Tumor-associated carbonic anhydrase 9 spatially coordinates intracellular pH in three-dimensional multicellular growths

CA9 is a membrane-tethered, carbonic anhydrase (CA) enzyme, expressed mainly at the external surface of cells, that catalyzes reversible CO(2) hydration. Expression is greatly enhanced in many tumors, particularly in aggressive carcinomas. The functional role of CA9 in tumors is not well established. Here we show that CA9, when expressed heterologously in cultured spheroids (0.5-mm diameter, ~25,000 cells) of RT112 cells (derived from bladder carcinoma), induces a near-uniform intracellular pH (pH(i)) throughout the structure. Dynamic pH(i) changes during displacements of superfusate CO(2) concentration are also spatially coincident (within 2 s). In contrast, spheroids of wild-type RT112 cells lacking CA9 exhibit an acidic core (~0.25 pH(i) reduction) and significant time delays (~9 s) for pH(i) changes in core versus peripheral regions. pH(i) non-uniformity also occurs in CA9-expressing spheroids after selective pharmacological inhibition of the enzyme. In isolated RT112 cells, pH(i) regulation is unaffected by CA9 expression. The influence of CA9 on pH(i) is thus only evident in multicellular tissue. Diffusion-reaction modeling indicates that CA9 coordinates pH(i) spatially by facilitating CO(2) diffusion in the unstirred extracellular space of the spheroid. We suggest that pH(i) coordination may favor survival and growth of a tumor. By disrupting spatial pH(i) control, inhibition of CA9 activity may offer a novel strategy for the clinical treatment of CA9-associated tumors.     

Fermentation of cellulose and cellobiose by Clostridium thermocellum in the absence of Methanobacterium thermoautotrophicum.

The fermentation of cellulose and cellobiose by Clostridium thermocellum monocultures and C. thermocellum/Methanobacterium thermoautotrophicum cocultures was studied. All cultures were grown under anaerobic conditions in batch culture at 60 degrees C. When grown on cellulose, the coculture exhibited a shorter lag before initiation and growth and celluloysis than did the monoculture. Cellulase activity appeared earlier in the coculture than in the monoculture; however, after growth had ceased, cellulase activity was greater in the monoculture. Monocultures produced primarily ethanol, acetic acid, H2 and CO2. Cocultures produced more H2 and acetic acid and less ethanol than did the monoculture. In the coculture, conversion of H2 to methane was usually complete, and most of the methane produced was derived from CO2 reduction rather than from acetate conversion. Agents of fermentation stoppage were found to be low pH and high concentrations of ethanol in the monoculture and low pH in the coculture. Fermentation of cellobiose was more rapid than that of cellulose. In cellobiose medium, the methanogen caused only slight changes in the fermentation balance of the Clostridium, and free H2 was produced.     

Inorganic carbon acquisition in red tide dinoflagellates

Carbon acquisition was investigated in three marine bloom-forming dinollagellates-Prorocentrum minimum, Heterocapsa triquetra and Ceratium lineatum. In vivo activities of extracellular and intracellular carbonic anhydrase (CA), photosynthetic O2 evolution, CO2 and HCO3- uptake rates were measured by membrane inlet mass spectrometry (MIMS) in cells acclimated to low pH (8.0) and high pH (8.5 or 9.1). A second approach used short-term 14C-disequilibrium incubations to estimate the carbon source utilized by the cells. All three species showed negligible extracellular CA (eCA) activity in cells acclimated to low pH and only slightly higher activity when acclimated to high pH. Intracellular CA (iCA) activity was present in all three species, but it increased only in P. minimum with increasing pH. Half-saturation concentrations (K1/2) for photosynthetic O2 evolution were low compared to ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) kinetics. Moreover, apparent affinities for inorganic carbon (Ci) increased with increasing pH in the acclimation, indicating the operation of an efficient CO2 concentration mechanism (CCM) in these dinoflagellates. Rates of CO2 uptake were comparably low and could not support the observed rates of photosynthesis. Consequently, rates of HCO3- uptake were high in the investigated species, contributing more than 80% of the photosynthetic carbon fixation. The affinity for HCO3- and maximum uptake rates increased under higher pH. The strong preference for HCO3- was also confirmed by the 14C-disequilibrium technique. Modes of carbon acquisition were consistent with the 13C-fractionation pattern observed and indicated a strong species-specific difference in leakage. These results suggest that photosynthesis in marine dinoflagellates is not limited by Ci even at high pH, which may occur during red tides in coastal waters.     

Acetate-inducible protein overexpression from the glnAp2 promoter of Escherichia coli.

The Ntr regulon in Escherichia coli has previously been engineered to control the expression of a heterologous metabolic pathway. In this study, we reengineered the same system for protein production. In the absence of NRII (glnL gene product), we showed that glnAp2 can be an effective promoter for protein production that is inducible by exogenous acetate, but both the induction ratio and the range of modulation are low. To deal with this issue, we inactivated phosphotransacetylase (pta gene product), which disrupts the acetate pathway and denies the cell the ability to synthesize acetate. With this additional modification, gene expression from glnAp2 can be controlled by directly adding acetate into the growth medium. Using a lacZ reporter fusion, we found that glnAp2 induction was modulatable over a range of potassium acetate concentrations, and the induction/noninduction ratio increased to 77 in the absence of pta. The extracellular acetate required for maximal induction is lower than the concentration that causes toxicity, and thus growth inhibition by acetate addition was not a matter of concern. Furthermore, compared to the P(tac) promoter, overexpression of a model protein using the modified glnAp2 promoter system did not cause significant growth inhibition, although a higher level of protein expression was achieved.     

Carbonic anhydrase activity in halophilic anaerobes from soda lakes

The activity and cellular localization of carbonic anhydrase (CA) in two alkaliphilic anaerobes growing in soda lakes at pH 9–10 were studied. CA activity in the cell extracts of the acetogenic bacterium Natroniella acetigena was comparable to that of neutrophilic acetogens. Hydrogenotrophically grown cells of Desulfonatronum lacustre exhibited higher CA activity compared to the cells grown on medium with formate. High CA activity in the cytoplasmic fraction and the absence of high activity in the extracellular fraction were demonstrated. We propose that the cytoplasmic CA in alkaliphilic sulfate-reducers participates in conversion of bicarbonate to CO₂, which is reduced in the cell to acetate via the acetyl-CoA pathway.     

Optimization of a cultivation process for recombinant protein production by Escherichia coli.

A single-stage fed-batch bioprocess for the production of a recombinant protein beta-galactosidase, by E. coli has been developed. The XL1-blue strain of E. coli which harbors a multi-number foreign plasmid PT was cultured in a reformulated medium. Critical medium components were selected and their respective concentrations were optimized with the Orthogonal Table method. An exponential substrate feeding schedule was used to maintain optimum conditions. Inhibition of growth and protein expression caused by excessive concentrations of glucose and acetate was investigated and subsequently minimized with an incremental nutrient feeding schedule which limited the specific growth rate of a culture. The program necessary to facilitate the control of substrate addition is fully described. This program has been used with a 2.5 l bioreactor and a commercially available software package for optimization without on-line or off-line measurement of optical density (OD), CO2, glucose or acetate. The optimized fed-batch process limited the acetate concentration to less than 20 mM; maintained an exponential growth phase for 50 h; and produced a cell density of 51 g l-1 dry cell weight (DCW) or 154 OD600 with a beta-galactosidase activity of 990 U ml-1.     

Hypoxia activates the capacity of tumor-associated carbonic anhydrase IX to acidify extracellular pH

Acidic extracellular pH (pHe) is a typical attribute of a tumor microenvironment, which has an impact on cancer development and treatment outcome. It was believed to result from an accumulation of lactic acid excessively produced by glycolysis. However, metabolic profiles of glycolysis-impaired tumors have revealed that CO2 is a significant source of acidity, thereby indicating a contribution of carbonic anhydrase (CA). The tumor-associated CA IX isoform is the best candidate, because its extracellular enzyme domain is highly active, expression is induced by hypoxia and correlates with poor prognosis. This study provides the first evidence for the role of CA IX in the control of pHe. We show that CA IX can acidify the pH of the culture medium in hypoxia but not in normoxia. This acidification can be perturbed by deletion of the enzyme active site and inhibited by CA IX-selective sulfonamides, which bind only to hypoxic cells containing CA IX. Our findings suggest that hypoxia regulates both expression and activity of CA IX in order to enhance the extracellular acidification, which may have important implications for tumor progression.     

Excretion and uptake of cadaverine by CadB and its physiological functions in Escherichia coli

The functions of the putative cadaverine transport protein CadB were studied in Escherichia coli. CadB had both cadaverine uptake activity, dependent on proton motive force, and cadaverine excretion activity, acting as a cadaverine-lysine antiporter. The Km values for uptake and excretion of cadaverine were 20.8 and 303 microM respectively. Both cadaverine uptake and cadaverine-lysine antiporter activities of CadB were functional in cells. Cell growth of a polyamine-requiring mutant was stimulated slightly at neutral pH by the cadaverine uptake activity and greatly at acidic pH by the cadaverine-lysine antiporter activity. At acidic pH, the operon containing cadB and cadA, encoding lysine decarboxylase, was induced in the presence of lysine. This caused neutralization of the extracellular medium and made possible the production of CO(2) and cadaverine and aminopropylcadaverine instead of putrescine and spermidine. The induction of the cadBA operon also generated a proton motive force. When the cadBA operon was not induced, the expression of the speF-potE operon, encoding inducible ornithine decarboxylase and a putrescine-ornithine antiporter, was increased. The results indicate that the cadBA operon plays important roles in cellular regulation at acidic pH.     

Metabolic flux modeling of detoxification of acetic acid by Ralstonia eutropha at slightly alkaline pH levels

Ralstonia eutropha grows on and produces polyhydroxyalkanoates (PHAs) from fermentation acids. Acetic acid, one major organic acid from acidogenesis of organic wastes, has an inhibitory effect on the bacterium at slightly alkaline pH (6 g HAc/L at pH 8). The tolerance of R. eutropha to acetate, however, was increased significantly up to 15 g/L at the slightly alkaline pH level with high cell mass concentration. A metabolic cell model with five fluxes is proposed to depict the detoxification mechanism including mass transfer and acetyl-CoA formation of acetic acid and the formation of three final metabolic products, polyhydroxybutyrate (PHB), active biomass, and CO(2). The fluxes were measured under different conditions such as cell mass concentration, acetic acid concentration, and medium composition. The experimental results indicate that the acetate detoxification by high cell mass concentration is attributed to the increased fluxes at high extracellular acetate concentrations. The fluxes could be doubled to reduce and hence detoxify the accumulated intracellular acetate anions.     

Effects of acetate on facultative autotrophy in Chlamydomonas reinhardtii assessed by photosynthetic measurements and stable isotope analyses

The green alga Chlamydomonas reinhardtii can grow photoautotrophically utilizing CO(2), heterotrophically utilizing acetate, and mixotrophically utilizing both carbon sources. Growth of cells in increasing concentrations of acetate plus 5% CO(2) in liquid culture progressively reduced photosynthetic CO(2) fixation and net O(2) evolution without effects on respiration, photosystem II efficiency (as measured by chlorophyll fluorescence), or growth. Using the technique of on-line oxygen isotope ratio mass spectrometry, we found that mixotrophic growth in acetate is not associated with activation of the cyanide-insensitive alternative oxidase pathway. The fraction of carbon biomass resulting from photosynthesis, determined by stable carbon isotope ratio mass spectrometry, declined dramatically (about 50%) in cells grown in acetate with saturating light and CO(2). Under these conditions, photosynthetic CO(2) fixation and O(2) evolution were also reduced by about 50%. Some growth conditions (e.g. limiting light, high acetate, solid medium in air) virtually abolished photosynthetic carbon gain. These effects of acetate were exacerbated in mutants with slowed electron transfer through the D1 reaction center protein of photosystem II or impaired chloroplast protein synthesis. Therefore, in mixotrophically grown cells of C. reinhardtii, interpretations of the effects of environmental or genetic manipulations of photosynthesis are likely to be confounded by acetate in the medium.     

Bacterial fermentation of cellulose: Effect of physical and chemical parameters

Acetivibrio celluloyticus converts cellulose directly to ethanol, acetate, H(2), and CO(2). The effects of various physical and chemical parameters, and their interdependence, including pH, temperature, redox, and ethanol toxicity on this fermentation, were studied. Controlling pH at 6.8 favored a predominance of ethanol over acetate. Supplementation of the medium with additional reductant, concomitant with a lower redox potential, incresed ethanol formation. Results from ethanol-challenged cultures indicated that cell lysis occurs with growing but not with nongrowing cells. A stable strain was adapted for growth in ethanol concentrations almost sevenfold greater than the parent organism.     

Relationship of formate to growth and methanogenesis by Methanococcus thermolithotrophicus

Methanococcus thermolithotrophicus is a methanogenic archaebacterium that can use either H2 or formate as its source of electrons for reduction of CO2 to methane. Growth and suspended-whole-cell experiments show that H2 plus CO2 methanogenesis was constitutive, while formate methanogenesis required adaptation time; selenium was necessary for formate utilization. Cells grown on formate had 20 to 100 times higher methanogenesis rates on formate than cells grown on H2-CO2 and transferred into formate medium. Enzyme assays with crude extracts and with F420 or methyl viologen as the electron acceptor revealed that hydrogenase was constitutive, while formate dehydrogenase was regulated. Cells grown on formate had 10 to 70 times higher formate dehydrogenase activity than cells grown on H2-CO2 with Se present in the medium; when no Se was added to H2-CO2 cultures, even lower activities were observed. Adaptation to and growth on formate were pH dependent, with an optimal pH for both about one pH unit above that optimal for H2-CO2 (pH 5.8 to 6.5). When cells were grown on H2-CO2 in the presence of formate, formate (greater than or equal to 50 mM) inhibited both growth and methanogenesis at pH 5.8 to 6.2, but not at pH greater than 6.6. Both acetate and propionate produced similar inhibition. Formate inhibition was also observed in Methanospirillum hungatei.     

Carbonic anhydrase is essential for growth of Ralstonia eutropha at ambient CO(2) concentrations

Mutant strain 25-1 of the facultative chemoautotroph Ralstonia eutropha H16 had previously been shown to exhibit an obligately high-CO(2)-requiring (HCR) phenotype. Although the requirement varied with the carbon and energy sources utilized, none of these conditions allowed growth at the air concentration of CO(2). In the present study, a gene designated can and encoding a beta-carbonic anhydrase (CA) was identified as the site altered in strain 25-1. The mutation caused a replacement of the highly conserved glycine residue 98 by aspartate in Can. A can deletion introduced into wild-type strain H16 generated mutant HB1, which showed the same HCR phenotype as mutant 25-1. Overexpression of can in Escherichia coli and mass spectrometric determination of CA activity demonstrated that can encodes a functional CA. The enzyme is inhibited by ethoxyzolamide and requires 40 mM MgSO(4) for maximal activity. Low but significant CA activities were detected in wild-type H16 but not in mutant HB1, strongly suggesting that the CA activity of Can is essential for growth of the wild type in the presence of low CO(2) concentrations. The HCR phenotype of HB1 was overcome by complementation with heterologous CA genes, indicating that growth of the organism at low CO(2) concentrations requires sufficient CA activity rather than the specific function of Can. The metabolic function(s) depending on CA activity remains to be identified.     

Relationship of formate to growth and methanogenesis by Methanococcus thermolithotrophicus.

Methanococcus thermolithotrophicus is a methanogenic archaebacterium that can use either H2 or formate as its source of electrons for reduction of CO2 to methane. Growth and suspended-whole-cell experiments show that H2 plus CO2 methanogenesis was constitutive, while formate methanogenesis required adaptation time; selenium was necessary for formate utilization. Cells grown on formate had 20 to 100 times higher methanogenesis rates on formate than cells grown on H2-CO2 and transferred into formate medium. Enzyme assays with crude extracts and with F420 or methyl viologen as the electron acceptor revealed that hydrogenase was constitutive, while formate dehydrogenase was regulated. Cells grown on formate had 10 to 70 times higher formate dehydrogenase activity than cells grown on H2-CO2 with Se present in the medium; when no Se was added to H2-CO2 cultures, even lower activities were observed. Adaptation to and growth on formate were pH dependent, with an optimal pH for both about one pH unit above that optimal for H2-CO2 (pH 5.8 to 6.5). When cells were grown on H2-CO2 in the presence of formate, formate (greater than or equal to 50 mM) inhibited both growth and methanogenesis at pH 5.8 to 6.2, but not at pH greater than 6.6. Both acetate and propionate produced similar inhibition. Formate inhibition was also observed in Methanospirillum hungatei.     

Role of extracellular electrolytes in the activation of ribosomal protein S6 kinase by epidermal growth factor, insulin-like growth factor 1, and insulin in ZR-75-1 cells

Activation of ribosomal protein S6 kinase by epidermal growth factor (EGF), insulin, and insulin-like growth factor 1 (IGF1) was studied in the human mammary tumor cell line ZR-75-1 in isotonic buffers. In contrast to growth factor-dependent S6 phosphorylation which is strongly dependent on extracellular pH (Chambard, J. C., and J. Pouyssegur. 1986. Exp. Cell Res. 164:282-294.) preincubation of cells in buffers with different pH values ranging from 7.5 to 6.5 had no effect on basal or EGF-stimulated S6 kinase activity. Replacement of extracellular Na+ with choline or replacement of extracellular Ca++ with EGTA also did not inhibit stimulation of S6 kinase by EGF. When intracellular Ca++ was buffered with the permeable Ca++ chelator quin2, EGF stimulation was reduced 50%. A similar inhibition of the EGF response was observed when cells were incubated in buffers with high K+ concentrations or in the presence of the K+ ionophore valinomycin. Insulin and IGF1 stimulation of S6 kinase were also inhibited by high K+ concentrations and by buffering intracellular Ca++. In contrast to the responses to EGF, insulin- and IGF1-activation of S6 kinase was enhanced when glucose was present and depended on the presence of bicarbonate in the medium. The results indicate that ionic signals generated by growth factors and insulin, such as increases in intracellular pH or Na+, do not seem to be involved in the activation of S6 kinase. However, effects of growth factors or insulin on membrane potential and/or K+ fluxes and redistribution of intracellular Ca++ may play a role in the activation process. Furthermore, the mechanism of insulin activation of S6 kinase is distinct from the growth factors by its dependency on extracellular bicarbonate.     

Carboanhydrase activity in halophilic anaerobes from soda lakes

The activity and cellular localization of carboanhydrase (CA) in two alkaliphilic anaerobes growing in soda lakes at pH 9-10 was studied. CA activity in the cell extracts of the acetogenic bacterium Natroniella acetigena was comparable to that of the neutrophilic acetogens. Hydrogenotrophically grown cells of Desulfonatronum lacustre exhibited higher CA activity compared to the cells grown on media with formate. High CA activity in the cytoplasmic fraction and the absence of high activity in the extracellular fraction were demonstrated. We propose that the cytoplasmic CA in alkaliphilic sulfate-reducers participates in conversion of bicarbonate to CO2, which is reduced in the cell to acetate via the acetyl-CoA pathway.