but adenosine 3′-phosphonate analogues showed inhibition of MurM, the most active being a 2′-deoxyadenosine analogue (IC₅₀ 100 μM). Structure/function studies upon this analogue established that modification of the amino group of the aminoalkylphosphonate resulted in loss of potency, and modification of the adenosine 5′-hydroxyl group with either a t-butyl dimethyl silyl or a carbamate functional group resulted in loss of activity. A library of 48 aryl sulfonamides was also screened against MurM using a radiochemical assay, and two compounds showed sub-millimolar inhibition. These compounds are the first small molecule inhibitors of the Fem ligase family of peptidyltransferases found in Gram-positive bacteria.     

Crystal structure of PBP2x from a highly penicillin-resistant Streptococcus pneumoniae clinical isolate: a mosaic framework containing 83 mutations.

Penicillin-binding proteins (PBPs) are the main targets for beta-lactam antibiotics, such as penicillins and cephalosporins, in a wide range of bacterial species. In some Gram-positive strains, the surge of resistance to treatment with beta-lactams is primarily the result of the proliferation of mosaic PBP-encoding genes, which encode novel proteins by recombination. PBP2x is a primary resistance determinant in Streptococcus pneumoniae, and its modification is an essential step in the development of high level beta-lactam resistance. To understand such a resistance mechanism at an atomic level, we have solved the x-ray crystal structure of PBP2x from a highly penicillin-resistant clinical isolate of S. pneumoniae, Sp328, which harbors 83 mutations in the soluble region. In the proximity of the Sp328 PBP2x* active site, the Thr(338) --> Ala mutation weakens the local hydrogen bonding network, thus abrogating the stabilization of a crucial buried water molecule. In addition, the Ser(389) --> Leu and Asn(514) --> His mutations produce a destabilizing effect that generates an "open" active site. It has been suggested that peptidoglycan substrates for beta-lactam-resistant PBPs contain a large amount of abnormal, branched peptides, whereas sensitive strains tend to catalyze cross-linking of linear forms. Thus, in vivo, an "open" active site could facilitate the recognition of distinct, branched physiological substrates.     

Distribution of the mosaic structured murM genes among natural populations of Streptococcus pneumoniae

The presence and sequence variation of the murM gene were studied in a large collection (814 strains) of genetically diverse Streptococcus pneumoniae isolates, which included 27 different serogroups and both penicillin-resistant (423 isolates, 67 pulsed-field gel electrophoretic [PFGE] types) and intermediately penicillin-resistant (165 isolates, 66 PFGE types) and penicillin-susceptible (226 isolates, 135 PFGE types) strains. Diversity of the murM sequences was tested by hybridization with mainly two kinds of probes: one derived from the amplification of the nucleotide sequence between nucleotides 201 and 624 in the penicillin-susceptible laboratory strain R36A (murMA probe) and a second probe that amplified the comparable, highly divergent sequence in the penicillin-resistant strain Pen6 (murMB probe). The great majority of the strains (761 of 814), including both penicillin-susceptible and penicillin-resistant isolates, reacted exclusively with the murMA probe. A smaller group of penicillin-resistant strains (48 of 814 isolates) reacted only with the murMB DNA probe, and an additional 5 isolates reacted with both probes. High-pressure liquid chromatography analysis of the peptidoglycan of strains hybridizing with murMB showed that they invariably contained an increased proportion of branched peptides. Complete sequencing of murM from a group of penicillin-resistant isolates allowed the identification of a number of different murMB alleles that differed in the length and exact position of the divergent (Pen6 type) sequences within the particular murM. The close similarity of these divergent sequences in the various murM alleles suggests a possible common heterologous origin.     

Insertion of an extra amino acid is the main cause of the low affinity of penicillin-binding protein 2 in penicillin-resistant strains of Neisseria gonorrhoeae

Non-beta-lactamase-producing, penicillin-resistant strains of Neisseria gonorrhoeae (CMRNG strains) produce altered forms of penicillin-binding protein 2 (PBP2) that have decreased affinity for penicillin. A feature of PBP2 from all CMRNG strains is the presence of an additional residue (Asp-345A) that is absent from PBP2 of penicillin-sensitive strains. The role of the additional aspartic acid residue in the decreased affinity of PBP2 is unclear as PBP2 of all previously examined CMRNG strains possess several other amino acid sequence alterations, in addition to the insertion of Asp-345A, compared to PBP2 of penicillin-sensitive strains. Site-directed mutagenesis has been used to insert the Asp-345A codon into the penA gene from a penicillin-sensitive gonococcus. The resulting penA gene expressed an altered form of PBP2 that had a decreased affinity for benzylpenicillin and was able to transform a penicillin-sensitive strain of N. gonorrhoeae to an increased level of resistance to benzylpenicillin. Insertion of amino acids other than aspartic acid did not produce forms of PBP2 that provided increased resistance to penicillin. Removal of the Asp-345A codon from the penA gene of a CMRNG strain reduced its ability to transform a penicillin-sensitive strain to an increased level of penicillin resistance. The reduction in the affinity of PBP2 in CMRNG strains is therefore largely, although not exclusively, due to the insertion of Asp-345A. Clinical isolates that produce altered forms of PBP2 that differ from that of penicillin-sensitive strains only in the insertion of Asp-345A have been identified.     

Cefonicid as Therapy for Uncomplicated Gonococcal Urethritis Caused by Penicillinase-Producing Neisseria gonorrhoeae

Young men with uncomplicated gonococcal urethritis were treated with 1 gram of cefonicid given intramuscularly plus 1 gram of probenecid by mouth. Of 53 evaluable patients, 33 (62%) had penicillinase-producing Neisseria gonorrhoeae. All but one of these patients were cured. All men who had penicillin-sensitive infections were cured. Cefonicid was highly effective in the treatment of both penicillin-sensitive and penicillin-resistant N gonorrhoeae. Other than moderate pain at the site of injection, there were no adverse side effects. Cefonicid can be added to the group of newer cephalosporins that are effective in the treatment of gonococcal urethritis caused by either penicillin-sensitive or penicillin-resistant strains.     

Relatedness between Streptococcus pneumoniae and viridans streptococci: transfer of penicillin resistance determinants and immunological similarities of penicillin-binding proteins

The occurrence of highly variable penicillin-binding proteins (PBPs) in penicillin-resistant Streptococcus pneumoniae suggested that transfer of homologous genes from related species may be involved in resistance development. Antiserum and monoclonal antibodies raised against PBPs 1a and 2b from the susceptible S. pneumoniae R6 strain were used to identify related PBPs in 41 S. mitis, S. sanguis I and S. sanguis II strains mostly isolated in South Africa with MIC values ranging from less than 0.15 to 16 mg/ml. Furthermore, the possibility of genetic exchange was examined with 30 penicillin-resistant strains of this collection (MIC greater than 0.06 mg/ml) as donors using S. pneumoniae R6 as recipient in transformation experiments. The majority of S. mitis and S. sanguis II strains but none of the S. sanguis I strains could transform penicillin resistance genes into S. pneumoniae R6. All positive donor strains and all susceptible isolates of S. mitis and S. sanguis II strains contained PBPs which cross-reacted with the anti-PBP 1a and/or anti-PBP 2b antibodies. On the other hand, only five of the 14 S. sanguis I strains contained a PBP that reacted with one of the antibodies. This strongly suggested the presence of genes homologous to the pneumococcal PBP 1a and 2b genes in viridans streptococci, and documents that penicillin resistance determinants can be transformed from viridans streptococci into the pneumococcus.     

Cell wall branches, penicillin resistance and the secrets of the MurM protein

Production of low-affinity forms of penicillin-binding proteins (PBPs), although essential, is not sufficient to protect pneumococci against the inhibitory action of penicillin. Resistance also requires the newly identified protein MurM which, together with MurN, is involved with the synthesis of short peptide branches in the pneumococcal cell wall. Cells in which murM was inactivated produced cell walls without branches and also completely lost penicillin resistance. To understand these surprising observations a 3D-model of MurM was constructed, which helped to put into structural context several of the biochemical and genetic observations made about this protein.     

Identification of essential residues in Thermus thermophilus DNA ligase.

DNA ligases play a pivotal role in DNA replication, repair and recombination. Reactions catalyzed by DNA ligases consist of three steps: adenylation of the ligase in the presence of ATP or NAD+, transferring the adenylate moiety to the 5'-phosphate of the nicked DNA substrate (deadenylation) and sealing the nick through the formation of a phosphodiester bond. Thermus thermophilus HB8 DNA ligase (Tth DNA ligase) differs from mesophilic ATP-dependent DNA ligases in three ways: (i) it is NAD+ dependent; (ii) its optimal temperature is 65 instead of 37 degrees C; (iii) it has higher fidelity than T4 DNA ligase. In order to understand the structural basis underlying the reaction mechanism of Tth DNA ligase, we performed site-directed mutagenesis studies on nine selected amino acid residues that are highly conserved in bacterial DNA ligases. Examination of these site-specific mutants revealed that: residue K118 plays an essential role in the adenylation step; residue D120 may facilitate the deadenylation step; residues G339 and C433 may be involved in formation of the phosphodiester bond. This evidence indicates that a previously identified KXDG motif for adenylation of eukaryotic DNA ligases [Tomkinson, A.E., Totty, N.F., Ginsburg, M. and Lindahl, T. (1991) Proc. Natl. Acad. Sci. USA, 88, 400-404] is also the adenylation site for NAD+-dependent bacterial DNA ligases. In a companion paper, we demonstrate that mutations at a different Lys residue, K294, may modulate the fidelity of Tth DNA ligase.     

Zif,the Zoocin A Immunity Factor,Is a FemABX-Like Immunity Protein with a Novel Mode of Action

Producer cell immunity to the streptococcolytic enzyme zoocin A, which is a d-alanyl-l-alanine endopeptidase, is due to Zif, the zoocin A immunity factor. Zif has high degrees of similarity to MurM and MurN (members of the FemABX family of proteins), which are responsible for the addition of amino acids to cross bridges during peptidoglycan synthesis in streptococci. In this study, purified peptidoglycans from strains with and without zif were compared to determine how Zif modifies the peptidoglycan layer to cause resistance to zoocin A. The peptidoglycan from each strain was hydrolyzed using the streptococcolytic phage lysin B30, and the resulting muropeptides were separated by reverse-phase high-pressure liquid chromatography, labeled with 4-sulfophenyl isothiocyanate, and analyzed by tandem mass spectrometry in the negative-ion mode. It was determined that Zif alters the peptidoglycan by increasing the proportion of cross bridges containing three l-alanines instead of two. This modification decreased binding of the recombinant target recognition domain of zoocin A to peptidoglycan. Zif-modified peptidoglycan also was less susceptible to hydrolysis by the recombinant catalytic domain of zoocin A. Thus, Zif is a novel FemABX-like immunity factor because it provides resistance to a bacteriolytic endopeptidase by lengthening the peptidoglycan cross bridge rather than by causing an amino acid substitution.During streptococcal peptidoglycan synthesis, monomer subunits are generated inside the cell, with nonribosomal peptidyl transferases responsible for the addition of amino acids onto the epsilon amino group of lysine in the subunits. These nonribosomal peptidyl transferases are part of the FemABX family of proteins, some of which have been implicated in penicillin resistance (5, 26). In Streptococcus pneumoniae peptidoglycan synthesis, MurM attaches either an l-alanine or an l-serine to the epsilon amino group of lysine, and MurN then adds an l-alanine (11, 26).Zoocin A is a d-alanyl-l-alanine endopeptidase produced by Streptococcus equi subsp. zooepidemicus 4881 that hydrolyzes peptidoglycan cross bridges of susceptible streptococci (12). Zoocin A has two functional domains (18). The N-terminal catalytic domain (CAT) has high degrees of similarity to several other bacteriolytic endopeptidases, including the staphylolytic enzyme lysostaphin. The C-terminal target recognition domain (TRD), which facilitates binding of the enzyme to peptidoglycan (1), has very little similarity to any characterized conserved domain.Producer cell immunity to zoocin A is due to zif (zoocin A immunity factor), which is adjacent to zooA on the chromosome and is transcribed divergently (4). Zif has high degrees of similarity to MurM and MurN and also to the lysostaphin resistance protein and other FemABX-like immunity proteins (23). Previously characterized FemABX-like immunity proteins provide resistance to peptidoglycan cross-bridge hydrolases by inserting an amino acid different from those specified by the normal FemABX-like proteins (6, 9, 15, 25), whereas Zif does not (4). It has been shown previously that Zif-specified resistance to zoocin A is an intrinsic characteristic of the peptidoglycan layer (12). Therefore, Zif must modify the peptidoglycan layer in a novel way that provides resistance to zoocin A. In the present study, Zif was shown to insert an additional l-alanine into the peptidoglycan cross bridges, which inhibited both binding of the zoocin A TRD and the ability of the zoocin A CAT to hydrolyze the cross bridge.