In order to overcome the challenges of insufficient restriction enzyme sites, and construct a fusion-expression vector with flexible fusion direction, we designed an LB cloning system based on the type IIS and type IIT restriction enzymes LguⅠ and BbvCⅠ. The LB cloning system is constructed by inserting the LB fragment (GCTCTTCCTCAGC) into the multiple cloning site region of the broad-host plasmid pBBR1MCS-3 using PCR. The LB fragment contains partially overlapped recognition sites of LguⅠ and BbvCⅠ. Therefore, the same non-palindromic sequence will be generated by these two restriction endonucleases digestion. This feature can be used to quickly and flexibly insert multiple genes into the expression vector in a stepwise and directed way. In order to verify the efficacy of the cloning system, two glycosyltransferase genes welB and welK of Sphingomonas sp. WG were consecutively fused to the LB cloning vector, and the recombinant plasmid was transferred into Sphingomonas sp. WG by triparental mating. The results showed that gene fusion expression has little effect on sphingan titer, but enhanced the viscosity of sphingan. The viscosity of the sphingan produced by recombinant strain Sphingomonas sp. WG/pBBR1MCS-3-LB-welKB was 24.7% higher than that of the wild strain after fermentation for 84 h, which would be beneficial for its application. In conclusion, the application of LB cloning system were verified using Sphingomonas sp. WG. The LB cloning system may provide an efficient tool for fusion expression of target genes.
Base Sequence ; Cloning, Molecular ; Fermentation ; Plasmids/genetics* ; Sphingomonas/metabolism*

Sphingomonas paucimobilis is an aerobic Gram-negative bacillus found in soil and water. Knowledge regarding the role of this infectious agent is limited because it is rarely isolated from human material. Furthermore, it is an unusual pathogen in cases of peritoneal dialysis (PD)-associated peritonitis. The clinical courses and outcomes of peritonitis caused by S. paucimobilis are variable. Whereas some patients were cured with appropriate antibiotic therapy, others required catheter removal. Cases of PD-associated peritonitis caused by S. paucimobilis have been reported worldwide, and there was a case report of coinfection with S. paucimobilis and Chryseobacterium indologenes in Korea. However, there has been no case caused by S. paucimobilis as a single pathogen. We report a case of PD-associated peritonitis due to S. paucimobilis in which the patient recovered after catheter removal.
Bacillus ; Catheters ; Chryseobacterium ; Coinfection ; Humans ; Korea ; Peritoneal Dialysis ; Peritonitis ; Soil ; Sphingomonas

A novel chitinolytic and chitosanolytic bacterium, Sphingomonas sp. CJ-5, has been isolated and characterized. It secretes both chitinase and chitosanase into surrounding medium in response to chitin or chitosan induction. To characterize the enzymes, both chitinase and chitosanase were purified by ammonium sulfate precipitation, Sephadex G-200 gel filtration and DEAE-Sepharose Fast Flow. SDS-PAGE analysis demonstrated molecular masses of chitinase and chitosanase were 230 kDa and 45 kDa respectively. The optimum hydrolysis conditions for chitinase were about pH 7.0 and 36 degrees C, and these for chitosanase were pH 6.5 and 56 degrees C, respectively. Both enzymes were quite stable up to 45 degrees C for one hour at pH 5~8. These results show that CJ-5 may have potential for industrial application particularly in recycling of chitin wastes.
Chitinases ; metabolism ; Enzyme Stability ; Fermentation ; Glycoside Hydrolases ; metabolism ; Hydrogen-Ion Concentration ; Sphingomonas ; enzymology

Eight distinct bacteria were isolated form diseased mycelia of the edible mushroom, Pleurotus eryngii. 16S rDNA sequence analysis showed that the isolates belonged to a variety of bacterial genera including Bacillus (LBS5), Enterobacter (LBS1), Sphingomonas (LBS8 and LBS10), Staphylococcus (LBS3, LBS4 and LBS9) and Moraxella (LBS6). Among them, 4 bacterial isolates including LBS1, LBS4, LBS5, and LBS9 evidenced growth inhibitory activity on the mushroom mycelia. The inhibitory activity on the growth of the mushroom fruiting bodies was evaluated by the treatment of the bacterial culture broth or the heat-treated cell-free supernatant of the broth. The treatment of the culture broths or the cell-free supernatants of LBS4 or LBS9 completely inhibited the formation of the fruiting body, thereby suggesting that the inhibitory agent is a heat-stable compound. In the case of LBS5, only the bacterial cell-containing culture broth was capable of inhibiting the formation of the fruiting body, whereas the cell-free supernatant did not, which suggests that an inhibitory agent generated by LBS5 is a protein or a heat-labile chemical compound, potentially a fungal cell wall-degrading enzyme. The culture broth of LBS1 was not inhibitory. However, its cell-free supernatant was capable of inhibiting the formation of fruiting bodies. This indicates that LBS1 may produce an inhibitory heat-stable chemical compound which is readily degraded by its own secreted enzyme.
Agaricales ; Bacillus ; Bacteria ; DNA, Ribosomal ; Enterobacter ; Fruit ; Moraxella ; Ostreidae ; Pleurotus ; Sequence Analysis ; Sphingomonas ; Staphylococcus

In our previous study, three bacterial strains, Bacillus megaterium KU143, Microbacterium testaceum KU313, and Pseudomonas protegens AS15, were selected as effective biocontrol agents against Aspergillus flavus on stored rice grains. In this study, we evaluated the inhibitory effects of the volatiles produced by the strains on A. flavus growth and aflatoxin production on stored rice grains. The three strains significantly reduced mycelial growth of A. flavus in dual-culture assays compared with the negative control strain, Sphingomonas aquatilis KU408, and an untreated control. Of these tested strains, volatiles produced by B. megaterium KU143 and P. protegens AS15 markedly inhibited mycelial growth, sporulation, and conidial germination of A. flavus on agar medium and suppressed the fungal populations in rice grains. Moreover, volatiles produced by these two strains significantly reduced aflatoxin production in the rice grains by A. flavus. To our knowledge, this is the first report of the suppression of A. flavus aflatoxin production in rice grains using B. megaterium and P. protegens volatiles.
Aflatoxins* ; Agar ; Aspergillus flavus* ; Aspergillus* ; Bacillus megaterium* ; Bacillus* ; Germination ; Pseudomonas* ; Sphingomonas

The purpose of our study is to compare the adhesion and biofilm formation abilities of isolates from water discharged from dental unit waterlines (DUWLs). Bacteria were isolated from a total of 15 DUWLs. Twelve isolates were selected for the experiment. To confirm the adhesion ability of the isolates, each isolate was attached to a glass coverslip using a 12-well plate. Plates were incubated at 26℃ for 7 days, and the degree of adhesion of each isolate was scored. To verify the biofilm formation ability of each isolate, biofilms were allowed to form on a 96-well polystyrene flat-bottom microtiter plate. The biofilm accumulations of all isolates formed at 26℃ for 7 days were identified and compared. A total of 56 strains were isolated from 15 water samples including 12 genera and 31 species. Of the 56 isolates, 12 isolates were selected according to the genus and used in the experiment. Sphingomonas echinoides, Methylobacterium aquaticum, and Cupriavidus pauculus had the highest adhesion ability scores of +3 among 12 isolates. Among these three isolates, the biofilm accumulation of C. pauculus was the highest and that of S. echinoides was the third-most abundant. The lowest biofilm accumulations were identified in Microbacterium testaceum and M. aquaticum. Most isolates with high adhesion ability also exhibited high biofilm formation ability. Analysis of adhesion and biofilm formation of the isolates from DUWLs can provide useful information to understand the mechanism of DUWL biofilm formation and development.
Bacteria* ; Bacterial Adhesion ; Biofilms* ; Cupriavidus ; Glass ; Infection Control, Dental ; Methylobacterium ; Polystyrenes ; Sphingomonas ; Water ; Water Microbiology

Indigo and indigo-like pigments are widely used in the industry of textile, food and medicine. Now people pays more and more attention to developing an alternative method of indigo production which is "environment-friendy", especially microbial biosynthesis of indigo. Many microorganisms involved in the biosynthesis of indigo have been isolated and characterized, and monooxygenase and dioxygenase have been identified to catalyze indigo biosynthesis. Some genes encoding for these enzymes have been cloned and used to construct "engineering bacteria". With this kind of bacteria, more efficient fermentation systems for indigo production have been exploited. In the meantime, biotransformation of the indigo produced by microorganisms has been under investigation. These progresses will bring us a greener method of indigo and indigo-like pigments production.
Biotechnology ; Coloring Agents ; metabolism ; Dioxygenases ; metabolism ; Fermentation ; Indigo Carmine ; Indoles ; metabolism ; Mixed Function Oxygenases ; metabolism ; Pseudomonas ; metabolism ; Sphingomonas ; metabolism

Bacterial peritonitis is a well-recognized complication of continuous ambulatory peritoneal dialysis (CAPD) in patients with end-stage renal failure. Chryseobacterium indologenes is a non-fermentative Gram-negative bacillus that is a rare pathogen in humans and Sphinomomas paucimobilis has rarely been reported as an opportunistic human pathogen. We present a case of peritonitis due to unusual pathogens, C. indologenes and S. paucimobilis, unresponsive to the standard antibiotics therapy. A 51-year-old diabetic man undergoing CAPD for 45 days developed the first peritonitis due to C. indolegenes. Although he had received intraperitoneal antibiotics with good in vitro activity against organism, the signs of peritonitis persisted. S. paucimobilis was isolated from dialysate sample. The Tenckhoff catheter was finally removed on 19th day of hospitalization and the fever and abdominal pain subsided.
Abdominal Pain ; Anti-Bacterial Agents ; Bacillus ; Catheters ; Chryseobacterium* ; Fever ; Hospitalization ; Humans ; Kidney Failure, Chronic ; Middle Aged ; Peritoneal Dialysis ; Peritoneal Dialysis, Continuous Ambulatory* ; Peritonitis* ; Sphingomonas*

Sphingomonas paucimobilis (S. paucimobilis) is a gram negative bacillus. It has existed in soil, drinking water and plants. It has been isolated from distilled water tanks, respirators, and hemodialysis devices at the hospital setting. Patients with chronic disorders or immune suppression may be susceptible to infections with it. This microorganism has also been reported to infect healthy persons. Both nosocomial and community-acquired infections have been reported. So far, a variety of infections have been reported, including sepsis, septic pulmonary embolism, septic arthritis, peritonitis, and endophthalmitis. Only 2 cases of meningitis have been reported so far in the literature. So far, no previous reports of culture proliferation have been reported in patients with external ventricular drains, as was the case in our patient. Therefore, our case is the first to have S. paucimobilis proliferation in cerebrospinal fluid culture during intensive care unit stay for an external ventricular drain.
Arthritis, Infectious ; Bacillus ; Bacteria ; Cerebrospinal Fluid* ; Community-Acquired Infections ; Drinking Water ; Endophthalmitis ; Humans ; Intensive Care Units ; Meningitis ; Peritonitis ; Pulmonary Embolism ; Renal Dialysis ; Sepsis ; Soil ; Sphingomonas* ; Ventilators, Mechanical ; Water

DNA pyrosequencing, one of the advanced methods for DNA sequencing, has been employed for phylogenetic analysis of bacterial communities using the conserved 16S rRNA gene. We performed a pilot study on a mother-neonate pair utilizing the DNA pyrosequencing assays to investigate the diversity of microbial communities in maternal amniotic fluid (AF), vagina, and rectum and newborn gastric fluid (GF) and stool. Phylum level analysis revealed that bacterial community was dominated by Firmicutes (63.2%) in maternal feces, and Actinobacteria (84.9%) in maternal vaginal swab. The bacterial communities in both the AF and GF were dominated by Proteobacteria (67.8%). Interestingly, the bacterial community in the newborn's meconium was quite similar to that in the AF. However, the composition of the bacterial community in newborn's feces was different on day 14 and dominated by Firmicutes (91.1%). Genus-level analysis revealed that the bacterial community in maternal feces was dominated by Anaerococcus (19.5%) and Prevotella (18.7%), whereas that in the maternal vaginal swab was dominated by Atopobium (83.6%). The bacterial communities in both the AF and GF were dominated by Sphingomonas (38.5%). The bacterial community in the newborn's meconium was quite similar to that in the AF, which was dominated by Sphingomonas (45.2%). However, the composition of bacterial community in the newborn's feces on day 14 was relatively different. Future studies with a large number of infants are needed to determine the factors involved in the changing profile of newborn's fecal bacterial communities.
Actinobacteria ; Amniotic Fluid ; DNA ; Feces ; Female ; Genes, rRNA ; Humans ; Infant ; Infant, Newborn* ; Infant, Premature* ; Meconium ; Microbiota ; Pilot Projects* ; Prevotella ; Proteobacteria ; Rectum ; Sequence Analysis, DNA ; Sphingomonas ; Vagina