Pseudomonas Pseudoalcaligenes Research Articles

Identification of Diazotrophs in the Culturable Bacterial Community Associated with Roots of Lasiurus sindicus, a Perennial Grass of Thar Desert, India

Lasiurus sindicus is a highly nutritive, drought-tolerant, perennial grass that is endemic to the Thar Desert of Rajasthan, India. Analysis of 16S rRNA coding genes of the bacterial isolates enriched in nitrogen-free semisolid medium, from the surface-sterilized roots of L. sindicus, showed predominance of Gram-negative over Gram-positive bacteria. According to comparative sequence analysis of 16S rDNA sequence data, Gram-positive bacteria with low GC content (Staphylococcus warneri and Bacillus sp.) and high GC content (Micrococcus luteus, Microbacterium sp.) were identified. Gram-negative bacteria included Azospirillum sp., Rhizobium sp., Agrobacterium tumefaciens, and Inquilinus limosus (alpha-proteobacteria); Ralstonia sp., Variovorax paradoxus, and Bordetella petrii (beta-proteobacteria); and Pseudomonas pseudoalcaligenes, Stenotrophomonas sp. (gamma-proteobacteria). The occurrence of nifH sequences in Azospirillum sp., Rhizobium sp., and P. pseudoalcaligenes showed the possibility of supplying biologically fixed nitrogen by the root-associated diazotrophs to the host plant.

Pseudomonas segetis sp. nov., isolated from soil

A Gram-negative, aerobic bacterium, designated strain FR1439T, was isolated from the soil of Dokdo in the Republic of Korea. The cells of strain FR1439T were catalase- and oxidase-positive, motile and rod-shaped. Phylogenetic analysis of the 16S rRNA gene sequence revealed that it represents a distinct line of descent within the genus Pseudomonas. The levels of DNA-DNA relatedness between strain FR1439T and type strains of phylogenetically related species, namely Pseudomonas flavescens, Pseudomonas mendocina, Pseudomonas pseudoalcaligenes and Pseudomonas straminea, ranged from 28 to 37%. Several phenotypic characteristics, together with the cellular fatty acid composition, can be used to differentiate strain FR1439T from related pseudomonads. On the basis of the polyphasic taxonomic evidence presented in this study, strain FR1439T represents a novel species, for which the name Pseudomonas segetis sp. nov. is proposed. The type strain is FR1439T (=IMSNU 14101T=CIP 108523T=KCTC 12331T).

Evidence for a tellurite-dependent generation of reactive oxygen species and absence of a tellurite-mediated adaptive response to oxidative stress in cells of Pseudomonas pseudoalcaligenes KF707

Tellurite (TeO3(2-)) is the most toxic and soluble oxyanion among tellurium (Te) compounds. The effects of the metalloid anion on the oxidative stress response of the obligate aerobe Pseudomonas pseudoalcaligenes KF707 were investigated. Cells treated with sub-lethal concentrations of TeO3(2-) showed neither adaptation to it nor cross-protection against oxidants such as 1,1'-4,4'-bipyridinium dichloride (paraquat, PQ2+), diazenedicarboxylic acid bis-N,N-dimethylamide (diamide), tert-butyl hydroperoxide (tBH) and hydrogen peroxide (H2O2). Notably, TeO3(2-) exerted a synergic effect on the toxicity of these latter oxidants. Tellurite was shown to decrease the cellular content of reduced thiols (RSH) with a consequent increase in the production of reactive oxygen species (ROS) and stimulation of the superoxide dismutase (SOD) activity. However, since the time course of ROS production by TeO3(2) (t1/2 > 30 min) was much slower than that with PQ2+ and/or diamide (t1/2 <or= 10 min), in the former case the SOD activity was poorly activated. We conclude that in P. pseudoalcaligenes KF707 cells: (1) the TeO3(2-) acts as a pro-oxidant by stimulating ROS production; (2) the release of superoxide oxyanions is directly linked to the mechanism of toxicity; (3) TeO3(2-) is unable to induce an adaptive response to oxidative stress.

Application of a Microfluidic Reactor for Screening Cancer Prodrug Activation Using Silica-Immobilized Nitrobenzene Nitroreductase

The nitroreductase-catalyzed conversion of a strong electron-withdrawing nitro group to the corresponding electron-donating hydroxylamine is useful in a variety of biotechnological applications. Activation of prodrugs for cancer treatments or antibiotic therapy are the most common applications. Here, we show that a bacterial nitrobenzene nitroreductase (NbzA) from Pseudomonas pseudoalcaligenes JS45 activates the dinitrobenzamide cancer prodrug CB1954 and the proantibiotic nitrofurazone. NbzA was purified by affinity chromatography and screened for substrate specificity with respect to prodrug activation. To facilitate screening of alternate potential prodrugs, polyethyleneimine-mediated silica formation was used to immobilize NbzA with high immobilization yields and high loading capacities. Greater than 80% of the NbzA was immobilized, and enzyme activity was significantly more stable than NbzA in solution. The resulting silica-encapsulated NbzA was packed into a microfluidic microreactor that proved suitable for continuous operation using nitrobenzene, CB1954, and the proantibiotic nitrofurazone. The flow-through system provides a rapid and reproducible screening method for determining the NbzA-catalyzed activation of prodrugs and proantibiotics.

Evaluation of pyrrolidonyl arylamidase for the identification of nonfermenting Gram-negative rods

To evaluate the activity of pyrrolidonyl arylamidase (PYR) for the differentiation and identification of nonfermenting gram negative rods (NFGNR), 293 isolates were tested. A 24 h culture of each test organism was prepared. From this a 108–109 cfu/mL suspension was added to 0.25 mL of sterile physiologic solution. A PYR disk was then added and the test was incubated for 30 minutes at 35–37°C, at environmental atmosphere. Reading was done by adding 1 drop of cinnamaldehyde reagent. Strains of Acinetobacter baumannii, Acinetobacter haemolyticus, Alcaligenes faecalis, Bergeyella zoohelcum, Bordetella bronchiseptica, Bordetella hinzii, Brevundimonas diminuta, Brevundimonas vesicularis, Brucella ovis, Brucella spp., Brucella suis, Burkholderia cepacia complex, Moraxella catarrhalis, Moraxella lacunata, Moraxella nonliquefaciens, Moraxella osloensis, Oligella ureolytica, Pseudomonas alcaligenes, Pseudomonas mendocina, Pseudomonas pseudoalcaligenes, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas Vb3, Psychrobacter phenylpyruvicus, and Stenotrophomonas maltophilia were PYR negative. On the other hand Achromobacter piechaudii, Achromobacter denitrificans, Achromobacter xylosoxidans, Burkholderia gladioli, Chryseobacterium gleum-indologenes, Comamonas testosroni, Cupriavidus pauculus, Delftia acidovorans, Elizabethkingia meningoseptica, Myroides spp., Ochrobactrum anthropi, Pseudomonas oryzihabitans, Ralstonia pickettii, Rhizobium radiobacter, Shewanella spp., Sphingobacterium multivorum, Sphingobacterium spiritivorum, and Weeksella virosa were PYR positive. Finally, Acinetobacter lwoffii, Pseudomonas aeruginosa, Pseudomonas fluorescens, Roseomonas spp., and Sphingomonas paucimobilis-parapaucimobilis were PYR variable. PYR testing should be considered as a useful tool to facilitate the identification of NFGNR.

Active-site engineering of biphenyl dioxygenase: effect of substituted amino acids on substrate specificity and regiospecificity

Biphenyl dioxygenase (Bph Dox) catalyzes the initial dioxygenation step in the metabolism of biphenyl. The large subunit (BphA1) of Bph Dox plays a crucial role in the determination of the substrate specificity of biphenyl-related compounds including polychlorinated biphenyls (PCBs). Previously, the substitution of Asn at Thr-376 near the active-site iron in the BphA1 of Pseudomonas pseudoalcaligenes KF707 expanded the oxidation range and altered the regiospecificity of Bph Dox for PCBs. In this study, we replaced Thr-376 with Gly, Ser, Gln, Tyr, Val, Phe, Asp, and Lys and expressed these enzymes in Escherichia coli. Bph Dox mutants of Thr376Asn, Thr376Val, Thr376Phe, and Thr376Lys showed novel degradation activity for dibenzofuran, which is a poor substrate for KF707 Bph Dox. All active Bph Dox mutants showed altered regiospecificity with 2,2'-dichlorobiphenyl and 2,5,4'-trichlorobiphenyl. The Thr376Gly, Thr376Val, Thr376Phe, and Thr376Asp Bph Dox mutants introduced molecular oxygen at the 2,3 position of 2,2'-dichlorobiphenyl, forming 2-chloro-2',3'-dihydroxybiphenyl with concomitant dechlorination. The Bph Dox mutants of Thr376Gly, Thr376Ser, Thr376Asp, and Thr376Lys attacked 2,5,4'-trichlorobiphenyl via both 2',3'- and 3,4-dioxygenation activities. In particular, the Thr376Phe Bph Dox mutant exhibited enhanced and expanded degradation activities toward all of the compounds tested. Further site-directed mutation was induced to change the oxidizing character of KF707 Bph Dox to that of the Bph Dox of Burkholderia xenovorans LB400 by the substitution of two amino acids, Ile335Phe and Thr376Asn, near the active-site.

Expression of organophosphorus hydrolase OPHC2 in Pichia pastoris: Purification and characterization

Organophosphorus hydrolase is able to hydrolyze phosphoester bonds and reduce the toxicity of organophosphorus compounds. In this work, recombinant organophosphorus hydrolase OPHC2 was successfully produced by Pichia pastoris at a high expression level (approximately 5.5 g/L) using 3 L high-cell-density fermentation. The expression level is higher than those produced in other expression systems. The results of the SDS–PAGE and the Western blot analyses showed a major 36 kDa polypeptide band, which was the same size as that from the original bacteria, Pseudomonas pseudoalcaligenes C2-1. The expressed enzyme was recovered from the culture supernatant and purified by a single-step purification procedure with a recovery rate of 72.78%. The main physiochemical features of the recombinant OPHC2, including its optimum temperature and pH for the reaction, its temperature and pH stability, and its sensitivity to some metal ions and chemical reagents, were also characterized. With methyl parathion as a substrate, the optimum temperature and pH for enzyme activity are 65°C and pH 9.0, respectively. It also shows good thermal and pH stability.

Identification of nif genes in N2‐fixing bacterial strains isolated from rice fields along the Yangtze River Plain

The aim of this research was to identify nifH and nifHDKYE ' genes in twenty strains of N2-fixing heterotrophic bacteria isolated from rice fields in the Yangtze River Plain. Southern hybridization of the total DNA from each strain was performed with the Klebsiella pneumoniae nifHDKYE ' gene probe (6.2 kb Eco RI fragment from pSA30) and the Azospirillum brasilense nifH gene probe (0.6 kb Eco RI-Hin dIII fragment from pHU8). We found that Eco RI fragments of total DNA from Aeromonas hydrophila HY2, Bacillus azotoformans FD, Bacillus licheniformis NCH1, NCH5, WH4, Bacillus brevis NC2, Bacillus pumilus NC12, Bacillus cereus NCH2, Citrobacter freundii HY5, HY9, Derxia gummosa HZ5, Pseudomonas mendocina HZ1 and Pseudomonas pseudoalcaligenes WH3 were positively hybridized with both of the probes. Agrobacterium radiobacter HY17, Corynebacterium sp. HY12, YZ and Pseudomonas sp. HY11 had Eco RI fragments hybridized with the K. pneumoniae nifHDKYE ' gene probe. An Eco RI fragment of total DNA from Bacillus megaterium YY4 was positively hybridized to the A. brasilense nifH gene probe. No hybridization sign was found in the total DNA fragments from Alcaligenes cupidus YY6 and Corynebacterium sp. NC11 hybridized with either of the gene probes. The data provide the number and size of EcoRI fragments of the total DNA hybridized with the nif gene probes for these strains of rarely studied species, suggesting additional evidence for N2 fixing and nif gene diversity of N2-fixing bacteria in rice fields along the Yangtze River Plain.

Cyanide metabolism of Pseudomonas pseudoalcaligenes CECT5344: role of siderophores

Cyanide is one of the most potent and toxic chemicals produced by industry. The jewelry industry of Córdoba (Spain) generates a wastewater (residue) that contains free cyanide, as well as large amounts of cyano-metal complexes. Cyanide is highly toxic to living systems because it forms very stable complexes with transition metals that are essential for protein function. In spite of its extreme toxicity, some organisms have acquired mechanisms to avoid cyanide poisoning. The biological assimilation of cyanide needs the concurrence of three separate processes: (i) a cyanide-insensitive respiratory chain, (ii) a system for iron acquisition (siderophores) and (iii) a cyanide assimilation pathway. Siderophores are low-molecular-mass compounds (600-1500 Da) that scavenge iron (Fe(3+)) ions (usually with extremely high affinity) from the environment under iron-limiting conditions. There are two main classes of siderophores: catechol and hydroxamate types. The catechol-type siderophores chelate ferric ion via a hydroxy group, whereas the hydroxamate-type siderophores bind iron via a carbonyl group with the adjacent nitrogen. In the presence of cyanide, bacterial proliferation requires this specific metal uptake system because siderophores are able to break down cyano-metal complexes. Pseudomonas pseudoalcaligenes CECT5344 is able to use free cyanide or cyano-metal complexes as nitrogen source. A proteomic approach was used for the isolation and identification, in this strain, of a protein that was induced in the presence of cyanide, namely CN0, that is involved in siderophore biosynthesis in response to cyanide. An overview of bacterial cyanide degradation pathways and the involvement of siderophores in this process are presented.

Screening of some traditionally used medicinal plants for potential antibacterial activity

In the present work an attempt has been made to carry out screening for the preliminary antibacterial activity of different plants used in Indian folk medicine. The aim of the study was to select an active plant extract which may be useful in developing new lead compounds to combat deadly diseases. Twelve plants were selected for preliminary screening for their antibacterial potentiality, viz., Abutilon indicum L., Acorous calamus L., Ammania baccifera L., Argyrea nervosa Burm. F., Bauhinia variegata L., Crataeva religiosa Forst., Hedychium spicatum L., Holarrhena antidysenterica L., Piper nigrum L., Plumbago zeylanica L., Psoralea corylifolia L., Saussurea lappa Costus. The antibacterial activity was done by both agar disc diffusion method and agar well diffusion method against five bacterial strains, viz., Bacillus cereus, Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Pseudomonas pseudoalcaligenes . The plant material was extracted with distilled water (aqueous) and methanol. The preliminary screening experiment revealed that methanol extracts were more potent than the aqueous extracts. The plant extracts were more active against gram-positive bacteria than gram-negative bacteria. The most susceptible bacteria were K. pneumoniae and the most resistant bacteria were E. coli . Bauhinia variegata L. exhibited remarkable antibacterial activity.

Steady-State Kinetic Characterization of Evolved Biphenyl Dioxygenase, Which Acquired Novel Degradation Ability for Benzene and Toluene

Biphenyl dioxygenase (Bph Dox) catalyzes initial oxygenation in the bacterial biphenyl degradation pathway. Bph Dox in Pseudomonas pseudoalcaligenes KF707 is a Rieske type three-component enzyme in which a large subunit (encoded by the bphA1 gene) plays an important role in the substrate specificity of Bph Dox. Steady-state kinetic assays using purified enzyme components demonstrated that KF707 Bph Dox had a kcat/Km of 33.1 x 10(3) (M(-1) s(-1)) for biphenyl. Evolved 1072 Bph Dox generated by the process of DNA shuffling (Suenaga, H. et al., J. Bacteriol., 184, 3682-3688 (2002)) exhibited enhanced degradation activity not only for biphenyl (kcat/Km of 62.2 x 10(3) [M(-1) s(-1)]) but also for benzene and toluene, compounds that are rarely attacked by KF707 Bph Dox. These results suggest that evolved 1072 Bph Dox acquires higher affinities and catalytic efficiencies for various substrates than the original KF707 enzyme.

Isolation of Bacteria Able to Metabolize High Concentrations of Formaldehyde

Nineteen bacterial strains able to degrade and metabolize formaldehyde as a sole carbon source were isolated from soil and wastewater of a formaldehyde production factory. The samples were cultured in complex and mineral salts media containing 370 mg formaldehyde/l. The bacterial strains were identified to be Pseudomonas pseudoalcaligenes, P. aeruginosa, P. testosteroni, P. putida, and Methylobacterium extorquens. After adaptation of these microorganisms to high concentrations of formaldehyde; two isolated strains of M. extorquens (strains ESS and PSS) and four strains of P. pseudoalcaligenes (strains LSW, SSW, NSW and OSS) degraded 1850 mg formaldehyde/l, where as P. pseudoalcaligenes strain OSS completely consumed 3700 mg of formaldehyde/l after 24 h and degraded 70% of 5920 mg of formaldehyde/l after 72 h.

Effect of temperature and additional carbon sources on phenol degradation by an indigenous soil Pseudomonad

A new indigenous soil bacterium Pseudomonas sp. growing on phenol and on a mixture of phenol, toluene, o-cresol, naphthalene and 1,2,3-trimethylbenzene (1,2,3-TMB) was isolated and characterized. Phylogenetic analysis suggested its classification to Pseudomonadaceae family and showed 99.8% DNA sequence identity to Pseudomonas pseudoalcaligenes species. The isolate was psychrotroph, with growth temperatures ranging from ca. 0 to 40 degrees C. The GC-MS structural analysis of metabolic products of phenol degradation by this microorganism indicated a possible ortho cleavage pathway for high concentrations (over 200 mg L(-1)) of phenol. Biodegradation rates by this species were found to be three times more effective than those previously reported by other Pseudomonas strains. The effect of temperature on phenol degradation was studied in batch cultures at temperatures ranging from 10 to 40 degrees C and different initial phenol concentrations (up to 500 mg L(-1)). Above 300 mg L(-1) of initial phenol concentration no considerable depletion was recorded at both 10 and 40 degrees C. Maximum degradation rates for phenol were recorded at 30 degrees C. The biodegradation rate of phenol was studied also in the presence of additional carbon sources (o-cresol, toluene, naphthalene, 1,2,3-TMB) at the optimum growth temperature and was found significantly lower by a factor of eight in respect to the strong competitive inhibition between the substrates and the more available sources of carbon and energy. The Haldane equation mu = mum S/(Ks + S + S2/K1) was found to best fit the experimental data at the optimum temperature of 30 degrees C than the Monod equation with kinetic constants mum = 0.27 h(-1), KS = 56.70 mg L(-1), KI = 249.08 mg L(-1).

Biodegradation of pyridine in a completely mixed activated sludge process

A potential bacterial culture (P1), isolated from garden soil and identified as Pseudomonas pseudoalcaligenes–KPN, was used as a starter seed to develop the biomass in a completely mixed activated sludge (CMAS) reactor and the system was evaluated for treatment of wastewater containing pyridine. The results of this study indicate that pyridine could be degraded efficiently at a loading of 0.251 kg pyridine kg MLSS −1 d −1 (0.156 kg TOC kg MLSS −1 d −1) and at an optimal hydraulic retention time (HRT) of 24 h. Pyridine was used as the sole source of carbon and nitrogen by the biomass. Ammonia-nitrogen (NH 3-N) was formed due to the metabolism of the pyridine ring. In the present investigation, the performance of CMAS with reference to pyridine biodegradation and the bio-kinetic constants for the biodegradation of pyridine, in a continuous system, were computed. The results indicate that a CMAS system inoculated with P. pseudoalcaligenes–KPN, under optimum conditions of HRT and pyridine loading, gives a yield coefficient of ( Y) 0.29, decay coefficient ( K d) 0.0011 d −1, maximum growth rate constant ( μ max) 0.108 d −1 and saturation rate constant ( K s) 5.37 mg L −1 for pyridine.

Increased mesquite gum formation in nodal explants cultures after treatment with a microbial biomass preparation

Prosopis laevigata nodal explants cultures were established in Murashige and Skoog medium. Simultaneously these cultures were subjected to stress with biotic elicitors and an environmental factor (temperature increase to promote heat stress) in order to promote and increase exuded mesquite gum production. The biotic elicitors were: Aspergillus nidulans and Pseudomonas pseudoalcaligenes both used in concentrations of 10, 20 and 30 mg, whereas the environmental condition was different incubation temperatures (25, 35 and 40 °C). The greatest gum production (~13 mg of pooled gum from 100 explants after 14 days incubation) took place when the culture medium was added 10, 20 and 30 mg of autoclaved fungal mycelium of A. nidulans or 30 mg of autoclaved bacterial biomass of P. pseudoalcaligenes in combination with an incubation temperature of 35 °C. These treatments were non-significantly different among themselves ( P < 0.05), but were significantly different to the rest of the treatments ( P > 0.05).

Biocatalytic synthesis of monocyclic arene-dihydrodiols and -diols by Escherichia coli cells expressing hybrid toluene/biphenyl dioxygenase and dihydrodiol dehydrogenase genes

The hybrid toluene/biphenyl dioxygenase, which is encoded by the todC1 gene of Pseudomonas putida F1 and the bphA2A3A4 genes of Pseudomonas pseudoalcaligenes KF707, has substrate ranges wider than toluene dioxygenase endoced by the todC1C2BA genes of P. putida F1. We carried out growing cell reactions by Escherichia coli expressing the todC1-bphA2A3A4 genes for the comprehensive production of monocyclic arene-dihydrodiols. As a result, we successfully biotranformed acetophenone-related compounds (acetophenone, propiophenone, and butyrophenone) to the corresponding cis-dihydrodiols. Furthermore, we performed the bioconversion experiments by E. coli cells expressing the bphB (dihydrodiol dehydrogenase) gene in addition to todC1-bphA2A3A4 to produce a series of monocyclic arene-diols. Consequently, toluene, benzene, stylene, p-xylene, acetophenone, propiophenone, butyrophenone, and trifluoroacetophenone were converted to the corresponding vicinal diols. The antioxidative activity of these generated diol compounds was markedly higher than that of the substrate used.

Diversity of Alkaliphilic and Alkalitolerant Bacteria Cultivated from Decomposing Reed Rhizomes in a Hungarian Soda Lake

Bacterial communities associated with decomposing rhizomes of Phragmites australis were investigated in Lake Ferto (Neusiedlersee, Hungary). Alkaliphilic and alkalitolerant strains were isolated on cellulose-containing alkaline medium spread with dilutions of scrapings taken from the surface of the decaying plant material. Fifty-one strains were grouped by numerical analysis based on physiological tests and BIOLOG sole carbon source utilization data. The strains identified by 16S rDNA sequence comparisons included members of low G+C Gram positives (Marinibacillus marinus, Bacillus cereus, and Exiguobacterium aurantiacum), high G+C Gram positives (Nesterenkonia halobia and Dietzia natronolimnea), alpha-proteobacteria (Pannonibacter phragmitetus), and gamma-proteobacteria (Pseudomonas pseudoalcaligenes and Halomonas venusta). Most of the strains were characterized by aerobic chemoorganotrophic respiratory metabolism and utilized several different carbon sources, although no direct cellulolytic activity was observed. Results of the pH and salt tolerance tests revealed optimuma in most cases at pH 11 and at the presence of 2.5-5% NaCl. These bacteria probably occupy niches in the aerobic, alkaline, water-influenced environments on the decomposing reed surfaces.

Detection of polychlorinated biphenyls employing chemical dechlorination followed by biphenyl whole cell sensing system

Polychlorinated biphenyls (PCBs) are a mixture of 209 individual chlorinated compounds commonly known as PCB congeners. These compounds are hydrophobic and are persistent in the environment. Their use was banned in the US a few decades ago because of harmful health effects. Therefore, detection of PCBs in environmental samples is increasingly important. To that end, we have developed a two-step simple and sensitive method for the detection of total PCBs. Specifically, our method involves dechlorination of PCBs to biphenyl followed by detection of biphenyl using a whole cell sensing system as the detection system. The whole cell sensing system consists of cells of the strain Pseudomonas pseudoalcaligenes KF707 harboring plasmid pSD7000. Plasmid pSD7000 contains the gene of lacZ, a reporter protein under the control of the bph operon. The detection is achieved through the emission of light afforded by the expression of reporter protein triggered by the presence of biphenyl. Due to the fact that this operon is activated only by few PCB congeners, a chemical dechlorination method was employed to convert all PCBs to biphenyl, and thus all the PCBs present in a given sample are able to be detected. The results showed that PCB congeners were rapidly (30 min) and efficiently (>98.5%) dechlorinated to biphenyl using a Mg/K2PdCl6 catalyst, and the biphenyl could be subsequently quantified using the whole cell sensing system. This hybrid analytical method that combines classical dechlorination with novel biosensing methods may find applications in the on-site monitoring of PCBs contamination levels.

Genetic Variation in Clones of Pseudomonas pseudoalcaligenes After Ten Months of Selection in Different Thermal Environments in the Laboratory

The random amplification of polymorphic DNA (RAPD) method was used to examine genetic variation in experimental clones of Pseudomonas pseudoalcaligenes in two experimental groups, as well as their common ancestor. Six clones derived from a single colony of P. pseudoalcaligenes were cultured in two different thermal regimes for 10 months. Three clones in the Control group were cultured at constant temperature of 35 degrees C and another three clones in the High Temperature (HT) group were propagated at incremental temperature ranging from 41 to 47 degrees C for 10 months. A total of 45 RAPD primers generated 146 polymorphic markers. Analysis of molecular variance (AMOVA) revealed mild (11%) but significant (P < 0.001) genetic difference between the Control and the HT clones. Phylogenetic analysis based on pairwise genetic distances showed that the HT clones were more divergent from the ancestor and from each other than the Control clones, implying that the HT clones of P. pseudoalcaligenes may have evolved faster than the Control clones.

Alcaligenes faecalis subsp . phenolicus subsp. nov. a phenol-degrading, denitrifying bacterium isolated from a graywater bioprocessor

A Gram (−) coccobacillary bacterium, J T, was isolated from a graywater bioprocessor. 16S rRNA and biochemical analysis has revealed strain J T closely resembles Alcaligenes faecalis ATCC 8750 T and A. faecalis subsp. parafaecalis DSM 13975 T, but is a distinct, previously uncharacterized isolate. Strain J T, along with the type strain of A. faecalis and its previously described subspecies share the ability to aerobically degrade phenol. The degradation rates of phenol for strain J T and reference phenol degrading bacteria were determined by photometrically measuring the change in optical density when grown on 0.1% phenol as the sole carbon source, followed by addition of Gibb's reagent to measure depletion of substrate. The phenol degradation rates of strain J T was found to exceed that of the phenol hydroxylase group III bacterium Pseudomonas pseudoalcaligenes, with isolate J T exhibiting a doubling time of 4.5 h. The presence of the large subunit of the multicomponent phenol hydroxylase gene in strain J T was confirmed by PCR. The presence of the nirK nitrite reductase gene as demonstrated by PCR as well as results obtained from nitrite media indicated denitrification at least to N 2O. Based on phenotypic, phylogenetic, fatty acid analysis and results from DNA–DNA hybridization, we propose assigning a novel subspecies of Alcaligenes faecalis, to be named Alcaligenes faecalis subsp. phenolicus with the type strain J T (=DSM 16503) (=NRRL B-41076).