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.

Modification of essential bacterial peptidoglycan (PG) containing cell walls can lead to antibiotic resistance, for example β-lactam resistance by L,D-transpeptidase activities. Predatory Bdellovibrio bacteriovorus are naturally antibacterial and combat infections by traversing, modifying and finally destroying walls of Gram-negative prey bacteria, modifying their own PG as they grow inside prey. Historically, these multi-enzymatic processes on two similar PG walls have proved challenging to elucidate. Here, with a PG labelling approach utilizing timed pulses of multiple fluorescent D-amino acids (FDAAs), we illuminate dynamic changes that predator and prey walls go through during the different phases of bacteria:bacteria invasion. We show formation of a reinforced circular port-hole in the prey wall; L,D-transpeptidaseBd mediated D-amino acid modifications strengthening prey PG during Bdellovibrio invasion and a zonal mode of predator-elongation. This process is followed by unconventional, multi-point and synchronous septation of the intracellular Bdellovibrio, accommodating odd- and even-numbered progeny formation by non-binary division.

Fluorescent d-amino acids (FDAAs) enable efficient in situ labeling of peptidoglycan in diverse bacterial species.

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. IMPORTANCEPG 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. P eptidoglycan (PG) biosynthesis and placement are dynamic processes that determine the shapes, sizes, chaining, and resistance to turgor of bacterial cells (1-6). In Gram-positive bacteria, PG also serves as the scaffolding for covalent attachment of surface wall teichoic acid, capsule, and sortase-attached proteins (7-9). The seminal work of Park and Uehara demonstrated that PG is rapidly turned over and the breakdown components recycled in...

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.

Electrochemical reduction of coumarin (1), 6-methylcoumarin (2), 7-methylcoumarin (3), 7-methoxycoumarin (4), and 5,7-dimethoxycoumarin (5) at carbon cathodes in dimethylformamide containing 0.10 M tetra-n-butylammonium tetrafluoroborate has been investigated by means of cyclic voltammetry and controlled-potential (bulk) electrolysis. Cyclic voltammograms for reduction of 1-5 exhibit two irreversible cathodic peaks: (a) the first peak arises from one-electron reduction of the coumarin to form a radical-anion intermediate, which is protonated by the medium to give a neutral radical; (b) although most of this radical undergoes self-coupling to yield a hydrodimer, reduction of the remaining radical (ultimately to produce a dihydrocoumarin) causes the second cathodic peak. At a potential corresponding to the first voltammetric peak, bulk electrolysis of 1-5 affords the corresponding hydrodimer as a mixture of meso and dl diastereomers. Although the meso form dominates, the dl-to-meso ratio varies, due to steric effects arising from substituents on the aromatic ring. Electroreduction of an equimolar mixture of 1 and 4 gives, along with the anticipated symmetrical hydrodimers, an unsymmetrical product derived from the two coumarins. A mechanistic scheme involving both radical-anion and radical intermediates is proposed to account for the formation of the various products.

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 concentrationdependent 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 inframe 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 apidaecinlike 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.

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.