Characterization of the novel dimethyl sulfide-degrading bacterium Alcaligenes sp. SY1 and its biochemical degradation pathway.

Recently, the biodegradation of volatile organic sulfur compounds (VOSCs) has become a burgeoning field, with a growing focus on the reduction of VOSCs. The reduction of VOSCs encompasses both organic emission control and odor control. Herein, Alcaligenes sp. SY1 was isolated from active sludge and found to utilize dimethyl sulfide (DMS) as a growth substrate in a mineral salt medium. Response surface methodology (RSM) analysis was applied to optimize the incubation conditions. The following conditions for optimal degradation were identified: temperature 27.03°C; pH 7.80; inoculum salinity 0.84%; and initial DMS concentration 1585.39 µM. Under these conditions, approximately 99% of the DMS was degraded within 30 h of incubation. Two metabolic compounds were detected and identified by gas chromatography-mass spectrometry (GC-MS): dimethyl disulfide (DMDS) and dimethyl trisulfide (DMTS). The DMS degradation kinetics for different concentrations were evaluated using the Haldane-Andrews model and the pseudo first-order model. The maximum specific growth rate and degradation rate of Alcaligenes sp. SY1 were 0.17 h(-1) and 0.63 gs gx(-1)h(-1). A possible degradation pathway is proposed, and the results suggest that Alcaligenes sp. SY1 has the potential to control odor emissions under aerobic conditions.

Kinetics and metabolic versatility of highly tolerant phenol degrading Alcaligenes strain TW1.

A bacterium that could completely metabolize phenol in batch culture supplied with up to 1200 mg phenol l(-1) at room temperature (25 degrees C) was isolated from the activated sludge of the industrial wastewater treatment plant of a Coke company (Cairo, Egypt). Morphological and physiological characterization showed strain TW1 was a motile, strictly aerobic, gram negative and short-rod occurring singly or in clusters. Partial 16S rRNA gene sequence analysis revealed strain TW1 belonged to the beta group of Proteobacteria, showing 100% identity to Alcaligenes SCTI. Strain TW1 aerobically grew on a number of monocyclic aromatic compounds (hydroquinone, catechol and o-cresol) as well as polycyclic aromatic compounds (pyrene, phenanthrene and naphthalene). The growth of Alcaligenes TW1 on phenol as sole carbon and energy source (25 degrees C) was well described by the Haldane kinetics model with a maximal specific growth rate of 0.58 h(-1), a half-saturation constant of 10 mg l(-1), and a substrate inhibition constant of 152-550 mg l(-1). The biomass yield coefficient ranged from 0.55 to 0.64 mg dry cell mass/mg phenol. Due to its high tolerance to phenol and high metabolic versatility, Alcaligenes sp. TW1 is considered an excellent candidate for the biotreatment of high strength phenol-laden industrial wastewaters.

Conversion of gamma-butyrobetaine to L-carnitine by Achromobacter cycloclast.

L-Carnitine is an ubiquitous substance that plays a major role in the transportation of long-chain fatty acids. We investigated crucial factors that influence microbial conversion of gamma-butyrobetaine to L-carnitine using an Achromobacter cycloclast strain. Two-stage culture results showed that gamma-butyrobetaine induced enzymes essential for the conversion, which suggests that the precursor should be present in the initial cell growth stage. The addition of yeast extract enhanced L-carnitine production whereas inorganic nitrogen sources inhibited it. Under nitrogen-limiting conditions, the cells accumulated poly-beta-hydroxybutyrate instead of L-carnitine. Among the trace elements tested, nickel addition enhanced L-carnitine production by almost twice that of the control and copper strongly inhibited the conversion. L-Carnitine production was reduced when the medium contained inorganic salts of sodium, potassium, and calcium at a concentration greater than 2 g l(-1). A higher L-carnitine yield was achieved when cells were incubated in a lower culture volume. The optimal pH for L-carnitine production was 5 to 5.5, whereas that of growth was 7.0, indicating that a pH shift was required. Under optimal conditions, L-carnitine concentrations as high as 15 g l(-1) were obtained in 62 h with a 45% molar conversion yield.

Production of two phase polyhydroxyalkanoic acid granules in Ralstonia eutropha.

To synthesize layered granules consisting of selected phases of polyhydroxybutyrate (PHB) homopolymer and PH(B-co-V) copolymer, Ralstonia eutropha was grown on fructose and limited quantities (1 g/l) of valeric acid. Exhaustion of the valerate resulted in a carbon source shift and a shift in the composition of polyhydroxyalkanoate (PHA) being synthesized within the cell. The synthesis rates were 0.030 g PH(B-co-V)/l per h and 0.033 g PHB/l per h, giving a copolymer composition of 48% HV. The valerate was exhausted at approximately 12 h at a rate of 0.0894 g/l per h after which only PHB was produced through the remaining 12 h at 0.033 g PHB/l per h from the remaining fructose, which was utilized at a constant rate of 0.0861 g/l per h throughout all 24 h of the experiment. Differential scanning calorimetry (DSC) of isolated granules showed two glass transitions, confirming the presence of two distinct polymer phases within the layered granules. Transmission electron microscopic images stained with RuO4 revealed a heavily stained copolymer core within a lighter stained PHB shell, confirming the expected morphology of granule composition. Thus, biosynthesis can be exploited for the control of domain sizes in layered granules, potentially providing metabolic control over the physical properties of the resultant polymer.

Immunoelectron microscopic studies indicate the existence of a cell shape preserving cytoskeleton in prokaryotes.

Immunoelectron microscopic studies of prokaryotes were performed with anti-actin antibodies directed against the C terminus of actin. Studies on ultrathin sections revealed high proportions of the overall label close to the cell periphery in Escherichia coli, Ralstonia eutropha, Thermoanaerobacterium thermosulfurigenes, T. thermosaccharolyticum, and Methanococcus jannaschii. Substantial label also in the cytoplasm was observed in Bacillus sp., Methanococcus voltae, and Methanobacterium thermoautotrophicum. Only very minor amounts of label were found in the nucleoid region of the cells. Whole-mount immunogold studies, combined with negative staining, revealed the existence of an intracellular network of fibrils which could be labeled by anti-actin antibodies. This network is assumed to be located below the cytoplasmic membrane all around the cytoplasm. It appears to have properties that would allow its function as a cytoskeleton-like structure preserving cell shape.

Antigenic determinants of the membrane-bound hydrogenase in Alcaligenes eutrophus are exposed toward the periplasm.

Electron microscopic immunogold labeling experiments were performed with ultrathin sections of plasmolyzed cells of Alcaligenes eutrophus and "whole-mount" samples of spheroplasts and protoplasts. They demonstrated that antigenic determinants of the membrane-bound hydrogenase are exposed, at the outside of the cytoplasmic membrane, to the periplasm.

Structural and immunological studies on the soluble formate dehydrogenase from Alcaligenes eutrophus.

During growth with formate as the sole energy source the autotrophic bacterium Alcaligenes eutrophus synthesizes a cytoplasmic formate dehydrogenase. The enzyme is a molybdo-iron-sulfur-flavo protein and the major NADH-producing system under these growth conditions, although it was estimated to constitute only 0.65% of the soluble cell protein. An electron microscopic analysis of the purified enzyme revealed that the particle is made up of four nonidentical submasses, corroborating previous structural data. The NH2-terminal amino acid sequences of the enzyme subunits exhibited significant similarities to those of only one other heteromeric formate dehydrogenase, the enzyme from the methane-utilizing bacterium Methylosinus trichosporium. Metal analyses yielded 21.5 g-atom iron, 2.18 g-atom nickel, 0.76 g-atom molybdenum, and 0.59 g-atom zinc per mol of enzyme. Initial electron paramagnetic resonance spectroscopic studies showed at least three distinct signals which appeared upon reduction of the enzyme with NADH or formate. The corresponding spin systems could be attributed to iron-sulfur centers of the enzyme. Comparative immunostaining and activity-staining experiments using cell extracts from various bacteria established immunological similarities between the soluble formate dehydrogenase of A. eutrophus and the soluble enzymes from all tested facultative autotrophs as well as from M. trichosporium.

Analysis of a 24-kilodalton protein associated with the polyhydroxyalkanoic acid granules in Alcaligenes eutrophus.

A 5.0-kbp genomic EcoRI restriction fragment which complemented a third subclass of polyhydroxyalkanoic acid (PHA)-leaky mutants of A. eutrophus that accumulated PHA at a lower rate than the wild type was cloned from Alcaligenes eutrophus H16. A 687-bp phaPAe gene on this fragment encoded a 24-kDa protein (M(r) = 23,963), which was referred to as the GA24 protein. The GA24 protein was solubilized from the granules and purified to electrophoretic homogeneity, and antibodies against the GA24 protein were obtained. The GA24 protein bound to the surface of PHA granules, as revealed by immunoelectron microscopy of whole cells and of artificial PHA granules. The GA24 protein contributed approximately 5% (wt/wt) of the total cellular protein, and it was the predominant protein present in the granules. It was synthesized only in cells accumulating PHA and only in amounts that could be bound to the granules; no soluble GA24 protein was detected. Tn5::mob-induced phaPAe mutants which were unable to synthesize intact GA24 protein formed only one large PHA granule per cell. The amino acid sequence of the GA24 protein revealed two closely related stretches consisting exclusively of nonhydrophilic amino acids at the C-terminal region, which are presumably involved in the binding of GA24 to the granules, as was recently proposed for a similar protein in Rhodococcus ruber. The GA24 protein seems to be a representative of phasins, which are a new class of protein that form a layer at the surface of PHA granules, like oleosins, which form a layer at the surface of triacylglycerol inclusions in oilseed plants.

Immunocytochemical analysis of poly-beta-hydroxybutyrate (PHB) synthase in Alcaligenes eutrophus H16: localization of the synthase enzyme at the surface of PHB granules.

Antibodies raised against the Alcaligenes eutrophus poly-beta-hydroxybutyrate (PHB) synthase polypeptide were used for immunocytochemical localization of the synthase enzyme in whole cells and purified PHB granules. The data presented demonstrate for the first time that the synthase enzyme is located on the surface of the PHB granule rather than being incorporated inside the granule during its formation. From these basic observations and data from the recent literature, a model of granule assembly is proposed.

A gene complex coding for the membrane-bound hydrogenase of Alcaligenes eutrophus H16.

One of the key enzymes in the chemolithoautotrophic metabolism of Alcaligenes eutrophus H16 is a dimeric, membrane-associated hydrogenase. The genetic determinants of this enzyme are located on the endogenous megaplasmid pHG1 (G. Eberz, C. Hogrefe, C. Kortlüke, A. Kamienski, and B. Friedrich, J. Bacteriol. 168:636-641, 1986). Complementation studies showed that the information required for the formation of active membrane-bound hydrogenase occupies more than 7.5 kb of megaplasmid DNA. We cloned and sequenced this region and identified the genes encoding the two hydrogenase subunits (hoxK and hoxG). The nucleotide sequence contains nine additional closely spaced open reading frames. Immunoelectron microscopy showed that the gene product of one of these open reading frames (hoxM) is involved in the process leading to the attachment of hydrogenase to the membrane. Other open reading frames may encode additional processing functions and components of a hydrogenase-linked electron transport chain. Analysis of Tn5-B21-mediated transcriptional fusions provided evidence that the structural genes and accessory functions belong to at least three coordinately regulated transcriptional units.

Immunoelectronmicroscopic study of the nucleoid structure of hydrogen bacteria.

Electron microscopical studies of the nucleoid structure of hydrogen bacteria using ultrahin sections and spread DNA from bacterial cell lysates revealed a different DNA packaging in the cell. A compact state of the major part of DNA at all growth stages and stability of nucleosome-like structures were shown. The use of antibodies to HU protein of E. coli labelled by protein A-colloidal gold demonstrated the immunological relationship between HU protein of E. coli and histone-like proteins of Alcaligenes eutrophus and their possible role in the nucleosome-like DNA packaging in procariotic genome.

Localization of the cytochrome cd1 and copper nitrite reductases in denitrifying bacteria.

The locations of cytochrome cd1 nitrite reductases in Pseudomonas aeruginosa and Pseudomonas fluorescens and copper nitrite reductases in Achromobacter cycloclastes and Achromobacter xylosoxidans were identified. Immunogold labeling with colloidal-gold probes showed that the nitrite reductases were synthesized exclusively in anaerobically grown (denitrifying) cells. Little immunogold label occurred in the cytoplasm of these four strains; most was found in the periplasmic space or was associated with cell membranes. Immunogold labeling of thin sections was superior to fractionation by osmotic shock for locating nitrite reductases. The results support models of dentrification energetics that require a periplasmic, not a cytoplasmic, location for nitrite reductases.

Staining bacterial flagella easily.

A wet-mount technique for staining bacterial flagella is highly successful when a stable stain and regular slides and cover slips are used. Although not producing a permanent mount, the technique is simple for routine use when the number and arrangement of flagella are critical in identifying species of motile bacteria.

Contribution of permeability and sensitivity to inhibition of DNA synthesis in determining susceptibilities of Escherichia coli, Pseudomonas aeruginosa, and Alcaligenes faecalis to ciprofloxacin.

To examine the correlation between bacterial cell susceptibility to ciprofloxacin and the magnitude of uptake and cell target sensitivity, the relative contribution of ciprofloxacin accumulation in intact cells and its ability to inhibit DNA synthesis were investigated among strains of Escherichia coli, Pseudomonas aeruginosa, and Alcaligenes faecalis. Uptake studies of [14C]ciprofloxacin demonstrated diffusion kinetics for P. aeruginosa and E. coli. Ciprofloxacin was more readily removed from E. coli J53 and A. faecalis ATCC 19018 by washing than from P. aeruginosa PAO503. These results indicate that the process of cell accumulation is different for P. aeruginosa in that the drug is firmly bound at an extracellular site. Whatever the washing conditions, A. faecalis accumulated less drug than either of the other two bacteria. Magnesium chloride (10 mM) caused a substantial decrease of ciprofloxacin accumulated and an increase in the MIC, depending upon the nature of the medium. The addition of carbonyl cyanide m-chlorophenylhydrazone caused a variable increase in drug accumulated, depending on the medium and the bacterial strain. The concentration of ciprofloxacin required to obtain 50% inhibition (ID50) of DNA synthesis for P. aeruginosa PAO503 and A. faecalis ATCC 19018 did not correlate with their corresponding MICs but did for E. coli J53. Treatment with EDTA decreased the ID50 of ciprofloxacin for P. aeruginosa PAO503 and its gyrA derivative by 5- and 2-fold, respectively, and decreased the ID50 for E. coli JB5R, a strain with a known decrease in OmpF, by 1.4-fold but did not decrease the ID50 for the normally susceptible E. coli J53. The ID(50) for P. aeruginosa obtained after EDTA treatment or in ether-permeabilized cells was higher than that obtained for the other two strains. The protonophore carbonyl cyanide m-chlorophenylhydrazone prevented killing by low ciprofloxacin concentrtaions, but sodium azide did not. The latter compound did not enhance killing in association with inhibition of a previously described energy-dependent efflux of ciprofloxacin susceptibility being the susceptibility to inhibition of DNA synthesis in E. coli, poor premeability associated with the small pore size of A. faecalis, and a combination of low permeability and reduced susceptibility of DNA synthesis to inhibition for P. aeruginosa.

The poly-beta-hydroxybutyrate granule in vivo. A new insight based on NMR spectroscopy of whole cells.

High resolution 13C NMR spectroscopy of live cells has been used to show that poly-beta-hydroxybutyrate (PHB) is predominantly in a mobile state within the storage granules of Alcaligenes eutrophus, Methylobacterium extorquens, and Methylobacterium AM1. Comparison of chemical and NMR analysis of PHB indicates that about 70% of the polymer in A. eutrophus gives sharp observable resonances. Temperature-dependent line widths and relaxation rates together with nuclear Overhauser effect measurements demonstrate that the observed material is effectively a mobile amorphous elastomer that is well above its glass transition temperature. The hydroxyvalerate-hydroxybutyrate copolymer produced by propionate-fed A. eutrophus has virtually the same mobility as the homopolymer. Evidence is presented indicating that water is an integral component of the PHB granule and that this component acts as a plasticizer for the polymer. These observations strongly suggest that the enzyme(s) responsible for PHB biosynthesis and consumption operate only on mobile hydrated material and that the solid granules characteristic of dried cells are partially artifactual. This model is supported by a reinterpretation of previously inexplicable biochemical results.

Size of diffusion pore of Alcaligenes faecalis.

The diffusion pore of the outer membrane of Alcaligenes faecalis was shown to be substantially smaller than the Escherichia coli porin pore. In experiments with intact cells, pentoses and hexoses penetrated into the NaCl-expanded periplasm, whereas saccharides of Mr greater than 342 did not. Cells treated with 0.5 M saccharides of Mr greater than 342 weighed 33 to 38% less than cells treated with isotonic solution, suggesting that these saccharides do not permeate through the outer membrane. The diffusion rates of various solutes through the liposome membranes reconstituted from the Mr-43,000 outer membrane protein showed the following characteristics. (i) The relative diffusion rates of pentoses, hexoses, and methylhexoses appeared to be about 1.0, 0.6, and negligibly small, respectively. (ii) The diffusion rate of glucose appeared to be about 1/10th that with the E. coli B porin. (iii) The diffusion rate of gluconic acid was five to seven times higher than that of glucose. (iv) The diffusion rates of beta-lactam antibiotics appeared to be 40 to less than 10% of those with the E. coli B porin.

Mathematical model for determining the effects of intracytoplasmic inclusions on volume and density of microorganisms.

Procaryotic microorganisms accumulate several polymers in the form of intracellular inclusions as a strategy to increase survival in a changing environment. Such inclusions avoid osmotic pressure increases by tightly packaging certain macromolecules into the inclusion. In the present paper, a model describing changes in volume and density of the microbial cell as a function of the weight of the macromolecule forming the inclusion is derived from simple theoretical principles. The model is then tested by linear regression with experimental data from glycogen accumulation in Escherichia coli, poly-beta-hydroxybutyrate accumulation in Alcaligenes eutrophus, and sulfur accumulation in Chromatium spp. The model predicts a certain degree of hydration of the polymer in the inclusion and explains both the linear relationship between volume of the cell and weight of the polymer and the hyperbolic relationship between density of the cell and weight of the polymer. Other implications of the model are also discussed.

Partial characterization of the hemagglutinin of Alcaligenes faecalis.

The hemagglutinin of Alcaligenes faecalis was partially characterized. Hemagglutination (HA) was blocked by enzymes inactivating proteins, by heat, and by antisera but not by sugar-blocking substances. Pili were not determined to be a factor in HA activity. There was no connection between virulence and HA activity.

The membrane-bound hydrogenase of Alcaligenes eutrophus: II. Localization and immunological comparison with other hydrogenase systems.

Immunological comparison of the soluble and the membrane-bound hydrogenase of Alcaligenes eutrophus revealed no common antigenic determinants shared by the native proteins, however, a small amount of cross-reacting material was detected after freezing and thawing. Immune precipitation assays supported previous observations indicating the membrane-bound hydrogenase to be localized in the outer surface of the cytoplasmic membrane. The membrane-bound hydrogenases of A. eutrophus and Pseudomonas pseudoflava showed close immunological relationship, and material cross-reacting to both antisera was found in membrane extracts of all hydrogen-oxidizing strains of Pseudomonas. Alcaligenes and Aquaspirillum. Material cross-reacting to the membrane-bound hydrogenase of Xanthobacter autotraphicus GZ 29 was found only in a few hydrogen-oxidizing bacteria. Material cross-reacting to the soluble hydrogenase of A. eutrophus was detected in strains of A. eutrophus and A. ruhlandii only. Comparison of the membrane-bound hydrogenase of A. eutrophus, P. pseudoflava and X. autotrophicus with hydrogenases of other physiological bacterial groups revealed serological relationship to the membrane-bound hydrogenases of the hydrogen bacteria and of Chromatium vinosum only. The results are discussed in terms of physiological, taxonomical, and evolutionary aspects.

Achromobacter species (CDC group Vd): morphological and biochemical characterization.

Twenty-three isolates of Achromobacter species (CDC group Vd) were examined morphologically and biochemically. Gram stains revealed gram-variable bacilli frequently curved or hooked at one pole and often coryneform in shape and arrangement. Electron microscopy revealed the presence of extracellular material in polar accumulations and demonstrated the polar flagella arrangement seen by light microscopy to be lateral. Two colony types were produced; one was minute and watery at 24 h (35 degrees C) progressing to large, mucoid colonies at 48 h, and the other type was shiny, glistening, opaque but nonmucoid. All isolates grew on MacConkey agar and produced catalase, oxidase, and urease. Most grew on salmonella-shigella agar, reduced nitrate to nitrite and gas, hydrolyzed esculin, deaminated phenylalanine (2 to 4 days) and produced H2S in triple sugar iron agar (4 to 12 days). Oxidation of carbohydrates was weak, delayed, and limited to glucose and xylose. Two isolates also oxidized maltose, mannitol, and sucrose. The ability of miniaturized "nonfermenter" kits to identify Achromobacter species was tested. The Minitek (Baltimore Biological Laboratory, Cockeysville, Md.) and N/F (Corning, Roslyn, N.Y.) systems, respectively, identified 21 and 19 of the 23 isolates, whereas the Oxi/Ferm (Roche, Nutley, N.J.) identified 13 and the API 20E (Analytab Products, Plainview, N.Y.) identified only 3.