Complete Genome Sequence of Pseudomonas sp. Strain MM213, an Isolate from a Brookside in Bielefeld, Germany

Here, we report the complete genome sequence of Pseudomonas sp. strain MM213 of the Pseudomonas mandelii group, which was isolated from a brookside in Bielefeld, Germany. The genome size is 6,746,355 bp, with a GC content of 59.4% and 6,145 predicted protein-coding sequences.

Lipopolysaccharide of Pseudomonas mandelii, Isolated from Antarctica

Representatives of the Pseudomonas mandelii species are able to exist and multiply in places where the temperature is constantly low. The optimum growth temperature for P. mandelii is 25–30°C, although this bacterium can grow at 4°C but not at 37°C. Therefore, P. mandelii is an excellent example of psychrotolerant bacterium which like psychrophilic bacteria is characterized by a number of structural and functional adaptations that facilitate survival at low temperatures. To understand these microorganisms’ role in Antarctica the characterization of its biopolymers is vital. One of these biopolymers is lipopolysaccharide (LPS), composition and structure of which are diagnostically significant. This determines the aim of the work – to isolate lipopolysaccharides from the cells of Antarctic strain of P. mandelii, grown at different temperatures, to characterize them chemically, and to study their functional and biological activity. Methods. The object of the study was Pseudomonas sp. U1, isolated from moss on Galindez Island in Antarctica. Lipopolysaccharides were extracted from dried cells by 45% phenol water solution at 65–68°С by Westphal and Jann method. The amount of carbohydrates was determined by phenol-sulfuric method. Carbohydrate content was determined in accordance to the calibration curve, which was built using glucose as a standard. The content of nucleic acids was determined by Spirin, protein − by Lowry method. Serological activity of LPS was investigated by double immunodiffusion in agar using the method of Ouchterlony. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAAG electrophoresis) was performed according to Laemmli. Results. As a result of phylogenetic analysis (programs ClustalX 2.1, Tree view, Mega v. 6.00) it was shown that the Antarctic bacterial strain Pseudomonas sp. U1 associated with green moss has a 99.4% homology with the type strain from the GenBank database NR024902 P. mandelii CIP 105273T. According to these data and proximity to the corresponding cluster of species, the studied isolate can be identified as P. mandelii. A characteristic feature of LPS isolated from P. mandelii cells, grown at different temperatures (20°C and 4°C) is their heterogeneity. This is evidenced by the data of the monosaccharide composition, electrophoretic distribution, which showed that P. mandelii produces S- and SR-forms of LPS, differed in the length of the O-specific polysaccharide chains. The R-form of LPS is also present, which does not contain an O-specific polysaccharide chains. Structural heterogeneity is also inherent in LPS lipid A. This is evidenced by the data of the fatty acid composition. In LPS grown at 4°C no unsaturated fatty acids were found, while such ones are synthesized in LPS of other bacteria grown in the cold, in response to a decrease in growth temperature. The study of the immunochemical properties of LPS was carried out using polyclonal O-antisera as antibodies, and LPS as antigens indicated that in homologous systems LPS exhibited serological activity. LPS obtained from P. mandelii U1 cells, grown at 20°C, had a complex antigenic composition and gave two clear lines of precipitation at a concentration of 1 mg/mL. LPS obtained from P. mandelii U1 cells, grown at 4°C, gave one line, which indicates their serological homogeneity. Conclusions. For the first time lipopolysaccharides were isolated from cells of P. mandelii U1, grown at 4°C and 20°С. A characteristic feature of these LPS is their heterogeneity. This is evidenced by the data of the monosaccharide and fatty acid composition, electrophoretic distribution, which showed that P. mandelii produces S- and SR-forms of LPS, differed in the length of the O-specific polysaccharide chains. LPS, obtained from cells, grown at different temperatures, are differed by serological activity.

Culturable nitrogen-transforming bacteria from sequential sedimentation biofiltration systems and their potential for nutrient removal in urban polluted rivers

Novel heterotrophic bacterial strains—Bzr02 and Str21, effective in nitrogen transformation, were isolated from sequential sedimentation-biofiltration systems (SSBSs). Bzr02, identified as Citrobacter freundii, removed up to 99.0% of N–NH4 and 70.2% of N–NO3, while Str21, identified as Pseudomonas mandelii, removed up to 98.9% of N–NH4 and 87.7% of N–NO3. The key functional genes napA/narG and hao were detected for Bzr02, confirming its ability to reduce nitrate to nitrite and remove hydroxylamine. Str21 was detected with the genes narG, nirS, norB and nosZ, confirming its potential for complete denitrification process. Nitrogen total balance experiments determined that Bzr02 and Str21 incorporated nitrogen into cell biomass (up to 94.7% and 74.7%, respectively), suggesting that nitrogen assimilation was also an important process occurring simultaneously with denitrification. Based on these results, both strains are suitable candidates for improving nutrient removal efficiencies in nature-based solutions such as SSBSs.

Pseudomonas piscium sp. nov., Pseudomonas pisciculturae sp. nov., Pseudomonas mucoides sp. nov. and Pseudomonas neuropathica sp. nov. isolated from rainbow trout

Six Gram negative, motile bacteria were isolated from rainbow trout (Oncorhynchus mykiss). The 16S rRNA sequence similarity values grouped them in the Pseudomonas mandelii (strains P49, P50T, 154aT and P154b), Pseudomonas fluorescens (strain P115T) and Pseudomonas koreensis (strain P155T) phylogenetic subgroups in the genus Pseudomonas . The DNA G+C content ranged from 58.5 to 60 mol%. The strains were characterized phenotypically using API 20NE and Biolog GENIII tests, and chemotaxonomically by their whole-cell MALDI-TOF MS protein profiles and fatty acid contents. Multi-locus sequence analysis with four housekeeping gene sequences (rpoD, rpoB, gyrB and 16S rRNA) together with genome comparisons by average nucleotide identity and genome-to-genome distance calculations were performed. Results showed that the similarity values of these strains to known species type strains were lower than the thresholds established for species in the genus Pseudomonas . Based on these data, we concluded that strains P49, P50T, P115T, P154aT, P154b and P155T belonged to four novel species. The names proposed are: Pseudomonas piscium sp. nov. for strains P49 and P50T with P50T (=CECT 30175T=CCUG 74871T) as the type strain; Pseudomonas pisciculturae sp. nov. for strain P115T (CECT 30173T=CCUG 74873T); Pseudomonas mucoides sp. nov. for strains P154aT and P154b with P154aT (=CECT 30177T=CCUG 74874T) as the type strain; and Pseudomonas neuropathica sp. nov. for strain P155T (=CECT 30178T=CCUG 74875T).

The First Step of Biodegradation of 7-Hydroxycoumarin in Pseudomonas mandelii 7HK4 Depends on an Alcohol Dehydrogenase-Type Enzyme

Coumarins are well known secondary metabolites widely found in various plants. However, the degradation of these compounds in the environment has not been studied in detail, and, especially, the initial stages of the catabolic pathways of coumarins are not fully understood. A soil isolate Pseudomonas mandelii 7HK4 is able to degrade 7-hydroxycoumarin (umbelliferone) via the formation of 3-(2,4-dihydroxyphenyl)propionic acid, but the enzymes catalyzing the α-pyrone ring transformations have not been characterized. To elucidate an upper pathway of the catabolism of 7-hydroxycoumarin, 7-hydroxycoumarin-inducible genes hcdD, hcdE, hcdF, and hcdG were identified by RT-qPCR analysis. The DNA fragment encoding a putative alcohol dehydrogenase HcdE was cloned, and the recombinant protein catalyzed the NADPH-dependent reduction of 7-hydroxycoumarin both in vivo and in vitro. The reaction product was isolated and characterized as a 7-hydroxy-3,4-dihydrocoumarin based on HPLC-MS and NMR analyses. In addition, the HcdE was active towards 6,7-dihydroxycoumarin, 6-hydroxycoumarin, 6-methylcoumarin and coumarin. Thus, in contrast to the well-known fact that the ene-reductases usually participate in the reduction of the double bond, an alcohol dehydrogenase catalyzing such reaction has been identified, and, for P. mandelii 7HK4, 7-hydroxycoumarin degradation via a 7-hydroxy-3,4-dihydrocoumarin pathway has been proposed.

Molecular cloning and biochemical characterization of a NAD-dependent sorbitol dehydrogenase from cold-adapted Pseudomonas mandelii

Sugar alcohols (polyols) have important roles as nutrients, anti-freezing agents, and scavengers of free radicals in cold-adapted bacteria, but the characteristics of polyol dehydrogenases in cold-adapted bacteria remain largely unknown. In this study, based on the observation that a cold-adapted bacterium Pseudomonas mandelii JR-1 predominantly utilized D-sorbitol as its carbon source, among the four polyols examined (D-galactitol, D-mannitol, D-sorbitol, or D-xylitol), we cloned and characterized a sorbitol dehydrogenase (SDH, EC 1.1.1.14) belonging to the short-chain dehydrogenase/reductase family from this bacterium (the SDH hereafter referred to as PmSDH). PmSDH contained Asn111, Ser140, Tyr153, and Lys157 as catalytic active site residues and existed as a ∼67 kDa dimer in size-exclusion chromatography. PmSDH converted D-sorbitol to D-fructose using NAD+ as a coenzyme and, vice versa, D-fructose to D-sorbitol using NADH as a coenzyme. PmSDH maintained its conformational flexibility, secondary and tertiary structures, and thermal stability at 4–25°C. At 40°C, PmSDH was rapidly denatured. These results indicate that PmSDH, which has a flexible structure and a high catalytic activity at colder temperatures, is well-suited to sorbitol utilization in the cold-adapted bacterium P. mandelii JR-1.

Study of the influence of external factors on the inheritance of forms of dissociants Pseudomonas mandelii

Differences in proteolytic, antioxidant, and endoglucanase activity were detected. One of the factors for switching dissociants of the P. mandelii strain IB-Ki14 is the pH of the medium; switching and inheritance schemes are drawn up.

Effects of Plant Growth Promoting Rhizobacteria on the Content of Abscisic Acid and Salt Resistance of Wheat Plants

Although salinity inhibits plant growth, application of appropriate rhizosphere bacteria can diminish this negative effect. We studied one possible mechanism that may underlie this beneficial response. Wheat plants were inoculated with Bacillus subtilis IB-22 and Pseudomonas mandelii IB-Ki14 and their consequences for growth, water relations, and concentrations of the hormone abscisic acid (ABA) were followed in the presence of soil salinity. Salinity alone increased ABA concentration in wheat leaves and roots and this was associated with decreased stomatal conductance, but also with chlorophyll loss. Bacterial treatment raised ABA concentrations in roots, suppressed accumulation of leaf ABA, decreased chlorophyll loss, and promoted leaf area and transpiration. However, water balance was maintained due to increased water uptake by inoculated plants, brought about in part by a larger root system. The effect may be the outcome of ABA action since the hormone is known to maintain root extension in stressed plants. Root ABA concentration was highest in salt-stressed plants inoculated with B. subtilis and this contributed to greater root hydraulic conductivity. We conclude that bacteria can raise salt resistance in wheat by increasing root ABA, resulting in larger root systems that can also possess enhanced hydraulic conductivity thereby supporting better-hydrated leaves.