Effect of plant growth promoting rhizobacteria,nematode parasitic fungi and root-nodule bacterium on root-knot nematodes Meloidogyne javanica and growth of chickpea

Effects of plant growth promoting rhizobacteria (Pseudomonas putida MTCC No. 3604 and Pseudomonas alcaligenes MTCC No. 493) and parasitic fungi (Pochonia chlamydosporia KIA and Paecilomyces lilacinus KIA) were studied, alone and together with Rhizobium sp. (charcoal commercial culture) on the growth of chickpea and multiplication of Meloidogyne javanica. Individually, P. putida 3604, P. alcaligenes 493 and Rhizobium caused a significant increase in the growth of chickpea in both nematode inoculated and uninoculated plants. Inoculation of Rhizobium with a parasitic fungus or with plant growth promoting rhizobaterium caused a greater increase in the growth of plants inoculated with nematodes than caused by either of them singly. Individually, P. lilacinus KIA caused a greater increase in the growth of nematode inoculated plants than caused by P. putida 3604 or P. alcaligenes 493. P. lilacinus KIA caused a greater reduction in galling and nematode multiplication followed by P. chlamydosporia KIA, P. putida 3604 and P. alcaligenes 493. Combined use of P. lilacinus KIA with Rhizobium was better in reducing galling and nematode multiplication than any other treatment. P. putida 3604 caused a greater colonization of root than P. alcaligenes 493 while P. lilacinus KIA was isolated from more nematodes than P. chlamydosporia KIA.     

Diversity of culturable sodium dodecyl sulfate (SDS) degrading bacteria isolated from detergent contaminated ponds situated in Varanasi city, India

In the present investigation, an attempt has been made to isolate and identify SDS-degrading bacteria from different detergent contaminated ponds situated in Varanasi city, UP, India. Initial survey of ponds indicated that these ponds were contaminated with detergents. Employing enrichment technique in minimal medium (PBM) with SDS as a sole carbon source, a total of 24 isolates were recovered from 7 detergent contaminated ponds. Studies on rates of SDS degradation indicated that the rate of SDS degradation varied from 97.2% to 19.6% after 12h incubation under identical conditions. An estimation of alkyl sulfatase activity indicated that the activity varied from 0.168 ± 0.004 to 0.024 ± 0.005 μmol SDS/mg protein/min. Molecular characterization of these isolates was performed on the basis of ARDRA and ERIC PCR, which indicated that these isolates were broadly divided in 8 groups. Some selected isolates were identified on the basis of 16S rDNA sequencing. It was found that these isolates belonged to Pseudomonas aeruginosa, Pseudomonas mendocina, Pseudomonas stutzeri, Pseudomonas alcaligenes, Pseudomonas pseudoalcaligenes, Pseudomonas putida and Pseudomonas otitidis respectively. Among these isolates P. aeruginosa, P. putida and P. otitidis have been previously shown to degrade and metabolize SDS, the rest of the isolates appear to be new.     

The Effects of Temperature and pH on the Growth of Eight Enteric and Nine Glucose Non-Fermenting Species of Gram-Negative Rods

We studied the heat resistance and the range of growth temperature o gram-negative rods to find one of the bacterial factors governing their infectivity in exogenous and endogenous infections in predisposed patients. Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Pseudomonas aeruginosa, and Acinetobacter calcoaceticus grew equally well at 25, 30, 37, and 42 C. Among other sugar non-fermenting gram-negative rods, six species showed suppressed growth at either or both ends of the incubation temperature range. All the bacterial species tested were killed within 30 min at 60 or 70 C. At 10 C, none of the bacterial strains multiplied, but all survived for 6 hr. Of 17 bacterial species tested, E. coli had the widest range of growth temperature (18–47 C), and also the shortest time necessary for growth to a certain population. Among the sugar non-fermenting rods, A. calcoaceticus had the widest range of growth temperature (20–45 C) and also multiplied rapidly. Pseudomonas strains exhibited slower growth at all temperatures and also had a narrower range of growth temperature than Enterobacteriaceae. Among Pseudomonas species, P. aeruginosa had the widest range of growth temperature (25–42 C) and also showed rapid growth. Pseudomonas cepacia, Achromobacter xylosoxidans, and Alcaligenes faecalis had a narrow range of growth temperature (28–37 C), and Pseudomonas fluorescens, Flavobacterium meningosepticum, and Moraxella grew most rapidly at 30 C. The above results are correlated fairly well with the incidence of clinical cases of infection. The growth attitude of a species of bacteria in response to temperature was considered to be one of the factors affecting the establishment of infection.     

Biocontrol of <Emphasis Type="Italic">Meloidogyne javanica</Emphasis> by <Emphasis Type="Italic">Rhizobium</Emphasis> and plant growth-promoting rhizobacteria on lentil

Biocontrol of the root-knot nematode Meloidogyne javanica was studied on lentil using plant growth-promoting rhizobacteria (PGPR) namely Pseudomonas putida, P. alcaligenes, Paenibacillus polymyxa and Bacillus pumilus and root nodule bacterium Rhizobium sp. Pseudomonas putida caused greater inhibitory effect on the hatching and penetration of M. javanica followed by P. alcaligenes, P. polymyxa and B. pumilus. Inoculation of any PGPR species alone or together with Rhizobium increased plant growth both in M. javanica-inoculated and -uninoculated plants. Inoculation of Rhizobum caused greater increase in plant growth than caused by any species of plant growth-promoting rhizobacteria in nematode-inoculated plants. Among PGPR, P. putida caused greater increase in plant growth and higher reduction in galling and nematode multiplication followed by P. alcaligenes, P. polymyxa and B. pumilus. Combined use of Rhizobium with any species of PGPR caused higher reduction in galling and nematode multiplication than their individual inoculation. Use of Rhizobium plus P. putida caused maximum reduction in galling and nematode multiplication followed by Rhizobium plus P. alcaligens. Pseudomonas putida caused greater root colonization and siderophore production followed by P. alcaligenes, P. polymyxa and B. pumilus. Analysis of the protein bands of these four species by SDS-PAGE revealed that P. putida had a different protein band profile compared to the protein profiles of P. alcaligenes, P. polymyxa and B. pumilus. However, the protein profiles of P. acaligenes, P. polymyxa and B. pumilus were similar.     

Effect of dissolved oxygen regime on growth dynamics of Pseudomonas spp during benzene degradation

We investigated the effect of different oxygen regimes on growth patterns of Pseudomonas spp. during benzene degradation in microcosm batch studies. Benzene degradation was induced by limiting oxygen available for microbial activity, which consists of three initial-dissolved oxygen (DO) levels of oxic, hypoxic, and anoxic conditions. Batch experiments were performed for cell growth and benzene degradation by inoculating three strains of Pseudomonas spp. (Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida) in mineral salt medium containing aqueous benzene. Results showed that all strains were capable to grow and degrade benzene under all oxygen regimes but in a different manner. The highest cell growth of P. aeruginosa and P. fluorescens was achieved under oxic and anoxic condition, respectively, but there was no substantial difference on benzene degradation between the oxygen treatments with about 25% reduction for both strains. P. putida showed a facultative process for both cell growth and benzene degradation. This reveals that care should be taken in selection of microorganisms with regard to environmental studies since they exhibit different responses for given environmental conditions such as DO levels.     

Modeling of growth kinetics for Pseudomonas spp. during benzene degradation

A modeling study was conducted on growth kinetics of three different strains of Pseudomonas spp. (Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida) during benzene degradation to determine optimum substrate concentrations for most efficient biodegradation. Batch tests were performed for eight different initial substrate concentrations to observe cell growth and associated substrate degradation using benzene-adapted cells. Kinetic parameters of both inhibitory (Haldane–Andrews, Aiba–Edwards) and noninhibitory (Monod) models were fitted to the relationship between specific growth rate and substrate concentration obtained from the growth curves. Results showed that half-saturation constant of P. fluorescens was the highest among the three strains, indicating that this strain could grow well at high concentration, while P. putida could grow best at low concentration. The inhibition constant of P. aeruginosa was the highest, implying that it could tolerate high benzene concentration and therefore could grow at a wider concentration range. Estimated specific growth rate of P. putida was lower, but half-saturation constant was higher than those from literature study due to high substrate concentration range used in this study. These two kinetic parameters resulted in substantial difference between Monod- and Haldane-type models, indicating that distinction should be made in applying those models.     

Thiosulfate oxidation by obligately heterotrophic bacteria

Thiosulfate was oxidized stoichiometrically to tetrathionate during growth on glucose byKlebsiella aerogenes, Bacillus globigii, B. megaterium, Pseudomonas putida, two strains each ofP. fluorescens andP. aeruginosa, and anAeromonas sp. A gram-negative, rod-shaped soil isolate, Pseudomonad H_w, converted thiosulfate to tetrathionate during growth on acetate. None of the organisms could use thiosulfate as sole energy source. The quantitative recovery of all the thiosulfate supplied to heterotrophic cultures either as tetrathionate alone or as tetrathionate and unused thiosulfate demonstrated that no oxidation to sulfate occurred with any of the strains tested. Two strains ofEscherichia coli did not oxidize thiosulfate. Thiosulfate oxidation in batch culture occurred at different stages of the growth cycle for different organisms:P. putida oxidized thiosulfate during lag and early exponential phase,K. aerogenes oxidized thiosulfate at all stages of growth, andB. megaterium andAeromonas oxidized thiosulfate during late exponential phase. The relative rates of oxidation byP. putida andK. aerogenes were apparently determined by different concentrations of thiosulfate oxidizing enzyme. Thiosulfate oxidation byP. aeruginosa grown in chemostat culture was inducible, since organisms pregrown on thiosulfate-containing media oxidized thiosulfate, but those pregrown on glucose only could not oxidize thiosulfate. Steady state growth yield ofP. aeruginosa in glucose-limited chemostat culture increased about 23% in the presence of 5–22 mM thiosulfate, with complete or partial concomitant oxidation to tetrathionate. The reasons for this stimulation are unclear. The results suggest that heterotrophic oxidation of thiosulfate to tetrathionate is widespread across several genera and may even stimulate bacterial growth in some organisms.     

N-butanoyl-l-homoserine lactone (BHL) deficient Pseudomonas aeruginosa isolates from an intensive care unit

Acylated homoserine lactones (AHLs) are self-generated diffusible signal molecules that mediate population density dependent gene expression (quorum sensing) in a variety of Gram-negative bacteria, and several virulence genes of human pathogens are known to be controlled by AHLs. In this study, strains of Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli and Klebsiella pneumoniae, isolated from intensive care patients, were screened for AHL production by using AHL responsive indicator strains of Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NT1. Positive reactions were recorded for all 50 isolates of P. aeruginosa and 10 isolates of Acinetobacter baumannii with Agrobacterium tumefaciens NT1. Surprisingly, most P. aeruginosa isolates gave negative results with C. violaceum CV026 in contrast to previous reports. This suggests that the new isolates of P. aeruginosa either failed to make short chain AHLs or the level of the signal molecule is very low.     

Characterization of type II protein secretion (xcp) genes in the plant growth-stimulatingPseudomonas putida, strain WCS358

InPseudomonas aeruginosa, the products of thexcp genes are required for the secretion of exoproteins across the outer membrane. Despite structural conservation of the Xcp components, secretion of exoproteins via the Xcp pathway is generally not found in heterologous organisms. To study the specificity of this protein secretion pathway, thexcp genes of another fluorescent pseudomonad, the plant growth-promotingPseudomonas putida strain WCS358, were cloned and characterized. Nucleotide sequence analysis revealed the presence of at least five genes, i.e.,xcpP, Q, R, S, andT, with homology toxcp genes ofP. aeruginosa. Unlike the genetic organization inP. aeruginosa, where thexcp cluster consists of two divergently transcribed operons, thexcp genes inP. putida are all oriented in the same direction, and probably comprise a single operon. Upstream ofxcpP inP. putida, an additional open reading frame, with no homolog inP. aeruginosa, was identified, which possibly encodes a lipoprotein. Mutational inactivation ofxcp genes inP. putida did not affect secretion, indicating that no proteins are secreted via the Xcp system under the growth conditions tested, and that an alternative secretion system is operative. To obtain some insight into the secretory pathway involved, the amino acid sequence of the N-terminus of the major extracellular protein was determined. The protein could be identified as flagellin. Mutations in thexcpQ andR genes ofP. aeruginosa could not be complemented by introduction of the correspondingxcp genes ofP. putida. However, expression of a hybrid XcpR protein, composed of the N-terminal one-third ofP. aeruginosa XcpR and the C-terminal two-thirds ofP. putida XcpR, did restore protein secretion in aP. aeruginosa xcpR mutant.     

Isolation of Bacteria Able to Metabolize High Concentrations of Formaldehyde

Summary Nineteen bacterial strains able to degrade and metabolize formaldehyde as a sole carbon source were isolated from soil and wastewater of a formaldehyde production factory. The samples were cultured in complex and mineral salts media containing 370 mg formaldehyde/l. The bacterial strains were identified to be Pseudomonas pseudoalcaligenes, P. aeruginosa, P. testosteroni, P. putida, and Methylobacterium extorquens. After adaptation of these microorganisms to high concentrations of formaldehyde; two isolated strains of M. extorquens (strains ESS and PSS) and four strains of P. pseudoalcaligenes (strains LSW, SSW, NSW and OSS) degraded 1850 mg formaldehyde/l, where as P. pseudoalcaligenes strain OSS completely consumed 3700 mg of formaldehyde/l after 24 h and degraded 70% of 5920 mg of formaldehyde/l after 72 h.     

Pseudomonas alcaligenes andPseudomonas testosteroni: Characterization and identification

Procedures used in most clinical laboratories do not clearly distinguish betweenPseudomonas alcaligenes andPseudomonas testosteroni. In an examination of 75 features of 31 strains, we found that only microscopic morphology definitively distinguished these two species.Pseudomonas alcalgenes is phenotypically heterogeneous;P. testosteroni is relatively homogeneous. Several additional features will distinguish most strains ofP. alcaligenes.     

Long-term analysis of diesel fuel consumption in a co-culture of Acinetobacter venetianus,Pseudomonas putida and Alcaligenes faecalis

The dynamics of a microbial population isolated from superficial waters of Venice Lagoon and the ability to utilise diesel fuel (n-alkanes mixture C₁₂-C₂₈) as the sole carbon and energy source were studied in a long-term reconstruction experiment. The reconstructed microbial population consisted of three bacterial strains belonging to the species Acinetobacter venetianus, Pseudomonas putida, and Alcaligenes faecalis, which were able to oxidise n-alkanes to alkanoates, n-alkanols to alkanoates, or only n-alkanoates, respectively. Three different approaches: plate counting, cell counting by epifluorescence microscopy with DAPI staining, and by fluorescence in situ hybridisation (FISH) by using a probe conjugate with fluoresceine isothiocyanate specifically targeted towards the 16S rRNA of bacteria belonging to the genus Acinetobacter were used to monitor the growth of the bacterial population. The growth of A. venetianus was stimulated by the presence of other strains, suggesting a beneficial interaction. After the first week of growth A. venetianus cells formed aggregates, as confirmed by confocal microscopy (CLSM), which allowed them to be distinguished from free cells. A relationship between cell number and measured areas (μm²) per aggregate was found. Each cell presented an average surface of 1.21 μm². Each aggregate was formed by a cellular monolayer biofilm consisting of up to several thousands of cells. The A. venetianus aggregates increased in number and size over time, but after two weeks fragmentation events, which had a beneficial effect on the growth of P. putida and A. faecalis, occurred. This revised version was published online in June 2006 with corrections to the Cover Date.     

Nucleotide sequence analysis and comparison of the lexA genes from Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa and Pseudomonas putida.

Summary The complete nucleotide sequences of the lexA genes from Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa and Pseudomonas putida were determined; the DNA sequences of the lexA genes from these bacteria were 86%, 76%, 61% and 59% similar, respectively, to the Escherichia coli K12 gene. The predicted amino acid sequences of the S. typhimurium, E. carotovora and P. putida LexA proteins are 202 residues long whereas that of P. aeruginosa is 204. Two putative LexA repressor binding sites were localized upstream of each of the heterologous genes, the distance between them being 5 by in S. typhimurium and E. carotovora, as in the lexA gene of E. coli, and 3 by in P. putida and P. aeruginosa. The first lexA site present in the lexA operator of all five bacteria is very well conserved. However, the second lexA box is considerably more variable. The Ala-84 — Gly-85 bond, at which the LexA repressor of E. coli is cleaved during the induction of the SOS response, is also found in the LexA proteins of S. typhimurium and E. carotovora. Likewise, the amino acids Ser-119 and Lys-156 are present in all of these three LexA repressors. These residues also exist in the LexA proteins of P. putida and P. aeruginosa, but they are displaced by 4 and 6 residues, respectively. Furthermore, the structure and sequence of the DNA-binding domain of the LexA repressor of E. coli are highly conserved in the S. typhimurium, E. carotovora, P. aeruginosa and P. putida LexA proteins.     

Pyrimidine base and ribonucleoside utilization by thePseudomonas alcaligenes group

Pyrimidine base and ribonucleoside utilization was investigated in the two type strains of thePseudomonas alcaligenes group. As sole sources of nitrogen, the pyrimidine bases uracil, thymine and cytosine as well as the dihydropyrimidine bases dihydrouracil and dihydrothymine supported the growth ofPseudomonas pseudoalcaligenes ATCC 17440 but neither these bases nor pyrimidine nucleosides supportedPseudomonas alcaligenes ATCC 14909 growth. Ribose, deoxyribose, pyrimidine and dihydropyrimidine bases as well as pyrimidine nucleosides failed to be utilized by eitherP. pseudoalcaligenes orP. alcaligenes as sole carbon sources. The activities of the pyrimidine salvage enzymes nucleoside hydrolase, cytosine deaminase, dihydropyrimidine dehydrogenase and dihydropyrimidinase were detected in cell-free extracts ofP. pseudoalcaligenes andP. alcaligenes. InP. pseudoalcaligenes, the levels of cytosine deaminase, dihydropyrimidine dehydrogenase and dihydropyrimidinase could be affected by the nitrogen source present in the culture medium.     

Horizontal transfer of catabolic plasmids and naphthalene biodegradation in open soil

The horizontal transfer of naphthalene biodegradation plasmids and the parallel process of its microbial degradation were studied for the first time. The tagged naphthalene-degrading strains bearing labeled biodegradation plasmids were used for the monitoring of horizontal plasmid transfer in open soil. The population kinetics of microorganisms, the survival rate and competitiveness of introduced strains, and the transfer of biodegradation plasmids to indigenous strains were investigated. The transfer of the labeled plasmid pNF142::TnMod-OTc to the introduced plasmid-free recipient P. putida KT2442 and to indigenous soil microorganisms of the genus Pseudomonas was shown both under selection pressure (in the presence of naphthalene) and in its absence. The 16S rRNA gene sequencing showed that the soil strains that had acquired plasmids were close to the species P. lini, P. frederiksbergensis, P. jessenii, P. graminis, P. putida, and P. alcaligenes. Thus, direct evidence of dissemination of the naphthalene biodegradation plasmids in microbial populations in open soil under selective and nonselective conditions has been obtained.     

Rhamnolipids produced by Pseudomonas: from molecular genetics to the market

Rhamnolipids are biosurfactants with a wide range of industrial applications that entered into the market a decade ago. They are naturally produced by Pseudomonas aeruginosa and some Burkholderia species. Occasionally, some strains of different bacterial species, like Pseudomonas chlororaphis NRRL B-30761, which have acquired RL-producing ability by horizontal gene transfer, have been described. P. aeruginosa, the ubiquitous opportunistic pathogenic bacterium, is the best rhamnolipids producer, but Pseudomonas putida has been used as heterologous host for the production of this biosurfactant with relatively good yields. The molecular genetics of rhamnolipids production by P. aeruginosa has been widely studied not only due to the interest in developing overproducing strains, but because it is coordinately regulated with the expression of different virulence-related traits by the quorum-sensing response. Here, we highlight how the research of the molecular mechanisms involved in rhamnolipid production have impacted the development of strains that are suitable for industrial production of this biosurfactant, as well as some perspectives to improve these industrial useful strains.     

Cell hydrophobicity of <Emphasis Type="Italic">Pseudomonas</Emphasis> spp. and <Emphasis Type="Italic">Bacillus</Emphasis> spp. bacteria and hydrocarbon biodegradation in the presence of Quillaya saponin

Biodegradation and hydrophobicity of Pseudomonas spp. and Bacillus spp. strains were tested at different concentrations of the biosurfactant Quillaya saponin. A model mixture of hydrocarbon (dodecane and hexadecane) was used for estimating the influence of surfactants on biodegradation. The bacterial adhesion to hydrocarbon method for determination of bacterial cell surface hydrophobicity was exploited. Among the tested bacterial strains the higher hydrophobicity was noticed for Pseudomonas aeruginosa TK. The hydrophobicity of this strain was 84%. The highest hydrocarbon biodegradation was observed for P. aeruginosa TK (49%) and Bacillus subtilis (35%) strains after 7 days of experiments. Generally the addition of Quillaya saponin increased hydrocarbon biodegradation remarkably. The optimal concentration proved to be 80 mg l⁻¹. The degree of hydrocarbon biodegradation was 75% for P. aeruginosa TK after the addition of saponin. However the most significant increase in biodegradation after addition of Quillaya saponin was in the case of P. aeruginosa 25 and Pseudomonas putida (the increase of biodegradation from 21 to 52% and from 31 to 66%, respectively). It is worth mentioning that decrease of hydrophobicity is correlated with the best biodegradation by P. aeruginosa strain. For the remaining strains, no significant hydrophobicity changes in relation to the system without surfactant were noticed.     

Expression and characterization of (<Emphasis Type="Italic">R</Emphasis>)-specific enoyl coenzyme A hydratases making a channeling route to polyhydroxyalkanoate biosynthesis in <Emphasis Type="Italic">Pseudomonas putida</Emphasis>

We investigated the expression of (R)-specific enoyl coenzyme A hydratase (PhaJ) in Pseudomonas putida KT2440 accumulating polyhydroxyalkanoate (PHA) from sodium octanoate in order to identify biosynthesis pathways of PHAs from fatty acids in pseudomonads. From a database search through the P. putida KT2440 genome, an additional phaJ gene homologous to phaJ4 _Pa from Pseudomonas aeruginosa, termed phaJ4 _Pp, was identified. The gene products of phaJ1 _Pp, which was identified previously, and phaJ4 _Pp were confirmed to be functional in recombinant Escherichia coli on PHA synthesis from sodium dodecanoate. Cytosolic proteins from P. putida grown on sodium octanoate were subjected to anion exchange chromatography and one of the eluted fractions with hydratase activity included PhaJ4_Pp, as revealed by western blot analysis. These results strongly suggest that PhaJ4_Pp forms a channeling route from β-oxidation to PHA biosynthesis in P. putida. Moreover, the substrate specificity of PhaJ1_Pp was suggested to be different from that of PhaJ1_Pa from P. aeruginosa although these two proteins share 67% amino acid sequence identity.