Peptidoglycan Biosynthesis Enzymes Research Articles

Transcriptomic analysis of cell envelope inhibition by prodigiosin in methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading threat to public health as it is resistant to most currently available antibiotics. Prodigiosin is a secondary metabolite of microorganisms with broad-spectrum antibacterial activity. This study identified a significant antibacterial effect of prodigiosin against MRSA with a minimum inhibitory concentration as low as 2.5 mg/L. The results of scanning electron microscopy, crystal violet staining, and confocal laser scanning microscopy indicated that prodigiosin inhibited biofilm formation in S. aureus USA300, while also destroying the structure of the cell wall and cell membrane, which was confirmed by transmission electron microscopy. At a prodigiosin concentration of 1.25 mg/L, biofilm formation was inhibited by 76.24%, while 2.5 mg/L prodigiosin significantly reduced the vitality of MRSA cells in the biofilm. Furthermore, the transcriptomic results obtained at 1/8 MIC of prodigiosin indicated that 235and 387 genes of S. aureus USA300 were significantly up- and downregulated, respectively. The downregulated genes were related to two-component systems, including the transcriptional regulator LytS, quorum sensing histidine kinases SrrB, NreA and NreB, peptidoglycan biosynthesis enzymes (MurQ and GlmU), iron-sulfur cluster repair protein ScdA, microbial surface components recognizing adaptive matrix molecules, as well as the key arginine synthesis enzymes ArcC and ArgF. The upregulated genes were mainly related to cell wall biosynthesis, as well as two-component systems including vancomycin resistance-associated regulator, lipoteichoic acid biosynthesis related proteins DltD and DltB, as well as the 9 capsular polysaccharide biosynthesis proteins. This study elucidated the molecular mechanisms through which prodigiosin affects the cell envelope of MRSA from the perspectives of cell wall synthesis, cell membrane and biofilm formation, providing new potential targets for the development of antimicrobials for the treatment of MRSA.

Synthesis of an Antimicrobial Enterobactin-Muraymycin Conjugate for Improved Activity Against Gram-Negative Bacteria.

Overcoming increasing antibiotic resistance requires the development of novel antibacterial agents that address new targets in bacterial cells. Naturally occurring nucleoside antibiotics (such as muraymycins) inhibit the bacterial membrane protein MraY, a clinically unexploited essential enzyme in peptidoglycan (cell wall) biosynthesis. Even though a range of synthetic muraymycin analogues has already been reported, they generally suffer from limited cellular uptake and a lack of activity against Gram-negative bacteria. We herein report an approach to overcome these hurdles: a synthetic muraymycin analogue has been conjugated to a siderophore, i. e. the enterobactin derivative EntKL , to increase the cellular uptake into Gram-negative bacteria. The resultant conjugate showed significantly improved antibacterial activity against an efflux-deficient E. coli strain, thus providing a proof-of-concept of this novel approach and a starting point for the future optimisation of such conjugates towards potent agents against Gram-negative pathogens.

Covalent inhibitors of bacterial peptidoglycan biosynthesis enzyme MurA with chloroacetamide warhead

MurA (UDP-N-acetylglucosamine enolpyruvyl transferase) catalyzes the first committed step in the cytoplasmic part of peptidoglycan biosynthesis and is a validated target enzyme for antibacterial drug discovery; the inhibitor fosfomycin has been used clinically for decades. Like fosfomycin, most MurA inhibitors are small heterocyclic compounds that inhibit the enzyme by forming a covalent bond with the active site cysteine. The reactive chloroacetamide group was selected from a series of suitable electrophilic thiol-reactive warheads. The predominantly one-step synthesis led to the construction of the final library of 47 fragment-sized chloroacetamide compounds. Several new E. coli MurA inhibitors were identified, with the most potent compound having an IC50 value in the low micromolar range. The electrophilic reactivity of all chloroacetamide fragments in our library was evaluated by a high-throughput spectrophotometric assay using the reduced Ellman reagent as a surrogate for the cysteine thiol. LC-MS/MS experiments confirmed the covalent binding of the most potent inhibitor to Cys115 of the digested MurA enzyme. The covalent binding was further investigated by a biochemical time-dependent assay and a dilution assay, which confirmed the irreversible and time-dependent mode of action. The efficacy of chloroacetamide derivatives against MurA does not correlate with their thiol reactivity, making the active fragments valuable starting points for fragment-based development of new antibacterial agents targeting MurA.

A defect in cell wall recycling confers antibiotic resistance and sensitivity in Staphylococcus aureus

WalKR is a two-component system that is essential for viability in Gram-positive bacteria that regulates the all-important autolysins in cell wall homeostasis. Further investigation of this essential system is important for identifying ways to address antibiotic resistance. Here, we show that a T101M mutation in walR confers a defect in autolysis, a thickened cell wall, and decreased susceptibility to antibiotics that target lipid II cycle, a phenotype that is reminiscent of the clinical resistance form known as vancomycin intermediate-resistant Staphylococcus aureus. Importantly, this is accompanied by dramatic sensitization to tunicamycin. We demonstrate that this phenotype is due to partial collapse of a pathway consisting of autolysins, AtlA and Sle1, a transmembrane sugar permease, MurP, and GlcNAc recycling enzymes, MupG and MurQ. We suggest that this causes a shortage of substrate for the peptidoglycan biosynthesis enzyme MraY, causing it to be hypersensitive to competitive inhibition by tunicamycin. In conclusion, our results constitute a new molecular model for antibiotic sensitivity in S. aureus and a promising new route for antibiotic discovery.

Ventilation induced evolution pattern of archaea, fungi, bacteria and their potential roles during co-bioevaporation treatment of concentrated landfill leachate and food waste

To obtain a favorable aeration type in co-bioevaporation treatment of concentrated landfill leachate and food waste, and to deeply understand the co-bioevaporation mechanisms, the temporal evolution differences of archaea, fungi and bacteria as well as the related microbial metabolism genes and functional enzymes under intermittent ventilation (IV) and continuous ventilation (CV) were investigated. Results through metagenomics analysis showed that the less sufficient oxygen and longer thermophilic phase in IV stimulated the vigorous growth of archaea, while CV was beneficial for fungal growth. Even genes of carbohydrates and lipids metabolism and ATP-associated enzymes (enzyme 2.7.13.3 and 3.6.4.12), as well as peptidoglycan biosynthesis enzyme (enzyme 3.4.16.4), were more abundant in CV, IV hold better DNA repair ability, higher microbial viability, and less dehydrogenase sensitivity to temperatures due to the critical contribution of Pseudomonas (3.1–45.9%). Furthermore, IV consumed a similar amount of heat for water evaporation with nearly half of the ventilation of CV and was a favorable aeration type in the practical application of co-bioevaporation.

Inhibition of Campylobacter jejuni Biofilm Formation by D-Amino Acids.

The ability of bacterial pathogens to form biofilms is an important virulence mechanism in relation to their pathogenesis and transmission. Biofilms play a crucial role in survival in unfavorable environmental conditions, acting as reservoirs of microbial contamination and antibiotic resistance. For intestinal pathogen Campylobacter jejuni, biofilms are considered to be a contributing factor in transmission through the food chain and currently, there are no known methods for intervention. Here, we present an unconventional approach to reducing biofilm formation by C. jejuni by the application of D-amino acids (DAs), and L-amino acids (LAs). We found that DAs and not LAs, except L-alanine, reduced biofilm formation by up to 70%. The treatment of C. jejuni cells with DAs changed the biofilm architecture and reduced the appearance of amyloid-like fibrils. In addition, a mixture of DAs enhanced antimicrobial efficacy of D-Cycloserine (DCS) up to 32% as compared with DCS treatment alone. Unexpectedly, D-alanine was able to reverse the inhibitory effect of other DAs as well as that of DCS. Furthermore, L-alanine and D-tryptophan decreased transcript levels of peptidoglycan biosynthesis enzymes alanine racemase (alr) and D-alanine-D-alanine ligase (ddlA) while D-serine was only able to decrease the transcript levels of alr. Our findings suggest that a combination of DAs could reduce biofilm formation, viability and persistence of C. jejuni through dysregulation of alr and ddlA.

Study of the cwaRS-ldcA Operon Coding a Two-Component System and a Putative L,D-Carboxypeptidase in Lactobacillus paracasei.

The cell surface is the primary recognition site between the bacterium and the host. An operon of three genes, LSEI_0219 (cwaR), LSEI_0220 (cwaS), and LSEI_0221 (ldcA), has been previously identified as required for the establishment of Lactobacillus paracasei in the gut. The genes cwaR and cwaS encode a predicted two-component system (TCS) and ldcA a predicted D-alanyl-D-alanine carboxypeptidase which is a peptidoglycan (PG) biosynthesis enzyme. We explored the functionality and the physiological role of these three genes, particularly their impact on the bacterial cell wall architecture and on the bacterial adaptation to environmental perturbations in the gut. The functionality of CwaS/R proteins as a TCS has been demonstrated by biochemical analysis. It is involved in the transcriptional regulation of several genes of the PG biosynthesis. Analysis of the muropeptides of PG in mutants allowed us to re-annotate LSEI_0221 as a putative L,D-carboxypeptidase (LdcA). The absence of this protein coincided with a decrease of two surface antigens: LSEI_0020, corresponding to p40 or msp2 whose implication in the host epithelial homeostasis has been recently studied, and LSEI_2029 which has never been functionally characterized. The inactivation of each of these three genes induces susceptibility to antimicrobial peptides (hBD1, hBD2, and CCL20), which could be the main cause of the gut establishment deficiency. Thus, this operon is necessary for the presence of two surface antigens and for a suitable cell wall architecture.

Inhibition of D-Ala:D-Ala ligase through a phosphorylated form of the antibiotic D-cycloserine

D-cycloserine is an antibiotic which targets sequential bacterial cell wall peptidoglycan biosynthesis enzymes: alanine racemase and D-alanine:D-alanine ligase. By a combination of structural, chemical and mechanistic studies here we show that the inhibition of D-alanine:D-alanine ligase by the antibiotic D-cycloserine proceeds via a distinct phosphorylated form of the drug. This mechanistic insight reveals a bimodal mechanism of action for a single antibiotic on different enzyme targets and has significance for the design of future inhibitor molecules based on this chemical structure.

Molecular architecture of the PBP2\u2013MreC core bacterial cell wall synthesis complex

Bacterial cell wall biosynthesis is an essential process that requires the coordinated activity of peptidoglycan biosynthesis enzymes within multi-protein complexes involved in cell division (the “divisome”) and lateral wall growth (the “elongasome”). MreC is a structural protein that serves as a platform during wall elongation, scaffolding other essential peptidoglycan biosynthesis macromolecules, such as penicillin-binding proteins. Despite the importance of these multi-partite complexes, details of their architecture have remained elusive due to the transitory nature of their interactions. Here, we present the crystal structures of the soluble PBP2:MreC core elongasome complex from Helicobacter pylori, and of uncomplexed PBP2. PBP2 recognizes the two-winged MreC molecule upon opening of its N-terminal region, revealing a hydrophobic zipper that serves as binding platform. The PBP2:MreC interface is essential both for protein recognition in vitro and maintenance of bacterial shape and growth. This work allows visualization as to how peptidoglycan machinery proteins are scaffolded, revealing interaction regions that could be targeted by tailored inhibitors.

Single-Pixel Densitometry Revealed the Presence of Peptidoglycan in the Intermembrane Space of the Moss Chloroplast Envelope in Conventional Electron Micrographs.

Chloroplasts are believed to be descendants of ancestral cyanobacteria that have a peptidoglycan layer between the outer and the inner membranes. In particular, cyanelles having peptidoglycan in Cyanophora paradoxa are considered as evidence for the endosymbiotic origin of chloroplasts. The moss Physcomitrella patens has a complete set of genes involved in the synthesis of peptidoglycan, but a peptidoglycan layer has not been observed by conventional electron microscopy to date. Recently, a new metabolic labeling technique using a fluorescent probe was applied to visualize putative peptidoglycan surrounding the chloroplasts. The exact localization of the peptidoglycan, however, has not been clearly identified. Here we examined conventional electron micrographs of two types of moss materials (mutants or ampicillin-treated plants), one presumably having peptidoglycan and the other presumably lacking peptidoglycan, and analyzed in detail, by single-pixel densitometry, the electron density of the chloroplast envelope membranes and the intermembrane space. Statistical analysis showed that the relative electron density within the intermembrane space with respect to that of the envelope membranes was significantly higher in the materials presumably having peptidoglycan than in the materials presumably devoid of peptidoglycan. We consider this difference as bona fide evidence for the presence of peptidoglycan between the outer and the inner envelope membranes in the wild-type chloroplasts of the moss, although its density is lower than that in bacteria and cyanelles. We will also discuss this low-density peptidoglycan in the light of the phylogenetic origin of peptidoglycan biosynthesis enzymes.

Insights into the function of FhaA, a cell division-associated protein in mycobacteria.

FhaA is a forkhead-associated domain-containing protein, the depletion of which leads to accumulation of peptidoglycan (PG) precursors at the septum and poles in Mycobacterium smegmatis (M. smegmatis), by a mechanism undefined thus far. To elucidate its function, we constructed an fhaA (MSMEG_0035) knockout (ΔfhaA) strain in M. smegmatis and demonstrated that this gene is dispensable for in vitro growth. The mutant showed a short cell length phenotype due to a probable defect in cell elongation/cell wall synthesis, which was reversed by complementation with both M. smegmatis and Mycobacterium tuberculosis (M. tb) fhaA (Rv0020c), confirming their association with the observed phenotype. The identification of penicillin binding protein A (PbpA), a PG biosynthesis enzyme as an interacting partner for mycobacterial FhaA, provided a hint into the functioning of FhaA. A drastic reduction in the levels of ectopically expressed PbpA in the ΔfhaA mutant vs wild-type M. smegmatis suggested that FhaA interacts with and stabilises PbpA. In addition, the fhaA deletion mutant was sensitive to multiple classes of antibiotics pointing to a general permeability defect. Our findings uncover a role for FhaA in PG biosynthesis and suggest its involvement in the maintenance of mycobacterial cell envelope integrity.

Total Synthesis of Dansylated Park's Nucleotide for High-Throughput MraY Assays.

The membrane protein translocase I (MraY) is a key enzyme in bacterial peptidoglycan biosynthesis. It is therefore frequently discussed as a target for the development of novel antibiotics. The screening of compound libraries for the identification of MraY inhibitors is enabled by an established fluorescence-based MraY assay. However, this assay requires a dansylated derivative of the bacterial biosynthetic intermediate Park's nucleotide as the MraY substrate. Isolation of Park's nucleotide from bacteria and subsequent dansylation only furnishes limited amounts of this substrate, thus hampering the high-throughput screening for MraY inhibitors. Accordingly, the efficient provision of dansylated Park's nucleotide is a major bottleneck in the exploration of this promising drug target. In this work, we present the first total synthesis of dansylated Park's nucleotide, affording an unprecedented amount of the target compound for high-throughput MraY assays.

Evidence-based green algal genomics reveals marine diversity and ancestral characteristics of land plants.

BackgroundPrasinophytes are widespread marine green algae that are related to plants. Cellular abundance of the prasinophyte Micromonas has reportedly increased in the Arctic due to climate-induced changes. Thus, studies of these unicellular eukaryotes are important for marine ecology and for understanding Viridiplantae evolution and diversification.ResultsWe generated evidence-based Micromonas gene models using proteomics and RNA-Seq to improve prasinophyte genomic resources. First, sequences of four chromosomes in the 22 Mb Micromonas pusilla (CCMP1545) genome were finished. Comparison with the finished 21 Mb genome of Micromonas commoda (RCC299; named herein) shows they share ≤8,141 of ~10,000 protein-encoding genes, depending on the analysis method. Unlike RCC299 and other sequenced eukaryotes, CCMP1545 has two abundant repetitive intron types and a high percent (26 %) GC splice donors. Micromonas has more genus-specific protein families (19 %) than other genome sequenced prasinophytes (11 %). Comparative analyses using predicted proteomes from other prasinophytes reveal proteins likely related to scale formation and ancestral photosynthesis. Our studies also indicate that peptidoglycan (PG) biosynthesis enzymes have been lost in multiple independent events in select prasinophytes and plants. However, CCMP1545, polar Micromonas CCMP2099 and prasinophytes from other classes retain the entire PG pathway, like moss and glaucophyte algae. Surprisingly, multiple vascular plants also have the PG pathway, except the Penicillin-Binding Protein, and share a unique bi-domain protein potentially associated with the pathway. Alongside Micromonas experiments using antibiotics that halt bacterial PG biosynthesis, the findings highlight unrecognized phylogenetic complexity in PG-pathway retention and implicate a role in chloroplast structure or division in several extant Viridiplantae lineages.ConclusionsExtensive differences in gene loss and architecture between related prasinophytes underscore their divergence. PG biosynthesis genes from the cyanobacterial endosymbiont that became the plastid, have been selectively retained in multiple plants and algae, implying a biological function. Our studies provide robust genomic resources for emerging model algae, advancing knowledge of marine phytoplankton and plant evolution.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2585-6) contains supplementary material, which is available to authorized users.

Inactivation of glutamate racemase (MurI) eliminates virulence in Streptococcus mutans

Inhibition of enzymes required for bacterial cell wall synthesis is often lethal or leads to virulence defects. Glutamate racemase (MurI), an essential enzyme in peptidoglycan biosynthesis, has been an attractive target for therapeutic interventions. Streptococcus mutans, one of the many etiological factors of dental caries, possesses a series of virulence factors associated with cariogenicity. However, little is known regarding the mechanism by which MurI influences pathogenesis of S. mutans. In this work, a stable mutant of S. mutans deficient in glutamate racemase (S. mutans FW1718) was constructed to investigate the impact of murI inactivation on cariogenic virulence in S. mutans UA159. Microscopy revealed that the murI mutant exhibited an enlarged cell size, longer cell chains, diminished cell⬜cell aggregation, and altered cell surface ultrastructure compared with the wild-type. Characterization of this mutant revealed that murI deficiency weakened acidogenicity, aciduricity, and biofilm formation ability of S. mutans (P<0.05). Real-time quantitative polymerase chain reaction (qRT-PCR) analysis demonstrated that the deletion of murI reduced the expression of the acidogenesis-related gene ldh by 44-fold (P<0.0001). The expression levels of the gene coding for surface protein antigen P (spaP) and the acid-tolerance related gene (atpD) were down-regulated by 99% (P<0.0001). Expression of comE, comD, gtfB and gtfC, genes related to biofilm formation, were down-regulated 8-, 43-, 85- and 298-fold in the murI mutant compared with the wild-type (P<0.0001), respectively. Taken together, the current study provides the first evidence that MurI deficiency adversely affects S. mutans virulence properties, making MurI a potential target for controlling dental caries.

Cyclic-di-AMP synthesis by the diadenylate cyclase CdaA is modulated by the peptidoglycan biosynthesis enzyme GlmM in Lactococcus lactis.

The second messenger cyclic-di-adenosine monophosphate (c-di-AMP) plays important roles in growth, virulence, cell wall homeostasis, potassium transport and affects resistance to antibiotics, heat and osmotic stress. Most Firmicutes contain only one c-di-AMP synthesizing diadenylate cyclase (CdaA); however, little is known about signals and effectors controlling CdaA activity and c-di-AMP levels. In this study, a genetic screen was employed to identify components which affect the c-di-AMP level in Lactococcus. We characterized suppressor mutations that restored osmoresistance to spontaneous c-di-AMP phosphodiesterase gdpP mutants, which contain high c-di-AMP levels. Loss-of-function and gain-of-function mutations were identified in the cdaA and gdpP genes, respectively, which led to lower c-di-AMP levels. A mutation was also identified in the phosphoglucosamine mutase gene glmM, which is commonly located within the cdaA operon in bacteria. The glmM I154F mutation resulted in a lowering of the c-di-AMP level and a reduction in the key peptidoglycan precursor UDP-N-acetylglucosamine in L. lactis. C-di-AMP synthesis by CdaA was shown to be inhibited by GlmM(I154F) more than GlmM and GlmM(I154F) was found to bind more strongly to CdaA than GlmM. These findings identify GlmM as a c-di-AMP level modulating protein and provide a direct connection between c-di-AMP synthesis and peptidoglycan biosynthesis.

Inhibitors of the peptidoglycan biosynthesis enzymes MurA-F

The widespread emergence of resistant bacterial strains is becoming a serious threat to public health. This thus signifies the need for the development of new antibacterial agents with novel mechanisms of action. Continuous efforts in the design of novel antibacterials remain one of the biggest challenges in drug development. In this respect, the Mur enzymes, MurA-F, that are involved in the formation of UDP-N-acetylmuramyl-pentapeptide can be genuinely considered as promising antibacterial targets. This review provides an in-depth insight into the recent developments in the field of inhibitors of the MurA-F enzymes. Special attention is also given to compounds that act as multiple inhibitors of two, three or more of the Mur enzymes. Moreover, the reasons for the lack of preclinically successful inhibitors and the challenges to overcome these hurdles in the next years are also debated.

Structure–activity relationships of new cyanothiophene inhibitors of the essential peptidoglycan biosynthesis enzyme MurF

Peptidoglycan is an essential component of the bacterial cell wall, and enzymes involved in its biosynthesis represent validated targets for antibacterial drug discovery. MurF catalyzes the final intracellular peptidoglycan biosynthesis step: the addition of d-Ala-d-Ala to the nucleotide precursor UDP-MurNAc-l-Ala-γ-d-Glu-meso-DAP (or l-Lys). As MurF has no human counterpart, it represents an attractive target for the development of new antibacterial drugs. Using recently published cyanothiophene inhibitors of MurF from Streptococcus pneumoniae as a starting point, we designed and synthesized a series of structurally related derivatives and investigated their inhibition of MurF enzymes from different bacterial species. Systematic structural modifications of the parent compounds resulted in a series of nanomolar inhibitors of MurF from S. pneumoniae and micromolar inhibitors of MurF from Escherichia coli and Staphylococcus aureus. Some of the inhibitors also show antibacterial activity against S. pneumoniae R6. These findings, together with two new co-crystal structures, represent an excellent starting point for further optimization toward effective novel antibacterials.

Alanine Scan of the Peptide Antibiotic Feglymycin: Assessment of Amino Acid Side Chains Contributing to Antimicrobial Activity

The antibiotic feglymycin is a linear 13-mer peptide synthesized by the bacterium Streptomyces sp. DSM 11171. It mainly consists of the nonproteinogenic amino acids 4-hydroxyphenylglycine and 3,5-dihydroxyphenylglycine. An alanine scan of feglymycin was performed by solution-phase peptide synthesis in order to assess the significance of individual amino acid side chains for biological activity. Hence, 13 peptides were synthesized from di- and tripeptide building blocks, and subsequently tested for antibacterial activity against Staphylococcus aureus strains. Furthermore we tested the inhibition of peptidoglycan biosynthesis enzymes MurA and MurC, which are inhibited by feglymycin. Whereas the antibacterial activity is significantly based on the three amino acids D-Hpg1, L-Hpg5, and L-Phe12, the inhibitory activity against MurA and MurC depends mainly on L-Asp13. The difference in the position dependence for antibacterial activity and enzyme inhibition suggests multiple molecular targets in the modes of action of feglymycin.

Chemical, biochemical and microbiological properties of a brominated nitrovinylfuran with broad-spectrum antibacterial activity

A di-bromo substituted nitrovinylfuran with reported broad-spectrum antibacterial activity was found to be a potent inhibitor of MurA, a key enzyme in peptidoglycan biosynthesis. Further characterization of the compound was carried out to assess its reactivity towards thiol nucleophiles, its stability and degradation under aqueous conditions, inhibitory potential at other enzymes, and antibacterial and cytotoxic activity. Our results indicate that the nitrovinylfuran derivative is reactive towards cysteine residues in proteins, as demonstrated by the irreversible inhibition of MurA and bacterial methionine aminopeptidase. Experiments with proteins and model thiols indicate that the compound forms covalent adducts with SH groups and induces intermolecular disulfide bonds, with the intermediate formation of a monobromide derivative. The parent molecule as well as most of its breakdown products are potent antibiotics with MIC values below 4μg/mL and are active against multiresistant strains such as methicillin-resistant Staphylococcus aureus (MRSA). Further development of the bromonitrovinyl scaffold towards antibiotics with clinical relevance, however, requires optimization of the antibiotic–cytotoxic selectivity profile.

Calcium-Dependent Complex Formation Between PBP2 and Lytic Transglycosylase SltB1 ofPseudomonas aeruginosa

In Gram-negative bacteria, the bacterial cell wall biosynthetic mechanism requires the coordinated action of enzymes and structural proteins located in the cytoplasm, within the membrane, and in the periplasm of the cell. Its main component, peptidoglycan (PG), is essential for cell division and wall elongation. Penicillin-binding proteins (PBPs) catalyze the last steps of PG biosynthesis, namely the polymerization of glycan chains and the cross-linking of stem peptides, and can be either monofunctional or bifunctional. Their action is coordinated with that of other enzymes essential for cell-wall biosynthesis, such as lytic transglycosylases (LT). Here, we have studied SltB1, an LT from Pseudomonas aeruginosa, and identified that it forms a complex with PBP2, a monofunctional enzyme, which requires the presence of Ca(2+). In addition, we have solved the structure of SltB1 to a high resolution, and identified that it harbors an EF-hand like motif containing a Ca(2+) ion displaying bipyramidal coordination. These studies provide initial structural details that shed light on the interactions between the PG biosynthesis enzymes in P. aeruginosa.