The RpoH (σ) Regulon and Its Role in Essential Cellular Functions, Starvation Survival, and Antibiotic Tolerance.

The bacterial heat-shock response is regulated by the alternative sigma factor, σ (RpoH), which responds to misfolded protein stress and directs the RNA polymerase to the promoters for genes required for protein refolding or degradation. In , RpoH is essential for viability under laboratory growth conditions. Here, we used a transcriptomics approach to identify the genes of the RpoH regulon, including RpoH-regulated genes that are essential for .

Role of Hibernation Promoting Factor in Ribosomal Protein Stability during Dormancy.

is an opportunistic pathogen that causes biofilm-associated infections. can survive in a dormant state with reduced metabolic activity in nutrient-limited environments, including the interiors of biofilms. When entering dormancy, the bacteria undergo metabolic remodeling, which includes reduced translation and degradation of cellular proteins.

Functional Characterization of the Pseudomonas aeruginosa Ribosome Hibernation-Promoting Factor.

Hibernation-promoting factor (HPF) is a ribosomal accessory protein that inactivates ribosomes during bacterial starvation. In , HPF protects ribosome integrity while the cells are dormant. The sequence of HPF has diverged among bacteria but contains conserved charged amino acids in its two alpha helices that interact with the rRNA.

DropSOAC: Stabilizing Microfluidic Drops for Time-Lapse Quantification of Single-Cell Bacterial Physiology.

The physiological heterogeneity of cells within a microbial population imparts resilience to stresses such as antimicrobial treatments and nutrient limitation. This resilience is partially due to a subpopulation of cells that can survive such stresses and regenerate the community. Microfluidic approaches now provide a means to study microbial physiology and bacterial heterogeneity at the single cell level, improving our ability to isolate and examine these subpopulations.

Conceptual Model of Biofilm Antibiotic Tolerance That Integrates Phenomena of Diffusion, Metabolism, Gene Expression, and Physiology.

Transcriptomic, metabolomic, physiological, and computational modeling approaches were integrated to gain insight into the mechanisms of antibiotic tolerance in an biofilm system. biofilms were grown in drip flow reactors on a medium composed to mimic the exudate from a chronic wound. After 4 days, the biofilm was 114 μm thick with 9.

Expression and regulation of the Pseudomonas aeruginosa hibernation promoting factor.

Bacterial biofilms contain subpopulations of cells that are dormant and highly tolerant to antibiotics. While dormant, the bacteria must maintain the integrity of macromolecules required for resuscitation. Previously, we showed that hibernation promoting factor (HPF) is essential for protecting Pseudomonas aeruginosa from ribosomal loss during dormancy.

Resuscitation of from dormancy requires hibernation promoting factor (PA4463) for ribosome preservation.

biofilm infections are difficult to treat with antibiotic therapy in part because the biofilms contain subpopulations of dormant antibiotic-tolerant cells. The dormant cells can repopulate the biofilms following alleviation of antibiotic treatments. While dormant, the bacteria must maintain cellular integrity, including ribosome abundance, to reinitiate the de novo protein synthesis required for resuscitation.

The Pseudomonas aeruginosa PAO1 Two-Component Regulator CarSR Regulates Calcium Homeostasis and Calcium-Induced Virulence Factor Production through Its Regulatory Targets CarO and CarP.

Unlabelled: Pseudomonas aeruginosa is an opportunistic human pathogen that causes severe, life-threatening infections in patients with cystic fibrosis (CF), endocarditis, wounds, or artificial implants. During CF pulmonary infections, P. aeruginosa often encounters environments where the levels of calcium (Ca(2+)) are elevated.

Microsensor and transcriptomic signatures of oxygen depletion in biofilms associated with chronic wounds.

Biofilms have been implicated in delayed wound healing, although the mechanisms by which biofilms impair wound healing are poorly understood. Many species of bacteria produce exotoxins and exoenzymes that may inhibit healing. In addition, oxygen consumption by biofilms and by the responding leukocytes, may impede wound healing by depleting the oxygen that is required for healing.

Contribution of stress responses to antibiotic tolerance in Pseudomonas aeruginosa biofilms.

Enhanced tolerance of biofilm-associated bacteria to antibiotic treatments is likely due to a combination of factors, including changes in cell physiology as bacteria adapt to biofilm growth and the inherent physiological heterogeneity of biofilm bacteria. In this study, a transcriptomics approach was used to identify genes differentially expressed during biofilm growth of Pseudomonas aeruginosa. These genes were tested for statistically significant overlap, with independently compiled gene lists corresponding to stress responses and other putative antibiotic-protective mechanisms.

Bile salts affect expression of Escherichia coli O157:H7 genes for virulence and iron acquisition, and promote growth under iron limiting conditions.

Bile salts exhibit potent antibacterial properties, acting as detergents to disrupt cell membranes and as DNA-damaging agents. Although bacteria inhabiting the intestinal tract are able to resist bile's antimicrobial effects, relatively little is known about how bile influences virulence of enteric pathogens. Escherichia coli O157:H7 is an important pathogen of humans, capable of causing severe diarrhea and more serious sequelae.

Heterogeneity in Pseudomonas aeruginosa biofilms includes expression of ribosome hibernation factors in the antibiotic-tolerant subpopulation and hypoxia-induced stress response in the metabolically active population.

Bacteria growing in biofilms are physiologically heterogeneous, due in part to their adaptation to local environmental conditions. Here, we characterized the local transcriptome responses of Pseudomonas aeruginosa growing in biofilms by using a microarray analysis of isolated biofilm subpopulations. The results demonstrated that cells at the top of the biofilms had high mRNA abundances for genes involved in general metabolic functions, while mRNA levels for these housekeeping genes were low in cells at the bottom of the biofilms.

Heterogeneous rpoS and rhlR mRNA levels and 16S rRNA/rDNA (rRNA gene) ratios within Pseudomonas aeruginosa biofilms, sampled by laser capture microdissection.

The local environmental conditions in biofilms are dependent on the impinging aqueous solution, chemical diffusion, and the metabolic activities of cells within the biofilms. Chemical gradients established in biofilms lead to physiological heterogeneities in bacterial gene expression. Previously, we used laser capture microdissection (LCM) and quantitative reverse transcription (RT)-PCR to target defined biofilm subpopulations for gene expression studies.

Localized gene expression in Pseudomonas aeruginosa biofilms.

Gene expression in biofilms is dependent on bacterial responses to the local environmental conditions. Most techniques for studying bacterial gene expression in biofilms characterize average values across the entire population. Here, we describe the use of laser capture microdissection microscopy (LCMM) combined with multiplex quantitative real-time reverse transcriptase PCR (qRT-PCR) to isolate and quantify RNA transcripts from small groups of cells at spatially resolved sites within biofilms.