Effect of osmotic stress on plant growth promoting Pseudomonas spp.

In this study we isolated and screened drought tolerant Pseudomonas isolates from arid and semi arid crop production systems of India. Five isolates could tolerate osmotic stress up to −0.73 MPa and possessed multiple PGP properties such as P-solubilization, production of phytohormones (IAA, GA and cytokinin), siderophores, ammonia and HCN however under osmotic stress expression of PGP traits was low compared to non-stressed conditions. The strains were identified as Pseudomonas entomophila, Pseudomonas stutzeri, Pseudomonas putida, Pseudomonas syringae and Pseudomonas monteilli respectively on the basis of 16S rRNA gene sequence analysis. Osmotic stress affected growth pattern of all the isolates as indicated by increased mean generation time. An increase level of intracellular free amino acids, proline, total soluble sugars and exopolysaccharides was observed under osmotic stress suggesting bacterial response to applied stress. Further, strains GAP-P45 and GRFHYTP52 showing higher levels of EPS and osmolytes (amino acids and proline) accumulation under stress as compared to non-stress conditions, also exhibited higher expression of PGP traits under stress indicating a relationship between stress response and expression of PGP traits. We conclude that isolation and screening of indigenous, stress adaptable strains possessing PGP traits can be a method for selection of efficient stress tolerant PGPR strains.     

Identification and characterization of Cr-, Cd-, and Ni-tolerant bacteria isolated from mine tailings

Eleven bacterial strains were isolated from soil samples collected from mine tailings. Bacterial strains were checked for tolerance against heavy metals (Cr, Cd, Ni), using the agar dilution method. All the strains showed multiple tolerances against heavy metals, but the most promising results appeared in strains BCr3, BCd33, and BNi11: they were tolerant to 15 mM of Cr⁶⁺, 7.5 mM of Cd²⁺, and 10 mM of Ni²⁺, respectively. The effect of heavy metals on bacterial growth was tested together with their ability to grow in different pH, NaCl, and temperature values. Bacterial isolates grew well between pH 7.5 and 8.5. The optimum temperature for maximum growth was between 35 and 37°C, and no significant change in bacterial growth was observed in the presence of 2% NaCl. In addition, the bioaccumulation potential of bacterial strains was investigated. Bacterial strains BCr3, BCd33, and BNi11 showed high bioaccumulation ability of Cr (68.7%), Cd (72.4%), and Ni (69.8%), respectively. All bacterial isolates were identified by 16S rRNA gene sequencing. Analysis of plasmid content revealed that all bacterial isolates contained a single plasmid. Further, polymerase chain reaction together with DNA sequence analysis was used to screen all bacterial strains for the presence metal tolerance genes (czcD, chrA, chrB, czcB, czcC, nccA, and cadA) on both plasmid and chromosomal genomes.     

The Genetic Diversity of Culturable Nitrogen-Fixing Bacteria in the Rhizosphere of Wheat

A total of 17 culturable nitrogen-fixing bacterial strains associated with the roots of wheat growing in different regions of Greece were isolated and characterized for plant-growth-promoting traits such as auxin production and phosphate solubilization. The phylogenetic position of the isolates was first assessed by the analysis of the PCR-amplified 16S rRNA gene. The comparative sequence analysis and phylogenetic analysis based on 16S rRNA gene sequences show that the isolates recovered in this study are grouped with Azospirillum brasilense, Azospirillum zeae, and Pseudomonas stutzeri. The diazotrophic nature of all isolates was confirmed by amplification of partial nifH gene sequences. The phylogenetic tree based on nifH gene sequences is consistent with 16S rRNA gene phylogeny. The isolates belonging to Azospirillum species were further characterized by examining the partial dnaK gene phylogenetic tree. Furthermore, it was demonstrated that the ipdC gene was present in all Azospirillum isolates, suggesting that auxin is mainly synthesized via the indole-3-pyruvate pathway. Although members of P. stutzeri and A. zeae are known diazotrophic bacteria, to the best of our knowledge, this is the first report of isolation and characterization of strains belonging to these bacterial genera associated with wheat.     

Biodegradation of chlorpyrifos by bacterial consortium isolated from agriculture soil

Organophosphorous pesticides are widely used in agriculture to control major insect pests. Chlorpyrifos is one of the major organophosphorous pesticides which is used to control insects including termites, beetles. The widespread use of these pesticides is hazardous to the environment and also toxic to mammals, thus it is essential to remove the same from the environment. From the chlorpyrifos contaminated soil nine morphologically different bacterial strains, one actinomycete and two fungal strains were isolated. Among those isolates four bacterial strains which were more efficient were developed as consortium. The four bacterial isolates namely Pseudomonas putida (NII 1117), Klebsiella sp., (NII 1118), Pseudomonas stutzeri (NII 1119), Pseudomonas aeruginosa (NII 1120) present in the consortia were identified on the basis of 16S rDNA analysis. The intracellular fractions of the consortium exhibited more organophosphorus hydrolase activity (0.171 ± 0.003 U/mL/min). The degradation studies were carried out at neutral pH and temperature 37°C with chlorpyrifos concentration 500 mg L⁻¹. LC-mass spectral analysis showed the presence of metabolites chlopyrifos-oxon and Diethylphosphorothioate. These results highlight an important potential use of this consortium for the cleanup of chlorpyrifos contaminated pesticide waste in the environment.     

Isolation and Identification of Polycyclic Aromatic Hydrocarbon–Degrading Bacteria from Crude Oil Exploration Bore Well Sludge

Two bacterial strains capable of degrading polycyclic aromatic hydrocarbons were isolated from the crude oil exploration bore well sludge and identified by 16s rRNA gene sequencing as Pseudomonas stutzeri and Bacillus subtilis. The bacterial strains Pseudomonas stutzeri and Bacillus subtilis were able to degrade 95.1% and 99.4% of naphthalene (100 mg L^?1) and 99.5% and 94.6% of anthracene (50 mg L^?1), respectively, as a sole carbon and energy source in the liquid phase within a period of 6 days. The specific growth rate was determined for both the species and found to be 0.169 and 0.124 day^?1.     

Selection of microbial consortia for treating metal-working fluids

The aim of this research was to determine the effectiveness of a strategy for constructing microbial consortia for treating chemically mixed industrial effluent, based on a more thorough understanding of communities within waste metal-working fluids (MWFs). Complementary phenotypic and genotypic methods revealed that the microbial communities in spent MWFs had low diversity and were very similar in species composition in samples originating from different locations and uses. Of 65 bacterial isolates studied, only 9 species were identified using fatty acid methyl ester (FAME) analysis. The results of genotypic analysis by denaturing gradient gel electrophoresis (DGGE) were congruent with observations made using FAME analysis. The metabolic potential of the isolates was assessed in terms of assimilation ability and tolerance of co-contaminants. The three isolates, selected (Clavibacter michiganensis, Methylobacterium mesophilicum, and Rhodococcus erythropolis) to form a consortium, were representative of three of the four most abundant populations and when combined could utilise'or tolerate all of the individual MWF components, including the biocide and the recalcitrant compound benzotriazole. Journal of Industrial Microbiology & Biotechnology (2002) 29, 20–27 doi:10.1038/sj.jim.7000271 Received 19 December 2001/ Accepted in revised form 02 May 2002     

Acid and heat tolerance of persistent and nonpersistent Listeria monocytogenes food plant strains

Aims: Acid and heat tolerance of 17 persistent and 23 nonpersistent Listeria monocytogenes strains, recovered from three meat‐processing plants, were investigated. Methods and Results: The isolates were genotyped by pulsed‐field gel electrophoresis and categorized into persistent strains according to the frequency of the strain and duration of the contamination. The persistent and nonpersistent strains were challenged to acidic conditions (pH 2·4 for 2 h, 1 mol l^?1 HCl were used to acidify the suspension) and to heat (55°C for 40 min) to receive a reduction in cell count. Listeria monocytogenes strains showed large variation in acid tolerance (over 6 log units) and in heat tolerance (3 log units). The persistent strains showed higher tolerance to acidic conditions than the nonpersistent strains (Student’s t‐test, P = 0·02), but significant differences in heat tolerance between persistent and nonpersistent strains were not observed. Conclusions: The results indicate that acid tolerance may have an effect on the persistence of L. monocytogenes contamination. Significance and Impact of the Study: This study highlights the fact that there are great differences in acid and heat tolerances between L. monocytogenes strains, and the preventive measures should be designed to be effective against the most tolerant strains.     

Reduction of chromate by microorganisms isolated from metal contaminated sites of Karachi, Pakistan

Three bacterial strains, two identified as Pseudomonas stutzeri and one as a strain of cucurbit yellow vine disease bacterium, isolated from a foundry soil and a tannery, respectively, in Pakistan, were resistant to up to 1 mM chromate and anaerobically reduced Cr(VI) up to 100 M. The highest removal was by P. stutzeri CMG463: 88 mol l^–1 (88% of that supplied; specific rate was 3.0 nmol mg^–1 protein h^–1), while 58 and 76 mol l^–1 (58% and 76%) were removed by P. stutzeri CMG462 and cucurbit yellow vine disease bacterium CMG480, respectively. These isolates were compared to strains isolated from an uncontaminated coastal site in the UK and designated as K2 (Pseudomonas synxantha) K3 (Bacillus sp.), and J3 (unidentified Gram-positive strain). Strain K3 was Cr-sensitive, partially lysed by Cr(VI), but had the highest removal of chromate anaerobically: 92 mol l^–1 (92% of that supplied) at a specific rate of 71 nmol mg^–1 protein h^–1. Analysis of cell sections using transmission electron microscopy with energy dispersive X-ray analysis showed intracellular chromium in P. stutzeri but the cucurbit yellow vine disease bacterium and the Bacillus sp. precipitated chromium extracellularly. The isolates from the Cr-contaminated sites did not remove more Cr(VI), overall, than Cr-unstressed bacteria, but their tolerance to Cr(VI) is potentially useful for bioremediation, particularly since other studies have shown that the two P. stutzeri strains can bioaccumulate Cu²⁺.     

Diversity of culturable denitrifying bacteria

Sequence divergence in the ribosomal genes of known strains and isolates of aquatic denitrifying bacteria was investigated using restriction fragment length polymorphism (RFLP) analysis. The same cultures were characterized for their homology with antibody and gene probes for nitrite reductase (NiR), a key enzyme in the denitrification pathway, and for amplification with a set of polymerase chain reaction primers designed to amplify a portion of the NiR gene. The NiR probes were developed from Pseudomonas stutzeri (ATCC 14405) and several P. stutzeri strains were included in the analyses. The RFLP analysis clustered most of the P. stutzeri strains together, but detected considerable diversity within this group. Isolates from three aquatic environments exhibited within —and among — habitat diversity by RFLP. Hybridization with the NiR probes and amplification with the NiR primers were not correlated with the clustering of strains by rDNA RFLP analysis. The relationships among strains deduced from ribosomal DNA RFLP reflect heterogeneity within the P. stutzeri group and among other pseudomonads, and the patterns differ from those inferred from specificity of the NiR probes.Abbreviations NiR Nitrite reductase - PCR polymerase chain reaction - RFLP restriction fragment length polymorphism     

Ornibactin production and transport properties in strains of Burkholderia vietnamiensis and Burkholderia cepacia (formerly Pseudomonas cepacia)

Several strains of Burkholderia vietnamiensis, isolated from the rhizosphere of rice plants, and four strains formerly known as Pseudomonas cepacia including two collection strains and two clinical isolates were compared for siderophore production and iron uptake. The B. vietnamiensis (TVV strains) as well as the B. cepacia strains (ATCC 25416 and ATCC 17759) and the clinical isolates K132 and LMG 6999 were all found to produce ornibactins under iron starvation. The two ATCC strains of B. cepacia additionally produced the previously described siderophores, pyochelin and cepabactin. Analysis of the ratio of isolated ornibactins (C4, C6 and C8) by HPLC revealed nearly identical profiles. Supplementation of the production medium with ornithine (20 mm) resulted in a 2.5-fold increase in ornibactin synthesis. Ornibactin-mediated iron uptake was independent of the length of the acyl side chain and was observed with all strains of B. vietnamiensis and B. cepacia, but was absent with strains of Pseudomonas aeruginosa, Pseudomonas fluorescens and Pseudomonas stutzeri, known to produce pyoverdines or desferriferrioxamines as siderophores. These results suggest that ornibactin production is a common feature of all Burkholderia strains and that these strains develop an ornibactin-specific iron transport system which is distinct from the pyoverdine-specific transport in Pseudomonas strains.     

Detection and characterization of natural transformation in the marine bacterium Pseudomonas stutzeri strain ZoBell

Soil isolates of Pseudomonas stutzeri have been shown previously to acquire genes by natural transformation. In this study a marine isolate, Pseudomonas stutzeri strain ZoBell, formerly Pseudomonas perfectomarina, was also shown to transform naturally. Transformation was detected by the Juni plate method and frequencies of transformation were determined by filter transformation procedures. Maximum frequencies of transformation were detected for three independent antibiotic resistance loci. Transformation frequencies were on the order of 4×10^-5 transformants per recipient, a frequency over 100 times that of spontancous antibiotic resistance. Transfer of antibiotic resistance was inhibited by DNase I digestion. Marine isolates achieved maximum competence 14 h after transfer of exponential cultures to filters on solid media, although lower levels of competence were detected immediately following filter immobilization. Like soil isolates, P. stutzeri strain ZoBell is capable of cell contact transformation, but unlike soil isolates where transformation frequencies are greater for cell contact transformation as compared to transformation with purified DNA, the maximum frequency of transformation achieved by cell contact in the marine strain was approximately 10-fold less than transformation frequencies with purified DNA. These studies establish the first marine model for the study of natural transformation.This paper is dedicated to John L. Ingraham, Professor Emeritus of Microbiology at the University of California, Davis. Professor Ingraham was the first person to recognize natural transformation in Pseudomonas stutzeri and has continued to contribute to our understanding of the process over the past eight years. This understanding of the genetics of P. stutzeri is only one of the many areas of microbiology to which Professor Ingraham has contributed in his exceptional career     

Exchange of chromosomal markers by natural transformation between the soil isolate,Pseudomonas stutzeri JM300, and the marine isolate,Pseudomonas stutzeri strain ZoBell

Both the soil isolate,Pseudomonas stutzeri JM300, and the marine isolate,Pseudomonas stutzeri strain ZoBell, have been shown previously to be naturally transformable. This study reports the detection of genetic exchange by natural transformation between these two isolates. Transformation frequency was determined by filter transformation procedures. Three independent antibiotic resistance loci were used as chromosomal markers to monitor this exchange event: resistance to rifampicin, streptomycin, and nalidixic acid. The maximum frequencies of transformation were on the order of 3.1 to 3.8×10^-6 transformants per recipient; frequencies over an order of magnitude greater than those for spontaneous antibiotic resistance, although they are lower than those observed for soil: soil or marine: marine strain crosses. This exchange was inhibited by DNase I. Transformation was observed between soil and marine strains, both by filter transformation using purified DNA solutions and when transforming DNA was added in the form of viable donor cells. The results from this study support the close genetic relationship betweenP. stutzeri JM300 andP. stutzeri strain ZoBell. These results also further validate the utility ofP. stutzeri as a benchmark organism for modeling gene transfer by natural transformation in both soil and marine habitats.     

The Nitrogen-Fixation Island Insertion Site Is Conserved in Diazotrophic Pseudomonas stutzeri and Pseudomonas sp. Isolated from Distal and Close Geographical Regions

The presence of nitrogen fixers within the genus Pseudomonas has been established and so far most isolated strains are phylogenetically affiliated to Pseudomonas stutzeri. A gene ortholog neighborhood analysis of the nitrogen fixation island (NFI) in four diazotrophic P. stutzeri strains and Pseudomonas azotifigens revealed that all are flanked by genes coding for cobalamin synthase (cobS) and glutathione peroxidise (gshP). The putative NFIs lack all the features characterizing a mobilizable genomic island. Nevertheless, bioinformatic analysis P. stutzeri DSM 4166 NFI demonstrated the presence of short inverted and/or direct repeats within both flanking regions. The other P. stutzeri strains carry only one set of repeats. The genetic diversity of eleven diazotrophic Pseudomonas isolates was also investigated. Multilocus sequence typing grouped nine isolates along with P. stutzeri and two isolates are grouped in a separate clade. A Rep-PCR fingerprinting analysis grouped the eleven isolates into four distinct genotypes. We also provided evidence that the putative NFI in our diazotrophic Pseudomonas isolates is flanked by cobS and gshP genes. Furthermore, we demonstrated that the putative NFI of Pseudomonas sp. Gr65 is flanked by inverted repeats identical to those found in P. stutzeri DSM 4166 and while the other P. stutzeri isolates harbor the repeats located in the intergenic region between cobS and glutaredoxin genes as in the case of P. stutzeri A1501. Taken together these data suggest that all putative NFIs of diazotrophic Pseudomonas isolates are anchored in an intergenic region between cobS and gshP genes and their flanking regions are designated by distinct repeats patterns. Moreover, the presence of almost identical NFIs in diazotrophic Pseudomonas strains isolated from distal geographical locations around the world suggested that this horizontal gene transfer event may have taken place early in the evolution.     

Biosynthesis of phytohormones from novel rhizobacterial isolates and their in vitro plant growth-promoting efficacy

The health of the plant and soil fertility is dependent on the plant–microbes interaction in the rhizosphere. Microbial life tends to endure various rhizosphere plant–microbe interactions. Phytohormones such as auxins, cytokinins, gibberellic acid, ethylene and abscisic acid are termed as the classical group of hormones. Out of the 70 rhizobacterial strains isolated from the Coleus rhizosphere, three different rhizobacterial strains Pseudomonas stutzeri MTP40, Stenotrophomonas maltophilia MTP42 and Pseudomonas putida MTP50 having plant growth-promoting attributes were isolated and characterized for its phytohormone-producing ability. The phytohormones such as indole 3-acetic acid (IAA), gibberellic acid and cytokinin (kinetin and 6-benzyladenosine) were affirmed in culture supernatant of the above isolates. IAA was detected in all the three isolates, where in highest production was found in S. maltophilia MTP42 (240?µg/mL) followed by P. stutzeri MTP40 (250?µg/mL) and P. putida MTP50 (233?µg/mL). Gibberellic acid production was found maximum in MTP40 (34?µg/mL), followed by MTP42 (31?µg/mL) and MTP50 (27?µg/mL). The cytokinin production from the isolates, namely, MTP40, MTP42 and MTP50 were 13, 11 and 7.5?µg/mL, respectively. The isolates showing the production of plant growth enhancing phytohormones can be commercialized as potent bioformulations.     

MBC tolerance in aggressive and non-aggressive isolates of Ceratocystis ulmi

In selection experiments, tolerance to 0–5 ppm methyl benzimidazole-2-ylcarbamate (MBC) occurred at a frequency of c. 1 in 1–3 ×10⁸ conidia in both aggressive and non-aggressive isolates of Ceratocystis ulmi. The tolerant strains were inhibited by 5 ppm MBC, however, and attempts to select strains tolerant to 10 ppm were unsuccessful. In each of three isolates examined, tolerance remained stable after fifteen successive transfers on fungicide-free medium. Genetic control was nuclear and probably conditioned by a single gene. It is thought unlikely that the appearance of tolerant strains in nature will jeopardize the use of MBC for the control of Dutch elm disease.     

Characterization of nitrogen-fixing bacteria isolated from field-grown barley,oat, and wheat

Diazotrophic bacteria were isolated from the rhizosphere of field-grown Triticum aestivum, Hordeum vulgare, and Avena sativa grown in various regions of Greece. One isolate, with the highest nitrogen-fixation ability from each of the eleven rhizospheres, was selected for further characterisation. Diazotrophic strains were assessed for plant-growth-promoting traits such as indoleacetic acid production and phosphate solubilisation. The phylogenies of 16S rRNA gene of the selected isolates were compared with those based on dnaK and nifH genes. The constructed trees indicated that the isolates were members of the species Azospirillum brasilense, Azospirillum zeae, and Pseudomonas stutzeri. Furthermore, the ipdC gene was detected in all A. brasilence and one A. zeae isolates. The work presented here provides the first molecular genetic evidence for the presence of culturable nitrogen-fixing P. stutzeri and A. zeae associated with field-grown A. sativa and H. vulgare in Greece.     

Silver resistance in Pseudomonas stutzeri

Silver resistance was studied in a silver-resistant Pseudomonas stutzeri AG259 strain and compared to a silver-sensitive P. stutzeri JM303 strain. Silver resistance was not due to silver complexation to intracellular polyphosphate or the presence of low molecular weight metal-binding protein(s). Both the silver-resistant and silver-sensitive P. stutzeri strains produced H₂S, with the silver-resistant AG259 strain producing lower amounts of H₂S than the silver-sensitive JM303 strain. However, intracellular acid-labile sulfide levels were generally higher in the silver-resistant P. stutzeri AG259 strain. Silver resistance may be due to formation of silver-sulfide complexes in the silver-resistant P. stutzeri AG259 strain.     

Characterization of novel diesel-degrading strains <Emphasis Type="Italic">Acinetobacter haemolyticus</Emphasis> MJ01 and <Emphasis Type="Italic">Acinetobacter johnsonii</Emphasis> MJ4 isolated from oil-contaminated soil

The diesel-degrading strains, designated as MJ01 and MJ4, were isolated from oil-contaminated soil in Daejeon (South Korea) and were taxonomically characterized using a polyphasic approach and their diesel oil degradation abilities were analyzed. The isolates MJ01 and MJ4 were identified as Acinetobacter haemolyticus and Acinetobacter johnsonii, respectively, based on their 16S rDNA gene sequences, DNA–DNA relatedness, fatty acid profiles and various physiological characteristics. Strains MJ01 and MJ4 were able to use diesel oil as the sole carbon and energy source. Both strains could degrade over 90% of diesel oil with an initial concentration of 20,000 mg/l after incubation for 7 days, the most significant degradation occurred during the first 3 days. To our knowledge, this is the first report on diesel oil-degrading microorganisms among bacterial strains belonging to A. haemolyticus and A. johnsonii.     

The Effect of Rhamnolipid Biosurfactant Produced by <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> on Model Bacterial Strains and Isolates from Industrial Wastewater

In this study, the effect of rhamnolipid biosurfactant produced by Pseudomonas fluorescens on bacterial strains, laboratory strains, and isolates from industrial wastewater was investigated. It was shown that biosurfactant, depending on the concentration, has a neutral or detrimental effect on the growth and protein release of model Gram (+) strain Bacillus subtilis 168. The growth and protein release of model Gram (−) strain Pseudomonas aeruginosa 1390 was not influenced by the presence of biosurfactant in the medium. Rhamnolipid biosurfactant at the used concentrations supported the growth of some slow growing on hexadecane bacterial isolates, members of the microbial community. Changes in cell surface hydrophobicity and permeability of some Gram (+) and Gram (−) isolates in the presence of rhamnolipid biosurfactant were followed in experiments in vitro. It was found that bacterial cells treated with biosurfactant became more or less hydrophobic than untreated cells depending on individual characteristics and abilities of the strains. For all treated strains, an increase in the amount of released protein was observed with increasing the amount of biosurfactant, probably due to increased cell permeability as a result of changes in the organization of cell surface structures. The results obtained could contribute to clarify the relationships between members of the microbial community as well as suggest the efficiency of surface properties of rhamnolipid biosurfactant from Pseudomonas fluorescens making it potentially applicable in bioremediation of hydrocarbon-polluted environments.     

Effects of 5-fluoro-2′-deoxyuridine on bacterial growth its use for DNA labelling

Experiments on the action of 5-fluoro-2′-deoxyuridine on growth ofEscherichia coli B, CECT 101;Pseudomonas fluorescens, CECT 318;Pseudomonas savastanoi, CECT 93;Micrococcus luteus, ATCC 4698;Bacillus cereus, CIP 52.58;Bacillus macerans, ClP 52.58 andBacillus subtilis, ATCC 6633, are described. The inhibition of growth is reversed by thymine plus uracil in all cases except inPseudomonas strains in which uracil alone is active, and in which no exogenous thymine is taken up, not even in the presece of 2′-deoxyguanosine. Growth conditions for improved labelling of bacterial DNA are discussed in the light of the results.