Pseudomonas sp. Strain 273, an Aerobic α,ω-DichloroalkaneDegrading Bacterium

A gram-negative, aerobic bacterium was isolated from soil; this bacterium grew in 50% (vol/vol) suspensions of 1,10-dichlorodecane (1,10-DCD) as the sole source of carbon and energy. Phenotypic and small-subunit ribosomal RNA characterizations identified the organism, designated strain 273, as a member of the genus Pseudomonas. After induction with 1,10-DCD, Pseudomonas sp. strain 273 released stoichiometric amounts of chloride from C₅ to C₁₂ α,ω-dichloroalkanes in the presence of oxygen. No dehalogenation occurred under anaerobic conditions. The best substrates for dehalogenation and growth were C₉ to C₁₂ chloroalkanes. The isolate also grew with nonhalogenated aliphatic compounds, and decane-grown cells dechlorinated 1,10-DCD without a lag phase. In addition, cells grown on decane dechlorinated 1,10-DCD in the presence of chloramphenicol, indicating that the 1,10-DCD-dechlorinating enzyme system was also induced by decane. Other known alkane-degrading Pseudomonas species did not grow with 1,10-DCD as a carbon source. Dechlorination of 1,10-DCD was demonstrated in cell extracts of Pseudomonas sp. strain 273. Cell-free activity was strictly oxygen dependent, and NADH stimulated dechlorination, whereas EDTA had an inhibitory effect.     

Determination of the sensitivity of bacteria to barium ions,a taxonomic marker of the genus <Emphasis Type="Italic">Pseudomonas</Emphasis>

Comparative efficacy of the determination of the sensitivity of bacterial cells to barium ions was evaluated on a synthetic nutrient medium, FMH agar, Mueller-Hinton agar, and AGV agar. The synthetic nutrient medium developed for this study contained L-proline and L-glutamine as the sole nitrogen and carbon source, which promoted growth of all Pseudomonas strains and ensured the minimal level of barium binding. The sensitivity of 80 strains belonging to 11 Pseudomonas species, including the type strains, as well as of 80 strains of 22 other bacterial species, was studied. The sensitivity of bacteria to barium ions was determined by using serial dilutions of barium chloride in the nutrient medium. The highest level of analytical sensitivity of pseudomonads to barium ions was determined on the synthetic nutrient medium: the minimal inhibitory concentration (MIC) values of barium chloride ranged from 0.5 to 6 g/L, the MIC₉₀ value was 2 g/L. At the same time, 86.1% of all strains of fluorescent Pseudomonas species produced fluorescein on the control BaCl₂-free synthetic nutrient medium. For representatives of other genera grown on all the studied nutrient media, the MIC values of barium chloride ranged from 20 to 50 g/L. The proposed method for determination of the sensitivity of bacteria to barium ions using the synthetic nutrient medium with 6 g/L of barium chloride as a criterion for the classification of barium-sensitive strains to the genus Pseudomonas is suitable for standardization.     

Taxonomic heterogeneity of the collection strains of fluorescent pseudomonads

Typing of 13 strains of fluorescent pseudomonads from the Belarusian collection of nonpathogenic microorganisms (BIM) by ERIC-PCR and BOX-PCR revealed high level of genetic heterogeneity in bacteria, most of which have been previously identified as Pseudomonas fluorescens according to the classical scheme. Evaluation of the similarities of the 16S rRNA gene sequences and their phylogenetic analysis excluded affiliation of the bacteria under study within the same species and allowed them to be distributed within three relatively distant clusters of the genus Pseudomonas phylogenetic tree. While eight strains fell into the phylogenetic group of P. fluorescens, only one of them could be identified as P. fluorescens. Four strains clustered within the P. vancouverensis phylogenetic group, formed by new species, which have been described mainly according to the evaluation of genome relationships. One bacterium was related to a stable branch that did not contain any type strains of the known Pseudomonas spp. These results indicate taxonomic heterogeneity of collection strains of the fluorescent pseudomonads and demonstrate the necessity of identification of them considering the requirements of phylogenetic bacterial taxonomy.     

Growth of Pseudomonas fluorescens in modified atmosphere packaged tofu

Aims: The objective was to study the growth of Pseudomonas in a food product (tofu) where it typically occurs as a spoilage organism, and when this product is stored under modified atmosphere. Methods and Results: A Pseudomonas strain was isolated from the endogenous microflora of tofu. Tofu was inoculated with the strain, packaged in different gas conditions (air, 100% N₂, 30% CO₂/70% N₂ or 100% CO₂) and stored under refrigerated conditions. Microbial loads and the headspace gas composition were monitored during storage. Conclusions: The strain was capable of growing in atmospheres containing no or limited amounts of oxygen and increased amounts of carbon dioxide. Even when 100% CO₂ was used, growth could not be inhibited completely. Significance and Impact of Study: In contrast to the general characteristics of the genus Pseudomonas (strictly aerobic, highly sensitive to CO₂), it should not be expected in the food industry that removing oxygen from the food package and increasing the carbon dioxide content, combined with cold storage, will easily avoid spoilage by Pseudomonas species. Guarantee of hygienic standards and combination of strategies with other microbial growth inhibiting measures should be implemented.     

Synthesis of 5-Alkyl-5-methyl-2-hydroxy-2-cyclopentenones

A hydrogen bacterium strain, N34, and its oxygen-resistant segregant strain, Y38, were subjected to a taxonomical study. Since both strains were capable of N₂-fixation, N₂-fixing facultative hydrogen autotrophs listed in “Bergey’s Manual of Systematic Bacteriology” were used for comparison. Both strains produced a water-insoluble carotenoid pigment, zeaxanthin dirhamnoside, indicating that both should be classified into the genus Xanthobacter. Then, the differential characteristics of the two species of the genus Xanthobacter, X. autotrophicus and X. flavus, were investigated as to both strains. The vitamin requirement, the sensitivity to oxygen under autotrophic conditions, the inducibility of hydrogenase, the substrate range of carbohydrates and N₂-fixing growth characteristics of both strains were almost completely opposite to those of X. flavus. Moreover, both strains coincided exactly with X. autotrophicus in morphological and other physiological characteristics. From these results both strains were identified as Xanthobacter autotrophicus.     

Characterization of a Facultatively Psychrophilic Bacterium, Vibrio rumoiensis sp. nov., That Exhibits High Catalase Activity

A novel facultatively psychrophilic bacterium, strain S-1, which exhibits extraordinarily high catalase activity was isolated from the drain pool of a fish product processing plant that uses H₂O₂ as a bleaching and microbicidal agent. The catalase activity of the isolate was 1 or 2 orders of magnitude higher than those of Corynebacterium glutamicum, Staphylococcus aureus, Pseudomonas fluorescens, and five other species tested in this study. The strain seemed to possess only one kind of catalase, according to the results of polyacrylamide gel electrophoresis of the cell extract. The optimum temperature for catalase activity was about 30°C, which was about 20°C lower than that for bovine catalase activity. Electron microscopic observation revealed that the surface of the microorganism was covered by blebs. Although the isolate was nonflagellated, its taxonomic position on the basis of physiological and biochemical characteristics and analysis of 16S rRNA sequence and DNA-DNA relatedness data indicated that strain S-1 is a new species belonging to the genus Vibrio. Accordingly, we propose the name Vibrio rumoiensis. The type strain is S-1 (FERM P-14531).     

Oxic and anoxic growth of a new Citrobacter species on amino acids

A facultatively anaerobic bacterium, strain P-88, was enriched selectively under dual limitation by glutamate and oxygen in a chemostat. The new strain is a gram-negative motile rod. The mol% guanine plus cytosine of the DNA is 51.4±0.6 mol%. The organism grows on citrate as a sole source of carbon and energy, does not form acetoin, does not induce lysine decarboxylase and was thus classified as a species of the genus Citrobacter. A remarkable characteristic of the new isolate is its ability to grow on several amino acids with either a respiratory or a fermentative type of metabolism. Under strictly anoxic conditions glutamate was fermented to acetate, H₂, CO₂ and ammonia. Asparagine, aspartate and serine could also be fermented. Furthermore, all type strains of the genus Citrobacter were shown to have the same fermentative abilities. Based on enzyme activities determined in cell-free extracts a combination of the methylaspartate pathway and the mixed acid fermentation of Enterobacteriaceae is proposed to explain the glutamate fermentation pattern observed in cultures of strain P-88. Analysis of the growth of strain P-88 in continuous culture with various degrees of oxygen supply, demonstrated that the bacterium can rapidly switch between oxic and anoxic metabolism. Cultures of strain P-88 grown under oxygen limitation simultaneously respire and ferment glutamate, suggesting that the organism is particularly well adapted to growth in microoxic environments.     

Utilization of simple phenolics for dinitrogen fixation by soil diazotrophic bacteria

Summary A microaerobic diazotrophic bacterium tentatively identified as aPseudomonas species was isolated from a forest soil. Its nitrogenase (C₂H₂ reduction) activity in liquid medium was significantly supported by phenolic compounds when compared with glucose-, mannitol- or malate-supported activity. The utilization of phenolics was dependent on substrate induction and the appropriate oxygen concentration. At a pO₂ of 0.05 protocatechuate was a better carbon source for N₂ fixation than glucose. In the case ofLignobacter protocatechuate was a better carbon source for N₂ fixation than glucose at pO₂ 0.2 but not at pO₂ 0.05. It is suggested that certain monomeric phenols can support nitrogenase activities in many carbon-limited soil environments.Contribution No. 1484 from the Chemistry and Biology Research Institute, Agriculture Canada, Ottawa, Canada.     

Gas Chromatographic Analysis of Volatile Esters in Wines

We isolated a new aerobic gram negative collagenolytic bacterium, strain CRZV₁. This strain is yellow pigmented, non motile; it poorly degrades the sugars and its proteolytic activity corresponds to one enzyme, a collagenase which is produced in media with collagen, or collagen like substrates. The strain CRZV_l, presents many similarities with Flavobacteria. However, the GC content of the DNA, which is 65%, excludes our collagenolytic strain from the genus Flavobacterium, where all the species have GC content between 30 and 42%. All the strains, already described as Flavobacteria, the GC contents of which are situated between 55 and 70%, are actually unclassified. They could be included in the genus Empedobacter. This genus is not in the approved list of bacterial names, so we only consider this new collagenolytic bacteria as a gram negative, aerobic, yellow pigmented bacteria.     

Classification of secondary alcohol-utilizing methanogens including a new thermophilic isolate

A thermophilic coccoid methanogenic bacterium, strain TCI, that grew optimally around 55° C was isolated with 2-propanol as hydrogen donor for methanogenesis from CO₂. H₂, formate or 2-butanol were used in addition. Each secondary alcohol was oxidized to its ketone. Growth occurred in defined freshwater as well as salt (2% NaCl, w/v) medium. Acetate was required as carbon source, and 4-aminobenzoate and biotin as growth factors. A need for molybdate or alternatively tungstate was shown.Strain TCI was further characterized together with two formerly isolated mesophilic secondary alcohol-utilizing methanogens, the coccoid strain CV and the spirilloid strain SK. The guanine plus cytosine content of the DNA of the three strains was 55,47, and 39 mol%, respectively. Determination of the molecular weights of the methylreductase subunits and sequencing of ribosomal 16S RNA of strains TCI and CV revealed close relationships to the genus Methanogenium. The new isolate TCI is classified as a strain of the existing species, Methanogenium thermophilum (thermophilicum). For strain CV, that uses ethanol or 1-propanol in addition, a classification as new species, Methanogenium organophilum, is proposed. Strain SK is affiliated with the existing species, Methanospirillum hungatei. The ability to use secondary alcohols was also tested with described species of methanogens. Growth with secondary alcohols was observed with Methanogenium marisnigri, Methanospirillum hungatei strain GP1 and Methanobacterium bryantii, but not with Methanospirillum strains JF1 and M1h, Methanosarcina barkeri, Methanococcus species or thermophilic strains or species other than the new isolate TCI.     

Induction of H2O2 and related enzymes in tomato roots infected with root knot nematode (M. javanica) by several chemical and microbial elicitors

The effects of chemical and microbial elicitors such as β-aminobutyric acid (BABA), Salicylic acid (SA), and Pseudomonas fluorecens CHAO on hydrogen peroxide generation and activity of the enzymes related to its metabolism, i.e., superoxide dismutase (SOD), guaiacol peroxidase (GPOX), and catalase (CAT) were investigated in tomato roots infected with root-knot nematode (Meloidogyne javanica). Results of this study show that treating the tomato seedlings with the above elicitors significantly reduces the nematode infection level. Among the tested elicitors, BABA has reduced the nematode galls, number of egg masses per plant and number of eggs per individual egg mass more than the others. Additionally, the amount of H₂O₂, a product of oxidative stress, SOD and GPOX specific activities were significantly increased in the elicitor treated plants in comparison to control. Our observation shows that BABA also increases the H₂O₂ accumulation and the SOD and GPOX activities more as compared with the other tested elicitors. Such increases have occurred in two phases and maximum levels of them were observed at 5 days after treatment. In contrast with the increase in SOD and GPOX activities, the CAT activity doesnot show any significant increase in treated plants as compared with the control and other tested elicitors. It can be concluded that BABA, SA, and Pseudomonas fluorescens CHAO induce oxidative stress in tomato roots through generation of reactive oxygen species (ROS) and the enzymes related to their metabolism.     

Influence of Ca<Superscript>2+</Superscript> ions on metabolism of active oxygen species in wheat calli cocultured with the bunt pathogen <Emphasis Type="Italic">Tilletia caries</Emphasis>

The effect of Ca²⁺ on morphophysiological parameters of wheat calli (Triticum aestivum L.) infected by the bunt pathogen Tilletia caries, in particular on the level of active oxygen species, activity of oxalate oxidase, peroxidase, and catalase is investigated. The concentration of O²⁻, H₂O₂, and activity of oxidoreductases (oxalate oxidase, peroxidase, and catalase) depended on the content of Ca²⁺ in the culture medium of calli. The increase of the concentration of Ca²⁺ ions in the culture medium led to forming of calli with high structure, induction of activity of oxalate oxidase and of some isoperoxidase, and to accumulation of active oxygen species. These changes contributed to inhibition of development of the fungus. So this dependence confirm the role of calcium as the intermediant in biochemical reactions related to the formation of the protective response of plant cells to biotic stress.     

Characterization of an O-desmethylangolensin-producing bacterium isolated from human feces

A bacterium that converted daidzein to O-desmethylangolensin was isolated from the feces of healthy humans. It was an obligately anaerobic, nonsporeforming, nonmotile and Gram-positive rod. The isolate used glucose, sucrose, raffinose, maltose, and fructose as carbon sources. It did not hydrolyze gelatin, esculin, or starch. The strain was urease, acid phosphatase, and arginine dihydrolase positive. It was catalase, oxidase, H₂S, and indole negative. The major products of glucose fermentation were butyrate and lactate. Its mol% G+C was 51.2. The major cellular fatty acids were C_16:0 DMA, C_16:0, and C_16:0 aldehyde. The structural type of cell wall peptidoglycan was suggested to be A1γ. The isolate was susceptible to β-lactam, cefem, and macrolide antibiotics and resistant to aminoglycoside and quinolone antibiotics. The bacterium was related to Eubacterium ramulus ATCC29099^T, Eubacterium rectale ATCC33656^T, and species of the genus Roseburia, but the highest 16S rRNA gene similarity to these described species was only 94.4%, consistent with its being classified as a novel genus. Based on the above, the isolate, named strain SY8519, was identified as belonging to a novel genus in the Clostridium rRNA cluster XIVa.     

Characteristics of a nitrogen-fixing methanotroph,Methylocystis T-1

A methane-oxidizing bacterium capable of nitrogen fixation was isolated from soil taken from an area which leaked methane gas. Strain T-1 was a catalase and oxidase-positive, gram-negative straight rod-shaped strictly aerobic bacterium which formed lipid cysts and type II intracytoplasmic membranes. The organism was a microaerophilic nitrogen-fixing methanotroph. Strain T-1 is considered to be classified intoMethylocystis. The organism evolved hydrogen gas when grown in the nitrogen-free medium of atmospheric oxygen concentrations of 1.5% or more. Below this level, however, hydrogen gas was not evolved. In addition to methanol, formaldehyde and formate, ethanol, acetate and hydrogen gas served as oxidizable substrates for the acetylene reduction test. H₂-stimulated nitrogenase activity was limited in a very narrow range of oxygen concentration and not detected at 2% O₂. With acetate as the substrate, however, about an 80% of the maximum acetylene reduction activity was detected at 2% O₂. These results suggest that strain T-1 is capable of recycling the hydrogen gas evolved during nitrogen fixation under low partial pressures of O₂.     

Substrate specificity of a long-chain alkylamine-degrading <Emphasis Type="Italic">Pseudomonas</Emphasis> sp isolated from activated sludge

A bacterium strain BERT, which utilizes primary long-chain alkylamines as nitrogen, carbon and energy source, was isolated from activated sludge. This rod-shaped motile, Gram-negative strain was identified as a Pseudomonas sp. The substrate spectrum of this Pseudomonas strain BERT includes primary alkylamines with alkyl chains ranging from C₃ to C₁₈, and dodecyl-1,3-diaminopropane. Amines with alkyl chains ranging from 8 to 14 carbons were the preferred substrates. Growth on dodecanal, dodecanoic acid and acetic acid and simultaneous adaptation studies indicated that this bacterium initiates degradation through a C_alkyl–N cleavage. The cleavage of alkylamines to the respective alkanals in Pseudomonas strain BERT is mediated by a PMS-dependent alkylamine dehydrogenase. This alkylamine dehydrogenase produces stoichiometric amounts of ammonium from octylamine. The PMS-dependent alkylamine was found to oxidize a broad range of long-chain alkylamines. PMS-dependent long-chain aldehyde dehydrogenase activity was also detected in cell-free extract of Pseudomonas strain BERT grown on octylamine. The proposed pathway for the oxidation of alkylamine in strain BERT proceeds from alkylamine to alkanal, and then to the fatty acid.     

A potential bacterial biocontrol agent,strain S2V2 against pathogenic marine <Emphasis Type="Italic">Vibrio</Emphasis> in aquaculture

We isolated a marine bacterium strain S2V2 which inhibited the growth of pathogenic marine Vibrio spp. The aims of this research were to identify a new antibiotic-producing marine bacterium strain S2V2, and evaluate its spectrum activity and pathogenic property. Analysis of 16S rDNA sequence placed strain S2V2 in the genus Pseudoalteromonas, but the sequence similarity was low (95.46%) implying the strain might be a new species in this genus. Strain S2V2 inhibited the growth of 67.9% of 28 Vibrio strains tested. This strain inhibited V. alginolyticus, V. anguillarum, V. fluvialis, V. harveyi, V. metschnikovii, V. splendidus, V. ordalii, V. parahaemolyticus, and V. vulnificus, but inactive against V. campbellii, Aeromonas hydrophyla and Staphylococcus aureus. Strain S2V2 produced extracellular non proteinaceous antibacterial substances. The highest antibacterial activity was found when strain S2V2 was cultured for 96 h in ZoBell broth medium. An artificial infection to post larvae of Lithopenaeus vanname indicated that strain S2V2 was a non pathogenic bacterium. Non pathogenic property and specific antibacterial activity against a broad range of fish pathogenic marine Vibrio of strain S2V2 suggest that this strain is a prospective source of unique antibiotic and a potential biocontrol agent in marine aquaculture.     

Pseudomonas as a frequent and important quorum quenching bacterium with biocontrol capability against many phytopathogens

The aim of the present study was to isolate a variety of quorum quenching bacteria (QB) from the rhizosphere and phyllosphere of three agricultural plants using minimal medium (MM)- and non-minimal medium (NM)-based methods. The members of the Pseudomonas genus constituted the most abundant QB genus, particularly in the rhizospheres of all plant samples and showed the highest quorum quenching (QQ) activity according to a screening assay using a biosensor and 3-oxo-C6-HSL (as an important quorum sensing signal in many phytopathogenic bacteria). In addition, QQ-Pseudomonas were recognised as versatile biocontrol agents against non-bacterial and bacterial plant pathogens, such as Pectobacterium carotovorum subsp. carotovorum (Pcc). Three types of quenching activities, including intracellular and extracellular enzymatic and non-enzymatic activities, were observed in QQ-Pseudomonas. Pseudomonas strains, particularly NM-isolated strains with extracellular activity, are the strongest QQ-based biocontrol agents.     

Biodegradation of propoxur by Pseudomonas species

A bacterium capable of degrading propoxur (2-isopropoxyphenyl-N-methylcarbamate) was isolated from soil by enrichment cultures and was identified as a Pseudomonas species. The organism grew on propoxur at 2 g/l as sole source of carbon and nitrogen, and accumulated 2-isopropoxyphenol as metabolite in the culture medium. The cell free extract of Pseudomonas sp. grown on propoxur contained the activity of propoxur hydrolase. The results suggest that the organism degraded propoxur by hydrolysis to yield 2-isopropoxyphenol and methylamine, which was further utilized as carbon source.     

Genotypic Characterization and Phylogenetic Relations of Pseudomonas sp. (Formerly P. stutzeri) OX1

Pseudomonas sp. OX1, an aromatic compound-degrading bacterium that was tentatively identified by conventional biochemical methods as P. stutzeri, has now been investigated at the molecular level to clarify its taxonomic position. Amplified ribosomal DNA restriction analysis and multiple enzyme restriction fragment length polymorphism (MERFLP) analysis suggested that Pseudomonas sp. OX1 could not be classified as P. stutzeri. Phylogenetic analyses based on 16S rRNA and gyrB genes further confirmed that this strain belongs to the Pseudomonas (sensu stricto) genus, but not to the stutzeri species. The data obtained demonstrated that Pseudomonas sp. OX1 belongs to intrageneric cluster II and is related to the P. fluorescens–P. syringae complex.