Peptidoglycan Biosynthesis Research Articles

The peptidoglycan synthase PBP interacts with PLASTID DIVISION2 to promote chloroplast division in Physcomitrium patens.

The peptidoglycan (PG) layer, a core component of the bacterial cell wall, has been retained in the Physcomitrium patens chloroplasts. The PG layer entirely encompasses the P. patens chloroplast, including the division site, but how PG biosynthesis cooperates with the constriction of two envelope membranes at the chloroplast division site remains elusive. Here, focusing on the PG synthase penicillin-binding protein (PBP), we performed cytological and molecular analyses to dissect the mechanism of chloroplast division in P. patens. We showed that PBP, acting in the final step of PG biosynthesis, is likely a chloroplast inner envelope protein that can aggregate at mid-chloroplasts during chloroplast division. Physcomitrium patens had five orthologs of PLASTID DIVISION2 (PDV2), an outer envelope component of the chloroplast division complex. Our data indicated that PpPDV2 proteins interact with PpPBP and are responsible for recruiting PpPBP to the chloroplast division site, in addition to PpDRP5B. Furthermore, we found that PBP deletion and carbenicillin application restrain constriction of the chloroplast division complex, rather than its assembly. This work provides direct molecular evidence for a link between chloroplast division of P. patens and PG biosynthesis and indicates that PG biosynthesis is required for the constriction of the chloroplast division apparatus in P. patens.

In-silico Screening of Novel Diterpene Derivatives for their Inhibitory Potentials against MurA Enzyme of Uropathogenic Escherichia coli

background: Enzyme UDP-NAG enolpyruvyl transferase (MurA), which aids in the formation of cell walls and is identified as the primary target of the antibiotic Fosfomycin, is a significant pharmacological target in the case of uropathogenic Escherichia coli (UPEC). Finding a potent drug with a distinct mode of action is required since UPEC treatment is challenging due to resistance and resurrection. methods: Here we have used virtual high-throughput screening and molecular dynamics simulation approach to explore the effectiveness of selected novel Diterpene derivatives against the therapeutic target MurA (both wild type and Cys115Asp mutant, which is responsible for lower effectiveness of Fosfomycin). results: The investigation showed improved binding efficacy of the ligands around the primary active site residue Cys115 as well as enhanced activity against Cys115Asp mutant. Against wildtype receptors, the docking score of best-docked diterpene derivative, DIT1 (-26.18) was found to be much better than that of the known drug, Fosfomycin (-23.72). There were 7 nos of hydrogen bonds formed by DIT1 and 5 nos by Fosfomycin, indicating a stronger interaction of DIT1 with MurA. The mutant form also showed similar findings, suggesting that DIT1 is much more effective than Fosfomycin. conclusion: The above result justifies the potential of Diterpene derivatives in blocking the MurA active site that can prevent peptidoglycan biosynthesis. Effectiveness of DIT1 against wild type as well as Cys115Asp mutant justifies that the selected ligands can be used as suitable drug candidates against MurA to add new molecules in the treatment pipeline. This establishes innovative frameworks for the creation of strong drugs that target MurA after suitable in-vitro and in-vivo trials.

Inhibition of MurA Enzyme from Escherichia coli by Flavonoids and Their Synthetic Analogues.

MurA catalyzes the first step of peptidoglycan (PG) biosynthesis and is a validated target for the development of new antimicrobial agents. In this study, a library of 49 plant flavonoids and their synthetic derivatives were evaluated for their inhibitory properties against MurA fromEscherichia coli. The compounds were tested with and without preincubation and with the addition of DTT to understand the mechanism of inhibition. Thirteen compounds were identified as reversible, time-dependent inhibitors, with inhibition levels ranging from 480 nM to 57 μM, and ampelopsin as the most potent compound. To investigate the major pharmacophore elements responsible for the activity, 2D-QSAR and docking analyzes were performed. The results showed that the catechol moiety and an additional aromatic system were the most important features contributing to the activity of the compounds. However, most of the compounds did not show antibacterial activity againstE. coli andStaphylococcus aureusstrains, suggesting that their inhibitory activity against MurA may not be strong enough to induce antibacterial effects. Nevertheless, our results suggest that flavonoids are a promising starting point to develop new inhibitors of MurA and show great potential for further steps in drug development.

Amidation of glutamate residues in mycobacterial peptidoglycan is essential for cell wall cross-linking.

Mycobacteria assemble a complex cell wall with cross-linked peptidoglycan (PG) which plays an essential role in maintenance of cell wall integrity and tolerance to osmotic pressure. We previously demonstrated that various hydrolytic enzymes are required to remodel PG during essential processes such as cell elongation and septal hydrolysis. Here, we explore the chemistry associated with PG cross-linking, specifically the requirement for amidation of the D-glutamate residue found in PG precursors. Synthetic fluorescent probes were used to assess PG remodelling dynamics in live bacteria. Fluorescence microscopy was used to assess protein localization in live bacteria and CRISPR-interference was used to construct targeted gene knockdown strains. Time-lapse microscopy was used to assess bacterial growth. Western blotting was used to assess protein phosphorylation. In Mycobacterium smegmatis, we confirmed the essentiality for D-glutamate amidation in PG biosynthesis by labelling cells with synthetic fluorescent PG probes carrying amidation modifications. We also used CRISPRi targeted knockdown of genes encoding the MurT-GatD complex, previously implicated in D-glutamate amidation, and demonstrated that these genes are essential for mycobacterial growth. We show that MurT-rseGFP co-localizes with mRFP-GatD at the cell poles and septum, which are the sites of cell wall synthesis in mycobacteria. Furthermore, time-lapse microscopic analysis of MurT-rseGFP localization, in fluorescent D-amino acid (FDAA)-labelled mycobacterial cells during growth, demonstrated co-localization with maturing PG, suggestive of a role for PG amidation during PG remodelling and repair. Depletion of MurT and GatD caused reduced PG cross-linking and increased sensitivity to lysozyme and β-lactam antibiotics. Cell growth inhibition was found to be the result of a shutdown of PG biosynthesis mediated by the serine/threonine protein kinase B (PknB) which senses uncross-linked PG. Collectively, these data demonstrate the essentiality of D-glutamate amidation in mycobacterial PG precursors and highlight the MurT-GatD complex as a novel drug target.

Structural basis of peptidoglycan synthesis by E. coli RodA-PBP2 complex

Peptidoglycan (PG) is an essential structural component of the bacterial cell wall that is synthetized during cell division and elongation. PG forms an extracellular polymer crucial for cellular viability, the synthesis of which is the target of many antibiotics. PG assembly requires a glycosyltransferase (GT) to generate a glycan polymer using a Lipid II substrate, which is then crosslinked to the existing PG via a transpeptidase (TP) reaction. A Shape, Elongation, Division and Sporulation (SEDS) GT enzyme and a Class B Penicillin Binding Protein (PBP) form the core of the multi-protein complex required for PG assembly. Here we used single particle cryo-electron microscopy to determine the structure of a cell elongation-specific E. coli RodA-PBP2 complex. We combine this information with biochemical, genetic, spectroscopic, and computational analyses to identify the Lipid II binding sites and propose a mechanism for Lipid II polymerization. Our data suggest a hypothesis for the movement of the glycan strand from the Lipid II polymerization site of RodA towards the TP site of PBP2, functionally linking these two central enzymatic activities required for cell wall peptidoglycan biosynthesis.

Mode of action of antimicrobial agents albofungins in eradicating penicillin- and cephalosporin-resistant Vibrio parahaemolyticus biofilm.

Albofungin is a promising broad-spectrum antimicrobial compound against multidrug-resistant bacteria. In the present study, we further investigated albofungin's biofilm eradication activity and its potential mode of action against drug-resistant Vibrio parahaemolyticus. Among all derivatives, albofungin exhibited the best antibiofilm and antibacterial activity with rapid killing effects at 0.12 µg mL-1. Confocal microscopy observation exhibited that albofungin disrupted V. parahaemolyticus biofilms by killing or dispersing biofilm cells. Meanwhile, scanning electron microscope and fluorescent staining experiments demonstrated that albofungin rapidly destroyed the integrity and permeability of the bacterial cell membrane. Moreover, this study revealed an antibiofilm mechanism of albofungin involving inhibition of peptidoglycan biosynthesis, flagella assembly pathways, and secretion system proteins in V. parahaemolyticus by quantitative proteomics and validation experiments. Our results highlighted albofungin's mechanism of action in planktonic cells and biofilms and suggested further development and potential applications of albofungin for treating infections caused by penicillins-and-cephalosporins-resistant V. parahaemolyticus. IMPORTANCE Infections caused by multidrug-resistant bacteria, as well as a scarcity of new antibiotics, have become a major health threat worldwide. To tackle the demand for new and effective treatments, we investigated the mechanism of action of albofungin, a natural product derived from Streptomyces, which exhibits potent antimicrobial activity against multidrug-resistant bacteria. Albofungin showed potent biofilm eradication activity against penicillins-and-cephalosporins-resistant Vibrio parahaemolyticus, which expresses a novel metallo-β-lactamase and, thus, reduces their sensitivity to various antibiotics. We observed membrane disruption and permeation mechanisms in planktonic cells and biofilms after albofungin treatment, while albofungin had a weak interaction with bacterial DNA. Moreover, the antibiofilm mechanism of albofungin included inhibition of peptidoglycan biosynthesis, flagellar assembly pathways, and secretion system proteins. Our finding suggested potential applications of albofungin as an antibacterial and antibiofilm therapeutic agent.

Novel expression system based on enhanced permeability of Vibrio natriegens cells induced by D,D- carboxypeptidase overexpression

Vibrio natriegens is a fast-growing, non-pathogenic marine bacterium with promising features for biotechnological applications such as high-level recombinant protein production or fast DNA propagation. A remarkable short generation time (< 10 min), robust proteosynthetic activity and versatile metabolism with abilities to utilise wide range of substrates contribute to its establishment as a future industrial platform for fermentation processes operating with high productivity.D,D-carboxypeptidases are membrane-associated enzymes involved in peptidoglycan biosynthesis and cell wall formation. This study investigates the impact of overexpressed D,D-carboxypeptidases on membrane integrity and the increased leakage of intracellular proteins into the growth medium in V. natriegens. Our findings confirm that co-expression of these enzymes can enhance membrane permeability, thereby facilitating the transport of target proteins into the extracellular environment, without the need for secretion signals, tags, or additional permeabilization methods. Using only a single step IMAC chromatography, we were able to purify AfKatG, MDBP or Taq polymerase in total yields of 117.9 ± 56.0 mg/L, 36.5 ± 12.9 mg/L and 26.5 ± 6.0 mg/L directly from growth medium, respectively. These results demonstrate the feasibility of our V. natriegens based system as a broadly applicable extracellular tag-less recombinant protein producer.

The effects of daptomycin on cell wall biosynthesis in Enterococcal faecalis

Daptomycin is a cyclic lipodepsipeptide antibiotic reserved for the treatment of serious infections by multidrug-resistant Gram-positive pathogens. Its mode of action is considered to be multifaceted, encompassing the targeting and depolarization of bacterial cell membranes, alongside the inhibition of cell wall biosynthesis. To characterize the daptomycin mode of action, 15N cross-polarization at magic-angle spinning NMR measurements were performed on intact whole cells of Staphylococcus aureus grown in the presence of a sub-inhibitory concentration of daptomycin in a chemically defined media containing l-[ϵ-15N]Lys. Daptomycin-treated cells showed a reduction in the lysyl-ε-amide intensity that was consistent with cell wall thinning. However, the reduced lysyl-ε-amine intensity at 10 ppm indicated that the daptomycin-treated cells did not accumulate in Park’s nucleotide, the cytoplasmic peptidoglycan (PG) precursor. Consequently, daptomycin did not inhibit the transglycosylation step of PG biosynthesis. To further elucidate the daptomycin mode of action, the PG composition of daptomycin-susceptible Enterococcus faecalis grown in the presence of daptomycin was analyzed using liquid chromatography-mass spectrometry. Sixty-nine muropeptide ions correspond to PG with varying degrees of modifications including crosslinking, acetylation, alanylation, and 1,6-anhydrous ring formation at MurNAc were quantified. Analysis showed that the cell walls of daptomycin-treated E. faecalis had a significant reduction in PG crosslinking which was accompanied by an increase in lytic transglycosylase activities and a decrease in PG-stem modifications by the carboxypeptidases. The changes in PG composition suggest that daptomycin inhibits cell wall biosynthesis by impeding the incorporation of nascent PG into the cell walls by transpeptidases and maturation by carboxypeptidases. As a result, the newly formed cell walls become highly susceptible to degradation by the autolysins, resulting in thinning of the cell wall.

Targeting the binding pocket of the fluorophore 8-anilinonaphthalene-1-sulfonic acid in the bacterial enzyme MurA.

8-Anilinonaphthalene-1-sulfonic acid (ANS) has been extensively used as a fluorescent probe to detect conformational changes of proteins. It has been cocrystallized with several of the proteins it is used to monitor, including the bacterial cell wall synthesis enzyme MurA. MurA catalyzes the first committed step of peptidoglycan biosynthesis, converting UDP-N-acetylglucosamine (UDP-GlcNAc) into enolpyruvyl UDP-GlcNAc. It has been reported before that ANS binds to MurA from Enterobacter cloacae without inhibiting the enzyme's activity up to a concentration of 1 mM ANS. In this study, we present evidence that ANS inhibits the activity of several isoforms of MurA with IC50 values of 18, 22, and 31 µM against wild-type Escherichia coli, C115D E. coli, and E. cloacae MurA, respectively. This prompted us to test a larger series of structural analogs of ANS for the inhibition of these MurA enzymes, which led to the discovery of compound 26. This ANS analog showed enhanced inhibition of MurA (WT and C115D MurA from E. coli, and E. cloacae MurA) with IC50 values of 2.7, 10, and 14 µM, respectively. Based on our results, the ANS binding pocket was identified as a novel target site for the development of potential antibiotics.

The mechanism of the phage-encoded protein antibiotic from ΦX174.

The historically important phage ΦX174 kills its host bacteria by encoding a 91-residue protein antibiotic called protein E. Using single-particle electron cryo-microscopy, we demonstrate that protein E bridges two bacterial proteins to form the transmembrane YES complex [MraY, protein E, sensitivity to lysis D (SlyD)]. Protein E inhibits peptidoglycan biosynthesis by obstructing the MraY active site leading to loss of lipid I production. We experimentally validate this result for two different viral species, providing a clear model for bacterial lysis and unifying previous experimental data. Additionally, we characterize the Escherichia coli MraY structure-revealing features of this essential enzyme-and the structure of the chaperone SlyD bound to a protein. Our structures provide insights into the mechanism of phage-mediated lysis and for structure-based design of phage therapeutics.

Transcriptional activation of ompA in Neisseria gonorrhoeae mediated by the XRE family member protein NceR.

Increasing antibiotic resistance of Neisseria gonorrhoeae, the causative agent of gonorrhea, is a growing global concern that has renewed vaccine development efforts. The gonococcal OmpA protein was previously identified as a vaccine candidate due to its surface exposure, conservation, stable expression, and involvement in host-cell interactions. We previously demonstrated that the transcription of ompA can be activated by the MisR/MisS two-component system. Interestingly, earlier work suggested that the availability of free iron also influences ompA expression, which we confirmed in this study. In the present study, we found that iron regulation of ompA was independent of MisR and searched for additional regulators. A DNA pull-down assay with the ompA promoter from gonococcal lysates obtained from bacteria grown in the presence or absence of iron identified an XRE (Xenobiotic Response Element) family member protein encoded by NGO1982. We found that an NGO1982 null mutant of N. gonorrhoeae strain FA19 displayed a reduced level of ompA expression compared to the wild-type (WT) parent strain. Given this regulation, and the capacity of this XRE-like protein to regulate a gene involved in peptidoglycan biosynthesis (ltgA), along with its presence in other Neisseria sp., we termed the NGO1982-encoded protein as NceR (Neisseria cell envelope regulator). Critically, results from DNA-binding studies indicated that NceR regulates ompA through a direct mechanism. Thus, ompA expression is subject to both iron-dependent (NceR) and -independent (MisR/MisS) pathways. Hence, levels of the vaccine antigen candidate OmpA in circulating gonococcal strains could be influenced by transcriptional regulatory systems and the availability of iron. IMPORTANCE Herein, we report that the gene encoding a conserved gonococcal surface-exposed vaccine candidate (OmpA) is activated by a heretofore undescribed XRE family transcription factor, which we term NceR. We report that NceR regulation of ompA expression in N. gonorrhoeae is mediated by an iron-dependent mechanism, while the previously described MisR regulatory system is iron-independent. Our study highlights the importance of defining the complexity of coordinated genetic and physiologic systems that regulate genes encoding vaccine candidates to better understand their availability during infection.

Unlocking the Secrets of Streptococcus suis: A peptidomics comparison of virulent and non-virulent serotypes 2, 14, 18, and 19.

Streptococcus suis (S. suis) is an important bacterial pathogen, that causes serious infections in humans and pigs. Although numerous virulence factors have been proposed, their particular role in pathogenesis is still inconclusive. The current study explored putative peptides responsible for the virulence of S. suis serotype 2 (SS2). Thus, the peptidome of highly virulent SS2, less prevalent SS14, and rarely reported serotypes SS18 and SS19 were comparatively analyzed using a high-performance liquid chromatography-mass spectrometry method (LC-MS/MS). Six serotype-specific peptides, 2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N-acetyltransferase (DapH), alanine racemase (Alr), CCA-adding enzyme (CCA), peptide chain release factor 3 (RF3), ATP synthase subunit delta (F0F1-ATPases) and aspartate carbamoyltransferase (ATCase), were expressed moderately to highly only in the SS2 peptidome with p-values of less than 0.05. Some of these proteins are responsible for bacterial cellular stability; especially, Alr was highly expressed in the SS2 peptidome and is associated with peptidoglycan biosynthesis and bacterial cell wall formation. This study indicated that these serotype-specific peptides, which were significantly expressed by virulent SS2, could serve as putative virulence factors to promote its competitiveness with other coexistences in a particular condition. Further in vivo studies of these peptides should be performed to confirm the virulence roles of these identified peptides.

Deconvolution of multichannel LC-MS/MS chromatograms of glucosamine-phosphates: Evidence of a GlmS regulatory difference between Staphylococcus aureus and Enterococcus faecium

Resolving isomeric analytes is challenging given their physical similarity – making chromatographic resolution difficult, and their identical masses – making simple mass resolution impossible. MS/MS data provides a means to resolve isomeric analytes if their MS/MS intensity profiles are sufficiently different. Glucosamine-6-phosphate (GlcN-6P) and glucosamine-1-phosphate (GlcN-1P) are early bacterial cell wall intermediates. These and other isomeric hexosamine-phosphates are highly polar and unretained on reverse-phase chromatography media. Three commercially available hexosamine-phosphate standards (GlcN-6P, GlcN-1P, and GalN-1P) were derivatized with octanoic anhydride, and chromatographic conditions were established to resolve these analytes on C18 columns. GlcN-1P and GalN-1P overlapped chromatographically under all tested chromatography conditions. Three MS/MS fragments (79, 97, and 199 m/z) were common to all three commercially available hexosamine-phosphates. Intensity ratios of the three MS/MS fragments from these three hexosamine-phosphate standards were used to deconvolute mixture chromatograms of these standards by non-negative linear regression. This approach allowed the complete resolution of these analytes. The chromatographically overlapping GlcN-1P and GalN-1P, which shared similar but modestly different MS/MS intensity profiles, were fully resolved with this non-negative deconvolution approach. This approach was then applied to MRSA, VSE, and VRE bacterial extracts before and after exposure to vancomycin. This demonstrated a substantial (3-fold) increase in GlcN-6P in vancomycin-treated MRSA samples but not in vancomycin-treated VSE or VRE samples. These observations appear to localize previously observed differences between MRSA and VRE/VSE peptidoglycan biosynthesis regulation to GlmS, which synthesizes GlcN-6P and is the product of a regulatory ribozyme sensitive to the levels of GlcN-6P

The role of CenKR in the coordination of Rhodobacter sphaeroides cell elongation and division.

Cell elongation and division are essential aspects of the bacterial life cycle that must be coordinated for viability and replication. The impact of misregulation of these processes is not well understood as these systems are often not amenable to traditional genetic manipulation. Recently, we reported on the CenKR two-component system (TCS) in the Gram-negative bacterium Rhodobacter sphaeroides that is genetically tractable, widely conserved in α-proteobacteria, and directly regulates the expression of components crucial for cell elongation and division, including genes encoding subunit of the Tol-Pal complex. In this work, we show that overexpression of cenK results in cell filamentation and chaining. Using cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET), we generated high-resolution two-dimensional (2D) images and three-dimensional (3D) volumes of the cell envelope and division septum of wild-type cells and a cenK overexpression strain finding that these morphological changes stem from defects in outer membrane (OM) and peptidoglycan (PG) constriction. By monitoring the localization of Pal, PG biosynthesis, and the bacterial cytoskeletal proteins MreB and FtsZ, we developed a model for how increased CenKR activity leads to changes in cell elongation and division. This model predicts that increased CenKR activity decreases the mobility of Pal, delaying OM constriction, and ultimately disrupting the midcell positioning of MreB and FtsZ and interfering with the spatial regulation of PG synthesis and remodeling.IMPORTANCEBy coordinating cell elongation and division, bacteria maintain their shape, support critical envelope functions, and orchestrate division. Regulatory and assembly systems have been implicated in these processes in some well-studied Gram-negative bacteria. However, we lack information on these processes and their conservation across the bacterial phylogeny. In R. sphaeroides and other α-proteobacteria, CenKR is an essential two-component system (TCS) that regulates the expression of genes known or predicted to function in cell envelope biosynthesis, elongation, and/or division. Here, we leverage unique features of CenKR to understand how increasing its activity impacts cell elongation/division and use antibiotics to identify how modulating the activity of this TCS leads to changes in cell morphology. Our results provide new insight into how CenKR activity controls the structure and function of the bacterial envelope, the localization of cell elongation and division machinery, and cellular processes in organisms with importance in health, host-microbe interactions, and biotechnology.

Efficient production of natural sunscreens shinorine, porphyra-334, and mycosporine-2-glycine in Saccharomyces cerevisiae

Mycosporine-like amino acids (MAAs) are promising natural sunscreens mainly produced in marine organisms. Until now, metabolic engineering efforts to produce MAAs in heterologous hosts have mainly focused on shinorine production, and the low production levels are still not suitable for industrial applications. In this study, we successfully developed Saccharomyces cerevisiae strains that can efficiently produce various disubstituted MAAs, including shinorine, porphyra-334, and mycosporine-2-glycine (M2G), which are formed by conjugating serine, threonine, and glycine to mycosporine-glycine (MG), respectively. We first generated an MG-producing strain by multiple integration of the biosynthetic genes from cyanobacteria and applying metabolic engineering strategies to increase sedoheptulose-7-phosphate pool, a substrate for MG production. Next, five mysD genes from cyanobacteria, which encode D-Ala-D-Ala ligase homologues that conjugate an amino acid to MG, were introduced into the MG-producing strain to determine the substrate preference of each MysD enzyme. MysDs from Lyngbya sp., Nostoclinckia, and Euhalothece sp. showed high specificity toward serine, threonine, and glycine, resulting in efficient production of shinorine, porphyra-334, and M2G, respectively. This is the first report on the production of porphyra-334 and M2G in S. cerevisiae. Furthermore, we identified that the substrate specificity of MysD was determined by the omega loop region of 43–45 amino acids predicted based on its structural homology to a D-Ala-D-Ala ligase from Thermus thermophilus involved in peptidoglycan biosynthesis. The substrate specificities of two MysD enzymes were interchangeable by swapping the omega loop region. Using the engineered strain expressing mysD from Lyngbya sp. or N. linckia, up to 1.53 g/L shinorine or 1.21 g/L porphyra-334 was produced by fed-batch fermentation in a 5-L bioreactor, the highest titer reported so far. These results suggest that S. cerevisiae is a promising host for industrial production of different types of MAAs, providing a sustainable and eco-friendly alternative for the development of natural sunscreens.

Improved species level bacterial characterization from rhizosphere soil of wilt infected Punica granatum

Pomegranate crops are prone to wilt complex disease, which is known to severely hamper the crop yield. There have been limited studies that have explored bacteria–plant–host associations in wilt complex disease affecting pomegranate crops. In the present study, wilt infected rhizosphere soil samples (ISI, ASI) in pomegranate were studied in comparison to a healthy control (HSC). The 16S metagenomics sequencing approach using the MinION platform was employed for screening of bacterial communities and predictive functional pathways. Altered physicochemical properties in the soil samples were recorded showing a comparatively acidic pH in the ISI (6.35) and ASI (6.63) soil samples to the HSC soil (7.66), along with higher electrical conductivity in the ISI (139.5 µS/cm), ASI soil (180 µS/cm), HSC soil sample (123.33 µS/cm). While concentration of micronutrients such as Cl and B were significantly higher in the ISI and ASI soil as compared to the HSC, Cu and Zn were significantly higher in the ASI soil. The effectiveness and accuracy of 16S metagenomics studies in identifying beneficial and pathogenic bacterial communities in multi-pathogen–host systems depend on the completeness and consistency of the available 16S rRNA sequence repositories. Enhancing these repositories could significantly improve the exploratory potential of such studies. Thus, multiple 16S rRNA data repositories (RDP, GTDB, EzBioCloud, SILVA, and GreenGenes) were benchmarked, and the findings indicated that SILVA yields the most reliable matches. Consequently, SILVA was chosen for further analysis at the species level. Relative abundance estimates of bacterial species showed variations of growth promoting bacteria, namely, Staphylococcus epidermidis, Bacillus subtilis, Bacillus megatarium, Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas stutzeri and Micrococcus luteus. Functional profiling predictions employing PICRUSt2 revealed a number of enriched pathways such as transporter protein families involved in signalling and cellular processes, iron complex transport system substrate binding protein, peptidoglycan biosynthesis II (staphylococci) and TCA cycle VII (acetate-producers). In line with past reports, results suggest that an acidic pH along with the bioavailability of micronutrients such as Fe and Mn could be facilitating the prevalence and virulence of Fusarium oxysporum, a known causative pathogen, against the host and beneficial bacterial communities. This study identifies bacterial communities taking into account the physicochemical and other abiotic soil parameters in wilt-affected pomegranate crops. The insights obtained could be instrumental in developing effective management strategies to enhance crop yield and mitigate the impact of wilt complex disease on pomegranate crops.

PBP2b Mutations Improve the Growth of Phage-Resistant Lactococcus cremoris Lacking Polysaccharide Pellicle.

Lactococcus lactis and Lactococcus cremoris are Gram-positive lactic acid bacteria widely used as starter in milk fermentations. Lactococcal cells are covered with a polysaccharide pellicle (PSP) that was previously shown to act as the receptor for numerous bacteriophages of the Caudoviricetes class. Thus, mutant strains lacking PSP are phage resistant. However, because PSP is a key cell wall component, PSP-negative mutants exhibit dramatic alterations of cell shape and severe growth defects, which limit their technological value. In the present study, we isolated spontaneous mutants with improved growth, from L. cremoris PSP-negative mutants. These mutants grow at rates similar to the wild-type strain, and based on transmission electron microscopy analysis, they exhibit improved cell morphology compared to their parental PSP-negative mutants. In addition, the selected mutants maintain their phage resistance. Whole-genome sequencing of several such mutants showed that they carried a mutation in pbp2b, a gene encoding a penicillin-binding protein involved in peptidoglycan biosynthesis. Our results indicate that lowering or turning off PBP2b activity suppresses the requirement for PSP and ameliorates substantially bacterial fitness and morphology. IMPORTANCE Lactococcus lactis and Lactococcus cremoris are widely used in the dairy industry as a starter culture. As such, they are consistently challenged by bacteriophage infections which may result in reduced or failed milk acidification with associated economic losses. Bacteriophage infection starts with the recognition of a receptor at the cell surface, which was shown to be a cell wall polysaccharide (the polysaccharide pellicle [PSP]) for the majority of lactococcal phages. Lactococcal mutants devoid of PSP exhibit phage resistance but also reduced fitness, since their morphology and division are severely impaired. Here, we isolated spontaneous, food-grade non-PSP-producing L. cremoris mutants resistant to bacteriophage infection with a restored fitness. This study provides an approach to isolate non-GMO phage-resistant L. cremoris and L. lactis strains, which can be applied to strains with technological functionalities. Also, our results highlight for the first time the link between peptidoglycan and cell wall polysaccharide biosynthesis.

DivIVA Interacts with the Cell Wall Hydrolase MltG To Regulate Peptidoglycan Synthesis in Streptococcus suis.

Bacterial morphology is largely determined by the spatial and temporal regulation of peptidoglycan (PG) biosynthesis. Ovococci possess a unique pattern of PG synthesis different from the well studied Bacillus, and the mechanism of the coordination of PG synthesis remains poorly understood. Several regulatory proteins have been identified to be involved in the regulation of ovococcal morphogenesis, among which DivIVA is an important one to regulate PG synthesis in streptococci, while its mechanism is largely unknown. Here, the zoonotic pathogen Streptococcus suis was used to investigate the regulation of DivIVA on PG synthesis. Fluorescent d-amino acid probing and 3D-structured illumination microscopy found that DivIVA deletion caused abortive peripheral PG synthesis, resulting in a decreased aspect ratio. The phosphorylation-depleted mutant (DivIVA3A) cells displayed a longer nascent PG and became longer, whereas the phosphorylation-mimicking mutant (DivIVA3E) cells showed a shorter nascent PG and became shorter, suggesting that DivIVA phosphorylation is involved in regulating peripheral PG synthesis. Several DivIVA-interacting proteins were identified, and the interaction was confirmed between DivIVA and MltG, a cell wall hydrolase essential for cell elongation. DivIVA did not affect the PG hydrolysis activity of MltG, while the phosphorylation state of DivIVA affected its interaction with MltG. MltG was mislocalized in the ΔdivIVA and DivIVA3E cells, and both ΔmltG and DivIVA3E cells formed significantly rounder cells, indicating an important role of DivIVA phosphorylation in regulating PG synthesis through MltG. These findings highlight the regulatory mechanism of PG synthesis and morphogenesis of ovococci. IMPORTANCE The peptidoglycan (PG) biosynthesis pathway provides a rich source of novel antimicrobial drug targets. However, bacterial PG synthesis and its regulation is a very complex process involving dozens of proteins. Moreover, unlike the well studied Bacillus, ovococci undergo unusual PG synthesis with unique mechanisms of coordination. DivIVA is an important regulator of PG synthesis in ovococci, while its exact role in regulating PG synthesis remains poorly understood. In this study, we determined the role of DivIVA in regulating lateral PG synthesis of Streptococcus suis and identified a critical interacting partner, MltG, in which DivIVA influenced the subcellular localizations of MltG through its phosphorylation. Our study characterizes the detailed role of DivIVA in regulating bacterial PG synthesis, which is very helpful for understanding the process of PG synthesis in streptococci.

Understanding Chromosome Replication and Segregation Unit of Mycobacterium and Its Comparative Analysis with Model Organisms: From Drug Targets to Drug Identification

Bacterium maintains its pathogenicity in the host by continuing replication and adopting temporal and spatial coordination of cell division steps such as cell wall synthesis, DNA replication, chromosome segregation, Z ring assembly, septum formation and finally cytokinesis. This multistep process requires spatiotemporal assembly of macromolecular complexes and is probably regulated by redundant and multifunctional activities of cell replication and division proteins. Two macromolecular assemblies of peptidoglycan biosynthesis, known as elongasome and divisome are known to drive the division of mother cell into two daughter cells and are characterized by the presence of signature protein complexes. Though the exact composition of macromolecular complexes is yet to be defined in Mycobacterium, the presence of some conserved proteins demonstrates the preservation of elementary units. Along with elongasome and divisome complexes, chromosome replication and segregation proteins are very important to understand as these proteins are very essential for bacilli survival, sustenance, and pathogenesis. In this review, along with presenting the differential features of Mycobacterium cell division process, we are comparing chromosome replication and segregation proteins of Mycobacterium with other bacterial species as we aim to identify structural and functional differences between these proteins in different species. In this review, we have also listed the potential drugs that can be tested to target Mycobacterium chromosome replication and segregation proteins. We expect that based on these differences identified, researchers would be able to direct their research in the characterization of Mycobacterium specific drug.

Architecture and genomic arrangement of the MurE–MurF bacterial cell wall biosynthesis complex

Peptidoglycan (PG) is a central component of the bacterial cell wall, and the disruption of its biosynthetic pathway has been a successful antibacterial strategy for decades. PG biosynthesis is initiated in the cytoplasm through sequential reactions catalyzed by Mur enzymes that have been suggested to associate into a multimembered complex. This idea is supported by the observation that in many eubacteria, mur genes are present in a single operon within the well conserved dcw cluster, and in some cases, pairs of mur genes are fused to encode a single, chimeric polypeptide. We performed a vast genomic analysis using >140 bacterial genomes and mapped Mur chimeras in numerous phyla, with Proteobacteria carrying the highest number. MurE-MurF, the most prevalent chimera, exists in forms that are either directly associated or separated by a linker. The crystal structure of the MurE-MurF chimera from Bordetella pertussis reveals a head-to-tail, elongated architecture supported by an interconnecting hydrophobic patch that stabilizes the positions of the two proteins. Fluorescence polarization assays reveal that MurE-MurF interacts with other Mur ligases via its central domains with KDs in the high nanomolar range, backing the existence of a Mur complex in the cytoplasm. These data support the idea of stronger evolutionary constraints on gene order when encoded proteins are intended for association, establish a link between Mur ligase interaction, complex assembly and genome evolution, and shed light on regulatory mechanisms of protein expression and stability in pathways of critical importance for bacterial survival.