Community-Based COVID-19 Vaccine Clinics in Medically Underserved Neighborhoods to Improve Access and Equity, Philadelphia, 2021-2022.

Vaccination remains key to reducing the risk of COVID-19-related severe illness and death. Because of historic medical exclusion and barriers to access, Black communities have had lower rates of COVID-19 vaccination than White communities. We describe the efforts of an academic medical institution to implement community-based COVID-19 vaccine clinics in medically underserved neighborhoods in Philadelphia, Pennsylvania.

Making the Enterobacterial Common Antigen Glycan and Measuring Its Substrate Sequestration.

The enterobacterial common antigen (ECA), a three-sugar repeat unit polysaccharide produced by Enterobacteriaceae family members, impacts bacterial outer membrane permeability, and its biosynthesis affects the glycan landscape of the organism. ECA synthesis impacts the production of other polysaccharides by reducing the availability of shared substrates, the most notable of which is the 55-carbon polyisoprenoid bactoprenyl phosphate (BP), which serves as a carrier for the production of numerous bacterial glycans including ECA, peptidoglycan, O-antigen, and more. Here, using a combination of enzymatic synthesis and liquid chromatography-mass spectrometry (LC-MS) analysis of bacterial lysates, we provide biochemical evidence for the effect on endogenous polyisoprenoid pools from cell culture that arises from glycan pathway disruption.

Sugar-Phosphate Metabolism Regulates Stationary-Phase Entry and Stalk Elongation in Caulobacter crescentus.

Bacteria have a variety of mechanisms for adapting to environmental perturbations. Changes in oxygen availability result in a switch between aerobic and anaerobic respiration, whereas iron limitation may lead to siderophore secretion. In addition to metabolic adaptations, many organisms respond by altering their cell shape.

Simultaneously inhibiting undecaprenyl phosphate production and peptidoglycan synthases promotes rapid lysis in Escherichia coli.

Peptidoglycan (PG) is a highly cross-linked polysaccharide that encases bacteria, resists the effects of turgor and confers cell shape. PG precursors are translocated across the cytoplasmic membrane by the lipid carrier undecaprenyl phosphate (Und-P) where they are incorporated into the PG superstructure. Previously, we found that one of our Escherichia coli laboratory strains (CS109) harbors a missense mutation in uppS, which encodes an enzymatically defective Und-P(P) synthase.

A Defective Undecaprenyl Pyrophosphate Synthase Induces Growth and Morphological Defects That Are Suppressed by Mutations in the Isoprenoid Pathway of Escherichia coli.

The peptidoglycan exoskeleton shapes bacteria and protects them against osmotic forces, making its synthesis the target of many current antibiotics. Peptidoglycan precursors are attached to a lipid carrier and flipped from the cytoplasm into the periplasm to be incorporated into the cell wall. In , this carrier is undecaprenyl phosphate (Und-P), which is synthesized as a diphosphate by the enzyme undecaprenyl pyrophosphate synthase (UppS).

YtfB, an OapA Domain-Containing Protein, Is a New Cell Division Protein in Escherichia coli.

While screening the Pfam database for novel peptidoglycan (PG) binding modules, we identified the OapA domain, which is annotated as a LysM-like domain. LysM domains bind PG and mediate localization to the septal ring. In the Gram-negative bacterium , an OapA domain is present in YtfB, an inner membrane protein of unknown function but whose overproduction causes cells to filament.

PBP1B Glycosyltransferase and Transpeptidase Activities Play Different Essential Roles during the Regeneration of Rod Morphology in Escherichia coli.

Peptidoglycan is a vital component of nearly all cell wall-bearing bacteria and is a valuable target for antibacterial therapy. However, despite decades of work, there remain important gaps in understanding how this macromolecule is synthesized and molded into a three-dimensional structure that imparts specific morphologies to individual cells. Here, we investigated the particularly enigmatic area of how peptidoglycan is synthesized and shaped during the first stages of creating cell shape , that is, in the absence of a preexisting template.

Interrupting Biosynthesis of O Antigen or the Lipopolysaccharide Core Produces Morphological Defects in Escherichia coli by Sequestering Undecaprenyl Phosphate.

Unlabelled: Undecaprenyl phosphate (Und-P) is a member of the family of essential polyprenyl phosphate lipid carriers and in the Gram-negative bacterium Escherichia coli is required for synthesizing the peptidoglycan (PG) cell wall, enterobacterial common antigen (ECA), O antigen, and colanic acid. Previously, we found that interruption of ECA biosynthesis indirectly alters PG synthesis by sequestering Und-P via dead-end intermediates, causing morphological defects. To determine if competition for Und-P was a more general phenomenon, we determined if O-antigen intermediates caused similar effects.

The Redundancy of Peptidoglycan Carboxypeptidases Ensures Robust Cell Shape Maintenance in Escherichia coli.

Unlabelled: Peptidoglycan (PG) is an essential structural component of the bacterial cell wall and maintains the integrity and shape of the cell by forming a continuous layer around the cytoplasmic membrane. The thin PG layer of Escherichia coli resides in the periplasm, a unique compartment whose composition and pH can vary depending on the local environment of the cell. Hence, the growth of the PG layer must be sufficiently robust to allow cell growth and division under different conditions.

Colanic Acid Intermediates Prevent De Novo Shape Recovery of Escherichia coli Spheroplasts, Calling into Question Biological Roles Previously Attributed to Colanic Acid.

Unlabelled: After losing their protective peptidoglycan, bacterial spheroplasts can resynthesize a cell wall to recreate their normal shape. In Escherichia coli, this process requires the Rcs response. In its absence, spheroplasts do not revert to rod shapes but instead form enlarged spheroids and lyse.

Dead-end intermediates in the enterobacterial common antigen pathway induce morphological defects in Escherichia coli by competing for undecaprenyl phosphate.

Bacterial morphology is determined primarily by the architecture of the peptidoglycan (PG) cell wall, a mesh-like layer that encases the cell. To identify novel mechanisms that create or maintain cell shape in Escherichia coli, we used flow cytometry to screen a transposon insertion library and identified a wecE mutant that altered cell shape, causing cells to filament and swell. WecE is a sugar aminotransferase involved in the biosynthesis of enterobacterial common antigen (ECA), a non-essential outer membrane glycolipid of the Enterobacteriaceae.

A new suite of tnaA mutants suggests that Escherichia coli tryptophanase is regulated by intracellular sequestration and by occlusion of its active site.

Background: The Escherichia coli enzyme tryptophanase (TnaA) converts tryptophan to indole, which triggers physiological changes and regulates interactions between bacteria and their mammalian hosts. Tryptophanase production is induced by external tryptophan, but the activity of TnaA is also regulated by other, more poorly understood mechanisms. For example, the enzyme accumulates as a spherical inclusion (focus) at midcell or at one pole, but how or why this localization occurs is unknown.

A cAMP-independent carbohydrate-driven mechanism inhibits tnaA expression and TnaA enzyme activity in Escherichia coli.

When Escherichia coli is grown in a medium lacking glucose or another preferred carbohydrate, the concentration of cAMP-cAMP receptor protein (cAMP-CRP) increases, and this latter complex regulates the expression of more than 180 genes. To respond rapidly to changes in carbohydrate availability, E. coli must maintain a suitable intracellular concentration of cAMP by either exporting or degrading excess cAMP.

Accumulation of periplasmic enterobactin impairs the growth and morphology of Escherichia coli tolC mutants.

TolC is the outer membrane component of tripartite efflux pumps, which expel proteins, toxins and antimicrobial agents from Gram-negative bacteria. Escherichia coli tolC mutants grow well and are slightly elongated in rich media but grow less well than wild-type cells in minimal media. These phenotypes have no physiological explanation as yet.

Eliminating a set of four penicillin binding proteins triggers the Rcs phosphorelay and Cpx stress responses in Escherichia coli.

Penicillin binding proteins (PBPs) are responsible for synthesizing and modifying the bacterial cell wall, and in Escherichia coli the loss of several nonessential low-molecular-weight PBPs gives rise to abnormalities in cell shape and division. To determine whether these proteins help connect the flagellar basal body to the peptidoglycan wall, we surveyed a set of PBP mutants and found that motility in an agar migration assay was compromised by the simultaneous absence of four enzymes: PBP4, PBP5, PBP7, and AmpH. A wild-type copy of any one of these restored migration, and complementation depended on the integrity of the PBP active-site serine.

The Rcs stress response and accessory envelope proteins are required for de novo generation of cell shape in Escherichia coli.

Interactions with immune responses or exposure to certain antibiotics can remove the peptidoglycan wall of many Gram-negative bacteria. Though the spheroplasts thus created usually lyse, some may survive by resynthesizing their walls and shapes. Normally, bacterial morphology is generated by synthetic complexes directed by FtsZ and MreBCD or their homologues, but whether these classic systems can recreate morphology in the absence of a preexisting template is unknown.

Indole production by the tryptophanase TnaA in Escherichia coli is determined by the amount of exogenous tryptophan.

The signalling molecule indole occurs in significant amounts in the mammalian intestinal tract and regulates diverse microbial processes, including bacterial motility, biofilm formation, antibiotic resistance and host cell invasion. In Escherichia coli, the enzyme tryptophanase (TnaA) produces indole from tryptophan, but it is not clear what determines how much indole E. coli can produce and excrete, making it difficult to interpret experiments that investigate the biological effects of indole at high concentrations.

Cell sorting enriches Escherichia coli mutants that rely on peptidoglycan endopeptidases to suppress highly aberrant morphologies.

Bacterial morphology imparts physiological advantages to cells in different environments and, judging by the fidelity with which shape is passed to daughter cells, is a tightly regulated characteristic. Surprisingly, only in the past 10 to 15 years has significant headway been made in identifying the mechanisms by which cells create and maintain particular shapes. One reason for this is that the relevant discoveries have relied heavily on the arduous, somewhat subjective process of manual microscopy.

ZipA is required for FtsZ-dependent preseptal peptidoglycan synthesis prior to invagination during cell division.

Rod-shaped bacteria grow by a repetitive cycle of elongation followed by division, and the mechanisms responsible for these two processes have been studied for decades. However, little is known about what happens during the transition between the two activities. At least one event occurs after elongation ends and before division commences, that being the insertion of new cell wall peptidoglycan into a narrowly circumscribed ribbon around midcell where septation is destined to take place.

Escherichia coli low-molecular-weight penicillin-binding proteins help orient septal FtsZ, and their absence leads to asymmetric cell division and branching.

Escherichia coli cells lacking low-molecular-weight penicillin-binding proteins (LMW PBPs) exhibit morphological alterations that also appear when the septal protein FtsZ is mislocalized, suggesting that peptidoglycan modification and division may work together to produce cell shape. We found that in strains lacking PBP5 and other LMW PBPs, higher FtsZ concentrations increased the frequency of branched cells and incorrectly oriented Z rings by 10- to 15-fold. Invagination of these rings produced improperly oriented septa, which in turn gave rise to asymmetric cell poles that eventually elongated into branches.

Isolation and identification of new inner membrane-associated proteins that localize to cell poles in Escherichia coli.

Several bacterial structures, processes and proteins are localized primarily to the poles of rod-shaped cells. To better understand this cellular organization, we devised a new method for identifying proteins that localize to the poles of Escherichia coli. Pole-derived membrane fragments were isolated by affinity capture of vesicles containing the chemotaxis protein, Tar; and for comparison, vesicles representing all parts of the cytoplasmic membrane were captured by expressing a Tar variant that was no longer pole-specific.