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.

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.

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.

Atacama desert is a source of new Micromonospora strains: description of Micromonospora sicca sp. nov.

Several strains were isolated from subsurface soil of the Atacama Desert and were previously assigned to the Micromonospora genus. A polyphasic study was designed to determine the taxonomic affiliation of isolates 4G51T, 4G53, and 4G57. All the strains showed chemotaxonomic properties in line with their classification in the genus Micromonospora, including meso-diaminopimelic acid in the cell wall peptidoglycan, MK-9(H4) as major respiratory quinone, iso-C15:0 and iso-C16:0 as major fatty acids and diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol as major polar lipids. The 16S rRNA gene sequences of strains 4G51T, 4G53, and 4G57 showed the highest similarity (97.9 %) with the type strain of Micromonospora costi CS1-12T, forming an independent branch in the phylogenetic gene tree. Their independent position was confirmed with genome phylogenies, being most closely related to the type strain of Micromonospora kangleipakensis. Digital DNA-DNA hybridization and average nucleotide identity analyses between the isolates and their closest phylogenomic neighbours confirmed that they should be assigned to a new species within the genus Micromonospora for which the name Micromonospora sicca sp. nov. (4G51T=PCM 3031T=LMG 30756T) is proposed.

Discovery and characterization of a novel LysinB from F2 sub-cluster mycobacteriophage RitSun.

The escalating antibiotic resistance in mycobacterial species poses a significant threat globally, necessitating an urgent need to find alternative solutions. Bacteriophage-derived endolysins, which facilitate phage progeny release by attacking bacterial cell walls, present promising antibacterial candidates due to their rapid lytic action, high specificity and low risk of resistance development. In mycobacteria, owing to the complex, hydrophobic cell wall, mycobacteriophages usually synthesize two endolysins: LysinA, which hydrolyzes peptidoglycan; LysinB, which delinks mycolic acid-containing outer membrane and arabinogalactan, releasing free mycolic acid. In this study, we conducted domain analysis and functional characterization of a novel LysinB from RitSun, an F2 sub-cluster mycobacteriophage from our phage collection. Several key properties of RitSun LysinB make it an important antimycobacterial agent: its ability to lyse Mycobacterium smegmatis from without, a higher than previously reported specific activity of 1.36 U/mg and its inhibitory effect on biofilm formation. Given the impermeable nature of the mycobacterial cell envelope, dissecting RitSun LysinB at the molecular level to identify its cell wall-destabilizing sequence could be utilized to engineer other native lysins as fusion proteins, broadening their activity spectrum.

Phage lysins for intestinal microbiome modulation: current challenges and enabling techniques.

The importance of the microbiota in the intestinal tract for human health has been increasingly recognized. In this perspective, microbiome modulation, a targeted alteration of the microbial composition, has gained interest. Phage lysins, peptidoglycan-degrading enzymes encoded by bacteriophages, are a promising new class of antibiotics currently under clinical development for treating bacterial infections. Due to their high specificity, lysins are considered microbiome-friendly. This review explores the opportunities and challenges of using lysins as microbiome modulators. First, the high specificity of endolysins, which can be further modulated using protein engineering or targeted delivery methods, is discussed. Next, obstacles and possible solutions to assess the microbiome-friendliness of lysins are considered. Finally, lysin delivery to the intestinal tract is discussed, including possible delivery methods such as particle-based and probiotic vehicles. Mapping the hurdles to developing lysins as microbiome modulators and identifying possible ways to overcome these hurdles can help in their development. In this way, the application of these innovative antimicrobial agents can be expanded, thereby taking full advantage of their characteristics.

Atomic structures of a bacteriocin targeting Gram-positive bacteria.

Due to envelope differences between Gram-positive and Gram-negative bacteria, engineering precision bactericidal contractile nanomachines requires atomic-level understanding of their structures; however, only those killing Gram-negative bacteria are currently known. Here, we report the atomic structures of an engineered diffocin, a contractile syringe-like molecular machine that kills the Gram-positive bacterium Clostridioides difficile. Captured in one pre-contraction and two post-contraction states, each structure fashions six proteins in the bacteria-targeting baseplate, two proteins in the energy-storing trunk, and a collar linking the sheath with the membrane-penetrating tube. Compared to contractile machines targeting Gram-negative bacteria, major differences reside in the baseplate and contraction magnitude, consistent with target envelope differences. The multifunctional hub-hydrolase protein connects the tube and baseplate and is positioned to degrade peptidoglycan during penetration. The full-length tape measure protein forms a coiled-coil helix bundle homotrimer spanning the entire diffocin. Our study offers mechanical insights and principles for designing potent protein-based precision antibiotics.

Vagococcus jeotgali sp. nov., isolated from cutlassfish jeotgal, a traditional Korean fermented seafood.

A novel, Gram-positive, facultatively anaerobic, and non-motile bacterial strain, designated B2T-5T, was isolated from jeotgal, a traditional Korean fermented seafood. Colonies grown on gifu anaerobic medium agar plates were cream-coloured, irregular, and umbonate with curled margins. Optimal growth of strain B2T-5T occurred at 20 °C, pH 8.0, and in the presence of 1% (w/v) NaCl. Strain B2T-5T was negative for oxidase and catalase activity. Hippurate was not hydrolysed and acetoin was not produced. The major cellular fatty acids were C18 : 1 ω9c and C16 : 0. The cell-wall peptidoglycan was of the A4α type containing l-Lys-d-Asp. The predominant respiratory quinone was menaquinone 7. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylcholine. According to the phylogenetic analysis based on 16S rRNA gene sequences, strain B2T-5T was most closely related to Vagococcus teuberi DSM 21459T, showing 98.2% sequence similarity. Genome sequencing of strain B2T-5T revealed a genome size of 2.0 Mbp and a G+C content of 33.8 mol%. The average nucleotide identities of strain B2T-5T with Vagococcus teuberi DSM 21459T, Vagococcus bubulae SS1994T, and Vagococcus martis D7T301T were 75.0, 74.7, and 75.1%, respectively. Based on the phenotypic, chemotaxonomic, and genotypic data, strain B2T-5T represents a novel species of the genus Vagococcus, for which the name Vagococcus jeotgali sp. nov. is proposed. The type strain is B2T-5T (=KCTC 21223T=JCM 35937T).

Actinomycetospora aeridis sp. nov., Actinomycetospora flava sp. nov. and Actinomycetospora aurantiaca sp. nov., endophytic actinobacteria isolated from wild orchid (Aerides multiflora Roxb).

Three novel mycelium-forming actinobacteria, designated OC33-EN06T, OC33-EN07T, and OC33-EN08T, were isolated from wild orchid (Aerides multiflora Roxb), collected from a hill evergreen forest in Northern Thailand. Strains OC33-EN06T and OC33-EN07T showed the highest 16S rRNA gene similarity with Actinomycetospora lutea TT00-04T, 99.17 and 99.45%, respectively. Strain OC33-EN08T showed high similarity with four species, namely 'Actinomycetospora termitidis Odt1-22T' (99.37%), Actinomycetospora chiangmaiensis DSM 45062T (99.02%), Actinomycetospora corticicola 014-5T (99.02%), and Actinomycetospora soli SF1T (98.81%). Comparative genome analysis of OC33-EN06T, OC33-EN07T, and OC33-EN08T with the closely related type strains showed that average nucleotide identity (ANI) based on blast, ANI based on MUMmer, and average amino acid identity values were less than 95% and the digital DNA-DNA hybridization values were less than 70%, all below the thresholds for species demarcation. The digital G+C content of OC33-EN06T, OC33-EN07T, and OC33-EN08T were 74.5, 74, and 74 mol%, respectively. These three strains developed bud-like chains of non-motile cylindrical spores with a smooth surface. The cell-wall peptidoglycan contained meso-diaminopimelic acid. The whole-cell sugars contained ribose, arabinose, and galactose. The predominant menaquinone was MK-8(H4). The phospholipid profile included phosphatidylcholine, phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, and phosphatidylinositol. Based on comparative analysis of genotypic, phenotypic and chemotaxonomic data, strains OC33-EN06T (=TBRC 18349T=NBRC 116543T), OC33-EN07T (=TBRC 18350T=NBRC 116544T), and OC33-EN08T (=TBRC 18318T=NBRC 116542T) represent the type strains of three novel species of the genus Actinomycetospora for which the names Actinomycetospora aeridis sp. nov., Actinomycetospora flava sp. nov., and Actinomycetospora aurantiaca sp. nov., are proposed.

Identification of a family of peptidoglycan transpeptidases reveals that Clostridioides difficile requires noncanonical cross-links for viability.

Most bacteria are surrounded by a cell wall that contains peptidoglycan (PG), a large polymer composed of glycan strands held together by short peptide cross-links. There are two major types of cross-links, termed 4-3 and 3-3 based on the amino acids involved. 4-3 cross-links are created by penicillin-binding proteins, while 3-3 cross-links are created by L,D-transpeptidases (LDTs). In most bacteria, the predominant mode of cross-linking is 4-3, and these cross-links are essential for viability, while 3-3 cross-links comprise only a minor fraction and are not essential. However, in the opportunistic intestinal pathogen Clostridioides difficile, about 70% of the cross-links are 3-3. We show here that 3-3 cross-links and LDTs are essential for viability in C. difficile. We also show that C. difficile has five LDTs, three with a YkuD catalytic domain as in all previously known LDTs and two with a VanW catalytic domain, whose function was until now unknown. The five LDTs exhibit extensive functional redundancy. VanW domain proteins are found in many gram-positive bacteria but scarce in other lineages. We tested seven non-C. difficile VanW domain proteins and confirmed LDT activity in three cases. In summary, our findings uncover a previously unrecognized family of PG cross-linking enzymes, assign a catalytic function to VanW domains, and demonstrate that 3-3 cross-linking is essential for viability in C. difficile, the first time this has been shown in any bacterial species. The essentiality of LDTs in C. difficile makes them potential targets for antibiotics that kill C. difficile selectively.

A Clostridioides difficile endolysin modulates toxin secretion without cell lysis.

The Clostridia produce and secrete Large Clostridial Glucosylating Toxins (LCGTs) responsible for disease symptoms, but the secretion mechanism is largely unknown. Recently, a holin-like protein was shown to be essential for toxin secretion. Holins, typically bacteriophage-specific proteins, are part of the holin-endo(lysin) system that releases phage progeny. To determine if the clostridia also use a lysin, we investigated two conserved putative lysins, M7404_01910 and M7404_02200, in the release of the LCGTs TcdA and TcdB from a Clostridioides difficile ribotype 027 strain, M7404. Sequence analysis and structural modelling indicates that both proteins are related to N-acetylmuramoyl-l-alanine amidases, similar to CD27L, a lysin from the C. difficile phage ΦCD27. Disruption of these genes reveal that only M7404_02200 contributes to toxin secretion and does so in a non-lytic fashion. Peptidoglycan hydrolysis assays show that recombinant M7404_02200 is an active peptidoglycan amidase, confirming its role in TcdA and TcdB secretion in C. difficile M7404.

Bacterial peptidoglycan signalling in microglia: Activation by MDP via the NF-κB/MAPK pathway.

Bacterial peptidoglycan (PGN) fragments are commonly studied in the context of bacterial infections. However, PGN fragments recently gained recognition as signalling molecules from the commensal gut microbiota in the healthy host. Here we focus on the minimal bioactive PGN motif muramyl dipeptide (MDP), found in both Gram-positive and Gram-negative commensal bacteria, which signals through the Nod2 receptor. MDP from the gut microbiota translocates to the brain and is associated with changes in neurodevelopment and behaviour, yet there is limited knowledge about the underlying mechanisms. In this study we demonstrate that physiologically relevant doses of MDP induce rapid changes in microglial gene expression and lead to cytokine and chemokine secretion. In immortalised microglial (IMG) cells, C-C Motif Chemokine Ligand 5 (CCL5/RANTES) expression is acutely sensitive to the lowest physiologically prevalent dose (0.1 µg/ml) of MDP. As CCL5 plays an important role in memory formation and synaptic plasticity, microglial CCL5 might be the missing link in elucidating MDP-induced alterations in synaptic gene expression. We observed that a higher physiological dose of MDP elevates the expression of cytokines TNF-α and IL-1ß, indicating a transition toward a pro-inflammatory phenotype in IMG cells, which was validated in primary microglial cultures. Furthermore, MDP induces the translocation of NF-κB subunit p65 into the nucleus, which is blocked by MAPK p38 inhibitor SB202190, suggesting that an interplay of both the NF-κB and MAPK pathways is responsible for the MDP-specific microglial phenotype. These findings underscore the significance of different MDP levels in shaping microglial function in the CNS and indicate MDP as a potential mediator for early inflammatory processes in the brain. It also positions microglia as an important target in the gut microbiota-brain-axis pathway through PGN signalling.

Pbp4 provides transpeptidase activity to the FtsW-PbpB peptidoglycan synthase to drive cephalosporin resistance in Enterococcus faecalis.

Enterococci exhibit intrinsic resistance to cephalosporins, mediated in part by the class B penicillin-binding protein (bPBP) Pbp4 that exhibits low reactivity toward cephalosporins and thus can continue crosslinking peptidoglycan despite exposure to cephalosporins. bPBPs partner with cognate SEDS (shape, elongation, division, and sporulation) glycosyltransferases to form the core catalytic complex of peptidoglycan synthases that synthesize peptidoglycan at discrete cellular locations, although the SEDS partner for Pbp4 is unknown. SEDS-bPBP peptidoglycan synthases of enterococci have not been studied, but some SEDS-bPBP pairs can be predicted based on sequence similarity. For example, FtsW (SEDS)-PbpB (bPBP) is predicted to form the catalytic core of the peptidoglycan synthase that functions at the division septum (the divisome). However, PbpB is readily inactivated by cephalosporins, raising the question-how could the FtsW-PbpB synthase continue functioning to enable growth in the presence of cephalosporins? In this work, we report that the FtsW-PbpB peptidoglycan synthase is required for cephalosporin resistance of Enterococcus faecalis, despite the fact that PbpB is inactivated by cephalosporins. Moreover, Pbp4 associates with the FtsW-PbpB synthase and the TPase activity of Pbp4 is required to enable growth in the presence of cephalosporins in an FtsW-PbpB-synthase-dependent manner. Overall, our results implicate a model in which Pbp4 directly interacts with the FtsW-PbpB peptidoglycan synthase to provide TPase activity during cephalosporin treatment, thereby maintaining the divisome SEDS-bPBP peptidoglycan synthase in a functional state competent to synthesize crosslinked peptidoglycan. These results suggest that two bPBPs coordinate within the FtsW-PbpB peptidoglycan synthase to drive cephalosporin resistance in E. faecalis.

Nocardioides bizhenqiangii sp. nov. and Nocardioides renjunii sp. nov., isolated from soil and faeces of Tibetan antelope (Pantholops hodgsonii) on the Qinghai-Tibet Plateau.

Two novel strain pairs (HM61T/HM23 and S-34T/S-58) were isolated from soil and the faeces of Tibetan antelope (Pantholops hodgsonii) collected at the Qinghai-Tibet Plateau of PR China. All four new isolates were aerobic, non-motile, Gram-stain-positive, catalase-positive, oxidase-negative, and short rod-shaped bacteria. The results of phylogenetic analysis based on the full-length 16S rRNA genes and 283 core genomic genes indicated that the four strains were separated into two independent branches belonging to the genus Nocardioides. Strains HM61T and HM23 were most closely related to Nocardioides pelophilus THG T63T (98.58 and 98.65 % 16S rRNA gene sequence similarity). Strains S-34T and S-58 were most closely related to Nocardioides okcheonensis MMS20-HV4-12T (98.89 and 98.89 % 16S rRNA gene sequence similarity). The G+C contents of the genomic DNA of strains HM61T and S-34T were 70.6 and 72.5 mol%, respectively. Strains HM61T, S-34T and the type strains of closely related species in the analysis had average nucleotide identity values of 75.4-90.5 % as well as digital DNA-DNA hybridization values between 20.1 and 40.8 %, which clearly indicated that the four isolates represent two novel species within the genus Nocardioides. The chemotaxonomic characteristics of strains HM61T and S-34T were consistent with the genus Nocardioides. The major fatty acids of all four strains were iso-C16 : 0, C17 : 1 ω8c or C18 : 1 ω9c. For strains HM61T and S-34T, MK-8(H4) was the predominant respiratory quinone, ll-2,6-diaminopimelic acid was the diagnostic diamino acid in the cell-wall peptidoglycan, and the polar lipids profiles were composed of diphosphatidylglycerol and phosphatidylglycerol. Based on phylogenetic, phenotypic, and chemotaxonomic data, we propose that strains HM61T and S-34T represent two novel species of the genus Nocardioides, respectively, with the names Nocardioides bizhenqiangii sp. nov. and Nocardioides renjunii sp. nov. The type strains are HM61T (=GDMCC 4.343T=JCM 36399T) and S-34T (=CGMCC 4.7664T=JCM 33792T).

Salinibacterium soli sp. nov., isolated from lakeside soil.

A Gram-stain-positive, rod-shaped, non-spore-forming and non-motile bacterium, designated strain WY-16T. Growth was observed at 20-42 °C (optimum, 30 °C), pH 6-9 (optimum, pH 7) and salinity of 0-3 % (w/v; optimum, 1 %). Phylogenetic analysis based on genome sequences indicated that WY-16T was affiliated to the family Microbacteriaceae and most closely related to Salinibacterium xinjiangense and Salinibacterium amurskyense. The average nucleotide identity values between strain WY-16T and S. xinjiangense and S. amurskyense were 74.7 and 72.5 %, respectively. The digital DNA-DNA hybridization values between strain WY-16T and S. xinjiangense and S. amurskyense were 19.6 and 18.6 %, respectively. The predominant fatty acids were anteiso-C15 : 0, iso-C16 : 0 and iso-C16 : 0 10-methyl. The major menaquinones were MK-12, MK-13, MK-14 and MK-15. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, one unidentified glycolipid and one unidentified phospholipid. The cell-wall peptidoglycan contained 2,4-diaminobutyric acid as the diamino acid and ribose, rhamnose, glucose and galactose were the major cell-wall sugars. Based on phenotypic, genotypic and phylogenetic evidence, strain WY-16T represents a novel species in the genus Salinibacterium, for which the name Salinibacterium soli sp. nov. is proposed. The type strain is WY-16T (=GDMCC 1.4011T=JCM 36421T).

Lysinibacillus pinottii sp. nov., a novel species with anti-mosquito and anti-mollusk activity.

An isolate of a Gram-positive, strictly aerobic, motile, rod-shaped, endospore forming bacterium was originally isolated from soil when screening and bioprospecting for plant beneficial microorganisms. Phylogenetic analysis of the 16S rRNA gene sequences indicated that this strain was closely related to Lysinibacillus fusiformis NRRL NRS-350T (99.7%) and Lysinibacillus sphaericus NRRL B-23268T (99.2%). In phenotypic characterization, the novel strain was found to grow between 10 and 45 °C and tolerate up to 8% (w/v) NaCl. Furthermore, the strain grew in media with pH 5 to 10 (optimal growth at pH 7.0). The predominant cellular fatty acids were observed to be iso-C15: 0 (52.3%), anteiso-C15: 0 (14.8%), C16:1ω7C alcohol (11.2%), and C16: 0 (9.5%). The cell-wall peptidoglycan contained lysine-aspartic acid, the same as congeners. A draft genome was assembled and the DNA G+C content was determined to be 37.1% (mol content). A phylogenomic analysis on the core genome of the new strain and 5 closest type strains of Lysinibacillus revealed this strain formed a distinct monophyletic clade with the nearest neighbor being Lysinibacillus fusiformis. DNA-DNA relatedness studies using in silico DNA-DNA hybridizations (DDH) showed this species was below the species threshold of 70%. Based upon the consensus of phylogenetic and phenotypic analyses, we conclude that this strain represents a novel species within the genus Lysinibacillus, for which the name Lysinibacillus pinottii sp. nov. is proposed, with type strain PB211T (= NRRL B-65672T, = CCUG 77181T).

PGFinder, an Open-Source Software for Peptidoglycomics: The Structural Analysis of Bacterial Peptidoglycan by LC-MS.

Peptidoglycan is a major and essential component of the bacterial cell envelope that confers cell shape and provides protection against internal osmotic pressure. This complex macromolecule is made of glycan strands cross-linked by short peptides, and its structure is continually modified throughout growth via a process referred to as "remodeling." Peptidoglycan remodeling allows cells to grow, adapt to their environment, and release fragments that can act as signaling molecules during host-pathogen interactions. Preparing peptidoglycan samples for structural analysis first requires purification of the peptidoglycan sacculus, followed by its enzymatic digestion into disaccharide peptides (muropeptides). These muropeptides can then be characterized by liquid chromatography coupled mass spectrometry (LC-MS) and used to infer the structure of intact peptidoglycan sacculi. Due to the presence of unusual crosslinks, noncanonical amino acids, and amino sugars, the analysis of peptidoglycan LC-MS datasets cannot be handled by traditional proteomics software. In this chapter, we describe a protocol to perform the analysis of peptidoglycan LC-MS datasets using the open-source software PGFinder. We provide a step-by-step strategy to deconvolute data from various mass spectrometry instruments, generate muropeptide databases, perform a PGFinder search, and process the data output.

Synthesis of a Borrelia burgdorferi-Derived Muropeptide Standard Fragment Library.

The interplay between the human innate immune system and bacterial cell wall components is pivotal in understanding diseases such as Crohn's disease and Lyme arthritis. Lyme disease, caused by Borrelia burgdorferi, is the most prevalent tick-borne illness in the United States, with a substantial number of cases reported annually. While antibiotic treatments are generally effective, approximately 10% of Lyme disease cases develop persistent arthritis, suggesting a dysregulated host immune response. We have previously identified a link between the immunogenic B. burgdorferi peptidoglycan (PG) and Lyme arthritis and showed that this pathogen sheds significant amounts of PG fragments during growth. Here, we synthesize these PG fragments, including ornithine-containing monosaccharides and disaccharides, to mimic the unique composition of Borrelia cell walls, using reproducible and rigorous synthetic methods. This synthetic approach allows for the modular preparation of PG derivatives, providing a diverse library of well-defined fragments. These fragments will serve as valuable tools for investigating the role of PG-mediated innate immune response in Lyme disease and aid in the development of improved diagnostic methods and treatment strategies.