Sphingomonas Strains Research Articles

Phylogeny, phenotypic characteristics and pathogenicity of Sphingomonas sp. and Erwinia persicina as bacterial causal agents of lettuce diseases in southwest of Iran

Bacterial-like symptoms, such as chlorosis, necrosis, and rot, have recently been observed on lettuce leaves in Khuzestan province, located in southwest Iran. To identify and specify the bacterial causal agents responsible for this outbreak, bacterial strains were purified from samples collected from lettuce fields in Dezful, Shushtar, and Shousha Daniel regions. The pathogenic bacterial strains were identified through hypersensitivity response (HR) and pathogenicity tests. By comparing the sequences of 16S rDNA of pathogenic strains, and phylogeny analysis, it was clarified that the pathogenic strains belonged to Erwinia persicina and genus Sphingomonas. Phenotypic study showed that Sphingomonas strains were resistant to streptomycin and gentamicin, and produced yellow pigment, and E. persicina strains fermented glucose, and produced pink soluble pigment. Additionally, the E. persicina strains were sensitive to streptomycin, gentamicin, levofloxacin, and erythromycin. The pathogenicity test showed that E. persicina strains induced necrosis, yellowing, and rot in the leaves of Romaine type of lettuce. Similarly, Sphingomonas strains caused necrosis, accompanied by a large yellow halo, in the leaves of Romaine type. Furthermore, the E. persicina and Sphingomonas strains induced necrosis of the veins of Romaine type, as well as brown spotting in the leaves of Iceberg type. In addition, the study of the host range of E. persicina and Sphingomonas strains showed that they infect the leaves of different crops (including cucumber, tomato, pepper, celery, and eggplant) and mallow weed. These crops are grown in rotation or simultaneously with lettuce in Khuzestan province. The diverse host range of mentioned pathogens facilitates their survival during the year in this area. This is the first report of the genus Sphingomonas in lettuce and the first report of E. persicina from Iran.

Pheno- and Genotyping of Three Novel Bacteriophage Genera That Target a Wheat Phyllosphere Sphingomonas Genus.

Bacteriophages are viral agents that infect and replicate within bacterial cells. Despite the increasing importance of phage ecology, environmental phages-particularly those targeting phyllosphere-associated bacteria-remain underexplored, and current genomic databases lack high-quality phage genome sequences linked to specific environmentally important bacteria, such as the ubiquitous sphingomonads. Here, we isolated three novel phages from a Danish wastewater treatment facility. Notably, these phages are among the first discovered to target and regulate a Sphingomonas genus within the wheat phyllosphere microbiome. Two of the phages displayed a non-prolate Siphovirus morphotype and demonstrated a narrow host range when tested against additional Sphingomonas strains. Intergenomic studies revealed limited nucleotide sequence similarity within the isolated phage genomes and to publicly available metagenome data of their closest relatives. Particularly intriguing was the limited homology observed between the DNA polymerase encoding genes of the isolated phages and their closest relatives. Based on these findings, we propose three newly identified genera of viruses: Longusvirus carli, Vexovirus birtae, and Molestusvirus kimi, following the latest ICTV binomial nomenclature for virus species. These results contribute to our current understanding of phage genetic diversity in natural environments and hold promising implications for phage applications in phyllosphere microbiome manipulation strategies.

Variation in Sphingomonas traits across habitats and phylogenetic clades.

Whether microbes show habitat preferences is a fundamental question in microbial ecology. If different microbial lineages have distinct traits, those lineages may occur more frequently in habitats where their traits are advantageous. Sphingomonas is an ideal bacterial clade in which to investigate how habitat preference relates to traits because these bacteria inhabit diverse environments and hosts. Here we downloaded 440 publicly available Sphingomonas genomes, assigned them to habitats based on isolation source, and examined their phylogenetic relationships. We sought to address whether: (1) there is a relationship between Sphingomonas habitat and phylogeny, and (2) whether there is a phylogenetic correlation between key, genome-based traits and habitat preference. We hypothesized that Sphingomonas strains from similar habitats would cluster together in phylogenetic clades, and key traits that improve fitness in specific environments should correlate with habitat. Genome-based traits were categorized into the Y-A-S trait-based framework for high growth yield, resource acquisition, and stress tolerance. We selected 252 high quality genomes and constructed a phylogenetic tree with 12 well-defined clades based on an alignment of 404 core genes. Sphingomonas strains from the same habitat clustered together within the same clades, and strains within clades shared similar clusters of accessory genes. Additionally, key genome-based trait frequencies varied across habitats. We conclude that Sphingomonas gene content reflects habitat preference. This knowledge of how environment and host relate to phylogeny may also help with future functional predictions about Sphingomonas and facilitate applications in bioremediation.

Biopolymers Produced by Sphingomonas Strains and Their Potential Applications in Petroleum Production.

The genus Sphingomonas was established by Yabuuchi et al. in 1990, and has attracted much attention in recent years due to its unique ability to degrade environmental pollutants. Some Sphingomonas species can secrete high-molecular-weight extracellular polymers called sphingans, most of which are acidic heteropolysaccharides. Typical sphingans include welan gum, gellan gum, and diutan gum. Most sphingans have a typical, conserved main chain structure, and differences of side chain groups lead to different rheological characteristics, such as shear thinning, temperature or salt resistance, and viscoelasticity. In petroleum production applications, sphingans, and their structurally modified derivatives can replace partially hydrolyzed polyacrylamide (HPAM) for enhanced oil recovery (EOR) in high-temperature and high-salt reservoirs, while also being able to replace guar gum as a fracturing fluid thickener. This paper focuses on the applications of sphingans and their derivatives in EOR.

Phenanthrene-degrading Sphingobium xenophagum are widely distributed in the western Pacific Ocean.

Six phenanthrene-degrading bacteria were isolated from surface seawater sampled from the western Pacific Ocean. They were identified as Sphingobium xenophagum (formerly Sphingomonas xenophaga) based on morphological, biochemical, and chemical characteristics, and 16S rRNA sequences. The salinity ranges for the growth of these isolates were broader than those of the seven reported Sphingomonas strains isolated from soil, and the optimum NaCl concentration in the growth medium was higher than that for soil sphingomonads. These isolates also exhibited higher phenanthrene-degrading activity under briny conditions than that of a phenanthrene-degrading Sphingomonas strain isolated from soil. A DNA fragment carrying nah genes, which are encoded on the naphthalene-catabolic plasmid NAH of Pseudomonas putida PpG7, hybridised less strongly with the total DNA of all isolates. Certain genes involved in phenanthrene degradation were also preliminarily characterised in all isolates. This is the first demonstration that S. xenophagum strains, which are able to degrade phenanthrene, are widely distributed in marine environments, and that the growth and phenanthrene-degrading activity of these strains are adapted to briny conditions. The results also suggest that genes for phenanthrene degradation, which are dissimilar to nah genes, were also ubiquitously distributed in marine strains.

Genomic Characterization of Potential Plant Growth-Promoting Features of Sphingomonas Strains Isolated from the International Space Station.

ABSTRACTIn 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.

A strategy for the synthesis of low-molecular-weight welan gum by eliminating capsule form of Sphingomonas strains

Welan gum is widely used in food, concrete additives, and oil recovery. Here we changed the capsule form of Sphingomonas strains by knocked out the sortase gene (srtW). The obtained welan gum was mainly composed of mannose, glucose, rhamnose, and glucuronic acid at a molar ratio of 4.0:5.8:1.6:1, respectively. Meanwhile, the molecular weight of welan gum decreased sharply (about 68 kDa). Moreover, the low molecular weight (LMW) welan gum was characterized by FT-IR and NMR spectroscopy. The rheological results revealed that the LMW welan gum solution is a pseudoplastic fluid with a lower apparent viscosity. Furthermore, the oscillation test illustrated stable dynamic viscoelasticity within the temperature range of 5–68 °C and frequency range of 0.01–15 rad/s. To the best of our knowledge, this is the first report of LMW welan gum production and characterization. These results provide references for LMW welan gum applications, and likely applicable for other biopolymers production.

Batch-Learning Self-Organizing Map Identifies Horizontal Gene Transfer Candidates and Their Origins in Entire Genomes.

Horizontal gene transfer (HGT) has been widely suggested to play a critical role in the environmental adaptation of microbes; however, the number and origin of the genes in microbial genomes obtained through HGT remain unknown as the frequency of detected HGT events is generally underestimated, particularly in the absence of information on donor sequences. As an alternative to phylogeny-based methods that rely on sequence alignments, we have developed an alignment-free clustering method on the basis of an unsupervised neural network “Batch-Learning Self-Organizing Map (BLSOM)” in which sequence fragments are clustered based solely on oligonucleotide similarity without taxonomical information, to detect HGT candidates and their origin in entire genomes. By mapping the microbial genomic sequences on large-scale BLSOMs constructed with nearly all prokaryotic genomes, HGT candidates can be identified, and their origin assigned comprehensively, even for microbial genomes that exhibit high novelty. By focusing on two types of Alphaproteobacteria, specifically psychrotolerant Sphingomonas strains from an Antarctic lake, we detected HGT candidates using BLSOM and found higher proportions of HGT candidates from organisms belonging to Betaproteobacteria in the genomes of these two Antarctic strains compared with those of continental strains. Further, an origin difference was noted in the HGT candidates found in the two Antarctic strains. Although their origins were highly diversified, gene functions related to the cell wall or membrane biogenesis were shared among the HGT candidates. Moreover, analyses of amino acid frequency suggested that housekeeping genes and some HGT candidates of the Antarctic strains exhibited different characteristics to other continental strains. Lys, Ser, Thr, and Val were the amino acids found to be increased in the Antarctic strains, whereas Ala, Arg, Glu, and Leu were decreased. Our findings strongly suggest a low-temperature adaptation process for microbes that may have arisen convergently as an independent evolutionary strategy in each Antarctic strain. Hence, BLSOM analysis could serve as a powerful tool in not only detecting HGT candidates and their origins in entire genomes, but also in providing novel perspectives into the environmental adaptations of microbes.

Draft Genome Sequences of Sphingomonas Species Associated with the International Space Station.

The draft genome sequences of three Sphingomonas strains isolated from the International Space Station (ISS) were assembled. These genomic sequences will help in understanding the influence of microgravity conditions on their potential bioactive compound production and other important characteristics compared to their Earth counterparts.

Antagonistic activity of combined bacteria strains against southern blight pathogen of Dendrobium officinale

AimThe aim of the study was to determine the in vitro and in vivo antagonistic potential of combined bacteria strains against Sclerotium rolfsii inducing southern blight on Dendrobium officinale. Methods and ResultsAntagonistic or growth-promoting bacteria such as Bacillus, Pseudomonas and Sphingomonas strains isolated from D. officinale and other medicinal herbs were tested for antagonistic activity against S. rolfsii, individually or combined. The results showed that compared to single strains, mixed strains effectively reduced the growth and infection of S. rolfsii, with an elevated inhibition efficacy (75–100%). The bacterial cell-free supernatant manifested a minimal antifungal activity on biocontrol tests in vitro, indicating that some antibiotics played a role in defending against the pathogen. Using Polymerase Chain Reaction (PCR), some antibiotic-encoding genes (Surfactin, Phenazine, and Iturin A) were detected in some strains. Other biocontrol mechanisms might include ecological niche colonization, enhancing the growth and resistance of the plant, as well as siderophore secretion. ConclusionsThe accumulation and combination of different antifungal mechanisms and activities of mixed strains make the strategy of suppressing the infection of S. rolfsii more effective.

Comprehensive Genome Analysis on the Novel Species Sphingomonas panacis DCY99T Reveals Insights into Iron Tolerance of Ginseng.

Plant growth-promoting rhizobacteria play vital roles not only in plant growth, but also in reducing biotic/abiotic stress. Sphingomonas panacis DCY99T 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 DCY99T with Sphingomonas sp. LK11 revealed plant growth-promoting potential of S. panacis DCY99T through indole acetic acid production, phosphate solubilizing, and antifungal abilities. Detailed genomic analysis has shown that S. panacis DCY99T contain various heavy metals resistance genes in its genome and the plasmid. Functional analysis with Sphingomonas paucimobilis EPA505 predicted that S. panacis DCY99T possess genes for degradation of polyaromatic hydrocarbon and phenolic compounds in rusty-ginseng root. Interestingly, when primed ginseng with S. panacis DCY99T during high concentration of iron exposure, iron stress of ginseng was suppressed. In order to detect S. panacis DCY99T in soil, biomarker was designed using spt gene. This study brings new insights into the role of S. panacis DCY99T as a microbial inoculant to protect ginseng plants against rusty root disease.

Endophytes and Epiphytes From the Grapevine Leaf Microbiome as Potential Biocontrol Agents Against Phytopathogens.

Plants harbor diverse microbial communities that colonize both below-ground and above-ground organs. Some bacterial members of these rhizosphere and phyllosphere microbial communities have been shown to contribute to plant defenses against pathogens. In this study, we characterize the pathogen-inhibiting potential of 78 bacterial isolates retrieved from endophytic and epiphytic communities living in the leaves of three grapevine cultivars. We selected two economically relevant pathogens, the fungus Botrytis cinerea causing gray mold and the oomycete Phytophthora infestans, which we used as a surrogate for Plasmopara viticola causing downy mildew. Our results showed that epiphytic isolates were phylogenetically more diverse than endophytic isolates, the latter mostly consisting of Bacillus and Staphylococcus strains, but that mycelial inhibition of both pathogens through bacterial diffusible metabolites was more widespread among endophytes than among epiphytes. Six closely related Bacillus strains induced strong inhibition (>60%) of Botrytis cinerea mycelial growth. Among these, five led to significant perturbation in spore germination, ranging from full inhibition to reduction in germination rate and germ tube length. Different types of spore developmental anomalies were observed for different strains, suggesting multiple active compounds with different modes of action on this pathogen. Compared with B. cinerea, the oomycete P. infestans was inhibited in its mycelial growth by a higher number and more diverse group of isolates, including many Bacillus but also Variovorax, Pantoea, Staphylococcus, Herbaspirillum, or Sphingomonas strains. Beyond mycelial growth, both zoospore and sporangia germination were strongly perturbed upon exposure to cells or cell-free filtrates of selected isolates. Moreover, three strains (all epiphytes) inhibited the pathogen’s growth via the emission of volatile compounds. The comparison of the volatile profiles of two of these active strains with those of two phylogenetically closely related, inactive strains led to the identification of molecules possibly involved in the observed volatile-mediated pathogen growth inhibition, including trimethylpyrazine, dihydrochalcone, and L-dihydroxanthurenic acid. This work demonstrates that grapevine leaves are a rich source of bacterial antagonists with strong inhibition potential against two pathogens of high economical relevance. It further suggests that combining diffusible metabolite-secreting endophytes with volatile-emitting epiphytes might be a promising multi-layer strategy for biological control of above-ground pathogens.

Complete genome sequence of Sphingomonas paucimobilis AIMST S2, a xenobiotic-degrading bacterium

Complete genomes of xenobiotic-degrading microorganisms provide valuable resources for researchers to understand molecular mechanisms involved in bioremediation. Despite the well-known ability of Sphingomonas paucimobilis to degrade persistent xenobiotic compounds, a complete genome sequencing is lacking for this organism. In line with this, we report the first complete genome sequence of Sphingomonas paucimobilis (strain AIMST S2), an organophosphate and hydrocarbon-degrading bacterium isolated from oil-polluted soil at Kedah, Malaysia. The genome was derived from a hybrid assembly of short and long reads generated by Illumina HiSeq and MinION, respectively. The assembly resulted in a single contig of 4,005,505 bases which consisted of 3,612 CDS and 56 tRNAs. An array of genes involved in xenobiotic degradation and plant-growth promoters were identified, suggesting its’ potential role as an effective microorganism in bioremediation and agriculture. Having reported the first complete genome of the species, this study will serve as a stepping stone for comparative genome analysis of Sphingomonas strains and other xenobiotic-degrading microorganisms as well as gene expression studies in organophosphate biodegradation.

Metagenomic Characterization Reveals Pronounced Seasonality in the Diversity and Structure of the Phyllosphere Bacterial Community in a Mediterranean Ecosystem.

We explore how the phyllosphere microbial community responds to a very seasonal environment such as the Mediterranean. For this, we studied the epiphytic bacterial community of a Mediterranean ecosystem in summer and winter, expecting to detect seasonal differences at their maximum. With high-throughput sequencing (HTS), we detected the operational taxonomic units (OTUs) present in the phyllosphere and also in the surrounding air. The epiphytic community is approximately five orders of magnitude denser than the airborne one and is made almost exclusively by habitat specialists. The two communities differ considerably but Proteobacteria and Actinobacteria are dominant in both. Of the five most abundant phyllosphere OTUs, two were closely related to Sphingomonas strains, one to Methylobacterium and the other two to Rhizobiales and Burkholderiales. We found the epiphytic community to become much richer, more distinct, even and diverse, denser and more connected in summer. In contrast, there was no difference in the level of bacterial colonization of the phyllosphere between the two seasons, although there were seasonal differences for individual taxonomic groups: Firmicutes, Gemmatimonadetes and Chlroroflexi had a higher participation in summer, whereas the major Proteobacteria classes presented reverse patterns, with Betaproteobacteria increasing in summer at the expense of the prominent Alphaproteobacteria.

An Effect of Salt Concentration and Inoculum Size on Poly(Vinyl Alcohol) Utilization by Two Sphingomonas Strains

Due to its widespread use and water solubility, poly(vinyl alcohol) (PVA) has the potential to find its way into various water or soil ecosystems. Despite the fact that many bacterial species with the capacity of utilizing PVA have been found and described, the influences of some environmental factors on their capabilities to biodegrade PVA have not been adequately studied. Therefore, study was made of the effects of two environmental factors on PVA degradation exhibited by two Sphingomonas strains. Both strains originated from common wastewater treatment plants, and proved to be considerably sensitive to increased inorganic salt concentrations; in brief, 13.3 mmol/l either of phosphate or chloride ions significantly delayed the degradation process or inhibited it entirely. In contrast to such halosensitivity, both strains were able to rapidly utilize PVA under suitable conditions, even when low inoculum sizes were applied. Initial cell densities, ranging from 100 to 107 cells/ml, were used in two series of degradation trials and PVA degradation occurred in all cases; merely delays extending over several days in the degradation process were noted when inoculum sizes of 100–103 cells/ml were applied.

Production, Characterization and Bioemulsifying Activity of an Exopolysaccharide Produced by Sphingomonas sp. Isolated from Freshwater

This study aimed to evaluate the emulsion stability of solutions containing exopolysaccharide and culture medium of a Sphingomonas sp. strain with various hydrophobic compounds. The exopolysaccharide characterized belongs to a sphingan group, however, not being a gellan gum as produced by certain Sphingomonas strains. In general, the emulsifying indexes found in this study were above 70% for gasoline, hexane, kerosene and used frying oil. Nonetheless, the best results were achieved in kerosene solutions, which showed an index of 80% after 24 h, remaining stable for more than 168 h in combinations with various EPS concentrations. Interestingly, diesel oil best results were singly achieved in solution pH of 11, showing an index of around 65%. Furthermore, hexane obtained an index of 100% after 24 h when culture medium was used. Thus, these findings highlight the use of EPS as a potential bioemulsifier agent to enhance hydrocarbon degradation and emulsification effects in environmental biotechnology.

Sphingomonas from petroleum-contaminated soils in Shenfu, China and their PAHs degradation abilities.

Members of the Sphingomonas genus are often isolated from petroleum-contaminated soils due to their unique abilities to degrade polycyclic aromatic hydrocarbons (PAHs), which are important for in situ bioremediation. In this study, a combined phenotypic and genotypic approach using streptomycin-containing medium and Sphingomonas-specific PCR was developed to isolate and identify culturable Sphingomonas strains present in petroleum-contaminated soils in the Shenfu wastewater irrigation zone. Of the 15 soil samples examined, 12 soils yielded yellow streptomycin-resistant colonies. The largest number of yellow colony-forming units (CFUs) could reach 105 CFUs g−1 soil. The number of yellow CFUs had a significant positive correlation (p < 0.05) with the ratio of PAHs to total petroleum hydrocarbons (TPH), indicating that Sphingomonas may play a key role in degrading the PAH fraction of the petroleum contaminants at this site. Sixty yellow colonies were selected randomly and analyzed by colony PCR using Sphingomonas-specific primers, out of which 48 isolates had PCR-positive signals. The 48 positive amplicons generated 8 distinct restriction fragment length polymorphism (RFLP) patterns, and 7 out of 8 phylotypes were identified as Sphingomonas by 16S rRNA gene sequencing of the representative strains. Within these 7 Sphingomonas strains, 6 strains were capable of using fluorene as the sole carbon source, while 2 strains were phenanthrene-degrading Sphingomonas. To the best of our knowledge, this is the first report to evaluate the relationship between PAHs contamination levels and culturable Sphingomonas in environmental samples.

Dendrobium nobile Lindl. seed germination in co-cultures with diverse associated bacteria

The conservation of orchids is challenging due to their strong biotic relations and tiny seeds, requiring mycorrhiza for germination. This is aggravated when tropical plants are maintained in artificial conditions of greenhouses. We aimed to select the plant growth promoting rhizobacteria (PGPR) for orchid seed germination, to study plant–microbial interactions, and to determine whether there is any specificity between two species of Dendrobium plants in choosing bacterial partners. By the isolation of rhizoplane and endophytic rhizobacteria from Dendrobium moschatum roots, the known PGPR (Azospirillum, Enterobacter, Streptomyces) and less popular (Roseomonas, Agrococcus) strains were tested for the production of biologically active auxin. The bacterization of another orchid, D. noblie, with several newly selected strains and previously isolated ones (Mycobacterium sp., Bacillus pumilus) revealed that the orchids did not express evident specificity in relations with favorable bacteria, but refused to establish associations with Streptomyces and Azospirillum. Endophytic Agrococcus and Sphingomonas strains showed significant promotion of orchid germination. Mycobacterium and B. pumilus were also stable in their positive influence on the acceleration of D. noblie seed development. The active colonization of the seed surface and the inner tissues by associated bacteria was observed under electron microscopy. The analysis of orchid–bacteria relations was made. Altogether, the data shows that selection provides a good strategy for choosing the active strains for orchid seeds’ bacterization, since not all known PGPR are useful and successful in building associative frameworks with orchid seeds. The stable activity of the strains guarantees their long-term and effective application in orchid in vitro biotechnology.

Development of genus‐specific primers for better understanding the diversity and population structure of Sphingomonas in soils

Genus Sphingomonas has received increasing attentions due to its somewhat unique metabolic versatilities in the contaminated environment. However, due to the lack of genus-specific primers, the ecological significance of Sphingomonas in polluted soils has been rarely documented by 16S rDNA finger-printing methods. In this study, three genus-specific primer sets targeted at the 16S rRNA gene of Sphingomonas were developed and their specificities were tested with four contaminated soils from Shenfu petroleum-wastewater irrigation zone by constructing clone libraries, amplified ribosomal DNA restriction analysis (ARDRA) and sequencing the represented ARDRA patterns. Meanwhile, the newly designed primer sets and a previously reported primer set were compared, and the results showed that the newly developed primer set SA/429f-933r could detect a larger spectrum (90%) of Sphingomonas strains with higher specificity. Despite the superiority of primer set SA/429f-933r in specifically detecting Sphingomonas from contaminated soils, we cannot blink the fact that different primer sets preferentially amplified different dominant species. Therefore, two or more primer sets are recommended for evaluating the diversity and population structure of genus Sphingomonas. Additionally, a proportion (9.7%) of the cloned sequences discovered in this study were different from known Sphingomonas sequences, suggesting that new Sphingomonas sequences might present in soils from Shenfu irrigation zone.

Cloning, Heterologous Expression, and Characterization of Novel Protease-Resistant α-Galactosidase from New Sphingomonas Strain

The α-galactosidase-coding gene agaAJB13 was cloned from Sphingomonas sp. JB13 showing 16S rDNA (1,343 bp) identities of < or =97.2% with other identified Sphingomonas strains. agaAJB13 (2,217 bp; 64.9% GC content) encodes a 738-residue polypeptide (AgaAJB13) with a calculated mass of 82.3 kDa. AgaAJB13 showed the highest identity of 61.4% with the putative glycosyl hydrolase family 36 α-galactosidase from Granulicella mallensis MP5ACTX8 (EFI56085). AgaAJB13 also showed <37% identities with reported protease-resistant or Sphingomonas alpha-galactosidases. A sequence analysis revealed different catalytic motifs between reported Sphingomonas α-galactosidases (KXD and RXXXD) and AgaAJB13 (KWD and SDXXDXXXR). Recombinant AgaAJB13 (rAgaAJB13) was expressed in Escherichia coli BL21 (DE3). The purified rAgaAJB13 was characterized using p-nitrophenyl-α-D-galactopyranoside as the substrate and showed an apparent optimum at pH 5.0 and 60 degrees C and strong resistance to trypsin and proteinase K digestion. Compared with reported proteaseresistant alpha-galactosidases showing thermolability at 50 degrees C or 60°C and specific activities of <71 U/mg with or without protease treatments, rAgaAJB13 exhibited a better thermal stability (half-life of >60 min at 60°C) and higher specific activities (225.0-256.5 U/mg). These sequence and enzymatic properties suggest AgaAJB13 is the first identified and characterized Sphingomonas α-galactosidase, and shows novel protease resistance with a potential value for basic research and industrial applications.