Maleic Acid Utilization by Mixed Cultures of Microorganisms

In a mixed batch culture, Alcaligenes xylosoxidans subsp. xylosoxidans 260 transformed maleic acid into malic acid. Bacillus subtilis 271 used malic acid as a substrate, thus stimulating further transformation of maleic acid. Both bacterial cultures dissociated with the formation of R, S, and M forms. At a concentration of 5.0 g/l, maleic acid was utilized maximally by RS and SS forms of the association A. xylosoxidans and B. subtilis. At concentrations 15.0 and 25.0 g/l, maleic acid was utilized maximally by SS and MS forms of the mixed culture, respectively. Association of bacteria A. xylosoxidans and B. subtilis was not stable under flow conditions of water.     

Optimum Conditions for Transformation of Maleic Acid by Immobilized Cells of Alcaligenes xylosoxidanssubsp. xylosoxidans260

Immobilized cells of Alcaligenes xylosoxidanssubsp. xylosoxidans260 transformed 98% of the maleic acid (initial concentration of 5.0 g/l medium) under periodic conditions for 48 h. Free cells transformed only 26% of the substrate in 96 h. Immobilized cells of a selected S-variant ofA. xylosoxidanstransformed the maleate (30.0 g/l) entirely in 96 h during batch cultivation and only 15.0 g/l of the maleate in continuous cultivation at a flow rate of 0.03 h^–1.     

Delivery of benzene to Alcaligenes xylosoxidans by solid polymers in a two-phase partitioning bioreactor

Toxic levels of benzene were decreased to sub-inhibitory levels in a bioreactor via absorption by polymer beads or cylinders (poly(ethylene-co-vinyl acetate) or poly(styrene-co-butadiene)). After inoculation with Alcaligenes xylosoxidans, the remaining benzene in the aqueous phase as well as the benzene taken up by the polymers was degraded to completion. The capacity of these polymers for benzene, and benzene diffusivity within the polymers were also determined.     

Detection and Molecular Analysis of Plant- and Insect-Associated Bacteria Harboring Aconitate Isomerase Involved in Biosynthesis of Trans-Aconitic Acid as Antifeedant in Brown Planthoppers

We isolated and characterized D,L-halidohydrolases from five different soil bacteria. Three of these bacterial strains bear plasmids with sizes of approximately 60 kb. Curing and mating experiments indicated that these three plasmids pFL160, pFL170, and pFL190 encoded a dehalogenase. Owing to their biochemical characterization, these halidohydrolases were closely related among each other and to the DhlIV halidohydrolase, encoded by plasmid pFL40 from Alcaligenes xylosoxidans ssp. denitrificans ABIV. Restriction enzyme patterns as well as DNA-hybridization experiments with an internal fragment of dhlIV revealed a high degree of homology among each of these four plasmids and their dehalogenase genes. Received: 5 July 1996 / Accepted: 1 August 1996     

Coordinate synthesis of azurin I and copper nitrite reductase in Alcaligenes xylosoxidans during denitrification

The denitrifying bacterium Alcaligenes xylosoxidans synthesises two azurins (Az), which are termed Az I and Az 2. Both function as effective electron donors to copper nitrite reductase (NiR) in vitro. As a first step towards identifying the physiological relevance of these electron transfer proteins in the denitrification process, the gene (azuA) encoding Az I was characterised and its expression with respect to denitrification determined. We show that the azuA gene from A. xylosoxidans is monocistronic and its expression is increased when cells are grown under denitrifying conditions in the presence of nitrate or nitrite. The expression pattern of azuA was similar, though not identical, to that of the monocistronic nirK gene, which encodes copper NiR, and is in accord with both gene products being synthesised when the bacterium denitrifies. Recombinant Az I was exported to the periplasm of the heterologous host Escherichia coli, was synthesised at very high levels (80 mg purified protein per litre) and was fully loaded with copper. Electron donation from reduced recombinant Az to NiR was indistinguishable from the activity determined with the native protein. Taken together, these findings indicate that in A. xylosoxidans azuA expression is coordinated with denitrification and recombinant Az I is processed and matured in the periplasm of E. coli in the same way it is in A. xylosoxidans.     

Selection of trichloroethene (TCE) degrading bacteria that resist inactivation by TCE

Two isoprene (2-methyl-1,3-butadiene) utilizing bacteria, Alcaligenes denitrificans ssp. xylosoxidans JE 75 and Rhodococcus erythropolis JE 77, were identified as highly efficient cooxidizers of TCE, cis- and transdichloroethene, 1,1-dichloroethene and vinylchloride. Isoprene grown cells eliminate chloride from TCE in stoichiometric amounts and tolerate high concentrations of TCE.     

Is Fecal Leukocyte Test a good predictor of Clostridium difficile associated diarrhea?

Alcaligenes xylosoxidans is an aerobic, motile, oxidase and catalase positive, nonfermentative Gram negative bacillus. This bacterium has been isolated from intestine of humans and from various hospital or environmental water sources. A.xylosoxidans is both waterborne and results from the poor-hygienic conditions healthcare workers are in. In this case report, the bacteremia which appeared in a patient with pancreas cancer after ERCP was described.     

Identification and characterization of the bacterial <Emphasis Type="SmallCaps">d</Emphasis>-gluconate dehydratase in <Emphasis Type="Italic">Achromobacter xylosoxidans</Emphasis>

Achromobacter xylosoxidans is known to utilize d-glucose via the modified Entner-Doudoroff pathway. Although d-gluconate dehydratase produced from this bacterium was purified and partially characterized previously, a gene that encodes this enzyme has not yet been identified. To obtain protein information on bacterial d-gluconate dehydratase, we partially purified d-gluconate dehydratase in A. xylosoxidans and investigated its biochemical properties. Two degenerate primers were designed based on the N-terminal amino acid sequence of the partially purified d-gluconate dehydratase. Through PCR performed using degenerate primers, a 1,782-bp DNA sequence encoding the A. xylosoxidans d-gluconate dehydratase (gnaD) was obtained. The deduced amino acid sequence of A. xylosoxidans gnaD showed strong similarity with that of proteins belonging to the dihydroxy-acid dehydratase/phosphogluconate dehydratase family (COG0129). This is in contrast to the archaeal d-gluconate dehydratase, which belongs to the enolase superfamily (COG4948). The phylogenetic tree showed that A. xylosoxidans d-gluconate dehydratase is closer to the 6-phosphogluconate dehydratase than the dihydroxy-acid dehydratase. Interestingly, a clade containing A. xylosoxidans enzyme was clustered with proteins annotated as a second and a third dihydroxy-acid dehydratase in the genomes of Clostridium acetobutylicum (Cac_ilvD2) and Streptomyces ceolicolor (Sco_ilvD2, Sco_ilvD3), indicating that the function of these enzymes is the dehydration of d-gluconate.     

Spectroscopic characterization and intramolecular electron transfer processes of native and type 2 Cu-depleted nitrite reductases

 Native nitrite reductases (NIRs) containing both type 1 and 2 Cu ions and type 2 Cu-depleted (T2D) NIRs from three denitrifying bacteria (Achromobacter cycloclastes IAM 1013, Alcaligenes xylosoxidans NCIB 11015, and Alcaligenes xylosoxidans GIFU 1051) have been characterized by electronic absorption, circular dichroism, and electron paramagnetic resonance spectra. The characteristic visible absorption spectra of these NIRs are due to the type 1 Cu centers, while the type 2 Cu centers hardly contribute in the same region. The intramolecular electron transfer (ET) process from the type 1 Cu to the type 2 Cu in native NIRs has been observed as the reoxidation of the type 1 Cu(I) center by pulse radiolysis, whereas no type 1 Cu in T2D NIRs exhibits the same reoxidation. The ET process obeys first-order kinetics, and observed rate constants are 1400–1900 s^–1 (t_1/2 = ca. 0.5 ms) at pH 7.0. In the presence of nitrite, the ET process also obeys first-order kinetics, with rate constants decreased by factors of 1/12–1/2 at the same pH. The redox potential of the type 2 Cu site is estimated to be +0.24 - +0.28 V, close to that of the type 1 Cu site. Nitrate and azide ions bound to the type 2 Cu site change the redox potential. Nitrite also would shift the redox potential of the type 2 Cu by coordination, and hence the intramolecular ET rate constant is decreased. Pulse radiolysis experiments on T2D NIRs in the presence of nitrite demonstrate that the type 1 Cu(I) site is slowly oxidized with a first-order rate constant of 0.03 s^–1 at pH 7.0, suggesting that nitrite bound to the protein accepts an electron from the type 1 Cu. This result is in accord with the finding that T2D NIRs show enzymatic activities, although they are lower than those of the native enzymes. Received: 9 July 1996 / Accepted: 30 January 1997     

Isolation and characterization of bacteria from soil contaminated with diesel oil and the possible use of these in autochthonous bioaugmentation

Two bacterial species (isolates N and O) were isolated from a paddy soil microcosm that had been artificially contaminated with diesel oil to which extrinsic Pseudomonas aeruginosa strain WatG, had been added exogenously. One bacterial species (isolate J) was isolated from a similar soil microcosm that had been biostimulated with Luria–Bertani (LB) medium. Isolates N and O, which were tentatively identified as Stenotrophomonas sp. and Ochromonas sp., respectively, by sequencing of their 16 S rRNA genes had no ability to degrade diesel oil on their own in any liquid medium. When each strain was cocultivated with P. aeruginosa strain WatG in liquid mineral salts medium (MSM) containing 1% diesel oil, isolate N enhanced the degradation of diesel oil by P. aeruginosa strain WatG, but isolate O inhibited it. In contrast, isolate J, which was tentatively identified as a Rhodococcus sp., degraded diesel oil contained not only in liquid LB and MSM, but also in paddy soil microcosms supplemented with LB medium. The bioaugmentation capacity of isolate J in soil microcosms contaminated with diesel oil was much higher than that of P. aeruginosa strain WatG. The possibility of using isolate J for autochthonous bioaugmentation is discussed.     

Biodegradation of endosulfan and endosulfan sulfate by <Emphasis Type="Italic">Achromobacter xylosoxidans</Emphasis> strain C8B in broth medium

Endosulfan is one of the most widely used wide spectrum cyclodiene organochlorine insecticide. In environment, endosulfan can undergo either oxidation or hydrolysis reaction to form endosulfan sulfate and endosulfan diol respectively. Endosulfan sulfate is as toxic and as persistent as its parent isomers. In the present study, endosulfan degrading bacteria were isolated from soil through selective enrichment technique using sulfur free medium with endosulfan as sole sulfur source. Out of the 8 isolated bacterial strains, strain C8B was found to be the most efficient endosulfan degrader, degrading 94.12% α-endosulfan and 84.52% β-endosulfan. The bacterial strain was identified as Achromobacter xylosoxidans strain C8B on the basis of 16S rDNA sequence similarity. Achromobacter xylosoxidans strain C8B was also found to degrade 80.10% endosulfan sulfate using it as sulfur source. No known metabolites were found to be formed in the culture media during the entire course of degradation. Besides, the bacterial strain was found to degrade all the known endosulfan metabolites. There was marked increase in the quantity of released CO₂ from the culture media with endosulfan as sulfur source as compared to MgSO₄ suggesting that the bacterial strain, Achromobacter xylosoxidans strain C8B probably degraded endosulfan completely through the formation of endosulfan ether.     

Strong Impact on the Polycyclic Aromatic Hydrocarbon (PAH)-Degrading Community of a PAH-Polluted Soil but Marginal Effect on PAH Degradation when Priming with Bioremediated Soil Dominated by Mycobacteria

Bioaugmentation of soil polluted with polycyclic aromatic hydrocarbons (PAHs) is often disappointing because of the low survival rate and low activity of the introduced degrader bacteria. We therefore investigated the possibility of priming PAH degradation in soil by adding 2% of bioremediated soil with a high capacity for PAH degradation. The culturable PAH-degrading community of the bioremediated primer soil was dominated by Mycobacterium spp. A microcosm containing pristine soil artificially polluted with PAHs and primed with bioremediated soil showed a fast, 100- to 1,000-fold increase in numbers of culturable phenanthrene-, pyrene-, and fluoranthene degraders and a 160-fold increase in copy numbers of the mycobacterial PAH dioxygenase gene pdo1. A nonpolluted microcosm primed with bioremediated soil showed a high rate of survival of the introduced degrader community during the 112 days of incubation. A nonprimed control microcosm containing pristine soil artificially polluted with PAHs showed only small increases in the numbers of culturable PAH degraders and no pdo1 genes. Initial PAH degradation rates were highest in the primed microcosm, but later, the degradation rates were comparable in primed and nonprimed soil. Thus, the proliferation and persistence of the introduced, soil-adapted degraders had only a marginal effect on PAH degradation. Given the small effect of priming with bioremediated soil and the likely presence of PAH degraders in almost all PAH-contaminated soils, it seems questionable to prime PAH-contaminated soil with bioremediated soil as a means of large-scale soil bioremediation.     

Characterization of the bottom sediments contaminated with polychlorinated biphenyls: Evaluation of ecotoxicity and biodegradability

At the locality of the former producer of PCBs Chemko Strá?ske in East Slovakia, a large amount of PCBs (the commercial mixture DELOR 103, an equivalent of AROCLOR 1242) is still persisting in sediments and negatively influences health of the population. The objective of this work was to provide a study of ecotoxicity and genotoxicity of PCBs in contaminated sediments. Toxicity of the PCB-contaminated sediments sampled from Zemplínska ?írava and Strá?sky canal (surroundings of the former producer of PCBs) was determined applying a standard aquatic plant toxicity test using Lemna minor. The endpoints for the test were frond numbers and frond areas. The sediment sampled from Zemplínska ?írava was more toxic to L. minor than the one sampled from Strá?sky canal. The results on genotoxicity showed that both sediments were not mutagenic toward the standard strains of the Ames test, Salmonella typhimurium TA98 and TA100. This work deals also with biodegradation of PCBs in two samples of the above mentioned contaminated sediments: a) in the natural sediments by autochthonous microbial consortium and b) in the bioaugmented sediments inoculated by allochthonous bacterial strains, two bacterial isolates from long-term PCB-contaminated soil Pseudomonas stutzeri and Alcaligenes xylosoxidans. Both approaches were applied under the biostimulation conditions, with addition of glucose or biphenyl as co-substrates, as well. The highest PCB degradation was observed in the bioaugmented sediment inoculated with bacterial strain P. stutzeri. Addition of biphenyl, as the co-substrate and the inducer, positively affected degradation of PCBs. The bphA1 gene, encoding enzyme biphenyldioxygenase, responsible for the start of PCB degradation, was identified in genome of P. stutzeri, a potential PCB-degrader isolated from long-term PCB-contaminated soil, but not in genome of A. xylosoxidans.     

Cooxidation of chloro- and methylphenols by Alcaligenes xylosoxidans JH1

Alcaligenes xylosoxidans subspecies denitrificans JH1 was enriched with 2-chlorophenol from a mixed culture degrading different chloro- and methylphenols. The strain used all monochloro- and monomethylphenols apart from 2-methylphenol as sole source of energy and carbon with stoichiometric release of chloride. 4-Chlorophenol was mineralized up to a concentration of 1.3 mM. Degradation of mixtures of monochloro- and monomethylphenols occurred at least partially except for the mixture of 2-chlorophenol and 3-methylphenol. Depending upon the growth substrates used, enzymes of the ortho and/or meta cleavage pathway catalysed the degradation of the phenols. The transformation of chlorophenols was concluded to occur exclusively via the ortho cleavage pathway because no chlorocatechol 2,3-dioxygenase activity was found in chlorophenol-grown cells. Degradation of 4-methylphenol in strain JH1 occurred both by the ortho and meta cleavage pathway as indicated by the finding that the ortho- and meta-cleaving dioxygenases were expressed in 4-methylphenol-grown cells. Transformation of methylphenols by the ortho cleavage pathway led to the accumulation of methyllactones as dead-end products. Mixtures of methyl- and chlorophenols were metabolized mainly by the ortho cleavage pathway because chlorocatechols formed inactivated the constitutive catechol 2,3-dioxygenase which caused channelling of methylphenols into the ortho cleavage pathway.     

Establishment of a Genetically Marked Insect-Derived Symbiont in Multiple Host Plants

Alcaligenes xylosoxidans subsp. denitrificans, originally isolated from the cibarial region of the foregut of the glassy-winged sharpshooter (Homalodisca coagulata), was transformed using the Himar1 transposition system to express EGFP. Seedlings of six potential host plants were inoculated with transformed bacteria and 2 weeks later samples were taken 5 cm away and analyzed by quantitative real-time PCR using primers designed to amplify the gene insert. The largest colony of 3,591,427 cells/2 cm of A. xylosoxidans subsp. denitrificans was found in Citrus limon, with almost all plants testing positive in both trials. The amount of colonization decreased in the other plants tested in the following order: orange (Citrus sinensis sweet orange) > chrysanthemum (Chrysanthemum grandiflora cv. White Diamond) > periwinkle (Vinca rosea) > crepe myrtle (Lagerstroemia indica) > grapevine (Vitis vinifera cv. Chardonnay). The bacteriums preference for citrus paralleled the host insects preference for this same plant. Additional tests determined that A. xylosoxidans subsp. denitrificans thrives as a nonpathogenic, xylem-associated endophyte.     

Purification and characterization of N-carbomoyl-l-amino acid amidohydrolase with broad substrate specificity from Alcaligenes xylosoxidans

N-Carbomoyl-L-amino acid amidohydrolase was purified to homogeneity for the first time from Alcaligenes xylosoxidans. The enzyme showed high affinity toward N-carbomoyl-L-amino acids with long-chain aliphatic or aromatic substituents, and hydrolyzed those with short-chain substituents quite well. The enzyme hydrolyzed N-formyl- and N-acetylamino acids quickly and very slowly, respectively. The enzyme did not hydrolyze -ureidopropionate and ureidosuccinate. The relative molecular mass of the native enzyme was about 135 000 and the enzyme consisted of two identical polypeptide chains. The enzyme activity was significantly inhibited by sulfhydryl reagents and required the following divalent metal ions: Mn²⁺, Ni²⁺ and Co²⁺.     

Association ofAlcaligenes faecalis with wetland rice

Alcaligenes faecalis isolated from rice roots is widespread in paddy soil of China. It was found to be a close association with rice.A. faecalis accumulate on the rice root surface, and part of them could enter into the rice root. It can grow in the intercellular space, especially inside the root cells, and multiply and fix dinitrogen there.A. faecalis could synthesize nitrogenase when it was grown in the medium containing a high concentration of ammonia. The mechanisms of association are also discussed.     

Consortia modulation of the stress response: proteomic analysis of single strain versus mixed culture

The high complexity of naturally occurring microbial communities is the major drawback limiting the study of these important biological systems. In this study, a comparison between pure cultures of Pseudomonas reinekei sp. strain MT1 and stable community cultures composed of MT1 plus the addition of Achromobacter xylosoxidans strain MT3 (in a steady‐state proportion 9:1) was used as a model system to study bacterial interactions that take place under simultaneous chemical and oxidative stress. Both are members of a real community isolated from a polluted sediment by enrichment in 4‐chlorosalicylate (4CS). The analysis of dynamic states was carried out at the proteome, metabolic profile and population dynamic level. Differential protein expression was evaluated under exposure to 4CS and high concentrations of toxic intermediates (4‐chlorocatechol and protoanemonin), including proteins from several functional groups and particularly enzymes of aromatic degradation pathways and outer membrane proteins. Remarkably, 4CS addition generated a strong oxidative stress response in pure strain MT1 culture led by alkyl hydroperoxide reductase, while the community showed an enhanced central metabolism response, where A. xylosoxidans MT3 helped to prevent toxic intermediate accumulation. A significant change in the outer membrane composition of P. reinekei MT1 was observed during the chemical stress caused by 4CS and in the presence of A. xylosoxidans MT3, highlighting the expression of the major outer membrane protein OprF, tightly correlated to 4CC concentration profile and its potential detoxification role.     

Nickel resistance of Alcaligenes denitrificans strain 4a-2 is chromosomally coded

A newly isolated aerobic hydrogen-oxidizing bacterium, Alcaligenes denitrificans strain 4a-2, differs from related autotrophic bacteria by containing only a single cytoplasmic, NAD-reducing hydrogenase, and by its high resistance to nickel ions, i.e. tolerance to 20 mM NiCl₂. Strain 4a-2 harbors a single plasmid of about 250 kb. On helper-assisted mating of 4a-2 with Alcaligenes eutrophus strains H16,G29, and M85 nickelresistant transconjugants were selected; these did not contain the donor plasmid, however. All three transconjugants tolerated 3 to 10 mM NiCl₂. The resistance was constitutively expressed. DNA/DNA hybridization showed homology with EcoRI-digested DNA of the wild type 4a-2 and transconjugants using a DNA probe containing nickel resistance genes of pMOL28. This indicated that the 4a-2 nickel resistance genes are located on the chromosome.