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.

In the model organism Bacillus subtilis, a signaling protease produced in the forespore, SpoIVB, is essential for the activation of the sigma factor σK, which is produced in the mother cell as an inactive pro-protein, pro-σK. SpoIVB has a second function essential to sporulation, most likely during cortex synthesis. The cortex is composed of peptidoglycan (PG) and is essential for the spore\'s heat resistance and dormancy. Surprisingly, the genome of the intestinal pathogen Clostridioides difficile, in which σK is produced without a pro-sequence, encodes two SpoIVB paralogs, SpoIVB1 and SpoIVB2. Here, we show that spoIVB1 is dispensable for sporulation, while a spoIVB2 in-frame deletion mutant fails to produce heat-resistant spores. The spoIVB2 mutant enters sporulation, undergoes asymmetric division, and completes engulfment of the forespore by the mother cell but fails to synthesize the spore cortex. We show that SpoIIP, a PG hydrolase and part of the engulfasome, the machinery essential for engulfment, is cleaved by SpoIVB2 into an inactive form. Within the engulfasome, the SpoIIP amidase activity generates the substrates for the SpoIID lytic transglycosylase. Thus, following engulfment completion, the cleavage and inactivation of SpoIIP by SpoIVB2 curtails the engulfasome hydrolytic activity, at a time when synthesis of the spore cortex peptidoglycan begins. SpoIVB2 is also required for normal late gene expression in the forespore by a currently unknown mechanism. Together, these observations suggest a role for SpoIVB2 in coordinating late morphological and gene expression events between the forespore and the mother cell.

A Gram-stain-positive, rod-shaped, aerobic, motile bacterium, J379T, was isolated from radioactive water spring C1, located in a former silver-uranium mine in the Czech Republic. This slow-growing strain exhibited optimal growth at 24-28 °C on solid media with <1 % salt concentration and alkaline pH 8-10. The only respiratory quinone found in strain J379T was MK-7(H4). C18 : 1 ω9c (60.9 %), C18 : 0 (9.4 %), C16 : 0 and alcohol-C18 : 0 (both 6.2 %) were found to be the major fatty acids. The peptidoglycan contained directly cross-linked meso-diaminopimelic acid. Phylogenetic reconstruction based on the 16S rRNA gene sequences and the core-genome analysis revealed that strain J379T forms a separate phylogenetic lineage within the recently amended order Solirubrobacterales. A comparison of the 16S rRNA gene sequences between strain J379T and other members of the order Solirubrobacterales showed <96 % similarity. This analysis revealed that the closest type strains were Parviterribacter kavangonensis D16/0 /H6T (95.2 %), Capillimicrobium parvum 0166_1T (94.9 %) and Conexibacter arvalis KV-962T (94.5 %). Whole-genome analysis showed that the closest type strain was Baekduia soli BR7-21T with an average nucleotide identity of 78 %, average amino acid identity of 63.2 % and percentage of conserved proteins of 48.2 %. The G+C content of the J379T genomic DNA was 71.7 mol%. Based on the phylogenetic and phylogenomic data, as well as its physiological characteristics, strain J379T is proposed to represent a type strain (DSM 113746T=CCM 9300T) of Svornostia abyssi gen. nov. sp. nov. within the family Baekduiaceae.

Acrylamide (AA) and 5-hydroxymethylfurfural (HMF), which are potentially carcinogenic to humans, are often produced during the hot processing of foods. This study first used a molecular docking model to simulate the binding behavior of four lactic acid bacteria peptidoglycans (PGNs) to AA/HMF, and the binding rate of LAB-based PGNs to AA/HMF was evaluated in vitro. In silico results show that interaction energy is the driving force responsible for the adsorption of LAB-derived PGNs to AA/HMF. In vitro results showed that the PGN of B. lactis B1-04 bound the most AA (28.7%) and HMF (48.0%), followed by L. acidophilus NCFM, B. breve CICC 6079, and L. plantarum CICC 22135. Moreover, an AA/HMF-bound layer on the cell surface of B. lactis B1-04 was observed via AFM and SEM due to adsorption. XPS analysis indicated the removal rate of AA/HMF by selected strains was positively correlated with the proportion of C-O, C=O, and N-H groups of PGNs. The atoms O1, O2, O3, O4, N1, N2, N3, H1, and H2 are involved in the adsorption of LAB-based PGNs to AA/HMF. Thus, the PGNs derived from these four Lactobacillus strains can be regarded as natural adsorbents for the binding of AA/HMF.

Pseudomonas aeruginosa encodes the beta-lactamase AmpC, which promotes resistance to beta-lactam antibiotics. Expression of ampC is induced by anhydro-muropeptides (AMPs) released from the peptidoglycan (PG) cell wall upon beta-lactam treatment. AmpC can also be induced via genetic inactivation of PG biogenesis factors such as the endopeptidase DacB that cleaves PG crosslinks. Mutants in dacB occur in beta-lactam-resistant clinical isolates of P. aeruginosa, but it has remained unclear why DacB inactivation promotes ampC induction. Similarly, the inactivation of lytic transglycosylase (LT) enzymes such as SltB1 that cut PG glycans has also been associated with ampC induction and beta-lactam resistance. Given that LT enzymes are capable of producing AMP products that serve as ampC inducers, this latter observation has been especially difficult to explain. Here, we show that ampC induction in sltB1 or dacB mutants requires another LT enzyme called MltG. In Escherichia coli, MltG has been implicated in the degradation of nascent PG strands produced upon beta-lactam treatment. Accordingly, in P. aeruginosa sltB1 and dacB mutants, we detected the MltG-dependent production of pentapeptide-containing AMP products that are signatures of nascent PG degradation. Our results therefore support a model in which SltB1 and DacB use their PG-cleaving activity to open space in the PG matrix for the insertion of new material. Thus, their inactivation mimics low-level beta-lactam treatment by reducing the efficiency of new PG insertion into the wall, causing the degradation of some nascent PG material by MltG to produce the ampC-inducing signal.
OBJECTIVE: Inducible beta-lactamases like the ampC system of Pseudomonas aeruginosa are a common determinant of beta-lactam resistance among gram-negative bacteria. The regulation of ampC is elegantly tuned to detect defects in cell wall synthesis caused by beta-lactam drugs. Studies of mutations causing ampC induction in the absence of drug therefore promise to reveal new insights into the process of cell wall biogenesis in addition to aiding our understanding of how resistance to beta-lactam antibiotics arises in the clinic. In this study, the ampC induction phenotype for mutants lacking a glycan-cleaving enzyme or an enzyme that cuts cell wall crosslinks was used to uncover a potential role for these enzymes in making space in the wall matrix for the insertion of new material during cell growth.