Alcaligenes Paradoxus Research Articles

Surveillance of the condition of bacteria and Amaranthus viridis in a gasoline contaminated soil

The condition of bacteria and Amaranthus viridis in gasoline-contaminated soil was surveyed. The soil was treated with different concentrations of petrol oil: 0 ml (control), 18, 56, 112, 168 and 224 ml. Each of the concentrations was in triplicates. Seventeen (17) bacterial isolates were recovered from the control soil while twenty (20) bacterial isolates were recovered from the polluted soil. A total of twenty four (24) different genera were recovered from the soil. The organisms areAcinetobacter ceticus, Actinomyces sp, Aerococcus viridians, Alcaligenes paradoxus, Arthrobacter, Bacillus cereus, Bacillus polymxa, Bacillus subtilis, Chromobacterium violaceum, Clostridium sp., Corynebacterium hydrocarbodastus, Corynebacterium kutsceri, Corynebacterium xerosis, Cytophaga sp., Erwinia sp., Flavobacterium sp., Micrococcus luteus, Proteus vulgaris, Pseudomonas fluorescens, Pseudomonas putida, Sarcina ventricule, Serratia sp., Staphylococcus aureus, and Streptococcus sp. The pH, temperature, organic matter content, moisture content and water holding capacity of the soil were determined. A. viridis had 100% germination in both the control (0 ml) and 18 ml gasoline-treated soils while it had delayed germination in the other treatments (56, 112, 168 and 224 ml). The results obtained in this study showed that gasoline spillage posses a great threat to the survival and development of plants. It also revealed that certain bacteria could survive the gasoline-contaminated soil and thus may be useful in biodegradation and/or bioremediation of gasoline contaminated soils. Key words: Surveillance, bacteria, Amaranthus viridis, gasoline, contaminated soil.

Biological control of phytophagous ladybird beetles Epilachna vigintioctopunctata (Col., Coccinellidae) by chitinolytic phylloplane bacteria Alcaligenes paradoxus entrapped in alginate beads

Abstract:The chitinase secreting strain KPM‐012A of Alcaligenes paradoxus was isolated from tomato leaves and vitally entrapped in sodium alginate gel beads to provide a new method for biocontrol of phytophagous ladybird beetles Epilachna vigintioctopunctata. First, the peritrophic membrane was dissected from the adult ladybird beetles that ingested the suspension of KPM‐012A after starvation to observe degradation of the midgut surface by the bacteria under electron microscopy. The peritrophic membrane around the bacteria was degraded, suggesting the release of chitinase from the ingested bacteria. Large amounts of chitinase were successfully released from KPM‐012A‐entrapped calcium alginate beads. This chitinase release from the microbial beads was sustained for 1 week and was sufficient to digest the peritrophic membrane. Daily supply of tomato leaves treated with the microbial beads caused considerable suppression of leaf feeding and oviposition by the adult ladybird beetles, indicating that this method is effective for decreasing population of insect pests in the subsequent generation. Thus, the present study provided an experimental basis for the biocontrol measures of herbivorous insect pests by the chitinolytic bacteria entrapped in alginate beads.

Enhancing bacterial transport for bioaugmentation of aquifers using low ionic strength solutions and surfactants

The transport of bacteria in contaminated aquifers over a distance of only one meter can require tens to thousands of unsuccessful collisions of bacteria with soil grains. Previous work has shown that low ionic strength (IS) solutions and the nonionic surfactant Tween 20 can reduce bacterial adhesion to ultraclean surfaces such as glass and quartz porous media. In this study, we examined whether these results could be generalized to soils and to other surfactants, by measuring the retention of two species of radiolabeled microbes over short (1 cm) distances in soil minicolumns. Calculations were also made, using the clean-bed filtration theory, to evaluate if bacterial transport distances are sufficient for bioagumentation to occur over a large region of the subsurface. Collision efficiencies were expressed using the filtration model in terms of the sticking coefficient, α, defined as the fraction of collisions that are successful. In glass bead columns, α's for monoclonal populations were reduced from α=0.19 ( Alcaligenes paradoxus) and α=0.01 (CD1), to α<0.008 for Tween 80-phosphate buffer solutions and α<0.0054 for low ionic strength (0.01 mM) solutions (Darcy velocity, U=10 −3 m s −1; Hammaker constant=10 −20 J; and fluid properties of water at 22°C). Low α's were also obtained using other nonionic surfactants (Tween 80, Triton 100 and 705, POE-10, Brij+35) and an anionic biosurfactant, all added at concentrations above their critical micelle concentration (CMC). Although sticking coefficients were also reduced by an order-of-magnitude for natural soils, sticking coefficients remained too high to permit wide dispersal of cells over distances of >1 m. For A. paradoxus, α was reduced using a low ionic strength solution from 0.72 to 0.083 for the Arizona soil and from 1.7 to 0.2 for the Ringold soil; for CD1, α was reduced from 0.57 to 0.09 for the Ringold soil. Based on the soil grain diameters of these soils (127 μm, Arizona soil; 224 μm, Ringold soil), α's in this range will permit transport distances (defined as 99.9% reduction in cell concentration) of ∼1 m ( U=10 m/d, 10°C) which may be sufficient for creating small, bioactive zones. However, in order to increase bacterial transport over distances >1 m, methods other than simple solution chemistry changes will be needed to enhance aquifer bioaugmentation operations.

Bacterial dichloropropene degradation in soil; screening of soils and involvement of plasmids carrying the dhlA gene

The nematocide cis-1,3-dichloropropene is widely used in intensive agricultural practice against root knot nematodes. As a result of the regular application of dichloropropene to six out of eight different soils, the compound was rapidly degraded to CO 2, H 2O and chloride ions in subsequent applications (adapted soils). Such degradation occurred at a lower rate in the same soil types without a history of dichloropropene use (unadapted soils). In two soils, the effect of repeated dichloropropene treatment was not found, since dichloropropene was already rapidly decomposed in the untreated controls. Since the dehalogenating enzyme haloalkane dehalogenase, encoded by the dhlA gene, is possibly involved in the degradation of dichloropropene, a specific PCR detection system combined with a dhlA probe was developed to detect dhlA in soil DNA extracts. Five of the eight adapted soils as well as three unadapted ones showed a response to this system, indicating the presence of DNA with sequence similarity to dhlA. Fifteen bacterial strains with dichloropropene-degradative capacity were isolated from enrichment cultures grown in the presence of this compound and inoculated with adapted soil (five soil types). Six selected isolates were identified as, respectively, Alcaligenes paradoxus, Pseudomonas cichorii I, Pseudomonas corrugata (2 ×), Pseudomonas putida and Pseudomonas sp. Each of the six strains harboured a plasmid of 50–60 kb in size and all but one carried resistance to HgCl 2. Filter matings were performed to investigate the possible cotransfer of biodegradative genes and Hg resistance to suitably-marked P. fluorescens R2f recipient strains. Three plasmid donor strains, P. cichorii I, P. corrugata and Pseudomonas sp., transferred the Hg resistance and the plasmid to the recipient used, suggesting that the Hg resistance marker was located on the plasmid. In addition, transconjugants produced with P. cichorii I and P. corrugata donor strains had also acquired 1,3-dichloropropene-degrading capability. PCR amplification with dhlA specific primers, of the plasmids obtained both from the original P. cichorii I and P. corrugata strains and from the respective transconjugant P. fluorescens R2f strains generated 400–500 bp amplification products. After cloning of the product of the P. cichorii I plasmid and sequencing of 180 bases, extensive homology with the dhlA gene was detected. These results suggest that a plasmid-located dhlA-like gene may be involved in dichloropropene degradation in soil by soil bacteria, but the extent to which the gene is involved in the process in situ is not clear.

Influence of different chemical treatments on transport of Alcaligenes paradoxus in porous media

Seven chemicals, three buffers, and a salt solution known to affect bacterial attachment were tested to quantify their abilities to enhance the penetration of Alcaligenes paradoxus in porous media. Chemical treatments included Tween 20 (a nonionic surfactant that affects hydrophobic interactions), sodium dodecyl sulfate (an anionic surfactant), EDTA (a cell membrane permeabilizer that removes outer membrane lipopolysaccharides), sodium PPi (a surface charge modifier), sodium periodate (an oxidizer that cleaves surface polysaccharides), lysozyme (an enzyme that cleaves cell wall components), and proteinase K (a nonspecific protease that cleaves peptide bonds). Buffers included MOPS [3-(N-morpholino)propanesulfonic acid], Tris, phosphate, and an unbuffered solution containing only NaCl. Transport characteristics in the porous media were compared by using a sticking coefficient, alpha, defined as the rate at which particles stick to a grain of medium divided by the rate at which they strike the grain. Tween 20 reduced alpha by 2.5 orders of magnitude, to alpha = 0.0016, and was the most effective chemical treatment for decreasing bacterial attachment to glass beads in buffered solutions. Similar reductions in alpha were achieved in unbuffered solutions by reducing the solution ionic strength to 0.01 mM. EDTA, protease, and other treatments designed to alter cell structures did not reduce alpha by more than an order of magnitude. The number of bacteria retained by the porous media was decreased by treatments that made A. paradoxus more hydrophobic and less electrostatically charged, although alpha was poorly correlated with electrophoretic mobility and hydrophobicity index measurements at lower alpha values.(ABSTRACT TRUNCATED AT 250 WORDS)

Secretion of human epidermal growth factor (EGF) in autotrophic culture by a recombinant hydrogen-utilizing bacterium, Pseudomonas pseudoflava, carrying broad-host-range EGF secretion vector pKSEGF2

We constructed the broad-host-range human epidermal growth factor (EGF) secretion plasmid pKSEGF2 by inserting the Escherichia coli tac promoter, the signal sequence of Pseudomonas stutzeri amylase, and the synthesized EGF gene into the broad-host-range vector pKT230. E. coli JM109 carrying pKSEGF2 secreted EGF into the periplasm and the culture medium under the control of the tac promoter. Pseudomonas aeruginosa PAO1161 carrying pKSEGF2 and Pseudomonas putida AC10 carrying pKSEGF2 secreted EGF into the culture medium constitutively. Four hydrogen-utilizing bacteria, Pseudomonas pseudoflava, Alcaligenes eutrophus, Alcaligenes paradoxus, and Paracoccus denitrificans, were transconjugated with pKSEGF2 from eight hydrogen-utilizing bacteria tested. In these transconjugated hydrogen-utilizing bacteria, P. pseudoflava carrying pKSEGF2 grew autotrophically and secreted EGF, confirmed by Western blot (immunoblot) analysis, into the culture medium constitutively.

Integration and excision of a 2,4-dichlorophenoxyacetic acid-degradative plasmid in Alcaligenes paradoxus and evidence of its natural intergeneric transfer

A self-transmissible 2,4-dichlorophenoxyacetic acid (2,4-D)-degradative plasmid, pKA2, has been identified in a new 2,4-D-degrading strain, Alcaligenes paradoxus 2811P, isolated from agricultural soil. pKA2 occurred as a 42.9-kb plasmid in strain 2811P. A derivative strain, 2811C, was isolated from a stock culture in which the entire pKA2 plasmid was apparently integrated into the host chromosome without loss of the 2,4-D+ phenotype. This interpretation is based on the disappearance of a free plasmid DNA band, a shift in the tfdA-hybridizing band to the chromosome, loss of transmissibility of the 2,4-D+ trait, and appropriate shifts in Southern hybridization bands of plasmid DNA compared with whole-cell DNA. The integrated plasmid of strain 2811C was excised either precisely or imprecisely after continued transfer on 2,4-D-containing medium. This suggests that a chromosome-free plasmid cycle may occur to optimize fitness under conditions of specific resource fluctuation. Another new 2,4-D-degrading strain, Pseudomonas pickettii 712, which was isolated from the same field plot but at a different time, was found to carry a plasmid that is nearly identical to pKA2. The plasmid of this strain, pKA4, is 40.9 kb long and has features in common with pKA2, such as high self-transmissibility, hybridization only to the tfdA gene among the 2,4-D-metabolic genes of 2,4-D-degradative plasmid pJP4, and similar restriction endonuclease-generated fragments. Furthermore, the genetic homology between the two plasmids was high since all fragments of pKA2 hybridized to pKA4. These results suggest that these two plasmids are closely related and thus their occurrence in two genera in nature is the result of natural horizontal gene transfer.

Degradation of veratraldehyde byAlcaligenes paradoxus

The degradation of veratraldehyde by Alcaligenes paradoxus was studied. Three products, veratric acid, vanillic acid and a minor amount of veratryl alcohol, were identified. The effect of various metabolic inhibitors on the uptake of veratraldehyde, veratric and vanillic acid showed the uptake process to be energy-dependent. The NAD+-dependent enzyme responsible for the conversion of veratraldehyde to veratric acid has been separated from veratryl alcohol-oxidizing enzyme.

NOTES: Comamonadaceae, a New Family Encompassing the Acidovorans rRNA Complex, Including Variovorax paradoxus gen. nov., comb. nov., for Alcaligenes paradoxus (Davis 1969)

A new family, the Comamonadaceae, is proposed for the organisms belonging to the acidovorans rRNA complex in the beta subclass of the Proteobacteria. This family includes the genera Comamonas, Acidovorax, Hydrogenophaga, Xylophilus, and Variovorax (formerly Alcaligenes paradoxus), as well as a number of phylogenetically misnamed Aquaspirillum and phytopathogenic Pseudomonas species.

The composition of the rhizosphere of chicory (Cichorium intybus L. var. foliosum Hegi)

The bacterial composition of the chicory rhizosphere (Cichorium intybus L. var. foliosum Hegi) was examined at four different growth stages in the field and also after 1 month storage of the roots. Based on protein fingerprints (SDS – polyacrylamide gel electrophoresis of total cell proteins) 233 isolates were grouped into 117 different groups. Forty percent of the isolates belonged to one of three groups: CH001, CH002, or CH213. Fingerprint type CH001 and CH002 were comprised of fluorescent pseudomonads. Fingerprint type CH213 was identified as Alcaligenes paradoxus. Fingerprint type CH213 strains, normally isolated early in the growing season, were inhibited in vitro by fingerprint type CH001 strains which appeared later in the growing season. Gram-negative isolates were predominant among the remaining fingerprint types: Pseudomonas paucimobilis, Xanthomonas maltophilia, Agrobacterium radiobacter, and Flavobacterium spp. A few Gram-positive isolates were found at the beginning of the growing season, i.e., Bacillus spp. and Streptomyces spp. The production of antifungal compounds was restricted to the 11 isolates among which were fluorescent Pseudomonas and Bacillus spp. Twenty-four fluorescent pseudomonad isolates from the rhizosphere were pathogenic on chicory leaves.

Transfer of Xanthomonas ampelina Panagopoulos 1969 to a New Genus, Xylophilus gen. nov., as Xylophilus ampelinus (Panagopoulos 1969) comb. nov.

Thirty-four strains of Xanthomonas ampelina, the causal agent of bacterial necrosis of grape vines, were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of their cellular proteins and by numerical analysis of 106 enzymatic features (API systems). These organisms formed a very homogeneous taxon. Generic and suprageneric relationships were determined by hybridizations between 23S 14C-labeled ribosomal ribonucleic acid from Xanthomonas ampelina NCPPB 2217T (T = type strain) and deoxyribonucleic acids from eight Xanthomonas ampelina strains, Xanthomonas campestris NCPPB 528T, and the type strains of 16 possibly related species. Xanthomonas ampelina was found to be a totally separate subbranch in ribosomal ribonucleic acid superfamily III, without any relatives at the generic level. It is not related to the genus Xanthomonas. Genetically its closest relatives are, among others, Pseudomonas acidovorans, Alcaligenes paradoxus, and Comamonas terrigena. We propose the transfer of Xanthomonas ampelina to a new genus, Xylophilus. The only species and thus the type species is Xylophilus ampelinus. The type strain is NCPPB 2217.

The taxonomic position of Xanthomonas ampelina1

The internal taxonomic structure and the suprageneric relations of the causal agent of bacterial blight and canker of grapevine, the misnamed Xanthomonas ampelina, were examined. By studying more than 30 strains of different geographical origin by polyacrylamide gel electrophoresis and DNA: DNA hybridization, we established the homogeneity of X. ampelina. [14C]‐labelled ribosomal RNA (rRNA) was prepared from the type strain of X. ampelina (NCPPB 2217) and hybridized with DNA from different representative strains from the Pseudomonas acidovorans rRNA complex in rRNA superfamily III. X. ampelina was found to be a separate branch in rRNA superfamily III, related at a suprageneric level to P. acidovorans, Alcaligenes paradoxus and the other organisms of the P. acidovorans rRNA complex.

The use of disposable gaas generating kits for the growth of hydrogen-oxidizing bacteria and the determination of hydrogen autotrophy

A simplified procedure for the determination of autotropic growth of hydrogen-oxidizing bacteria has been developed. The method uses commercially available disposable hydrogen and carbon dioxide kits, commonly used in anaerobic bacteriology, to produce a gaseous atmosphre containing by volume approximately 41% hydrogen, 6% carbon dioxide, 11% oxygen and 42% nitrogen. The atmosphere was suitable for the growth of strains assigned to the species Alcaligenes eutrophus, Alcaligenes paradoxus, Paracoccus denitrificans, Pseudomonas facilis, Pseudomonas flava, Pseudomonas palleronii, Pseudomonas saccahrophilia and Rhodococcus sp. ( ‘Nocardia opaca’). The method can also be used for the screening of hydrogen-oxidizing ability in bacterial isolates, thus eliminating the need for complex gas mixing devices or expensive gas mixtures.

Deoxyribonucleic Acid Relationships Among Hydrogen-Oxidizing Strains of the Genera Pseudomonas, Alcaligenes, and Paracoccus

Optical determination of deoxyribonucleic acid (DNA)-DNA reassociation kinetics was applied to the classification of 32 selected strains of hydrogen-oxidizing bacteria belonging to the genera Pseudomonas, Alcaligenes, and Paracoccus. The renaturation studies revealed a high intraspecies DNA homology for some strains of the species Pseudomonas palleronii, Pseudomonas pseudoflava, and Alcaligenes paradoxus, supporting former taxonomic concepts of different authors. The 12 denitrifying strains belonging to the genus Paracoccus were shown to be interrelated at various levels of percent degree of binding, and at least two clusters of very high genome-DNA relatedness have been found. DNA-DNA reassociation kinetics were also used for the calculation of the average molecular weight of genome DNA. The genome molecular weight of the hydrogen-oxidizing bacteria investigated in this study ranged from 3 X 109 to 5 X 109.

Isolation and characterization of the pesticide-degrading plasmid pJP1 from Alcaligenes paradoxus.

A strain of Alcaligenes paradoxus, unable to degrade phenoxyacetic acid, was shown to degrade two synthetic derivatives of this molecule, the herbicides 2,4-dichlorophenoxyacetic acid and 2-methyl-4-chlorophenoxyacetic acid. The ability to degrade these pesticides is encoded by a 58-megadalton conjugal plasmid, pJP1.

Intra- and Intergeneric Similarities of Chromobacterium and Janthinobacterium Ribosomal Ribonucleic Acid Cistrons

We prepared hybrids between 14C-labeled ribosomal ribonucleic acid (rRNA) from either Chromobacterium violaceum NCTC 9757 or Chromobacterium lividum NCTC 9796 and deoxyribonucleic acid (DNA) from a great variety of named gram-negative bacteria, including many type and reference strains. Each hybrid was described by two parameters: (i) Tm(e) , the temperature at which 50% of the hybrid was denatured; (ii) the percent rRNA binding, the amount (micrograms) of [14C]rRNA duplexed in stringent conditions per 100 μg of filter-fixed homologous or heterologous DNA. Each taxon occupied a definite area on the rRNA similarity maps. All C. lividum and C. violaceum strains formed tight separate clusters around their neotype strains. The rRNA parameters of both taxa were about as different from each other as was the case for many genera. Both taxa are phenotypically and genotypically (by DNA-DNA hybridization) likewise very different. We proposed to elevate each cluster to genus rank as C. violaceum and Janthinobacterium lividum. The following strains were misnamed and belonged in neither genus: “C. lividum” = “C.” folium NCTC 10590 and 10591; “C. lividum” GA; “C.” marismortui ATCC 17056; “Pseudomonas” (“Chromobacterium”) iodinum ATCC 9897, 15728, and 15729; “C.” indicum-rubrum (now Serratia marcescens) NCTC 2847; and “C.” viscosum (now Corynebacterium sp.) NCTC 2416. The rRNA cistrons of Chromobacterium and Janthinobacterium resembled most closely those of Pseudomonas section II and III, the authentic Alcaligenes, Bordetella bronchiseptica, the H2-oxidizing Alcaligenes eutrophus and Alcaligenes paradoxus, and Comamonas percolans NCIB 8193. These taxa displayed a number of phenotypical similarities. We suggested that all these taxa are the closest taxonomic relatives of Chromobacterium and Janthinobacterium.

Micromorphology of Gram-negative hydrogen bacteria

The cell morphology, the arrangement and fine structure of flagella and the piliation of the following Gram-negative aerobic hydrogen bacteria have been studied: Alcaligenes eutrophus, Alcaligenes paradoxus, Alcaligenes ruhlandii, Pseudomonas flava, Pseudomonas pseudoflava, Pseudomonas palleronii, Pseudomonas facilis, Aquaspirillum autotrophicum, Paracoccus denitrificans, Corynebacterium autotrophicum, and strains MA 2 and SA 35. The identity of the bacteria was examined by their substrate spectra and type of flagellation. Three types of flagellar fine structure were differentiated. The presence of pili was noted in strains of Alcaligenes paradoxus, Pseudomonas flava, P.pseudoflava, P.palleronii, and P.facilis.

Localization and stability of hydrogenases from aerobic hydrogen bacteria.

Alcaligenes eutrophus strains H 16, B 19, G 27 and N9A contained two different hydrogenases. One enzyme catalyzed the reduction of NAD by hydrogen and was strictly localized in the soluble cell fraction. While the second enzyme was found to be particulate and unable to react with NAD. All other tested strains, Alcaligenes paradoxus SA 29, Pseudomonas facilis, P. palleronii RH 2, Pseudomonas sp. strain GA 3, Paracoccus denitrificans, Aquaspirillum autotrophicum SA 32, and Corynebacterium autotrophicum 14g and 7C contained only a single enzyme exclusively bound to membranes. This was established using fractional centrifugation, indicator enzyme systems, gently methods of cell disintegration and discontinuous sucrose density gradient centrifugation. In cell-free extracts obtained by rough disruption (sonication) of cells, hydrogenase was associated to particles of different size and sedimentation velocity. A partial solubilization of hydrogenase caused by sonication was observed with P. facilis. Without exception, the particulate hydrogenases were found (1) to be unable to reduce pyridine nucleotides, and (2) to reduce methylene blue at an extremely high activity. The eminent reaction rate of 34 micronmoles H2 oxidized per min and mg protein has been determined in particle suspensions of Pseudomonas sp. strain GA 3. All hydrogenases were stable during storage under hydrogen atmosphere, except the soluble enzyme for A. eutrophus H 16 which was shown to be more stable under aerobic conditions.