Genome analysis to decipher syntrophy in the bacterial consortium 'SCP' for azo dye degradation.

BACKGROUND: A bacterial consortium SCP comprising three bacterial members, viz. Stenotrophomonas acidaminiphila APG1, Pseudomonas stutzeri APG2 and Cellulomonas sp. APG4 was developed for degradation of the mono-azo dye, Reactive Blue 28. The genomic analysis of each member of the SCP consortium was done to elucidate the catabolic potential and role of the individual organism in dye degradation. RESULTS: The genes for glycerol utilization were detected in the genomes of APG2 and APG4, which corroborated with their ability to grow on a minimal medium containing glycerol as the sole co-substrate. The genes for azoreductase were identified in the genomes of APG2 and APG4, while no such trait could be determined in APG1. In addition to co-substrate oxidation and dye reduction, several other cellular functions like chemotaxis, signal transduction, stress-tolerance, repair mechanisms, aromatic degradation, and copper tolerance associated with dye degradation were also annotated. A model for azo dye degradation is postulated, representing the predominant role of APG4 and APG2 in dye metabolism while suggesting an accessory role of APG1. CONCLUSIONS: This exploratory study is the first-ever attempt to divulge the genetic basis of azo-dye co-metabolism by cross-genome comparisons and can be harnessed as an example for demonstrating microbial syntrophy.

Treatment of petroleum refinery sludge by petroleum degrading bacterium Stenotrophomonas pavanii IRB19 as an efficient novel technology.

Petroleum hydrocarbons (PHCs) in petroleum refinery sludge (PRS) are the most adverse components because of their toxic nature, which are harmful to human health and the aquatic ecosystem. This study aimed to identify and characterize an indigenous bacterium isolated from PRS of Indian oil corporation ltd. (IOCL), Haldia, India, and evaluate its performance for biodegradation of total petroleum hydrocarbon (TPH) of PRS. The bacterium molecularly characterized as Stenotrophomonas sp. IRB19 by 16S rRNA sequencing and phylogenetic analysis. The strain IRB19 showed a significant ability to utilize four different oils (kerosene, diesel, petrol and hexadecane) in-vitro. IRB19 could able to degrade up to 65 ± 2.4% of TPH in 28 d of incubation. Solvent extraction study showed that PRS contain 180.57 ± 3.44 g kg-1 of TPH and maltene fraction composed of aliphatic, aromatics and polar components of 52 ± 4, 39 ± 2 and 9 ± 1%, respectively. The TPH degradation best fitted for the Gompertz model and followed the first-order kinetics having the rate constant (k) and half-life period (t 1/2) of 0.036 d-1 and 19 d, respectively. Results of this study verified the suitability of the novel strain IRB19 for the biodegradation of PHCs.

Biodegradation of phenol by cold-tolerant bacteria isolated from alpine soils of Binaloud Mountains in Iran.

Degradation of phenol is considered to be a challenge because of harsh environments in cold regions and ground waters. Molecular characterization of phenol degrading bacteria was investigated to gain an insight into the biodegradation in cold areas. The psychrotolerant and psychrophiles bacteria were isolated from alpine soils in the northeast of Iran. These strains belonged to Pseudomonas sp., Stenotrophomonas spp. and Shinella spp. based on analysis of the 16S rRNA gene. These strains were capable of the complete phenol degradation at a concentration of 200 mg L-1 at 20 °C. Moreover, the strains could degrade phenol at a concentration of 400 and 600 mg L-1 at a higher time. Effects of environmental factors were studied using one factor at a time (OFAT) approach for Pseudomonas sp.ATR208. When the bacterium was grown in a liquid medium with 600 mg L-1 of concentration supplemented with optimum carbon and nitrogen sources, more than 99% of phenol removal was obtained at 20 °C and 24 h. Therefore, the present study indicated the potential of the local cold tolerant bacteria in the phenol bioremediation.

Analysis of growth and lipid production characteristics of Chlorella vulgaris in artificially constructed consortia with symbiotic bacteria.

The aim was to study the effect of artificially constructed consortia of microalgae-bacterial symbionts on growth and lipid production by Chlorella vulgaris (C. vulgaris), as well as the inter-relationship between microalgae and bacterial in a photoautotrophic system. The results showed that compared to an axenic culture of C. vulgaris, H1 co-culture system (axenic C. vulgaris-Stenotrophomona smaltophilia) had the strongest effect on the C. vulgaris growth. The biomass, specific growth rate and maximum productivity of C. vulgaris were increased by 21.9, 20.4, and 18%, respectively. The bacteria in co-culture system had a significant effect on the accumulation of lipid and fatty acid components of C. vulgaris: the content of lipid was increased by 8.2-33.83%, and the components of the saturated fatty acids and oleic acids also had an obvious improvement. The results indicate that the microalgae-bacterial co-culture system can improve microalgal biomass and the quality of biodiesel.

Low-abundant species facilitates specific spatial organization that promotes multispecies biofilm formation.

Microorganisms frequently co-exist in matrix-embedded multispecies biofilms. Within biofilms, interspecies interactions influence the spatial organization of member species, which likely play an important role in shaping the development, structure and function of these communities. Here, a reproducible four-species biofilm, composed of Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans and Paenibacillus amylolyticus, was established to study the importance of individual species spatial organization during multispecies biofilm development. We found that the growth of species that are poor biofilm formers, M. oxydans and P. amylolyticus, were highly enhanced when residing in the four-species biofilm. Interestingly, the presence of the low-abundant M. oxydans (0.5% of biomass volume) was observed to trigger changes in the composition of the four-species community. The other three species were crucially needed for the successful inclusion of M. oxydans in the four-species biofilm, where X. retroflexus was consistently positioned in the top layer of the mature four-species biofilm. These findings suggest that low abundance key species can significantly impact the spatial organization and hereby stabilize the function and composition of complex microbiomes.

Optimization of medium components and physicochemical parameters to simultaneously enhance microbial growth and production of lypolitic enzymes by Stenotrophomonas sp.

The optimization of lipase and esterase production (LP and EP) and bacterial growth (BG) of a Stenotrophomonas sp. strain was developed. For this purpose, the effect of five different medium components and three physicochemical parameters were evaluated using a Plackett-Burman statistical design. Among eight variables, stirring speed, pH, and peptone concentration were found to be the most effective factors on the three responses under evaluation. An optimization study applying Box-Behnken response surface methodology was used to study the interactive effects of the three selected variables on LP/EP and microorganism growth. Predicted models were found to be significant with high regression coefficients (90%-99%). By using the desirability function approach, the optimum condition applying simultaneous optimization of the three responses under study resulted to be: stirring speed of 100 rpm, pH of 7.5, and a peptone concentration of 10 g/L, with a desirability value of 0.977. Under these optimal conditions, it is possible to achieve in the optimized medium a 15-fold increase in esterase productivity, a 117-fold increase in lipase production, and a 9-log CFU/mL increase in BG, compared with the basal medium without agitation.

Arsenic bioremediation potential of a new arsenite-oxidizing bacterium Stenotrophomonas sp. MM-7 isolated from soil.

A new arsenite-oxidizing bacterium was isolated from a low arsenic-containing (8.8 mg kg(-1)) soil. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that the strain was closely related to Stenotrophomonas panacihumi. Batch experiment results showed that the strain completely oxidized 500 µM of arsenite to arsenate within 12 h of incubation in a minimal salts medium. The optimum initial pH range for arsenite oxidation was 5-7. The strain was found to tolerate as high as 60 mM arsenite in culture media. The arsenite oxidase gene was amplified by PCR with degenerate primers. The deduced amino acid sequence showed the highest identity (69.1 %) with the molybdenum containing large subunit of arsenite oxidase derived from Bosea sp. Furthermore the amino acids involved in binding the substrate arsenite, were conserved with the arsenite oxidases of other arsenite oxidizing bacteria such as Alcaligenes feacalis and Herminnimonas arsenicoxydans. To our knowledge, this study constitutes the first report on arsenite oxidation using Stenotrophomonas sp. and the strain has great potential for application in arsenic remediation of contaminated water.

Production of phytohormones, siderophores and population fluctuation of two root-promoting rhizobacteria in Eucalyptus globulus cuttings.

Vegetative propagation by stem cuttings and mini-cuttings has been used worldwide for growing Eucalyptus plants. However, clones and hybrids of this plant present a great variability in their rooting capacity, apart from a gradual decrease in the rooting potential due to the ontogenetic age of the mother plant. Several studies have demonstrated that some bacteria promote plant growth and rooting through the action of direct and indirect mechanisms that are not still completely clear. Considering this, the objective of this study was to assess the production of auxins, abscisic acid and siderophores in Bacillus subtilis and Stenotrophomona maltophilia, which in previous studies increased rooting of E. globulus cuttings. Additionally, the population of these bacteria in the rhizosphere, superficial tissues of the stem-base and callus of the mini-cuttings was identified, and quantified by real-time PCR. Only S. maltophilia produced IAA in the presence of tryptophan; none of the bacterial strains produced ABA, but both produced siderophores. A comparative analysis of the separation profiles showed that there is a diverse microbial community in the rhizosphere, and only S. maltophilia was capable of keeping its population at a density of 2.03 × 10(7) cells/mg in different tissues of the mini-cuttings. The results would indicate that the rooting stimulus in E. globulus could be related to the action of one or several mechanisms such as the production of auxins and siderophores, and it could also be associated with the ability of bacteria to stay in the rhizosphere or in plant callus tissues.

Conversion of oleic acid to 10-hydroxystearic acid by whole cells of Stenotrophomonas nitritireducens.

The optimal reaction conditions for the conversion of oleic acid to 10-hydroxystearic acid by whole cells of Stenotrophomonas nitritireducens were: pH 7.5, 35 °C, 0.05% (w/v) Tween 80, 20 g cells l(-1), and 30 g oleic acid l(-1) in an anaerobic atmosphere. Under these conditions, the cells produced 31.5 g 10-hydroxystearic acid l(-1) over 4 h with a conversion yield of 100% (mol/mol) and a productivity of 7.9 g l(-1) h(-1), indicating that oleic acid was converted completely to 10-hydroxystearic acid, with no detectable byproduct. This is the highest concentration, productivity, and yield of 10-hydroxystearic acid from oleic acid reported thus far.

High-throughput screening alternative to crystal violet biofilm assay combining fluorescence quantification and imaging.

The crystal violet assay is widely used for biofilm quantitation despite its toxicity and variability. Here, we instead combine fluorescence labelling with the Cytation 5 multi-mode plate reader, to enable simultaneous acquisition of both quantitative and imaging biofilm data. This high-throughput method produces more robust data and provides information about morphology and spatial species organization within the biofilm.

Bacterial and fungal endophyte communities in healthy and diseased oilseed rape and their potential for biocontrol of Sclerotinia and Phoma disease.

Phoma stem canker (caused by the ascomycetes Leptosphaeria maculans and Leptosphaeria biglobosa) is an important disease of oilseed rape. Its effect on endophyte communities in roots and shoots and the potential of endophytes to promote growth and control diseases of oilseed rape (OSR) was investigated. Phoma stem canker had a large effect especially on fungal but also on bacterial endophyte communities. Dominant bacterial genera were Pseudomonas, followed by Enterobacter, Serratia, Stenotrophomonas, Bacillus and Staphylococcus. Achromobacter, Pectobacter and Sphingobacterium were isolated only from diseased plants, though in very small numbers. The fungal genera Cladosporium, Botrytis and Torula were dominant in healthy plants whereas Alternaria, Fusarium and Basidiomycetes (Vishniacozyma, Holtermaniella, Bjerkandera/Thanatephorus) occurred exclusively in diseased plants. Remarkably, Leptosphaeria biglobosa could be isolated in large numbers from shoots of both healthy and diseased plants. Plant growth promoting properties (antioxidative activity, P-solubilisation, production of phytohormones and siderophores) were widespread in OSR endophytes. Although none of the tested bacterial endophytes (Achromobacter, Enterobacter, Pseudomonas, Serratia and Stenotrophomonas) promoted growth of oilseed rape under P-limiting conditions or controlled Phoma disease on oilseed rape cotyledons, they significantly reduced incidence of Sclerotinia disease. In the field, a combined inoculum consisting of Achromobacter piechaudii, two pseudomonads and Stenotrophomonas rhizophila tendencially increased OSR yield and reduced Phoma stem canker.

Functional consortium for denitrifying sulfide removal process.

Denitrifying sulfide removal (DSR) process simultaneously converts sulfide, nitrate, and chemical oxygen demand from industrial wastewaters to elemental sulfur, nitrogen gas, and carbon dioxide, respectively. This investigation utilizes a dilution-to-extinction approach at 10(-2) to 10(-6) dilutions to elucidate the correlation between the composition of the microbial community and the DSR performance. In the original suspension and in 10(-2) dilution, the strains Stenotrophomonas sp., Thauera sp., and Azoarcus sp. are the heterotrophic denitrifiers and the strains Paracoccus sp. and Pseudomonas sp. are the sulfide-oxidizing denitrifers. The 10(-4) dilution is identified as the functional consortium for the present DSR system, which comprises two functional strains, Stenotrophomonas sp. strain Paracoccus sp. At 10(-6) dilution, all DSR performance was lost. The functions of the constituent cells in the DSR granules were discussed based on data obtained using the dilution-to-extinction approach.

Reductive degradation of pyrazine-2-carboxylate by a newly isolated Stenotrophomonas sp. HCU1.

A bacterium growing on pyrazine-2-carboxylate broth was isolated, purified and identified as a strain of Stenotrophomonas sp. based on polyphasic taxonomic analyses and designated as strain HCU1. 16S rRNA gene sequence of strain HCU1 showed 98.7% sequence similarity with the type strain of Stenotrophomonas maltophilia belonging to Gammaproteobacteria. Growth of strain HCU1 was demonstrated when pyrazine-2-carboxylate was used as a sole source of nitrogen. Ring reduction of pyrazine-2-carboxylate was shown as increase in absorbance at 268 nm and the reduced product was confirmed as 1,2,5,6-tetrahydropyrazine-2-carboxylate, while a ring opened product, 2-amino-2-hydroxy-3-(methylamino) propanoic acid (with a loss in carbon atom), indicated a reductive degradation of pyrazine-2-carboxylate by strain HCU1.

Metabolism of acenaphthylene via 1,2-dihydroxynaphthalene and catechol by Stenotrophomonas sp. RMSK.

Stenotrophomonas sp. RMSK capable of degrading acenaphthylene as a sole source of carbon and energy was isolated from coal sample. Metabolites produced were analyzed and characterized by TLC, HPLC and mass spectrometry. Identification of naphthalene-1,8-dicarboxylic acid, 1-naphthoic acid, 1,2-dihydroxynaphthalene, salicylate and detection of key enzymes namely 1,2-dihydroxynaphthalene dioxygenase, salicylaldehyde dehydrogenase and catechol-1,2-dioxygenase in the cell free extract suggest that acenaphthylene metabolized via 1,2-dihydroxynaphthalene, salicylate and catechol. The terminal metabolite, catechol was then metabolized by catechol-1,2-dioxygenase to cis,cis-muconic acid, ultimately forming TCA cycle intermediates. Based on these studies, the proposed metabolic pathway in strain RMSK is, acenaphthylene --> naphthalene-1,8-dicarboxylic acid --> 1-naphthoic acid --> 1,2-dihydroxynaphthalene --> salicylic acid --> catechol --> cis,cis-muconic acid.

Multiple Lines of Evidences Reveal Mechanisms Underpinning Mercury Resistance and Volatilization by Stenotrophomonas sp. MA5 Isolated from the Savannah River Site (SRS), USA.

A largely understudied microbially mediated mercury (Hg) bioremediative pathway includes the volatilization of Hg2+ to Hg°. Therefore, studies on Hg resistant bacteria (HgR), isolated from historically long-term contaminated environments, can serve as models to understand mechanisms underpinning Hg cycling. Towards this end, a mercury resistant bacterial strain, identified as Stenotrophomonas sp., strain MA5, was isolated from Mill Branch on the Savannah River Site (SRS); an Hg-impacted ecosystem. Minimum inhibitory concentration (MIC) analysis showed Hg resistance of up to 20 µg/mL by MA5 with 95% of cells retaining viability. Microcosm studies showed that the strain depleted more than 90% of spiked Hg2+ within the first 24 h of growth and the detection of volatilized mercury indicated that the strain was able to reduce Hg2+ to Hg°. To understand molecular mechanisms of Hg volatilization, a draft whole genome sequence was obtained, annotated and analyzed, which revealed the presence of a transposon-derived mer operon (merRTPADE) in MA5, known to transport and reduce Hg2+ into Hg°. Based on the whole genome sequence of strain MA5, qRT-PCR assays were designed on merRTPADE, we found a ~40-fold higher transcription of merT, P, A, D and E when cells were exposed to 5 µg/mL Hg2+. Interestingly, strain MA5 increased cellular size as a function of increasing Hg concentrations, which is likely an evolutionary response mechanism to cope with Hg stress. Moreover, metal contaminated environments are shown to co-select for antibiotic resistance. When MA5 was screened for antibiotic resistance, broad resistance against penicillin, streptomycin, tetracycline, ampicillin, rifampicin, and erythromycin was found; this correlated with the presence of multiple gene determinants for antibiotic resistance within the whole genome sequence of MA5. Overall, this study provides an in-depth understanding of the underpinnings of Stenotrophomonas-mercury interactions that facilitate cellular survival in a contaminated soil habitat.

The plant-associated bacterium Stenotrophomonas rhizophila expresses a new enzyme for the synthesis of the compatible solute glucosylglycerol.

The rhizobacterium Stenotrophomonas rhizophila accumulates the compatible solutes glucosylglycerol (GG) and trehalose under salt stress conditions. The complete gene for the GG synthesis enzyme was cloned and sequenced. This enzyme from S. rhizophila represented a novel fusion protein composed of a putative C-terminal GG-phosphate synthase domain and an N-terminal putative GG-phosphate phosphatase domain, which was named GgpPS. A similar gene was cloned from Pseudomonas sp. strain OA146. The ggpPS gene was induced after a salt shock in S. rhizophila cells. After the salt-loaded cells reached stationary phase, the ggpPS mRNA content returned to the low level characteristic of the control cells, and GG was released into the medium. The complete ggpPS gene and a truncated version devoid of the phosphatase part were obtained as recombinant proteins. Enzyme activity tests revealed the expected abilities of the full-length protein to synthesize GG and the truncated GgpPS to synthesize GG-phosphate. However, dephosphorylation of GG-phosphate was detected only with the complete GgpPS protein. These enzyme activities were confirmed by complementation experiments using defined GG-defective mutants of the cyanobacterium Synechocystis sp. strain PCC 6803. Genes coding for proteins very similar to the newly identified fusion protein GgpPS for GG synthesis in S. rhizophila were found in genome sequences of related bacteria, where these genes are often linked to a gene coding for a transporter of the Mfs superfamily.

Conversion of linoleic acid into 10-Hydroxy-12(Z)-octadecenoic acid by whole cells of Stenotrophomonas nitritireducens.

The conversion of linoleic acid into 10-hydroxy-12(Z)-octadecenoic acid by whole cells of Stenotrophomonas nitritireducens as an isolated bacterium was optimized, and the optimal temperature, pH, and cell and substrate concentrations were 30 degrees C, 7.5, and 20 and 20 g/L, respectively. Under these conditions, whole cells in a bioreactor produced 15 g/L 10-hydroxy-12(Z)-octadecenoic acid in 2 h of reaction time without detectable byproducts. Using 2 g/L linoleic acid, the cells produced 1.92 g/L 10-hydroxy-12(Z)-octadecenoic acid. These are the highest concentration and yield of 10-hydroxy-12(Z)-octadecenoic acid ever reported.

Selection for biocontrol bacteria antagonistic toward Rosellinia necatrix by enrichment of competitive avocado root tip colonizers.

Biological control of soil-borne pathogens is frequently based on the application of antagonistic microorganisms selected solely for their ability to produce in vitro antifungal factors. The aim of this work was to select bacteria that efficiently colonize the roots of avocado plants and display antagonism towards Rosellinia necatrix, the causal agent of avocado white root rot. A high frequency of antagonistic strains (ten isolates, 24.4%) was obtained using a novel procedure based on the selection of competitive avocado root tip colonizers. Amplification and sequencing of the 16S rRNA gene, in combination with biochemical characterization, showed that eight and two of the selected isolates belonged to the genera Pseudomonas and Stenotrophomonas, respectively. Characterization of antifungal compounds produced by the antagonistic strains showed variable production of exoenzymes and HCN. Only one of these strains, Pseudomonas sp. AVO94, produced a compound that could be related to antifungal antibiotics. All of the ten selected strains showed twitching motility, a cell movement involved in competitive colonization of root tips. Production of N-acyl-homoserine lactones and indole-3-acetic acid was also reported for some of these isolates. Resistance to several bacterial antibiotics was tested, and three strains showing resistance to only one of them were selected for biocontrol assays. The three selected strains persisted in the rhizosphere of avocado plants at levels considered crucial for efficient biocontrol, 10(5)-10(6) colony forming units/g of root; two of them, Pseudomonas putida AVO102 and Pseudomonas pseudoalcaligenes AVO110, demonstrated significant protection of avocado plants against white root rot.

Removal of cadmium ions during stationary growth phase by an extremely cadmium-resistant strain of Stenotrophomonas sp.

Stenotrophomonas sp. CD02 was isolated from a site that previously had been contaminated with high concentrations of the heavy metals cadmium (3 mg kg(-1)) and chromium (115 mg kg(-1)). This strain was able to grow on complex (Luria Bertani) medium containing high concentrations of cadmium ion (up to 4 mM). Additionally, it could remove up to 80% of the dissolved ions but only after reaching stationary growth phase. Strain CD02 also tolerated high concentrations of other heavy metals such as chromium, zinc, copper, nickel, and lead at levels more than 2 mM. Although strain CD02 can tolerate much higher cadmium concentrations than the three Stenotrophomonas maltophilia strains tested, they all possess resistance to the same range of antibiotics. This suggests that strain CD02 possesses a mechanism that allows it to tolerate and remove cadmium differently from those conferring resistance to antibiotics. Strain CD02 can be a suitable candidate for heavy metal bioremediation in contaminated environment because it is able to tolerate high concentration of heavy metals and remove cadmium aerobically.