An improved genome editing system for Sphingomonadaceae.

The sphingomonads encompass a diverse group of bacteria within the family Sphingomonadaceae, with the presence of sphingolipids on their cell surface instead of lipopolysaccharide as their main common feature. They are particularly interesting for bioremediation purposes due to their ability to degrade or metabolise a variety of recalcitrant organic pollutants. However, research and development on their full bioremediation potential has been hampered because of the limited number of tools available to investigate and modify their genome. Here, we present a markerless genome editing method for Sphingopyxis granuli TFA, which can be further optimised for other sphingomonads. This procedure is based on a double recombination triggered by a DNA double-strand break in the chromosome. The strength of this protocol lies in forcing the second recombination rather than favouring it by pressing a counterselection marker, thus avoiding laborious restreaking or passaging screenings. Additionally, we introduce a modification with respect to the original protocol to increase the efficiency of the screening after the first recombination event. We show this procedure step by step and compare our modified method with respect to the original one by deleting ecfG2, the master regulator of the general stress response in S. granuli TFA. This adds to the genetic tool repertoire that can be applied to sphingomonads and stands as an efficient option for fast genome editing of this bacterial group.

Regulation of quorum sensing activities by the stringent response gene rsh in sphingomonads is species-specific and culture condition dependent.

Stringent response and quorum sensing (QS) are two essential mechanisms that control bacterial global metabolism for better survival. Sphingomonads are a clade of bacteria that survive successfully in diverse ecosystems. In silico survey indicated that 36 out of 79 investigated sphingomonads strains contained more than one luxI homolog, the gene responsible for the biosynthesis of QS signal acyl homoserine lactones (AHLs). Investigation of the regulatory effects of the stringent response gene rsh on QS related bioactivities were carried out using rsh mutants of Sphingobium japonicum UT26 and Sphingobium sp. SYK-6, both had three luxI homologs. Results indicated that deletion of rsh upregulated the overall production of AHLs and extracellular polymeric substances (EPS) in both UT26 and SYK-6 in rich medium, but affected expressions of these luxI/luxR homologs in different ways. In the poor medium (1% LB), rsh mutant of SYK-6 significantly lost AHLs production in broth cultivation but not in biofilm cultivation. The regulatory effects of rsh on QS activities were growth phase dependent in UT26 and culture condition dependent in SYK-6. Our results demonstrated the negative regulatory effect of rsh on QS activities in sphingomonads, which were very different from the positive effect found in sphingomonads containing only one luxI/R circuit. This study extends the current knowledge on the intricate networks between stringent response and QS system in sphingomonads, which would help to understand their survival advantage.

Engineering Novosphingobium aromaticivorans to produce cis,cis-muconic acid from biomass aromatics.

The platform chemical cis,cis-muconic acid (ccMA) provides facile access to a number of monomers used in the synthesis of commercial plastics. It is also a metabolic intermediate in the ß-ketoadipic acid pathway of many bacteria and, therefore, a current target for microbial production from abundant renewable resources via metabolic engineering. This study investigates Novosphingobium aromaticivorans DSM12444 as a chassis for the production of ccMA from biomass aromatics. The N. aromaticivorans genome predicts that it encodes a previously uncharacterized protocatechuic acid (PCA) decarboxylase and a catechol 1,2-dioxygenase, which would be necessary for the conversion of aromatic metabolic intermediates to ccMA. This study confirmed the activity of these two enzymes in vitro and compared their activity to ones that have been previously characterized and used in ccMA production. From these results, we generated one strain that is completely derived from native genes and a second that contains genes previously used in microbial engineering synthesis of this compound. Both of these strains exhibited stoichiometric production of ccMA from PCA and produced greater than 100% yield of ccMA from the aromatic monomers that were identified in liquor derived from alkaline pretreated biomass. Our results show that a strain completely derived from native genes and one containing homologs from other hosts are both capable of stoichiometric production of ccMA from biomass aromatics. Overall, this work combines previously unknown aspects of aromatic metabolism in N. aromaticivorans and the genetic tractability of this organism to generate strains that produce ccMA from deconstructed biomass.IMPORTANCEThe production of commodity chemicals from renewable resources is an important goal toward increasing the environmental and economic sustainability of industrial processes. The aromatics in plant biomass are an underutilized and abundant renewable resource for the production of valuable chemicals. However, due to the chemical composition of plant biomass, many deconstruction methods generate a heterogeneous mixture of aromatics, thus making it difficult to extract valuable chemicals using current methods. Therefore, recent efforts have focused on harnessing the pathways of microorganisms to convert a diverse set of aromatics into a single product. Novosphingobium aromaticivorans DSM12444 has the native ability to metabolize a wide range of aromatics and, thus, is a potential chassis for conversion of these abundant compounds to commodity chemicals. This study reports on new features of N. aromaticivorans that can be used to produce the commodity chemical cis,cis-muconic acid from renewable and abundant biomass aromatics.

The variations of native plasmids greatly affect the cell surface hydrophobicity of sphingomonads.

IMPORTANCE: Microbial cell surface hydrophobicity (CSH) reflects nonspecific adhesion ability and affects various physiological processes, such as biofilm formation and pollutant biodegradation. Understanding the regulation mechanisms of CSH will contribute to illuminating microbial adaptation strategies and provide guidance for controlling CSH artificially to benefit humans. Sphingomonads, a common bacterial group with great xenobiotic-degrading ability, generally show higher CSH than typical Gram-negative bacteria, which plays a positive role in organic pollutant capture and cell colonization. This study verified that the variations of two native plasmids involved in synthesizing outer membrane proteins and polysaccharides greatly affected the CSH of sphingomonads. It is feasible to control their CSH by changing the plasmid copy number and sequences. Additionally, considering that plasmids are likely to evolve faster than chromosomes, the CSH of sphingomonads may evolve quickly to respond to environmental changes. Our results provide valuable insights into the CSH regulation and evolution of sphingomonads.

Degradation of DDT by γ-hexachlorocyclohexane dehydrochlorinase LinA.

1,1,1-Trichloro-2,2-bis(4-chlorophenyl)-ethane (DDT) is the first synthetic insecticide and one of the most widely used pesticides. The use of DDT has been banned, but it remains one of the most notorious environmental pollutants around the world. In this study, we found that γ-hexachlorocyclohexane (γ-HCH) dehydrochlorinase LinA from a γ-HCH-degrading bacterium, Sphingobium japonicum UT26, converts DDT to 1,1-dichloro-2,2-bis(4-chlorophenyl)-ethylene (DDE). Because of the weak DDT degradation activity of LinA, we could not detect such activity in UT26 cells expressing LinA constitutively. However, the linA-deletion mutant of UT26 harboring a plasmid for the expression of LinA, in which LinA was expressed at a higher level than UT26, showed the DDT degradation activity. This outcome highlights the potential for constructing DDT-degrading sphingomonad cells through elevated LinA expression.

Comparative Genomic Analysis of Carbofuran-Degrading Sphingomonads Reveals the Carbofuran Catabolism Mechanism in Sphingobium sp. Strain CFD-1.

The worldwide use of the carbamate insecticide carbofuran has caused considerable concern about its environmental fate. Degradation of carbofuran by Sphingobium sp. strain CFD-1 is initiated via the hydrolysis of its ester bond by carbamate hydrolase CehA to form carbofuran phenol. In this study, another carbofuran-degrading strain, Sphingobium sp. CFD-2, was isolated. Subsequently, a cfd gene cluster responsible for the catabolism of carbofuran phenol was predicted by comparing the genomes of strains CFD-1, CFD-2, and Novosphingobium sp. strain KN65.2. The key genes verified to be involved in the catabolism of carbofuran phenol within the cfd cluster include the hydroxylase gene cfdC, epoxide hydrolase gene cfdF, and ring cleavage dioxygenase gene cfdE and are responsible for the successive conversion of carbofuran phenol, resulting in complete ring cleavage. These carbofuran-catabolic genes (cehA and the cfd cluster) are distributed on two plasmids in strain CFD-1 and are highly conserved among the carbofuran-degrading sphingomonad strains. The mobile genetic element IS6100 flanks cehA and the cfd gene cluster, indicating the importance of horizontal gene transfer in the formation of carbofuran degradation gene clusters. The elucidation of the molecular mechanism of carbofuran catabolism provides insights into the evolutionary scenario of the conserved carbofuran catabolic pathway. IMPORTANCE Owing to the extensive use of carbofuran over the past 50 years, bacteria have evolved catabolic pathways to mineralize this insecticide, which plays an important role in eliminating carbofuran residue in the environment. In this study, the cfd gene cluster, responsible for the catabolism of carbofuran phenol, was predicted by comparing sphingomonad genomes. The function of key enzymatic genes in this gene cluster was identified. Furthermore, the carbamate hydrolase gene cehA and the cfd gene cluster are highly conserved in different carbofuran-degrading strains. Additionally, the horizontal gene transfer elements flanking the cfd gene cluster were investigated. These findings help elucidate the molecular mechanism of microbial carbofuran degradation and enhance our understanding of the evolutionary mechanism of the carbofuran catabolic pathway.

First Case Report of Sphingobium lactosutens as a Human Pathogen Causing Peritoneal Dialysis-Related Peritonitis.

Novel pathogens keep evolving from time to time. In this article, we describe a rare case of the bacterium Sphingobium lactosutens causing peritoneal dialysis-related peritonitis in a patient who presented to our dialysis clinic with typical features of abdominal pain and diffuse abdominal tenderness and was successfully treated with the intraperitoneal antibiotic therapy. There were only very few cases of infections caused by Sphingobium species before. Here, we discuss the infections caused by other Sphingobium species, probable sources of infection, clinical findings, and susceptibility patterns. We also aim to create awareness about this rare bacterial pathogen and emphasize the need for more research to successfully treat and prevent future infections.

Opposite selection effects of nZVI and PAHs on bacterial community composition revealed by universal and sphingomonads-specific 16S rRNA primers.

Next-generation sequencing method employing the sphingomonads-specific and universal 16s rRNA primers were applied to analyze the response of soil bacterial community to continuous stress of nanoscale zero-valent iron (nZVI) and/or polycyclic aromatic hydrocarbons (PAHs) under suspended conditions. The group specific primer greatly improves taxonomic resolution, the overall OTU numbers increased from 12 by universal primer to 42 by sphingomonads-specific primer. It was found that nZVI and PAHs had opposite selection effects on bacterial community composition, furthermore, the abundance of Sphingopyxis was positively related to nZVI while negatively related to PAHs, whereas abundances of Sphingobium and Sphingosinicella were positively related to PAHs (p < 0.01). The improved taxonomic resolution helps identify the potential keystone taxa, which provides valuable information for future bioremediation technology such as synthetic microbiome.

Genome evolution related to γ-hexachlorocyclohexane metabolic function in the soil microbial population.

γ-Hexachlorocyclohexane (γ-HCH)-degrading strain, Sphingobium sp. TA15, was newly isolated from an experimental field soil from which the archetypal γ-HCH-degrading strain, S. japonicum UT26, was isolated previously. Comparison of the complete genome sequences of these 2 strains revealed that TA15 shares the same basic genome backbone with UT26, but also has the variable regions that are presumed to have changed either from UT26 or from a putative common ancestor. Organization and localization of lin genes of TA15 were different from those of UT26. It was inferred that transposition of IS6100 had played a crucial role in these genome rearrangements. The accumulation of toxic dead-end products in TA15 was lower than in UT26, suggesting that TA15 utilizes γ-HCH more effectively than UT26. These results suggested that genome evolution related to the γ-HCH metabolic function in the soil microbial population is ongoing.

Aromatic Dimer Dehydrogenases from Novosphingobium aromaticivorans Reduce Monoaromatic Diketones.

Lignin is a potential source of valuable chemicals, but its chemical depolymerization results in a heterogeneous mixture of aromatics and other products. Microbes could valorize depolymerized lignin by converting multiple substrates into one or a small number of products. In this study, we describe the ability of Novosphingobium aromaticivorans to metabolize 1-(4-hydroxy-3-methoxyphenyl)propane-1,2-dione (G-diketone), an aromatic Hibbert diketone that is produced during formic acid-catalyzed lignin depolymerization. By assaying genome-wide transcript levels from N. aromaticivorans during growth on G-diketone and other chemically-related aromatics, we hypothesized that the Lig dehydrogenases, previously characterized as oxidizing ß-O-4 linkages in aromatic dimers, were involved in G-diketone metabolism by N. aromaticivorans. Using purified N. aromaticivorans Lig dehydrogenases, we found that LigL, LigN, and LigD each reduced the Cα ketone of G-diketone in vitro but with different substrate specificities and rates. Furthermore, LigL, but not LigN or LigD, also reduced the Cα ketone of 2-hydroxy-1-(4-hydroxy-3-methoxyphenyl)propan-1-one (GP-1) in vitro, a derivative of G-diketone with the Cß ketone reduced, when GP-1 was provided as a substrate. The newly identified activity of these Lig dehydrogenases expands the potential range of substrates utilized by N. aromaticivorans beyond what has been previously recognized. This is beneficial both for metabolizing a wide range of natural and non-native depolymerized lignin substrates and for engineering microbes and enzymes that are active with a broader range of aromatic compounds. IMPORTANCE Lignin is a major plant polymer composed of aromatic units that have value as chemicals. However, the structure and composition of lignin have made it difficult to use this polymer as a renewable source of industrial chemicals. Bacteria like Novosphingobium aromaticivorans have the potential to make chemicals from lignin not only because of their natural ability to metabolize a variety of aromatics but also because there are established protocols to engineer N. aromaticivorans strains to funnel lignin-derived aromatics into valuable products. In this work, we report a newly discovered activity of previously characterized dehydrogenase enzymes with a chemically modified by-product of lignin depolymerization. We propose that the activity of N. aromaticivorans enzymes with both native lignin aromatics and those produced by chemical depolymerization will expand opportunities for producing industrial chemicals from the heterogenous components of this abundant plant polymer.

Expression of an alcohol dehydrogenase gene in a heterotrophic bacterium induces carbon dioxide-dependent high-yield growth under oligotrophic conditions.

Sphingobium japonicum strain UT26, whose γ-hexachlorocyclohexane-degrading ability has been studied in detail, is a typical aerobic and heterotrophic bacterium that needs organic carbon sources for its growth, and cannot grow on a minimal salt agar medium prepared without adding any organic carbon sources. Here, we isolated a mutant of UT26 with the ability to grow to visible state on such an oligotrophic medium from a transposon-induced mutant library. This high-yield growth under oligotrophic conditions (HYGO) phenotype was CO2-dependent and accompanied with CO2 incorporation. In the HYGO mutant, a transposon was inserted just upstream of the putative Zn-dependent alcohol dehydrogenase (ADH) gene (adhX) so that the adhX gene was constitutively expressed, probably by the transposon-derived promoter. The adhX-deletion mutant (UT26DAX) harbouring a plasmid carrying the adhX gene under the control of a constitutive promoter exhibited the HYGO phenotype. Moreover, the HYGO mutants spontaneously emerged among the UT26-derived hypermutator strain cells, and adhX was highly expressed in these HYGO mutants, while no HYGO mutant appeared among UT26DAX-derived hypermutator strain cells, indicating the necessity of adhX for the HYGO phenotype. His-tagged AdhX that was expressed in Escherichia coli and purified to homogeneity showed ADH activity towards methanol and other alcohols. Mutagenesis analysis of the adhX gene indicated a correlation between the ADH activity and the HYGO phenotype. These results demonstrated that the constitutive expression of an adhX-encoding protein with ADH activity in UT26 leads to the CO2-dependent HYGO phenotype. Identical or nearly identical adhX orthologues were found in other sphingomonad strains, and most of them were located on plasmids, suggesting that the adhX-mediated HYGO phenotype may be an important adaptation strategy to oligotrophic environments among sphingomonads.

TonB-Dependent Transporters in Sphingomonads: Unraveling Their Distribution and Function in Environmental Adaptation.

TonB-dependent transport system plays a critical role in the transport of nutrients across the energy-deprived outer membrane of Gram-negative bacteria. It contains a specialized outer membrane TonB-dependent transporter (TBDT) and energy generating (ExbB/ExbD) and transducing (TonB) inner membrane multi-protein complex, called TonB complex. Very few TonB complex protein-coding sequences exist in the genomes of Gram-negative bacteria. Interestingly, the TBDT coding alleles are phenomenally high, especially in the genomes of bacteria surviving in complex and stressful environments. Sphingomonads are known to survive in highly polluted environments using rare, recalcitrant, and toxic substances as their sole source of carbon. Naturally, they also contain a huge number of TBDTs in the outer membrane. Out of them, only a few align with the well-characterized TBDTs. The functions of the remaining TBDTs are not known. Predictions made based on genome context and expression pattern suggest their involvement in the transport of xenobiotic compounds across the outer membrane.

Transfer of Sphingorhabdus marina, Sphingorhabdus litoris, Sphingorhabdus flavimaris and Sphingorhabdus pacifica corrig. into the novel genus Parasphingorhabdus gen. nov. and Sphingopyxis baekryungensis into the novel genus Novosphingopyxis gen. nov. within the family Sphingomonadaceae.

During a phylogenetic analysis of Sphingorhabdus and its closely related genera in the family Sphingomonadaceae, we found that the genus Sphingorhabdus and the species Sphingopyxis baekryungensis might not be properly assigned in the taxonomy. Phylogenetic, phenotypic and chemotaxonomic characterizations clearly showed that the genus Sphingorhabdus should be reclassified into two genera (Clade I and Clade II), for which the original genus name, Sphingorhabdus, is proposed to be retained only for Clade I, and a new genus named as Parasphingorhabdus gen. nov. is proposed for Clade II with four new combinations: Parasphingorhabdus marina comb. nov., Parasphingorhabdus litoris comb. nov., Parasphingorhabdus flavimaris comb. nov. and Parasphingorhabdus pacifica comb. nov. Moreover, Sphingopyxis baekryungensis should represent a novel genus in the family Sphingomonadaceae, for which the name Novosphingopyxis gen. nov. is proposed, with a combination of Novosphingopyxis baekryungensis comb. nov. The study provides a new insight into the taxonomy of closely related genera in the family Sphingomonadaceae.

Whole genome sequences reveal the presence of 11 heterotypic synonyms in the genus Sphingobium and emended descriptions of Sphingobium indicum, Sphingobium fuliginis, Sphingobium xenophagum and Sphingobium cupriresistens.

Phylogenetic analysis based on 16S rRNA gene sequences of the genus Sphingobium showed the presence of four distinguishable clusters, in each of which the species shared almost the same evolutionary distance. They were Sphingobium indicum, Sphinogbium lucknowense, Sphinogbium chinhatense, Sphinogbium francense and Sphinogbium japonicum in cluster I, Sphinogbium barthaii and Sphinogbium fuliginis in cluster II, Sphinogbium hydrophobicum and Sphinogbium xenophagum in cluster III and Sphinogbium czechense and Sphinogbium cupriresistens in cluster IV. The 16S rRNA gene sequence similarities between the species in each cluster were all higher than 98 %. Genome-based average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) relatedness values between the species of each cluster were all higher than the threshold values of 95-96 % ANI and 70 % dDDH for species discrimination, respectively, suggesting that each cluster represents only one species of the genus Sphingobium. Due to priority of publication, S. lucknowense, S. chinhatense, S. francense and S. japonicum should be taken as later heterotypic synonyms of S. indicum, S. barthaii as a later heterotypic synonym of S. fuliginis, S. hydrophobicum as a later heterotypic synonym of S. xenophagum and S. czechense as a later heterotypic synonym of S. cupriresistens. Correspondingly, the descriptions of S. indicum, S. fuliginis, S. xenophagum and S. cupriresistens are also emended based on this study.

Comparative genome analysis reveals the evolution of chloroacetanilide herbicide mineralization in Sphingomonas wittichii DC-6.

The environmental fate of the extensively used chloroacetanilide herbicides (CH) has been a cause of increasing concern in the past decade because of their carcinogenic properties. Although microbes play important roles in CH degradation, Sphingomonas wittichii DC-6 was the first reported CH-mineralizing bacterium. In this study, the complete genome of strain DC-6 was sequenced and comparative genomic analysis was performed using strain DC-6 and other three partial CH-degrading bacteria, Sphingobium quisquiliarum DC-2, Sphingobium baderi DE-13, and Sphingobium sp. MEA3-1. 16S rDNA phylogenetic analysis indicated that strain DC-2, MEA3-1, and DE-13 are closely related and DC-6 has relatively distant genetic relationship with the other three strains. The identified CH degradation genes responsible for the upstream and downstream pathway, including cndA, cmeH, meaXY, and meaAB, were all located in conserved DNA fragments (or genetic islands) in the vicinity of mobile element proteins. Protein BLAST in the NCBI database showed that cndA and cmeH were present in the genomes of other sequenced strains isolated from various habitats; however, the gene compositions in these host strains were completely different from those of other sphingomonads, and codon usage of genes for upstream pathway were also different from that of downstream pathway. These results showed that the upstream and downstream pathways of CH degradation in strain DC-6 have evolved by horizontal gene transfer and gene combination. In addition, the genes of the ring-cleavage pathway were not conserved and may have evolved directly from bacterial degradation of hydroxyquinol. The present study provides insights into the evolutionary strategy and microbial catabolic pathway of CH mineralization.

A heterodimeric glutathione S-transferase that stereospecifically breaks lignin's ß(R)-aryl ether bond reveals the diversity of bacterial ß-etherases.

Lignin is a heterogeneous polymer of aromatic subunits that is a major component of lignocellulosic plant biomass. Understanding how microorganisms deconstruct lignin is important for understanding the global carbon cycle and could aid in developing systems for processing plant biomass into valuable commodities. Sphingomonad bacteria use stereospecific glutathione S-transferases (GSTs) called ß-etherases to cleave the ß-aryl ether (ß-O-4) bond, the most common bond between aromatic subunits in lignin. Previously characterized bacterial ß-etherases are homodimers that fall into two distinct GST subclasses: LigE homologues, which cleave the ß(R) stereoisomer of the bond, and LigF homologues, which cleave the ß(S) stereoisomer. Here, we report on a heterodimeric ß-etherase (BaeAB) from the sphingomonad Novosphingobium aromaticivorans that stereospecifically cleaves the ß(R)-aryl ether bond of the di-aromatic compound ß-(2-methoxyphenoxy)-γ-hydroxypropiovanillone (MPHPV). BaeAB's subunits are phylogenetically distinct from each other and from other ß-etherases, although they are evolutionarily related to LigF, despite the fact that BaeAB and LigF cleave different ß-aryl ether bond stereoisomers. We identify amino acid residues in BaeAB's BaeA subunit important for substrate binding and catalysis, including an asparagine that is proposed to activate the GSH cofactor. We also show that BaeAB homologues from other sphingomonads can cleave ß(R)-MPHPV and that they may be as common in bacteria as LigE homologues. Our results suggest that the ability to cleave the ß-aryl ether bond arose independently at least twice in GSTs and that BaeAB homologues may be important for cleaving the ß(R)-aryl ether bonds of lignin-derived oligomers in nature.

Transcriptome Analysis of Novosphingobium pentaromativorans US6-1 Reveals the Rsh Regulon and Potential Molecular Mechanisms of N-acyl-l-homoserine Lactone Accumulation.

In most bacteria, a bifunctional Rsh responsible for (p)ppGpp metabolism is the key player in stringent response. To date, no transcriptome-wide study has been conducted to investigate the Rsh regulon, and the molecular mechanism of how Rsh affects the accumulation of N-acyl-l-homoserine lactone (AHL) remains unknown in sphingomonads. In this study, we identified an rshUS6⁻1 gene by sequence analysis in Novosphingobium pentaromativorans US6-1, a member of the sphingomonads. RNA-seq was used to determine transcription profiles of the wild type and the ppGpp-deficient rshUS6⁻1 deletion mutant (∆rsh). There were 1540 genes in the RshUS6⁻1 regulon, including those involved in common traits of sphingomonads such as exopolysaccharide biosynthesis. Furthermore, both RNA-seq and quantitative real-time polymerase chain reaction (qRT-PCR) showed essential genes for AHL production (novI and novR) were positively regulated by RshUS6⁻1 during the exponential growth phase. A degradation experiment indicated the reason for the AHL absence in ∆rsh was unrelated to the AHL degradation. According to RNA-seq, we proposed σE, DksA, Lon protease and RNA degradation enzymes might be involved in the RshUS6⁻1-dependent expression of novI and novR. Here, we report the first transcriptome-wide analysis of the Rsh regulon in sphingomonads and investigate the potential mechanisms regulating AHL accumulation, which is an important step towards understanding the regulatory system of stringent response in sphingomonads.

Hydrolase CehA and Monooxygenase CfdC Are Responsible for Carbofuran Degradation in Sphingomonas sp. Strain CDS-1.

Carbofuran, a broad-spectrum systemic insecticide, has been extensively used for approximately 50 years. Diverse carbofuran-degrading bacteria have been described, among which sphingomonads have exhibited an extraordinary ability to catabolize carbofuran; other bacteria can only convert carbofuran to carbofuran phenol, while all carbofuran-degrading sphingomonads can degrade both carbofuran and carbofuran phenol. However, the genetic basis of carbofuran catabolism in sphingomonads has not been well elucidated. In this work, we sequenced the draft genome of Sphingomonas sp. strain CDS-1 that can transform both carbofuran and carbofuran phenol but fails to grow on them. On the basis of the hypothesis that the genes involved in carbofuran catabolism are highly conserved among carbofuran-degrading sphingomonads, two such genes, cehACDS-1 and cfdCCDS-1, were predicted from the 84 open reading frames (ORFs) that share ≥95% nucleic acid similarities between strain CDS-1 and another sphingomonad Novosphingobium sp. strain KN65.2 that is able to mineralize the benzene ring of carbofuran. The results of the gene knockout, genetic complementation, heterologous expression, and enzymatic experiments reveal that cehACDS-1 and cfdCCDS-1 are responsible for the conversion of carbofuran and carbofuran phenol, respectively, in strain CDS-1. CehACDS-1 hydrolyzes carbofuran to carbofuran phenol. CfdCCDS-1, a reduced flavin mononucleotide (FMNH2)- or reduced flavin adenine dinucleotide (FADH2)-dependent monooxygenase, hydroxylates carbofuran phenol at the benzene ring in the presence of NADH, FMN/FAD, and the reductase CfdX. It is worth noting that we found that carbaryl hydrolase CehAAC100, which was previously demonstrated to have no activity toward carbofuran, can actually convert carbofuran to carbofuran phenol, albeit with very low activity.IMPORTANCE Due to the extensive use of carbofuran over the past 50 years, bacteria have evolved catabolic pathways to mineralize this insecticide, which plays an important role in eliminating carbofuran residue in the environment. This study revealed the genetic determinants of carbofuran degradation in Sphingomonas sp. strain CDS-1. We speculate that the close homologues cehA and cfdC are highly conserved among other carbofuran-degrading sphingomonads and play the same roles as those described here. These findings deepen our understanding of the microbial degradation mechanism of carbofuran and lay a foundation for the better use of microbes to remediate carbofuran contamination.

Novel 16S rDNA primers revealed the diversity and habitats-related community structure of sphingomonads in 10 different niches.

It is believed that sphingomonads are ubiquitously distributed in environments. However detailed information about their community structure and their co-relationship with environmental parameters remain unclear. In this study, novel sphingomonads-specific primers based on the 16S rRNA gene were designed to investigate the distribution of sphingomonads in 10 different niches. Both in silico and in-practice tests on pure cultures and environmental samples showed that Sph384f/Sph701r was an efficient primer set. Illumina MiSeq sequencing revealed that community structures of sphingomonads were significantly different among the 10 samples, although 12 sphingomonad genera were present in all samples. Based on RDA analysis and Monte Carlo permutation test, sphingomonad community structure was significantly correlated with limnetic and marine habitat types. Among these niches, the genus Sphingomicrobium showed strong positive correlation with marine habitats, whereas genera Sphingobium, Novosphingobium, Sphingopyxis, and Sphingorhabdus showed strong positive correlation with limnetic habitats. Our study provided direct evidence that sphingomonads are ubiquitously distributed in environments, and revealed for the first time that their community structure can be correlated with habitats.

The Two-Component Monooxygenase MeaXY Initiates the Downstream Pathway of Chloroacetanilide Herbicide Catabolism in Sphingomonads.

Due to the extensive use of chloroacetanilide herbicides over the past 60 years, bacteria have evolved catabolic pathways to mineralize these compounds. In the upstream catabolic pathway, chloroacetanilide herbicides are transformed into the two common metabolites 2-methyl-6-ethylaniline (MEA) and 2,6-diethylaniline (DEA) through N-dealkylation and amide hydrolysis. The pathway downstream of MEA is initiated by the hydroxylation of aromatic rings, followed by its conversion to a substrate for ring cleavage after several steps. Most of the key genes in the pathway have been identified. However, the genes involved in the initial hydroxylation step of MEA are still unknown. As a special aniline derivative, MEA cannot be transformed by the aniline dioxygenases that have been characterized. Sphingobium baderi DE-13 can completely degrade MEA and use it as a sole carbon source for growth. In this work, an MEA degradation-deficient mutant of S. baderi DE-13 was isolated. MEA catabolism genes were predicted through comparative genomic analysis. The results of genetic complementation and heterologous expression demonstrated that the products of meaX and meaY are responsible for the initial step of MEA degradation in S. baderi DE-13. MeaXY is a two-component flavoprotein monooxygenase system that catalyzes the hydroxylation of MEA and DEA using NADH and flavin mononucleotide (FMN) as cofactors. Nuclear magnetic resonance (NMR) analysis confirmed that MeaXY hydroxylates MEA and DEA at the para-position. Transcription of meaX was enhanced remarkably upon induction of MEA or DEA in S. baderi DE-13. Additionally, meaX and meaY were highly conserved among other MEA-degrading sphingomonads. This study fills a gap in our knowledge of the biochemical pathway that carries out mineralization of chloroacetanilide herbicides in sphingomonads.IMPORTANCE Much attention has been paid to the environmental fate of chloroacetanilide herbicides used for the past 60 years. Microbial degradation is considered an important mechanism in the degradation of these compounds. Bacterial degradation of chloroacetanilide herbicides has been investigated in many recent studies. Pure cultures or consortia able to mineralize these herbicides have been obtained. The catabolic pathway has been proposed, and most key genes involved have been identified. However, the genes responsible for the initiation step (from MEA to hydroxylated MEA or from DEA to hydroxylated DEA) of the downstream pathway have not been reported. The present study demonstrates that a two-component flavin-dependent monooxygenase system, MeaXY, catalyzes the para-hydroxylation of MEA or DEA in sphingomonads. Therefore, this work finds a missing link in the biochemical pathway that carries out the mineralization of chloroacetanilide herbicides in sphingomonads. Additionally, the results expand our understanding of the degradation of a special kind of aniline derivative.