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In an ongoing microbial tracking investigation of the International Space Station (ISS), several Sphingomonas strains were isolated. Based on the 16S rRNA gene sequence, phylogenetic analysis identified the ISS strains as Sphingomonas sanguinis ( n  = 2) and one strain isolated from the Kennedy Space Center cleanroom (used to assemble various Mars mission spacecraft components) as Sphingomonas paucimobilis. Metagenomic sequence analyses of different ISS locations identified 23 Sphingomonas species. An abundance of shotgun metagenomic reads were detected for S. sanguinis in the location from where the ISS strains were isolated. A complete metagenome-assembled genome was generated from the shotgun reads metagenome, and its comparison with the whole-genome sequences (WGS) of the ISS S. sanguinis isolates revealed that they were highly similar. In addition to the phylogeny, the WGS of these Sphingomonas strains were compared with the WGS of the type strains to elucidate genes that can potentially aid in plant growth promotion. Furthermore, the WGS comparison of these strains with the well-characterized Sphingomonas sp. LK11, an arid desert strain, identified several genes responsible for the production of phytohormones and for stress tolerance. Production of one of the phytohormones, indole-3-acetic acid, was further confirmed in the ISS strains using liquid chromatography-mass spectrometry. Pathways associated with phosphate uptake, metabolism, and solubilization in soil were conserved across all the S. sanguinis and S. paucimobilis strains tested. Furthermore, genes thought to promote plant resistance to abiotic stress, including heat/cold shock response, heavy metal resistance, and oxidative and osmotic stress resistance, appear to be present in these space-related S. sanguinis and S. paucimobilis strains. Characterizing these biotechnologically important microorganisms found on the ISS and harnessing their key features will aid in the development of self-sustainable long-term space missions in the future. IMPORTANCE Sphingomonas is ubiquitous in nature, including the anthropogenically contaminated extreme environments. Members of the Sphingomonas genus have been identified as potential candidates for space biomining beyond earth. This study describes the isolation and identification of Sphingomonas members from the ISS, which are capable of producing the phytohormone indole-3-acetic acid. Microbial production of phytohormones will help future in situ studies, grow plants beyond low earth orbit, and establish self-sustainable life support systems. Beyond phytohormone production, stable genomic elements of abiotic stress resistance, heavy metal resistance, and oxidative and osmotic stress resistance were identified, rendering the ISS Sphingomonas isolate a strong candidate for biotechnology-related applications.

Objective: To investigate the Interaction between chronic endometritis (CE) caused endometrial microbiota disorder and endometrial immune environment change in recurrent implantation failure (RIF)., Method: Transcriptome sequencing analysis of the endometrial of 112 patients was preform by using High-Throughput Sequencing. The endometrial microbiota of 43 patients was analyzed by using 16s rRNA sequencing technology., Result: In host endometrium, CD4 T cell and macrophage exhibited significant differences abundance between CE and non-CE patients. The enrichment analysis indicated differentially expressed genes mainly enriched in immune-related functional terms. Phyllobacterium and Sphingomonas were significantly high infiltration in CE patients, and active in pathways related to carbohydrate metabolism and/or fat metabolism. The increased synthesis of lipopolysaccharide, an important immunomodulator, was the result of microbial disorders in the endometrium., Conclusion: The composition of endometrial microorganisms in CE and non-CE patients were significantly different. Phyllobacterium and Sphingomonas mainly regulated immune cells by interfering with the process of carbohydrate metabolism and/or fat metabolism in the endometrium. CE endometrial microorganisms might regulate Th17 response and the ratio of Th1 to Th17 through lipopolysaccharide (LPS)., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chen, Chen, Guo, Fang and Li.)

Background: Numerous studies have reported the health-promoting effects of exopolysaccharides (EPSs) in in vitro models; however, a functional evaluation of EPSs will provide additional knowledge of EPS-microbe interactions by in vivo intestinal microbial model. In the present study, high-throughput amplicon sequencing, short-chain fatty acid (SCFAs) and intestinal inflammation evaluation were performed to explore the potential benefits of exopolysaccharides (EPSs) and EPS-producing Lactobacillus (HNUB20 group) using the healthy zebrafish (Danio rerio) model., Results: The results based on microbial taxonomic analysis revealed that the abundance of four genera, Ochrobactrum, Sediminibacterium, Sphingomonas and Sphingobium, were increased in the control group in comparison to HNUB20 group. Pelomonas spp. levels were significantly higher and that of the genera Lactobacillus and Brachybacterium were significantly decreased in EPS group compared with control group. PICRUSt based functional prediction of gut microbiota metabolic pathways indicated that significantly lower abundance was found for transcription, and membrane transport, whereas folding, sorting and degradation and energy metabolism had significantly higher abundance after HNUB20 treatment. Two metabolic pathways, including metabolism and endocrine functions, were more abundant in the EPS group than control group. Similar to the HNUB20 group, transcription was also decreased in the EPS group compared with the control group. However, SCFAs and immune indexes indicated EPS and HNUB20 performed limited efficacy in the healthy zebrafish., Conclusions: The present intestinal microbial model-based study indicated that EPSs and high-yield EPS-producing Lactobacillus can shake the structure of intestinal microbiota, but cannot change SCFAs presence and intestinal inflammation.

Alphaproteobacteria belonging to the group of the sphingomonads are frequently found in biofilms colonizing pure-water systems, where they cause technical and hygienic problems. In this study, physiological properties of sphingomonads for biofilm formation on plastic surfaces were analysed. Sphingomonas sp. strain S2M10 was isolated from a used water-filtration membrane and submitted to transposon mutagenesis for isolating mutants with altered biofilm formation. Mutants showing strongly decreased biofilm formation carried transposon insertions in genes for the biosynthesis of the polysaccharide sphingan and for flagellar motility. Flagella-mediated attachment was apparently important for biofilm formation on plastic materials of intermediate hydrophobicity, while a mutant with defect in spnB , encoding the first enzyme in sphingan biosynthesis, showed no biofilm formation on all tested materials. Sphingan-dependent biofilm formation was induced in the presence of specific carbon sources while it was not induced in complex medium with yeast extract and tryptone. The regulation of sphingan-based biofilm formation was investigated by interfering with the CckA/ChpT/CtrA phosphorelay, a central signal-transduction pathway in most Alphaproteobacteria. Construction and ectopic expression of a kinase-deficient histidine kinase CckA caused cell elongation and massive sphingan-dependent cell aggregation. In addition, it caused increased activity of the promotor of spnB . In conclusion, these results indicate that sphingan-based biofilm formation by sphingomonads might be triggered by specific carbon sources under prototrophic conditions resembling a milieu that often prevails in pure-water systems.

Plant growth-promoting rhizobacteria play vital roles not only in plant growth, but also in reducing biotic/abiotic stress. Sphingomonas panacis DCY99 T is isolated from soil and root of Panax ginseng with rusty root disease, characterized by raised reddish-brown root and this is seriously affects ginseng cultivation. To investigate the relationship between 159 sequenced Sphingomonas strains, pan-genome analysis was carried out, which suggested genomic diversity of the Sphingomonas genus. Comparative analysis of S. panacis DCY99 T with Sphingomonas sp. LK11 revealed plant growth-promoting potential of S. panacis DCY99 T through indole acetic acid production, phosphate solubilizing, and antifungal abilities. Detailed genomic analysis has shown that S. panacis DCY99 T contain various heavy metals resistance genes in its genome and the plasmid. Functional analysis with Sphingomonas paucimobilis EPA505 predicted that S. panacis DCY99 T possess genes for degradation of polyaromatic hydrocarbon and phenolic compounds in rusty-ginseng root. Interestingly, when primed ginseng with S . panacis DCY99 T during high concentration of iron exposure, iron stress of ginseng was suppressed. In order to detect S. panacis DCY99 T in soil, biomarker was designed using spt gene. This study brings new insights into the role of S . panacis DCY99 T as a microbial inoculant to protect ginseng plants against rusty root disease.

Background: Microbes isolated from hyperaccumulating plants have been reported to be effective in achieving higher phytoextraction efficiency. The plant growth-promoting bacteria (PGPB) SaMR12 from the cadmium (Cd)/zinc hyperaccumulator Sedum alfredii Hance could promote the growth of a non-host plant, oilseed rape, under Cd stress. However, the effect of SaMR12 on Brasscia juncea antioxidative response under Cd exposure was still unclear., Results: A hydroponic experiment was conducted to study the effects of Sphingomonas SaMR12 on its non-host plant Brassica juncea (L.) Czern. under four different Cd treatments. The results showed that SaMR12 could colonize and aggregate in the roots and then move to the shoots. SaMR12 inoculation promoted plant growth by up to 71% in aboveground biomass and 81% in root biomass over that of the non-inoculated plants. SaMR12-inoculated plants significantly enhanced root Cd accumulation in the 10 and 20 μM Cd treatments, with 1.72- and 0.86-fold increases, respectively, over that of the non-inoculated plants. SaMR12 inoculation not only decreased shoot hydrogen peroxide (H 2 O 2 ) content by up to 38% and malondialdehyde (MDA) content by up to 60% but also reduced proline content by 7-30% in shoots and 17-32% in roots compared to the levels in non-inoculated plants. Additionally, SaMR12 inoculation promoted the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) and facilitated the relative gene expression levels of dehydroascorbate reductase (DHAR) and glutathione reductase (GR) involved in the glutathione (GSH)-ascorbic acid (AsA) cycle., Conclusions: The results demonstrated that, under Cd stress, SaMR12 inoculation could activate the antioxidative response of B. juncea by decreasing the concentrations of H 2 O 2 , MDA and proline, increasing the activities of antioxidative enzymes, and regulating the GSH-AsA cycle. These results provide a theoretical foundation for the potential application of hyperaccumulator endophytic bacteria as remediating agents to improve heavy metal tolerance within non-host plant species, which could further improve phytoextraction efficiency.

The general stress response (GSR) represents an important trait to survive in the environment by leading to multiple stress resistance. In alphaproteobacteria, the GSR is under the transcriptional control of the alternative sigma factor EcfG. Here we performed transcriptome analyses to investigate the genes controlled by EcfG of Sphingomonas melonis Fr1 and the plasticity of this regulation under stress conditions. We found that EcfG regulates genes for proteins that are typically associated with stress responses. Moreover, EcfG controls regulatory proteins, which likely fine-tune the GSR. Among these, we identified a novel negative GSR feedback regulator, termed NepR2, on the basis of gene reporter assays, phenotypic analyses, and biochemical assays. Transcriptional profiling of signaling components upstream of EcfG under complex stress conditions showed an overall congruence with EcfG-regulated genes. Interestingly however, we found that the GSR is transcriptionally linked to the regulation of motility and biofilm formation via the single domain response regulator SdrG and GSR-activating histidine kinases. Altogether, our findings indicate that the GSR in S. melonis Fr1 underlies a complex regulation to optimize resource allocation and resilience in stressful and changing environments.

Among the plethora of unusual secondary metabolites isolated from Stachylidium bicolor are the tetrapeptidic endolides A and B. Both tetrapeptides contain 3-(3-furyl)-alanine residues, previously proposed to originate from bacterial metabolism. Inspired by this observation, we aimed to identify the presence of endosymbiotic bacteria in S. bicolor and to discover the true producer of the endolides. The endobacterium Burkholderia contaminans was initially detected by 16S rRNA gene amplicon sequencing from the fungal metagenome and was subsequently isolated. It was confirmed that the tetrapeptides were produced by the axenic B. contaminans only when in latency. Fungal colonies unable to produce conidia and the tetrapeptides were isolated and confirmed to be free of B. contaminans A second endosymbiont identified as related to Sphingomonas leidyi was also isolated. In situ imaging of the mycelium supported an endosymbiotic relationship between S. bicolor and the two endobacteria. Besides the technical novelty, our in situ analyses revealed that the two endobacteria are compartmentalized in defined fungal cells, prevailing mostly in latency when in symbiosis. Within the emerging field of intracellular bacterial symbioses, fungi are the least studied eukaryotic hosts. Our study further supports the Fungi as a valuable model for understanding endobacterial symbioses in eukaryotes. IMPORTANCE The discovery of two bacterial endosymbionts harbored in Stachylidium bicolor mycelium, Burkholderia contaminans and Sphingomonas leidyi , is described here. Production of tetrapeptides inside the mycelium is ensured by B. contaminans , and fungal sporulation is influenced by the endosymbionts. Here, we illustrate the bacterial endosymbiotic origin of secondary metabolites in an Ascomycota host., (Copyright © 2018 American Society for Microbiology.)

Plant growth-promoting rhizobacteria (PGPR) increase plant growth and crop productivity. The inoculation of plants with a bacterial mixture (consortium) apparently provides greater benefits to plant growth than inoculation with a single bacterial strain. In the present work, a bacterial consortium was formulated containing four compatible and desiccation-tolerant strains with potential as PGPR. The formulation had one moderately (Pseudomonas putida KT2440) and three highly desiccation-tolerant (Sphingomonas sp. OF178, Azospirillum brasilense Sp7 and Acinetobacter sp. EMM02) strains. The four bacterial strains were able to adhere to seeds and colonize the rhizosphere of plants when applied in both mono-inoculation and multi-inoculation treatments, showing that they can also coexist without antagonistic effects in association with plants. The effects of the bacterial consortium on the growth of blue maize were evaluated. Seeds inoculated with either individual bacterial strains or the bacterial consortium were subjected to two experimental conditions before sowing: normal hydration or desiccation. In general, inoculation with the bacterial consortium increased the shoot and root dry weight, plant height and plant diameter compared to the non-inoculated control or mono-inoculation treatments. The bacterial consortium formulated in this work had greater benefits for blue maize plants even when the inoculated seeds underwent desiccation stress before germination, making this formulation attractive for future field applications.

The CckA-ChpT-CtrA phosphorelay is central to the regulation of the cell cycle in Caulobacter crescentus. The three proteins are conserved in Alphaproteobacteria, but little is known about their roles in most members of this class. Here, we characterized the system in Sphingomonas melonis. We found that the transcription factor CtrA is the master regulator of flagella synthesis genes, the hierarchical transcriptional organization of which is herein described. CtrA also regulates genes involved in exopolysaccharide synthesis and cyclic-di-GMP signaling, and is important for biofilm formation. In addition, the ctrA mutant exhibits an aberrant morphology, suggesting a role for CtrA in cell division. An analysis of the regulation of CtrA indicates that the phosphorelay composed of CckA and ChpT is conserved and that the absence of the bifunctional kinase/phosphatase CckA apparently results in overactivation of CtrA through ChpT. Suppressors of this phenotype identified the hybrid histidine kinase CcsA. Phosphorelays initiated by CckA or CcsA were reconstituted in vitro, suggesting that in S. melonis, CtrA phosphorylation is controlled by a branched pathway upstream of ChpT. This study thus suggests that signals can directly converge at the level of ChpT phosphorylation through multiple hybrid kinases to coordinate a number of important physiological processes., (© 2015 John Wiley & Sons Ltd.)

The endophytic bacterium, Sphingomonas SaMR12, isolated from Sedum alfredii Hance, appears to increase plant biomass and zinc-extraction from contaminated soil; however, the mechanism by which this occurs is not clear. Here, the ability of SaMR12 to promote zinc extraction and its effects on root morphology and exudation were examined in hydroponics. Zinc treatment increased shoot biomass by 30 to 45%, and by a further 10 to 19% when combined with SaMR12 inoculation. Zinc treatment also increased zinc accumulation modestly and this too was enhanced with SaMR12. Both biomass and zinc levels increased in a dose-dependent manner with significant effects seen at 50 µM zinc and apparent saturation at 500 µM. Zinc and the endophyte also increased levels of auxin but not at 50 µM and zinc increased levels of superoxide and hydrogen peroxide but mainly at 500 µM. As for root morphology, SaMR12 increased root branching, the number of root tips, and surface area. Zinc and SaMR12 also increased the exudation of oxalic acid. For most assays the effects of the endophyte and zinc were additive, with the notable exception of SaMR12 strongly reducing the production of reactive oxygen species at 500 µM zinc. Taken together, these results suggest that the promotion of growth and zinc uptake by exposure to zinc and to SaMR12 are independent of reactive oxygen and do not involve increases in auxin.

Background: Pyomyoma is a life-threatening complication of uterine leiomyoma. It may occur in post- menopausal women, during pregnancy and in the postpartum period. Fever may be the only manifestation during the early stages of the disease. We detail the first reported case of postpartum pyomyoma-related sepsis due to Sphingomonas paucimobilis, a Gram-negative bacillus that is gaining recognition as an important human pathogen., Case Presentation: A woman presented with an asymptomatic uterine fibroid and a two-week history of fever during the postpartum period. Suppurative uterine leiomyoma was diagnosed, and blood cultures grew Sphingomonas paucimobilis. The myoma was surgically removed from the uterus without hysterectomy. Intravenous antimicrobial therapy was given for fifteen days, and the patient was discharged from hospital in good condition., Conclusion: Pyomyoma should be considered in broad differential diagnosis of postpartum fever. This case highlights a unique disease manifestation of S. paucimobilis, an emerging opportunistic pathogen with increasing significance in the nosocomial setting.

The survival, physiology and gene expression profile of the phenanthrene-degrading Sphingomonas sp. LH128 was examined after an extended period of complete nutrient starvation and compared with a non-starved population that had been harvested in exponential phase. After 6 months of starvation in an isotonic solution, only 5 % of the initial population formed culturable cells. Microscopic observation of GFP fluorescent cells, however, suggested that a larger fraction of cells (up to 80 %) were still alive and apparently had entered a viable but non-culturable (VBNC) state. The strain displayed several cellular and genetic adaptive strategies to survive long-term starvation. Flow cytometry, microscopic observation and fatty acid methyl ester (FAME) analysis showed a reduction in cell size, a change in cell shape and an increase in the degree of membrane fatty acid saturation. Transcriptome analysis showed decreased expression of genes involved in ribosomal protein biosynthesis, chromosomal replication, cell division and aromatic catabolism, increased expression of genes involved in regulation of gene expression and efflux systems, genetic translocations, and degradation of rRNA and fatty acids. Those phenotypic and transcriptomic changes were not observed after 4 h of starvation. Despite the starvation situation, the polycyclic aromatic hydrocarbon (PAH) catabolic activity was immediate upon exposure to phenanthrene. We conclude that a large fraction of cells maintain viability after an extended period of starvation apparently due to tuning the expression of a wide variety of cellular processes. Due to these survival attributes, bacteria of the genus Sphingomonas, like strain LH128, could be considered as suitable targets for use in remediation of nutrient-poor PAH-contaminated environments.

Members of the genus Sphingomonas are important catalysts for removal of polycyclic aromatic hydrocarbons (PAHs) in soil, but their activity can be affected by various stress factors. This study examines the physiological and genome-wide transcription response of the phenanthrene-degrading Sphingomonas sp. strain LH128 in biofilms to solute stress (invoked by 450 mM NaCl solution), either as an acute (4-h) or a chronic (3-day) exposure. The degree of membrane fatty acid saturation was increased as a response to chronic stress. Oxygen consumption in the biofilms and phenanthrene mineralization activities of biofilm cells were, however, not significantly affected after imposing either acute or chronic stress. This finding was in agreement with the transcriptomic data, since genes involved in PAH degradation were not differentially expressed in stressed conditions compared to nonstressed conditions. The transcriptomic data suggest that LH128 adapts to NaCl stress by (i) increasing the expression of genes coping with osmolytic and ionic stress such as biosynthesis of compatible solutes and regulation of ion homeostasis, (ii) increasing the expression of genes involved in general stress response, (iii) changing the expression of general and specific regulatory functions, and (iv) decreasing the expression of protein synthesis such as proteins involved in motility. Differences in gene expression between cells under acute and chronic stress suggest that LH128 goes through changes in genome-wide expression to fully adapt to NaCl stress, without significantly changing phenanthrene degrading activity.

Sphingomonas sp. strain Fr1 has recently been shown to protect Arabidopsis thaliana against the bacterial leaf pathogen Pseudomonas syringae DC3000. Here, we describe a forward genetic in planta screen to identify genes in Sphingomonas sp. Fr1 necessary for this effect. About 5,000 Sphingomonas sp. Fr1 mini-Tn5 mutants were assayed for a defect in plant protection against a luxCDABE-tagged P. syringae DC3000 derivative in a space-saving 24-well plate system. The bioluminescence of the pathogen was used as the indicator of pathogen proliferation and allowed for the identification of Sphingomonas sp. Fr1 mutants that had lost the ability to restrict pathogen growth before disease symptoms were visible. Potential candidates were validated using the same miniaturized experimental system. Of these mutants, 10 were confirmed as plant protection defective yet colonization competent. The mutants were subsequently evaluated in a previously described standard microbox system, and plants showed enhanced disease phenotypes after pathogen infection relative to those inoculated with the parental strain as a control. However, the disease severities were lower than those observed for control plants that were grown axenically prior to pathogen challenge, which suggests that several traits may contribute to plant protection. Transposon insertion sites of validated mutants with defects in plant protection were determined and mapped to 7 distinct genomic regions. In conclusion, the established screening protocol allowed us to identify mutations that affect plant protection, and it opens the possibility to uncover traits important for in planta microbe-microbe interactions.

Sphingomonas sp. strain PAMC 26617 has been isolated from an Arctic lichen Umbilicaria sp. on the Svalbard Islands. Here we present the draft genome sequence of this strain, which represents a valuable resource for understanding the symbiotic mechanisms between endosymbiotic bacteria and lichens surviving in extreme environments.

Reprogramming gene expression is an essential component of adaptation to changing environmental conditions. In bacteria, a widespread mechanism involves alternative sigma factors that redirect transcription toward specific regulons. The activity of sigma factors is often regulated through sequestration by cognate anti-sigma factors; however, for most systems, it is not known how the activity of the anti-sigma factor is controlled to release the sigma factor. Recently, the general stress response sigma factor in Alphaproteobacteria, σ(EcfG), was identified. σ(EcfG) is inactivated by the anti-sigma factor NepR, which is itself regulated by the response regulator PhyR. This key regulator sequesters NepR upon phosphorylation of its PhyR receiver domain via its σ(EcfG) sigma factor-like output domain (PhyR(SL)). To understand the molecular basis of the PhyR-mediated partner-switching mechanism, we solved the structure of the PhyR(SL)-NepR complex using NMR. The complex reveals an unprecedented anti-sigma factor binding mode: upon PhyR(SL) binding, NepR forms two helices that extend over the surface of the PhyR(SL) subdomains. Homology modeling and comparative analysis of NepR, PhyR(SL), and σ(EcfG) mutants indicate that NepR contacts both proteins with the same determinants, showing sigma factor mimicry at the atomic level. A lower density of hydrophobic interactions, together with the absence of specific polar contacts in the σ(EcfG)-NepR complex model, is consistent with the higher affinity of NepR for PhyR compared with σ(EcfG). Finally, by reconstituting the partner switch in vitro, we demonstrate that the difference in affinity of NepR for its partners is sufficient for the switch to occur.

A Gram-reaction-negative, oxidase- and catalase-positive, non-gliding motile bacteriun, designated DCY58(T), was isolated from a ginseng field in Pocheon Province, South Korea. Colonies of strain DCY58(T) were circular, 0.5-1.5 mm in diameter, yellow, and convex on an R2A agar plate after 2 days. The 16S rRNA gene sequence analysis revealed that strain DCY58(T) belongs to the family of the Sphingomonadaceae and is most closely related to the species of genus Sphingomonas with similarity levels of 95.7-96.4%. Strain DCY58(T) contained Q-10 as the predominant ubiquinone. The major cellular fatty acids included Summed Feature 8 (containing C(18:1ω)6c and/or C(18:1ω)7c), C(14:0) 2-OH and C(16:0). The DNA G+C content was 65.1 mol%. Furthermore, strain DCY58(T) differed from other related Sphingomonas species by a number of phenotypic characteristics. On the basis of the evidence presented in this study, strain DCY58(T) is described as a novel species of genus Sphingomonas, for which the name Sphingomonas ginsengisoli sp. nov., is proposed. The type strain is DCY58(T) (=KCTC 23762(T) =JCM 18016(T)).

Biofilms cause runnability problems in paper machines and are therefore controlled with biocides. Peracetic acid is usually effective in preventing bulky biofilms. This study investigated the microbiological status of a paper machine where low concentrations (≤ 15 ppm active ingredient) of peracetic acid had been used for several years. The paper machine contained a low amount of biofilms. Biofilm-forming bacteria from this environment were isolated and characterized by 16S rRNA gene sequencing, whole-cell fatty acid analysis, biochemical tests, and DNA fingerprinting. Seventy-five percent of the isolates were identified as members of the subclades Sphingomonas trueperi and S. aquatilis, and the others as species of the genera Burkholderia (B. cepacia complex), Methylobacterium, and Rhizobium. Although the isolation media were suitable for the common paper machine biofoulers Deinococcus, Meiothermus, and Pseudoxanthomonas, none of these were found, indicating that peracetic acid had prevented their growth. Spontaneous, irreversible loss of the ability to form biofilm was observed during subculturing of certain isolates of the subclade S. trueperi. The Sphingomonas isolates formed monoculture biofilms that tolerated peracetic acid at concentrations (10 ppm active ingredient) used for antifouling in paper machines. High pH and low conductivity of the process waters favored the peracetic acid tolerance of Sphingomonas sp. biofilms. This appears to be the first report on sphingomonads as biofilm formers in warm water using industries.

Diverse bacterial taxa live in association with plants without causing deleterious effects. Previous analyses of phyllosphere communities revealed the predominance of few bacterial genera on healthy dicotyl plants, provoking the question of whether these commensals play a particular role in plant protection. Here, we tested two of them, Methylobacterium and Sphingomonas, with respect to their ability to diminish disease symptom formation and the proliferation of the foliar plant pathogen Pseudomonas syringae pv. tomato DC3000 on Arabidopsis thaliana. Plants were grown under gnotobiotic conditions in the absence or presence of the potential antagonists and then challenged with the pathogen. No effect of Methylobacterium strains on disease development was observed. However, members of the genus Sphingomonas showed a striking plant-protective effect by suppressing disease symptoms and diminishing pathogen growth. A survey of different Sphingomonas strains revealed that most plant isolates protected A. thaliana plants from developing severe disease symptoms. This was not true for Sphingomonas strains isolated from air, dust, or water, even when they reached cell densities in the phyllosphere comparable to those of the plant isolates. This suggests that plant protection is common among plant-colonizing Sphingomonas spp. but is not a general trait conserved within the genus Sphingomonas. The carbon source profiling of representative isolates revealed differences between protecting and nonprotecting strains, suggesting that substrate competition plays a role in plant protection by Sphingomonas. However, other mechanisms cannot be excluded at this time. In conclusion, the ability to protect plants as shown here in a model system may be an unexplored, common trait of indigenous Sphingomonas spp. and may be of relevance under natural conditions.

The genes required for tetralin biodegradation by Sphingomonas macrogolitabida strain TFA are clustered in two divergent and closely linked operons. ThnR, a LysR-type regulator, activates transcription from each operon in response to tetralin. The regulatory thnR gene is co-transcribed with the catabolic genes thnC, thnA3 and thnA4, resulting in positive autoregulation. ThnR binds with different affinity to two primary binding sites, designated B and C, in the intervening region between the two operons and makes additional contact with secondary sites that extend towards the promoters. In addition, ThnR may interact with itself when bound to each site via the formation of a DNA loop, as evidenced by the distortion of the DNA between the primary binding sites and the elimination of the higher-order complexes following the introduction of a half-turn of the DNA helix between the primary binding sites. Transcription from each promoter is not fully independent since mutations in each binding site affected transcription from both promoters. Based on these results, we propose a model of transcription activation that involves the formation of a complex structure by interactions between ThnR molecules bound to distant binding sites and favours transcription from one promoter to the detriment of the other.

Coaggregation is hypothesized to enhance freshwater biofilm development. To investigate this hypothesis, the ability of the coaggregating bacterium Sphingomonas natatoria to form single- and dual-species biofilms was studied and compared to that of a naturally occurring spontaneous coaggregation-deficient variant. Attachment assays using metabolically inactive cells were performed using epifluorescence and confocal laser scanning microscopy. Under static and flowing conditions, coaggregating S. natatoria 2.1gfp cells adhered to glass surfaces to form diaphanous single-species biofilms. When glass surfaces were precoated with coaggregation partner Micrococcus luteus 2.13 cells, S. natatoria 2.1gfp cells formed densely packed dual-species biofilms. The addition of 80 mM galactosamine, which reverses coaggregation, mildly reduced adhesion to glass but inhibited the interaction and attachment to glass-surface-attached M. luteus 2.13 cells. As opposed to wild-type coaggregating cells, coaggregation-deficient S. natatoria 2.1COGgfp variant cells were retarded in colonizing glass and did not interact with glass-surface-attached M. luteus 2.13 cells. To determine if coaggregation enhances biofilm growth and expansion, viable coaggregating S. natatoria 2.1gfp cells or the coaggregation-deficient variant S. natatoria 2.1COGgfp cells were coinoculated in flow cells with viable M. luteus 2.13 cells and allowed to grow together for 96 h. Coaggregating S. natatoria 2.1gfp cells outcompeted M. luteus 2.13 cells, and 96-h biofilms were composed predominantly of S. natatoria 2.1gfp cells. Conversely, when coaggregation-deficient S. natatoria 2.1COGgfp cells were coinoculated with M. luteus 2.13 cells, the 96-h biofilm contained few coaggregation-deficient S. natatoria 2.1 cells. Thus, coaggregation promotes biofilm integration by facilitating attachment to partner species and likely contributes to the expansion of coaggregating S. natatoria 2.1 populations in dual-species biofilms through competitive interactions.

Chlorinated organophosphate ester (OPE)-degrading enrichment cultures were obtained using tris(2-chloroethyl) phosphate (TCEP) or tris(1,3-dichloro-2-propyl) phosphate (TDCPP) as the sole phosphorus source. In cultures with 46 environmental samples, significant TCEP and TDCPP degradation was observed in 10 and 3 cultures, respectively, and successive subcultivation markedly increased their degradation rates. 67E and 45D stable enrichment cultures obtained with TCEP and TDCPP, respectively, completely degraded 20 muM of the respective compounds within 6 h and also the other, although the degradation rate of TCEP by 45D was relatively slow. We confirmed chloride ion generation on degradation in both cases and the generation of 2-chloroethanol (2-CE) and 1,3-dichloro-2-propanol (1,3-DCP) as metabolites of TCEP and TDCPP, respectively. 67E and 45D also showed dehalogenation ability toward 2-CE and 1,3-DCP, respectively. Addition of inorganic phosphate did not significantly influence their ability to degrade the chlorinated OPEs but markedly increased their dehalogenation ability, which was maximum at 0.2 mM of inorganic phosphate and decreased at a higher concentration. Denaturing gradient gel electrophoresis analysis showed that dominant bacteria in 67E are related to Acidovorax spp. and Sphingomonas spp. and those in 45D are Acidovorax spp., Aquabacterium spp., and Sphingomonas spp. This analysis indicated the relationship of the Sphingomonas- and Acidovorax-related bacteria with the cleavage of the phosphoester bond and dehalogenation, respectively, in both cultures. This is the first report on bacterial enrichment cultures capable of degrading both TCEP and TDCPP.

Sphingomonas species A1 is a newly identified pit-forming bacterium that directly incorporates a macromolecule (alginate) into its cytoplasm through a pit-dependent transport system, which we termed a superchannel. A pit is a novel, high-dimensional organ acquired through the fluidity and reconstitution of cell surface molecules, including flagellin, and through cooperation with the transport machinery in the cells, which confers upon bacterial cells a more efficient way to secure and assimilate macromolecules. The analysis of the superchannel changes general ideas regarding the fluidity and function of the cell surface, evolution and origin of cell-surface organs, including flagella, transport, and assimilation systems of macromolecules, and the divergence and energetics of metabolism.

Sphingobium yanoikuyae B1 is able to degrade a range of polycyclic aromatic hydrocarbons (PAHs) and as a sphingomonad belongs to one of the dominant genera found in PAH-contaminated soils. We examined the ecological effect that soil inoculation with S. yanoikuyae B1 has on the native bacterial community in three different soils: aged PAH-contaminated soil from an industrial site, compost freshly contaminated with PAHs and un-contaminated compost. Survival of S. yanoikuyae B1 was dependent on the presence of PAHs, and the strain was unable to colonize un-contaminated compost. Inoculation with S. yanoikuyae B1 did not cause extensive changes in the native bacterial community of either soil, as assessed by denaturing gel electrophoresis, but its presence led to an increase in the population level of two other species in the aged contaminated soil community and appeared to have an antagonistic affect on several members of the contaminated compost community, indicating niche competition.

In the present study, water was circulated in a simulated dental unit water line with electrifying a small current. The morphology of the biofilm developed on inner surface of the water line and the number of heterotrophic bacteria were investigated to elucidate the effect of a low level electric current on the biofilms formation associated with bacteria reproduction. Destruction and malconformation of biofilms by electrification was observed using SEM, in addition to deformation and hypertrophy of the bacteria. By naked eye observation, small pieces, which were possibly exfoliated biofilms, were detected in electrified water. While an adherent, yellow gel was demonstrated on the inner surface of the water line without electrification. With electrification, the number of bacteria decreased during the first week, however the bacteria increased gradually after that. The number of bacteria without electrification was consistently greater than that with electrification and the difference was statistically significant (P<0.05). The predominant bacteria were identified as Sphingomonas paucimobilis. The excess chlorine levels decreased to a minimum value within one week. The small current appeared to have effects on biofilm formation of heterotrophic bacteria that resulted in enhanced chlorine sterilization of dental unit water. Thus, electrification has considerable potential for the extermination of bacterial biofilms in dental unit water lines.

Many chlorophenols tend to persist in the environment, and they may become public health hazards. Among chlorophenols, pentachlorophenol (PCP) is a priority pollutant that has been used widely as a general biocide in commercial wood treatment. Owing to the rapid industrial growth, serious soil and water pollutions by chlorophenols has been reported in Taiwan. In this study, 10 indigenous PCP-degrading bacterial strains were isolated from a PCP-degrading mixed culture, and the potential of both the pure and mixed cultures for PCP degradation compared. Moreover, the physiological characteristics and optimum growth conditions of the PCP-degrading bacteria were investigated. One of the isolated bacterial strains with good potential for PCP degradation was characterized and identified as Sphingomonas chlorophenolica by 16S rDNA gene analysis. The result of the optimum growth temperatures revealed that this organism was a mesophile. The optimum pH for PCP removal by S. chlorophenolica was between 6.9 and 7.6. Increase in concentration of PCP has a negative effect on the biodegradation potential of S. chlorophenolica and PCP concentration above 600 mg l(-1) was inhibitory to its growth. The results of this study indicate that this S. chlorophenolica strain has a better potential for PCP degradation compared to the enriched mixed culture. The physiological characterization of the isolates also indicates the possible application of this strain for bioremediation of sites contaminated with PCP.

The architecture of a Sphingomonas biofilm was studied during early phases of its formation, using strain L138, a gfp-tagged derivative of Sphingomonas sp. strain LB126, as a model organism and flow cells and confocal laser scanning microscopy as experimental tools. Spatial and temporal distribution of cells and exopolymer secretions (EPS) within the biofilm, development of microcolonies under flow conditions representing varied Reynolds numbers, and changes in diffusion length with reference to EPS production were studied by sequential sacrificing of biofilms grown in multichannel flow cells and by time-lapse confocal imaging. The area of biofilm in terms of microscopic images required to ensure representative sampling varied by an order of magnitude when area of cell coverage (2 x 10(5) microm(2)) or microcolony size (1 x 10(6) microm(2)) was the biofilm parameter under investigation. Hence, it is necessary to establish the inherent variability of any biofilm metric one is attempting to quantify. Sphingomonas sp. strain L138 biofilm architecture consisted of microcolonies and extensive water channels. Biomass and EPS distribution were maximal at 8 to 9 mum above the substratum, with a high void fraction near the substratum. Time-lapse confocal imaging and digital image analysis showed that growth of the microcolonies was not uniform: adjacently located colonies registered significant growth or no growth at all. Microcolonies in the biofilm had the ability to move across the attachment surface as a unit, irrespective of fluid flow direction, indicating that movement of microcolonies is an inherent property of the biofilm. Width of water channels decreased as EPS production increased, resulting in increased diffusion distances in the biofilm. Changing hydrodynamic conditions (Reynolds numbers of 0.07, 52, and 87) had no discernible influence on the characteristics of microcolonies (size, shape, or orientation with respect to flow) during the first 24 h of biofilm development. Inherent factors appear to have overriding influence, vis-a-vis environmental factors, on early stages of microcolony development under these laminar flow conditions.

The Sphingomonas genus hosts many interesting pollutant-degrading strains. Sphingomonas sp. EPA505 is the best studied polycyclic aromatic hydrocarbon (PAH)-degrading Sphingomonas strain. Based on 16S rRNA gene sequence analysis, Sphingomonas sp. strain EPA505 forms a separate branch in the Sphingomonas phylogenetic tree grouping exclusively PAH-degrading isolates. For specific PCR detection and monitoring of Sphingomonas sp. EPA505 and related strains in PAH-contaminated soils, a new 16S rRNA gene-based primer set was designed. The new primer set was shown to be highly selective for Sphingomonas sp. strain EPA505 as it only amplified DNA from strain EPA505 and not from other tested Sphingomonas strains or soil bacteria not belonging to the Sphingomonas genus. Using DNA extracts of a variety of inoculated PAH-contaminated soils, the primer pair was able to detect EPA505 in concentrations as low as 10(2) cells per gram of soil. Applying the new primer set, 16S rRNA gene fragments which were 99-100% similar to the corresponding gene of strain EPA505 were amplified from four of five PAH-contaminated soils. On the other hand, no PCR products were obtained from any of five tested uncontaminated soils. The preferential presence of EPA505 related Sphingomonas strains in PAH-contaminated soils with very different contamination profiles and different origin suggests an important role of this type of Sphingomonas in the natural Sphingomonas community colonizing PAH-contaminated sites.

Bacteria of the genus Sphingomonas are environmental organisms that have recently been implicated in a variety of community-acquired and nosocomial infections. During studies on bacteria-cell interactions, we incurred a microorganism contaminating our HeLa cell culture, possibly from water utilized for reagent preparation; this bacterium appeared to tightly adhere to cell monolayers and to survive, with only limited growth rate, which did not seem to alter cells as far as shape, growth rate or survival were concerned. The contaminating organism was isolated and partially characterized by morphological, genetic, and biochemical assays. Mechanisms of cell interaction and entry into epithelial cells were investigated by electron microscopy, immunofluorescence, and biochemical inhibitors. Morphological and biochemical features indicated that the microorganism belonged to the genus Sphingomonas. Electron microscopy showed that contact between the Sphingomonas bacterium and epithelial cells leads to a dramatic alteration of the cell surface, with formation of numerous microvillar extensions plus membrane ruffling. Confocal microscopy and the use of inhibitors showed that actin microfilaments were involved during attachment and entry into HeLa cells. Macropinosome formation and an inhibitory effect by amiloride indicate that internalization occurs in part via a macropinocytosis mechanism. Moreover, cholesterol distribution at the site of bacterial binding suggests that Sphingomonas bacteria could use the lipid rafts as initial binding sites.

The application of Fourier Transform near infrared spectroscopy (FT-NIRS) to near real-time monitoring of polysaccharide and biomass concentration was investigated using a gellan-producing strain of Sphingomonas paucimobilis grown in a stirred tank reactor. Successful models for both biomass and gellan were constructed despite the physichochemical complexity of the viscous process fluid. Modelling of biomass proved more challenging than for gellan, partly because of the low range of biomass concentration but a model with a good correlation coefficient (0.94) was formulated based on second derivative spectra. The gellan model was highly satisfactory, with an excellent correlation coefficient (0.98), again based on second derivative spectra. No sample pre-treatment was required and all spectral scanning was carried out on whole broth. Additionally, both models should be robust in practice since both were formulated using low numbers of factors. Thus, the near real time simultaneous monitoring of gellan and biomass in this highly complex matrix using FT-NIRS potentially opens the way to greatly improved process control strategies.

The oceans of the world are nutrient-limited environments that support a dynamic diversity of microbial life. Heterotrophic prokaryotes proliferate in oligotrophic regions and affect nutrient transformation and remineralization thereby impacting directly on the all marine biota. An important challenge in studying the microbial ecology of oligotrophic environments has been the isolation of ecologically important species. This goal has been recognized not only for its relevance in defining the dynamics of community composition, but for enabling physiological studies of competitive species and inferring their impact on the microbial food web. This review describes the successful isolation attempts of the ultramicrobacterium, Sphingopyxis alaskensis (formerly described as Sphingomonas alaskensis) using extinction dilution culturing methods. It then provides a comprehensive perspective of the unique physiological and genetic properties that have been identified that distinguish it from typical copiotrophic species. These properties are described through studies of the growth phase and growth rate control of macromolecular synthesis, stress resistance and global gene expression (proteomics). We also discuss the importance of integrating ecological and physiological approaches for studying microorganisms in marine environments.

The chlorophenol degradation pathway in Sphingobium chlorophenolicum is initiated by the pcpB gene product, pentachlorophenol-4-monooxygenase. The distribution of the gene was studied in a phylogenetically diverse group of polychlorophenol-degrading bacteria isolated from contaminated groundwater in Kärkölä, Finland. All the sphingomonads isolated were shown to share pcpB gene homologs with 98.9 to 100% sequence identity. The gene product was expressed when the strains were induced by 2,3,4,6-tetrachlorophenol. A comparative analysis of the 16S rDNA and pcpB gene trees suggested that a recent horizontal transfer of the pcpB gene was involved in the evolution of the catabolic pathway in the Kärkölä sphingomonads. The full-length Kärkölä pcpB gene allele had approximately 70% identity with the three pcpB genes previously sequenced from sphingomonads. It was very closely related to the environmental clones obtained from chlorophenol-enriched soil samples (M. Beaulieu, V. Becaert, L. Deschenes, and R. Villemur, Microbiol. Ecol. 40:345-355, 2000). The gene was not present in polychlorophenol-degrading nonsphingomonads isolated from the Kärkölä source.

Fluorinated compounds are known to be more resistant to microbial degradation than other halogenated chemicals. A microbial consortium capable of aerobic biodegradation of fluorobenzene (FB) as the sole source of carbon and energy was isolated by selective enrichment from sediments collected in a drain near an industrial site. A combination of three microbial strains recovered from the enriched consortium was shown to be necessary for complete FB mineralization. Two of the strains (F1 and F3) were classified by 16S rRNA analysis as belonging to the Sphingobacterium/Flavobacterium group, while the third (F4) falls in the beta-Proteobacteria group, clustering with Alcaligenes species. Strain F4 was consistently found in the liquid cultures in a much greater proportion than strains F1 and F3 (86:8:6 for F4, F1, and F3, respectively). Stoichiometric release of fluoride ions was measured in batch and fed-batch cultures. In batch cultures, the consortium was able to use FB up to concentrations of 400 mg liter(-1) and was able to utilize a range of other organic compounds, including 4-fluorophenol and 4-fluorobenzoate. To our knowledge this is the first time biodegradation of FB as a sole carbon source has been reported.

Sphingomonas sp. strain Ant 17 was isolated from fuel-contaminated soil collected at Scott Base, Ross Island, Antarctica. We anticipated that Ant 17 would be a good model organism for studying cold climate bioremediation, and therefore determined its biodegradation capabilities and tolerance of potentially growth-limiting environmental conditions. Sphingomonas sp. Ant 17 degrades the aromatic fraction of several different crude oils, jet fuel, and diesel fuel at low temperatures and without nutrient amendment. It utilizes or transforms a broad range of pure aromatic substrates, including hydrocarbons, heterocycles, and aromatic acids and alcohols. Ant 17 grows at temperatures of 1 degree C to 35 degrees C and mineralizes radiolabeled phenanthrene over a range of more than 24 degrees C. This psychrotolerant isolate appears to utilize hydrocarbons more efficiently at low temperatures than would be predicted by mesophilic enzyme kinetics. The optimum pH for growth was 6.4 at 22 degrees C, with extended lag phases observed in more alkaline media. However, there was less effect of pH on lag phase at lower temperatures. Ant 17 displayed greater tolerance to UV irradiation and freeze-thaw cycles than the hydrocarbon-degrading isolate Sphingomonas sp. WPO-1, which may reflect adaptation to its Antarctic soil environment. However, it was more sensitive than expected to desiccation and to low concentrations of NaCl and CaCl(2). Ant 17 was phenotypically stable and lacked detectable plasmids, suggesting a chromosomal location for genes encoding aromatic degradation enzymes. Its broad aromatic substrate range and tolerance of low and fluctuating temperature and low nutrients make Sphingomonas sp. Ant 17 a valuable microbe for examining fuel spill bioremediation in cold soils.

Free and agarose-encapsulated pentachlorophenol (PCP)-degrading Sphingomonas sp. isolates UG25 and UG30 were compared to Sphingomonas chlorophenolica ATCC 39723 with respect to the ability to degrade PCP. Pretreatment of the UG25 and UG30 strains with 50 microg of PCP per ml enabled the cells to subsequently degrade higher levels of this environmental pollutant. Similar treatment of ATCC 39723 cells had no effect on the level of PCP degraded by this strain. Phosphorus-31 nuclear magnetic resonance spectra of agarose-immobilized strains UG25 and UG30 grown in the absence of PCP showed that there was marked deenergization of the cells upon exposure to a nonlethal concentration of PCP (120 microg/ml). For example, no transmembrane pH gradient was observed, and the ATP levels were lower than the levels obtained in the absence of PCP. The transmembrane pH gradient and ATP levels were restored once the immobilized cells had almost completely degraded the PCP in the perfusion medium. PCP-pretreated cells, on the other hand, maintained their transmembrane pH gradient and ATP levels even in the presence of high levels of PCP. The ability of PCP-pretreated strain UG25 and UG30 cells to remain energized in the presence of PCP was shown to correlate with an altered membrane phospholipid profile; these cells had a higher concentration of cardiolipin than cells cultured in the absence of PCP. Strain ATCC 39723, which did not degrade higher levels of PCP after PCP pretreatment, did not show this response.

Two different procedures were compared to isolate polycyclic aromatic hydrocarbon (PAH)-utilizing bacteria from PAH-contaminated soil and sludge samples, i.e., (i) shaken enrichment cultures in liquid mineral medium in which PAHs were supplied as crystals and (ii) a new method in which PAH degraders were enriched on and recovered from hydrophobic membranes containing sorbed PAHs. Both techniques were successful, but selected from the same source different bacterial strains able to grow on PAHs as the sole source of carbon and energy. The liquid enrichment mainly selected for Sphingomonas spp., whereas the membrane method exclusively led to the selection of Mycobacterium spp. Furthermore, in separate membrane enrichment set-ups with different membrane types, three repetitive extragenic palindromic PCR-related Mycobacterium strains were recovered. The new Mycobacterium isolates were strongly hydrophobic and displayed the capacity to adhere strongly to different surfaces. One strain, Mycobacterium sp. LB501T, displayed an unusual combination of high adhesion efficiency and an extremely high negative charge. This strain may represent a new bacterial species as suggested by 16S rRNA gene sequence analysis. These results indicate that the provision of hydrophobic sorbents containing sorbed PAHs in the enrichment procedure discriminated in favor of certain bacterial characteristics. The new isolation method is appropriate to select for adherent PAH-degrading bacteria, which might be useful to biodegrade sorbed PAHs in soils and sludge.

Sphingomonas sp. strain RB2256 is representative of the ultramicrobacteria that proliferate in oligotrophic marine waters. While this class of bacteria is well adapted for growth with low concentrations of nutrients, their ability to respond to complete nutrient deprivation has not previously been investigated. In this study, we examined two-dimensional protein profiles for logarithmic and stationary-phase cells and found that protein spot intensity was regulated by up to 70-fold. A total of 72 and 177 spots showed increased or decreased intensity, respectively, by at least twofold during starvation. The large number of protein spots (1,500) relative to the small genome size (ca. 1.5 Mb) indicates that gene expression may involve co- and posttranslational modifications of proteins. Rates of protein and RNA synthesis were examined throughout the growth phase and up to 7 days of starvation and revealed that synthesis was highly regulated. Rates of protein synthesis and cellular protein content were compared to ribosome content, demonstrating that ribosome synthesis was not directly linked to protein synthesis and that the function of ribosomes may not be limited to translation. By comparing the genetic capacity and physiological responses to starvation of RB2256 to those of the copiotrophic marine bacterium Vibrio angustum S14 (J. Ostling, L. Holmquist, and S. Kjelleberg, J. Bacteriol. 178:4901-4908, 1996), the characteristics of a distinct starvation response were defined for Sphingomonas strain RB2256. The capacity of this ultramicrobacterium to respond to starvation is discussed in terms of the ecological relevance of complete nutrient deprivation in an oligotrophic marine environment. These studies provide the first evidence that marine oligotrophic ultramicrobacteria may be expected to include a starvation response and the capacity for a high degree of gene regulation.