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 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.     

Metabolism of sitosterol by a Pseudomonas species.

Fermentation of sitosterol by a Pseudomonas species (SK-25) resulted in the formation of 5-stigmastene-3 beta, 7 alpha-diol; 5,6 alpha-epoxy-5 alpha-stigmastan-3 beta-ol; 5,6 beta-epoxy-5 beta-stigmastan-3 beta-ol and 5 alpha-stigmastan-3 beta, 5,6 beta-triol. The metabolites were characterized by a variety of conventional chemical and spectrometric techniques.     

Manganese binding and oxidation by spores of a marine bacillus.

Mature, dormant spores of a marine bacillus, SG-1, bound and oxidized (precipitated) manganese on their surfaces. The binding and oxidation occurred under dormant conditions, with mature spores suspended in natural seawater. These heat-stable spores were formed in the absence of added manganese in the growth medium. The rate and amount of manganese bound by SG-1 spores was a function of spore concentration. Temperatures greater than 45 degrees C, pH values below 6.5, or the addition of EDTA or the metabolic inhibitors sodium azide, potassium cyanide, and mercuric chloride inhibited manganese binding and oxidation. However, SG-1 spores bound and oxidized manganese after treatment with glutaraldehyde, formaldehyde, ethylene oxide gas, or UV light, all of which killed the spores. Manganese oxidation never occurred in the absence of manganese binding to spores. The data suggest that Mn2+ was complexed by a spore component, perhaps an exosporium or a spore coat protein: once bound, the manganese was rapidly oxidized.     

Regulation of intracellular signalling through cysteine oxidation by reactive oxygen species

Reactive oxygen species (ROS) have been regarded as harmful molecules that damage various molecules inside cells by oxidation and are responsible for ageing and various human diseases. However, recent studies have revealed an opposite aspect of ROS that these are actively generated in cells and mediate physiological intracellular signalling as second messengers. Several proteins have been shown to function as effectors for ROS, which are sensitively and reversibly oxidized by ROS. Such ROS-effector proteins commonly possess a highly reactive cysteine (Cys) residue, of which oxidation changes the protein function, thus enabling signal transmission to downstream targets. Among the ROS effectors, protein tyrosine phosphatase (PTP), thioredoxin (TRX) and peroxiredoxin (PRX) family proteins possess special domains/motifs to maintain the reactivity of Cys and utilize them to respond to ROS. Progressively advancing identification of ROS-effector proteins reveals the pleiotropic functions of ROS in physiological and pathological cell biology.     

Pathway of degradation of nitrilotriacetate by a Pseudomonas species.

The pathway of degradation of nitrilotriacetate (NTA) was determined by using cell-free extracts and a 35-fold purification of NTA monooxygenase. The first step in the breakdown was an oxidative cleavage of the tertiary amine by the monooxygenase to form the aldo acid, glyoxylate, and the secondary amine, iminodiacetate (IDA). NTA N-oxide acted as a substrate analog for induction of the monooxygenase and was slowly metabolized by the enzyme, but was not an intermediate in the pathway. No intermediate before IDA was found, but an unstable alpha-hydroxy-NTA intermediate was postulated. IDA did undergo cleavage in the presence of the purified monooxygenase to give glyoxylate and glycine, but was not metabolized in cell-free extracts. Glyoxylate was further metabolized by cell-free extracts to yield CO2 and glycerate or glycine, products also found from NTA metabolism. Of the three bacterial isolates in which the NTA pathway has been studied, two strains, one isolated from a British soil and ours from a Michigan soil, appear to be almost identical.     

SNARE protein mimicry by an intracellular bacterium

Many intracellular pathogens rely on host cell membrane compartments for their survival. The strategies they have developed to subvert intracellular trafficking are often unknown, and SNARE proteins, which are essential for membrane fusion, are possible targets. The obligate intracellular bacteria Chlamydia replicate within an intracellular vacuole, termed an inclusion. A large family of bacterial proteins is inserted in the inclusion membrane, and the role of these inclusion proteins is mostly unknown. Here we identify SNARE-like motifs in the inclusion protein IncA, which are conserved among most Chlamydia species. We show that IncA can bind directly to several host SNARE proteins. A subset of SNAREs is specifically recruited to the immediate vicinity of the inclusion membrane, and their accumulation is reduced around inclusions that lack IncA, demonstrating that IncA plays a predominant role in SNARE recruitment. However, interaction with the SNARE machinery is probably not restricted to IncA as at least another inclusion protein shows similarities with SNARE motifs and can interact with SNAREs. We modelled IncA's association with host SNAREs. The analysis of intermolecular contacts showed that the IncA SNARE-like motif can make specific interactions with host SNARE motifs similar to those found in a bona fide SNARE complex. Moreover, point mutations in the central layer of IncA SNARE-like motifs resulted in the loss of binding to host SNAREs. Altogether, our data demonstrate for the first time mimicry of the SNARE motif by a bacterium.     

Degradation of 4-chlorophenylacetic acid by a Pseudomonas species.

Pseudomonas sp. strain CBS3 was able to utilize 4-chlorophenylacetic acid as the sole source of carbon and energy. When this strain was grown with 4-chlorophenylacetic acid, homoprotocatechuic acid was found to be an intermediate which was further metabolized by the meta-cleavage pathway. Furthermore, three isomers of chlorohydroxyphenylacetic acid, two of them identified as 3-chloro-4-hydroxyphenylacetic acid and 4-chloro-3-hydroxyphenylacetic acid, were isolated from the culture medium. 4-Hydroxyphenylacetic acid was catabolized in a different manner by the glutathione-dependent homogentisate pathway. Degradation enzymes of both of these pathways were inducible.     

Temperature-induced alanine oxidation in a psychrotrophic Pseudomonas.

A psychrotrophic pseudomonad isolated from iced fish oxidized alanine at temperatures close to 0 degrees C and grew over the range 0 degrees C-35 degrees C. The rate of oxidation of alanine, measured manometrically, by cells grown at 2 degrees C was lower than that of cells grown at 22 degrees C. However, the consumption of oxygen after heat treatment at 35 degrees for 35 min was reduced considerably by 2 degrees C grown cells. Alanine oxidase activity was tested in an extract from cells grown at 2 degrees C and 22 degrees C with alanine as the sole carbon, nitrogen, and energy source. Cells grown at 2 degrees C produced an alanine oxidase with a temperature optimum of 35 degrees C and pH optimum of 8, which lost about 80% activity by heat treatment at 40 degrees C for 30 min. There was no change in activity after dialysis at pH 7, 8, or 9. Extracts from cells grown at 22 degrees C contained an alanine oxidase system with an optimum temperature of 45 degrees C, a pH optimum above 8, and only about 30% reduction of activity after heat treatment. This enzyme activity was concentrated in the 0.5 M elution fraction from a Sephadex column, and dialysis reduced the activity at pH 7 and 8. Mesophilic enzyme synthesis apparently started around a growth temperature of 10 degrees C. The crude alanine oxidase systems of Pseudomonas aeruginosa derived from cells grown at 13 degrees C and 37 degrees C had a common optimum temperature of 45 degrees C. These data suggest that one mechanism of psychrophilic growth by psychrotrophic bacteria may be the induction of enzymes with low optimum temperatures in response to low temperature conditions.     

Biodegradation of acyclic isoprenoids by Pseudomonas species.

The ability of various pseudomonads to utilize acyclic isoprenoids as a sole carbon source was investigated. Tests for utilization of acyclic isoprenols such as citronellol and geraniol were complicated by toxic effects of these alcohols, and most species tested were killed by exposure to citronellol or geraniol (0.1%, vol/vol) in liquid culture. In the case of Pseudomonas citronellolis, sensitivity to isoprenols is reduced by prior induction of the isoprenoid degradative pathway via either growth on succinate in the presence of citronellol or growth on citronellic acid. For this species, citronellic acid proved to be the best isoprenoid growth substrate tested. Geraniol utilization as a taxonomic indicator for different subgroups of pseudomonads is discussed. Only a few of the species tested were able to utilize acyclic isoprenoids. Two species which utilize C10 acyclic isoprenoids, P. aeruginosa and P. mendocina, were shown to contain the inducible enzyme geranyl-coenzyme A carboxylase, one of the unique enzymes in the isoprenol degradative pathway known to occur in P. citronellolis. Of the species which utilized geranitol, none showed definite growth on the homologous C15 and C20 isoprenols.     

Plasmid-mediated degradation of dibenzothiophene by Pseudomonas species.

The microbial transformation of dibenzothiophene (DBT) is of interest in the potential desulfurization of oil. We isolated three soil Pseudomonas species which oxidized DBT to characteristic water-soluble, sulfur-containing products. Two of our isolates harbored a 55-megadalton plasmid; growth in the presence of novobiocin resulted in both loss of the plasmid and loss of the ability to oxidize DBT. Reintroduction of the plasmid restored the ability to oxidize DBT to water-soluble products. The products resulting from the oxidation of DBT were characterized and included 3-hydroxy-2-formyl benzothiophene, 3-oxo-[3'-hydroxy-thionaphthenyl-(2)-methylene]-dihydrothionaph thene, and the hemiacetal and trans forms of 4-[2-(3-hydroxy)-thianaphthenyl]-2-oxo-3-butenoic acid. The products of DBT oxidation were inhibitory to cell growth and further DBT oxidation. DBT oxidation in our soil isolates was induced by naphthalene or salicylate and to a much lesser extent by DBT and was repressed by succinate.     

Cyanide production from glycine by a homogenate from a Pseudomonas species.

A cell-free preparation with cyanide-producing activity was obtained from a bacterium, strain C, of the genus Pseudomonas. To preserve activity, an oxidizing agent, e.g., phenazine methosulphage (PMS), had to be added to the cell suspension before disruption by sonic treatment. By the procedure described, a total homogenate made from a 15% (wet weight) bacterial suspension in tris(hydroxymethyl)aminomethane-hydrochloride buffer (0.05 M, pH 8.2) and with PMS (0.4mM) exhibited about 8% of the activity obtained from a suspension of untreated bacteria. In the presence of flavine-adenine dinucleotide (0.3 mM) and PMS (0.4mM), the activity was augmented to about 16% of that of the intact cells. By gradient centrifugation the homogenate was separated into three fractions. The main enzyme activity was associated with those fractions which by electron microscopy were found to consist of membranous structures.     

Arginine decarboxylase from a Pseudomonas species.

An arginine decarboxylase has been isolated from a Pseudomonas species. The enzyme is constitutive and did not appear to be repressed by a variety of carbon sources. After an approximately 40-fold purification, the enzyme appeared more similar in its properties to the Escherichia coli biosynthetic arginine decarboxylase than to the E. coli inducible (biodegradative) enzyme. The Pseudomonas arginine decarboxylase exhibited a pH optimum of 8.1 and an absolute requirement of Mg2+ and pyridoxal phosphate, and was inhibited significantly at lower Mg2+ concentrations by the polyamines putrescine, spermidine, and cadaverine. The Km for L-arginine was about 0.25 mM at pH 8.1 AND 7.2. The enzyme was completely inhibited by p-chloromercuribenzoate. The inhibition was prevented by dithiothreitol, a feature that suggests the involvement of an -SH group. Of a variety of labeled amino acids tested, only L-arginine, but not D-arginine was decarboxylated. D-Arginine was a potent inhibitor of arginine decarboxylase with a Ki of 3.2 muM.