Macromolecular intelligence in microorganisms.

Biochemistry and molecular biology have been focusing on the structural, catalytic, and regulatory properties of individual macromolecules from the perspective of clarifying the mechanisms of metabolism and gene expression. Complete genomes of 'primitive' living organisms seem to be substantially larger than necessary for metabolism and gene expression alone. This is in line with the findings of silent phenotypes for supposedly important genes, apparent redundancy of functions, and variegated networks of signal transduction and transcription factors. Here we propose that evolutionary optimization has been much more intensive than to lead to the bare minima necessary for autonomous life. Much more complex organisms prevail. Much of this complexity arises in the nonlinear interactions between cellular macromolecules and in subtle differences between paralogs (isoenzymes). The complexity can only be understood when analyzed quantitatively, for which quantitative experimentation is needed in living systems that are as simple and manipulatable as possible, yet complex in the above sense. We illustrate this for the glutamine synthetase cascade in Escherichia coli. By reviewing recent molecular findings, we show that this cascade is much more complex than necessary for simple regulation of ammonia assimilation. Simulations suggest that the function of this complexity may lie in quasi-intelligent behavior, including conditioning and learning.

atp Mutants of Escherichia coli fail to grow on succinate due to a transport deficiency.

Escherichia coli atp mutants, which lack a functional H+-ATPase complex, are capable of growth on glucose but not on succinate or other C4-dicarboxylates (Suc- phenotype). Suc+ revertants of an atp deletion strain were isolated which were capable of growth on succinate even though they lack the entire H+-ATPase complex. Complementation in trans with the yhiF gene suppressed the growth of the Suc+ mutants on succinate, which implicates the yhiF gene product in the regulation of C4-dicarboxylate metabolism. Indeed, when the E. coli C4-dicarboxylate transporter (encoded by the dctA gene) was expressed in trans, the Suc- phenotype of the atp deletion strain reverted to Suc+, which shows that the reason why the E. coli atp mutant is unable to grow aerobically on C4-dicarboxylates is insufficient transport capacity for these substrates.

Coproporphyrin excretion by Azorhizobium caulinodans under micro-aerobic conditions.

Azorhizobium caulinodans ORS571 was found to excrete moderate amounts of a fluorescent pigment into the culture medium in response to dissolved oxygen tensions below 1.0 kPa. The pigment was identified as coproporphyrin, on the basis of its optical and fluorescence spectra. FixLJ and fixK mutant derivatives of ORS571 were found to excrete 25-fold higher amounts of coproporphyrin under micro-aerobic conditions than the wild type strain. These observations suggest that A. caulinodans switches from an aerobic to an anaerobic coproporphyrinogen oxidase when the dissolved oxygen tension falls below 1.0 kPa and that the fixLJ and fixK genes are involved in the regulation of expression of the anaerobic coproporphyrinogen oxidase.

Influence of environmental factors on endo-beta-1,4-glucanase production by Bacillus HR68, isolated from a Zimbabwean hot spring.

The production of endo-beta-1,4-glucanase by a Bacillus strain isolated from a hot spring in Zimbabwe was studied in batch culture, chemostat culture, and carbon dioxide-regulated auxostat (CO2-auxostat). The bacteria produced the enzyme in the presence of excess glucose or sucrose, but not under carbon-limited conditions in a chemostat using mineral medium. There was a specific growth rate dependent linear increase in enzyme production in glucose excess, nitrogen-limited chemostat cultures. A high specific growth rate of 2.2 h-1 and a high rate of enzyme production of 362 nkat (mg dry mass.h)-1 were attained under nutrient rich conditions in the CO2-auxostat. The bacteria had the highest specific growth rate and endo-beta-1,4-glucanase enzyme production at 50 degrees C. The maximum specific growth rate and the rate of enzyme production increased when yeast extract and tryptone were added in increasing amounts to the mineral medium used for cultivation in separate experiments. Increasing the glucose concentration in the CO2-auxostat cultures increased the rate of enzyme production but did not affect the specific growth rate.

Genetic and phenetic analyses of Bradyrhizobium strains nodulating peanut (Arachis hypogaea L.) roots.

Seventeen Bradyrhizobium sp. strains and one Azorhizobium strain were compared on the basis of five genetic and phenetic features: (i) partial sequence analyses of the 16S rRNA gene (rDNA), (ii) randomly amplified DNA polymorphisms (RAPD) using three oligonucleotide primers, (iii) total cellular protein profiles, (iv) utilization of 21 aliphatic and 22 aromatic substrates, and (v) intrinsic resistances to seven antibiotics. Partial 16S rDNA analysis revealed the presence of only two rDNA homology (i.e., identity) groups among the 17 Bradyrhizobium strains. The partial 16S rDNA sequences of Bradyrhizobium sp. strains form a tight similarity (> 95%) cluster with Rhodopseudomonas palustris, Nitrobacter species, Afipia species, and Blastobacter denitrificans but were less similar to other members of the alpha-Proteobacteria, including other members of the Rhizobiaceae family. Clustering the Bradyrhizobium sp. strains for their RAPD profiles, protein profiles, and substrate utilization data revealed more diversity than rDNA analysis. Intrinsic antibiotic resistance yielded strain-specific patterns that could not be clustered. High rDNA similarity appeared to be a prerequisite, but it did not necessarily lead to high similarity values between RAPD profiles, protein profiles, and substrate utilization. The various relationship structures, coming forth from each of the studied features, had low compatibilities, casting doubt on the usefulness of a polyphasic approach in rhizobial taxonomy.

Nicotinate catabolism is dispensable and nicotinate anabolism is crucial in Azorhizobium caulinodans growing in batch culture and chemostat culture on N2 as The N source.

When Azorhizobium caulinodans was grown in chemostat cultures with N2 as the N source at a constant dilution rate of 0.1 h-1 in media with a constant concentration (50 mM) of succinate and variable concentrations (1.5 to 585 microM) of nicotinate, neither the growth yield on succinate, the specific rate of O2 consumption, nor the specific rate of CO2 production showed linear regression with the concentration of nicotinate. Moreover, for transient continuous cultures in which the nicotinate concentration was gradually lowered, growth parameters remained unchanged until an apparently critical level of 0.7 microM nicotinate was reached. Below this nicotinate level, an immediate washout of the chemostat population began. A. caulinodans nicotinate hydroxylase-negative mutant 61007, unable to catabolize nicotinate, and the wild type behaved similarly. Thus, for continuous cultures supplied with N2 as the N source, submicromolar concentrations of nicotinate both sustained pyridine nucleotide biosynthesis at sufficient levels and precluded the use of nicotinate as a catabolic substrate. Furthermore, when more nicotinate was provided, dual succinate-nicotinate limitation in continuous cultures did not occur. Finally, when nicotinate is present in suboptimal concentrations, the specific growth rate is directly proportional to the amount of nicotinate present per unit of biomass. By contrast, in batch cultures with different nicotinate concentrations and with either succinate or lactate as the carbon and energy source, anomalous growth curves were obtained. With a low concentration (1.5 microM) of nicotinate, growth on N2 occurred, albeit at low rates. With a high concentration (195 microM) of nicotinate, growth on N2 was temporarily stimulated, but nicotinate was quickly exhausted and growth was thereafter nicotinate limited. Continuous supplementation of batch cultures with nicotinate allowed only transient exponential growth followed by linear growth. Thus, also for batch cultures, nicotinate catabolism is dispensable, although a high concentration of nicotinate temporarily stimulates growth on N2. Ut us concluded that A. caulinodans is a true diazotroph.

Nitrogen Fixation and Hydrogen Metabolism in Relation to the Dissolved Oxygen Tension in Chemostat Cultures of the Wild Type and a Hydrogenase-Negative Mutant of Azorhizobium caulinodans.

Both the wild type and an isogenic hydrogenase-negative mutant of Azorhizobium caulinodans growing ex planta on N(2) as the N source were studied in succinate-limited steady-state chemostat cultures under 0.2 to 3.0% dissolved O(2) tension. Production or consumption of O(2), H(2), and CO(2) was measured with an on-line-connected mass spectrometer. In the range of 0.2 to 3.0%, growth of both the wild type and the mutant was equally dependent on the dissolved O(2) tension: the growth yield decreased, and the specific O(2) consumption and CO(2) production increased. A similar dependency on the dissolved O(2) tension was found for the mutant with 2.5% H(2) in the influent gas. The H(2)/N(2) ratio (moles of H(2) evolved per mole of N(2) consumed via nitrogenase) of the mutant, growing with or without 2.5% H(2), increased with increasing dissolved O(2) tensions. This increase in the H(2)/N(2) ratio was small but significant. The dependencies of the ATP/N(2) ratio (moles of ATP consumed per mole of N(2) fixed) and the ATP/2e ratio [moles of ATP consumed per mole of electron pairs transferred from NAD(P)H to nitrogenase] on the dissolved O(2) tension were estimated. These dependencies were interpreted in terms of the physiological concepts of respiratory protection and autoprotection.

In situ determination of the reduction levels of cytochromes b and c in growing bacteria: a case study with N2-fixing Azorhizobium caulinodans.

The determination of the in situ reduction levels of cytochromes b and c in growing bacteria is achieved by coupling a chemostat with a dual wavelength spectrophotometer. Visible light absorption spectra of cytochromes present in bacterial cells actively growing in a chemostat at a specific growth rate of 0.1 h-1 are recorded. This is accomplished by transporting the emitted light from the spectrophotometer via glass fibers to one side of the chemostat vessel and detecting the transmitted light via a photomultiplier at the other side. The vessel itself is enclosed in a dark box, which contains mirrors on the inside surfaces. The reduction levels of cytochromes b and c during steady state in chemostat cultures are expressed as percentage absorbance of fully reduced cytochromes in the alpha-region of the spectrum. Steady state spectra are recorded in N2-fixing, succinate-limited continuous cultures of Azorhizobium caulinodans at dissolved oxygen tensions in the range between 0.1 and 3.5% O2. Spectra of fully reduced cytochromes are obtained on the basis of spectra recorded after having reached anoxic conditions by sparging pure nitrogen gas through the culture. These spectra of cytochromes b and c reduced by endogenous substrates are corrected as to give the spectrum of fully reduced cytochromes. The respective contributions of cytochromes b and c to spectra in the alpha-region are estimated by deconvolution using best-fit analysis. Using this in situ technique it is observed that at each dissolved oxygen tension the reduction level of the cytochromes b is higher than that of the cytochromes c.(ABSTRACT TRUNCATED AT 250 WORDS)

Relative contributions of biological and chemical reactions to the overall rate of pyrite oxidation at temperatures between 30 degrees C and 70 degrees C.

The stoichiometry and kinetics of the spontaneous, chemical reaction between pyrite and ferric iron was studied at 30, 45, and 70 degrees C in shake flasks at pH 1.5 by monitoring the ferrous iron, total iron, elemental sulfur, and sulfate concentration profiles in time. It was found that the sulfur moiety of pyrite was oxidized completely to sulfate. Elemental sulfur was not produced in detectable amounts. The iron moiety of pyrite was released as ferrous iron. All observed initial reaction rates could be fitted into an empirical equation. This equation includes the concentrations of ferric iron and pyrite, and a constant which is dependent on the temperature and the nature of the main anion present. It was observed that ferrous iron formed during the reaction slowed down the oxidation of pyrite by ferric iron. The extent of this effect decreased with increasing temperature. With the aid of the empirical equation, the contribution of the chemical oxidation of pyrite by ferric iron to the overall oxidation in a hypothetical plug-flow reactor, in which biologically mediated oxdidation of pyrite and ferrous iron by oxygen also takes place, can be assessed. At 30, 45, and 70 degrees C, respectively, 2, 8-17, and 43% of the pyrite was oxidized chemically by ferric iron. Therefore, it is expected that only in reactors operating at high temperatures with extremely thermophilic bacteria, will chemical oxidation cause a significant deviation from the apparent first order overall kinetics of biological pyrite oxidation.

Floating filters, a novel technique for isolation and enumeration of fastidious, acidophilic, iron-oxidizing, autotrophic bacteria.

Nuclepore polycarbonate filters floating on a liquid, FeSO(4)-containing medium (pH 1.6) were used to isolate a moderately thermophilic bacterium from a pyrite-oxidizing enrichment culture. The isolate failed to grow on any of the conventional solid media tried. To test the general applicability of the method, the enumeration of a fastidious acidophilic organism, Thiobacillus ferrooxidans, was carried out and the results compared with those obtained with other filters, solid media, and the most probable number technique. T. ferrooxidans showed better viability on the floating polycarbonate filters and grew in a much shorter time (4 to 5 days) than with the other techniques.

Oxygen and carbon dioxide mass transfer and the aerobic, autotrophic cultivation of moderate and extreme thermophiles: a case study related to the microbial desulfurization of coal.

Mass transfers of O(2), CO(2), and water vapor are among the key processes in the aerobic, autotrophic cultivation of moderate and extreme thermophiles. The dynamics and kinetics of these processes are, in addition to the obvious microbial kinetics, of crucial importance for the industrial desulfurization of high-pyritic coal by such thermophiles. To evaluate the role of the temperature on the gas mass transfer, k(L)a measurements have been used to supplement the existing published data. Oxygen mass transfer from gas (air) to liquid (5 mM H(2)SO(4) in water) phase as a function of the temperature has been studied in a laboratory-scale fermentor. At 15, 30, 45, and 70 degrees C, (k(L)a)(o) values (for oxygen) were determined under three different energy input conditions by the dynamic gassing in/out method. The (k(L)a)(o) was shown to increase under these conditions with increasing temperature, and straight lines were obtained when the logarithm of (k(L)a)(o) was plotted against the temperature. By multiplying the equilibrium concentration of O(2) in water with (k(L)a)(o) maximal, O(2) transfer capacities were calculated. It appeared that in finite of a decreased solubility of O(2) at elevated temperature in mechanically mixed fermentors the calculated transfer capacities showed only minor changes for the range between 15 and 70 degrees C. However, in an air-mixed fermentor the transfer capacity of O(2) decreased slowly but steadily.Carbon dioxide mass transfer was predicted by calculations on the basis of the data for oxygen transfer. The maximal CO(2) transfer capacity, calculated as the product of the equilibrium CO(2) concentration times (k(L)a)(c), decreased slowly as the temperature increased over the range 15-70 degrees C under all three energy input conditions. Subsequent process design calculations showed that for aerobic, autotrophic cultures, CO(2) limitation is more likely to occur than O(2) limitation.

Manganese oxidation by Leptothrix discophora.

Cells of Leptothrix discophora SS1 released Mn2+-oxidizing factors into the medium during growth in batch culture. Manganese was optimally oxidized when the medium was buffered with HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) at pH 7.5. Manganese-oxidizing activity in the culture medium in which this strain had been grown previously was sensitive to heat, phosphate, Tris, NaN3, HgCl2 NaCl, sodium dodecyl sulfate, and pronase; 0.5 mol of O2 was consumed per mol of MnO2 formed. During Mn2+ oxidation, protons were liberated. With sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two protein-containing bands were detected in the spent culture medium. One band had an apparent molecular weight of 110,000 and was predominant in Mn2+-oxidizing activity. The second product (Mr 85,000) was only detected in some cases and probably represents a proteolytic breakdown moiety of the 110,000-Mr protein. The Mn2+-oxidizing factors were associated with the MnO2 aggregates that had been formed in spent culture medium. After solubilization of this MnO2 with ascorbate, Mn2+-oxidizing activity could be recovered.

Manganese oxidation by spores and spore coats of a marine bacillus species.

Bacillus sp. strain SG-1 is a marine bacterial species isolated from a near-shore manganese sediment sample. Its mature dormant spores promote the oxidation of Mn to MnO(2). By quantifying the amounts of immobilized and oxidized manganese, it was established that bound manganese was almost instantaneously oxidized. When the final oxidation of manganese by the spores was partly inhibited by NaN(3) or anaerobiosis, an equivalent decrease in manganese immobilization was observed. After formation of a certain amount of MnO(2) by the spores, the oxidation rate decreased. A maximal encrustment was observed after which no further oxidation occurred. The oxidizing activity could be recovered by reduction of the MnO(2) with hydroxylamine. Once the spores were encrusted, they could bind significant amounts of manganese, even when no oxidation occurred. Purified spore coat preparations oxidized manganese at the same rate as intact spores. During the oxidation of manganese in spore coat preparations, molecular oxygen was consumed and protons were liberated. The data indicate that a spore coat component promoted the oxidation of Mn in a biologically catalyzed process, after adsorption of the ion to incipiently formed MnO(2). Eventually, when large amounts of MnO(2) were allowed to accumulate, the active sites were masked and further oxidation was prevented.

Manganese reduction by a marine Bacillus species.

Mature dormant spores of marine Bacillus sp. strain SG1 catalyze the oxidation of Mn(II) to MnO2. We report that vegetative cells of the same strain reduced MnO2 under low-oxygen conditions. The rate of reduction was a function of cell concentration. The process had a pH optimum of 7.5 to 8.0 and was inhibited by HgCl2, by preheating of the cells at 80 degrees C for 5 min, by antimycin A, and by N-heptyl-hydroxy-quinoline-N-oxide. At a nonlimiting O2 concentration, little MnO2 reduction was observed. Under these conditions, the process could be induced by the addition of NaN3. Spectrophotometric analysis of the Bacillus cells indicated the presence of type b and c cytochromes. Both types can be oxidized in situ by addition of MnO2 to the cells.

The bioenergetics of denitrification.

In anaerobically grown Paracoccus denitrificans the dissimilatory nitrate reductase is linked to the respiratory chain at the level of cytochromes b. Electron transport to nitrite and nitrous oxide involves c-type cytochromes. During electron transport from NADH to nitrate one phosphorylation site is passed, whereas two sites are passed during electron transport from NADH to oxygen, nitrite and nitrous oxide. The presentation of a respiratory chain as a linear array of electron carriers gives a misleading picture of the efficiency of energy conservation since the location of the reductases is not taken into account. For the reduction of nitrite and nitrous oxide, protons are utilized from the periplasmic space, whereas for the reduction of oxygen and nitrate, protons are utilized from the cytoplasmic side of the inner membrane. Evidence for two transport systems for nitrate was obtained. One is driven by the proton motive force; this system is used to initiate nitrate reduction. The second system is a nitrate-nitrite antiport system. A scheme for proton translocation and electron transport to nitrate, nitrite, nitrous oxide and oxygen is presented. The number of charges translocated across the membrane during flow of two electrons from NADH is the same for all nitrogenous oxides and is 67-71% of that during electron transfer to oxygen via cytochrome o. These findings are in accordance with growth yield studies. YMAX electron values determined in chemostat cultures for growth with various substrates and hydrogen acceptors are proportional to the number of charges translocated to these hydrogen acceptors during electron transport.

Respiration-driven proton translocation with nitrite and nitrous oxide in Paracoccus denitrificans.

(1)H+ leads to/electron acceptor ratios have been determined with the oxidant pulse method for cells of denitrifying Paracoccus denitrificans oxidizing endogenous substrates during reduction of O2, NO2- or N2O. Under optimal H+-translocation conditions, the ratios leads to H+/O, H+ leads to/N2O, H+ leads to/NO2- for reduction to N2 and H+ leads to/NO2- for reduction to N2O were 6.0-6.3, 4.02, 5.79 and 3.37, respectively. (2) With ascorbate/N,N,N,'N'-tetramethyl-p-phenylene-diamine as exogenous substrate, addition of NO2- or N2O to an anaerobic cell suspension resulted in rapid alkalinization of the outer bulk medium. H+/N2O, H+/NO2- for reduction to N2 and H+/NO2- for reduction to N2O were -0.84, -2.33 and -1.90, respectively. (3) The H+/oxidant ratios, mentioned in item 2, were not altered in the presence of valinomycin/K+ and the triphenylmethylphosphonium cation. (4) A simplified scheme of electron transport to O2, NO2- and N2O is presented which shows a periplasmic orientation of the nitrite reductase as well as the nitrous oxide reductase. Electrons destined for NO2-, N2O or O2 pass two H+-trans-locating sites. The H+ leads to/electron acceptor ratios predicted by this scheme are in good agreement with the experimental values.