• Korean Journal of Microbiology
• /
• v.46 no.3
• /
• pp.233-239
• /
• 2010

Four genes (yqfR, yfmL, ydbR, deaD) were identified as putative DEAD-box RNA helicase genes in the genomic sequence of Bacillus subtilis by homology search. To understand the function of these genes, each of the genes was deleted and the constructed strains were tested for their growth charateristics at different temperatures. The growth rate of ydbR deletion mutant ($T_d$=53 min) was a little bit reduced at $37^{\circ}C$ as compared to that of wild type strain (CU1065). But the growth rate of other three (yqfR, yfmL, deaD) deletion mutants ($T_d$=30-40 min) is nearly equal to the growth rate of wild type ($T_d$=32 min). On the other hands, the growth rate of deletion mutants were reduced at $22^{\circ}C$ in order of yqfR ($T_d$=151 min), yfmL ($T_d$=214 min), ydbR ($T_d$=343 min), which showed cold-sensitive phenotype. The deletion mutant of deaD ($T_d$=109 min) grew equally as compared to the growth rate ($T_d$=102 min) of the wild type at $22^{\circ}C$ and did not show cold-sensitive growth. Double, triple and quadruple deletion mutants of these genes were constructed, and growth rate of these mutants were measured at various temperature conditions ($22^{\circ}C$, $37^{\circ}C$, $42^{\circ}C$) using LB broth. Multiple deletion mutations showed more severe cold-sensitive growth than single deletion mutations, and double deletion of ydbR and yfmL ($T_d$=984 min) showed most cold-sensitive growth than any other double mutants. Such a cold-sensitive growth of these mutations is quite similar to the result of csdA or srmB deletion in E. coli and suggested that physiological role of ydbR and yfmL is related with ribosome assembly.

• Korean Journal of Soil Science and Fertilizer
• /
• v.27 no.2
• /
• pp.136-146
• /
• 1994

To research the effect of chemotaxis of Rhizobia toward the root exudate on nitrogen fixing ability in soybean Rhizobia symbiosis system. Root exudate from seedlings of Glycine max. L was collected aseptic conditions. B. japonicum KCTC 2422 induced the formation of symbiotic nitrogen fixing nodules on the root of soybean plant and possessed motility and chemotaxis toward the 2mM proline. LPN-100 mutant was $Nod^-$, $Che^+$, and LPN-101 was $Che^-$, $Nod^+$ strains. Physiological properties of mutants were similar to parent strain. The crude root exudate was tested for its chemotactic ability using the capillary tube method. Chemotactic responses of RCR 3407 toward crude root exudate were 2.2, 2.6, 2.9, those of KCTC 2422 were 2.3, 2.9, 3.0, respectively. The crude root exudate was fractionated into neutral, cationic and anionic fractions. Chemotactic responses of KCTC 2422 was least with anionic fraction, most with neutral and intermediate with cationic fraction. B. japonicum KCTC 2422 was attracted by carbohydrates, amino acids and carboxylic acid. Carbohydrates and amino acids were good chemoattractants and carboxylic acids were intermediate chemoattractants. The peak concentration was $10^{-3}M$ for ribose, glucose, glutamine, aspartic acid and carboxylic acids, with exception of xylose, arabinose, tryptophan, which elicited maximum responses at $10^{-4}M$. The formation of nodules and nitrogenase activity of soybean inoculated with KCTC 2422 was determined in 7days after inoculation, and those of LPN-101 was detected in 15days after inoculation, but LPN-100 didn't form of nodules in soybean plants.

• Microbiology and Biotechnology Letters
• /
• v.37 no.2
• /
• pp.140-146
• /
• 2009

Cyclosporin, an immunosuppressant, is a representative group of biologically active secondary metabolites produced by the fungus Tolypocladium inflatum. The amount and ratio of cyclosporin derivatives in the culture broth are an important factors for the production of cyclosporin A and the purification in the industrial process. Therefore, we studied the effect of amino acids and complex organic nitrogen sources using Tolypocladium inflatum mutants on the productivity of cyclosporin A and the ratio of cyclosporin derivatives. Overproducing mutant YHC-004 having seven times higher productivity than mother strain's could be obtained through the artificial mutation by UV irradiation. The concentration and kind of organic nitrogens and amino acids shows the profound effect on the productivity of cyclosporin A and ratio of cyclosporin derivatives. As a result, it was possible to raise the productivity and the ratio of cyclosporin A up to 3,430 mg/L and 93% respectively, but on the other hand the other cyclosporin derivatives decreased less than 2% in the culture broth.

• Korean Journal of Microbiology
• /
• v.31 no.2
• /
• pp.141-147
• /
• 1993

Plasmid DNAs were detected from the mitochondrial fraction of four strains of whiterot fungus, Pleurotus ostreatus. The size of the plasmids were 10.2 and 7.2 kb in strain NFFA 2, 10.2 kb in NFFA 4001, 11.2 kb in NFFA 4501, and 10.2 and 11.2 kb in KFCC 11635. The two strains,NFFA 2ml and NFFA 2m2, which are mutant derivatives of NFFA 2, did not contain any plasmids. The cleavage by proteinase K indicated that these plasmids have DNA ends associated with proteins. In digestion with proteinase K all the plasm ids remained resistant to lambda exonuclease which hydrolyzes DNA from 5' ends and were sensitive to exonuclease III which hydrolyzes DNA from 3' ends. This suggests that the plasmids are linear double-stranded DNA and the terminal proteins are covalently linked to 5' ends of plasm ids. In order to find relationship between these plasmids, hybridization of plasm ids by each separate plasmid DNA was done. The result indicated that the plasmids can be classified into at least 3 groups. Plasmids of group I were present in all the P ostreatus. More mitochondrial plasmids were detected in P cornucopiae. P ,florida, P pulmonarius, P sajor-caju, and P spodoleucus. The size of plasmids ranged between 7.2 kb and 14 kb. All the species except P cornucopiae contained plasmids of approximately 10 kb which hybridized with the 10.2 kb plasmid (group I) of P ostreatus NFFA 2.

• Journal of Microbiology and Biotechnology
• /
• v.16 no.11
• /
• pp.1690-1698
• /
• 2006

In order to investigate the effect of dissolved oxygen (DO) level on AVM $B_{1a}$ production by a high yielding mutant of Streptomyces avermitilis, five sets of bioreactor cultures were performed under variously controlled DO levels. Using an online computer control system, the agitation speed and aeration rate were automatically controlled in an adaptive manner, responding timely to the oxygen requirement of the producer microorganism. In the two cultures of DO limitation, the onset of AVM $B_{1a}$ biosynthesis was observed to casually coincide with the fermentation time when oxygen-limited conditions were overcome by the producing microorganism. In contrast, this phenomenon did not occur in the parallel fermentations with DO levels controlled at around 30% and 40% throughout the entire fermentation period, showing an almost growth-associated mode of AVM $B_{1a}$ production: AVM $B_{1a}$ biosynthesis under the environments of high DO levels started much earlier than the corresponding oxygen-limited cultures, leading to a significant enhancement of AVM $B_{1a}$ production during the exponential stage. Consequently, approximately 6-fold and 9-fold increases in the final AVM $B_{1a}$ production were obtained in 30% and 40% DO-controlled fermentations, respectively, especially when compared with the culture of severe DO limitation (the culture with 0% DO level during the exponential phase). The production yield ($Y_{p/x}$), volumetric production rate (Qp), and specific production rate (${\bar{q}}_p$) of the 40% DO-controlled culture were observed to be 14%, 15%, and 15% higher, respectively, than those of the parallel cultures that were performed under an excessive agitation speed (350 rpm) and aeration rate (1 vvm) to maintain sufficiently high DO levels throughout the entire fermentation period. These results suggest that high shear damage of the high-yielding strain due to an excessive agitation speed is the primary reason for the reduction of the AVM $B_{1a}$ biosynthetic capability of the producer. As for the cell growth, exponential growth patterns during the initial 3 days were observed in the fermentations of sufficient DO levels, whereas almost linear patterns of cell growth were observed in the other two cultures of DO limitation during the identical period, resulting in apparently lower amounts of DCW. These results led us to conclude that maintenance of optimum DO levels, but not too high to cause potential shear damage on the producer, was crucial not only for the cell growth, but also for the enhanced production of AVM $B_{1a}$ by the filamentous mycelial cells of Streptomyces avermitilis.

• Microbiology and Biotechnology Letters
• /
• v.32 no.1
• /
• pp.52-59
• /
• 2004

When both fructose and galactose were added to a production medium as carbon sources, the productivity of PAFS (Psedomonas Antifungal Substance) biosynthesized by Pseudomonas aeruginosa was observed to be reduced significantly due to the well-known phenomenon of catabolite repression. In order to overcome this phenomenon by use of fermentation bioprocess, fed-batch cultivation method was examined. In addition, a high producer mutant strain, AP-20 obtained by a rational screening method was tested for its productivity of PAFS in both batch and fed-batch fermentation processes. Notably fed-batch operation showed approximately 4 fold higher PAFS productivity than traditional batch operation process. It was appeared that galactose was utilized principally for the cell growth of Pseudomonas aeruginosa whereas large portion of fructose was used for the biosynthesis of PAFS. Furthermore it was observed that composition and feeding rate of production media should be optimized even in the fed-batch fermentation bioprocess. As an example, very slow feeding of carbon sources gave rather negative effect on the production of PAFS due to significant limitation of carbon and energy sources available for the producer microorganism.

• Journal of Microbiology and Biotechnology
• /
• v.25 no.9
• /
• pp.1467-1475
• /
• 2015

The use of microbial extracts containing plant hormones is a promising technique to improve crop growth. Little is known about the effect of bacterial cell-free extracts on plant growth promotion. This study, based on phytohormonal analyses, aimed at exploring the potential mechanisms by which Enterococcus faecium LKE12 enhances plant growth in oriental melon. A bacterial strain, LKE12, was isolated from soil, and further identified as E. faecium by 16S rDNA sequencing and phylogenetic analysis. The plant growth-promoting ability of an LKE12 bacterial culture was tested in a gibberellin (GA)-deficient rice dwarf mutant (waito-C) and a normal GA biosynthesis rice cultivar (Hwayongbyeo). E. faecium LKE12 significantly improved the length and biomass of rice shoots in both normal and dwarf cultivars through the secretion of an array of gibberellins (GA₁, GA₃, GA₇, GA₈, GA₉, GA₁₂, GA₁₉, GA₂₀, GA₂₄, and GA₅₃), as well as indole-3-acetic acid (IAA). To the best of our knowledge, this is the first study indicating that E. faecium can produce GAs. Increases in shoot and root lengths, plant fresh weight, and chlorophyll content promoted by E. faecium LKE12 and its cell-free extract inoculated in oriental melon plants revealed a favorable interaction of E. faecium LKE12 with plants. Higher plant growth rates and nutrient contents of magnesium, calcium, sodium, iron, manganese, silicon, zinc, and nitrogen were found in cell-free extract-treated plants than in control plants. The results of the current study suggest that E. faecium LKE12 promotes plant growth by producing GAs and IAA; interestingly, the exogenous application of its cell-free culture extract can be a potential strategy to accelerate plant growth.

• Microbiology and Biotechnology Letters
• /
• v.14 no.6
• /
• pp.479-485
• /
• 1986

A 5200 basepair DNA fragment containing the Bacillus amyloliquefaciens amyE gene, encoding liquefying $\alpha$-amylase (1,4-$\alpha$-1)-glucan glucanohydrolase, EC 3.2.1.1), has been inserted into BamHI site of the pUB110 and the hybrid plasmid was designated as pSKS3. The pSKS3 was transformed into the Bacillus subtilis KM2l3 as a host which is a saccharifying $\alpha$-amylase deficient mutant of Bacillus subtilis NA64, and the plasmid in the transformed cell was expressed $\alpha$-amylase production and kanamycin resistance. The $\alpha$-amylase production of the transformed cell was reduced to one fifth of that of the donor strain. The Bacillus subtilis KM2l3 tarring pSKS3 indicated that the amyE gene product is a polypeptide which has the same electrophoretic mobility with that of the Bacillus amyloliquefaciens, but different from the saccharifying $\alpha$-amylase of Bacillus subtilis NA64. It means that the amyE gene of pSKS3 originales from the Bacillus amyloliquefaciens.

• BMB Reports
• /
• v.40 no.3
• /
• pp.315-324
• /
• 2007

The novel $\alpha$-amylase purified from locally isolated strain, Bacillus sp. KR-8104, (KRA) (Enzyme Microb Technol; 2005; 36: 666-671) is active in a wide range of pH. The enzyme maximum activity is at pH 4.0 and it retains 90% of activity at pH 3.5. The irreversible thermoinactivation patterns of KRA and the enzyme activity are not changed in the presence and absence of $Ca^{2+}$ and EDTA. Therefore, KRA acts as a $Ca^{2+}$-independent enzyme. Based on circular dichroism (CD) data from thermal unfolding of the enzyme recorded at 222 nm, addition of $Ca^{2+}$ and EDTA similar to its irreversible thermoinactivation, does not influence the thermal denaturation of the enzyme and its Tm. The amino acid sequence of KRA was obtained from the nucleotide sequencing of PCR products of encoding gene. The deduced amino acid sequence of the enzyme revealed a very high sequence homology to Bacillus amyloliquefaciens (BAA) (85% identity, 90% similarity) and Bacillus licheniformis $\alpha$-amylases (BLA) (81% identity, 88% similarity). To elucidate and understand these characteristics of the $\alpha$-amylase, a model of 3D structure of KRA was constructed using the crystal structure of the mutant of BLA as the platform and refined with a molecular dynamics (MD) simulation program. Interestingly enough, there is only one amino acid substitution for KRA in comparison with BLA and BAA in the region involved in the calcium-binding sites. On the other hand, there are many amino acid differences between BLA and KRA at the interface of A and B domains and around the metal triad and active site area. These alterations could have a role in stabilizing the native structure of the loop in the active site cleft and maintenance and stabilization of the putative metal triad-binding site. The amino acid differences at the active site cleft and around the catalytic residues might affect their pKa values and consequently shift its pH profile. In addition, the intrinsic fluorescence intensity of the enzyme at 350 nm does not show considerable change at pH 3.5-7.0.

• The Journal of the Korean Society for Microbiology
• /
• v.35 no.3
• /
• pp.251-261
• /
• 2000

V. vulnificus is an estuarine bacterium which causes septicemia and shock in susceptible patients. The organism produces a hemolytic cytolysin (VvH), which has a membrane damaging effect on erythrocytes. To clarify the mechanisms by which VvH might contribute to virulence, we examined its effect on macrophages. When mouse peritoneal macrophages were harvested and co-cultured with hemolysin-positive V. vulnificus strains (100 bacteria/cell), about 60% of the macrophages were killed; macrophages were not killed when co-cultured V. vulnificus strain CVD 707, a VvH-negative deletion mutant. Exposure of macrophages to filtered culture supernatants (2.5 HU/ml) and purified VvH (3 HU/ml) resulted in an increase in dead cells (80 and 90%, respectively), as determined by the trypan blue dye exclusion method and LDH release from macrophages was also increased (70 and 65.5%, respectively). The cytotoxic effect of VvH on macrophages was both the dose- and time-dependent. The VvH caused damage to the macrophage membrane and was blocked significantly by preincubation with cholesterol (p<0.01). Fetal bovine serum showed remarkable inhibition of VvH synthesis by V. vulnificus and inhibited VvH activity in culture supernatant. Cell viability was increased by 35% (p<0.01) and LDH release decreased by 28% (p<0.01) when macrophages were incubated with V. vulnificus (100 bacterial cell) in DMEM-10% FBS for 2 hr. Bacterial clearance activity of mice against V. vulnificus CVD 707 was decreased by pretreatment with 10 HU of VvH. This result suggests that the VvH can impair the membrane of macrophages and may playa role in the pathogenesis of V. vulnificus septicemia.