Sphingobacterium Spiritivorum Research Articles

Investigating the bacterial consortia properties of electrogenic anodic biofilm in a double-chamber microbial fuel cell: electrochemical, physical, biochemical and molecular characterization

This work evaluated the electrochemical, physical, biochemical, and molecular characterization of electrogens from a graphite felt anode when zinc oxide on activated carbon (ZnO/AC) was used as a cathodic electrocatalyst in a double-chambered microbial fuel cell (DCMFC). The electrochemical polarization behavior of the DCMFC showed that ZnO/AC had a higher power density (PDmax) of 89 mW m−2 with a corresponding cell current density (CD) of 248 mA m−2 and a voltage output of 395 mV, which was higher than those of the blank electrode used as a benchmark (PDmax of 68 mW m−2 at a CD of 161 mA m−2 and a voltage of 421 mV). Furthermore, scanning electron microscopy and transmission electron microscopy revealed that the morphology and interior properties of the strains varied among the rods (bacilli), spirals (vibrios), and spheres (diplococci, staphylococci and streptococci). In addition, biochemical characterization via the Vitek2 compact system and molecular analysis via 16 S rRNA and 18 S rRNA gene sequencing revealed the occurrence of nine prevalent species that were correlated with Sphingobacterium spiritivorum, Ochrobactrum anthropicus, Pseudomonas mendocina, Stenotrophomonas maltophilia, Leuconostoc mesenteroides, Staphylococcus equorum, Bacillus subtilis HQ334981.1, Kocuria kristinae KC581674.1 and Saccharomyces cerevisiae NR111007.1. Consequently, the present study outlines different characterization strategies for electrogenic microbes that play an important role in the overall performance of DCMFC for scaling up and managing existing environmental pollution for sustainable energy generation.

Potential of the Bioaugmentation Method to Enhance the Remediation Process in the Solidification/Stabilization Method

In the published literature, Stabilization/Solidification (S/S) method was excellent in remediating heavy metals, whereas bioremediation was excellent in remediating organic contaminants. However, tremendous papers onlyfocus on specific contaminants, while the polluted environment is not due to specific contaminants. In this paper, both methods were combined and targeted to remediate both group contaminants, namely organic contaminants and heavy metals. The study focused on the effect of the bioaugmentation method on S/S method with the use of Portland cement as a binder in the S/S method. Sphingobacterium spiritivorum(bacteria) and Aspergillus brasiliensis(fungi) as degrading agents in the bioaugmentation method. The S/S matrices were tested for leaching behaviour which is the concentration of Chemical Oxygen Demand (COD) and heavy metals after 14 and 28 days of hydration. Bacteria augmentation showed a better COD removal compared to without augmentation (S/S method only). Bacteria augmentation also shows better removal compared to fungi augmentation for both hydration days. In addition, by using the S/S method only, COD concentration was increased from 14 to 28 days. For heavy metals, chromium shows a positive result; bacteria augmentation shows the lowest concentration after 14 and 28 days of hydration. While other metals, S/S method only shows the lower concentration compared to samples that augment with bacteria and fungi. Nonetheless, the concentrations of heavy metals in all samples did not exceed the USEPA limit. Therefore, this study contributed to the possibility of combining the S/S and bioaugmentation methods by adding degrading agents to remediate contaminants in the sludge, especially for sludge that containshigh organic contaminants.

Bioremediation of fibreboard sludge by using bacteria and fungi

Sludge produced from the fibreboard industry releases hazardous substances that may damage the environment as well as human health via inhaled air, drinking water, and food consumed. Correspondingly, the aim of this study was to focus on the bioremediation to treat industrial wastewater sludge produced from Evergreen Fibreboard, Parit Raja, Batu Pahat, Johor by using bacteria (Sphingobacterium spiritivorum) and fungi (Aspergillus brasiliensis). Three stages of the investigation were performed, which are the collection of raw materials, the preparation of microbes, and sample analysis, including the determination of COD and heavy metals including Chromium (Cr), Copper (Cu), Arsenic (As), Lead (Pb) and Zink (Zn). From the Chemical Oxygen Demand (COD) test, Aspergillus brasiliensis shows higher potential for sludge bioremediation as 61 mg/L COD was reduced in 14 days compared to the Sphingobacterium spiritivorum which only 31 mg/L was reduced. Meanwhile, the leaching result for heavy metals shows that Zn had the highest concentrations and As was the lowest for 14 and 28 days. Inoculation of Aspergillus brasiliensis shows the lowest leaching concentration for heavy metal elements such as Cr, Cu, As, Pb and Zn compared to Sphingobacterium spiritivorum. The reduction concentrations of these elements are 13%, 22%, 17%, 33% and 6%, respectively. The result shows that the fungi strain has the highest capability to absorb metals compared to the bacteria strain. Indigenous microorganisms in sludge have the lowest potential to reduce COD and heavy metals. However, a combination with the other method such as physical and chemical processes would be recommended to improve the results in the future.

Hemodialysis catheter-associated infection by Sphingobacterium Spiritivorum

Introduction: Sphingobacterium spiritivorum is a rare cause of human infections worldwide. It occurs in immunocompromised patients, and its treatment is a clinical challenge. Clinical case: A 46-year-old woman was admitted to a hospital in Medellín with acute hypoxemic respiratory failure secondary to severe pneumonia due to SARS-CoV-2 and requiring mechanical ventilation. He developed acute renal failure, requiring hemodialysis through a right jugular catheter. At 48 hours after insertion, signs of a systemic inflammatory response developed, and Sphingobacterium spiritivorum was cultured in all cultures. Evolution: Intravenous ciprofloxacin was started for seven days with favorable clinical evolution and control culture at 48 hours without microbiological isolation, so it did not require the removal of the hemodialysis catheter. After this, with improvement in her admission condition, she was transferred to the general hospitalization ward and later discharged. Conclusion: In this case, severe SARS/CoV-2 was aggravated by acute renal failure and septicemia superadded by S. Spiritivorum.

A cross sectional study on airborne inhalable microorganisms, endotoxin, and particles in pigeon coops – Risk assessment of exposure

Pigeon breeding is associated with symptoms of the airways. The aim of this study is to illuminate the bacteriological and toxicological characteristics of airborne dust in pigeon coops. Airborne dust was sampled in 31 urban pigeon coops with homing and fancy pigeons, and following the dust was characterized. In total 141 different bacterial species were identified using MALDI-TOF MS, and of these 11 species are classified in risk group 2. Of the cultivable bacteria, Staphylococcus equorum was present in the highest concentration. Microorganisms in the dust were able to form biofilm, and the amount correlated positively with the number of bacteria. Next generation sequencing showed 180 genera with Acinetobacter in highest reads. On average 999 ± 225 ZOTUs were observed per sample with a Shannon-Wiener biodiversity index of 6.17 ± 0.24. Of the identified species the following have previously been suggested as causative agents of extrinsic allergic alveolitis: Alcaligenes faecalis, Bacillus subtilis, Pantoea agglomerans, Sphingobacterium spiritivorum, Thermoactinomyces sp., and Streptomyces albus. Staphylococcus was present on particles with sizes between 1.1 and > 7.0 μm with a geometric mean diameter of particles on 4.7 ± 1.1 μm. Concentrations of airborne endotoxin and dust were elevated compared to references, and the geometric mean concentrations were 102 EU/m3 and 1.07 mg dust/m3, respectively. Upon exposure to the airborne dust human granulocytes produced Reactive Oxidative Species during the first 5 min, and then no further reaction was observed. The concentrations of bacteria in general, Staphylococcus spp., and endotoxin and biodiversity were associated significantly with season, temperature and/or relative humidity, but not with type or density of pigeons. The bacterial composition and biodiversity indices were not affected by type of pigeon. In conclusion, the exposure to bacteria and endotoxin in pigeon houses should not be neglected in the evaluation of causative agents of airways symptoms among pigeon breeders.

Bacteria occurring in onion (Allium cepa L.) foliage in Puerto Rico.

Bacteria associated with foliar symptoms of onion (Allium cepa L.) were examined in the southern region of Puerto Rico from January through April 2004. Different symptoms were observed in onion foliage of cultivars 'Mercedes' and 'Excalibur' at Juana Díaz and Santa Isabel, Puerto Rico. Ellipsoidal sunken lesions with soft rot and disruption of tissue were the most common symptoms observed in onion foliage in field conditions. From a total of 39 bacterial strains isolated from diverse symptoms in onion foliage, 38% were isolated from soft rotting lesions. Ninety-two percent of the bacteria isolated from onion foliage was Gram negative. Pantoea spp. with 25%, was the most frequently isolated genus, followed by Pasteurella spp. and Serratia rubidae with 10% each. Fifty- six percent of the strains held plant pathogenic potential; these strains belong to the genera Acidovorax sp., Burkholderia sp., Clavibacter sp., Curtobacterium sp., Enterobacter sp., Pantoea spp., Pseudomonas spp., and Xanthomonas spp. Pathogenicity tests showed that seven out of eight tested bacterial strains evaluated under field conditions caused symptoms in onion foliage for both cultivars. Acidovorax avenae subsp. citrulli, Burkholderia glumae, Pantoea agglomerans, P. dispersa, Pseudomonas sp., Xanthomonas sp., and Xanthomonas-like sp. were pathogenic to leaf tissues. Clavibacter michiganensis was not pathogenic to leaf tissues. Other bacteria identified as associated with onion leaf tissue were Curtobacterium flaccumfaciens, Cytophaga sp., Enterobacter cloacae, Flavimonas oryzihabitans, Mannheimia haemolytica, Pantoea stewartii, Pasteurella anatis, P. bettyae, P. langaaensis, Photobacterium damselae, Pseudomonas syringae pv. aptata, Rhizobium radiobacter, Serratia rubidae, Sphingobacterium spiritivorum, Sphingomonas sanguinis, and an unknown strain. This paper is the first survey of bacteria associated with onion foliage in Puerto Rico. The role of non- phytopathogenic bacteria associated with the life cycle of onion under field conditions remains unknown.

Circulating microbial small RNAs are altered in patients with rheumatoid arthritis

ObjectivesTo determine if plasma microbial small RNAs (sRNAs) are altered in patients with rheumatoid arthritis (RA) compared with control subjects, associated with RA disease-related features, and altered by disease-modifying antirheumatic...

A Case of Sphingobacterium spiritivorum Bacteremia and Literature Review

Abstract High sphingophospholipids in the cell membrane characterize the genus Sphingobacterium. Sphingobacterium spiritivorum is a gram-negative, non–spore-forming, nonmotile bacterium that is ubiquitous in nature and rarely involved in human disease. It is intrinsically resistant to many antimicrobials, which if underrecognized can be potentially life-threatening. We present a case of a 72-year-old white man who presented with cellulitis and sepsis secondary to S. spiritivorum.

Sphingobacterium spiritivorum bacteremia due to cellulitis in an elderly man with chronic obstructive pulmonary disease and congestive heart failure: a case report

BackgroundSphingobacterium spiritivorum is a glucose non-fermenting Gram-negative rod, formerly classified as one of the Flavobacterium species. It is characterized by a large number of cellular membrane sphingophospholipids. Sphingobacterium species are ubiquitous and isolated from natural environments, such as soil and water. However, they rarely cause infections in humans. Only a limited number of cases have been reported in elderly and immunocompromised patients with underlying diseases and predisposing factors.Case presentationAn 80-year-old Japanese man with chronic obstructive pulmonary disease and congestive heart failure visited the Kariya Toyota General Hospital, Aichi, Japan with the chief complaint of fever accompanied by chills and left leg pain. At initial presentation, he was distressed and dyspneic. He was febrile (38.8 °C), and his left foot was swollen with reddening and tenderness. We diagnosed him as having cellulitis, and he was hospitalized for antibiotic therapy. Initially, he was treated with intravenously administered cefazolin, but after the isolation of a glucose non-fermenting Gram-negative rod from blood cultures, we decided to switch cefazolin to intravenously administered meropenem on day 4, considering the antibiotic resistance of the causative organism. The causative organism was identified as S. spiritivorum on day 6. His condition gradually stabilized after admission. Meropenem was switched to orally administered levofloxacin on day 12. He was discharged on day 16 and treated successfully without any complications.ConclusionsAlthough S. spiritivorum is rare, with limited cases isolated from cellulitis, it should be considered as a causative organism in elderly and immunocompromised patients with cellulitis. Blood cultures are the key to correct diagnosis and appropriate treatment.

Sphingobacterium cibi sp. nov., isolated from the food-waste compost and emended descriptions of Sphingobacterium spiritivorum (Holmes et al. 1982) Yabuuchi et al. 1983 and Sphingobacteriumthermophilum Yabe et al. 2013.

A novel, pale yellow-coloured, Gram-staining-negative, aerobic, rod-shaped and non-motile bacterium, designated as strain CC-YY411T, was isolated from the food-waste compost and subjected to polyphasic taxonomy. Strain CC-YY411T exhibited the highest pairwise 16S rRNA gene sequence similarity to Sphingobacterium thermophilum JCM 17858T (94.6 %) while sharing 94.1 to 89.7 % similarities with other Sphingobacterium species. Novel strain established a discrete phylogenetic lineage within the clade that accommodated validly established members of the genus Sphingobacterium. The polar lipid profile of strain CC-YY411T contained major amounts of phosphatidylethanolamine, one unidentified lipid and two unidentified aminolipids besides accommodating trace amounts of a sphingolipid, two phospholipids, an unidentified aminophospholipid and an unidentified glycolipid. The DNA G+C content of strain CC-YY411T was 34.5 mol%. The major and minor respiratory quinones were MK-7 (89.1 %) and MK-6 (10.9 %), respectively. The predominant fatty acids were iso-C15 : 0 (24.0 %), iso-C17 : 0 3-OH (11.9 %), iso-C15 : 0 3-OH (6.9 %), C15 : 1ω5c (5.5 %) and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) (32.0 %). Based on the phylogenetic, phenotypic and chemotaxonomic distinctiveness, strain CC-YY411T represents a novel species of Sphingobacterium, for which the name Sphingobacteriumcibi sp. nov. is proposed. The type strain is CC-YY411T (=BCRC 80430T=JCM 18407T). Amended species descriptions of Sphingobacterium spiritivorum(Holmes et al. 1982) Yabuuchi et al. 1983 and Sphingobacterium thermophilum Yabe et al. 2013 are also proposed based on new data obtained in this study.

Enhancing Performance of Sphingobacterium spiritivorum in Bioremediation Phenanthrene Contaminated Sand

Many biodegradation studies have focused on survival of isolated bacteria to increase the bacteria population and subsequently enhance the efficiency of polycyclic aromatic hydrocarbon (PAH) biodegradation. However, there is limited research on enhancing the performance of isolated bacteria through reinoculation. Thus, this study was designed to evaluate the effects of reinoculation on the performance of Sphingobacterium spiritivorum in degradation of phenanthrene contaminated sand. Experiments were performed in three different reactors. Inoculation was performed once (day 0) in reactor 1. In reactor 2, inoculation was performed twice (day 0 and day 5). The bacteria was isolated from reactor 2 and inoculated into reactor 3. The study results show reactor 3 having the highest degradation rate (13.61 mg/kg/day) and percentage removal (95.36 percent). In contrast, without reinoculation in reactor 1, 68.93 percent of phenanthrene was removed. Thus, the performance of S. spiritivorum in phenanthrene degradation can be enhanced through reinoculation. © 2014 Wiley Periodicals, Inc.

Application of Biosurfactant from Sphingobacterium spiritivorum AS43 in the Biodegradation of Used Lubricating Oil

This study aimed at investigating the application of biosurfactant from Sphingobacterium spiritivorum AS43 using molasses as a substrate and fertilizer to enhance the biodegradation of used lubricating oil (ULO). The cell surface hydrophobicity of bacteria, the emulsification activity, and the biodegradation efficiency of ULO were measured. The bacterial adhesion in the hydrocarbon test was used to denote the cell surface hydrophobicity of the used bacterial species. The results indicate a strong correlation between cell surface hydrophobicity, emulsification activity, and the degree of ULO biodegradation. The maximum degradation of ULO (62 %) was observed when either 1.5 % (w/v) of biosurfactant or fertilizer was added. The results also revealed that biosurfactants alone are capable of promoting biodegradation to a large extent without added fertilizer. The data indicate the potential for biosurfactant production by using low-cost substrate for application in the bioremediation of soils contaminated with petroleum hydrocarbons or oils.

The First Korean Case ofSphingobacterium spiritivorumBacteremia in a Patient with Acute Myeloid Leukemia

Sphingobacterium spiritivorum has been rarely isolated from clinical specimens of immunocompromised patients, and there have been no case reports of S. spiritivorum infection in Korea to our knowledge. We report a case of S. spiritivorum bacteremia in a 68-yr-old woman, who was diagnosed with acute myeloid leukemia and subsequently received chemotherapy. One day after chemotherapy ended, her body temperature increased to 38.3℃. A gram-negative bacillus was isolated in aerobic blood cultures and identified as S. spiritivorum by an automated biochemical system. A 16S rRNA sequencing analysis confirmed that the isolate was S. spiritivorum. The patient received antibiotic therapy for 11 days but died of septic shock. This is the first reported case of human S. spiritivorum infection in Korea. Although human infection is rare, S. spiritivorum can be a fatal opportunistic pathogen in immunocompromised patients.

Bacterial sphingophospholipids containing non-hydroxy fatty acid activate murine macrophages via Toll-like receptor 4 and stimulate bacterial clearance

Sphingobacterium spiritivorum has five unusual sphingophospholipids (SPLs). Our previous study determined the complete chemical structures of these SPLs. The compositions of the long-chain bases/fatty acids in the ceramide portion, isoheptadecasphingosine/isopentadecanoate or isoheptadecasphingosine/2-hydroxy isopentadecanoate, are characteristic. The immune response against bacterial lipid components is considered to play important roles in microbial infections. It is reported that several bacterial sphingolipids composed of ceramide are recognized by CD1-restricted T and NKT cells and that a non-peptide antigen is recognized by γδ T cells. In this study, we demonstrated that these bacterial SPLs activated murine bone marrow macrophages (BMMs) via Toll-like receptor (TLR) 4 but not TLR2, although they slightly activated CD1d-restricted NKT and γδT cells. Interestingly, this TLR 4-recognition pathway of bacterial SPLs involves the fatty acid composition of ceramide in addition to the sugar moiety. A non-hydroxy fatty acid composed of ceramide was necessary to activate murine BMMs. The bacterial survival was significantly higher in TLR4-KO mice than in TLR2-KO and wild-type mice. The results indicate that activation of the TLR4-dependent pathway of BMMs by SPLs induced an innate immune response and contributed to bacterial clearance.

Identification and susceptibility to multipurpose disinfectant solutions of bacteria isolated from contact lens storage cases of patients with corneal infiltrative events

Corneal infiltrative events (CIEs) are being reported with increasing frequency in lens wearers and may be related to specific multipurpose disinfecting solution (MPDS), contact lens type or bacterial bio-burden. Here, the efficacy of MPDS's against bacteria from contact lens storage cases (CLSC) of patients with CIEs was investigated. Eighteen CLSC from patients with CIEs were cultured. All reported using the same MPDS based on PQ-1+Aldox+nonanoyl-EDTA prior to experiencing CIEs. Bacteria were identified and tested for sensitivity to MPDS-1 and three other MPSDs. 16/18 CLSC (89%) contained bacterial counts of ≥10(4)-10(8)/mL. Achromobacter spp. was most frequently identified and was found in 11/18 cases (61%). This was followed by 4/18 (22%) Stenotrophomonas maltophilia, 3/18 (17%) Serratia marcescens, 3/18 (17%) Delftia spp., 2/18 (11%) Elizabethkingia spp., 2/18 (11%) Chryseobacterium indologenes and 1/18 Sphingobacterium spiritivorum. Acanthamoeba was not isolated. All of the Achromobacter strains were resistant to MPDS-1 with <1log10 kill up to 14 days exposure and the solution also showed reduced efficacy against the other isolates at the manufacturer's recommended disinfection time of 6h. Two strains of S. maltophilia and Delftia spp. grew in the solution over 14 days. Factors responsible for causing adverse events such as CIEs in contact lens wearers remain unclear. However, the presence of significant bio-burden in the contact lens storage case and lens may initiate an immunological response resulting in CIEs either directly or through the release of endotoxins (e.g. lipopolysaccharides) from the bacterial outer cell membrane.

Effects of rhizobacteria on parasitism by Meloidogyne ethiopica on grapevines

Meloidogyne ethiopica is one of the most important plant-parasitic nematodes affecting vines in Chile and is very aggressive and difficult to control. This study evaluated 16 strains of rhizobacteria, originally isolated from roots of grapevines, for their effects on parasitism and nematode damage to potted vine plants. The antagonistic effect of rhizobacteria was assessed by treating 2-month-old plants of cv. Chardonnay in 3-l pots with a suspension containing 1 × 106 cfu ml−1 of the bacteria and 1,000 nematode eggs. After 6 months of growth, the plants were cut and root and canopy weights, nematode populations and root damage determined. The effect of rhizobacterial culture filtrate on hatching of nematode eggs was also assessed in vitro. Seven strains of rhizobacteria proved effective in inhibiting damage or reproduction of the nematode. These were strains of Serratia marcescens, Comamonas acidovorans, Pantoea agglomerans, Sphingobacterium spiritivorum, Bacillus mycoides, Alcaligenes piechaudii and Serratia plymuthica. A further three strains, of Bacillus megaterium, P. agglomerans and Pseudomonas savastanoi, significantly increased root weight, but did not decrease nematode damage or population density. The supernatant of all strains significantly decreased hatching of juvenile nematodes after 24 h of immersion, with isolates of P. putida and B. megaterium being the most effective.

Structure and apoptosis-inducing activity of sphingolipids isolated from Sphingobacterium spiritivorum

Structure and apoptosis-inducing activity of sphingolipids isolated from Sphingobacterium spiritivorum

Sphingobacterium anhuiense sp. nov., isolated from forest soil

A Gram-negative bacterium, designated strain CW 186(T), was isolated from forest soil in Anhui province, China. Cells of strain CW 186(T) were strictly aerobic, non-motile and rod-shaped. The strain grew optimally at 25-30 degrees C and pH 6.0-8.0. The major cellular fatty acids of strain CW 186(T) were iso-C(15:0) (32.2%), iso-C(17:0) 3-OH (9.8%) and summed feature 3 (iso-C(15:0) 2-OH and/or C(16:1)omega7c; 33.7%). The predominant isoprenoid quinone was MK-7. The G+C content of the genomic DNA was 36.3 mol%. Phylogenetic analysis using 16S rRNA gene sequences showed that strain CW 186(T) formed a monophyletic cluster with Sphingobacterium daejeonense LMG 23402(T), Sphingobacterium composti LMG 23401(T), Sphingobacterium composti DSM 18850(T), Sphingobacterium mizutaii ATCC 33299(T) and Sphingobacterium spiritivorum ATCC 33861(T). Sequence similarities were less than 94% (the maximal similarity was about 93.9% to S. composti LMG 23401(T)) to Sphingobacterium species with validly published names. A polyphasic taxonomic study including chemotaxonomic and phylogenetic analyses demonstrated that strain CW 186(T) should be classified as representing a novel species of the genus Sphingobacterium, for which the name Sphingobacterium anhuiense sp. nov. is proposed. The type strain is CW 186(T) (=KCTC 22209(T)=CCTCC AB 207197(T)).

Acceleration of the Substrate Cα Deprotonation by an Analogue of the Second Substrate Palmitoyl-CoA in Serine Palmitoyltransferase

Serine palmitoyltransferase (SPT) is a key enzyme of sphingolipid biosynthesis and catalyzes the pyridoxal 5'-phosphate (PLP)-dependent decarboxylative condensation reaction of l-serine with palmitoyl-CoA to generate 3-ketodihydrosphingosine. The binding of l-serine alone to SPT leads to the formation of the external aldimine but does not produce a detectable amount of the quinonoid intermediate. However, the further addition of S-(2-oxoheptadecyl)-CoA, a nonreactive analogue of palmitoyl-CoA, caused the apparent accumulation of the quinonoid. NMR studies showed that the hydrogen-deuterium exchange at Calpha of l-serine is very slow in the SPT-l-serine external aldimine complex, but the rate is 100-fold increased by the addition of S-(2-oxoheptadecyl)-CoA, showing a remarkable substrate synergism in SPT. In addition, the observation that the nonreactive palmitoyl-CoA facilitated alpha-deprotonation indicates that the alpha-deprotonation takes place before the Claisen-type C-C bond formation, which is consistent with the accepted mechanism of the alpha-oxamine synthase subfamily enzymes. Structural modeling of both the SPT-l-serine external aldimine complex and SPT-l-serine-palmitoyl-CoA ternary complex suggests a mechanism in which the binding of palmitoyl-CoA to SPT induced a conformation change in the PLP-l-serine external aldimine so that the Calpha-H bond of l-serine becomes perpendicular to the plane of the PLP-pyridine ring and is favorable for the alpha-deprotonation. The model also proposed that the two alternative hydrogen bonding interactions of His(159) with l-serine and palmitoyl-CoA play an important role in the conformational change of the external aldimine. This is the unique mechanism of SPT that prevents the formation of the reactive intermediate before the binding of the second substrate.

Molecular characterization of membrane-associated soluble serine palmitoyltransferases from Sphingobacterium multivorum and Bdellovibrio stolpii.

Serine palmitoyltransferase (SPT) is a key enzyme in sphingolipid biosynthesis and catalyzes the decarboxylative condensation of l-serine and palmitoyl coenzyme A (CoA) to form 3-ketodihydrosphingosine (KDS). Eukaryotic SPTs comprise tightly membrane-associated heterodimers belonging to the pyridoxal 5'-phosphate (PLP)-dependent alpha-oxamine synthase family. Sphingomonas paucimobilis, a sphingolipid-containing bacterium, contains an abundant water-soluble homodimeric SPT of the same family (H. Ikushiro et al., J. Biol. Chem. 276:18249-18256, 2001). This enzyme is suitable for the detailed mechanistic studies of SPT, although single crystals appropriate for high-resolution crystallography have not yet been obtained. We have now isolated three novel SPT genes from Sphingobacterium multivorum, Sphingobacterium spiritivorum, and Bdellovibrio stolpii, respectively. Each gene product exhibits an approximately 30% sequence identity to both eukaryotic subunits, and the putative catalytic amino acid residues are conserved. All bacterial SPTs were successfully overproduced in Escherichia coli and purified as water-soluble active homodimers. The spectroscopic properties of the purified SPTs are characteristic of PLP-dependent enzymes. The KDS formation by the bacterial SPTs was confirmed by high-performance liquid chromatography/mass spectrometry. The Sphingobacterium SPTs obeyed normal steady-state ordered Bi-Bi kinetics, while the Bdellovibrio SPT underwent a remarkable substrate inhibition at palmitoyl CoA concentrations higher than 100 microM, as does the eukaryotic enzyme. Immunoelectron microscopy showed that unlike the cytosolic Sphingomonas SPT, S. multivorum and Bdellovibrio SPTs were bound to the inner membrane of cells as peripheral membrane proteins, indicating that these enzymes can be a prokaryotic model mimicking the membrane-associated eukaryotic SPT.