Whole genome analysis of Pseudomonas mandelii SW-3 and the insights into low-temperature adaptation.

Pseudomonas mandelii SW-3, isolated from the Napahai plateau wetland, can survive in cold environments. The mechanisms underlying the survival of bacteria in low temperatures and high altitudes are not yet fully understood. In this study, the whole genome of SW-3 was sequenced to identify the genomic features that may contribute to survival in cold environments. The results showed that the genome size of strain SW-3 was 6,538,059 bp with a GC content of 59%. A total of 67 tRNAs, a 34,110 bp prophage sequence, and a large number of metabolic genes were found. Based on 16S rRNA gene phylogeny and average nucleotide identity analysis among P. mandelii, SW-3 was identified as a strain belonging to P. mandelii. In addition, we clarified the mechanisms by which SW-3 survived in a cold environment, providing a basis for further investigation of host-phage interaction. P. mandelii SW-3 showed stress resistance mechanisms, including glycogen and trehalose metabolic pathways, and antisense transcriptional silencing. Furthermore, cold shock proteins and glucose 6-phosphate dehydrogenase may play pivotal roles in facilitating adaptation to cold environmental conditions. The genome-wide analysis provided us with a deeper understanding of the cold-adapted bacterium.

Pseudomonas hormoni sp. nov., a plant hormone producing bacterium isolated from Arctic grass, Ellesmere Island, Canada.

Bacterial strain G20-18T was previously isolated from the rhizosphere of an Arctic grass on Ellesmere Island, Canada and was characterized and described as Pseudomonas fluorescens. However, new polyphasic analyses coupled with phenotypic, phylogenetic and genomic analyses reported here demonstrate that the affiliation to the species P. fluorescens was incorrect. The strain is Gram-stain-negative, rod-shaped, aerobic and displays growth at 5-25 °C (optimum, 20-25 °C), at pH 5-9 (optimum, pH 6-7) and with 0-4 % NaCl (optimum, 2 % NaCl). The major fatty acids are C16 : 0 (35.6 %), C17 : 0 cyclo ω7c (26.3 %) and summed feature C18 : 1/C18 : 1 ω7c (13.6 %). The respiratory quinones were determined to be Q9 (93.5 %) and Q8 (6.5 %) and the major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Strain G20-18T was shown to synthesize cytokinin and auxin plant hormones and to produce 1-aminocyclopropane-1-carboxylate deaminase. The DNA G+C content was determined to be 59.1 mol%. Phylogenetic analysis based on the 16S rRNA gene and multilocus sequence analysis (concatenated 16S rRNA, gyrB, rpoB and rpoD sequences) showed that G20-18T was affiliated with the Pseudomonas mandelii subgroup within the genus Pseudomonas. Comparisons of the G20-18T genome sequence and related Pseudomonas type strain sequences showed an average nucleotide identity value of ≤93.6 % and a digital DNA-DNA hybridization value of less than 54.4 % relatedness. The phenotypic, phylogenetic and genomic data support the hypothesis that strain G20-18T represents a novel species of the genus Pseudomonas. As strain G20-18T produces or modifies hormones, the name Pseudomonas hormoni sp. nov. is proposed. The type strain is G20-18T (=LMG 33086T=NCIMB 15469T).

Corrigendum: In-depth characterization of phytase-producing plant growth promotion bacteria isolated in alpine grassland of Qinghai-Tibetan Plateau.

[This corrects the article DOI: 10.3389/fmicb.2022.1019383.].

Pseudomonas nunensis sp. nov. isolated from a suppressive potato field in Greenland.

The bacterial strain In5T was previously isolated from a suppressive potato field in southern Greenland and has been characterized and described as Pseudomonas fluorescens. However, the results of new polyphasic analyses coupled with those of phenotypic, phylogenetic and genomic analyses reported here demonstrate that the affiliation to the species P. fluorescens was incorrect. The strain is Gram-stain-negative, rod-shaped, aerobic and displays growth at 4-28 °C (optimum temperature 20-25 °C) and at pH 5-9 (optimum pH 6-7). Major fatty acids were C16 : 0 (38.2 %), a summed feature consisting of C16 : 1ω6c and/or C16 : 1ω7c) (20.7 %), C17 : 0cyclo ω7c (14.3 %) and a summed feature consisting of C18 : 1ω6c and/or C18 : 1ω7c (11.7 %). The respiratory quinones were determined to be Q9 (95.5 %) and Q8 (4.5 %) and major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The DNA G+C content was determined to be 59.4 mol%. The results of phylogenetic analysis based on the 16S rRNA gene and multi-locus sequence analysis (MLSA; concatenated 16S rRNA, gyrB, rpoB and rpoD sequences) indicated that In5T was affiliated with the Pseudomonas mandelii subgroup within the genus Pseudomonas. Comparison of the genome sequence of In5T and those of related type strains of species of the genus Pseudomonas revealed an average nucleotide identity (ANI) of 87.7 % or less and digital DNA-DNA hybridization (dDDH) of less than 34.5 % relatedness, respectively. Two more strains, In614 and In655, isolated from the same suppressive soil were included in the genome analysis. The ANI and dDDH of In614 and In655 compared with In5T were ANI: 99.9 and 97.6 and dDDH (GGDC) 99.9 and 79.4, respectively, indicating that In5T, In614 and In655 are representatives of the same species. The results of the phenotypic, phylogenetic and genomic analyses support the hypothesis that strain In5T represents a novel species of the genus Pseudomonas, for which the name Pseudomonas nunensis sp. nov. is proposed. The type strain is In5T(=LMG 32653T=NCIMB 15428T).

Effects of a Pseudomonas Strain on the Lipid Transfer Proteins, Appoplast Barriers and Activity of Aquaporins Associated with Hydraulic Conductance of Pea Plants.

Lipid transfer proteins (LTPs) are known to be involved in suberin deposition in the Casparian bands of pea roots, thereby reinforcing apoplast barriers. Moreover, the Pseudomonas mandelii IB-Ki14 strain accelerated formation of the Casparian bands in wheat plants, although involvement of LTPs in the process was not studied. Here, we investigated the effects of P. mandelii IB-Ki14 on LTPs, formation of the Casparian bands, hydraulic conductance and activity of aquaporins (AQPs) in pea plants. RT PCR showed a 1.6-1.9-fold up-regulation of the PsLTP-coding genes and an increase in the abundance of LTP proteins in the phloem of pea roots induced by the treatment with P. mandelii IB-Ki14. The treatment was accompanied with increased deposition of suberin in the Casparian bands. Hydraulic conductance did not decrease in association with the bacterial treatment despite strengthening of the apoplast barriers. At the same time, the Fenton reagent, serving as an AQPs inhibitor, decreased hydraulic conductance to a greater extent in treated plants relative to the control group, indicating an increase in the AQP activity by the bacteria. We hypothesize that P. mandelii IB-Ki14 stimulates deposition of suberin, in the biosynthesis of which LTPs are involved, and increases aquaporin activity, which in turn prevents a decrease in hydraulic conductance due to formation of the apoplast barriers in pea roots.

The Effects of Rhizosphere Inoculation with Pseudomonas mandelii on Formation of Apoplast Barriers, HvPIP2 Aquaporins and Hydraulic Conductance of Barley.

Pseudomonas mandelii strain IB-Ki14 has recently been shown to strengthen the apoplastic barriers of salt-stressed plants, which prevents the entry of toxic sodium. It was of interest to find out whether the same effect manifests itself in the absence of salinity and how this affects the hydraulic conductivity of barley plants. Berberine staining confirmed that the bacterial treatment enhanced the deposition of lignin and suberin and formation of Casparian bands in the roots of barley plants. The calculation of hydraulic conductance by relating transpiration to leaf water potential showed that it did not decrease in bacteria-treated plants. We hypothesized that reduced apoplastic conductivity could be compensated by the higher conductivity of the water pathway across the membranes. This assumption was confirmed by the results of the immunolocalization of HvPIP2;5 aquaporins with specific antibodies, showing their increased abundance around the areas of the endodermis and exodermis of bacteria-treated plants. The immunolocalization with antibodies against auxins and abscisic acid revealed elevated levels of these hormones in the roots of plants treated with bacteria. This root accumulation of hormones is likely to be associated with the ability of Pseudomonas mandelii IB-Ki14 to synthesize these hormones. The involvement of abscisic acid in the control of aquaporin abundance and auxins-in the regulation of and formation of apoplast barriers-is discussed.

In-depth characterization of phytase-producing plant growth promotion bacteria isolated in alpine grassland of Qinghai-Tibetan Plateau.

The use of plant growth promoting bacteria (PGPB) express phytase (myo-inositol hexakisphosphate phosphohydrolase) capable of hydrolyzing inositol phosphate in soil was a sustainable approach to supply available phosphorus (P) to plants. A total of 73 bacterial isolates with extracellular phytase activity were selected from seven dominant grass species rhizosphere in alpine grassland of Qinghai-Tibetan Plateau. Then, the plant growth promoting (PGP) traits of candidate bacteria were screened by qualitative and quantitative methods, including organic/inorganic Phosphorus solubilization (P. solubilization), plant hormones (PHs) production, nitrogen fixation, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity and antimicrobial activity. Further experiment were conducted to test their growth promoting effect on Lolium perenne L. under P-limitation. Our results indicated that these bacteria as members of phyla Proteobacteria (90.41%) and Actinobacteria (9.59%) were related to 16 different genera. The isolates of Pseudomonas species showed the highest isolates number (36) and average values of phytase activity (0.267 ± 0.012 U mL-1), and showed a multiple of PGP traits, which was a great candidate for PGPBs. In addition, six strains were positive in phytase gene (ß-propeller phytase, bpp) amplification, which significantly increased the shoot length, shoot/root fresh weight, root average diameter and root system phytase activity of Lolium perenne L. under P-limitation, and the expression of phytase gene (bppP) in root system were verified by qPCR. Finally, the PHY101 gene encoding phytase from Pseudomonas mandelii GS10-1 was cloned, sequenced, and recombinantly expressed in Escherichia coli. Biochemical characterization demonstrated that the recombinant phytase PHY101 revealed the highest activity at pH 6 and 40°C temperature. In particular, more than 60% of activity was retained at a low temperature of 15°C. This study demonstrates the opportunity for commercialization of the phytase-producing PGPB to developing localized microbial inoculants and engineering rhizobacteria for sustainable use in alpine grasslands.

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 and 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 an ∼67-kDa dimer in size-exclusion chromatography. PmSDH converted d-sorbitol to d-fructose using nicotinamide adenine dinucleotide (NAD+) as a cofactor and, vice versa, d-fructose to d-sorbitol using nicotinamide adenine dinucleotide reduced (NADH) as a cofactor. PmSDH maintained its conformational flexibility, secondary and tertiary structures, and thermal stability at 4-25°C. 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.

Nano-sized biopolymer levan: Its antimicrobial, anti-biofilm and anti-cancer effects.

Among other polysaccharides, levan is a fructan with great potential in biotechnological applications due to its functional properties. The levan has only been available in small quantities because of its high cost. Here, a levan-producing microorganism was isolated from soil and identified as Pseudomonas mandelii. TGA, SEM, FTIR, 1H NMR, 13C NMR and Zetasizer analyses were used to characterize thermal properties and the morphology of the levan. It is the first time, the levan synthesized from P. mandelii is reported as nano sized. The culture conditions were optimized. It is the most comprehensive optimization study regarding levan production of P. mandelii up to the present. The optimum conditions found for levan production were 37 °C, at pH 8, under static conditions, with 15% sucrose and 1.5% mannitol as the C sources and yeast extract as the N source. In addition, the levan production yield was calculated at optimum conditions. Antibacterial activity of levan was evaluated against bacteria and the largest zone of inhibition was observed against E. coli at a concentration of 1000 µg/mL. Antibiofilm activity of levan was evaluated, and we found that all levan concentrations inhibited biofilm formation of all microorganisms in the study. We have shown that the levan from Pseudomonas mandelii induce cytotoxicity breast (MCF-7) cells in a dose-dependent manner.

Biochemical comparison of two glucose 6-phosphate dehydrogenase isozymes from a cold-adapted Pseudomonas mandelii.

Cold-adapted bacteria primarily have two glucose 6-phosphate dehydrogenase isozymes (G6PD, also known as zwf), zwf-1 for the Entner-Doudoroff pathway and zwf-2 for the oxidative pentose phosphate pathway. Although the roles of zwfs in carbon metabolism and antioxidant defense have been reported, the biochemical properties of zwfs at low and moderate temperatures have not been fully described. In this study, we cloned and characterized zwf-1 (Pmzwf-1) and zwf-2 (Pmzwf-2) from a cold-adapted bacterium Pseudomonas mandelii JR-1. Pmzwf-1 and Pmzwf-2 were expressed in Escherichia coli BL21 (DE3) as soluble tetrameric proteins. Both Pmzwf proteins were active at 4 °C, but Pmzwf-1 exhibited overall better biochemical properties than those of Pmzwf-2, including 10-30% higher specific activity at 4-40 °C as well as consistent conformational flexibility and thermal stability in the 4-40 °C range. Pmzwf-2 showed reduced thermal stability at moderate temperatures. Furthermore, the mRNA expression of Pmzwf-1 was higher than that of Pmzwf-2 at both 4 °C and 25 °C. These results indicate that Pmzwfs are cold-adapted enzymes, but Pmzwf-1 can function at both low to moderate temperatures while Pmzwf-2 is primarily functional at low temperatures. Our results suggest distinct temperature adaptations of two G6PD isozymes in P. mandelii JR-1, adaptations that are metabolic pathway dependent.

Biodegradation of 7-Hydroxycoumarin in Pseudomonas mandelii 7HK4 via ipso-Hydroxylation of 3-(2,4-Dihydroxyphenyl)-propionic Acid.

A gene cluster, denoted as hcdABC, required for the degradation of 3-(2,4-dihydroxyphenyl)-propionic acid has been cloned from 7-hydroxycoumarin-degrading Pseudomonas mandelii 7HK4 (DSM 107615), and sequenced. Bioinformatic analysis shows that the operon hcdABC encodes a flavin-binding hydroxylase (HcdA), an extradiol dioxygenase (HcdB), and a putative hydroxymuconic semialdehyde hydrolase (HcdC). The analysis of the recombinant HcdA activity in vitro confirms that this enzyme belongs to the group of ipso-hydroxylases. The activity of the proteins HcdB and HcdC has been analyzed by using recombinant Escherichia coli cells. Identification of intermediate metabolites allowed us to confirm the predicted enzyme functions and to reconstruct the catabolic pathway of 3-(2,4-dihydroxyphenyl)-propionic acid. HcdA catalyzes the conversion of 3-(2,4-dihydroxyphenyl)-propionic acid to 3-(2,3,5-trihydroxyphenyl)-propionic acid through an ipso-hydroxylation followed by an internal (1,2-C,C)-shift of the alkyl moiety. Then, in the presence of HcdB, a subsequent oxidative meta-cleavage of the aromatic ring occurs, resulting in the corresponding linear product (2E,4E)-2,4-dihydroxy-6-oxonona-2,4-dienedioic acid. Here, we describe a Pseudomonas mandelii strain 7HK4 capable of degrading 7-hydroxycoumarin via 3-(2,4-dihydroxyphenyl)-propionic acid pathway.

A bacterium with close genetic identity to Pseudomonas mandelii associated with spring fish kills in wild bluegill Lepomis macrochirus Rafinesque and pumpkinseed sunfish Lepomis gibbosus (Linnaeus).

Pseudomonas fluorescens are known bacterial pathogens in fish. The P. fluorescens group contains at least nine different bacterial species, although species from fish have rarely been differentiated. Two isolated fish kills affecting wild bluegills, Lepomis macrochirus Rafinesque, and pumpkinseed sunfish, Lepomis gibbosus (Linnaeus), occurred in the spring of 2015 during cool water temperatures (12.5°C-15.5°C). Disease signs included severe bacteraemia with rare gross external signs. Pure bacterial cultures isolated from kidneys of all affected fish were identified as P. fluorescens using the API 20NE system, while no bacteria were isolated from asymptomatic fish. To further identify the species of bacterium within the P. fluorescens complex, genetic analysis of the 16S rRNA, rpoD and gyrB genes was conducted. DNA sequences of bacterial isolates from both mortality events were identical and had close identity (≥99.7%) to Pseudomonas mandelii. Although likely widespread in the aquatic environment, this is the first report of a bacterium closely resembling P. mandelii infecting and causing disease in fish. The bacterium grew at temperatures between 5°C and 30°C, but not at 37°C. It is possible that infections in fish were a result of immunosuppression associated with spring conditions combined with the psychrotrophic nature of the bacterium.

Enhanced catalytic site thermal stability of cold-adapted esterase EstK by a W208Y mutation.

Hydrophobic interactions are known to play an important role for cold-adaptation of proteins; however, the role of amino acid residue, Trp, has not been systematically investigated. The extracellular esterase, EstK, which was isolated from the cold-adapted bacterium Pseudomonas mandelii, has 5 Trp residues. In this study, the effects of Trp mutation on thermal stability, catalytic activity, and conformational change of EstK were investigated. Among the 5 Trp residues, W(208) was the most crucial in maintaining structural conformation and thermal stability of the enzyme. Surprisingly, mutation of W(208) to Tyr (W(208)Y) showed an increased catalytic site thermal stability at ambient temperatures with a 13-fold increase in the activity at 40°C compared to wild-type EstK. The structure model of W(208)Y suggested that Y(208) could form a hydrogen bond with D(308), which is located next to catalytic residue H(307), stabilizing the catalytic domain. Interestingly, Tyr was conserved in the corresponding position of hyper-thermophilic esterases EstE1 and AFEST, which are active at high temperatures. Our study provides a novel insight into the engineering of the catalytic site of cold-adapted enzymes with increased thermal stability and catalytic activity at ambient temperatures.

Structure of the O-polysaccharide of Pseudomonas mandelii CYar1 containing 3,6-dideoxy-4-C-[(S)-1-hydroxyethyl]-D-xylo-hexose (yersiniose A).

The O-polysaccharide isolated by mild acid hydrolysis of the lipopolysaccharide of Pseudomonas mandelii CYar1 was studied by sugar analysis and 1D and 2D (1)H and (13)C NMR spectroscopies. The following structure of the O-polysaccharide was established:

Psychrotrophic Pseudomonas mandelii CBS-1 produces high levels of poly-ß-hydroxybutyrate.

A novel facultative psychrotroph (strain CBS-1), which accumulates poly-ß-hydroxybutyrate (PHB), was isolated from soil samples taken from Changbai Mountain, China. Phylogenetic analysis based on 16S rRNA sequence data and Biolog analysis identified strain CBS-1 as Pseudomonas mandelii. Transmission electron micrographs revealed abundant electron-transparent intracellular granules. (1)H-nuclear magnetic resonance analysis revealed that the granules were composed of PHB. P. mandelii CBS-1 grew optimally at 20°C. When cultured aerobically for 48 h with sucrose as the sole carbon source, strain CBS-1 yielded a maximum cell density of 29.3 g/L cell dry weight and synthesized 22.3 g/L of PHB. The ability of strain CBS-1 to grow at a low temperature and rapidly synthesize high levels of PHB may reduce the costs of industrial PHB production.