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*

Chryseobacterium hominis is non-fermenting Gram-negative rod that was first identified as a novel species in 2007. Here, we report the first clinical case of C. hominis bacteremia, which was confirmed by MALDI-TOF MS and 16S rRNA gene sequencing. A 16-year-old boy diagnosed with acute lymphoblastic leukemia was hospitalized for three months. Two sets of blood culture test through a peripherally inserted central catheter (PICC), which was inserted a month ago, was performed when his white blood cell count declined and he had a high fever. Colonies of medium sizes that looked round, mucoid, sticky, and grayish on blood and chocolate agar plates were observed. Identification of bacteria using the VITEK MALDI-TOF MS system (BioMérieux, France) was not successful and the VITEK 2 system (BioMérieux, USA) indicated Sphingomonas paucimobilis, with a questionable level of confidence (92%). However, Microflex LT Biotyper (Bruker Daltonics, Germany) showed C. homins (log score: 1.81) and sequence of 16S rRNA showed a 100% identity with C. hominis. Piperacillin-tazobactam was administered since the isolate was susceptible to piperacillin-tazobactam but C. hominis showed growth in the next four follow-up culture of blood drawn through PICC. The fever subsided only after PICC was changed. The clinical prognosis and antimicrobial susceptibility test of C. hominis should be further studied.
Adolescent ; Agar ; Bacteremia ; Bacteria ; Cacao ; Catheters ; Chryseobacterium ; Fever ; Follow-Up Studies ; Genes, rRNA ; Humans ; Leukocyte Count ; Male ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; Prognosis ; 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

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

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

Microorganisms found in bioaerosols from animal confinement buildings not only foster the risk of spreading diseases among livestock buildings, but also pose health hazards to farm workers and nearby residents. This study identified the various microorganisms present in the air of swine, chicken, and cattle farms with different kinds of ventilation conditions in Korea. Microbial air samples were collected onto Petri dishes with bacterial or fungal growth media using a cascade impactor. Endotoxin levels in total dust were determined by the limulus amebocyte lysate kinetic QCL method. Prevalent Gram-positive bacteria were Staphylococcus (S.) lentus, S. chromogenes, Bacillus (B.) cereus, B. licheniformis, and Enterococcus faecalis, while the dominant fungi and Gram-negative bacteria were Candida albicans and Sphingomonas paucimobilis, respectively. Considering no significant relationship between the indoor dust endotoxin levels and the isolation of Gram-negative bacteria from the indoor air, monitoring the indoor airborne endotoxin level was found to be also critical for risk assessment on health for animals or workers. The present study confirms the importance of microbiological monitoring and control on animal husbandry indoor air to ensure animal and worker welfare.
Agriculture ; Animal Husbandry ; Animals ; Bacillus ; Bacteria* ; Candida albicans ; Cattle ; Chickens* ; Dust ; Enterococcus faecalis ; Farmers ; Fungi* ; Gram-Negative Bacteria ; Gram-Positive Bacteria ; Horseshoe Crabs ; Korea ; Livestock ; Methods ; Risk Assessment ; Sphingomonas ; Staphylococcus ; Swine ; Ventilation

Coenzyme Q10 (CoQ10) is a lipophilic antioxidant that improves human immunity, delays senility and enhances the vitality of the human body and has wide applications in pharmaceutical and cosmetic industries. Microbial fermentation is a sustainable way to produce CoQ10, and attracts increased interest. In this work, the native CoQ8 synthetic pathway of Escherichia coli was replaced by the CoQ10 synthetic pathway through integrating decaprenyl diphosphate synthase gene (dps) from Rhodobacter sphaeroides into chromosome of E. coli ATCC 8739, followed by deletion of the native octaprenyl diphosphate synthase gene (ispB). The resulting strain GD-14 produced 0.68 mg/L CoQ10 with a yield of 0.54 mg/g DCW. Modulation of dxs and idi genes of the MEP pathway and ubiCA genes in combination led to 2.46-fold increase of CoQ10 production (from 0.54 to 1.87 mg/g DCW). Recruiting glucose facilitator protein of Zymomonas mobilis to replace the native phosphoenolpyruvate: carbohydrate phosphotransferase systems (PTS) further led to a 16% increase of CoQ10 yield. Finally, fed-batch fermentation of the best strain GD-51 was performed, which produced 433 mg/L CoQ10 with a yield of 11.7 mg/g DCW. To the best of our knowledge, this was the highest CoQ10 titer and yield obtained for engineered E. coli.
Alkyl and Aryl Transferases ; genetics ; Bacterial Proteins ; genetics ; Batch Cell Culture Techniques ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Gene Deletion ; Industrial Microbiology ; Metabolic Engineering ; Rhodobacter sphaeroides ; enzymology ; genetics ; Ubiquinone ; analogs & derivatives ; biosynthesis ; Zymomonas ; genetics

Uterine sterilization is important for improving fertility in cattle. This study compared bacterial flora in the uterus between healthy and repeat breeder cows (RBCs). The uterine flushing of six heifers, 13 healthy HanWoo cows and eight RBCs (HanWoo) were sampled, and 15 frozen semen samples were selected. Overall, 35 bacteria were identified from in HanWoo uterine flushing and semen. The bacterial genera identified from HanWoo uterine flushing were Alloiococcus, Bacillus, Enterobacter, Enterococcus, Erysipelothrix, Gardnerella, Granulicatella, Kocuria, Pantoea, Pasteurella, Rothia, Serratia, Sphingomonas, Staphylococcus, Stenotrophomonas and Streptococcus. The bacterial genera identified from HanWoo semen were Bacillus, Escherichia, Kocuria, Oligella, Pseudomonas, Serratia, Sphingomonas, Staphylococcus, Stenotrophomonas and Streptococcus. The prevalence and presence of the identified bacteria between healthy cows and RBCs differed significantly. Further studies are needed to determine the role of these bacteria in the uterus of HanWoo cattle with reproductive disorder.
Animals ; Bacillus ; Bacteria ; Cattle* ; Enterobacter ; Enterococcus ; Erysipelothrix ; Escherichia ; Fertility ; Flushing ; Gardnerella ; Pantoea ; Pasteurella ; Prevalence ; Pseudomonas ; Semen ; Semen Preservation ; Serratia ; Sphingomonas ; Staphylococcus ; Stenotrophomonas ; Sterilization ; Streptococcus ; Uterus*

We constructed several recombinant Escherichia coli strains to transform phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS system) and compared the characteristics of growth and metabolism of the mutants. We knocked-out the key genes ptsI and ptsG in PTS system by using Red homologous recombination in E. coli and meanwhile we also knocked-in the glucose facilitator gene glf from Zymomonas mobilis in the E. coli chromosome. Recombinant E. coli strains were constructed and the effects of cell growth, glucose consumption and acetic acid accumulation were also evaluated in all recombinant strains. The deletion of gene ptsG and ptsI inactivated some PTS system functions and inhibited the growth ability of the cell. Expressing the gene glf can help recombinant E. coli strains re-absorb the glucose through Glf-Glk (glucose facilitator-glucokinase) pathway as it can use ATP to phosphorylate glucose and transport into cell. This pathway can improve the availability of glucose and also reduce the accumulation of acetic acid; it can also broaden the carbon flux in the metabolism pathway.
Biological Transport ; Escherichia coli ; enzymology ; genetics ; Gene Deletion ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Glucose ; metabolism ; Phosphoenolpyruvate Sugar Phosphotransferase System ; genetics ; Zymomonas ; genetics

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