The deep marine sediments represent a major repository of organic matter whilst hosting a great number of uncultivated microbes. Microbial metabolism plays a key role in the recycling of organic matter in the deep marine sediments. D-amino acids (DAAs) and DAA-containing muropeptides, an important group of organic matter in the deep marine sediments, are primarily derived from bacterial peptidoglycan decomposition. Archaea are abundant in the deep ocean microbiome, yet their role in DAA metabolism remains poorly studied. Here, we report bioinformatic investigation and enzymatic characterization of deep marine sedimentary archaea involved in DAA metabolism. Our analyses suggest that a variety of archaea, particularly the Candidatus Bathyarchaeota and the Candidatus Lokiarchaeaota, can metabolize DAAs. DAAs are converted into L-amino acids via amino acid racemases (Ala racemase, Asp racemase and broad substrate specificity amino acid racemase), and converted into α-keto acid via d-serine ammonia-lyase, whereas DAA-containing di-/tri-muropeptides can be hydrolyzed by peptidases (dipeptidase and D-aminopeptidase). Overall, this study reveals the identity and activity of deep marine sedimentary archaea involved in DAA metabolism, shedding light on the mineralization and biogeochemical cycling of DAAs in the deep marine sediments.

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).

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.

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-1β, 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.

Across living organisms, division is necessary for cell survival and passing heritable information to the next generation. For this reason, cell division is highly conserved among eukaryotes and prokaryotes. Among the most highly conserved cell division proteins in eukaryotes are tubulin and actin. Tubulin polymerizes to form microtubules, which assemble into cytoskeletal structures in eukaryotes, such as the mitotic spindle that pulls chromatids apart during mitosis. Actin polymerizes to form a morphological framework for the eukaryotic cell, or cytoskeleton, that undergoes reorganization during mitosis. In prokaryotes, two of the most highly conserved cell division proteins are the tubulin homolog FtsZ and the actin homolog FtsA. In this chapter, the functions of the essential bacterial cell division proteins FtsZ and FtsA and their roles in assembly of the divisome at the septum, the site of cell division, will be discussed. In most bacteria, including Escherichia coli, the tubulin homolog FtsZ polymerizes at midcell, and this step is crucial for recruitment of many other proteins to the division site. For this reason, both FtsZ abundance and polymerization are tightly regulated by a variety of proteins. The actin-like FtsA protein polymerizes and tethers FtsZ polymers to the cytoplasmic membrane. Additionally, FtsA interacts with later stage cell division proteins, which are essential for division and for building the new cell wall at the septum. Recent studies have investigated how actin-like polymerization of FtsA on the lipid membrane may impact division, and we will discuss this and other ways that division in bacteria is regulated through FtsZ and FtsA.

Phagocytosis is an essential mechanism of the human immune system where pathogens are eliminated by immune cells. The CCN1 protein plays an important role in the phagocytosis of Staphylococcus aureus by favoring the bridging of the αVβ3 integrin to the bacterial peptidoglycan (PG), through mechanical forces that remain unknown. Here, we employ single-molecule experiments to unravel the nanomechanics of the PG-CCN1-αVβ3 ternary complex. While CCN1 binds αVβ3 integrins with moderate force (∼60 pN), much higher binding strengths (up to ∼800 pN) are observed between CCN1 and PG. Notably, the strength of both CCN1-αVβ3 and CCN1-PG bonds is dramatically enhanced by tensile loading, favoring a model in which mechanical stress induces the exposure of cryptic integrin binding sites in CCN1 and multivalent binding between CCN1 lectin sites and monosaccharides along the PG glycan chains.

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).

Keratinicyclins and keratinimicins are recently discovered glycopeptide antibiotics. Keratinimicins show broad-spectrum activity against Gram-positive bacteria, while keratinicyclins form a new chemotype by virtue of an unusual oxazolidinone moiety and exhibit specific antibiosis against Clostridioides difficile. Here we report the mechanism of action of keratinicyclin B (KCB). We find that steric constraints preclude KCB from binding peptidoglycan termini. Instead, KCB inhibits C. difficile growth by binding wall teichoic acids (WTAs) and interfering with cell wall remodeling. A computational model, guided by biochemical studies, provides an image of the interaction of KCB with C. difficile WTAs and shows that the same H-bonding framework used by glycopeptide antibiotics to bind peptidoglycan termini is used by KCB for interacting with WTAs. Analysis of KCB in combination with vancomycin (VAN) shows highly synergistic and specific antimicrobial activity, and that nanomolar combinations of the two drugs are sufficient for complete growth inhibition of C. difficile, while leaving common commensal strains unaffected.

Peptidoglycan (PG) sacculi surround the cytoplasmic membrane, maintaining cell integrity by withstanding internal turgor pressure. During cell growth, PG endopeptidases cleave the crosslinks of the fully closed sacculi, allowing for the incorporation of new glycan strands and expansion of the peptidoglycan mesh. Outer-membrane-anchored NlpI associates with hydrolases and synthases near PG synthesis complexes, facilitating spatially close PG hydrolysis. Here, we present the structure of adaptor NlpI in complex with the endopeptidase MepS, revealing atomic details of how NlpI recruits multiple MepS molecules and subsequently influences PG expansion. NlpI binding elicits a disorder-to-order transition in the intrinsically disordered N-terminal of MepS, concomitantly promoting the dimerization of monomeric MepS. This results in the alignment of two asymmetric MepS dimers respectively located on the two opposite sides of the dimerization interface of NlpI, thus enhancing MepS activity in PG hydrolysis. Notably, the protein level of MepS is primarily modulated by the tail-specific protease Prc, which is known to interact with NlpI. The structure of the Prc-NlpI-MepS complex demonstrates that NlpI brings together MepS and Prc, leading to the efficient MepS degradation by Prc. Collectively, our results provide structural insights into the NlpI-enabled avidity effect of cellular endopeptidases and NlpI-directed MepS degradation by Prc.

Most rod-shaped bacteria elongate by inserting new cell wall material into the inner surface of the cell sidewall. This is performed by class A penicillin binding proteins (PBPs) and a highly conserved protein complex, the elongasome, which moves processively around the cell circumference and inserts long glycan strands that act as barrel-hoop-like reinforcing structures, thereby giving rise to a rod-shaped cell. However, it remains unclear how elongasome synthesis dynamics and termination events are regulated to determine the length of these critical cell-reinforcing structures. To address this, we developed a method to track individual elongasome complexes around the entire circumference of Bacillus subtilis cells for minutes-long periods using single-molecule fluorescence microscopy. We found that the B. subtilis elongasome is highly processive and that processive synthesis events are frequently terminated by rapid reversal or extended pauses. We found that cellular levels of RodA regulate elongasome processivity, reversal and pausing. Our single-molecule data, together with stochastic simulations, show that elongasome dynamics and processivity are regulated by molecular motor tug-of-war competition between several, likely two, oppositely oriented peptidoglycan synthesis complexes associated with the MreB filament. Altogether these results demonstrate that molecular motor tug-of-war is a key regulator of elongasome dynamics in B. subtilis, which likely also regulates the cell shape via modulation of elongasome processivity.