Sphingomonas Strains Research Articles

Carbofuran degradation mediated by three related plasmid systems

Two carbofuran-metabolizing Sphingomonas strains, TA and CD, were isolated from soils with differing histories of exposure to carbofuran. These strains were compared with a previously described strain, Sphingomonas sp. CFO6, with regard to growth rate, formation of metabolites, and plasmid content and structure. Extensive regions of similarity were observed between the three different plasmid systems as evidenced by cross hybridization. In addition, all three systems harbor IS 1412, an insertion sequence (IS) element involved in heat-induced loss of carbofuran phenotype in CFO6, and heat-induced carbofuran deficient mutants of all three strains correlated with loss of IS 1412. A carbofuran deficient mutant of TA generated by induction of IS elements was complemented by reintroduction of the wild-type plasmid, confirming the presence of genes required for carbofuran metabolism on this plasmid. Carbofuran metabolism in these three strains is clearly linked via plasmids of different numbers and sizes that share extensive common regions, and carbofuran-degrading genes may be associated with active IS elements.

Chemotaxonomic and phylogenetic analyses of Sphingomonas strains isolated from ears of plants in the family Gramineae and a proposal of Sphingomonas roseoflava sp. nov.

Chemotaxonomic and phylogenetic characteristics of Sphingomonas strains isolated from plants of the family Gramineae were investigated. All strains contained the monosaccharide (glucuronic acid) type of glycosphingolipid (GSL-1). Most were found also to contain the oligosaccharide-type glycosphingolipids. Fatty acid and sphingosine profiles of the isolates were identical, although the ratio of the contents varies among the isolates. They all contained ubiquinone Q-10, and the G1C contents were from 66 to 68%. Phylogenetic analysis using 16S rRNA gene base sequences revealed that all the isolates were placed in the phylogenetic group of Sphingomonas paucimobilis in the alpha-4 subclass of Proteobacteria. By DNA-DNA hybridization experiments, the plant isolates were divided into five genotypic groups (groups 1 to 5). The strains of group 5 showed common physiological characteristics and formed pink-yellow colored colonies. Based on these results, Sphingomonas roseoflava sp. nov. was proposed for that homology group.

Ecology, physiology, and phylogeny of deep subsurface Sphingomonas sp.

Several new species of the genus Sphingomonas including S. aromaticivorans, S. stygia, and S. subterranea that have the capacity for degrading a broad range of aromatic compounds including toluene, naphthalene, xylenes, p-cresol, fluorene, biphenyl, and dibenzothiophene, were isolated from deeply-buried (>200 m) sediments of the US Atlantic coastal plain (ACP). In S. aromaticivorans F199, many of the genes involved in the catabolism of these aromatic compounds are encoded on a 184-kb conjugative plasmid; some of the genes involved in aromatic catabolism are plasmid-encoded in the other strains as well. Members of the genus Sphingomonas were common among aerobic heterotrophic bacteria cultured from ACP sediments and have been detected in deep subsurface environments elsewhere. The major source of organic carbon for heterotrophic metabolism in ACP deep aquifers is lignite that originated from plant material buried with the sediments. We speculate that the ability of the subsurface Sphingomonas strains to degrade a wide array of aromatic compounds represents an adaptation for utilization of sedimentary lignite. These and related subsurface Sphingomonas spp may play an important role in the transformation of sedimentary organic carbon in the aerobic and microaerobic regions of the deep aquifers of the ACP.

Differential detection of key enzymes of polyaromatic-hydrocarbon-degrading bacteria using PCR and gene probes.

Bacteria with ability to degrade polyaromatic hydrocarbons (PAHs), isolated from wastewater and soil samples, were investigated for their taxonomic, physiological and genetic diversity. Eighteen isolates able to metabolize naphthalene or phenanthrene as sole carbon source were taxonomically affiliated to different subclasses of the Proteobacteria (Sphingomonas spp., Acidovorax spp., Comamonas spp. and Pseudomonas spp.) and to phyla of Gram-positive bacteria with low and high DNA G + C content (Paenibacillus sp. and Rhodococcus spp., respectively). Representatives of the genera Pseudomonas and Sphingomonas formed a remarkably high fraction of these isolates; 9 out of 18 strains belonged to these groups. Tests for enzyme activities showed that the majority of the isolates growing with PAHs as sole sources of carbon and energy had an active catechol 2,3-dioxygenase (C230). C230 specific activities were very diverse, ranging from 0.1 to 650 mU (mg protein)-1. Pseudomonas and Sphingomonas strains showed considerably higher activities than the other isolates. All PAH degraders were examined for the presence of an initial PAH dioxygenase and C230, which catalyse key steps of PAH degradation, by PCR amplification of gene fragments and subsequent hybridization. PCR primers and internal oligonucleotide probes were developed for the specific detection of the genes of Pseudomonas and Sphingomonas strains.

Response of fluoranthene-degrading bacteria to surfactants

A prerequisite for surfactant-enhanced biodegradation is that the microorganisms survive, take up substrate and degrade it in the presence of the surfactant. Two Mycobacterium and two Sphingomonas strains, degrading fluoranthene, were investigated for their sensitivity towards non-ionic chemical surfactants. The effect of Triton X-100 and Tween 80 above their critical micelle concentration on mineralization of [14C]-glucose and [14C]-fluoranthene was measured in shaker cultures. Tween 80 had no toxic effect on any of the tested strains. The surfactant inhibited fluoranthene mineralization by the hydrophobic Mycobacterium spp. slightly, but more than doubled that by the two less hydrophobic Sphingomonas strains. Triton X-100 inhibited fluoranthene mineralization by all strains, yet this was more pronounced for the Sphingomonas spp. Both surfactants caused cell wall permeabilization, as shown by transient colouring of surfactant-containing media. Inhibition of glucose mineralization, indicating non-specific toxic effects of Triton X-100, was observed only for the Sphingomonas strains and the toxicity was caused by micelle-to-cell interactions. These strains, however, appeared to recover from initial Triton X-100 toxicity within 50–500 h of exposure. The ratio of surfactant concentration to initial cell density was found to determine critically the bacterial response to surfactants. For both Sphingomonas and Mycobacterium strains, this work indicates that fluoranthene solubilized in surfactant micelles is only partially available for mineralization by the bacteria tested. However, our results suggest that optimal conditions for polycyclic aromatic hydrocarbon mineralization can be developed by selection of the proper surfactant, bacterial strains, cell density and incubation conditions.

Amplification of putative chlorocatechol dioxygenase gene fragments from α- and β-Proteobacteria

Redundant primers were designed for the PCR amplification of DNA from chlorocatechol dioxygenase genes. These primers were used successfully to amplify 270- to 279-bp fragments from a variety of 2,4-dichlorophenoxyacetate- and chlorobenzoate-degrading strains, including species of Sphingomonas. Three groups of closely related sequences were amplified: one from chlorobenzoate degraders that was 86% similar to the amino acid sequence of the protein coded by the tfdC gene of Ralstonia eutropha JMP134 (pJP4), a second from Sphingomonas strains that was 70% similar to this amino acid sequence, and a third from diverse 2,4-D degraders that showed only 53% similarity to the product coded by tfdC from pJP4 but 88-100% similarity to the product of the tfdC gene of the plasmid pEST4011 from a Pseudomonas putida strain. The primers should be useful in further study of this gene and in tracking a variety of degraders of chloroaromatic compounds in natural systems.Key words: 2,4-dichlorophenoxyacetate, chlorobenzoate, biodegradation, ortho cleavage, detection.

Amplification of putative chlorocatechol dioxygenase gene fragments from alpha- and beta-Proteobacteria.

Redundant primers were designed for the PCR amplification of DNA from chlorocatechol dioxygenase genes. These primers were used successfully to amplify 270- to 279-bp fragments from a variety of 2,4-dichlorophenoxyacetate- and cholorobenzoate-degrading strains, including species of Sphingomonas. Three groups of closely related sequences were amplified: one from chlorobenzoate degraders that was 86% similar to the amino acid sequence of the protein coded by the tfdC gene of Ralstonia eutropha JMP134 (pJP4), a second from Sphingomonas strains that was 70% similar to this amino acid sequence, and a third from diverse 2,4-D degraders that showed only 53% similarity to the product coded by tfdC from pJP4 but 88-100% similarity to the product of the tfdC gene of the plasmid pEST4011 from a Pseudomonas putida strain. The primers should be useful in further study of this gene and in tracking a variety of degraders of chloroaromatic compounds in natural systems.

Polysaccharide lyases from gellan-producing Sphingomonas spp.

A number of Sphingomonas strains capable of synthesizing the bacterial exopolysaccharide gellan and related polymers were shown to possess constitutive gellanase activity. In each case, the degradation of deacylated gellan was due to extracellular, eliminase-type enzymes (lyases) which cleave the sequence ... beta-D-glucosyl 1,4-beta-D-glucuronosyl ... in the tetrasaccharide repeat unit of the substrate polysaccharides. Deacetylated rhamsan was an alternative substrate but there was little or no action against most other polysaccharides with similar structures. Slight differences were found between the specificities of the lyases from different strains. Activities of gellan lyase preparations were generally low. As well as the extracellular 'gellanase' activity, all the bacteria possessed varying amounts of beta-D-glucosidase and beta-D-glucuronidase activities apparently located in the periplasm. The products from deacylated gellan and the chemically deacylated form of polysaccharide S194 (rhamsan gum), which is effectively a gentiobiosylated form of gellan, closely resembled those recently obtained by the authors from other, gellan-degrading, non-gellan-producing bacteria. The enzymes had negligible activity against the natural, acylated gellan and rhamsan polysaccharides from bacteria now designated as strains of Sphingomonas.

Mechanism of bacitracin resistance in gram-negative bacteria that synthesize exopolysaccharides.

Four representative species from three genera of gram-negative bacteria that secrete exopolysaccharides acquired resistance to the antibiotic bacitracin by stopping synthesis of the exopolysaccharide. Xanthomonas campestris, Sphingomonas strains S-88 and NW11, and Escherichia coli K-12 secrete xanthan gum, sphingans S-88 and NW11, and colanic acid, respectively. The gumD gene in X. campestris is required to attach glucose-P to C55-isoprenyl phosphate, the first step in the assembly of xanthan. A recombinant plasmid carrying the gumD gene of X. campestris restored polysaccharide synthesis to bacitracin-resistant exopolysaccharide-negative mutants of X. campestris and Sphingomonas strains. Similarly, a newly cloned gene (spsB) from strain S-88 restored xanthan synthesis to the same X. campestris mutants. However, the intergeneric complementation did not extend to mutants of E. coli that were both resistant to bacitracin and nonproducers of colanic acid. The genetic results also suggest mechanisms for assembling the sphingans which have commercial potential as gelling and viscosifying agents.

Isolation ofSphingomonasStrains from Ears of Rice and Other Plants of Family Gramineae

Sphingomonas strains were isolated in high frequency from ears of rice (Oryza sativa), Echinochloa crus-galli, and Setaria viridis. Isolates were identified by the rapid method of cellular fatty acid analysis. Isolated Sphingomonas strains have 2-hydroxymyristate as a sole hydroxy fatty acid, ubiquinone Q-10, and glycosphingolipid. This study demonstrated that sphingomonads are members of a natural flora of microorganisms in ears of rice and taxonomically related plants.