Characterization of esculin-positive<Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> strains isolated from an underground brook

A group of sixteen esculin-positive fluorescent pseudomonads isolated from an underground brook flowing through a cave complex was characterized by biotyping, multiple enzyme restriction fragment length polymorphism analysis of 16S rDNA (MERFLP), ribotyping and whole-cell fatty-acid methyl-esters analysis (FAME). All strains were phenotypically close to Pseudomonas fluorescens, but they revealed high biochemical variability as well as some reactions atypical for P. fluorescens species. Because identification of pseudomonads by of biochemical testing is often unclear, further techniques were employed. Fingerprints obtained by MERFLP clearly showed that all strains represent P. fluorescens species. Ribotyping separated the strains analyzed into four groups corresponding almost completely (with the exception of one strain) to the clustering based on biochemical profiles. FAME analysis grouped all the strains into one cluster together with the P. putida (biotype A, B), P. chlororaphis and P. fluorescens biotype F representatives, but differentiated them from other FAME profiles of all pseudomonads included in the standard library TSBA 40 provided by MIDI, Inc.     

Three new species of brevibacteria--Brevibacterium antiquum sp. nov., Brevibacterium aurantiacum sp. nov. and Brevibacterium permense sp. nov

This work deals with the taxonomic study of 12 orange-pigmented bacteria isolated from permafrost sediments, rice plots, and soils contaminated with wastes from the chemical and salt industries, which were assigned to the genus Brevibacterium on the basis of phenotypic characteristics, as well as of some strains described previously as Brevibacterium linens. The study revealed three genomic species, whose members and the type strains of the closest species of Brevibacterium had DNA similarity levels between 24 and 59%. The strains of the genomic species differed from each other and from the known species of Brevibacterium in some physiological and biochemical characteristics, as well as in the sugar and polyol composition of their teichoic acids. The 16S rDNA sequence analysis confirmed the assignment of the environmental isolates to the genus Brevibacterium and showed the phylogenetic distinction of the three genomic species. The results obtained in this study allow three new Brevibacterium species to be described: Brevibacterium antiquum (type strain VKM Ac-2118T = UCM Ac-411T), Brevibacterium aurantiacum (type strain VKM Ac-2111T = NCDO 739T = ATCC 9175T), and Brevibacterium permense (type strain VKM Ac-2280T = UCM Ac-413T).     

Methylovorus mays--novel species of aerobic, obligatory methylotrophic bacteria associated with plants

A bacterial strain utilizing methanol as the sole source of carbon and energy was isolated from the maize phyllosphere. Cells are nonpigmented gram-negative motile rods that do not form spores or prosthecae and reproduce by binary fission. The strain does not require vitamins or supplementary growth factors. It is obligately aerobic and urease-, oxidase-, and catalase-positive. The optimum growth temperature is 35-40 degrees C; the optimum pH is 7.0-7.5. The doubling time is 2 h. The bacterium implements the ribulose monophosphate pathway and possesses NAD(+)-dependent 6-phosphogluconate dehydrogenase and enzymes of the glutamate cycle. alpha-Ketoglutarate dehydrogenase and enzymes of the glyoxylate cycle (isocitrate lyase and malate synthase) are absent. Fatty acids are dominated by palmitic (C16:0) and palmitoleic (C16:1) acids. The major phospholipids are phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylcholine. Cardiolipin is present in minor amounts. The dominant ubiquinone is Q8. The bacterial genome contains genes controlling the synthesis and secretion of cytokinins. The G + C content of DNA is 57.2 mol %, as determined from the DNA thermal denaturation temperature (Tm). The bacterium shows low DNA homology (< 10%) with restricted facultative methylotrophic bacteria of the genus Methylophilus (M. methylotrophus NCIMB 10515T and M. leisingerii VKM B-2013T) and with the obligate methylotrophic bacterium (Methylobacillus glycogenes ATCC 29475T). DNA homology with the type representative of the genus Methylovorus, M. glucosetrophus VKM B-1745T, is high (58%). The new isolate was classified as a new species, Methylovorus mays sp. now.     

Introduction of the phzH gene of Pseudomonas chlororaphis PCL1391 extends the range of biocontrol ability of phenazine-1-carboxylic acid-producing Pseudomonas spp. strains

Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici. Its biocontrol activity is mediated by the production of phenazine-1-carboxamide (PCN). In contrast, the take-all biocontrol strains P. fluorescens 2-79 and P. aureofaciens 30-84, which produce phenazine-1-carboxylic acid (PCA), do not control this disease. To determine the role of the amide group in biocontrol, the PCN biosynthetic genes of strain PCL1391 were identified and characterized. Downstream of phzA through phzG, the novel phenazine biosynthetic gene phzH was identified and shown to be required for the presence of the 1-carboxamide group of PCN because a phzH mutant of strain PCL1391 accumulated PCA. The deduced PhzH protein shows homology with asparagine synthetases that belong to the class II glutamine amidotransferases, indicating that the conversion of PCA to PCN occurs via a transamidase reaction catalyzed by PhzH. Mutation of phzH caused loss of biocontrol activity, showing that the 1-carboxamide group of PCN is crucial for control of tomato foot and root rot. PCN production and biocontrol activity of the mutant were restored by complementing the phzH gene in trans. Moreover, transfer of phzH under control of the tac promoter to the PCA-producing biocontrol strains P. fluorescens 2-79 and P. aureofaciens 30-84 enabled these strains to produce PCN instead of PCA and suppress tomato foot and root rot. Thus, we have shown, for what we believe is the first time, that the introduction of a single gene can efficiently extend the range of the biocontrol ability of bacterial strains.     

Tryptophan 7-halogenase from Pseudomonas aureofaciens ACN strain: gene cloning and sequencing and the enzyme expression

The gene of tryptophan 7-halogenase was isolated from the Pseudomonas aureofaciens ACN strain producing pyrrolnitrin, a chlorocontaining antibiotic, and sequenced. A high homology degree (over 95%) was established for the genes and the corresponding halogenases from P. aureofaciens ACN and P. fluorescens BL915. The tryptophan 7-halogenase gene was amplified by PCR, and the corresponding enzyme was expressed in Escherichia coli cells using the pBSII SK+ vector.     

<Emphasis Type="Italic">Methylovorus menthalis</Emphasis>, a novel species of aerobic obligate methylobacteria associated with plants

A bacterial strain (MM) utilizing methanol as the only carbon and energy source was isolated from corn mint rhizoplane. The cells of the strain were gram-negative colorless motile rods. Spores and prosthecae were not formed, reproduced by binary fission, and did not require vitamins and growth factors. The organism was strictly aerobic, urease-, oxidase-, and catalase-positive. Used the KDPG variant of the ribulose monophosphate pathway. Possessed NAD⁺ dependent 6-phosphogluconate dehydrogenase activity and enzymes of the glutamate cycle. The activities of α-ketoglutarate dehydrogenase and of the glyoxylate bypass enzymes (isocitrate lyase and malate synthase) were absent. Palmitic (C_16:0) and palmitoleic (C_16:1) acids were predominant in the cell fatty-acid composition. The dominant phospholipids were phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylcholine. The dominant ubiquinone was Q₈. The strain formed indole from tryptophan. The DNA G + C content was 54.5 mol % (T _m). According to the data of the 16S rRNA gene sequencing, strain MM showed high similarity (98–99%) to Methylovorus glucosotrophus VKM B-1745^T and Methylovorus mays VKM B-2221^T, but the level of DNA-DNA homology with these cultures was only 40 and 58%, respectively. The strain was classified as a new species, Methylovorus menthalis sp. nov. (VKM B-2663^T).     

Comparative Immunological Studies of Two Pseudomonas Enzymes

Crystalline preparations of muconate lactonizing enzyme and muconolactone isomerase, two inducible enzymes that catalyze successive steps in the catechol branch of the beta-ketoadipate pathway, were used to prepare antisera. Both enzymes were isolated from a strain of Pseudomonas putida biotype A. The antisera did not cross-react with enzymes of the same bacterial strain that catalyze the chemically analogous steps in the protocatechuate branch of the beta-ketoadipate pathway, carboxymuconate lactonizing enzyme and carboxymuconolactone decarboxylase. The antisera gave heterologous cross-reactions of varying intensities with the muconate lactonizing enzymes and muconolactone isomerases of P. putida biotype B, P. aeruginosa, P. stutzeri, and all biotypes of P. fluorescens, but did not cross-react with the isofunctional enzymes of P. acidovorans, of P. multivorans, and of two bacterial species that belong to other genera. The evolutionary and taxonomic implications of the findings are discussed.     

Numerical systematics of bacteria of the genus Pseudomonas

In 290 strains of bacteria belonging to the genus Pseudomonas, 120 morphological and physiologo-biochemical characters were studied and the results obtained thereby were analyzed by the methods of numerical taxonomy using computers. The majority of strains were subdivided into 11 clusters: Ps. aeruginosa (1), Ps. putida (2), Ps. rathonis (5), Ps. syringae (8), Ps. pseudoalcaligenes (9), Ps. maltophilia (10), Ps. acidovorans (11), Ps. testosteroni (12), Ps. mendocina (13), Ps. cepacia (14), Ps. fluorescens (3). The latter cluster included also the strains identified earlier as Ps. aurantiaca, Ps. lemonnieri, Ps. fluoro-violaceus, and Ps. aureofaciens. Three clusters contained strains which could not be identified and probably should be regarded as distinct species. The characteristics have been selected useful for diagnostics of the above Pseudomonas bacteria and the subgroups of Ps. fluorescens.     

Deoxyribonucleic Acid Homologies Among Some Pseudomonas Species

Phylogenetic relationships among a number of strains belonging to the genus Pseudomonas were explored by the use of in vitro deoxyribonucleic acid (DNA) hybridization. The fluorescent nomenspecies (P. fluorescens, P. putida, P. aeruginosa, P. cichorii, P. syringae, and related species), as well as the nonfluorescent species P. stutzeri, P. mendocina, P. alcaligenes, and P. pseudoalcaligenes, were shown to belong to a single DNA homology complex which is isolated from other Pseudomonas species that have been studied [P. cepacia (= P. multivorans), P. caryophylli, P. marginata (= P. alliicola), P. pseudomallei, P. acidovorans, P. testosteroni, P. solanacearum, P. diminuta, P. facilis, P. delafieldii, P. saccharophila, P. palleronii]. A limited numerical analysis of the phenotypic properties of the examined strains supported, with some exceptions, their previous allocation to nomenspecies and biotypes. The internal structure and nomenclature of the "P. fluorescens homology complex" are discussed.     

Three New Species of Brevibacteria,Brevibacterium antiquum sp. nov., Brevibacterium aurantiacum sp. nov., and Brevibacterium permense sp. nov.

This work deals with the taxonomic study of orange-pigmented bacteria isolated from permafrost sediments, rice plots, and soils contaminated with wastes from the chemical and salt industries that were assigned to the genus Brevibacterium on the basis of phenotypic characteristics, as well as of some strains described previously as Brevibacterium linens. The study revealed three genomic species, whose members and the type strains of the closest species of Brevibacterium had DNA similarity levels between 24 and 59%. The strains of the genomic species differed from each other and from the known species of Brevibacterium in some physiological and biochemical characteristics, as well as in the sugar and polyol composition of their teichoic acids. The 16S rDNA sequence analysis confirmed the assignment of the environmental isolates to the genus Brevibacterium and showed the phylogenetic distinction of the three genomic species. The results obtained in this study allow three new Brevibacterium species to be described: Brevibacterium antiquum (type strain VKM Ac-2118^T = UCM Ac-411^T), Brevibacterium aurantiacum (type strain VKM Ac-2111^T = NCDO 739^T = ATCC 9175^T), and Brevibacterium permense (type strain VKM Ac-2280^T = UCM Ac-413^T).     

Biological Control of Crown Gall in Grapevine and Raspberry by Two Pseudomonas spp. with a Wide Spectrum of Antagonistic Activity

Pseudomonas aureofaciens B-4117 and P. fluorescens CR330D inhibited the growth of a wide range of plant pathogens, including Agrobacterium tumefaciens , when tested on agar media. In a series of nursery-based trials with natural pathogen inoculum, application of either B-4117 or CR330D significantly reduced the incidence and severity of crown gall caused by A. tumefaciens on grapevine and raspberry. The extent of disease control depended upon the variety tested. Both bacteria reduced disease during seedling root production and grafting. The disease incidence on root cuttings of three grapevine varieties was reduced by 56-80% and the disease severity index (DSI) was decreased by 75-86%. Depending on the scion variety, the number of healthy rooted grafts increased by 2-3.5-fold, while the DSI was reduced by 1.5-3-fold. The results suggest that there is potential in using these antagonists to diminish the influence of latent rootstock infection on graft sensitivity to crown gall. Pretreatment of rooted raspberry seedlings with P. aureofaciens B-4117 prevented the development of crown galls caused by A. tumefaciens strain K24 or by a mixture of A. tumefaciens pathogenic strains previously isolated from raspberry. Both Pseudomonas spp. persisted on the root surfaces of inoculated vine cuttings and in non-sterile soil. The advantages of using the antagonistic bacteria as biocontrol agents of crown gall are discussed.     

Halophilic and halotolerant aerobic methylobacteria from the technogenic Solikamsk biotopes

Seven strains of moderately halophilic and halotolerant aerobic methylobacteria from the technogenic Solikamsk biotopes (Perm krai, Russia) were isolated in pure cultures and characterized. The isolates were represented by gram-negative and gram-positive (strain 2395B) cells. All the cells were shown to multiply by binary fission without formation of spores or prosthecae. All isolates except strain 2395B were able to oxidize methanol by a classical methanol dehydrogenase. The ribulose monophosphate (RMP) (strain LS), serine (strains S12, S3, 2395A), or ribulose bisphosphate (strains SK15 and S3270) pathways of C₁-assimilation were used. In strain 2395B, the key enzymes of the RMP and serine metabolic pathways were determined. Using polyphasic taxonomy, three strains were identified as representatives of the known species: Arthrobacter protophormiae 2395B, Methylophaga thalassica LS, and Ancylobacter rudongensis S3270. Three more strains were identified as members of new species: Methylopila oligotropha sp. nov. (strain 2395AT; VKM B-2788^T = CCUG 63805^T), Ancylobacter defluvii sp. nov. (strain SK15^T; VKM B-2789^T = CCUG 63806^T), and Paracoccus communis sp. nov. (strain S3^T; VKM B-2787^T = CCUG 63804^T). According to the results of 16S rRNA gene sequencing, the obligately methylotrophic strain S12 had less than 94% similarity with the known genera of the Proteobacteria and was probably a representative of a novel genus.     

Alkylresorcinols are abundant lipid components in different strains of Azotobacter chroococcum and Pseudomonas spp.

The occurrence of various amounts of 5-n-alkylresorcinols was shown in lipids extracted from 14 bacterial strains of Azotobacter chroococcum as well as from strains of Pseudomonas aureofaciens, P. chlororapsis, and P. fluorescens. The amount of alkylresorcinols found varied from 2.3 to 56.2 microg/mg (dry weight) of cells in A. chroococum and from 0.2 to 0.8 microg/mg (dry weight) of cells in Pseudomonas spp. Strains of both genera produce saturated homologs with C13 to C27 side chains. C19, C21, and C23 homologs are predominant in and characteristic for A. chroococum strains, the C15 homolog is predominant in and characteristic for P. chlororapsis and P. fluorescens, and the C17 homolog is predominant in and characteristic for P. aureofaciens. The presence of 5-n-(2-ketoalkyl)resorcinols, not previously observed, was demonstrated in lipids isolated from the cells of A. chroococum Az5.     

Identification of Bacillus subtilis NRRL B-3275 as a Strain of Bacillus pumilus

The physiological and biochemical properties of a species of Bacillus previously identified as B. subtilis NRRL B-3275 (B-3275) were compared with those of seven strains of B. pumilus and five strains of B. subtilis. The biotin requirement of B-3275, its inability to hydrolyze starch, and its failure to reduce nitrate indicate that the organism is more closely related to the B. pumilus strains than to those of B. subtilis. Hybridization of deoxyribonucleic acid (DNA) from B-3275 with that of the strains of B. pumilus showed a binding efficiency (compared with the homologous reaction) of 58 to 99%, depending on the strain. Hybridization with the DNA from any of the strains of B. subtilis did not exceed 24%. DNA from B-3275 was unable to transform two amino acid auxotrophic markers to prototrophy in a highly competent strain of B. subtilis 168. We conclude that B-3275 is a strain of B. pumilus which we designate as B. pumilus NRRL B-3275.     

Vitamin B12-independent strains of Methylophaga marina isolated from Red Sea algae

Two strains (KM3 and KM5) of halophilic methylobacteria isolated from Red Sea algae do not require vitamin B12 for growth and can use methanol, methylamine, dimethylamine, trimethylamine, dimethyl sulfide, and fructose as sources of carbon and energy. The cells of these strains are gram-negative motile monotrichous (strain KM3) or peritrichous (strain KM5) rods. The strains are strictly aerobic and require Na+ ions but not growth factors for growth. They are oxidase- and catalase-positive and reduce nitrates to nitrites. Both strains can grow in a temperature range of 4 to 37 degrees C (with optimal growth at 29-34 degrees C), at pH between 5.5 and 8.5 (with optimal growth at pH 7.5-8.0), and in a range of salt concentrations between 0.5 and 15% NaCl (with optimal growth at 5-9% NaCl). The phospholipids of these strains are dominated by phosphatidylethanolamine and phosphatidylglycerol and also include phosphatidylcholine, phosphatidylserine, and cardiolipin. The dominant fatty acids are C(16:1omega7c) and C(16:0). The major ubiquinone is Q8. The cells accumulate ectoin, glutamate, and sucrose as intracellular osmoprotectants. The strains implement the 2-keto-3-deoxy-6-phosphogluconate-dependent variant of the ribulose monophosphate pathway. The G+C content of the DNA is 44.4-44.7 mol %. Analysis of the 16S rRNA genes showed that both strains belong to Gammaproteobacteria and have a high degree of homology (99.4%) to Methylophaga marina ATCC 35842T . Based on the data of polyphasic taxonomy, strains KM3 and KM5 are identified as new strains M. marina KM3 (VKM B-2386) and M. marina KM5 (VKM B-2387). The ability of these strains to produce auxins (indole-3-acetic acid) suggests their metabolic association with marine algae.     

Antibiotic and biosurfactant properties of cyclic lipopeptides produced by fluorescent Pseudomonas spp. from the sugar beet rhizosphere

Cyclic lipopeptides (CLPs) with antibiotic and biosurfactant properties are produced by a number of soil bacteria, including fluorescent Pseudomonas spp. To provide new and efficient strains for the biological control of root-pathogenic fungi in agricultural crops, we isolated approximately 600 fluorescent Pseudomonas spp. from two different agricultural soils by using three different growth media. CLP production was observed in a large proportion of the strains (approximately 60%) inhabiting the sandy soil, compared to a low proportion (approximately 6%) in the loamy soil. Chemical structure analysis revealed that all CLPs could be clustered into two major groups, each consisting of four subgroups. The two major groups varied primarily in the number of amino acids in the cyclic peptide moiety, while each of the subgroups could be differentiated by substitutions of specific amino acids in the peptide moiety. Production of specific CLPs could be affiliated with Pseudomonas fluorescens strain groups belonging to biotype I, V, or VI. In vitro analysis using both purified CLPs and whole-cell P. fluorescens preparations demonstrated that all CLPs exhibited strong biosurfactant properties and that some also had antibiotic properties towards root-pathogenic microfungi. The CLP-producing P. fluorescens strains provide a useful resource for selection of biological control agents, whether a single strain or a consortium of strains was used to maximize the synergistic effect of multiple antagonistic traits in the inoculum.     

Analysis of the anaerobic microbial community capable of degrading p-toluene sulphonate

Three strains of Clostridium sp., 14 (VKM B-2201), 42 (VKM B-2202), and 21 (VKM B-2279), two methanogens, Methanobacterium formicicum MH (VKM B-2198) and Methanosarcina mazei MM (VKM B-2199), and one sulfate-reducing bacterium, Desulfovibrio sp. SR1 (VKM B-2200), were isolated in pure cultures from an anaerobic microbial community capable of degrading p-toluene sulfonate. Strain 14 was able to degrade p-toluene sulfonate in the presence of yeast extract and bactotryptone and, like strain 42, to utilize p-toluene sulfonate as the sole sulfur source with the production of toluene. p-Toluene sulfonate stimulated the growth of Ms. mazei MM on acetate. The sulfate-reducing strain Desulfovibrio sp. SR1 utilized p-toluene sulfonate as an electron acceptor. The putative scheme of p-toluene sulfonate degradation by the anaerobic microbial community is discussed.     

Spatial organization of dual-species bacterial aggregates on leaf surfaces

The spatial organization of cells within bacterial aggregates on leaf surfaces was determined for pair-wise mixtures of three different bacterial species commonly found on leaves, Pseudomonas syringae, Pantoea agglomerans, and Pseudomonas fluorescens. Cells were coinoculated onto bean plants and allowed to grow under moist conditions, and the resulting aggregates were examined in situ by epifluorescence microscopy. Each bacterial strain could be localized because it expressed either the green or the cyan fluorescent protein constitutively, and the viability of individual cells was assessed by propidium iodide staining. Each pair of bacterial strains that was coinoculated onto leaves formed mixed aggregates. The degree of segregation of cells in mixed aggregates differed between the different coinoculated pairs of strains and was higher in mixtures of P. fluorescens A506 and P. agglomerans 299R and mixtures of P. syringae B728a and P. agglomerans 299R than in mixtures of two isogenic strains of P. agglomerans 299R. The fractions of the total cell population that were dead in mixed and monospecific aggregates of a gfp-marked strain of P. agglomerans 299R and a cfp-marked strain of P. agglomerans 299R, or of P. fluorescens A506 and P. agglomerans 299R, were similar. However, the proportion of dead cells in mixed aggregates of P. syringae B728a and P. agglomerans 299R was significantly higher (13.2% +/- 8.2%) than that in monospecific aggregates of these two strains (1.6% +/- 0.7%), and it increased over time. While dead cells in such mixed aggregates were preferentially found at the interface between clusters of cells of these strains, cells of these two strains located at the interface did not exhibit equal probabilities of mortality. After 9 days of incubation, about 77% of the P. agglomerans 299R cells located at the interface were dead, while only about 24% of the P. syringae B728a cells were dead. The relevance of our results to understanding bacterial interactions on leaf surfaces and the implications for biological control of pathogenic and other deleterious microorganisms is discussed.     

Rapid identification of environmental isolates of Pseudomonas aeruginosa, P. fluorescens and P. putida by SDS-PAGE analysis of whole-cell protein patterns

Abstract Environmental isolates of fluorescent pseudomonads grown to early stationary phase in glucose-enriched Luria broth were treated with proteinase K in sodium dodecylsulphate (SDS) lysis buffer and subsequently analyzed by polyacrylamide gel electrophoresis (PAGE). Four silver-staining protein-fragment bands could be used for rapid identification at the species level. Pseudomonas aeruginosa isolates were easily recognized by a unique banding pattern. Isolates considered to be P. fluorescen from biochemical and physiological tests (classical biotypes I, II, III, IV and V) also had a characteristic banding pattern, which in turn was different from that of P. putida isolates (classical biotype A). A residual group representing intermediate isolates of P. fluorescens (new biotype VI of Barrett et al., J. Gen. Microbiol. 132, 1986) or P. putida (biotype B) had a banding pattern similar to that of classical P. fluorescens biotypes. On the other hand, a group representing other intermediate isolates of P. putida (new biotype C of Barrett et al., J. Gen. Microbiol. 132, 1986) had a unique banding pattern resembling that of classical P. putida biotype A. A small number of protein fragment bands appearing in SDS-PAGE analysis of whole-cell lysates seems adequate for a rapid identification at the species level of P. aeruginosa, P. fluorescens and P. putida isolated from natural environments.