Genome-based taxonomy and functional prediction of Sphingomonas fuzhouensis sp. nov. and Massilia phyllosphaerae sp. nov. isolated from Pennisetum sp. with plant growth-promoting potential.

Two facultatively aerobic strains, designated SGZ-02T and SGZ-792T, were isolated from plant Pennisetum sp., exhibiting the highest 16S rRNA gene sequence similarities with the type strains of Sphingomonas zeae LMG 28739T (98.6%) and Massilia forsythiae NBRC 114511T (98.4%), respectively. SGZ-02T grew between 5 and 45 °C, pH 5.0-11.0 and tolerated NaCl concentrations of 0-4% (w/v), whereas SGZ-792T thrived at 5-40 °C, pH 5.0-11.0 and NaCl tolerance to 0-3.5% (w/v). The major quinone of SGZ-02T was ubiquinone-10, with the dominant fatty acids being C16:0 (13.5%), Summed Feature 3 (6.3%), C14:02-OH (5.3%) and Summed Feature 8 (66.3%). SGZ-792T predominantly contained ubiquinone-8, with major fatty acids being C16:0 (20.3%), Summed Feature 3 (5.0%) and Summed Feature 8 (54.7%). Average nucleotide identity and digital DNA-DNA hybridization values between two strains and their closest references strains were below the bacterial species threshold. Based on genotypic and phenotypic characteristics, strains SGZ-02T and SGZ-792T are proposed as novel species within the genera Sphingomonas and Massilia, respectively. The suggested names for the new species are Sphingomonas fuzhouensis sp. nov. (SGZ-02T = GDMCC 1.4033T = JCM 36769T) and Massilia phyllosphaerae sp. nov. (SGZ-792T = GDMCC 1.4211T = JCM 36643T), respectively.

Deciphering the triclosan degradation mechanism in Sphingomonas sp. strain YL-JM2C: Implications for wastewater treatment and marine resources.

Triclosan (TCS), an antimicrobial agent extensively incorporated into pharmaceuticals and personal care products, poses significant environmental risks because of its persistence and ecotoxicity. So far, a few microorganisms were suggested to degrade TCS, but the microbial degradation mechanism remains elusive. Here, a two-component angular dioxygenase (TcsAaAb) responsible for the initial TCS degradation was characterized in Sphingomonas sp. strain YL-JM2C. Whole-cell biotransformation and crude enzyme assays demonstrated that TcsAaAb catalyzed the conversion of TCS to 4-chlorocatechol and 3,5-dichlorocatechol rather than the commonly suggested product 2,4-dichlorophenol. Then two intermediates were catabolized by tcsCDEF cluster via an ortho-cleavage pathway. Critical residues (N262, F279, and F391) for substrate binding were identified via molecular docking and mutagenesis. Further, TcsAaAb showed activity toward methyl triclosan and nitrofen, suggesting its versatile potential for bioremediation. In addition, TCS-degrading genes were also present in diverse bacterial genomes in wastewater, ocean and soil, and a relatively high gene abundance was observed in marine metagenomes, revealing the transformation fate of TCS in environments and the microbial potential in pollutant removal. These findings extend the understanding of the microbe-mediated TCS degradation and contribute to the mining of TCS-degrading strains and enzymes, as well as their application in the bioremediation of contaminated environments.

Comprehensive review on Haloalkane dehalogenase (LinB): a ß-hexachlorocyclohexane (HCH) degrading enzyme.

Haloalkane dehalogenase, LinB, is a member of the α/ß hydrolase family of enzymes. It has a wide range of halogenated substrates, but, has been mostly studied in context of degradation of hexachlorocyclohexane (HCH) isomers, especially ß-HCH (5-12% of total HCH isomers), which is the most recalcitrant and persistent among all the HCH isomers. LinB was identified to directly act on ß-HCH in a one or two step transformation which decreases its toxicity manifold. Thereafter, many studies focused on LinB including its structure determination using X-ray crystallographic studies, structure comparison with other haloalkane dehalogenases, substrate specificity and kinetic studies, protein engineering and site-directed mutagenesis studies in search of better catalytic activity of the enzyme. LinB was mainly identified and characterized in bacteria belonging to sphingomonads. Detailed sequence comparison of LinB from different sphingomonads further revealed the residues critical for its activity and ability to catalyze either one or two step transformation of ß-HCH. Association of LinB with IS6100 elements is also being discussed in detail in sphingomonads. In this review, we summarized vigorous efforts done by different research groups on LinB for developing better bioremediation strategies against HCH contamination. Also, kinetic studies, protein engineering and site directed mutagenesis studies discussed here forms the basis of further exploration of LinB's role as an efficient enzyme in bioremediation projects.

Diversity of bacteria of the genus Sphingomonas associated with sugarcane (Saccharum spp.) culm apoplast fluid and their agrotechnological potential.

In sugarcane, sequences related to the genus Sphingomonas have been widely detected by microbiome studies. In this work, the presence of bacteria of this genus was confirmed using culture-dependent and independent techniques. A collection of thirty isolates was obtained using semispecific cultivation conditions, and a specific PCR assay was applied to help confirm the isolates as belonging to the genus. A series of laboratory evaluations were carried out to identify potential properties among the isolates in the collection, which consequently allowed the identification of some most promising isolates for the development of new agricultural bioinputs. In a separate analysis, the culture-independent fluorescence in situ hybridization (FISH) methodology was applied to demonstrate the natural occurrence of Sphingomonas in different organs and tissues of sugarcane. The results showed the presence of bacteria of the genus in the spaces between cells (apoplast) of the culm parenchyma, in vessels in the region of the leaf vein, on the adaxial surface of the leaf blade, and on the root surface, sometimes close to the base of root hairs, which suggests extensive colonization on the host plant. In summary, the present study corroborates previous metagenomic amplicon sequencing results that indicated a high occurrence of Sphingomonas associated with sugarcane. This is the first study that uses approaches other than amplicon sequencing to confirm the occurrence of the genus in sugarcane and, at the same time, demonstrates potentially beneficial activities to be explored by sugarcane cultivation.

An exopolysaccharide pathway from a freshwater Sphingomonas isolate.

Bacteria embellish their cell envelopes with a variety of specialized polysaccharides. Biosynthesis pathways for these glycans are complex, and final products vary greatly in their chemical structures, physical properties, and biological activities. This tremendous diversity comes from the ability to arrange complex pools of monosaccharide building blocks into polymers with many possible linkage configurations. Due to the complex chemistry of bacterial glycans, very few biosynthetic pathways have been defined in detail. As part of an initiative to characterize novel polysaccharide biosynthesis enzymes, we isolated a bacterium from Lake Michigan called Sphingomonas sp. LM7 that is proficient in exopolysaccharide (EPS) production. We identified genes that contribute to EPS biosynthesis in LM7 by screening a transposon mutant library for colonies displaying altered colony morphology. A gene cluster was identified that appears to encode a complete wzy/wzx-dependent polysaccharide assembly pathway. Deleting individual genes in this cluster caused a non-mucoid phenotype and a corresponding loss of EPS secretion, confirming the role of this gene cluster in polysaccharide production. We extracted EPS from LM7 cultures and determined that it contains a linear chain of 3- and 4-linked glucose, galactose, and glucuronic acid residues. Finally, we show that the EPS pathway in Sphingomonas sp. LM7 diverges from that of sphingan-family EPSs and adhesive polysaccharides such as the holdfast that are present in other Alphaproteobacteria. Our approach of characterizing complete biosynthetic pathways holds promise for engineering polysaccharides with valuable properties. IMPORTANCE: Bacteria produce complex polysaccharides that serve a range of biological functions. These polymers often have properties that make them attractive for industrial applications, but they remain woefully underutilized. In this work, we studied a novel polysaccharide called promonan that is produced by Sphingomonas sp. LM7, a bacterium we isolated from Lake Michigan. We extracted promonan from LM7 cultures and identified which sugars are present in the polymer. We also identified the genes responsible for polysaccharide production. Comparing the promonan genes to those of other bacteria showed that promonan is distinct from previously characterized polysaccharides. We conclude by discussing how the promonan pathway could be used to produce new polysaccharides through genetic engineering.

Sphingomonas sp. Hbc-6 alters Arabidopsis metabolites to improve plant growth and drought resistance by manipulating the microbiome.

Drought significantly affects crop productivity and poses a considerable threat to agricultural ecosystems. Plant growth-promoting bacteria (PGPB) and plant microbiome play important roles in improving drought resistance and plant performance. However, the response of the rhizosphere microbiota to PGPB during the development of plants and the interaction between inoculum, microbiota, and plants under drought stress remain to be explored. In the present study, we used culturomic, microbiomic, and metabonomic analyses to uncover the mechanisms by which Sphingomonas sp. Hbc-6, a PGPB, promotes Arabidopsis growth and enhances drought resistance. We found that the rhizosphere microbiome assembly was interactively influenced by developmental stage, Hbc-6, and drought; the bacterial composition exhibited three patterns of shifts with developmental stage: resilience, increase, and decrease. Drought diminished microbial diversity and richness, whereas Hbc-6 increased microbial diversity and helped plants recruit specific beneficial bacterial taxa at each developmental stage, particularly during the bolting stage. Some microorganisms enriched by Hbc-6 had the potential to promote carbon and nitrogen cycling processes, and 86.79 % of the isolated strains exhibited PGP characteristics (for example Pseudomonas sp. TA9). They jointly regulated plant physiological metabolism (i.e., upregulated drought resistant-facilitating substances and reduced harmful substances), thereby stimulating the growth of Arabidopsis and increasing plant biomass under drought stress conditions. Collectively, these results indicate that Hbc-6 mediates plant growth and drought resistance by affecting the microbiome. The study thus provides novel insights and strain resources for drought-resistant, high-yielding crop cultivation and breeding.

Ancient and remote quartzite caves as a novel source of culturable microbes with biotechnological potential.

Quartzite caves located on table-top mountains (tepuis) in the Guyana Shield, are ancient, remote, and pristine subterranean environments where microbes have evolved peculiar metabolic strategies to thrive in silica-rich, slightly acidic and oligotrophic conditions. In this study, we explored the culturable fraction of the microbiota inhabiting the (ortho)quartzite cave systems in Venezuelan tepui (remote table-top mountains) and we investigated their metabolic and enzymatic activities in relation with silica solubilization and extracellular hydrolytic activities as well as the capacity to produce antimicrobial compounds. Eighty microbial strains were isolated with a range of different enzymatic capabilities. More than half of the isolated strains performed at least three enzymatic activities and four bacterial strains displayed antimicrobial activities. The antimicrobial producers Paraburkholderia bryophila CMB_CA002 and Sphingomonas sp. MEM_CA187, were further analyzed by conducting chemotaxonomy, phylogenomics, and phenomics. While the isolate MEM_CA187 represents a novel species of the genus Sphingomonas, for which the name Sphingomonas imawarii sp. nov. is proposed, P. bryophila CMB_CA002 is affiliated with a few strains of the same species that are antimicrobial producers. Chemical analyses demonstrated that CMB_CA002 produces ditropolonyl sulfide that has a broad range of activity and a possibly novel siderophore. Although the antimicrobial compounds produced by MEM_CA187 could not be identified through HPLC-MS analysis due to the absence of reference compounds, it represents the first soil-associated Sphingomonas strain with the capacity to produce antimicrobials. This work provides first insights into the metabolic potential present in quartzite cave systems pointing out that these environments are a novel and still understudied source of microbial strains with biotechnological potential.

Sphingomonas Immobilis sp. nov., and Sphingomonas natans sp. nov. bacteria isolated from soil.

Two novel strains of bacteria, CA1-15T and BIUV-7T, were isolated from soil samples gathered in Cheonan-si, Republic of Korea, and Inje-gun, Republic of Korea, respectively. These bacteria are Gram-negative, aerobic, and non-motile. Phylogenetic evaluations, using the sequence of the 16S rRNA gene, showed that strains CA1-15T and BIUV-7T belong to a distinctive clade within the family Sphingomonadaceae (order Sphingomonadales, class Alphaproteobacteria). The strains exhibited the highest similarity in their genetic makeup with representatives of the genus Sphingomonas. Strain CA1-15T was closely related to Sphingomonas echinoides NRRL B-3126T (97.8% similarity in 16S rRNA gene sequence), Sphingomonas oligophenolica JCM 12,082T (97.8%), Sphingomonas glacialis C16yT (97.6%) and Sphingomonas psychrolutea MDB1-AT (97.3%). Strain BIUV-7T was closely related to Sphingomonas nostoxanthinifaciens AK-PDB1-5T (97.0%), Sphingomonas vulcanisoli SN6-13T (96.3%), Sphingomonas naphthae DKC-5-1T (96.2%), and Sphingomonas prati W18RDT (95.7%). The optimal growth conditions for strains CA1-15T and BIUV-7T were determined to be at pH 7.0 and a temperature of 25 °C. Analysis of the cellular fatty acids of strain CA1-15T and BIUV-7T revealed that summed feature 8 (C18:1ω7c/C18:1ω6c) (60.4%), summed feature 8 (C18:1ω7c/C18:1ω6c) (62.9%) were the major component, respectively. Additionally, both strains exhibited ubiquinone Q-10 as their major respiratory quinone, and diphosphatidylglycerol (DPG), glycosphingolipid (SGL), and phosphatidylethanolamine (PE) as the major polar lipid. The genome of strain CA1-15T measures 4,133,944 bp, comprising 4,026 coding sequences (CDSs) and 46 tRNA genes. Similarly, the genome of strain BIUV-7T is 4,563,252 bp, characterized by 4,226 CDSs and 44 tRNA genes. The average nucleotide identity (ANI) analysis and digital DNA-DNA hybridization (dDDH) values between strain CA1-15T and other Sphingomonas species range from 73.2 to 79.9% and 19.4-22.9%, respectively. Comparatively, ANI and dDDH values between strain BIUV-7T and other Sphingomonas species are in the range of 72.9-76.5% and 19.3-20.9%, respectively. Based on the biochemical, chemotaxonomic, and phylogenetic analyses, it is evident that strains CA1-15T and BIUV-7T represent two novel bacterial species within the genus Sphingomonas. Accordingly, the names Sphingomonas immobilis sp. nov. and Sphingomonas natans sp. nov. are proposed. also, CA1-15T(= KCTC 92960T = NBRC 116547T) is the type strain of Sphingomonas immobilis and BIUV-7T(= KCTC 92961T = NBRC 116546T) is the type strain of Sphingomonas natans.

Functional analysis, diversity, and distribution of the ean cluster responsible for 17ß-estradiol degradation in sphingomonads.

17ß-estradiol (E2) is a natural endocrine disruptor that is frequently detected in surface and groundwater sources, thereby threatening ecosystems and human health. The newly isolated E2-degrading strain Sphingomonas colocasiae C3-2 can degrade E2 through both the 4,5-seco pathway and the 9,10-seco pathway; the former is the primary pathway supporting the growth of this strain and the latter is a branching pathway. The novel gene cluster ean was found to be responsible for E2 degradation through the 4,5-seco pathway, where E2 is converted to estrone (E1) by EanA, which belongs to the short-chain dehydrogenases/reductases (SDR) superfamily. A three-component oxygenase system (including the P450 monooxygenase EanB1, the small iron-sulfur protein ferredoxin EanB2, and the ferredoxin reductase EanB3) was responsible for hydroxylating E1 to 4-hydroxyestrone (4-OH-E1). The enzymatic assay showed that the proportion of the three components is critical for its function. The dioxygenase EanC catalyzes ring A cleavage of 4-OH-E1, and the oxidoreductase EanD is responsible for the decarboxylation of the ring A-cleavage product of 4-OH-E1. EanR, a TetR family transcriptional regulator, acts as a transcriptional repressor of the ean cluster. The ean cluster was also found in other reported E2-degrading sphingomonads. In addition, the novel two-component monooxygenase EanE1E2 can open ring B of 4-OH-E1 via the 9,10-seco pathway, but its encoding genes are not located within the ean cluster. These results refine research on genes involved in E2 degradation and enrich the understanding of the cleavages of ring A and ring B of E2.IMPORTANCESteroid estrogens have been detected in diverse environments, ranging from oceans and rivers to soils and groundwater, posing serious risks to both human health and ecological safety. The United States National Toxicology Program and the World Health Organization have both classified estrogens as Group 1 carcinogens. Several model organisms (proteobacteria) have established the 4,5-seco pathway for estrogen degradation. In this study, the newly isolated Sphingomonas colocasiae C3-2 could degrade E2 through both the 4,5-seco pathway and the 9,10-seco pathway. The novel gene cluster ean (including eanA, eanB1, eanC, and eanD) responsible for E2 degradation by the 4,5-seco pathway was identified; the novel two-component monooxygenase EanE1E2 can open ring B of 4-OH-E1 through the 9,10-seco pathway. The TetR family transcriptional regulator EanR acts as a transcriptional repressor of the ean cluster. The cluster ean was also found to be present in other reported E2-degrading sphingomonads, indicating the ubiquity of the E2 metabolism in the environment.

Sphingomonas clade and functional distribution with simulated climate change.

Microbes are essential for the functioning of all ecosystems, and as global warming and anthropogenic pollution threaten ecosystems, it is critical to understand how microbes respond to these changes. We investigated the climate response of Sphingomonas, a widespread gram-negative bacterial genus, during an 18-month microbial community reciprocal transplant experiment across a Southern California climate gradient. We hypothesized that after 18 months, the transplanted Sphingomonas clade and functional composition would correspond with site conditions and reflect the Sphingomonas composition of native communities. We extracted Sphingomonas sequences from metagenomic data across the gradient and assessed their clade and functional composition. Representatives of at least 12 major Sphingomonas clades were found at varying relative abundances along the climate gradient, and transplanted Sphingomonas clade composition shifted after 18 months. Site had a significant effect (PERMANOVA; P < 0.001) on the distribution of both Sphingomonas functional (R2 = 0.465) and clade composition (R2 = 0.400), suggesting that Sphingomonas composition depends on climate parameters. Additionally, for both Sphingomonas clade and functional composition, ordinations revealed that the transplanted communities shifted closer to the native Sphingomonas composition of the grassland site compared with the site they were transplanted into. Overall, our results indicate that climate and substrate collectively determine Sphingomonas clade and functional composition.IMPORTANCESphingomonas is the most abundant gram-negative bacterial genus in litter-degrading microbial communities of desert, grassland, shrubland, and forest ecosystems in Southern California. We aimed to determine whether Sphingomonas responds to climate change in the same way as gram-positive bacteria and whole bacterial communities in these ecosystems. Within Sphingomonas, both clade composition and functional genes shifted in response to climate and litter chemistry, supporting the idea that bacteria respond similarly to climate at different scales of genetic variation. This understanding of how microbes respond to perturbation across scales may aid in future predictions of microbial responses to climate change.

Sphingomonas lacusdianchii sp. nov., an attached bacterium inhibited by metabolites from its symbiotic cyanobacterium.

An alpha-proteobacterial strain JXJ CY 53 T was isolated from the cyanosphere of Microcystis sp. FACHB-905 (MF-905) collected from Lake Dianchi, China. JXJ CY 53 T was observed to be an aerobic, Gram-stain-negative, oval shaped, and mucus-secreting bacterium. It had C18:1ω7c and C16:0 as the major cellular fatty acids, Q-10 as the predominant ubiquinone, and sphingoglycolipid, diphosphatidylglycerol, phosphatidylcholine, and phosphatidylmethylethanolamine as the polar lipids. The G + C content of DNA was 65.85%. The bacterium had 16S rRNA gene sequence identities of 98.9% and 98.7% with Sphingomonas panni DSM 15761 T and Sphingomonas hankookensis KCTC 22579 T, respectively, while less than 97.4% identities with other members of the genus. Further taxonomic analysis indicated that JXJ CY 53 T represented a new member of Sphingomonas, and the species epithet was proposed as Sphingomonas lacusdianchii sp. nov. (type strain JXJ CY 53 T = KCTC 72813 T = CGMCC 1.17657 T). JXJ CY 53 T promoted the growth of MF-905 by providing bio-available phosphorus and nitrogen, plant hormones, vitamins, and carotenoids. It could modulate the relative abundances of nonculturable bacteria associated with MF-905 and influence the interactions of MF-905 and other bacteria isolated from the cyanobacterium, in addition to microcystin production characteristics. Meanwhile, MF-905 could provide JXJ CY 53 T dissolved organic carbon for growth, and control the growth of JXJ CY 53 T by secreting specific chemicals other than microcystins. Overall, these results suggest that the interactions between Microcystis and its attached bacteria are complex and dynamic, and may influence the growth characteristics of the cyanobacterium. This study provided new ideas to understand the interactions between Microcystis and its attached bacteria. KEY POINTS: • A novel bacterium (JXJCY 53 T) was isolated from the cyanosphere of Microcystis sp. FACHB-905 (MF-905) • JXJCY 53 T modulated the growth and microcystin production of MF-905 • MF-905 could control the attached bacteria by specific chemicals other than microcystins (MCs).

Sphingomonas bacteria could serve as an early bioindicator for the development of chlorantraniliprole resistance in Spodoptera frugiperda.

The fall armyworm (Spodoptera frugiperda) was found to have invaded China in December 2018, and in just one year, crops in 26 provinces were heavily affected. Currently, the most effective method for emergency control of fulminant pests is to use of chemical pesticides. Recently, most fall armyworm populations in China were begining to exhibite low level resistance to chlorantraniliprole. At present, it is not possible to sensitively reflect the low level resistance of S. frugiperda by detecting target mutation and detoxification enzyme activity. In this study we found that 12 successive generations of screening with chlorantraniliprole caused S. frugiperda to develop low level resistance to this insecticide, and this phenotype was not attribute to genetic mutations in S. frugiperda, but rather to a marked increase in the relative amount of the symbiotic bacteria Sphingomonas. Using FISH and qPCR assays, we determined the amount of Sphingomonas in the gut of S. frugiperda and found Sphingomonas accumulation to be highest in the 3rd-instar larvae. Additionally, Sphingomonas was observed to provide a protective effect to against chlorantraniliprole stress to S. frugiperda. With the increase of the resistance to chlorantraniliprole, the abundance of bacteria also increased, we propose Sphingomonas monitoring could be adapted into an early warning index for the development of chlorantraniliprole resistance in S. frugiperda populations, such that timely measures can be taken to delay or prevent the widespread propagation of resistance to this highly useful agricultural chemical in S. frugiperda field populations.

Sphingomonas flavescens sp. nov., isolated from soil.

An aerobic bacterium, designated as PT-12T, was isolated from soil collected from agriculture field, and its taxonomic position was validated through a comprehensive polyphasic methodology. The strain was identified as Gram-stain-negative, non-motile, rod-shaped, and catalase- and oxidase-positive. The yellow-colored colonies showed growth ability at temperature range of 18-37 °C, NaCl content of 0-1.0% (w/v), and at a pH of 6.0-8.0. The 16S rRNA gene and phylogenetic analysis showed that strain PT-12T affiliated with the genus Sphingomonas in the family Sphingomonadaceae, and displayed the highest 16S rRNA nucleotide sequence similarity with Sphingomonas limnosediminicola 03SUJ6T (98.4%). The genome size of strain PT-12T was 2,656,862 bp and the DNA G + C content estimated from genome was 63.5%. The highest values of average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) were observed between strain PT-12T and Sphingomonas segetis YJ09T, accounting to 76.2% and 20.2%, respectively. In addition, both ANI and dDDH values between strain PT-12T and other phylogenetically related neighbors ranged between 69.6% and 76.2% and 18.4% and 20.2%, respectively. Chemotaxonomic features exhibited Q-10 as the only ubiquinone; homospermidine as the major polyamine; summed feature 8 (C18:1ω7c and/or C18:1ω6c), C16:0, and 10-methyl C18:0 as the notable fatty acids; and phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine, and sphingoglycolipid as dominating polar lipids. Overall, the comprehensive polyphasic data supported that strain PT-12T represents a novel bacterial species within the genus Sphingomonas. Accordingly, we propose the name Sphingomonas flavescens sp. nov. The type strain is PT-12T (= KCTC 92114T = NBRC 115717T).

sRNA molecules participate in hyperosmotic stress response regulation in Sphingomonas melonis TY.

Drought and salinity are ubiquitous environmental factors that pose hyperosmotic threats to microorganisms and impair their efficiency in performing environmental functions. However, bacteria have developed various responses and regulatory systems to cope with these abiotic challenges. Posttranscriptional regulation plays vital roles in regulating gene expression and cellular homeostasis, as hyperosmotic stress conditions can lead to the induction of specific small RNA molecules (sRNAs) that participate in stress response regulation. Here, we report a candidate functional sRNA landscape of Sphingomonas melonis TY under hyperosmotic stress, and 18 sRNAs were found with a clear response to hyperosmotic stress. These findings will help in the comprehensive analysis of sRNA regulation in Sphingomonas species. Weighted correlation network analysis revealed a 263 nucleotide sRNA, SNC251, which was transcribed from its own promoter and showed the most significant correlation with hyperosmotic response factors. Deletion of snc251 affected biofilm formation and multiple cellular processes, including ribosome-related pathways, aromatic compound degradation, and the nicotine degradation capacity of S. melonis TY, while overexpression of SNC251 facilitated biofilm formation by TY under hyperosmotic stress. Two genes involved in the TonB system were further verified to be activated by SNC251, which also indicated that SNC251 is a trans-acting sRNA. Briefly, this research reports a landscape of sRNAs participating in the hyperosmotic stress response in S. melonis and reveals a novel sRNA, SNC251, which contributes to the S. melonis TY biofilm formation and thus enhances its hyperosmotic stress response ability.IMPORTANCESphingomonas species play a vital role in plant defense and pollutant degradation and survive extensively under drought or salinity. Previous studies have focused on the transcriptional and translational responses of Sphingomonas under hyperosmotic stress, but the posttranscriptional regulation of small RNA molecules (sRNAs) is also crucial for quickly modulating cellular processes to adapt dynamically to osmotic environments. In addition, the current knowledge of sRNAs in Sphingomonas is extremely scarce. This research revealed a novel sRNA landscape of Sphingomonas melonis and will greatly enhance our understanding of sRNAs' acting mechanisms in the hyperosmotic stress response.

The production of ultrahigh molecular weight xanthan gum from a Sphingomonas chassis capable of co-utilising glucose and xylose from corn straw.

Corn straw is an abundant and renewable alternative for microbial biopolymer production. In this paper, an engineered Sphingomonas sanxanigenens NXG-P916 capable of co-utilising glucose and xylose from corn straw total hydrolysate to produce xanthan gum was constructed. This strain was obtained by introducing the xanthan gum synthetic operon gum as a module into the genome of the constructed chassis strain NXdPE that could mass produce activated precursors of polysaccharide, and in which the transcriptional levels of gum genes were optimised by screening for a more appropriate promoter, P916 . As a result, strain NXG-P916 produced 9.48 ± 0.34 g of xanthan gum per kg of fermentation broth (g/kg) when glucose was used as a carbon source, which was 2.1 times improved over the original engineering strain NXdPE::gum. Furthermore, in batch fermentation, 12.72 ± 0.75 g/kg xanthan gum was produced from the corn straw total hydrolysate containing both glucose and xylose, and the producing xanthan gum showed an ultrahigh molecular weight (UHMW) of 6.04 × 107 Da, which was increased by 15.8 times. Therefore, the great potential of producing UHMW xanthan gum by Sphingomonas sanxanigenens was proved, and the chassis NXdPE has the prospect of becoming an attractive platform organism producing polysaccharides derived from biomass hydrolysates.

Metagenomic and proteomic insights into the self-adaptive cell surface hydrophobicity of Sphingomonas sp. strain PAH02 reducing the migration of cadmium-phenanthrene co-pollutant in rice.

Cell surface hydrophobicity (CSH) dominates the interactions between rhizobacteria and pollutants at the soil-water interface, which is critical for understanding the dissipation of pollutants in the rhizosphere microzone of rice. Herein, we explored the effects of self-adaptive CSH of Sphingomonas sp. strain PAH02 on the translocation and biotransformation behaviour of cadmium-phenanthrene (Cd-Phe) co-pollutant in rice and rhizosphere microbiome. We evidenced that strain PAH02 reduced the adsorption of Cd-Phe co-pollutant on the rice root surface while enhancing the degradation of Phe and adsorption of Cd via its self-adaptive CSH in the hydroponic experiment. The significant upregulation of key protein expression levels such as MerR, ARHDs and enoyl-CoA hydratase/isomerase, ensures self-adaptive CSH to cope with the stress of Cd-Phe co-pollutant. Consistently, the bioaugmentation of strain PAH02 promoted the formation of core microbiota in the rhizosphere soil of rice (Oryza sativa L.), such as Bradyrhizobium and Streptomyces and induced gene enrichment of CusA and PobA that are strongly associated with pollutant transformation. Consequently, the contents of Cd and Phe in rice grains at maturity decreased by 17.2% ± 0.2% and 65.7% ± 0.3%, respectively, after the bioaugmentation of strain PAH02. These findings present new opportunities for the implementation of rhizosphere bioremediation strategies of co-contaminants in paddy fields.

Mutagenesis enhances gellan gum production by a novel Sphingomonas spp.: upstream optimization, kinetic modeling, and structural and physico-functional evaluation.

Gellan gum (GG) has gained tremendous attention owing to its diversified applications. However, its high production and hence market cost are still a bottleneck in its widespread utilization. In the present study, high GG producing mutant of Sphingomonas spp. was developed by random mutagenesis using ethyl methylsulphonate (EMS) for industrial fermentation and identified as Sphingomonas trueperi after 16S rRNA and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) analysis. The fermentation conditions such as pH, temperature, and inoculum ratio were optimized by one factor at a time (OFAT) followed by screening of medium components by the Plackett-Burman statistical design. The most critical nutrients were further optimized by response surface methodology for maximizing GG production. The effect of dissolved oxygen tension in bioreactor on cell growth, substrate consumption, GG production, and batch productivity was elucidated. The highest GG titer (23 ± 2.4 g/L) was attained in optimized medium at 10% inoculum (6.45 ± 0.5 log cfu/mL) under controlled fermentation conditions of pH (7), temperature (30 °C), agitation (300-600 rpm), and aeration (0.5-2.0 SLPM) at 22 ± 2% dissolved oxygen tension in a 10-L bioreactor. Kinetic modeling of optimized batch process revealed that logistic growth model could best explain biomass accumulation, while GG formation and substrate consumption were best explained by Luedeking-Piret and exponential decay model, respectively. Structural and physico-functional features of GG produced by mutant Sphingomonas spp. were characterized by HPLC, FTIR, NMR, DSC, TGA, GPC, SEM, and rheological analysis. The higher productivity (0.51 g/L/h) under optimized fermentation conditions suggests potential consideration of mutant and process for commercial utilization.

Multifaceted photoreceptor compositions in dual phototrophic systems - A genomic analysis.

For microbes and their hosts, sensing of external cues is essential for their survival. For example, in the case of plant associated microbes, the light absorbing pigment composition of the plant as well as the ambient light conditions determine the well-being of the microbe. In addition to light sensing, some microbes can utilize xanthorhodopsin based proton pumps and bacterial photosynthetic complexes that work in parallel for energy production. They are called dual phototrophic systems. Light sensing requirements in these type of systems are obviously demanding. In nature, the photosensing machinery follows mainly the same composition in all organisms. However, the specific role of each photosensor in specific light conditions is elusive. In this study, we provide an overall picture of photosensors present in dual phototrophic systems. We compare the genomes of the photosensor proteins from dual phototrophs to those from similar microbes with "single" phototrophicity or microbes without phototrophicity. We find that the dual phototrophic bacteria obtain a larger variety of photosensors than their light inactive counterparts. Their rich domain composition and functional repertoire remains similar across all microbial photosensors. Our study calls further investigations of this particular group of bacteria. This includes protein specific biophysical characterization in vitro, microbiological studies, as well as clarification of the ecological meaning of their host microbial interactions.

Structural conservation in the glutathione binding in Sphingomonas sp. glutaredoxin Grx3 and variations for cold adaptation.

Glutaredoxin 3 (Grx3), a redox protein with a thioredoxin-fold structure, maintains structural integrity and glutathione (GSH) binding capabilities across varying habitat temperatures. The cis-Pro loop, essential for GSH binding, relies on the Arg-Asp salt bridge (α2-α3) and Gln-His hydrogen bond (ß3-ß4) for its conformation. In some psychrophilic Grx3 variants, Arg in α2 is replaced with Tyr, and His in ß4 is replaced with Phe. This study examines the roles of these bonds in Grx3's structure, function, and cold adaptation, using SpGrx3 from the Arctic bacterium Sphingomonas sp. Despite its cold habitat, SpGrx3 maintains the Arg51-Asp69 salt bridge and Gln56-His63 hydrogen bond. The R51Y substitution disrupts the α2-α3 salt bridge, while the H63F and H63Y substitutions hinder the salt bridge through cation-π interactions with Arg51, involving Phe63/Tyr63, thereby enhancing flexibility. Conversely, mutations that disrupt the hydrogen bond (Q56A, H63A, and H63F) reduce thermal stability. In the psychrophilic Grx3 configuration A48T/R51Y/H63F, a Thr48-Gln56 hydrogen bond stabilizes the cis-Pro loop, enhancing flexibility by disrupting both bonds. Furthermore, all mutants exhibit reduced α-helical content and catalytic efficiency. In summary, the highly conserved Arg51-Asp69 salt bridge and Gln56-His63 hydrogen bond are crucial for stabilizing the cis-Pro loop and catalytic activity in SpGrx3. His63 is favored as it avoids cation-π interactions with Arg51, unlike Phe63/Tyr63. Psychrophilic Grx3 variants have adapted to cold environments by reducing GSH binding and increasing structural flexibility. These findings deepen our understanding of the structural conservation in Grx3 for GSH binding and the critical alterations required for cold adaptation.

A novel UV-resistant bacterium Sphingomonas endolithica sp. nov., and genomic analysis, isolated from the north slope of Mount Everest.

Gram-stain-negative, aerobic, rod-shaped, non-motile bacterium strain ZFBP2030T was isolated from a rock on the North slope of Mount Everest. This strain contained a unique ubiquinone-10 (Q-10) as a predominant respiratory quinone. Among the tested fatty acids, the strain contained summed feature 8, C14:0 2OH, and C16:0, as major cellular fatty acids. The polar lipid profile contained phosphatidyl glycerol, phosphatidyl ethanolamine, three unidentified phospholipids, two unidentified aminolipids, and six unidentified lipids. The cell-wall peptidoglycan was a meso-diaminopimelic acid, and cell-wall sugars were ribose and galactose. Phylogenetic analyses based on 16S rRNA gene sequence revealed that strain ZFBP2030T was a member of the genus Sphingomonas, exhibiting high sequence similarity to the 16S rRNA gene sequences of Sphingomonas aliaeris DH-S5T (97.9%), Sphingomonas alpina DSM 22537T (97.3%) and Sphingomonas hylomeconis CCTCC AB 2013304T (97.0%). The 16S rRNA gene sequence similarity between ZFBP2030T and other typical strains was less than 97.0%. The average amino acid identity values, average nucleotide identity, and digital DNA-DNA hybridization values between strain ZFBP2030T and its highest sequence similarity strains were 56.9-79.9%, 65.1-82.2%, and 19.3-25.8%, respectively. The whole-genome size of the novel strain ZFBP2030T was 4.1 Mbp, annotated with 3838 protein-coding genes and 54 RNA genes. Moreover, DNA G + C content was 64.7 mol%. Stress-related functions predicted in the subsystem classification of the strain ZFBP2030T genome included osmotic, oxidative, cold/heat shock, detoxification, and periplasmic stress responses. The overall results of this study clearly showed that strain ZFBP2030T is a novel species of the genus Sphingomonas, for which the name Sphingomonas endolithica sp. nov. is proposed. The type of strain is ZFBP2030T (= EE 013T = GDMCC 1.3123T = JCM 35386T).