Proteomic characterization of acid stress response in Synechocystis sp. PCC 6803

A comparative proteomic analysis using 2-DE coupled with MALDI-MS and LC-MS/MS was performed in Synechocystis sp. PCC 6803 to identify protein candidates involved in acid stress response in cyanobacteria. Comparison of soluble proteins from the cytoplasmic fraction of cells grown on media set at pH 7.5 and 5.5 using 2-DE identified four proteins, which showed significant changes in the abundance. Surprisingly, several general stress proteins, either the heat shock family proteins or chaperonins, did not show perceptible fold changes in response to acidity. Compared to the cytoplasmic proteome, the periplasmic proteome showed remarkable changes as a function of external pH. Protein expression profiling at different external pH, i.e., 9.0, 7.5, 6.0 and 5.5, allowed classifying the periplasmic proteins depending on their preferential expression patterns towards acidity or alkalinity. Among the acid- and base-induced proteins, oxalate decarboxylase and carbonic anhydrase were already known for their role in pH homeostasis. Several unknown proteins from the periplasm, that showed significant changes in response to pH, provide ideal targets for further studies in understanding pH stress response in cyanobacteria. This study also identified 14 novel proteins, hitherto unknown from the periplasmic space of Synechocystis.     

Unidirectional polar growth of cells of Seliberia stellata and aquatic seliberia-like bacteria revealed by immunoferritin labeling

When grown in a complex peptone-yeast extract culture medium, Seliberia stellata and related morphologically similar aquatic bacterial strains typically divided asymmetrically, giving rise to a motile swarmer and a longer sessile rod. Indirect immunoferritin labeling of these bacteria, followed by incubation during which cell growth occurred, has provided evidence that antigenic cell-surface components are synthesized de novo in a sharply demarcated zone at one pole of the growing parent cells. Cell elongation occurred unidirectionally from the pole showing the de novo surface synthesis; it was this end of the elongating, helically sculptured (i.e., screw-like) rod that became the daughter swarmer cell. The daughter swarmers, produced after polar growth and division of the immunoferritinlabeled parent cells, were not labeled. The immunoferritin label remaining on the parent cell did not appear to be diluted or disturbed by the cell growth and division process. Under the cultural conditions used in this study, the growth and division events which led to production of swarmer cells in the seliberia strains examined met two major criteria of accepted definitions of budding (de novo cell surface synthesis and transverse asymmetry of division). However, the developing daughter cell was not initially narrower than the parent and thus did not increase in cell diameter during growth.In memory: R. Y. Stanier     

The Role of auxin,pH, and stress in the activation of embryogenic cell division in leaf protoplast-derived cells of alfalfa

Culturing leaf protoplast-derived cells of the embryogenic alfalfa (Medicago sativa subsp. varia A2) genotype in the presence of low (1 microM) or high (10 microM) 2, 4-dichlorophenoxyacetic acid (2,4-D) concentrations results in different cell types. Cells exposed to high 2,4-D concentration remain small with dense cytoplasm and can develop into proembryogenic cell clusters, whereas protoplasts cultured at low auxin concentration elongate and subsequently die or form undifferentiated cell colonies. Fe stress applied at nonlethal concentrations (1 mM) in the presence of 1 microM 2,4-D also resulted in the development of the embryogenic cell type. Although cytoplasmic alkalinization was detected during cell activation of both types, embryogenic cells could be characterized by earlier cell division, a more alkalic vacuolar pH, and nonfunctional chloroplasts as compared with the elongated, nonembryogenic cells. Buffering of the 10 microM 2,4-D-containing culture medium by 10 mM 2-(N-morpholino)ethanesulfonic acid delayed cell division and resulted in nonembryogenic cell-type formation. The level of endogenous indoleacetic acid (IAA) increased transiently in all protoplast cultures during the first 4 to 5 d, but an earlier peak of IAA accumulation correlated with the earlier activation of the division cycle in embryogenic-type cells. However, this IAA peak could also be delayed by buffering of the medium pH by 2-(N-morpholino)ethanesulfonic acid. Based on the above data, we propose the involvement of stress responses, endogenous auxin synthesis, and the establishment of cellular pH gradients in the formation of the embryogenic cell type.     

The centrosome and asymmetric cell division

Asymmetric stem cell division is a mechanism widely employed by the cell to maintain tissue homeostasis, resulting in the production of one stem cell and one differentiating cell. However, asymmetric cell division is not limited to stem cells and is widely observed even in unicellular organisms as well as in cells that make up highly complex tissues. In asymmetric cell division, cells must organize their intracellular components along the axis of asymmetry (sometimes in the context of extracellular architecture). Recent studies have described cell asymmetry in many cell types and in many cases such asymmetry involves the centrosome (or spindle pole body in yeast) as the center of cytoskeleton organization. In this review, I summarize recent discoveries in cellular polarity that lead to an asymmetric outcome, with a focus on centrosome function.Key words: stem cell, asymmetric division, niche, centrosome, spindle orientation     

Pole cap formation in Escherichia coli following induction of the maltose-binding protein

After induction with maltose, 30–40% of the total protein in the osmotic shock fluid consist of maltose-binding protein while the induction ratio (maltose versus glycerol grown cells) for the amount of binding protein synthesized as well as for maltose transport is in the order of 10. Induction of maltose transport does not occur during all times of the cell cycle, but only shortly before cell division. Electronmicroscopic analysis of cells grown logarithmically on glycerol or maltose revealed in the latter the formation of large pole caps. These pole caps arise from an enlargement of the periplasmic space. Small cells contain one pole cap, large cells contain two. Pulse label studies with strain BUG-6, a mutant that is temperature sensitive for cell division reveal the following: Growth at the non-permissive temperature prevents maltose-binding protein synthesis and formation of new transport capacity.After shifting to the permissive temperature the cells regain both functions. Simultaneously, the newly formed cells exhibit pole caps.We conclude that the induction of maltose-binding protein is responsible for the formation of pole caps. In addition, beside the presence of inducer, cell cycle events occuring during division are necessary for the synthesis of maltose-binding protein.Non Standard Abbreviations GLPT periplasmic protein, related to transport of glycerolphosphate in Escherichia coli (Silhavy et al., 1976b)     

The centrosome and asymmetric cell division

Asymmetric stem cell division is a mechanism widely employed by the cell to maintain tissue homeostasis, resulting in the production of one stem cell and one differentiating cell. However, asymmetric cell division is not limited to stem cells and is widely observed even in unicellular organisms as well as in cells that make up highly complex tissues. In asymmetric cell division, cells must organize their intracellular components along the axis of asymmetry(sometimes in the context of extracellular architecture). Recent studies have described cell asymmetry in many cell types, and in many cases such asymmetry involves the centrosome (or spindle pole body in yeast) as the center of cytoskeleton organization. In this review, I summarize recent discoveries in cellular polarity that lead to an asymmetric outcome, with a focus on centrosome function.     

Cytoplasmic pH response to acid stress in individual cells of Escherichia coli and Bacillus subtilis observed by fluorescence ratio imaging microscopy

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 (range of pH 6.2 to 6.8), but a majority of cells recovered (to pH 7.0 to 7.5) within 2 min. In an E. coli biofilm, cells shifted from pH 7.5 to pH 5.5 failed to recover cytoplasmic pH. Following a smaller shift (from pH 7.5 to pH 6.0), most biofilm cells recovered fully, although the pH decreased further than that of isolated adherent cells, and recovery took longer (7 min or longer). Some biofilm cells began to recover pH and then failed, a response not seen in isolated cells. B. subtilis cells were acid shifted from pH 7.5 to pH 6.0. In B. subtilis, unlike the case with E. coli, cytoplasmic pH showed no "overshoot" but fell to a level that was maintained. This level of cytoplasmic pH post-acid shift varied among individual B. subtilis cells (range of pH, 7.0 to 7.7). Overall, the cytoplasmic pHs of individual bacteria show important variation in the acid stress response, including novel responses in biofilms.     

Inhibition by ultraviolet light of pole cell formation in Smittia sp (Chironomidae,Diptera): Action spectrum and photoreversibility

The formation of pole cells (primordial germ cells) in Smittia sp can be inhibited by ultraviolet (uv) irradiation without causing significant mortality. Until 70 min after egg deposition, pole cells are suppressed by low uv doses applied to the posterior pole region. Microbeam irradiation of a target area including the oosome inhibits pole cell formation; this is not observed after irradiation of other target areas. The action spectrum for uv inhibition of pole cells shows a distinct peak at 260 nm; its shape suggests that a nucleic acid or nucleic acid-protein complex acts as an effective target. Independent evidence for the involvement of a nucleic acid moiety is derived from the fact that uv inhibition of pole cell formation is photoreversible. The results are discussed in the context of pole cell determination by localized cytoplasmic components.     

The developmental fate of fission yeast cells is determined by the pattern of inheritance of parental and grandparental DNA strands.

A key feature for development consists of producing sister cells that differ in their potential for cellular differentiation. Following two cell divisions, a haploid Schizosaccharomyces pombe cell produces one cell in four 'granddaughters' with a changed mating cell type, implying nonequivalence of sister cells in each of two consecutive cell divisions. The observed pattern of switching is analogous to the mammalian 'stem cell' lineage by which a cell produces one daughter like itself while the other daughter is advanced in its developmental program. It is tested here whether sisters differ because of unequal distribution of cytoplasmic and/or nuclear components to them or due to inheriting a specific parental DNA chain at the mating type locus. Only the DNA strand-segregation model predicts that those cells engineered to contain an inverted tandem duplication of the mating type locus should produce equivalent sisters. Consequently, two 'cousins' in four related granddaughter cells should switch. The results verified the prediction, thus establishing that all cells otherwise fully possess the potential to switch. Therefore, the program of cell type change in S.pombe cell lineages is determined by the pattern of DNA strand inheritance at the mating type locus. A specific DNA sequence present at the mating type locus is postulated to be the cause of developmental asymmetry between sister cells. A general model for cellular differentiation is proposed in which the act of DNA replication itself is hypothesized to produce developmentally nonequivalent sister genomes.     

Artificial control of cytoplasmic pH and its bearing on cytoplasmic streaming,electrogenesis and excitability of characeae cells

Effects of changing the cytoplasmic pH on the cytoplasmic streaming, membrane potential and membrane excitability were studied in tonoplast-free cells ofChara australis andNitellopsis obtusa. The cytoplasmic pH was varied by internal perfusion of pH-buffered media.Nitellopsis cells were perfused only once, whileChara cells were perfused twice to control the pH more accurately. In both materials the rate of cytoplasmic streaming was maximum at about pH 7, low at pH 8.5–9 and almost zero at pH 5–5.5. The membrane potential was most negative at about pH 7. InChara the membrane potential supported by Mg·ATP was strongly inhibited at pH 5.5, and almost zero at pH 9, supporting the results obtained by Fujiiet al. (1979) on cells ofChara australis which were perfused once. The action potential could be induced by electrical stimulation inChara at pH 6.0–9.0 and inNitellopsis at pH 6.6–7.9. The membrane resistance ofNitellopsis was high at acidic and neutral pH values and low at alkaline pH, while that ofChara was low at both acidic and alkaline pH values.     

Cell contact-dependent positioning of the D cleavage plane restricts eye development in the Ilyanassa embryo

In embryos of the gastropod Ilyanassa obsoleta, the first-quartet micromeres of the A, B and C lineages (1a, 1b, and 1c) are each competent to form an eye in response to signaling from the 3D cell. The first-quartet micromere of the dorsal D lineage (1d) is smaller than the others, divides at a slower rate, and lacks the ability to form an eye. These properties of 1d all depend on inheritance of vegetal polar lobe cytoplasm by its mother cell D at second cleavage. I show that they depend also on the presence of cells adjacent to D during the late four-cell stage: after ablation of the A and/or C cells before this stage, 1d inherits more cytoplasm than normal, divides more rapidly, and frequently forms an eye. In non-D lineages, cleavage plane positioning and micromere division rates are relatively insensitive to cell contacts. Compressing whole embryos during third cleavage also leads to an increase in 1d volume correlated with abnormal eye formation; this suggests that the normal effect of cell contacts is to position the D cell cleavage furrow closer to the animal pole, and the enhanced division asymmetry of the D cell contributes to the suppression of eye development.     

Localization of Cell Division Protein FtsQ by Immunofluorescence Microscopy in Dividing and Nondividing Cells of Escherichia coli

The localization of cell division protein FtsQ in Escherichia coli wild-type cells was studied by immunofluorescence microscopy with specific monoclonal antibodies. FtsQ could be localized to the division site in constricting cells. FtsQ could also localize to the division site in ftsQ1(Ts) cells grown at the permissive temperature. A hybrid protein in which the cytoplasmic domain and the transmembrane domain were derived from the γ form of penicillin-binding protein 1B and the periplasmic domain was derived from FtsQ was also able to localize to the division site. This result indicates that the periplasmic domain of FtsQ determines the localization of FtsQ, as has also been concluded by others for the periplasmic domain of FtsN. Noncentral FtsQ foci were found in the area of the cell where the nucleoid resides and were therefore assumed to represent sites where the FtsQ protein is synthesized and simultaneously inserted into the cytoplasmic membrane.     

pH of the cytoplasm and periplasm of Escherichia coli: rapid measurement by green fluorescent protein fluorimetry

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 control, KCl addition had no effect on fluorescence. In the presence of 5 to 10 mM acetate or benzoate, recovery from external acidification was diminished. Addition of benzoate at pH 7.0 resulted in cytoplasmic acidification with only slow recovery. Periplasmic pH was observed using TorA-GFPmut3* exported to the periplasm through the Tat system. The periplasmic location of the fusion protein was confirmed by the observation that osmotic shock greatly decreased the periplasmic fluorescence signal by loss of the protein but had no effect on the fluorescence of the cytoplasmic protein. Based on GFPmut3* fluorescence, the pH of the periplasm equaled the external pH under all conditions tested, including rapid acid shift. Benzoate addition had no effect on periplasmic pH. The cytoplasmic pH of E. coli was measured with 4-s time resolution using a method that can be applied to any strain construct, and the periplasmic pH was measured directly for the first time.     

Is Escherichia coli getting old?

Whether or not bacteria divide symmetrically, the inheritance of cell poles is always asymmetrical. Because each cell carries an old and a new pole, its daughters will not be the same. Tracking poles of cells and measuring their lengths and doubling times in micro-colonies, Stewart et al.1 observed that growth rate diminished in cells inheriting old poles and concluded that these cells are susceptible to aging. Here, their results are compared with studies on the variabilities of length and age at division. It is argued that the decreased growth rate in old pole cells falls within the expected variation and may therefore be sufficiently far from a catastrophe-like cell death through aging.     

FtsL, an Essential Cytoplasmic Membrane Protein Involved in Cell Division in Escherichia coli

We have identified a gene involved in bacterial cell division, located immediately upstream of the ftsI gene in the min 2 region of the Escherichia coli chromosome. This gene, which we named ftsL, was detected through characterization of TnphoA insertions in a plasmid containing this chromosomal region. TnphoA topological analysis and fractionation of alkaline phosphatase fusion proteins indicated that the ftsL gene product is a 13.6-kDa cytoplasmic membrane protein with a cytoplasmic amino terminus, a single membrane-spanning segment, and a periplasmic carboxy terminus. The ftsL gene is essential for cell growth and division. A null mutation in ftsL resulted in inhibition of cell division, formation of long, nonseptate filaments, ultimate cessation of growth, and lysis. Under certain growth conditions, depletion of FtsL or expression of the largest ftsL-phoA fusion produced a variety of cell morphologies, including Y-shaped bacteria, indicating a possible general weakening of the cell wall. The FtsL protein is estimated to be present at about 20 to 40 copies per cell. The periplasmic domain of the protein displays a sequence with features characteristic of leucine zippers, which are involved in protein dimerization.     

The changes of proteomes components of Helicobacter pylori in response to acid stress without urea

Acid stress is the most obvious challenge Helicobacter pylori encounters in human stomach. The urease system is the basic process used to maintain periplasmic and cytoplasmic pH near neutrality when H. pylori is exposed to acidic condition. However, since the urea concentration in gastric juice is approximately 1 mM, considered possibly insufficient to ensure the survival of H. pylori, it is postulated that additional mechanisms of pH homeostasis may contribute to the acid adaptation in H. pylori. In order to identify the acid-related proteins other than the urease system we have compared the proteome profiles of H. pylori strain 26695 exposed to different levels of external pH (7.4, 6.0, 5.0, 4.0, 3.0, and 2.0) for 30 min in the absence of urea using 2-DE. Differentially expressed proteins were identified by MALDI-TOF-TOF-MS analysis, which turned out to be 36 different proteins. The functions of these proteins included ammonia production, molecular chaperones, energy metabolism, cell envelope, response regulator and some proteins with unknown function. SOM analysis indicated that H. pylori responds to acid stress through multi-mechanisms involving many proteins, which depend on the levels of acidity the cells encounter.     

Growth‐driven displacement of protein aggregates along the cell length ensures partitioning to both daughter cells in Caulobacter crescentus

All living cells must cope with protein aggregation, which occurs as a result of experiencing stress. In previously studied bacteria, aggregated protein is collected at the cell poles and is retained throughout consecutive cell divisions only in old pole‐inheriting daughter cells, resulting in aggregation‐free progeny within a few generations. In this study, we describe the in vivo kinetics of aggregate formation and elimination following heat and antibiotic stress in the asymmetrically dividing bacterium Caulobacter crescentus. Unexpectedly, in this bacterium, protein aggregates form as multiple distributed foci located throughout the cell volume. Time‐lapse microscopy revealed that under moderate stress, the majority of these protein aggregates are short‐lived and rapidly dissolved by the major chaperone DnaK and the disaggregase ClpB. Severe stress or genetic perturbation of the protein quality control machinery induces the formation of long‐lived aggregates. Importantly, the majority of persistent aggregates neither collect at the cell poles nor are they partitioned to only one daughter cell type. Instead, we show that aggregates are distributed to both daughter cells in the same ratio at each division, which is driven by the continuous elongation of the growing mother cell. Therefore, our study has revealed a new pattern of protein aggregate inheritance in bacteria.     

The hyperpolarizing and depolarizing effects of 2,4-dinitrophenol on ehrlich cells

The ability of glucose to reverse the effects of dinitrophenol on amino acid uptake in Ehrlich cells is a function of pH. At pH 6.0, the presence of glucose does not reverse the inhibitory action of the uncoupler. Nearly complete restoration occurs with glucose at pH 7.4. At pH 8, the presence of glucose may cause a modest increase in amino acid uptake in presence of dinitrophenol. At all pH values, glucose restores ATP and cellular K⁺ to the control levels at the same pH. Although the cytoplasmic pH changes with changes in the external pH, the cell interior is more alkaline than the medium near pH 6.0 and more acid than the medium at pH 7.8 even after 45 min incubation at 37°C. With dinitrophenol and in presence of glucose the difference in pH between the medium and the cell is minimal at both pH 6.0 and 7.8.     

Cosegregation of asymmetric features during cell division

Cellular asymmetry plays a major role in the ageing and evolution of multicellular organisms. However, it remains unknown how the cell distinguishes ‘old’ from ‘new’ and whether asymmetry is an attribute of highly specialized cells or a feature inherent in all cells. Here, we investigate the segregation of three asymmetric features: old and new DNA, the spindle pole body (SPB, the centrosome analogue) and the old and new cell ends, using a simple unicellular eukaryote, Schizosaccharomyces pombe. To our knowledge, this is the first study exploring three asymmetric features in the same cells. We show that of the three chromosomes of S. pombe, chromosome I containing the new parental strand, preferentially segregated to the cells inheriting the old cell end. Furthermore, the new SPB also preferentially segregated to the cells inheriting the old end. Our results suggest that the ability to distinguish ‘old’ from ‘new’ and to segregate DNA asymmetrically are inherent features even in simple unicellular eukaryotes.     

Repair of Injury in Freeze-Dried Salmonella anatum

Repair of injury induced by freeze-drying Salmonella anatum in nonfat milk solids occurred rapidly after rehydration. Injury in surviving cells was defined as the inability to form colonies on a plating medium containing deoxycholate. Death was defined as inability to form colonies in the same medium without this selective agent. The rate of repair of injury was reduced by lowering the temperature from 35 C to 10 C and was extremely low at 1 C. Repair was independent of influence of pH between 6.0 and 7.0. Repair did not require synthesis of protein, ribonucleic acid, or cell wall mucopeptide, but did require energy in the form of adenosine triphosphate (ATP) synthesized through oxidative phosphorylation. The requirement for ATP was based on dinitrophenol or cyanide interference with repair. Dinitrophenol activity was pH-dependent; no repair occurred at pH 6.0 and some repair was observed at pH 6.5 and above. Injured cells were extremely sensitive to low concentrations of ethylenedinitrilotetraacetate. This indicated that freeze-drying injury of S. anatum may involve the lipopolysaccharide portion of the cell wall and that repair of this damage requires ATP synthesis.