희소방선균 SeaR 유전자가 Streptomyces virginiae의 virginiamycins 생산에 미치는 영향

류재기,김현경,김병원,김동찬,이형선,Ryu, Jae-Ki,Kim, Hyun-Kyung,Kim, Byung-Won,Kim, Dong-Chan,Lee, Hyeong-Seon 한국미생물학회 2015 미생물학회지 Vol.51 No.3

본 연구는 희소방선균 Saccharopolyspora erythreae receptor gene (SeaR)의 기능을 연구하기 위해 다른 속의 균주인 Streptomyces virginiae에 SeaR 유전자를 도입하였다. S. virginiae의 형질전환은 oriT, attP, $ermEp^{\ast}$과 SeaR 유전자 단편을 가지고 있는 ${\varphi}C31$ 유래의 integration vector인 pEV615 (6.6 kb)를 이용하여 Escherichia coli ET12567/pUZ8002를 DNA 공여체(donor)로 이용한 접합전달법(conjugal transfer)을 사용하여 확립하였다. SeaR 유전자의 삽입 유무는 PCR방법으로 확인하였고, SeaR 유전자의 전사 발현은 RT-PCR방법으로 확인하였다. S. virginiae의 경우, virginiamycins 생산은 wild type (S. virginiae)와 transformants (C1, C3) 모두 최초생산시기가 14시간으로 같았다. $VB-C_6$ 첨가시기에 따른 항생물질 유도능 확인결과 본 배양 4시간에 $VB-C_6$ 첨가 시 wild type과 transformants (C1, C3) 모두 $VB-C_6$에 의한 virginiamycins 생성이 유도되지 않았다. 본배양 6시간, 8시간에 $VB-C_6$ 첨가하였을 시 $VB-C_6$에 의한 virginiamycins 생성이 유도되는 것을 확인하였다. 이 결과는 $VB-C_6$에 의한 유도의 경우 S. virginiae 내의 BarA에 의해 VMs 생산시기가 2-4시간 단축되었다고 사료되나, transformants C1, C3의 경우 $VB-C_6$ 첨가 시 virginiamycins 생산이 억제되는 것은 SeaR이 virginiamycins 생합성 유전자에 결합하여 억제자로 기능 한다고 추정 되었다. 이러한 결과로 인하여 외부에서 도입된 SeaR gene이 virginiamycins 생산에 영향을 주는 것으로 확인되었다. In order to study the effect of the receptor protein (SeaR), which is isolated from Saccharopolyspora erythraea, we introduced the SeaR gene to Streptomyces virginiae as host strains. An effective transformation procedure for S. virginiae was established based on transconjugation by Escherichia coli ET12567/pUZ8002 with a ${\varphi}C31$-derived integration vector, pSET152, which contained int, oriT, attP, and $ermEp^{\ast}$ (erythromycin promotor). Therefore, the pEV615 was introduced into S. virginiae by conjugation and integrated at the attB locus in the chromosome of the recipients by the ${\varphi}C31$ integrase (int) function. Transformants of S. virginiae containing the SeaR gene were confirmed by PCR and transcriptional expression of the SeaR gene in the transformants was analyzed by RT-PCR, respectively. And, we examined the production time of virginiamycins in the culture media of both the transformants and the wild type. The production time of virginiamycins in the wild type and transformants was the same. When 100 ng/ml of synthetic $VB-C_6$ was added to the state of 6 or 8 hour cultivation of wild type and transformants, respectively, the virginiamycins production was induced, meaning that the virginiamycins production in the wild type was detected 2 h early than transformants. From these results, SeaR expression was also affected to virginiamycins production in transformants derived from S. virginiae. In this study, we showed that the SeaR protein worked as a repressor in transformants.

Effect of energy density and virginiamycin supplementation in diets on growth performance and digestive function of finishing steers

Navarrete, Juan D.,Montano, Martin F.,Raymundo, Constantino,Salinas-Chavira, Jaime,Torrentera, Noemi,Zinn, Richard A. Asian Australasian Association of Animal Productio 2017 Animal Bioscience Vol.30 No.10

Objective: This study was determined the influence of virginiamycin supplementation on growth-performance and characteristics of digestion of cattle with decreasing dietary net energy value of the diet for maintenance ($NE_m$) from 2.22 to 2.10 Mcal/kg. Methods: Eighty crossbred beef steers ($298.2{\pm}6.3kg$) were used in a 152-d performance evaluation consisting of a 28-d adaptation period followed by a 124-d growing-finishing period. During the 124-d period steers were fed either a lesser energy dense (LED, $2.10Mcal/kg\;NE_m$) or higher energy dense (HED, $2.22Mcal/kg\;NE_m$) diet. Diets were fed with or without 28 mg/kg (dry matter [DM] basis) virginiamycin in a $2{\times}2$ factorial arrangement. Four Holstein steers ($170.4{\pm}5.6kg$) with cannulas in the rumen (3.8 cm internal diameter) and proximal duodenum were used in $4{\times}4$ Latin square experiment to study treatment effects on characteristics of digestion. Results: Neither diet energy density nor virginiamycin affected average daily gain (p>0.10). As expected, dry matter intake and gain efficiency were greater (p<0.01) for LED- than for HED-fed steers. Virginiamycin did not affect estimated net energy value of the LED diet. Virginiamycin increased estimated NE of the HED diet. During daylight hours when the temperature humidity index averaged $81.3{\pm}2.7$, virginiamycin decreased (p<0.05) ruminal temperature. Virginiamycin did not influence (p>0.10) ruminal or total tract digestion. Ruminal (p = 0.02) and total tract digestion (p<0.01) of organic matter, and digestible energy (p<0.01) were greater for HED vs LED. Ruminal microbial efficiency was lower (p<0.01) for HED vs LED diets. Conclusion: The positive effect of virginiamycin on growth performance of cattle is due to increased efficiency of energy utilization, as effects of virginiamycin on characteristics of digestion were not appreciable. Under conditions of high ambient temperature virginiamycin may reduce body temperature.

Virginiamycin 생산유도에 관여하는 Virginiae Butanolide C(VB-C) 및 Receptor의 상관관계

김현수,현지숙,유대식 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.1

S. virginiae에서 virginiamycin 생산촉진에 관여하는 호르몬성 물질인 Virginiae butanolide C(VB-C) 및 receptor의 유도기구를 규명하기 위해, NTG 처리에 의해 분리한 3종류의 변이주를 사용하여 유도능의 관계를 검토하였다. 분리한 3가지 변이주 중 No.1은 VB-C, receptor 및 항생물질 생산 지연, No.3은 receptor 대량생산, No.4는 VB-C 결손, receptor 및 항생물질 생산시기 지연 등의 특성을 나타내었다. 이들 변이주의 합성 VB-C 첨가에 의한 유도능의 특징은, No.1의 경우 receptor 생산시기 지연으로 인한 항생물질 생산 억제, No.3의 경우 다량의 receptor 생산으로 인한 유도 초기 항생물질의 양적 증가가 나타났으며, No.4의 경우 적당한 양의 receptor를 생산함에 따라 정상적인 유도능을 가졌음이 시사되었으나 VB-C 결손으로 인한 항생물질 생산량의 감소 및 생산시기 지연 등의 특성도 함께 나타내었다. 또한 이들 변이주가 생산한 항생물질의 향균 spectrum 및 HPLC 분석 결과, virginiamycin M 또는 S의 생산량의 변화 및 유도체 혹은 새로운 항생물질의 생산 가능성, 그리고 VB-C에 의한 VM-M 및 S의 특이적인 유도가 시사되었다. 이들 결과로부터 virginiamycin 생산 촉진을 위해서는 VB-C가 필수적이며, receptor 존재시에 VB-C의 신호 전달에 따른 항생물질 유도가 일어난다고 판단된다. 또한 앞으로 VB-C^+, receptor^- 및 VB-C^-, receptor^- 등의 변이주가 분리되어 다양한 변이주에 대한 유도능의 검토가 이루어져야 한다고 사료되며 VB-C같은 2차 대사산물 생산 촉진인자의 다양한 할용이 기대된다. Virginiae butanolide C(VB-C) is one of the butyrolactone autoregulators, which triggers the production of virginiamycin in Streptomyces virginiae. To further understand the mechanism of virginiamycin induction, we isolated three mutants from S. virginiae by N-methyl-N'-nitro-N-nitrosoguanidine (NTG) treatment. The characteristics of the three mutants were confirmed as follows: the mutant No.1 delayed the production of the VB-C, receptor and antiiotics; the mutant No. 3 hyperproduced receptor; the mutant No. 4failed to produce the VB-C. The addition of synthetic VB-C couldn't induce the production of antibiotics in the mutant No.1 due to delayed production of receptor, could provoke the production of larger amount of antibiotics than parental wild type strain in the mutant No.3 due to the presence of large amount of receptor, and could induce production of very small amount of antibiotics in the mutant No.4 due to the absence of VB-C. Antimicrobial spectrum and HPLC analysis of the mutant No.1 and No.3 suggested that the VB-C might have a specific ability to induce the production of virginiamycin M and S. These results imply that the VB-C has an ability to trigger the production of virginiamycin under receptor existence in S. virginiae.

Virginiamycin 생산유도에 관여하는 Virginiae Butanolide C(VB-C) 및 Receptor의 기능

김현수,현지숙 한국미생물학회 1997 미생물학회지 Vol.33 No.2

Virginiamycin(VM) 생산 유도에 관여하는 virginiae butanolide C(VB-C)및 receptor의 기능을 명확히 규명하기 위해 S. virginiae로부터 NTG및 hydroxylamine 처리를 통해 두 개의 변이주를 분리하였으며, VM을 생산하는 균중에서 VB, receptor를 모두 생산하지 않는 S. ostreogriseus와, receptor는 생산하나 VB를 생산하지 않는 S. graminofaciens를 대상으로 하여 유도능의 관계를 검토하였다. 분리한 S. virginiae mutant N-25와 H-05는 VB가 receptor보다 먼저 생산되는 특성을 나타내었으며, VM 생산시기가 현저히 지연되었다. 이는 S. virginiae 모균주에서 receptor가 생사노디기 이전에 합성 VB-C를 첨가하였을 때 VM 생산이 억제되는 것과 같은 현상인것으로 판단되었다. 한편, VM 생산균인 S. ostreogriseus 및 S. graminofaciens는 모두 VB를 생산하지 않으며 합성 VB-C 첨가에 의한 유도능을 검토한 결과, receptor를 생산하지 않는 S. ostreogriseus의 경우 VM 생산이 억제되는 반면, receptor를 생산하는 S. graminofaciens의 경우에는 VM 생산이 촉진되었다. 이들 결과에서 볼 때, VM 생산 촉진에 VB가 필수적이며 VB의 신호전달에 따른 VM 생산 촉진 효과는 반드시 receptor의 존재하에서 일어난다고 사료되었다. S. graminofaciens의 경우 합성 VB-C 첨가에 의해 생산된 하생물질을 HPLC를 이용하여 분석한 결과, 항생물질 생산량이 증가하였으며, 새로운 항생물질 생상 유도도 가능한 것으로 시사되었다. Streptomyces virginiae produces a set of autoregulators termed virginiae butanolide A-E(VB-A-E) which trigger virginiamycin production, and possesses a high-affinity virginiae butanolide receptor. To elucidate the functions of VB-C and VB-C receptor, we isolated two mutants from S. virginiae by N-methyl-N'-nitro-N-nitrosoguanidine and hydroxylamine. The characteristics of the mutants showed that the producing time of antibiotics was very delayed due to a slower production of VB-C receptor than that of VB. In S. ostreogriseus(VB', receptor -) and S. graminofaciens(VBU, receptor+), which produce the virginiamycin, the addition of synthetic VB-C repressed the production of antibiotics in S. ostreogriseus but induced tbe production in S. graminofaciens. HPLC analysis of S. graminofaciens suggested that the VB-C might have an ability to induce the production of virginiamycin and other antibiotics. These results imply that the VB-C has an ability to trigger the production of other secondary metabolites as well as virginiamycin under VB-C receptor existence.

Virginiamycin 생합성 유도인자 Virginiae Butanolide C에 이한 Erythromycin 생산 유도

김현수,성림식,Kim, Hyun-Soo,Seong, Lim-Shik 한국생물공학회 1999 KSBB Journal Vol.14 No.6

Streptomyces virginiae 유래의 virginiamycin 생합성 유도인자인 VB-C를 이용하여 다른 방선균에의 항생물질 생산 유도 기능을 검토하였다. Erythromycin 생산균인 Streptomyces erythraeus를 공시균주로 사용하였으며, VB-C 결합 receptor 유전자가 포함된 plasmid(pARS 701, 9 kb)를 공시균주에 도입하여 transformant로 사용하였다. Parent와 transformant 모두 유도인자인 VB류를 생산하였으며, VB-C의 signal 전달에 관여하는 결합 단백질의 존재가 확인되었다. Parent는 본배양 0, 20, 44시간째에 합성 VB-C 300 ng/ml를 첨가시 초기 항생물질 생산시기가 약 8시간 이상 단축되었다. Transformant는 본배양 44시간에 첨가시 6시간 이상 항생물질 생산시기가 단축되었고 항생물질 생산량도 증가되었으며, parent에 비해 B. subtilis에도 강한 항균력을 나타내었다. 또한 항균 spectrum은 parent보다는 transformant가 생산한 항생물질의 항균호과가 더 큰 것으로 나타났으며, VB-C를 첨가한 경우 미첨가 경우보다 넓은 항균 spectrum을 보였다. 이들 결과에서 공시균인 S. erythraeus에서 VB signal 전달에 따른 항생물질 생산유도가 입증되었으며, VB-C receptor gene의 도입에 따른 발현과 함께 VBs의 유도, 항생물질의 생산시기의 단축 및 생산량의 증가가 예상됨에 따라 다양한 Streptomyces sp. 균주를 대상으로 항생물질의 대량생산을 비롯한 새로운 2차 대사산물 생산에의 응용이 예상되었다. Virginiae butanolide C(VB-C) is one of the butyrolactone autoregulators, which triggers the production of virginiamycin in Streptomyces virginiae. In order to investigate the function of VB-C as inducer in other strains, Streptomyces erythraeus was used as a test strain(parent). VB-C binding receptor gene was introduced into S. erythraeus(transformant) and the production of VBs and specific VB-C binding protein were analysed in parent and transformant. When 300ng/ml of the synthetic VB-C was added at 0, 20, 44 h cultivation of the parent and at 44 h cultivation of the transformant, the initial production times a antibiotics were shortened by more than 8 and 6 h, respectively. The transformant showed strong antibiotic activity against B. subtilis. These results suggest that the VB-C might have an ability to induce the production of secondary metabolites in S. erythraeus.

유산균과 버지니아마이신의 급여가 육계의 생산성 및 장내 미생물에 미치는 영향

김상호,박수영,유동조,이상진,류경선 한국가금학회 2001 韓國家禽學會誌 Vol.28 No.1

This experiment was conducted to investigate the effect of feeding two strains of Lactobacillus and virginiamycin on performance, nutrients digestibility and intestinal microflora of broiler chicks(Abor acres$\times$Abor Acres) were randomly allocated into six treatments with four replications for five weeks. Control(no supplement), 0.05% virginiamycin(VM), Lactobacillus crispatus avibro1(LC), Lactobacillus reuteri avibro2(LR), LC+0.05% VM(LC+VM), LR+0.05% VM(LR+VM) were supplemented into basal diets, which contained ME 3,100kcal/kg and CP 22.0, 20.0% for starting and finishing period, respectively. Weight gain, feed intake and feed conversion(FC) were weekly measured. Nutrients digestibility, intestinal microflora and fecal noxious gas were examined at the end of experiment. Weight gains of chicks fed Lactobacillus or VM was significantly higher than control(P〈0.05). Feed intake increased significantly in those supplemental groups(P〈0.05). FC of chicks fed Lactobacillus or VM significantly lower than control(P〈0.05). Degestibility of crude protein, calcium, and phosphorus improved significantly in alone or combined Lactobacillus treatments(P〈0.05). Whereas DM, crude fat and ash digestibility were not statistically different. Feeding Lactobacilli tended to increase the total Lactobacillus spp. in ileum at one and three weeks of age(WOA) and showed significantly higher in cecum than control at 5 WOA. Total yeast were not shown difference at 1 and 3 WOA, but significantly increased at 5 WOA(P〈0.05). The ileal and cecal anaerobes were started to increase from the first WOA. Fecal NH$_3$gas tended to decrease in Lactobacillus treatments compared to that of other treatments.

Influence of Monensin and Virginiamycin on In Vitro Ruminal Fermentation of Ammoniated Rice Straw

Kook, K.,Sun, S.S.,Yang, C.J.,Myung, K.H. Asian Australasian Association of Animal Productio 1999 Animal Bioscience Vol.12 No.4

The object of this study was to determine the influence of monensin and virginiamycin (VM) on in vitro ruminal fermentation of rice straw or ammoniated rice straw. Rumen fluid was collected from 4 wethers fed 200 g of concentrate supplement with 400 g of untreated (U) or ammoniated (A) rice straw once daily for 28 days. Mixed ruminal microorganisms were incubated in anaerobic media that contained 20% (vol/vol) ruminal fluid and 0.3 g of either U or A rice straw. Monensin and/or VM, dissolved in ethanol, were added in centrifuge tubes at final concentrations of 0, 15, 30, 15+15 and 30+30 ppm of culture fluid. The addition of monensin and VM combination to A rice straw fermentation decreased (p<0.05) the acetate to propionate ratio, total VFA and lactate production, but increased (p<0.05) pH. Total gas production tended to be decreased by the addition of monensin plus VM. Antimicrobial agents decreased $NH_3$ N concentration and dry matter digestibility.

EFFECT OF BASAL DIET AND FEED ADDITIVES ON THE SUSCEPTIBILITY OF SHEEP RUMEN FLUID TO LACTIC ACID PRODUCTION

Rowe, J.B.,Winslow, S.G.,Murray, P.J. Asian Australasian Association of Animal Productio 1989 Animal Bioscience Vol.2 No.3