Dissecting the Enterococcal Polysaccharide Antigen (EPA) structure to explore innate immune evasion and phage specificity.

Streptococci, Lactococci and Enterococci all produce L-rhamnose-containing cell wall polysaccharides which define Lancefield serotypes and represent promising candidates for the design of glycoconjugate vaccines. The L-rhamnose containing Enterococcal Polysaccharide Antigen (EPA), produced by the opportunistic pathogen Enterococcus faecalis, plays a critical role in normal growth, division, biofilm formation, antimicrobial resistance, phage susceptibility, and innate immune evasion. Despite the critical role of this polymer in E. faecalis physiology and host-pathogen interactions, little information is available on its structure and biosynthesis. Here, using an NMR approach, we elucidate the structure of EPA and propose a model for biosynthesis. We report the structure of the EPA-peptidoglycan linkage unit and reveal an unprecedented complexity of the EPA rhamnose backbone and decoration subunits. Finally, we explore the impact of several EPA structural modifications on innate immune evasion and recognition by bacteriophages. This work represents a first step towards the functional characterisation of EPA and the rational design of therapeutic strategies against a group of important pathogens.

Reassessing the substrate specificities of the major Staphylococcus aureus peptidoglycan hydrolases lysostaphin and LytM.

Orchestrated action of peptidoglycan (PG) synthetases and hydrolases is vital for bacterial growth and viability. Although the function of several PG synthetases and hydrolases is well understood, the function, regulation, and mechanism of action of PG hydrolases characterised as lysostaphin-like endopeptidases have remained elusive. Many of these M23 family members can hydrolyse glycyl-glycine peptide bonds and show lytic activity against Staphylococcus aureus whose PG contains a pentaglycine bridge, but their exact substrate specificity and hydrolysed bonds are still vaguely determined. In this work, we have employed NMR spectroscopy to study both the substrate specificity and the bond cleavage of the bactericide lysostaphin and the S. aureus PG hydrolase LytM. Yet, we provide substrate-level evidence for the functional role of these enzymes. Indeed, our results show that the substrate specificities of these structurally highly homologous enzymes are similar, but unlike observed earlier both LytM and lysostaphin prefer the D-Ala-Gly cross-linked part of mature peptidoglycan. However, we show that while lysostaphin is genuinely a glycyl-glycine hydrolase, LytM can also act as a D-alanyl-glycine endopeptidase.

Adlercreutzia wanghongyangiae sp. nov., and Adlercreutzia shanghongiae sp. nov., two new members of the genus Adlercreutzia isolated from plateau pika (Ochotona curzoniae).

Four anaerobic, Gram-stain-positive, non-motile, non-sporulating rod-shaped bacterial strains (R7T, R21, R22 and R25T) were isolated from the intestinal contents of plateau pika (Ochotona curzoniae) collected from the Qinghai-Tibet Plateau, PR China. The four isolates grew at between 25 and 42 °C (optimally at 35-37 °C), and with 0.3-3.3% NaCl (w/v) [optimum, 1.3% (w/v)]. Adding l-arginine to the medium could promote their growth. Strains R7T and R21 were most closely related to Adlercreutzia caecimuris B7T (97.48% 16S rRNA gene sequence similarity). Strains R25T and R22 were most closely related to Adlercreutzia equolifaciens DSM 19450T (98.25% 16S rRNA gene sequence similarity). The genome sequences of R7T and R25T were 2.89 and 2.90 Mb in size with 63.6 and 62.8 mol% DNA G+C contents, respectively. Phylogenetic analysis based on 16S rRNA gene sequences and core genes revealed that R7T and R21 were most closely related to A. caecimuris B7T and Adlercreutzia mucosicola DSM 19490T, whereas R25T and R22 were most closely related to A. equolifaciens DSM 19450T and Adlercreutzia rubneri ResAG-91T. R7T, R25T and the closely related species had average nucleotide identity (ANI) values of 81.9-83.2% as well as digital DNA-DNA hybridisation (dDDH) values between 27.3 and 27.9%, which clearly indicated that they represent two novel species within the genus Adlercreutzia. For R7T and R25T, meso-diaminopimelic acid was the diagnostic diamino acid in the cell-wall peptidoglycan, and the whole cell sugars included galactose, glucose and ribose. On the basis of these results, we propose that strains R7T and R25T represent two novel species of the genus Adlercreutzia, namely Adlercreutzia wanghongyangiae sp. nov. and Adlercreutzia shanghongiae sp. nov., respectively. The type strains are R7T (=GDMCC 1.4459T=KCTC 25860T) and R25T (=GDMCC 1.4458T=KCTC 25861T).

Leuconostoc koreense sp. nov., isolated from kimchi, a fermented vegetable.

The novel strain CBA3628T was isolated from kimchi, a Korean fermented vegetable. CBA3628T is a cocci-shaped, Gram-stain-positive, catalase- and oxidase-negative and facultatively anaerobic bacterium. The results of phylogenetic analysis based on 16S rRNA gene sequencing indicated that CBA3628T represented a member of the genus Leuconostoc of the family Leuconostocaceae. CBA3628T has a circular chromosomal genome and three plasmids of 1 864 558 bp (37% DNA G+C content), containing 1,887 genes, 1,762 predicted protein-coding genes, 4 complete rRNA loci and 70 tRNA genes. The cells were non-haemolytic, non-motile and non-spore forming. The optimal growth of CBA3628T occurred at 30 °C, pH 6.0 and with 0-2% (w/v) NaCl. The major polar lipids of CBA3628T were diphosphatidylglycerol and phosphatidylglycerol. The major fatty acids (>10%) of CBA3628T were C16  :  0, C20  :  0 and C19 : 0 cyclo ω8c. CBA3628T contained A3α-type peptidoglycans. CBA3628T was most closely related to Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293T, L. mesenteroides subsp. dextranicum DSM 20484T and L. suionicum DSM 20241T with 99.52% 16S rRNA gene sequence similarity. However, the average nucleotide identities of 91.9%, 91.7% and 91.1% and the digital DNA-DNA hybridisation values of 45.6%, 45.4% and 45.4% indicated that the novel isolate represented a distinct species. Phylogenetic analyses of both the 16S rRNA gene and genome sequences revealed that CBA3628T formed a distinct phylogenetic lineage within the genus Leuconostoc and was most closely related to Leuconostoc litchii MB7T. The ANI and dDDH values between CBA3628T and L. litchii MB7T were 84.9 and 22.8%, respectively. Functional genes belonging to COG categories E, J and K were enriched in the genome of CBA3628T (>7.9%). On the basis of its physiological, chemotaxonomic, phylogenetic and genomic properties, strain CBA3628T represents a novel species from the genus Leuconostoc, for which we propose the name Leuconostoc koreense sp. nov., with the type strain CBA3628T (= KACC 23049T = DSM 116836T).

Peptidoglycan-Chi3l1 interaction shapes gut microbiota in intestinal mucus layer.

The balanced gut microbiota in intestinal mucus layer plays an instrumental role in the health of the host. However, the mechanisms by which the host regulates microbial communities in the mucus layer remain largely unknown. Here, we discovered that the host regulates bacterial colonization in the gut mucus layer by producing a protein called Chitinase 3-like protein 1 (Chi3l1). Intestinal epithelial cells are stimulated by the gut microbiota to express Chi3l1. Once expressed, Chi3l1 is secreted into the mucus layer where it interacts with the gut microbiota, specifically through a component of bacterial cell walls called peptidoglycan. This interaction between Chi3l1 and bacteria is beneficial for the colonization of bacteria in the mucus, particularly for Gram-positive bacteria like Lactobacillus. Moreover, a deficiency of Chi3l1 leads to an imbalance in the gut microbiota, which exacerbates colitis induced by dextran sodium sulfate. By performing fecal microbiota transplantation from Villin-cre mice or replenishing Lactobacillus in IEC∆Chil1 mice, we were able to restore their colitis to the same level as that of Villin-cre mice. In summary, this study shows a 'scaffold model' for microbiota homeostasis by interaction between intestinal Chi3l1 and bacteria cell wall interaction, and it also highlights that an unbalanced gut microbiota in the intestinal mucus contributes to the development of colitis.

Muropeptides and muropeptide transporters impact on host immune response.

In bacteria, the cell envelope is the key element surrounding and protecting the bacterial content from mechanical or osmotic damages. It allows the selective interchanges of solutes, ions, cellular debris, and drugs between the cellular compartments and the external environment, thanks to the presence of transmembrane proteins called transporters. The major component of the cell envelope is the peptidoglycan, consisting of long linear glycan strands cross-linked by short peptide stems. During cell growth or under stress conditions, peptidoglycan fragments, the muropeptides, are released by bacteria and recognized by the host Pattern Recognition Receptor, promoting the activation of their innate defense mechanisms. The review sums up the salient aspects of microbiota-host interaction with a focus on the NOD-dependent immune response to bacterial peptidoglycan and on the accountability of muropeptide transporters in the crosstalk with the host and in antibiotic resistance. Furthermore, it retraces the discoveries and applications of microorganisms-derived components such as vaccines or vaccine adjuvants.

A lytic transglycosylase connects bacterial focal adhesion complexes to the peptidoglycan cell wall.

The Gram-negative bacterium Myxococcus xanthus glides on solid surfaces. Dynamic bacterial focal adhesion complexes (bFACs) convert proton motive force from the inner membrane into mechanical propulsion on the cell surface. It is unclear how the mechanical force transmits across the rigid peptidoglycan (PG) cell wall. Here, we show that AgmT, a highly abundant lytic PG transglycosylase homologous to Escherichia coli MltG, couples bFACs to PG. Coprecipitation assay and single-particle microscopy reveal that the gliding motors fail to connect to PG and thus are unable to assemble into bFACs in the absence of an active AgmT. Heterologous expression of E. coli MltG restores the connection between PG and bFACs and thus rescues gliding motility in the M. xanthus cells that lack AgmT. Our results indicate that bFACs anchor to AgmT-modified PG to transmit mechanical force across the PG cell wall.

LD-transpeptidation is crucial for fitness and polar growth in Agrobacterium tumefaciens.

Peptidoglycan (PG), a mesh-like structure which is the primary component of the bacterial cell wall, is crucial to maintain cell integrity and shape. While most bacteria rely on penicillin binding proteins (PBPs) for crosslinking, some species also employ LD-transpeptidases (LDTs). Unlike PBPs, the essentiality and biological functions of LDTs remain largely unclear. The Hyphomicrobiales order of the Alphaproteobacteria, known for their polar growth, have PG which is unusually rich in LD-crosslinks, suggesting that LDTs may play a more significant role in PG synthesis in these bacteria. Here, we investigated LDTs in the plant pathogen Agrobacterium tumefaciens and found that LD-transpeptidation, resulting from at least one of 14 putative LDTs present in this bacterium, is essential for its survival. Notably, a mutant lacking a distinctive group of 7 LDTs which are broadly conserved among the Hyphomicrobiales exhibited reduced LD-crosslinking and tethering of PG to outer membrane ß-barrel proteins. Consequently, this mutant suffered severe fitness loss and cell shape rounding, underscoring the critical role played by these Hyphomicrobiales-specific LDTs in maintaining cell wall integrity and promoting elongation. Tn-sequencing screens further revealed non-redundant functions for A. tumefaciens LDTs. Specifically, Hyphomicrobiales-specific LDTs exhibited synthetic genetic interactions with division and cell cycle proteins, and a single LDT from another group. Additionally, our findings demonstrate that strains lacking all LDTs except one displayed distinctive phenotypic profiles and genetic interactions. Collectively, our work emphasizes the critical role of LD-crosslinking in A. tumefaciens cell wall integrity and growth and provides insights into the functional specialization of these crosslinking activities.

Two codependent routes lead to high-level MRSA.

Methicillin-resistant Staphylococcus aureus (MRSA), in which acquisition of mecA [which encodes the cell wall peptidoglycan biosynthesis component penicillin-binding protein 2a (PBP2a)] confers resistance to ß-lactam antibiotics, is of major clinical concern. We show that, in the presence of antibiotics, MRSA adopts an alternative mode of cell division and shows an altered peptidoglycan architecture at the division septum. PBP2a can replace the transpeptidase activity of the endogenous and essential PBP2 but not that of PBP1, which is responsible for the distinctive native septal peptidoglycan architecture. Successful division without PBP1 activity requires the alternative division mode and is enabled by several possible chromosomal potentiator (pot) mutations. MRSA resensitizing agents differentially interfere with the two codependent mechanisms required for high-level antibiotic resistance, which provides opportunities for new interventions.

Janibacter alittae sp. nov., an actinobacterium isolated from the gut of marine sandworm (Alitta virens).

A novel Gram-stain-positive, facultatively anaerobic, non-motile, catalase-positive, oxidase-negative and ovoid cocci, designated as A1S7T, was isolated from the gut of a marine sandworm (Alitta virens). Strain A1S7T exhibited optimal growth at temperatures of 20-30 ℃, pH 6-8 and in the presence of 2-4% (w/v) NaCl. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain A1S7T belonged to the genus Janibacter, exhibiting a similarity of 99.0% to Janibacter cremeus KCTC 49873T, followed by Janibacter anophelis KCTC 19282T (98.8%), Janibacter hoylei KCTC 49872T (98.4%), Janibacter limosus KACC 20518T (98.2%) and Janibacter corallicola KACC 21120T (97.2%). The complete genome sequence of strain A1S7T revealed a genome size of 3360920 bp with a genomic G+C content of 70.1 mol%. The orthologous average nucleotide identity and the digital DNA-DNA hybridization values between strain A1S7T and Janibacter cremeus KCTC 49873T were determined to be 89.5 and 37.2%, respectively. The major respiratory quinone was MK-8(H4). The predominant fatty acids (>10%) included iso-C16:0, C17:1 ω8c, C18:1 ω9c and C17:0. Polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, one unknown phosphoglycolipid and three unknown polar lipids. The cell-wall peptidoglycan type was A1γ. The major whole-cell sugars were ribose, mannose and glucose. Based on phenotypic, phylogenetic, genotypic and chemotaxonomic properties, strain A1S7T represents a novel species in the genus Janibacter, for which the name Janibacter alittae sp. nov. is proposed. The type strain is A1S7T (=KCTC 49714T = JCM 36706T).

ER stress aggravates NOD1-mediated inflammatory response leading to impaired nutrient metabolism in hepatoma cells.

Nucleotide-binding Oligomerization Domain 1 (NOD1) is a cytosolic pattern recognition receptor that senses specific bacterial peptidoglycan moieties, leading to the induction of inflammatory response. Besides, sensing peptidoglycan, NOD1 has been reported to sense metabolic disturbances including the ER stress-induced unfolded protein response (UPR). However, the underpinning crosstalk between the NOD1 activating microbial ligands and the metabolic cues to alter metabolic response is not yet comprehensively defined. Here, we show that underlying ER stress aggravated peptidoglycan-induced NOD1-mediated inflammatory response in hepatoma cells. The HepG2 cells, undergoing ER stress induced by thapsigargin exhibited an amplified inflammatory response induced by peptidoglycan ligand of NOD1 (i.e. iE-DAP). This aggravated inflammatory response disrupted lipid and glucose metabolism, characterized by de novo lipogenic response, and increased gluconeogenesis in HepG2 cells. Further, we characterized that the aggravation of NOD1-induced inflammatory response was dependent on inositol-requiring enzyme 1-α (IRE1-α) and protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) activation, in conjunction with calcium flux. Altogether, our findings suggest that differential UPR activation makes liver cells more sensitive towards bacterial-derived ligands to pronounce inflammatory response in a NOD1-dependent manner that impairs hepatic nutrient metabolism.

Peptidoglycan-tethered and free forms of the Braun lipoprotein are in dynamic equilibrium in Escherichia coli.

Peptidoglycan (PG) is a giant macromolecule that completely surrounds bacterial cells and prevents lysis in hypo-osmotic environments. This net-like macromolecule is made of glycan strands linked to each other by two types of transpeptidases that form either 4→3 (PBPs) or 3→3 (LDTs) cross-links. Previously, we devised a heavy isotope-based PG full labeling method coupled to mass spectrometry to determine the mode of insertion of new subunits into the expanding PG network (Atze et al., 2022). We showed that PG polymerization operates according to different modes for the formation of the septum and of the lateral cell walls, as well as for bacterial growth in the presence or absence of ß-lactams in engineered strains that can exclusively rely on LDTs for PG cross-linking when drugs are present. Here, we apply our method to the resolution of the kinetics of the reactions leading to the covalent tethering of the Braun lipoprotein (Lpp) to PG and the subsequent hydrolysis of that same covalent link. We find that Lpp and disaccharide-peptide subunits are independently incorporated into the expanding lateral cell walls. Newly synthesized septum PG appears to contain small amounts of tethered Lpp. LDTs did mediate intense shuffling of Lpp between PG stems leading to a dynamic equilibrium between the PG-tethered and free forms of Lpp.

Halostreptopolyspora alba gen. nov., sp. nov., a halophilic actinobacterium isolated from saline soil of Xinjiang, Northwest of China.

A Gram-stain-positive, aerobic, moderate halophilic actinobacterium, designated strain YIM 96095T, was isolated from a saline soil sample collected from Aiding Lake, Xinjiang, North-western China. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the isolate belonged to the family Nocardiopsidaceae, formed a distinct subclade, and was most closely related to Lipingzhangella halophila DSM 102030T and Allosalinactinospora lopnorensis DSM 45697T with sequence identity values of 95.8 and 95.1%, respectively. Optimal growth occurred at 37 °C, pH 7.0-8.0 and with 5-16% (w/v) NaCl, with well-developed, non-fragmented substrate mycelia and single-, double-, or triple-wrinkled spore(s) on the mature aerial hyphae. The chemical analysis presented meso-diaminopimelic acid as the diagnostic diamino acid of the cell-wall peptidoglycan, and glucose, galactose and rhamnose as the major whole-cell sugars, and iso-C15 : 0 and anteiso-C15 : 0 as the major fatty acids. The phospholipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, unidentified phospholipids and unidentified glycolipid. The menaquinones were MK-10(H8), MK-10(H6) and MK-9(H10). Its G+C content was 69.7 mol% in the determined genome sequence. Based on phenotypic, chemotaxonomic and phylogenetic characteristics, a novel genus and species named Halostreptopolyspora alba gen. nov., sp. nov. is proposed for isolate YIM 96095T (=KCTC 49266T=CGMCC 4.7636T).

Control of bacterial cell wall autolysins by peptidoglycan crosslinking mode.

To withstand their internal turgor pressure and external threats, most bacteria have a protective peptidoglycan (PG) cell wall. The growth of this PG polymer relies on autolysins, enzymes that create space within the structure. Despite extensive research, the regulatory mechanisms governing these PG-degrading enzymes remain poorly understood. Here, we unveil a novel and widespread control mechanism of lytic transglycosylases (LTs), a type of autolysin responsible for breaking down PG glycan chains. Specifically, we show that LD-crosslinks within the PG sacculus act as an inhibitor of LT activity. Moreover, we demonstrate that this regulation controls the release of immunogenic PG fragments and provides resistance against predatory LTs of both bacterial and viral origin. Our findings address a critical gap in understanding the physiological role of the LD-crosslinking mode in PG homeostasis, highlighting how bacteria can enhance their resilience against environmental threats, including phage attacks, through a single structural PG modification.

The hidden base of the iceberg: gut peptidoglycome dynamics is foundational to its influence on the host.

The intestinal microbiota of humans includes a highly diverse range of bacterial species. All these bacteria possess a cell wall, composed primarily of the macromolecule peptidoglycan. As such, the gut also harbors an abundant and varied peptidoglycome. A remarkable range of host physiological pathways are regulated by peptidoglycan fragments that originate from the gut microbiota and enter the host system. Interactions between the host system and peptidoglycan can influence physiological development and homeostasis, promote health, or contribute to inflammatory disease. Underlying these effects is the interplay between microbiota composition and enzymatic processes that shape the intestinal peptidoglycome, dictating the types of peptidoglycan generated, that subsequently cross the gut barrier. In this review, we highlight and discuss the hidden and emerging functional aspects of the microbiome, i.e. the hidden base of the iceberg, that modulate the composition of gut peptidoglycan, and how these fundamental processes are drivers of physiological outcomes for the host.

Antimicrobial activity of compounds identified by artificial intelligence discovery engine targeting enzymes involved in Neisseria gonorrhoeae peptidoglycan metabolism.

BACKGROUND: Neisseria gonorrhoeae (Ng) causes the sexually transmitted disease gonorrhoea. There are no vaccines and infections are treated principally with antibiotics. However, gonococci rapidly develop resistance to every antibiotic class used and there is a need for developing new antimicrobial treatments. In this study we focused on two gonococcal enzymes as potential antimicrobial targets, namely the serine protease L,D-carboxypeptidase LdcA (NgO1274/NEIS1546) and the lytic transglycosylase LtgD (NgO0626/NEIS1212). To identify compounds that could interact with these enzymes as potential antimicrobials, we used the AtomNet virtual high-throughput screening technology. We then did a computational modelling study to examine the interactions of the most bioactive compounds with their target enzymes. The identified compounds were tested against gonococci to determine minimum inhibitory and bactericidal concentrations (MIC/MBC), specificity, and compound toxicity in vitro. RESULTS: AtomNet identified 74 compounds that could potentially interact with Ng-LdcA and 84 compounds that could potentially interact with Ng-LtgD. Through MIC and MBC assays, we selected the three best performing compounds for both enzymes. Compound 16 was the most active against Ng-LdcA, with a MIC50 value < 1.56 µM and MBC50/90 values between 0.195 and 0.39 µM. In general, the Ng-LdcA compounds showed higher activity than the compounds directed against Ng-LtgD, of which compound 45 had MIC50 values of 1.56-3.125 µM and MBC50/90 values between 3.125 and 6.25 µM. The compounds were specific for gonococci and did not kill other bacteria. They were also non-toxic for human conjunctival epithelial cells as judged by a resazurin assay. To support our biological data, in-depth computational modelling study detailed the interactions of the compounds with their target enzymes. Protein models were generated in silico and validated, the active binding sites and amino acids involved elucidated, and the interactions of the compounds interacting with the enzymes visualised through molecular docking and Molecular Dynamics Simulations for 50 ns and Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA). CONCLUSIONS: We have identified bioactive compounds that appear to target the N. gonorrhoeae LdcA and LtgD enzymes. By using a reductionist approach involving biological and computational data, we propose that compound Ng-LdcA-16 and Ng-LtgD-45 are promising anti-gonococcal compounds for further development.

Biochemical and crystallographic studies of L,D-transpeptidase 2 from Mycobacterium tuberculosis with its natural monomer substrate.

The essential L,D-transpeptidase of Mycobacterium tuberculosis (LdtMt2) catalyses the formation of 3 → 3 cross-links in cell wall peptidoglycan and is a target for development of antituberculosis therapeutics. Efforts to inhibit LdtMt2 have been hampered by lack of knowledge of how it binds its substrate. To address this gap, we optimised the isolation of natural disaccharide tetrapeptide monomers from the Corynebacterium jeikeium bacterial cell wall through overproduction of the peptidoglycan sacculus. The tetrapeptides were used in binding / turnover assays and biophysical studies on LdtMt2. We determined a crystal structure of wild-type LdtMt2 reacted with its natural substrate, the tetrapeptide monomer of the peptidoglycan layer. This structure shows formation of a thioester linking the catalytic cysteine and the donor substrate, reflecting an intermediate in the transpeptidase reaction; it informs on the mode of entrance of the donor substrate into the LdtMt2 active site. The results will be useful in design of LdtMt2 inhibitors, including those based on substrate binding interactions, a strategy successfully employed for other nucleophilic cysteine enzymes.

Tip extension and simultaneous multiple fission in a filamentous bacterium.

Organisms display an immense variety of shapes, sizes, and reproductive strategies. At microscopic scales, bacterial cell morphology and growth dynamics are adaptive traits that influence the spatial organization of microbial communities. In one such community-the human dental plaque biofilm-a network of filamentous Corynebacterium matruchotii cells forms the core of bacterial consortia known as hedgehogs, but the processes that generate these structures are unclear. Here, using live-cell time-lapse microscopy and fluorescent D-amino acids to track peptidoglycan biosynthesis, we report an extraordinary example of simultaneous multiple division within the domain Bacteria. We show that C. matruchotii cells elongate at one pole through tip extension, similar to the growth strategy of soil-dwelling Streptomyces bacteria. Filaments elongate rapidly, at rates more than five times greater than other closely related bacterial species. Following elongation, many septa form simultaneously, and each cell divides into 3 to 14 daughter cells, depending on the length of the mother filament. The daughter cells then nucleate outgrowth of new thinner vegetative filaments, generating the classic "whip handle" morphology of this taxon. Our results expand the known diversity of bacterial cell cycles and help explain how this filamentous bacterium can compete for space, access nutrients, and form important interspecies interactions within dental plaque.

RNA interference-mediated silencing of ctl13 inhibits innate immunity and development in stored pest Tribolium castaneum.

C-type lectins (CTLs) play a pivotal role in the regulation of insect immunity and growth, making them potential molecular targets for RNA interference (RNAi)-mediated pest control. Although multiple CTLs have been identified in the genomes of various insects, their specific functions and underlying molecular mechanisms remain unclear. In the present study, a novel CTL, Tcctl13 with a single CRD, was identified in Tribolium castaneum. Tcctl13 is expressed in diverse immune-related tissues and developmental stages, with a notable increase in its expression upon exposure to lipopolysaccharides (LPS) and peptidoglycan (PGN). Molecular docking and enzyme-linked immunosorbent assay (ELISA) analyses revealed that TcCTL13 possesses the ability interacted with LPS and PGN. The binding and agglutinating activities of recombinant TcCTL13 (rTcCTL13) were demonstrated against both gram-negative and positive bacteria. After using RNAi to silence Tcctl13, the expression of the eight antimicrobial peptide (AMP) genes was significantly reduced. In addition, knocking down Tcctl13 during the early larval or pupal stage hindered, the normal metamorphosis process in T. castaneum, ultimately leading to the demise of all beetles. Further research showed that Tcctl13 and nine AMPs were significantly downregulation after 20-Hydroxyecdysone (20E) injection. Instead, the up-regulation of Tcctl13 and six AMPs was observed following interference with the 20E receptor (ecdysone receptor, EcR), indicating that the function of Tcctl13 is regulated by 20E in T. castaneum. Collectively, these findings suggest that Tcctl13 plays a role in the regulation of innate immunity and development in T. castaneum, offering a promising molecular target for managing insect pests using RNAi-based approaches.

Cytosolic Factors Controlling PASTA Kinase-Dependent ReoM Phosphorylation.

Bacteria adapt the biosynthesis of their envelopes to specific growth conditions and prevailing stress factors. Peptidoglycan (PG) is the major component of the cell wall in Gram-positive bacteria, where PASTA kinases play a central role in PG biosynthesis regulation. Despite their importance for growth, cell division and antibiotic resistance, the mechanisms of PASTA kinase activation are not fully understood. ReoM, a recently discovered cytosolic phosphoprotein, is one of the main substrates of the PASTA kinase PrkA in the Gram-positive human pathogen Listeria monocytogenes. Depending on its phosphorylation, ReoM controls proteolytic stability of MurA, the first enzyme in the PG biosynthesis pathway. The late cell division protein GpsB has been implicated in PASTA kinase signalling. Consistently, we show that L. monocytogenes prkA and gpsB mutants phenocopied each other. Analysis of in vivo ReoM phosphorylation confirmed GpsB as an activator of PrkA leading to the description of structural features in GpsB that are important for kinase activation. We further show that ReoM phosphorylation is growth phase-dependent and that this kinetic is reliant on the protein phosphatase PrpC. ReoM phosphorylation was inhibited in mutants with defects in MurA degradation, leading to the discovery that MurA overexpression prevented ReoM phosphorylation. Overexpressed MurA must be able to bind its substrates and interact with ReoM to exert this effect, but the extracellular PASTA domains of PrkA or MurJ flippases were not required. Our results indicate that intracellular signals control ReoM phosphorylation and extend current models describing the mechanisms of PASTA kinase activation.