Are gram-positive bacteria capable of electron transfer across their cell wall without an externally available electron shuttle?

The extensive contributions by Terry Beveridge to our understanding of the differences in cell wall organization with respect to structure, chemistry and compartmentalization between gram-positive and gram-negative bacteria are summarized. These contributions greatly aided in conceptualization of recent discoveries concerning electron export and import across cell walls of some gram-negative bacteria. Although electron export and import across the cell wall by any gram-positive has not been documented so far, Beveridge's observations and concepts concerning cell walls of gram-positive bacteria suggest potential mechanisms by which such electron transfer may occur.

Chromate resistance plasmid in Pseudomonas fluorescens.

Chromate resistance of Pseudomonas fluorescens LB300, isolated from chromium-contaminated sediment in the upper Hudson River, was found to be plasmid specified. Loss of the plasmid (pLHB1) by spontaneous segregation or mitomycin C curing resulted in a simultaneous loss of chromate resistance. Subsequent transformation of such strains with purified pLHB1 plasmid DNA resulted in a simultaneous re-acquisition of the chromate resistance phenotype and the plasmid. When pLHB1 was transferred by conjugation to Escherichia coli, the plasmid still conferred chromate resistance.

A comparison of manganese oxidation by growing and resting cells of a freshwater bacterial isolate, strain FMn 1.

A bacterial isolate, strain FMn 1, from reservoir sediment oxidized MN2+ when tested in growing culture and resting-cell suspension. The oxidation was biologically mediated and not the result of autoxidation because at a MnSO4 . H2O concentration of 0.05%, the pH remained below 7.5 for the duration of the experiments. The production of manganese oxide was qualitatively and quantitatively demonstrated. The manganese-oxidizing activity of this organism was found to be inducible.

A novel MN2+-oxidizing enzyme system in a freshwater bacterium.

Manganese oxidation by cell suspensions and cell extracts of a freshwater bacterium, designated strain FMn 1, was investigated. Manganese appeared to be oxidized in the periplasmic space. A conventional, membrane-bound-electron transport system was not utilized. An enzyme or enzyme complex and a cofactor, each of different molecular size, were located in different parts of the cell envelope. Results suggest that the cofactor reacts with manganese in the periplasmic space and that in the presence of oxygen it is reoxidized by the enzyme. The enzyme is probably loosely bound to the membrane. A combination of enzyme and cofactor in a crude preparation exhibited a pH optimum at around 7.0. The enzyme exhibited a temperature optimum at around 30 degrees C. No temperature optimum was found for the cofactor. The enzyme was heat-stable and could oxidize manganese under anaerobic conditions. The enzyme system appears to be different from others so far described.

Cytochrome Involvement in Mn(II) Oxidation by Two Marine Bacteria.

Two marine, Mn(II)-oxidizing bacterial cultures, BIII 45 and BIII 82, were examined spectrophotometrically at ambient temperature for their cytochrome complements. Membrane preparations from an ethylenediaminetetracetate-lysozyme treatment of 48-h cultures of both strains contained type b, c, and o cytochromes. No evidence for a type a cytochrome was noted. "Periplasmic" fractions of both strains also contained small amounts of cytochrome, including cytochrome o, but "intracellular" fractions did not. Type c cytochrome in membrane preparations of culture BIII 45 was consistently reduced by Mn(II) when the membranes were suspended in the periplasmic fraction of the culture. In the case of culture BIII 82, type c cytochrome in membrane preparations was consistently reduced by Mn(II) when the membranes were suspended in either periplasmic or intracellular fractions of the strain. Although, based on previous inhibitor studies, type b cytochrome was also expected to be reduced by Mn(II), no spectrophotometric evidence for its reduction was found, probably because not enough of it was reduced under the steady-state conditions of the experiments.

Influence of hydrostatic pressure on the effects of the heavy metal cations of manganese, copper, cobalt, and nickel on the growth of three deep-sea bacterial isolates.

Increases hydrostatic pressure varied the 72-h growth yield of three bacterial isolates from the deep sea in the presence of heavy metal cations of Mn, Cu, Co, and Ni, depending on the bacterial isolate, the metal cation and its concentration, and the level of hydrostatic pressure. Above atmospheric, hydrostatic pressure was found to have one of the following four effects on the response of culture growth to a heavy metal cation. (i) It could be without effect; (ii) it could enhance inhibition by a metal cation; (iii) it could increase the 72-h growth yield by a metal cation; or (iv) it could protect against a growth inhibitory effect noted at a lower pressure. Possible reasons for these varied responses are discussed.

Electron transport components of the MnO2 reductase system and the location of the terminal reductase in a marine Bacillus.

The response of MnO2 reduction by uninduced and induced whole cells and cell extracts of Bacillus 29 to several electron transport inhibitors was compared. MnO2 reduction with glucose by uninduced whole cells and cell extracts was strongly inhibited at 0.1 mM dicumarol, 100 mM azide, and 8 mM cyanide but not by atebrine or carbon monoxide, suggesting the involvement of a vitamin K--type quinone and a metalloenzyme in the electron transport chain. MnO2 reduction with ferrocyanide by uninduced cell extracts was inhibited by 5 mM cyanide and 100 mM azide but not by atebrine, dicumarol, or carbon monoxide, suggesting that the metalloenzyme was associated with the terminal oxidase activity. MnO2 reduction with glucose by induced whole cells and cell extracts, was inhibited by 1 mM atebrine, 0.1 mM dicumarol, and 10 mM cyanide but not by antimycin A, 2n-nonyl-4-hydroxyguinoline-N-oxide) (NOQNO), 4,4,4-trifluoro-1-(2-thienyl),1,3-butanedione, or carbon monoxide. Induced cell extract was also inhibited by 100 mM azide, but stimulated by 1 mM and 10 mM azide. Induced whole cells were stimulated by 10 mM and 100 mM azide. These results suggested that electron transport from glucose to MnO2 in induced cells involved such components as flavoprotein, a vitamin K-type quinone, and metalloenzyme. The stimulatory effect of azide on induced cells was explained on the basis of a branching in the terminal part of the electron transport chain, one branch involving a metalloenzyme for the reduction of MnO2 and the other involving a metalloenzyme for the reduction of oxygen. The latter was assumed to be the more azide sensitive. Spectral studies showed the presence of a-, b-, and c-type cytochromes in membrane but not in soluble fractions. Of these cytochromes, only the c type may be involved in electron transport of MnO2, owing to the lack of inhibition by antimycin A or 2n-nonyl-4-hydroxyquinoline-N-oxide. The terminal MnO2 reductase appears to be loosely attached to the cell membrane of Bacillus 29 because of cell fractionation it is found associated with both particulate and soluble fractions. Electron photomicrographs of bacilli attached to synthetic Fe-Mn oxide revealed an intimate contact of the cell walls with the oxide particles.

Observations of bacterial microcolonies on the surface of ferromanganese nodules from blake plateau by scanning electron microscopy.

Examination of the surface of freshly collected ferromanganese nodules by scanning electron microscopy revealed the presence of microcolonies of rod- and coccus-shaped bacteria which appeared to be anchored to the nodule surface by slime. The attachment of microcolonies by slime to the surface of freshly collected nodules argues against their being contaminants introduced during nodule collection or processing. These results corroborate cultural and biochemical detection of bacteria on ferromanganese nodules.

Effects of seawater cations and temperature on manganese dioxide-reductase activity in a marine Bacillus.

The seawater cations, Na(+), K(+), Mg(2+), and Ca(2+), each stimulated MnO(2)-reductase activity of whole cells and cell extracts of Bacillus 29. Concentrations of Na(+) and K(+) which stimulated whole cells and cell extracts maximally were equivalent to those in two- to fivefold diluted seawater. Cell-extract activity was strongly stimulated by Ca(2+) and Mg(2+) up to a concentration of 0.01 M Mg(2+) and 0.002 M Ca(2+), with little additional stimulation above these concentrations. Whole-cell activity was stimulated biphasically with increasing concentrations of Ca(2+) and Mg(2+). Comparison of the effects of individual cations or mixtures of them at concentrations equivalent to their concentration in fivefold diluted seawater showed that more activity was obtained with 0.01 M Mg(2+) or 0.002 M Ca(2+) than with 0.1 M Na(+), and more with 0.1 M Na(+) than with 0.0022 M K(+). Fivefold diluted seawater permitted as much or more activity as solutions of individual or synthetic mixtures of the cations. Pre-exposure experiments showed that the ionic history of whole cells was important to their ultimate activity. The MnO(2)-reductase activity of induced whole cells exhibited a temperature optimum near 40 C. Cell extracts had different temperature optima (T(opt)), depending on whether induced glucose-linked activity (T(opt) = 25 C), uninduced glucose-linked, ferricyanide-dependent activity (T(opt) = 30 C), or uninduced ferrocyanide-linked activity (T(opt) = 40 C) were being measured. Some of these optima are higher than previously reported.

Bacteriology of manganese nodules. V. Effect of hydrostatic pressure on bacterial oxidation of MnII and reduction of MnO2.

It was experimentally demonstrated that two strains of Arthrobacter 37, one growing at 25 C and the other at 5 C, could catalyze Mn(II) oxidation at hydrostatic pressures well in excess of the pressure encountered by the parent culture in its original habitat in the ocean (80 atm). The strain grown at 5 C showed an increase in temperature optimum for manganese oxidation with increase in pressure. It was like-wise experimentally shown that induced Bacillus 29 without added ferricyanide and uninduced Bacillus 29 with added ferricyanide could catalyze MnO(2) reduction at hydrostatic pressures in excess of the pressure encountered by this organism in its original habitat (187 atm). The uninduced Bacillus 29, in the presence of ferricyanide, was active over a wider range of pressures (1 to 1,000 atm) than the induced Bacillus 29 in the absence of ferricyanide (1 to 467 atm). At corresponding pressures, the uninduced culture was also considerably more active than the induced culture. Special techniques were developed for measuring Mn(II)-oxidizing and MnO(2)-reducing activity under pressure.

Bacteriology of manganese nodules. IV. Induction of an MnO2-reductase system in a marine bacillus.

Bacillus 29, isolated from a ferromanganese nodule from the Atlantic Ocean, was shown to possess an MnO(2)-reductase system which is induced in the presence of manganous ion. Maximal activity of the enzyme system was induced in about 5 hr in the presence of 4.35 mm MnSO(4) and was minimally dependent on the presence of either glucose or peptone and oxygen. Induction of optimal activity required the simultaneous presence of glucose and peptone. At least 30% of maximal activity was induced in 5 hr in the presence of 0.4 mum MnSO(4). Actinomycin D (5 mug/ml) or chloramphenicol (35 mug/ml), when added to the induction medium, inhibited approximately 90% of MnO(2)-reductase synthesis and incorporation of uracil-2-(14)C or leucine-1-(14)C. Cell-free extracts having MnO(2)-reductase activity were prepared by sonic disruption of cell suspensions of induced Bacillus 29. Such extracts used glucose metabolism as a source of electrons. They had an average specific activity of 1.15 nmoles of Mn(II) produced per mg of protein per hr at 25 C. They had a temperature optimum of 18 C for reductase activity and retained 50% of their activity at 4 C, the approximate temperature of the natural habitat of the organism. Extracts were stable for several days at 4 C but rapidly lost over 50% of their activity on freezing and thawing. Over 90% of the activity of the extract could be destroyed by heating in a boiling-water bath for 5 min. At a concentration of 1 mm, HgCl(2) and atebrine dihydrochloride inhibited MnO(2)-reductase activity by at least 50%, but sodium azide was ineffective. The MnO(2)-reductase activity of induced cells and extracts from them was no greater in the absence of oxygen than in its presence, confirming an earlier observation that MnO(2) and O(2) do not compete as terminal electron acceptors in the respiratory activity of this organism.

Bacteriology of manganese nodules: III. Reduction of MnO(2) by two strains of nodule bacteria.

MnO(2) reduction by aerobic growing cultures of Bacillus 29 and coccus 32, isolated from ferromanganese nodules, was assessed for 7 days. A 1-day lag was observed before the onset of MnO(2) reduction by either culture. Addition of HgCl(2) to a final concentration of about 10 M caused a rapid cessation of MnO(2) reduction by the growing cultures. Neither culture reduced MnO(2) when grown under continued anaerobiosis from the start of an experiment. However, if conditions were made anaerobic after MnO(2) reduction was initiated, reduction continued at a rate only slightly lower than that under aerobic conditions. Resting-cell cultures reduced MnO(2) equally well aerobically and anaerobically, provided that ferricyanide was present to serve as electron carrier. These findings showed that oxygen is needed for culture adaptation to MnO(2) reduction, and that oxygen does not interfere with microbial MnO(2) reduction itself. Both cultures caused sharp drops in the pH of the medium during MnO(2) reduction: with coccus 32, during the entire incubation time; with Bacillus 29, for the first 3 days. The E(h) of the medium fluctuated with either culture and never fell below 469 mv with Bacillus 29 and below 394 mv with coccus 32. The rates of glucose consumption and Mn release by Bacillus 29 and coccus 32 were fairly constant, but the rates of lactate and pyruvate production were not. Although acid production undoubtedly helped in the reduction of pyrolusite (MnO(2)) by the bacteria, it did not appear to be important in the reduction of manganese oxide in ferromanganese nodules, as shown by the results with a nodule enrichment.

Bacteriology of manganese nodules. II. Manganese oxidation by cell-free extract from a manganese nodule bacterium.

A cell-free extract from Arthrobacter 37, isolated from a manganese nodule from the Atlantic Ocean, exhibited enzymatic activity which accelerated manganese accretion to synthetic Mn-Fe oxide as well as to crushed manganese nodule. The reaction required oxygen and was inhibited by HgCl(2) and p-chloromercuribenzoate but not by Atebrine dihydrochloride. The rate of enzymatic action depended on the concentration of cell-free extract used. The enzymatic activity had a temperature optimum around 17.5 C and was destroyed by heating at 100 C. The amount of heat required for inactivation depended on the amount of nucleic acid in the preparation. In the cell-free extract, unlike the whole-cell preparation, peptone could not substitute for NaHCO(3) in the reaction mixture. An enzyme-containing protein fraction and a nucleic acid fraction could be separated from cell extract by gel filtration, when prepared in 3% NaCl but not in seawater. The nucleic acid fraction was not required for enzymatic activity.

Copper sulfide precipitation by yeasts from Acid mine-waters.

Two strains of Rhodotorula and one of Trichosporon precipitated dissolved copper with H(2)S formed by reducing elemental sulfur with glucose. Iron stimulated this activity under certain conditions. In the case of Rhodotorula strain L, iron stimulated copper precipitation aerobically at a copper concentration of 18 but not 180 mug/ml. Anaerobically, the L strain required iron for precipitation of copper from a medium with 180 mug of copper per ml. Rhodotorula strain L was able to precipitate about five times as much copper anaerobically as aerobically. The precipitated copper was identified as copper sulfide, but its exact composition could not be ascertained. Iron was not precipitated by the H(2)S formed by any of the yeasts. Added as ferric iron, it was able to redissolve copper sulfide formed aerobically by Rhodotorula strain L from 18 but not 180 mug of copper per ml of medium. Since the yeasts were derived from acid mine-waters, their ability to precipitate copper may be of geomicrobial importance.