Cell shape and cell-envelope integrity of bacteria are determined by the peptidoglycan cell wall. In rod-shaped Escherichia coli, two conserved sets of machinery are essential for cell-wall insertion in the cylindrical part of the cell: the Rod complex and the class-A penicillin-binding proteins (aPBPs). While the Rod complex governs rod-like cell shape, aPBP function is less well understood. aPBPs were previously hypothesized to either work in concert with the Rod complex or to independently repair cell-wall defects. First, we demonstrate through modulation of enzyme levels that aPBPs do not contribute to rod-like cell shape but are required for mechanical stability, supporting their independent activity. By combining measurements of cell-wall stiffness, cell-wall insertion, and PBP1b motion at the single-molecule level, we then present evidence that PBP1b, the major aPBP, contributes to cell-wall integrity by repairing cell wall defects.

Bacteria exhibit a myriad of different morphologies, through the synthesis and modification of their essential peptidoglycan (PG) cell wall. Our discovery of a fluorescent D-amino acid (FDAA)-based PG labeling approach provided a powerful method for observing how these morphological changes occur. Given that PG is unique to bacterial cells and a common target for antibiotics, understanding the precise mechanism(s) for incorporation of (F)DAA-based probes is a crucial determinant in understanding the role of PG synthesis in bacterial cell biology and could provide a valuable tool in the development of new antimicrobials to treat drug-resistant antibacterial infections. Here, we systematically investigate the mechanisms of FDAA probe incorporation into PG using two model organisms Escherichia coli (Gram-negative) and Bacillus subtilis (Gram-positive). Our in vitro and in vivo data unequivocally demonstrate that these bacteria incorporate FDAAs using two extracytoplasmic pathways: through activity of their D,D-transpeptidases, and, if present, by their L,D-transpeptidases and not via cytoplasmic incorporation into a D-Ala-D-Ala dipeptide precursor. Our data also revealed the unprecedented finding that the DAA-drug, D-cycloserine, can be incorporated into peptide stems by each of these transpeptidases, in addition to its known inhibitory activity against D-alanine racemase and D-Ala-D-Ala ligase. These mechanistic findings enabled development of a new, FDAA-based, in vitro labeling approach that reports on subcellular distribution of muropeptides, an especially important attribute to enable the study of bacteria with poorly defined growth modes. An improved understanding of the incorporation mechanisms utilized by DAA-based probes is essential when interpreting results from high resolution experiments and highlights the antimicrobial potential of synthetic DAAs.

In the environment bacteria share their habitat with a great diversity of organisms, from microbes to humans, animals and plants. In these complex communities, the production of extracellular effectors is a common strategy to control the biodiversity by interfering with the growth and/or viability of nearby microbes. One of such effectors relies on the production and release of extracellular D-amino acids which regulate diverse cellular processes such as cell wall biogenesis, biofilm integrity, and spore germination. Non-canonical D-amino acids are mainly produced by broad spectrum racemases (Bsr). Bsr’s promiscuity allows it to generate high concentrations of D-amino acids in environments with variable compositions of L-amino acids. However, it was not clear until recent whether these molecules exhibit divergent functions. Here we review the distinctive biological roles of D-amino acids, their mechanisms of action and their modulatory properties of the biodiversity of complex eco-systems.

The bacterial cell wall is critical for maintenance of cell shape and survival. Following exposure to antibiotics that target enzymes required for cell wall synthesis, bacteria typically lyse. Although several cell envelope stress response systems have been well described, there is little knowledge of systems that modulate cell wall synthesis in response to cell wall damage, particularly in Gram-negative bacteria. Here we describe WigK/WigR, a histidine kinase/response regulator pair that enables Vibrio cholerae, the cholera pathogen, to survive exposure to antibiotics targeting cell wall synthesis in vitro and during infection. Unlike wild-type V. cholerae, mutants lacking wigR fail to recover following exposure to cell-wall–acting antibiotics, and they exhibit a drastically increased cell diameter in the absence of such antibiotics. Conversely, overexpression of wigR leads to cell slimming. Overexpression of activated WigR also results in increased expression of the full set of cell wall synthesis genes and to elevated cell wall content. WigKR-dependent expression of cell wall synthesis genes is induced by various cell-wall–acting antibiotics as well as by overexpression of an endogenous cell wall hydrolase. Thus, WigKR appears to monitor cell wall integrity and to enhance the capacity for increased cell wall production in response to damage. Taken together, these findings implicate WigKR as a regulator of cell wall synthesis that controls cell wall homeostasis in response to antibiotics and likely during normal growth as well.

Synthesis and hydrolysis of septal peptidoglycan (PG) are critical processes at the conclusion of cell division that enable separation of daughter cells. Cleavage of septal PG is mediated by PG amidases, hydrolytic enzymes that release peptide side chains from the glycan strand. Most gammaproteobacteria, including Escherichia coli, encode several functionally redundant periplasmic amidases. However, members of the Vibrio genus, including the enteric pathogen Vibrio cholerae, encode only a single PG amidase, AmiB. Here, we show that V. cholerae AmiB is crucial for cell division and growth. Genetic and biochemical analyses indicated that AmiB is regulated by two activators, EnvC and NlpD, at least one of which is required for AmiB's localization to the cell division site. Localization of the activators (and thus of AmiB) is dependent upon the cell division protein FtsN. These factors mediate septal PG cleavage in E. coli as well; however, their precise roles vary between the two organisms in a number of ways. Notably, even though V. cholerae EnvC and NlpD appear to be functionally redundant under most growth conditions tested, NlpD is specifically required for intestinal colonization in the infant mouse model of cholera and for V. cholerae resistance against bile salts, perhaps due to environmental regulation of AmiB or its activators. Collectively, our findings reveal that although the cellular components that enable cleavage of septal PG appear to be generally conserved between E. coli and V. cholerae, they can be combined into diverse functional regulatory networks.

Broad-spectrum amino-acid racemases (Bsrs) enable bacteria to generate noncanonical D-amino acids, the roles of which in microbial physiology, including the modulation of cell-wall structure and the dissolution of biofilms, are just beginning to be appreciated. Here, extensive crystallographic, mutational, biochemical and bioinformatic studies were used to define the molecular features of the racemase BsrV that enable this enzyme to accommodate more diverse substrates than the related PLP-dependent alanine racemases. Conserved residues were identified that distinguish BsrV and a newly defined family of broad-spectrum racemases from alanine racemases, and these residues were found to be key mediators of the multispecificity of BrsV. Finally, the structural analysis of an additional Bsr that was identified in the bioinformatic analysis confirmed that the distinguishing features of BrsV are conserved among Bsr family members.

ABSTRACTeXTP is a science mission designed to study the state of matter under extreme conditions of density, gravity and magnetism. Primary goals are the determination of the equation of state of matter at supra-nuclear density, the measurement of QED effects in highly magnetized star, and the study of accretion in the strong-field regime of gravity. Primary targets include isolated and binary neutron stars, strong magnetic field systems like magnetars, and stellar-mass and supermassive black holes. The mission carries a unique and unprecedented suite of state-of-the-art scientific instruments enabling for the first time ever the simultaneous spectral-timing-polarimetry studies of cosmic sources in the energy range from 0.5-30 keV (and beyond). Key elements of the payload are: the Spectroscopic Focusing Array (SFA) -a set of 11 X-ray optics for a total effective area of ∼0.9 m 2 and 0.6 m 2 at 2 keV and 6 keV respectively, equipped with Silicon Drift Detectors offering <180 eV spectral resolution; the Large Area Detector (LAD) -a deployable set of 640 Silicon Drift Detectors, for a total effective area of ∼3.4 m 2 , between 6 and 10 keV, and spectral resolution better than 250 eV; the Polarimetry Focusing Array (PFA) -a set of 2 X-ray telescope, for a total effective area of 250 cm 2 at 2 keV, equipped with imaging gas pixel photoelectric polarimeters; the Wide Field Monitor (WFM) -a set of 3 coded mask wide field units, equipped with position-sensitive Silicon Drift Detectors, each covering a 90 degrees x 90 degrees field of view. The eXTP international consortium includes major institutions of the Chinese Academy of Sciences and Universities in China, as well as major institutions in several European countries and the United States. The predecessor of eXTP, the XTP mission concept, has been selected and funded as one of the so-called background missions in the Strategic Priority Space Science Program of the Chinese Academy of Sciences since 2011. The strong European participation has significantly enhanced the scientific capabilities of eXTP. The planned launch date of the mission is earlier than 2025.

Peptidoglycan (PG) is an essential cell wall component that maintains the morphology and viability of nearly all bacteria. Its biosynthesis requires periplasmic transpeptidation reactions which construct peptide cross-linkages between polysaccharide chains to endow mechanical strength. However, tracking transpeptidation reaction in vivo and in vitro is challenging, mainly due to the lack of efficient, biocompatible probes. Here, we report the design, synthesis, and application of rotor-fluorogenic D-amino acids (RfDAAs) enabling real-time, continuous tracking of transpeptidation reactions. These probes enable monitoring PG biosynthesis in real time through visualizing transpeptidase reactions in live cells, as well as real-time activity assays of D,D-, L,D-transpeptidases, and sortases in vitro . The unique ability of RfDAAs to become fluorescent when incorporated into PG provides a powerful new tool to study PG biosynthesis with high temporal resolution and prospectively enable high-throughput screening for inhibitors of PG biosynthesis.