The mechanism of force transmission at bacterial focal adhesion complexes.

Various rod-shaped bacteria mysteriously glide on surfaces in the absence of appendages such as flagella or pili. In the deltaproteobacterium Myxococcus xanthus, a putative gliding motility machinery (the Agl-Glt complex) localizes to so-called focal adhesion sites (FASs) that form stationary contact points with the underlying surface. Here we show that the Agl-Glt machinery contains an inner-membrane motor complex that moves intracellularly along a right-handed helical path; when the machinery becomes stationary at FASs, the motor complex powers a left-handed rotation of the cell around its long axis. At FASs, force transmission requires cyclic interactions between the molecular motor and the adhesion proteins of the outer membrane via a periplasmic interaction platform, which presumably involves contractile activity of motor components and possible interactions with peptidoglycan. Our results provide a molecular model of bacterial gliding motility.

Engineering posttranslational proofreading to discriminate nonstandard amino acids.

Incorporation of nonstandard amino acids (nsAAs) leads to chemical diversification of proteins, which is an important tool for the investigation and engineering of biological processes. However, the aminoacyl-tRNA synthetases crucial for this process are polyspecific in regard to nsAAs and standard amino acids. Here, we develop a quality control system called "posttranslational proofreading" to more accurately and rapidly evaluate nsAA incorporation. We achieve this proofreading by hijacking a natural pathway of protein degradation known as the N-end rule, which regulates the lifespan of a protein based on its amino-terminal residue. We find that proteins containing certain desired N-terminal nsAAs have much longer half-lives compared with those proteins containing undesired amino acids. We use the posttranslational proofreading system to further evolve a Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (TyrRS) variant and a tRNATyr species for improved specificity of the nsAA biphenylalanine in vitro and in vivo. Our newly evolved biphenylalanine incorporation machinery enhances the biocontainment and growth of genetically engineered Escherichia coli strains that depend on biphenylalanine incorporation. Finally, we show that our posttranslational proofreading system can be designed for incorporation of other nsAAs by rational engineering of the ClpS protein, which mediates the N-end rule. Taken together, our posttranslational proofreading system for in vivo protein sequence verification presents an alternative paradigm for molecular recognition of amino acids and is a major advance in our ability to accurately expand the genetic code.

MapZ marks the division sites and positions FtsZ rings in Streptococcus pneumoniae.

In every living organism, cell division requires accurate identification of the division site and placement of the division machinery. In bacteria, this process is traditionally considered to begin with the polymerization of the highly conserved tubulin-like protein FtsZ into a ring that locates precisely at mid-cell. Over the past decades, several systems have been reported to regulate the spatiotemporal assembly and placement of the FtsZ ring. However, the human pathogen Streptococcus pneumoniae, in common with many other organisms, is devoid of these canonical systems and the mechanisms of positioning the division machinery remain unknown. Here we characterize a novel factor that locates at the division site before FtsZ and guides septum positioning in pneumococcus. Mid-cell-anchored protein Z (MapZ) forms ring structures at the cell equator and moves apart as the cell elongates, therefore behaving as a permanent beacon of division sites. MapZ then positions the FtsZ ring through direct protein-protein interactions. MapZ-mediated control differs from previously described systems mostly on the basis of negative regulation of FtsZ assembly. Furthermore, MapZ is an endogenous target of the Ser/Thr kinase StkP, which was recently shown to have a central role in cytokinesis and morphogenesis of S. pneumoniae. We show that both phosphorylated and non-phosphorylated forms of MapZ are required for proper Z-ring formation and dynamics. Altogether, this work uncovers a new mechanism for bacterial cell division that is regulated by phosphorylation and illustrates that nature has evolved a diversity of cell division mechanisms adapted to the different bacterial clades.

Pathogenic Chlamydia Lack a Classical Sacculus but Synthesize a Narrow, Mid-cell Peptidoglycan Ring, Regulated by MreB, for Cell Division.

The peptidoglycan (PG) cell wall is a peptide cross-linked glycan polymer essential for bacterial division and maintenance of cell shape and hydrostatic pressure. Bacteria in the Chlamydiales were long thought to lack PG until recent advances in PG labeling technologies revealed the presence of this critical cell wall component in Chlamydia trachomatis. In this study, we utilize bio-orthogonal D-amino acid dipeptide probes combined with super-resolution microscopy to demonstrate that four pathogenic Chlamydiae species each possess a ≤ 140 nm wide PG ring limited to the division plane during the replicative phase of their developmental cycles. Assembly of this PG ring is rapid, processive, and linked to the bacterial actin-like protein, MreB. Both MreB polymerization and PG biosynthesis occur only in the intracellular form of pathogenic Chlamydia and are required for cell enlargement, division, and transition between the microbe's developmental forms. Our kinetic, molecular, and biochemical analyses suggest that the development of this limited, transient, PG ring structure is the result of pathoadaptation by Chlamydia to an intracellular niche within its vertebrate host.

Interplay of the serine/threonine-kinase StkP and the paralogs DivIVA and GpsB in pneumococcal cell elongation and division.

Despite years of intensive research, much remains to be discovered to understand the regulatory networks coordinating bacterial cell growth and division. The mechanisms by which Streptococcus pneumoniae achieves its characteristic ellipsoid-cell shape remain largely unknown. In this study, we analyzed the interplay of the cell division paralogs DivIVA and GpsB with the ser/thr kinase StkP. We observed that the deletion of divIVA hindered cell elongation and resulted in cell shortening and rounding. By contrast, the absence of GpsB resulted in hampered cell division and triggered cell elongation. Remarkably, ΔgpsB elongated cells exhibited a helical FtsZ pattern instead of a Z-ring, accompanied by helical patterns for DivIVA and peptidoglycan synthesis. Strikingly, divIVA deletion suppressed the elongated phenotype of ΔgpsB cells. These data suggest that DivIVA promotes cell elongation and that GpsB counteracts it. Analysis of protein-protein interactions revealed that GpsB and DivIVA do not interact with FtsZ but with the cell division protein EzrA, which itself interacts with FtsZ. In addition, GpsB interacts directly with DivIVA. These results are consistent with DivIVA and GpsB acting as a molecular switch to orchestrate peripheral and septal PG synthesis and connecting them with the Z-ring via EzrA. The cellular co-localization of the transpeptidases PBP2x and PBP2b as well as the lipid-flippases FtsW and RodA in ΔgpsB cells further suggest the existence of a single large PG assembly complex. Finally, we show that GpsB is required for septal localization and kinase activity of StkP, and therefore for StkP-dependent phosphorylation of DivIVA. Altogether, we propose that the StkP/DivIVA/GpsB triad finely tunes the two modes of peptidoglycan (peripheral and septal) synthesis responsible for the pneumococcal ellipsoid cell shape.

Minimal Peptidoglycan (PG) Turnover in Wild-Type and PG Hydrolase and Cell Division Mutants of Streptococcus pneumoniae D39 Growing Planktonically and in Host-Relevant Biofilms.

UNLABELLED: We determined whether there is turnover of the peptidoglycan (PG) cell wall of the ovococcus bacterial pathogen Streptococcus pneumoniae (pneumococcus). Pulse-chase experiments on serotype 2 strain D39 radiolabeled with N-acetylglucosamine revealed little turnover and release of PG breakdown products during growth compared to published reports of PG turnover in Bacillus subtilis. PG dynamics were visualized directly by long-pulse-chase-new-labeling experiments using two colors of fluorescent d-amino acid (FDAA) probes to microscopically detect regions of new PG synthesis. Consistent with minimal PG turnover, hemispherical regions of stable "old" PG persisted in D39 and TIGR4 (serotype 4) cells grown in rich brain heart infusion broth, in D39 cells grown in chemically defined medium containing glucose or galactose as the carbon source, and in D39 cells grown as biofilms on a layer of fixed human epithelial cells. In contrast, B. subtilis exhibited rapid sidewall PG turnover in similar FDAA-labeling experiments. High-performance liquid chromatography (HPLC) analysis of biochemically released peptides from S. pneumoniae PG validated that FDAAs incorporated at low levels into pentamer PG peptides and did not change the overall composition of PG peptides. PG dynamics were also visualized in mutants lacking PG hydrolases that mediate PG remodeling, cell separation, or autolysis and in cells lacking the MapZ and DivIVA division regulators. In all cases, hemispheres of stable old PG were maintained. In PG hydrolase mutants exhibiting aberrant division plane placement, FDAA labeling revealed patches of inert PG at turns and bulge points. We conclude that growing S. pneumoniae cells exhibit minimal PG turnover compared to the PG turnover in rod-shaped cells. IMPORTANCE: PG cell walls are unique to eubacteria, and many bacterial species turn over and recycle their PG during growth, stress, colonization, and virulence. Consequently, PG breakdown products serve as signals for bacteria to induce antibiotic resistance and as activators of innate immune responses. S. pneumoniae is a commensal bacterium that colonizes the human nasopharynx and opportunistically causes serious respiratory and invasive diseases. The results presented here demonstrate a distinct demarcation between regions of old PG and regions of new PG synthesis and minimal turnover of PG in S. pneumoniae cells growing in culture or in host-relevant biofilms. These findings suggest that S. pneumoniae minimizes the release of PG breakdown products by turnover, which may contribute to evasion of the innate immune system.

Pbp2x localizes separately from Pbp2b and other peptidoglycan synthesis proteins during later stages of cell division of Streptococcus pneumoniae†D39.

The relative localization patterns of class B penicillin-binding proteins Pbp2x and Pbp2b were used as positional indicators of septal and peripheral (side-wall-like) peptidoglycan (PG) synthesis, respectively, in the mid-cell regions of Streptococcus pneumoniae cells at different stages of division. We confirm that Pbp2x and Pbp2b are essential in the strain D39 genetic background, which differs from that of laboratory strains. We show that Pbp2b, like Pbp2x and class A Pbp1a, follows a different localization pattern than FtsZ and remains at division septa after FtsZ reappears at the equators of daughter cells. Pulse-experiments with fluorescent D-amino acids (FDAAs) were performed in wild-type cells and in cells in which Pbp2x activity was preferentially inhibited by methicillin or Pbp2x amount was depleted. These experiments show that Pbp2x activity separates from that of other PBPs to the centres of constricting septa in mid-to-late divisional cells resolved by high-resolution 3D-SIM microscopy. Dual-protein and protein-fluorescent vancomycin 2D and 3D-SIM immunofluorescence microscopy (IFM) of cells at different division stages corroborate that Pbp2x separates to the centres of septa surrounded by an adjacent constricting ring containing Pbp2b, Pbp1a and regulators, StkP and MreC. The separate localization of Pbp2x suggests distinctive roles in completing septal PG synthesis and remodelling.

Peptidoglycan transformations during Bacillus subtilis sporulation.

While vegetative Bacillus subtilis cells and mature spores are both surrounded by a thick layer of peptidoglycan (PG, a polymer of glycan strands cross-linked by peptide bridges), it has remained unclear whether PG surrounds prespores during engulfment. To clarify this issue, we generated a slender ΔponA mutant that enabled high-resolution electron cryotomographic imaging. Three-dimensional reconstructions of whole cells in near-native states revealed a thin PG-like layer extending from the lateral cell wall around the prespore throughout engulfment. Cryotomography of purified sacculi and fluorescent labelling of PG in live cells confirmed that PG surrounds the prespore. The presence of PG throughout engulfment suggests new roles for PG in sporulation, including a new model for how PG synthesis might drive engulfment, and obviates the need to synthesize a PG layer de novo during cortex formation. In addition, it reveals that B. subtilis can synthesize thin, Gram-negative-like PG layers as well as its thick, archetypal Gram-positive cell wall. The continuous transformations from thick to thin and back to thick during sporulation suggest that both forms of PG have the same basic architecture (circumferential). Endopeptidase activity may be the main switch that governs whether a thin or a thick PG layer is assembled.

Inserting "OFF-to-ON" BODIPY Tags into Cytokines: A Fluorogenic Interleukin IL-33 for Real-Time Imaging of Immune Cells.

The essential functions that cytokine/immune cell interactions play in tissue homeostasis and during disease have prompted the molecular design of targeted fluorophores to monitor their activity in real time. Whereas activatable probes for imaging immune-related enzymes are common, many immunological functions are mediated by binding events between cytokines and their cognate receptors that are hard to monitor by live-cell imaging. A prime example is interleukin-33 (IL-33), a key cytokine in innate and adaptive immunity, whose interaction with the ST2 cell-surface receptor results in downstream signaling and activation of NF-κB and AP-1 pathways. In the present work, we have designed a chemical platform to site-specifically introduce OFF-to-ON BODIPY fluorophores into full cytokine proteins and generate the first nativelike fluorescent analogues of IL-33. Among different incorporation strategies, chemical aminoacylation followed by bioorthogonal derivatization led to the best labeling results. Importantly, the BODIPY-labeled IL-33 derivatives-unlike IL-33-GFP constructs-exhibited ST2-specific binding and downstream bioactivity profiles comparable to those of the wild-type interleukin. Real-time fluorescence microscopy assays under no wash conditions confirmed the internalization of IL-33 through ST2 receptors and its intracellular trafficking through the endosomal pathway. We envision that the modularity and versatility of our BODIPY labeling platform will facilitate the synthesis of minimally tagged fluorogenic cytokines as the next generation of imaging reagents for real-time visualization of signaling events in live immune cells.

In†Situ probing of newly synthesized peptidoglycan in live bacteria with fluorescent D-amino acids.

Tracking a bug's life: Peptidoglycan (PG) of diverse bacteria is labeled by exploiting the tolerance of cells for incorporating different non-natural D-amino acids. These nontoxic D-amino acids preferably label the sites of active PG synthesis, thereby enabling fine spatiotemporal tracking of cell-wall dynamics in phylogenetically and morphologically diverse bacteria. HCC = 7-hydroxycoumarin, NBD = 7-nitrobenzofurazan, TAMRA = carboxytetramethylrhodamine.

Unipolar Peptidoglycan Synthesis in the Rhizobiales Requires an Essential Class A Penicillin-Binding Protein.

Members of the Rhizobiales are polarly growing bacteria that lack homologs of the canonical Rod complex. To investigate the mechanisms underlying polar cell wall synthesis, we systematically probed the function of cell wall synthesis enzymes in the plant pathogen Agrobacterium tumefaciens. The development of fluorescent d-amino acid dipeptide (FDAAD) probes, which are incorporated into peptidoglycan by penicillin-binding proteins in A. tumefaciens, enabled us to monitor changes in growth patterns in the mutants. Use of these fluorescent cell wall probes and peptidoglycan compositional analysis demonstrate that a single class A penicillin-binding protein is essential for polar peptidoglycan synthesis. Furthermore, we find evidence of an additional mode of cell wall synthesis that requires ld-transpeptidase activity. Genetic analysis and cell wall targeting antibiotics reveal that the mechanism of unipolar growth is conserved in Sinorhizobium and Brucella. This work provides insights into unipolar peptidoglycan biosynthesis employed by the Rhizobiales during cell elongation. IMPORTANCE While the structure and function of the bacterial cell wall are well conserved, the mechanisms responsible for cell wall biosynthesis during elongation are variable. It is increasingly clear that rod-shaped bacteria use a diverse array of growth strategies with distinct spatial zones of cell wall biosynthesis, including lateral elongation, unipolar growth, bipolar elongation, and medial elongation. Yet the vast majority of our understanding regarding bacterial elongation is derived from model organisms exhibiting lateral elongation. Here, we explore the role of penicillin-binding proteins in unipolar elongation of Agrobacterium tumefaciens and related bacteria within the Rhizobiales. Our findings suggest that penicillin-binding protein 1a, along with a subset of ld-transpeptidases, drives unipolar growth. Thus, these enzymes may serve as attractive targets for biocontrol of pathogenic Rhizobiales.

Designing efficient genetic code expansion in Bacillus subtilis to gain biological insights.

Bacillus subtilis is a model gram-positive bacterium, commonly used to explore questions across bacterial cell biology and for industrial uses. To enable greater understanding and control of proteins in B. subtilis, here we report broad and efficient genetic code expansion in B. subtilis by incorporating 20 distinct non-standard amino acids within proteins using 3 different families of genetic code expansion systems and two choices of codons. We use these systems to achieve click-labelling, photo-crosslinking, and translational titration. These tools allow us to demonstrate differences between E. coli and B. subtilis stop codon suppression, validate a predicted protein-protein binding interface, and begin to interrogate properties underlying bacterial cytokinesis by precisely modulating cell division dynamics in vivo. We expect that the establishment of this simple and easily accessible chemical biology system in B. subtilis will help uncover an abundance of biological insights and aid genetic code expansion in other organisms.

Photoactivatable metabolic warheads enable precise and safe ablation of target cells in vivo.

Photoactivatable molecules enable ablation of malignant cells under the control of light, yet current agents can be ineffective at early stages of disease when target cells are similar to healthy surrounding tissues. In this work, we describe a chemical platform based on amino-substituted benzoselenadiazoles to build photoactivatable probes that mimic native metabolites as indicators of disease onset and progression. Through a series of synthetic derivatives, we have identified the key chemical groups in the benzoselenadiazole scaffold responsible for its photodynamic activity, and subsequently designed photosensitive metabolic warheads to target cells associated with various diseases, including bacterial infections and cancer. We demonstrate that versatile benzoselenadiazole metabolites can selectively kill pathogenic cells - but not healthy cells - with high precision after exposure to non-toxic visible light, reducing any potential side effects in vivo. This chemical platform provides powerful tools to exploit cellular metabolic signatures for safer therapeutic and surgical approaches.

Laboratory-Generated DNA Can Cause Anomalous Pathogen Diagnostic Test Results.

The coronavirus disease 2019 (COVID-19) pandemic has brought about the unprecedented expansion of highly sensitive molecular diagnostics as a primary infection control strategy. At the same time, many laboratories have shifted focus to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research and diagnostic development, leading to large-scale production of SARS-CoV-2 nucleic acids that can interfere with these tests. We have identified multiple instances, in independent laboratories, in which nucleic acids generated in research settings are suspected to have caused researchers to test positive for SARS-CoV-2 in surveillance testing. In some cases, the affected individuals did not work directly with these nucleic acids but were exposed via a contaminated surface or object. Though researchers have long been vigilant of DNA contaminants, the transfer of these contaminants to SARS-CoV-2 testing samples can result in anomalous test results. The impact of these incidents stretches into the public sphere, placing additional burdens on public health resources, placing affected researchers and their contacts in isolation and quarantine, removing them from the testing pool for 3 months, and carrying the potential to trigger shutdowns of classrooms and workplaces. We report our observations as a call for increased stewardship over nucleic acids with the potential to impact both the use and development of diagnostics. IMPORTANCE To meet the challenges imposed by the COVID-19 pandemic, research laboratories shifted their focus and clinical diagnostic laboratories developed and utilized new assays. Nucleic acid-based testing became widespread and, for the first time, was used as a prophylactic measure. We report 15 cases of researchers at two institutes testing positive for SARS-CoV-2 on routine surveillance tests, in the absence of any symptoms or transmission. These researchers were likely contaminated with nonhazardous nucleic acids generated in the laboratory in the course of developing new SARS-CoV-2 diagnostics. These contaminating nucleic acids were persistent and widespread throughout the laboratory. We report these findings as a cautionary tale to those working with nucleic acids used in diagnostic testing and as a call for careful stewardship of diagnostically relevant molecules. Our conclusions are especially relevant as at-home COVID-19 testing gains traction in the marketplace and these amplicons may impact on the general public.

Fluorescent amino acids as versatile building blocks for chemical biology.

Fluorophores have transformed the way we study biological systems, enabling non-invasive studies in cells and intact organisms, which increase our understanding of complex processes at the molecular level. Fluorescent amino acids have become an essential chemical tool because they can be used to construct fluorescent macromolecules, such as peptides and proteins, without disrupting their native biomolecular properties. Fluorescent and fluorogenic amino acids with unique photophysical properties have been designed for tracking protein-protein interactions in situ or imaging nanoscopic events in real time with high spatial resolution. In this Review, we discuss advances in the design and synthesis of fluorescent amino acids and how they have contributed to the field of chemical biology in the past 10 years. Important areas of research that we review include novel methodologies to synthesize building blocks with tunable spectral properties, their integration into peptide and protein scaffolds using site-specific genetic encoding and bioorthogonal approaches, and their application to design novel artificial proteins, as well as to investigate biological processes in cells by means of optical imaging.

Release Factor Inhibiting Antimicrobial Peptides Improve Nonstandard Amino Acid Incorporation in Wild-type Bacterial Cells.

We report a tunable chemical genetics approach for enhancing genetic code expansion in different wild-type bacterial strains that employ apidaecin-like, antimicrobial peptides observed to temporarily sequester and thereby inhibit Release Factor 1 (RF1). In a concentration-dependent matter, these peptides granted a conditional lambda phage resistance to a recoded Escherichia coli strain with nonessential RF1 activity and promoted multisite nonstandard amino acid (nsAA) incorporation at in-frame amber stop codons in vivo and in vitro. When exogenously added, the peptides stimulated specific nsAA incorporation in a variety of sensitive, wild-type (RF1+) strains, including Agrobacterium tumefaciens, a species in which nsAA incorporation has not been previously reported. Improvement in nsAA incorporation was typically 2-15-fold in E. coli BL21, MG1655, and DH10B strains and A. tumefaciens with the >20-fold improvement observed in probiotic E. coli Nissle 1917. In-cell expression of these peptides promoted multisite nsAA incorporation in transcripts with up to 6 amber codons, with a >35-fold increase in BL21 showing moderate toxicity. Leveraging this RF1 sensitivity allowed multiplexed partial recoding of MG1655 and DH10B that rapidly resulted in resistant strains that showed an additional approximately twofold boost to nsAA incorporation independent of the peptide. Finally, in-cell expression of an apidaecin-like peptide library allowed the discovery of new peptide variants with reduced toxicity that still improved multisite nsAA incorporation >25-fold. In parallel to genetic reprogramming efforts, these new approaches can facilitate genetic code expansion technologies in a variety of wild-type bacterial strains.

Distinct cytoskeletal proteins define zones of enhanced cell wall synthesis in Helicobacter pylori.

Helical cell shape is necessary for efficient stomach colonization by Helicobacter pylori, but the molecular mechanisms for generating helical shape remain unclear. The helical centerline pitch and radius of wild-type H. pylori cells dictate surface curvatures of considerably higher positive and negative Gaussian curvatures than those present in straight- or curved-rod H. pylori. Quantitative 3D microscopy analysis of short pulses with either N-acetylmuramic acid or D-alanine metabolic probes showed that cell wall growth is enhanced at both sidewall curvature extremes. Immunofluorescence revealed MreB is most abundant at negative Gaussian curvature, while the bactofilin CcmA is most abundant at positive Gaussian curvature. Strains expressing CcmA variants with altered polymerization properties lose helical shape and associated positive Gaussian curvatures. We thus propose a model where CcmA and MreB promote PG synthesis at positive and negative Gaussian curvatures, respectively, and that this patterning is one mechanism necessary for maintaining helical shape.

Round spheres, straight rods, and twisting corkscrews, bacteria come in many different shapes. The shape of bacteria is dictated by their cell wall, the strong outer barrier of the cell. As bacteria grow and multiply, they must add to their cell wall while keeping the same basic shape. The cells walls are made from long chain-like molecules via processes that are guided by protein scaffolds within the cell. Many common antibiotics, including penicillin, stop bacterial infections by interrupting the growth of cell walls. Helicobacter pylori is a common bacterium that lives in the gut and, after many years, can cause stomach ulcers and stomach cancer. H. pylori are shaped in a twisting helix, much like a corkscrew. This shape helps H. pylori to take hold and colonize the stomach. It remains unclear how H. pylori creates and maintains its helical shape. The helix is much more curved than other bacteria, and H. pylori does not have the same helpful proteins that other curved bacteria do. If H. pylori grows asymmetrically, adding more material to the cell wall on its long outer side to create a twisting helix, what controls the process? To find out, Taylor et al. grew H. pylori cells and watched how the cell walls took shape. First, a fluorescent dye was attached to the building blocks of the cell wall or to underlying proteins that were thought to help direct its growth. The cells were then imaged in 3D, and images from hundreds of cells were reconstructed to analyze the growth patterns of the bacteria's cell wall. A protein called CcmA was found most often on the long side of the twisting H. pylori. When the CcmA protein was isolated in a dish, it spontaneously formed sheets and helical bundles, confirming its role as a structural scaffold for the cell wall. When CcmA was absent from the cell of H. pylori, Taylor et al. observed that the pattern of cell growth changed substantially. This work identifies a key component directing the growth of the cell wall of H. pylori and therefore, a new target for antibiotics. Its helical shape is essential for H. pylori to infect the gut, so blocking the action of the CcmA protein may interrupt cell wall growth and prevent stomach infections.

Mechanisms of Incorporation for D-Amino Acid Probes That Target Peptidoglycan Biosynthesis.

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.

Fluorogenic D-amino acids enable real-time monitoring of peptidoglycan biosynthesis and high-throughput transpeptidation assays.

Peptidoglycan 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 crosslinkages between polysaccharide chains to endow mechanical strength. However, tracking the 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 allow peptidoglycan biosynthesis to be monitored in real time by visualizing transpeptidase reactions in live cells, as well as real-time activity assays of D,D- and L,D-transpeptidases and sortases in vitro. The unique ability of RfDAAs to become fluorescent when incorporated into peptidoglycan provides a powerful new tool to study peptidoglycan biosynthesis with high temporal resolution and prospectively enable high-throughput screening for inhibitors of peptidoglycan biosynthesis.

Host-Polarized Cell Growth in Animal Symbionts.

To determine the fundamentals of cell growth, we must extend cell biological studies to non-model organisms. Here, we investigated the growth modes of the only two rods known to widen instead of elongating, Candidatus Thiosymbion oneisti and Thiosymbion hypermnestrae. These bacteria are attached by one pole to the surface of their respective nematode hosts. By incubating live Ca. T. oneisti and T. hypermnestrae with a peptidoglycan metabolic probe, we observed that the insertion of new cell wall starts at the poles and proceeds inward, concomitantly with FtsZ-based membrane constriction. Remarkably, in Ca. T. hypermnestrae, the proximal, animal-attached pole grows before the distal, free pole, indicating that the peptidoglycan synthesis machinery is host oriented. Immunostaining of the symbionts with an antibody against the actin homolog MreB revealed that it was arranged medially-that is, parallel to the cell long axis-throughout the symbiont life cycle. Given that depolymerization of MreB abolished newly synthesized peptidoglycan insertion and impaired divisome assembly, we conclude that MreB function is required for symbiont widening and division. In conclusion, our data invoke a reassessment of the localization and function of the bacterial actin homolog.