RESUMO
Bacterial biofilms are surface-associated communities of bacterial cells enmeshed in an extracellular matrix (ECM). The biofilm lifestyle results in physiological heterogeneity across the community, promotes persistence, and protects cells from external insults such as antibiotic treatment. Escherichia coli was recently discovered to produce a chemically modified form of cellulose, phosphoethanolamine (pEtN) cellulose, which contributes to the formation of its extracellular matrix and elaboration of its hallmark wrinkled macrocolony architectures. Both pEtN cellulose and unmodified cellulose bind dyes such as calcofluor white and Congo red (CR). Here, we present the use of CR fluorescence to distinguish between pEtN cellulose and unmodified cellulose producers. We demonstrate the utility of this tool in the evaluation of a uropathogenic E. coli clinical isolate that appeared to produce curli and a cellulosic component but did not exhibit macrocolony wrinkling. We determined that lack of macrocolony wrinkling was attributed to a single-nucleotide mutation and introduction of a stop codon in bcsG, abrogating production of BcsG, the pEtN transferase. Thus, this work underscores the important contribution of the pEtN cellulose modification to the E. coli agar-based macrocolony wrinkling phenotype and introduces a facile approach to distinguish between modified and unmodified cellulose.IMPORTANCEE. coli bacteria produce amyloid fibers, termed curli, and a cellulosic component to assemble biofilm communities. Cellulose is the most abundant biopolymer on Earth, and we recently discovered that the cellulosic component in E. coli biofilms was not standard cellulose, but a newly identified cellulosic polymer, phosphoethanolamine cellulose. Studies involving the biological and functional impact of this cellulose modification among E. coli and other organisms are just beginning. Convenient methods for distinguishing pEtN cellulose from unmodified cellulose in E. coli and for estimating production are needed to facilitate further research. Dissecting the balance of pEtN cellulose and curli production by E. coli commensal strains and clinical isolates will improve our understanding of the host microbiome and of factors contributing to bacterial pathogenesis.
Assuntos
Celulose/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Etanolaminas/metabolismo , Coloração e Rotulagem/métodos , Celulose/química , Vermelho Congo/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Etanolaminas/química , Matriz Extracelular/química , Matriz Extracelular/metabolismo , FluorescênciaRESUMO
Pursuing polymers that can transform from a nonconjugated to a conjugated state under mechanical stress to significantly change their properties, we developed a new generation of ladder-type mechanophore monomers, bicyclo[2.2.0]hex-5-ene-peri-naphthalene (BCH-Naph), that can be directly and efficiently polymerized by ring-opening metathesis polymerization (ROMP). BCH-Naphs can be synthesized in multigram quantities and functionalized with a wide range of electron-rich and electron-poor substituents, allowing tuning of the optoelectronic and physical properties of mechanically generated conjugated polymers. Efficient ROMP of BCH-Naphs yielded ultrahigh molecular weight polymechanophores with controlled MWs and low dispersity. The resulting poly(BCH-Naph)s can be mechanically activated into conjugated polymers using ultrasonication, grinding, and even simple stirring of the dilute solutions, leading to changes in absorption and fluorescence. Poly(BCH-Naph)s represent an attractive polymechanophore system to explore multifaceted mechanical response in solution and solid states, owing to the synthetic scalability, functional diversity, efficient polymerization, and facile mechanoactivation.
RESUMO
Whole-cell protein profiling, spatial localization, and quantification of activities such as gene transcription and protein translation are possible with modern biochemical and biophysical techniques. Yet, addressing questions of overall compositional changes within a cell - capturing the relative amounts of protein and ribosomal RNA levels and lipid content simultaneously - would require extractions and purifications with caveats due to isolation yields and detection methods. A holistic view of cellular composition would aid in the study of cellular composition and function. Here, solid state NMR is used to identify 13C NMR signatures for cellular organelles in HeLa cells without the use of any isotopic labeling. Comparisons are made with carbon spectra of subcellular assemblies including DNA, lipids, ribosomes, nuclei and mitochondria. Whole-cell comparisons are made with different mammalian cells lines, with red blood cells that lack nuclei and organelles, and with Gram-negative and Gram-positive bacteria. Furthermore, treatment of mammalian cells with cycloheximide, a commonly used protein synthesis inhibitor, revealed unanticipated changes consistent with a significant increase in protein glycosylation, obvious at the whole cell level. Thus, we demonstrate that solid-state NMR serves as a unique analytical tool to catalog and compare the ratios of distinct carbon types in cells and serves as a discovery tool to reveal the workings of inhibitors such as cycloheximide on whole-cell biochemistry.
Assuntos
Núcleo Celular/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Mitocôndrias/metabolismo , Cicloeximida/farmacologia , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Escherichia coli/química , Escherichia coli/isolamento & purificação , Glicoproteínas/metabolismo , Células HEK293 , Células HeLa , Humanos , Substâncias Macromoleculares/isolamento & purificação , Substâncias Macromoleculares/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Staphylococcus aureus/química , Staphylococcus aureus/isolamento & purificaçãoRESUMO
The introduction of new and improved antibacterial agents based on facile synthetic modifications of existing antibiotics represents a promising strategy to deliver urgently needed antibacterial candidates to treat multi-drug resistant bacterial infections. Using this strategy, vancomycin was transformed into a highly active agent against antibiotic-resistant Gram-negative organisms in vitro and in vivo through the addition of a single arginine to yield vancomycin-arginine (V-R). Here, we report detection of the accumulation of V-R in E. coli by whole-cell solid-state NMR using 15N-labeled V-R. 15N CPMAS NMR revealed that the conjugate remained fully amidated without loss of arginine, demonstrating that intact V-R represents the active antibacterial agent. Furthermore, C{N}REDOR NMR in whole cells with all carbons at natural abundance 13C levels exhibited the sensitivity and selectivity to detect the directly bonded 13C-15N pairs of V-R within E. coli cells. Thus, we also present an effective methodology to directly detect and evaluate active drug agents and their accumulation within bacteria without the need for potentially perturbative cell lysis and analysis protocols.
RESUMO
Biofilms are multicellular bacterial communities encased in a self-secreted extracellular matrix comprised of polysaccharides, proteinaceous fibers, and DNA. Organization of these components lends spatial organization to the biofilm community such that biofilm residents can benefit from the production of common goods while being protected from exogenous insults. Spatial organization is driven by the presence of chemical gradients, such as oxygen. Here we show that two quinol oxidases found in Escherichia coli and other bacteria organize along the biofilm oxygen gradient and that this spatially coordinated expression controls architectural integrity. Cytochrome bd, a high-affinity quinol oxidase required for aerobic respiration under hypoxic conditions, is the most abundantly expressed respiratory complex in the biofilm community. Depletion of the cytochrome bd-expressing subpopulation compromises biofilm complexity by reducing the abundance of secreted extracellular matrix as well as increasing cellular sensitivity to exogenous stresses. Interrogation of the distribution of quinol oxidases in the planktonic state revealed that â¼15% of the population expresses cytochrome bd at atmospheric oxygen concentration, and this population dominates during acute urinary tract infection. These data point toward a bet-hedging mechanism in which heterogeneous expression of respiratory complexes ensures respiratory plasticity of E. coli across diverse host niches.IMPORTANCE Biofilms are multicellular bacterial communities encased in a self-secreted extracellular matrix comprised of polysaccharides, proteinaceous fibers, and DNA. Organization of these components lends spatial organization in the biofilm community. Here we demonstrate that oxygen gradients in uropathogenic Escherichia coli (UPEC) biofilms lead to spatially distinct expression programs for quinol oxidases-components of the terminal electron transport chain. Our studies reveal that the cytochrome bd-expressing subpopulation is critical for biofilm development and matrix production. In addition, we show that quinol oxidases are heterogeneously expressed in planktonic populations and that this respiratory heterogeneity provides a fitness advantage during infection. These studies define the contributions of quinol oxidases to biofilm physiology and suggest the presence of respiratory bet-hedging behavior in UPEC.