Biochemistry, Biophysics, and Structural Biology Commons^™

Cytoplasmic Ph Measurement And Homeostasis In Bacteria And Archaea., Joan Slonczewski

Joan Slonczewski

Of all the molecular determinants for growth, the hydronium and hydroxide ions are found naturally in the widest concentration range, from acid mine drainage below pH 0 to soda lakes above pH 13. Most bacteria and archaea have mechanisms that maintain their internal, cytoplasmic pH within a narrower range than the pH outside the cell, termed "pH homeostasis." Some mechanisms of pH homeostasis are specific to particular species or groups of microorganisms while some common principles apply across the pH spectrum. The measurement of internal pH of microbes presents challenges, which are addressed by a range of techniques under varying …

Ph Of The Cytoplasm And Periplasm Of Escherichia Coli: Rapid Measurement By Green Fluorescent Protein Fluorimetry, Joan Slonczewski, Jessica C. Wilks

Joan Slonczewski

Cytoplasmic pH and periplasmic pH of Escherichia coli cells in suspension were observed with 4-s time resolution using fluorimetry of TorA-green fluorescent protein mutant 3* (TorA-GFPmut3*) and TetR-yellow fluorescent protein. Fluorescence intensity was correlated with pH using cell suspensions containing 20 mM benzoate, which equalizes the cytoplasmic pH with the external pH. When the external pH was lowered from pH 7.5 to 5.5, the cytoplasmic pH fell within 10 to 20 s to pH 5.6 to 6.5. Rapid recovery occurred until about 30 s after HCl addition and was followed by slower recovery over the next 5 min. As a …

Cytoplasmic Ph Response To Acid Stress In Individual Cells Of Escherichia Coli And Bacillus Subtilis Observed By Fluorescence Ratio Imaging Microscopy, Joan Slonczewski, Keith A. Martinez Ii, Ryan D. Kitko, J. Patrick Mershon, Haley E. Adcox, Kotiba A. Malek, Melanie B. Berkmen

Joan Slonczewski

The ability of Escherichia coli and Bacillus subtilis to regulate their cytoplasmic pH is well studied in cell suspensions but is poorly understood in individual adherent cells and biofilms. We observed the cytoplasmic pH of individual cells using ratiometric pHluorin. A standard curve equating the fluorescence ratio with pH was obtained by perfusion at a range of external pH 5.0 to 9.0, with uncouplers that collapse the transmembrane pH difference. Adherent cells were acid stressed by switching the perfusion medium from pH 7.5 to pH 5.5. The E. coli cytoplasmic pH fell to a value that varied among individual cells …

Ph Regulates Genes For Flagellar Motility, Catabolism, And Oxidative Stress In Escherichia Coli K-12†, Joan Slonczewski

Joan Slonczewski

Gene expression profiles of Escherichia coli K-12 W3110 were compared as a function of steady-state external pH. Cultures were grown to an optical density at 600 nm of 0.3 in potassium-modified Luria-Bertani medium buffered at pH 5.0, 7.0, and 8.7. For each of the three pH conditions, cDNA from RNA of five independent cultures was hybridized to Affymetrix E. coli arrays. Normalized expression indices were calculated for each gene and intergenic region (IG). Differential expression among the three pH classes was observed for 763 genes and 353 IGs. Hierarchical clustering yielded six well-defined clusters of pH profiles, designated Acid High …

Generalization Of Dna Microarray Dispersion Properties: Microarray Equivalent Of T-Distribution, Joan Slonczewski

Joan Slonczewski

Background: DNA microarrays are a powerful technology that can provide a wealth of gene expression data for disease studies, drug development, and a wide scope of other investigations. Because of the large volume and inherent variability of DNA microarray data, many new statistical methods have been developed for evaluating the significance of the observed differences in gene expression. However, until now little attention has been given to the characterization of dispersion of DNA microarray data. Results: Here we examine the expression data obtained from 682 Affymetrix GeneChips® with 22 different types and we demonstrate that the Gaussian (normal) frequency distribution …

Escherichia Coli K-12 Survives Anaerobic Exposure At Ph 2 Without Rpos, Gad, Or Hydrogenases, But Shows Sensitivity To Autoclaved Broth Products, Joan Slonczewski, Daniel P. Riggins, Maria J. Narvaez, Keith A. Martinez, Mark M. Harden

Joan Slonczewski

Escherichia coli and other enteric bacteria survive exposure to extreme acid (pH 2 or lower) in gastric fluid. Aerated cultures survive via regulons expressing glutamate decarboxylase (Gad, activated by RpoS), cyclopropane fatty acid synthase (Cfa) and others. But extreme-acid survival is rarely tested under low oxygen, a condition found in the stomach and the intestinal tract. We observed survival of E. coli K-12 W3110 at pH 1.2–pH 2.0, conducting all manipulations (overnight culture at pH 5.5, extreme-acid exposure, dilution and plating) in a glove box excluding oxygen (10% H2, 5% CO2, balance N2). With dissolved O2 concentrations maintained below 6 …

Hydrogenase-3 Contributes To Anaerobic Acid Resistance Of Escherichia Coli, Joan Slonczewski, Ken Noguchi, Daniel P. Riggins, Khalid C. Eldahan, Ryan D. Kitko

Joan Slonczewski

Background: Hydrogen production by fermenting bacteria such as Escherichia coli offers a potential source of hydrogen biofuel. Because H2 production involves consumption of 2H+ , hydrogenase expression is likely to involve pH response and regulation. Hydrogenase consumption of protons in E. coli has been implicated in acid resistance, the ability to survive exposure to acid levels (pH 2–2.5) that are three pH units lower than the pH limit of growth (pH 5–6). Enhanced survival in acid enables a larger infective inoculum to pass through the stomach and colonize the intestine. Most acid resistance mechanisms have been defined using aerobic cultures, …

Osmolytes Contribute To Ph Homeostasis Of Escherichia Coli, Joan Slonczewski, Ryan D. Kitko, Jessica C. Wilks, Gian M. Garduque

Joan Slonczewski

Background: Cytoplasmic pH homeostasis in Escherichia coli includes numerous mechanisms involving pH-dependent catabolism and ion fluxes. An important contributor is transmembrane K+ flux, but the actual basis of K+ compensation for pH stress remains unclear. Osmoprotection could mediate the pH protection afforded by K+ and other osmolytes. Methods and Principal Findings: The cytoplasmic pH of E. coli K-12 strains was measured by GFPmut3 fluorimetry. The wild-type strain Frag1 was exposed to rapid external acidification by HCl addition. Recovery of cytoplasmic pH was enhanced equally by supplementation with NaCl, KCl, proline, or sucrose. A triple mutant strain TK2420 defective for the …

Oxygen Limitation Modulates Ph Regulation Of Catabolism And Hydrogenases, Multidrug Transporters, And Envelope Composition In Escherichia Coli K-12, Joan Slonczewski, Everett T. Hayes, Jessica C. Wilks, Piero Sanfilippo, Elizabeth Yohannes, Daniel P. Tate, Brian D. Jones, Michael D. Radmacher, Sandra S. Bondurant

Joan Slonczewski

Background: In Escherichia coli, pH regulates genes for amino-acid and sugar catabolism, electron transport, oxidative stress, periplasmic and envelope proteins. Many pH-dependent genes are co-regulated by anaerobiosis, but the overall intersection of pH stress and oxygen limitation has not been investigated. Results: The pH dependence of gene expression was analyzed in oxygen-limited cultures of E. coli K-12 strain W3110. E. coli K-12 strain W3110 was cultured in closed tubes containing LBK broth buffered at pH 5.7, pH 7.0, and pH 8.5. Affymetrix array hybridization revealed pH-dependent expression of 1,384 genes and 610 intergenic regions. A core group of 251 genes …

Ph-Dependent Expression Of Periplasmic Proteins And Amino Acid Catabolism In Escherichia Coli, Joan Slonczewski, Lauren M. Stancik, Dawn M. Stancik, Brian Schmidt, D. Michael Barnhart, Yuliya N. Yoncheva

Joan Slonczewski

Escherichia coli grows over a wide range of pHs (pH 4.4 to 9.2), and its own metabolism shifts the external pH toward either extreme, depending on available nutrients and electron acceptors. Responses to pH values across the growth range were examined through two-dimensional electrophoresis (2-D gels) of the proteome and through lac gene fusions. Strain W3110 was grown to early log phase in complex broth buffered at pH 4.9, 6.0, 8.0, or 9.1. 2-D gel analysis revealed the pH dependence of 19 proteins not previously known to be pH dependent. At low pH, several acetate-induced proteins were elevated (LuxS, Tpx, …

Ph-Dependent Catabolic Protein Expression During Anaerobic Growth Of Escherichia Coli K-12, Joan Slonczewski, Elizabeth Yohannes, D. Michael Barnhart

Joan Slonczewski

During aerobic growth of Escherichia coli, expression of catabolic enzymes and envelope and periplasmic proteins is regulated by pH. Additional modes of pH regulation were revealed under anaerobiosis. E. coli K-12 strain W3110 was cultured anaerobically in broth medium buffered at pH 5.5 or 8.5 for protein identification on proteomic two-dimensional gels. A total of 32 proteins from anaerobic cultures show pH-dependent expression, and only four of these proteins (DsbA, TnaA, GatY, and HdeA) showed pH regulation in aerated cultures. The levels of 19 proteins were elevated at the high pH; these proteins included metabolic enzymes (DhaKLM, GapA, TnaA, HisC, …

Polyamine Stress At High Ph In Escherichia Coli K-12, Joan Slonczewski, Elizabeth Yohannes, Amy E. Thurber, Jessica C. Wilks, Daniel P. Tate

Joan Slonczewski

Background: Polyamines such as spermine and spermidine are required for growth of Escherichia coli; they interact with nucleic acids, and they bind to ribosomes. Polyamines block porins and decrease membrane permeability, activities that may protect cells in acid. At high concentrations, however, polyamines impair growth. They impair growth more severely at high pH, probably due to their increased uptake as membrane-permeant weak bases. The role of pH is critical in understanding polyamine stress. Results: The effect of polyamines was tested on survival of Escherichia coli K-12 W3110 in extreme acid or base (pH conditions outside the growth range). At pH …

Homeotic Gene Teashirt (Tsh) Has A Neuroprotective Function In Amyloid-Beta 42 Mediated Neurodegeneration, Michael T. Moran, Meghana Tare, Madhuri Kango-Singh, Amit Singh

Madhuri Kango-Singh

Background: Alzheimer's disease (AD) is a debilitating age related progressive neurodegenerative disorder characterized by the loss of cognition, and eventual death of the affected individual. One of the major causes of AD is the accumulation of Amyloid-beta 42 (Aβ42) polypeptides formed by the improper cleavage of amyloid precursor protein (APP) in the brain. These plaques disrupt normal cellular processes through oxidative stress and aberrant signaling resulting in the loss of synaptic activity and death of the neurons. However, the detailed genetic mechanism(s) responsible for this neurodegeneration still remain elusive. Methodology/Principal Findings: We have generated a transgenic Drosophila eye model where …

Homeotic Gene Teashirt (Tsh) Has A Neuroprotective Function In Amyloid-Beta 42 Mediated Neurodegeneration, Michael T. Moran, Meghana Tare, Madhuri Kango-Singh, Amit Singh

Amit Singh

Background: Alzheimer's disease (AD) is a debilitating age related progressive neurodegenerative disorder characterized by the loss of cognition, and eventual death of the affected individual. One of the major causes of AD is the accumulation of Amyloid-beta 42 (Aβ42) polypeptides formed by the improper cleavage of amyloid precursor protein (APP) in the brain. These plaques disrupt normal cellular processes through oxidative stress and aberrant signaling resulting in the loss of synaptic activity and death of the neurons. However, the detailed genetic mechanism(s) responsible for this neurodegeneration still remain elusive. Methodology/Principal Findings: We have generated a transgenic Drosophila eye model where …

Identification Of Ctla2a, Defb29, Wfdc15b, Serpina1f And Mup19 As Novel Tissue-Specific Secretory Factors In Mouse..Pdf, Jibin Zhang, Jinsoo Ahn, Yeunsu Suh, Seongsoo Hwang, Michael E. Davis, Kichoon Lee

Jibin Zhang

Heterogeneous Dynamics In Dna Site Discrimination By The Structurally Homologous Dna-Binding Domains Of Ets-Family Transcription Factors, Gaofei He, Ana Tolic, James K. Bashkin, Gregory M. K. Poon

James Bashkin

Conditional Regulation Of Puf1p, Puf4p, And Puf5p Activity Alters Yhb1 Mrna Stability For A Rapid Response To Toxic Nitric Oxide Stress In Yeast, Joseph Russo, Wendy M. Olivas

Wendy Olivas