What is Sphingomonas?

Sphingomonas is a rich new type of microbial resources that can be used for biodegradation of aromatic compounds. The strain of this genus has great potential for application in environmental protection and industrial production due to its high metabolic capacity and multifunctional physiological characteristics. However, due to the late understanding of Sphingomonas, the ecological and economic value of this bacterium has been paid little attention, and its research has remained at the primary stage.

Sphingomonas

Japanese scholar Yabuuchi et al. First proposed Sphingomonas in 1990 , and renamed the strain Pseudomonas paucimobilis isolated from hospital clinical samples by Holmes et al. In 1977 as Sphingomonas paucimobilis and identified it as Sphingomonas Of the typical strains and describes the physiological and biochemical characteristics of the genus. Takeuchi revised this in 1993. Based on 16S rRNA sequence comparisons, Sphingomonas belongs to the 4 subclass of Proteus. The strains of this genus are all Gram-negative bacteria without spores. They move with unilateral polar flagella, mostly yellow, obligate aerobic and can produce catalase. In addition to inulin, Sphingomonas can convert pentose, hexose, and disaccharides to acids. The main respiratory chain in the bacteria is ubiquinone Q-10, and the fatty acids in cell lipids are mainly 18: 1 and 2OH14: 0. The G + C content of DNA is between 61.6% and 67.8%. The glycosphingolipid component in the cell membrane is Sphingomonas, which is an important feature that distinguishes it from other typical Gram-negative bacteria.

Because Sphingomonas has a very extensive metabolic capacity for aromatic compounds, and some species of this genus can synthesize valuable extracellular biopolymers. Therefore, Sphingomonas has become a focus of attention and research in recent years.

Degradation characteristics

Sphingomonas are ubiquitous in the environment, and they are found in river water, rhizosphere, surface and deep underground sediments, oceans, and even polar soils. A large number have been separated from the environment. The degradation characteristics of sphingomonas are related to the degradation of biomass polymers and the like. Sphingomonas have unique advantages in the degradation of polycyclic aromatic hydrocarbons (PAHs) and hexahexan (HCH) isomers. S. Yanoikuyae B1 strain can degrade monocyclic aromatic hydrocarbons, biphenyls, substituted aromatic compounds and PAHs, and is a model strain of Sphingomonas. The B1 strain metabolizes monocyclic aromatic hydrocarbons, such as xylene and toluene, using the TOL-plasmid metabolism pathway; benzoic acid is metabolized by the meta-cleavage pathway to generate acetaldehyde and pyruvate, as shown in Figure 1. Although many microorganisms can degrade the HCH isomer, HCH, only S. The degradation pathway of HCH by japonicum UT26 is the most detailed. The most important step of HCH degradation is the dechlorination reaction, as shown in Figure 2: The degradation of y-HCH to 2,5-dichlorohydroquinone (2,5-DCHQ) involves the upstream pathway, and the continued degradation of 2,5-dichlorohydroquinone belongs to the downstream pathway. Sphingomonas

Bacteria can also degrade anthraquinone dyes and their intermediates.

Taxonomic research

With the development of microbial community analysis technology, taxonomic studies of Sphingomonas have also gone through three stages. The first stage is to identify the taxonomy of Sphingomonas by the traditional method of culture and isolation, that is, the comparison of morphology, culture and physiological and biochemical characteristics. However, due to the similarity of phenotypic characteristics of Sphingomonas to other species, it is easy to be mistakenly attributed to other genus, so this method is gradually replaced by the second-stage biomarker taxonomy. The biomarker classification method is a classification method for determining the belonging of a microorganism by qualitative and quantitative analysis by extracting chemical components (ie, biomarkers) unique to the microorganism. According to the characteristics of Sphingomonas, several unique biomarker classification methods have gradually formed, namely: color analysis, respiratory quinone system analysis, polyamine mode analysis, and polar lipid and fatty acid profile analysis.

Color analysis

Most Sphingomonas strains are yellow. This pigment is easily extracted with acetone and usually has characteristic absorption peaks at 452 nm and 480 nm. S. The yellow pigment of paucimobilis was identified as nostoxan-thin. In contrast, S. Yanoikuyae strain contains less pigment, RW1 and Alcaligenes sp. The A175 strain does not contain pigment. In recent years, researchers have also isolated some orange Sphingomonas. Therefore, yellow cannot be a characteristic of Sphingomonas and should be used in combination with other analytical methods.

Respiratory quinone system analysis

Respiratory quinones are the constituents of the cell membrane that play an electron transfer function. There are mainly two types of respiratory quinones: ubiquinone (coquinone Q) and menaquinone (vitamin K). Each microorganism contains a predominant quinone. Analysis of the respiratory quinone system of Sphingomonas species revealed that they all contained 10 isoprene-like ubiquinone groups on the side chain. 10 Although this feature is not limited to sheaths

Ammoniomonas are also contained in most strains of the subclass Proteus, but from the homology of the respiratory quinone system of Sphingomonas, it is possible that all members of the genus Sphingomyelin contain ubiquinone Q-10.

Polyamine mode and shape analysis of polar lipids and fatty acids

In the genus Sphingomonas, two main polyamine patterns have been observed: one pattern contains a large amount of triamine spermidine and a small amount of variable putrescine, spermidine, and spermine; in The main polyamines in the second mode are triamine spermidine and a small amount of putrescine and spermine. These two modes divide Sphingomonas into two broad categories. The first mode exists only in the Cluster I and RW1 and A175 strains, and the second mode exists in the remaining strains (Figure 3). Analysis of polar lipids and fatty acid profiles showed that all the lipids of Sphingomonas contained phosphatidylethanolamine (PE), phosphatidylglycerol (PG), bisphosphatidylglycerol (DPG), and sphingomyelin (SGL) ). The fatty acids in cell lipids are mainly 18: 1 and 20H14: 0. Busse et al. Found that the analysis of polyamine mode and quinone system is only suitable for the preliminary identification of Sphingomyces strains. In contrast, the analysis of polar lipids and fatty acid profiles is based on the detection of 80% of relatives of sphingosine glycolipids. Identification is a better method. In general, when these two types of methods are used in combination, sphingomonas can be identified at the species level.

However, the classification of biomarkers still has certain limitations. With the development of molecular biology, modern molecular biology taxonomy has gradually matured, and the taxonomic research of Sphingomonas has entered the third stage. Therefore, according to the comparison of 16S rRNA sequences of Sphingomonas and some proteus species, Sphingomonas can be divided into at least 4 clusters.

Research on Degrading Enzymes and Genes

Among the aerobic degradation pathways of aromatic compounds, aromatic ring hydroxylated dioxygenase and cleaved dioxygenase are considered to be the most critical enzymes. They are related to the degradability and degree of degradability of the compound. Gibson and other associates believe that dioxygenase is a multi-component and NADH-dependent enzyme system, consisting of three parts: Fe-S flavin protein, ferredoxin, and Rieske-type Fe-S oxidase.

For bacteria related to the degradation of polycyclic aromatic hydrocarbons (PAHs), the most studied are the genes that degrade naphthalene and phenanthrene. PAHs degrading genes using naphthalene or phenanthrene often have high homology among PAHs degrading genes, especially those encoding the dioxygenase component. Because sphingomonas can utilize both high molecular weight and low molecular weight aromatic compounds, it has been considered as a type of microbial resource with metabolic diversity in recent years. Studying the genetics of Sphingomonas degrading PAHs can better explain why Sphingomonas can grow in various aromatic compounds. In some Sphingomonas, the sequences of genes involved in the degradation of PAHs are clearly similar, but these genes are different from those in the previously described Pseudomonas. Use S. The PAHs deletion mutant of yanoikuyae B1 strain assisted gene identification. It was found that several mutant strains accumulated dihydrodiol and the expression of dehydrogenase gene was blocked. The two types of meta-cleavage dioxygenases are transcribed in opposite directions, suggesting that one inter-cleavage dioxygenase functions in the upstream pathway and the other acts in the downstream pathway, which is in contrast to previously reported false The operons of the upstream and downstream pathways of naphthalene in the genus Monomonas are similar. In addition, an operon similar to the TOL of Pseudomonas was found in the chromosome of B1. Although this gene is arranged differently from the TOL plasmid, B1 and TOL of Pseudomonas

Genes have a high degree of nucleic acid homology. S. aromaticivorans F199 is a typical representative of Sphingomonas isolated from deep underground. The sequence of its 184 kb metabolic plasmid PNL1 has been obtained. Approximately half of the DNA sequence encodes genes for aromatic metabolism, aromatic Transport and detoxification (eg, glutathione-s-transferase), and the other half encodes for plasmid replication, conjugation, transfer and maintenance. The gene sequence and sequence of F199 plasmid DNA is highly conserved with chromosomes on PAHs degrading operons in Bl. In addition, according to the inferred amino acid sequence, the enzyme related to aromatic conversion in F199 has been detached from Pseudomonas, and it may partially explain the metabolic diversity of Sphingomonas. Recent genetic homology studies have found that the genes for biphenyl and m-xylene degradation in strain Bl isolated from underground soil are very similar to the genes for five deep underground isolates (F199, B0522, B0695, B0478, B0712). similar. However, the degradation genes of the underground strains are located on the chromosome, while the degradation genes of the deep underground strains are on the plasmid. Few genes have been reported about Sphingomonas that degrade high molecular weight PAHs. Low molecular weight PAHs, such as naphthalene, can be easily degraded by bacteria, but the more stubborn high molecular PAHs have little research on their biodegradation pathways, but it has been proven that their primary metabolism is catalyzed by dioxygenase. Sandrine et al. CHY-1 related PAHs degrading enzymes. Sequencing the genes encoding two aromatic ring hydroxylated dioxygenases (Phn I and Phn lI) found that the co-cluster metabolic genes at two different loci are corresponding to those in F199. Gene similarity is high, and the single enzyme Phn I may be related to the initial steps of CHY-1 metabolizing PAHs.

Study of sphingolipids

Sphingomyelin is a lipid formed by sphingosine and fatty acids and is widely found in the cell membranes of mammals and certain bacteria and fungi. At present, some mature methods for purifying sphingolipids from bacteria have been developed. Sphingomonas contains an unusual envelope, that is, there is no lipopolysaccharide in other Gram-negative bacteria in the outer membrane, but there are glycolipids. Studies have found that these glycolipids are composed of dihydrosphingosine, 2 monohydroxy fatty acids, and glucuronic acid, which is why Sphingomonas is named. Later, nuclear magnetic resonance spectroscopy and chemical analysis finally proved that this glycolipid is a sphingoglycolipid, and its structure is shown in Figure 4. Due to the highly hydrolyzable nature of sphingosine, its detection is more difficult. Some scholars have detected the chemical structure of the glycosphingolipid component through improved analytical purification methods, and found that sphingosine-1 in all Sphingomyces (C-SL-1) is the main component of the outer membrane structure.

In addition, Kawahara et al. Studied the cell membrane structure of spauci-mobilis and its glycosphingolipid function. It was found that, because Sphingomonas is different from other Gram-negative cell membranes in its membrane characteristics, the hydrophobic surface formed is more beneficial to the survival of these bacteria in the ecological environment and the uptake of aroma compounds. More research indicates that despite the fat

Carbohydrates and glycosphingolipids differ in details, but their functions are basically similar as part of the surface structure and outer membrane structure of the antigen. Since the content of sphingolipids is different between genus and species, this component also serves as a biomarker to assist other methods for identifying and classifying Sphingomonas.

Research on Produced Polymers

Many bacterial cells can produce polysaccharides, which are either adsorbed on the cell membrane surface as the O-antigen of lipopolysaccharide (LPS), or form a capsule around the cell, or are secreted completely as extracellular polysaccharides (EPS). Microbial EPS is a long-chain high-molecular polymer. Its unique physical and rheological properties, as well as its safety in use, make it popular in the food and other industries. Sphingomonas has attracted wide attention for its ability to produce extracellular biopolymers. It has been reported that some Sphingomonas sp. Can produce jelly-like extracellular polysaccharides. With its own acid stability and structural diversity, jelly gum can replace agar and is widely used in the food and production industries. The current research work mainly focuses on how to increase the output of jelly gum and realize industrial production. Studies have shown that Xanthomonas campestris strain can produce another important extracellular polymer, xanthan gum, and found 12 co-cluster chromosome genes encoding this response. Pollock et al. Used gene recombination technology to implant these genes into sphingomonas to achieve the synthesis of xanthan gum in the genus Sphingomyces. These xanthan gums are not structurally and functionally similar to natural xanthan gums. the difference. The above research proves that the production of extracellular polymers can be achieved through the mutual transfer of genes between bacteria, and also shows the technical feasibility of synthesizing xanthan gum and other extracellular polymers in non-hosts.

Conclusion and Outlook

Aromatic compounds are common pollutants in the environment, and biological methods to remove such pollutants are promising methods. The widespread distribution of Sphingomonas, its specific effect on refractory aromatic compounds with complex structures, and its superiority in the production of biopolymers have attracted more and more attention. However, due to its late understanding, most of the research on Sphingomonas remains in the primary stage. Currently, the main reports mostly focus on the isolation of the strain, degradation pathway, optimization of degradation conditions, and analysis of degradation products. On the other hand, the enzyme systems and related genes that degrade aromatic compounds are still less involved, especially the high-molecular-weight aromatic compounds. Combined with relevant international research, the following aspects need to be further explored in the future.

(1) In the production of extracellular polymers, the removal of proteins from the gum is a technical obstacle to the purification of gum polymers. The commonly used Sevag method, alkaline protease method, papain and neutral protease method each have certain limitations. Sex. Therefore, the development of an efficient deproteinization method can ensure the quality of the polymer gum, and at the same time look for functional genes for the production of polymer gum by Sphingomonas and express them for large-scale production;

(2) As an important feature of Sphingomonas, the physiological significance of sphingolipids in pollutant metabolism needs to be studied;

(3) The cloning and expression of related functional genes in the process of biodegradation of Sphingomonas, and the use of biotechnological means to construct efficient genetic engineering bacteria and apply them to the treatment of environmental pollution. In addition, the phenomenon that some Sphingomonas spp. Can cause corrosion of water distribution pipes and infect plant pathogens should attract researchers' attention. With further research on the physiological and ecological potential of this genus, Sphingomonas will have broad application prospects.