- Email: [email protected]
Microsatellite DNA analysis of Strongylocentrotus intermedius from three natural populations from Japan, Russia and North Korea and an introduced population from China
Abstracts / Journal of Biotechnology 136S (2008) S22–S71
fermentation. The results indicated that the final succinic acid concentration in E. coli (BL21) carrying pET-cyanoca was increased from 1.624 g/L to 3.486 g/L, while at the same time the activity of phosphoenolpyruvate carboxylase (PEPC) was also five folds raised. This increased succinic acid production came at the availability of higher concentration of HCO3 − , which was derived from CO2 catalyzed by the overexpression of CA. Acting as direct substrate and supplying as another carbon source other than glucose, HCO3 − promoted the PEPCs activity and pushed the carboxylation of phosphoenolpyruvate (PEP) ahead (Lin et al., 2004). The expression of CA did not affect the maximum glucose uptake rate. However, the maximum specific growth rate was decreased from 0.86 h−1 to 0.74 h−1 . Plasmid pTrc-cyanoca was also constructed and introduced into an E. coli mutant strain DC1515 deficient in ptsG, lactate dehydrogenase (ldh) and pyruvate: formate lyase (pfl) (Chatterjee et al., 2001). Results showed that cyanobacterial CA was effective in fixing CO2 in the cytoplasm and the molar yield of the recombinant strain increased to 1.15 mol/mol glucose. Acknowledgements This research was supported in part by grants from the Knowledge Innovation Program of Chinese Academy of Sciences (Grant NO. KSCX2-YW-G-021). We thank Professor P. Clark for strain DC1515 and kindly guidance. References Chatterjee, R., Sanville Millard, C., Champion, K., Clark, D.P., Donnelly, M.I., 2001. Mutation of the ptsG gene results in increased production of succinate in fermentation of glucose by Escherichia coli. Appl. Environ. Microbiol. 67, 148–154. Kai, Y., Matsumura, H., Inoue, T., Terada, K., Nagara, Y., Yoshinaga, T., Kihara, A., Tsumura, K., Katsura, Izui, 1999. Three-dimensional structure of phosphoenolpyruvate carboxylase: a proposed mechanism for allosteric inhibition. Proc. Natl. Acad. Sci. U.S.A. 96, 823–828. Lin, H., San, K.-Y., Bennett, G.N., 2004. Effect of Sorghum vulgare phosphoenolpyruvate carboxylase and Lactococcus lactis pyruvate carboxylase coexpression on succinate production in mutant strains of Escherichia coli. Appl. Microbiol. Biotechnol. 67, 515–523.
S27
increases ldh activity (Garrigues et al., 1997). It was found that 0.05 mole mannitol increases lactic acid yield significantly and cell yield increases up to 0.2 h−1 while total lactic acid concentration increases up to 22 g/l. Mannitol in the medium also found to reduce the growth associated coefficient (alpha) from 8.3 to 6.3 and nongrowth associated (maintenance energy) coefficient (beta) from 0.3 to 0.07. Mannitol also reduces ethanol concentration up to 93 g/l. From the result obtained it can be concluded finally that only a fixed concentration of mannitol is involved in enhancement of cell growth and lactic acid. Mannitol is also helpful in rapid transport of glucose and thus relieves substrate inhibition. Mannitol as a catabolite is utilized for cell growth and minimizes the ethanol byproduct during lactate formation by regenerating additional molecule of NADH as confirmed by gas chromatography. Thus the insight into mannitol and glucose co-fermentation gained from the present work discloses potential affect of mannitol in repressing substrate inhibition and ethanol leads to increase in cell mass and lactic acid and thus can be aimed toward scale up of lactic acid production at industrial scale by taking advantage of metabolism itself without undergoing major genetic change that are very costly and decreases lactic acid yield. Mannitol is well known osmolyte and acts as a protector of Lactococcus lactis cells (Efiuvwevwere et al., 1999). In spite of its physiological and biotechnological interest, mannitol metabolism has not been investigated in lactic acid bacteria, except in Lactobacillus acidophilus and Lactococcus lactis that utilizes mannitol as primary energy source for growth and increases NADH/NAD+ ratio. Therefore role of mannitol over lactic acid production were studied. As par from our knowledge this is the first report about reliving of substrate inhibition by using mannitol. References
doi:10.1016/j.jbiotec.2008.07.048
Efiuvwevwere, B.J.O., Gorris, L.G.M., Smid, E.J., Kets, E.P.W., 1999. Mannitol-enhanced survival of Lactococcus lactis subjected to drying. Appl. Microbiol. Biotechnol. 51, 100–104. Garrigues, C.R.P.L., Lindley, N.D., Bousquet, M.C., 1997. Control of the shift from homolactic to mixed acid fermentation in Lactococcus lactis: predominant role of the NADH/NAD ratio. J. Bacteriol. 79, 5282–5287. Siegumfeldt, H., Rechinger, K.B.R., Mogens, J., 2000. Dynamic changes of intracellular pH in individual lactic bacterium cells in response to a rapid drop in extracellular pH. Appl. Environ. Microb. 66, 2330–2335.
I1-Y-007
doi:10.1016/j.jbiotec.2008.07.049
Mannitol increases lactic acid production NADH/NAD+ ratio inhibiting ethanol production
by
shifting
S.M. Bhatt 1,∗ , S.K. Srivastava 2 1
Amity Institute of Biotechnology, Amity University Uttar Pradesh, Sector 125, Noida 210303, India 2 School of Biochemical Engineering, Institute of Technology, Banaras Hindu University, Varanasi 221005, India E-mail address: Sheel [email protected] (S.M. Bhatt). Co-fermentation kinetics of glucose and mannitol with Lactobacillus delbrueckii NCIM 2025 has been examined in MRS media in aim to enhance lactic acid production since glucose is not fully consumed during fermentation. The major problems associated with lactic acid production are substrate inhibition, end product inhibition and byproduct formation. End product inhibition occurs due to rise in pH of extracellular medium during lactic acid production (Siegumfeldt et al., 2000) that results in disturbance of NADH/NAD+ ratio. It has been evidenced that conversion of pyruvate to lactic acid requires high cytosolic NADH and a high NADH/NAD+ ratio
∗ Corresponding author. Tel.: +91 542 2307070; fax: +91 542 23688428.
I1-Y-052 Microsatellite DNA analysis of Strongylocentrotus intermedius from three natural populations from Japan, Russia and North Korea and an introduced population from China Ding Jun, Yu Jiaping ∗ , Feng Zhigang, Chang Yaqing ∗ , Zhao Feng, Li Yunfeng Dalian Fisheries University, Ministry of Agriculture, Key Laboratory of Marine Culture and Biotechnology, Dalian, China E-mail addresses: [email protected] (Y. Jiaping), [email protected] (C. Yaqing). Four populations of Strongylocentrotus intermedius were analyzed by 17 microsatellite loci (16 pairs of primer, primer SUX59 amplified 2 loci). Three populations were natural populations from Chongjing, North Korea (KC), Hakotade, Japan (JH) and Vladivostok, Russia (RV), respectively. One was an introduced population from Rongcheng, China (CR), which was introduced from Hokkaido, Japan in 1989. The results indicate that the effective number of alleles (Ne) ranged from 1.0000 to 12.3559. The observed heterozygosity (Ho) and the expected heterozygosity (He) at each locus range from 0.000 to 0.9000 and 0.0333 to 0.9329, respectively. All genetic parameters of
S28
Abstracts / Journal of Biotechnology 136S (2008) S22–S71
CR population are lower than those of the three natural populations, but a one-way ANOVA of the parameters at each locus indicates no significant differences. Although the decline of the genetic parameters in the CR population has not reached a significant level, it is of importance and should be monitored. Cluster analysis shows two branches in the UPGMA tree. The CR and JH populations form one branch and the KC and RV populations form another. The results are consistent with the geographic distribution of the populations. The research provides basic data for monitoring and protection of the populations, and provides direction for the development of sea urchin aquaculture. References Addison, J.A., Hart, M.W., 2004. Analysis of population genetic structure of the green sea urchin (Strongylocentrotus droebachiensis) using microsatellites. Mar. Biol. 144, 243–251. Chang, Y.Q., Chen, X.X., Ding, J., Cao, X.B., Li, R.L., Sun, X.W., 2007. Genetic diversity in five scallop populations of the Japanese scallop (Patinopecten yessoensis). Acta Ecol. Sin. 27, 0001–0007. Sato, M., Kawamata, K., Zaslavskaya, N., Nakamura, A., Ohta, T., Nishikiori, T., Brykov, V., Nagashima, K., 2005. Development of microsatellite markers for Japanese scallop (Mizuhopecten yessoensis) and their application to a population genetic study. Mar. Biotechnol. 7, 713–728. Sea Urchin Genome Sequencing Consortium, 2006. The genome of the sea urchin Strongylocentrotus purpuratus. Science 314, 941–952. Yu, H.T., Lee, Y.J., Huang, S.W., Chiu, T.S., 2002. Genetic analysis of the populations of Japanese anchovy (Engraulidae: Engraulis japonicus) using microsatellite DNA. Mar. Biotechnol. 4, 471–479.
doi:10.1016/j.jbiotec.2008.07.050
carboxylic acid (PCA) and the prenylated phenazine endophenazine A. This provided functional proof that the cluster contained all necessary genes for the biosynthesis of prenylated phenazines. To identify the function of the gene ppz1, PCR targeting was used to inactivate this gene, and the resulting construct was heterologously expressed in S. coelicolor M512. In this case, the resulting mutant could produce PCA but no prenylated phenazines. This proves that ppz1 codes for the prenyltransferase of endophenazine biosynthesis. References Laursen, J.B., Nielsen, J., 2004. Phenazine natural products: biosynthesis, synthetic analogues, and biological activity. Chem. Rev. 104, 1663–1686. Mavrodi, D.V., Blankenfeldt, W., Thomashow, L.S., 2006. Phenazine compounds in fluorescent Pseudomonas spp. biosynthesis and regulation. Annu. Rev. Phytopathol. 44, 417–447. Tello, M., Kuzuyama, T., Heide, L., Noel, J.P., Richard, S.B., 2008. The ABBA family of aromatic prenyltransferases: broadening natural product diversity. Cell. Mol. Life Sci. [Epub ahead of print].
doi:10.1016/j.jbiotec.2008.07.051 I1-Y-065 Antimicrobial activity of amino acids and dipeptide-based amphiphiles Nilanjan Kayal 1,∗ , Sangita Roy 2 , Rajendra Narayan Mitra 2 , Prasanta Kumar Das 2
A gene cluster for prenylated phenazine biosynthesis in Streptomyces anulatus 9663
1 Department of Biotechnology, School of Biotechnology Chemical and Biomedical Engineering, Vellore Institute of Technology University (VITU), Vellore 632014, India 2 Department of Biological Chemistry, Indian Association for Cultivation of Science (IACS), Kolkata 700032, India
Orwah Saleh 1 , Bertolt Gust 1 , Hans-Peter Fiedler 2 , Lutz Heide 1,∗
E-mail address: nilanjan [email protected] (N. Kayal).
I1-Y-064
1
Pharmazeutische Biologie, Eberhard-Karls-Universität Tübingen, auf der Morgenstelle 8, 72076 Tübingen, Germany 2 Mikrobiologisches Institut, Eberhard-Karls-Universität Tübingen, auf der Morgenstelle 28, 72076 Tübingen, Germany E-mail address: [email protected] (L. Heide). Naturally occurring phenazines are primarily isolated from Pseudomonas and Streptomyces. The biological properties of this class of natural products include antibiotic, antitumor, antimalarial and antiparasitic activities (Laursen and Nielsen, 2004). Several gene clusters for phenazine biosynthesis have been isolated from Pseudomonas, but no complete cluster from Streptomyces could be isolated yet (Mavrodi et al., 2006). To study the genetic basis of the phenazine biosynthesis in Streptomyces, we isolated a phenazine biosynthetic cluster from Streptomyces anulatus 9663. A cosmid library was prepared and screened for phenazine and mevalonate biosynthetic genes by Southern blot and PCR. One cosmid was selected for total sequencing. Sequence analysis revealed 30 open reading frames, including orthologues of all seven genes involved in the biosynthesis of phenazine-1-carboxylic acid in Pseudomonas (Mavrodi et al., 2006). Furthermore, this cluster contained a set of six genes for the biosynthesis of the prenyl moiety via the mevalonate pathway, a putative gene (ppz1) for an aromatic prenyltransferase from the ABBA family (Tello et al., 2008) and a putative gene for the N-methyltransferase in endophenazine B biosynthesis (ppzM). PCR targeting was used to introduce the integration functions of phage ФC31 and the resulting cosmid, containing the entire cluster, was heterologously expressed in Streptomyces coelicolor M512. HPLC and LC–MS analysis revealed in the formation of phenazine-1-
Cationic surfactants bear anti-bacterial activity. Surfactants are usually organic compounds which are amphiphilic in nature; they contain both hydrophobic groups (their “tails”) and hydrophilic groups (their “heads”). Thus they are soluble in both organic solvents and water. Quaternary ammonium compounds are found to be quite effective against both Gram’s positive and Gram’s negative bacteria, but they are also toxic. Their toxicity is related primarily to the various biological effects of the quaternary ammonium head and its metabolism (such as oxidative dealkylation), but it is also believed that the surfactant characteristics of the molecules, particularly in liver, causes additional alterations in a number of chemical, biological and transport phenomena. The mechanism of action of cationic surfactants on bacteria is understood to be purely electrostatic interaction and physical disruption. The cationic site of the agent is able to bind to the anionic sites of the cell wall surface. With a significant lipophilic component present, it is then able to diffuse through the cell wall and bind to membrane. As a surfactant it is able to disrupt the membrane and permit the release of electrolytes and nucleic materials, leading to cell death. Amino acid (Tryptophan)based cationic surfactants having carbon lengths C14 and C16 were tested with Klebsiela aerogenes (Gram negative) and Bacillus subtilis (Gram positive) and also give rise to semi-solid materials i.e. Gels, which can in turn have antimicrobial properties and can have a variety of uses in terms of antibiotics. Mainly peptides and dipeptides have been used which contains a combination of a number of amino acids. It was found that both the surfactants have their MIC (minimum inhibitory concentration) values far below their CMC (critical micellar concentration) and MGC (minimum gelation concentration) values hence thereby showing that they have antimicrobial activity.
fermentation. The results indicated that the final succinic acid concentration in E. coli (BL21) carrying pET-cyanoca was increased from 1.624 g/L to 3.486 g/L, while at the same time the activity of phosphoenolpyruvate carboxylase (PEPC) was also five folds raised. This increased succinic acid production came at the availability of higher concentration of HCO3 − , which was derived from CO2 catalyzed by the overexpression of CA. Acting as direct substrate and supplying as another carbon source other than glucose, HCO3 − promoted the PEPCs activity and pushed the carboxylation of phosphoenolpyruvate (PEP) ahead (Lin et al., 2004). The expression of CA did not affect the maximum glucose uptake rate. However, the maximum specific growth rate was decreased from 0.86 h−1 to 0.74 h−1 . Plasmid pTrc-cyanoca was also constructed and introduced into an E. coli mutant strain DC1515 deficient in ptsG, lactate dehydrogenase (ldh) and pyruvate: formate lyase (pfl) (Chatterjee et al., 2001). Results showed that cyanobacterial CA was effective in fixing CO2 in the cytoplasm and the molar yield of the recombinant strain increased to 1.15 mol/mol glucose. Acknowledgements This research was supported in part by grants from the Knowledge Innovation Program of Chinese Academy of Sciences (Grant NO. KSCX2-YW-G-021). We thank Professor P. Clark for strain DC1515 and kindly guidance. References Chatterjee, R., Sanville Millard, C., Champion, K., Clark, D.P., Donnelly, M.I., 2001. Mutation of the ptsG gene results in increased production of succinate in fermentation of glucose by Escherichia coli. Appl. Environ. Microbiol. 67, 148–154. Kai, Y., Matsumura, H., Inoue, T., Terada, K., Nagara, Y., Yoshinaga, T., Kihara, A., Tsumura, K., Katsura, Izui, 1999. Three-dimensional structure of phosphoenolpyruvate carboxylase: a proposed mechanism for allosteric inhibition. Proc. Natl. Acad. Sci. U.S.A. 96, 823–828. Lin, H., San, K.-Y., Bennett, G.N., 2004. Effect of Sorghum vulgare phosphoenolpyruvate carboxylase and Lactococcus lactis pyruvate carboxylase coexpression on succinate production in mutant strains of Escherichia coli. Appl. Microbiol. Biotechnol. 67, 515–523.
S27
increases ldh activity (Garrigues et al., 1997). It was found that 0.05 mole mannitol increases lactic acid yield significantly and cell yield increases up to 0.2 h−1 while total lactic acid concentration increases up to 22 g/l. Mannitol in the medium also found to reduce the growth associated coefficient (alpha) from 8.3 to 6.3 and nongrowth associated (maintenance energy) coefficient (beta) from 0.3 to 0.07. Mannitol also reduces ethanol concentration up to 93 g/l. From the result obtained it can be concluded finally that only a fixed concentration of mannitol is involved in enhancement of cell growth and lactic acid. Mannitol is also helpful in rapid transport of glucose and thus relieves substrate inhibition. Mannitol as a catabolite is utilized for cell growth and minimizes the ethanol byproduct during lactate formation by regenerating additional molecule of NADH as confirmed by gas chromatography. Thus the insight into mannitol and glucose co-fermentation gained from the present work discloses potential affect of mannitol in repressing substrate inhibition and ethanol leads to increase in cell mass and lactic acid and thus can be aimed toward scale up of lactic acid production at industrial scale by taking advantage of metabolism itself without undergoing major genetic change that are very costly and decreases lactic acid yield. Mannitol is well known osmolyte and acts as a protector of Lactococcus lactis cells (Efiuvwevwere et al., 1999). In spite of its physiological and biotechnological interest, mannitol metabolism has not been investigated in lactic acid bacteria, except in Lactobacillus acidophilus and Lactococcus lactis that utilizes mannitol as primary energy source for growth and increases NADH/NAD+ ratio. Therefore role of mannitol over lactic acid production were studied. As par from our knowledge this is the first report about reliving of substrate inhibition by using mannitol. References
doi:10.1016/j.jbiotec.2008.07.048
Efiuvwevwere, B.J.O., Gorris, L.G.M., Smid, E.J., Kets, E.P.W., 1999. Mannitol-enhanced survival of Lactococcus lactis subjected to drying. Appl. Microbiol. Biotechnol. 51, 100–104. Garrigues, C.R.P.L., Lindley, N.D., Bousquet, M.C., 1997. Control of the shift from homolactic to mixed acid fermentation in Lactococcus lactis: predominant role of the NADH/NAD ratio. J. Bacteriol. 79, 5282–5287. Siegumfeldt, H., Rechinger, K.B.R., Mogens, J., 2000. Dynamic changes of intracellular pH in individual lactic bacterium cells in response to a rapid drop in extracellular pH. Appl. Environ. Microb. 66, 2330–2335.
I1-Y-007
doi:10.1016/j.jbiotec.2008.07.049
Mannitol increases lactic acid production NADH/NAD+ ratio inhibiting ethanol production
by
shifting
S.M. Bhatt 1,∗ , S.K. Srivastava 2 1
Amity Institute of Biotechnology, Amity University Uttar Pradesh, Sector 125, Noida 210303, India 2 School of Biochemical Engineering, Institute of Technology, Banaras Hindu University, Varanasi 221005, India E-mail address: Sheel [email protected] (S.M. Bhatt). Co-fermentation kinetics of glucose and mannitol with Lactobacillus delbrueckii NCIM 2025 has been examined in MRS media in aim to enhance lactic acid production since glucose is not fully consumed during fermentation. The major problems associated with lactic acid production are substrate inhibition, end product inhibition and byproduct formation. End product inhibition occurs due to rise in pH of extracellular medium during lactic acid production (Siegumfeldt et al., 2000) that results in disturbance of NADH/NAD+ ratio. It has been evidenced that conversion of pyruvate to lactic acid requires high cytosolic NADH and a high NADH/NAD+ ratio
∗ Corresponding author. Tel.: +91 542 2307070; fax: +91 542 23688428.
I1-Y-052 Microsatellite DNA analysis of Strongylocentrotus intermedius from three natural populations from Japan, Russia and North Korea and an introduced population from China Ding Jun, Yu Jiaping ∗ , Feng Zhigang, Chang Yaqing ∗ , Zhao Feng, Li Yunfeng Dalian Fisheries University, Ministry of Agriculture, Key Laboratory of Marine Culture and Biotechnology, Dalian, China E-mail addresses: [email protected] (Y. Jiaping), [email protected] (C. Yaqing). Four populations of Strongylocentrotus intermedius were analyzed by 17 microsatellite loci (16 pairs of primer, primer SUX59 amplified 2 loci). Three populations were natural populations from Chongjing, North Korea (KC), Hakotade, Japan (JH) and Vladivostok, Russia (RV), respectively. One was an introduced population from Rongcheng, China (CR), which was introduced from Hokkaido, Japan in 1989. The results indicate that the effective number of alleles (Ne) ranged from 1.0000 to 12.3559. The observed heterozygosity (Ho) and the expected heterozygosity (He) at each locus range from 0.000 to 0.9000 and 0.0333 to 0.9329, respectively. All genetic parameters of
S28
Abstracts / Journal of Biotechnology 136S (2008) S22–S71
CR population are lower than those of the three natural populations, but a one-way ANOVA of the parameters at each locus indicates no significant differences. Although the decline of the genetic parameters in the CR population has not reached a significant level, it is of importance and should be monitored. Cluster analysis shows two branches in the UPGMA tree. The CR and JH populations form one branch and the KC and RV populations form another. The results are consistent with the geographic distribution of the populations. The research provides basic data for monitoring and protection of the populations, and provides direction for the development of sea urchin aquaculture. References Addison, J.A., Hart, M.W., 2004. Analysis of population genetic structure of the green sea urchin (Strongylocentrotus droebachiensis) using microsatellites. Mar. Biol. 144, 243–251. Chang, Y.Q., Chen, X.X., Ding, J., Cao, X.B., Li, R.L., Sun, X.W., 2007. Genetic diversity in five scallop populations of the Japanese scallop (Patinopecten yessoensis). Acta Ecol. Sin. 27, 0001–0007. Sato, M., Kawamata, K., Zaslavskaya, N., Nakamura, A., Ohta, T., Nishikiori, T., Brykov, V., Nagashima, K., 2005. Development of microsatellite markers for Japanese scallop (Mizuhopecten yessoensis) and their application to a population genetic study. Mar. Biotechnol. 7, 713–728. Sea Urchin Genome Sequencing Consortium, 2006. The genome of the sea urchin Strongylocentrotus purpuratus. Science 314, 941–952. Yu, H.T., Lee, Y.J., Huang, S.W., Chiu, T.S., 2002. Genetic analysis of the populations of Japanese anchovy (Engraulidae: Engraulis japonicus) using microsatellite DNA. Mar. Biotechnol. 4, 471–479.
doi:10.1016/j.jbiotec.2008.07.050
carboxylic acid (PCA) and the prenylated phenazine endophenazine A. This provided functional proof that the cluster contained all necessary genes for the biosynthesis of prenylated phenazines. To identify the function of the gene ppz1, PCR targeting was used to inactivate this gene, and the resulting construct was heterologously expressed in S. coelicolor M512. In this case, the resulting mutant could produce PCA but no prenylated phenazines. This proves that ppz1 codes for the prenyltransferase of endophenazine biosynthesis. References Laursen, J.B., Nielsen, J., 2004. Phenazine natural products: biosynthesis, synthetic analogues, and biological activity. Chem. Rev. 104, 1663–1686. Mavrodi, D.V., Blankenfeldt, W., Thomashow, L.S., 2006. Phenazine compounds in fluorescent Pseudomonas spp. biosynthesis and regulation. Annu. Rev. Phytopathol. 44, 417–447. Tello, M., Kuzuyama, T., Heide, L., Noel, J.P., Richard, S.B., 2008. The ABBA family of aromatic prenyltransferases: broadening natural product diversity. Cell. Mol. Life Sci. [Epub ahead of print].
doi:10.1016/j.jbiotec.2008.07.051 I1-Y-065 Antimicrobial activity of amino acids and dipeptide-based amphiphiles Nilanjan Kayal 1,∗ , Sangita Roy 2 , Rajendra Narayan Mitra 2 , Prasanta Kumar Das 2
A gene cluster for prenylated phenazine biosynthesis in Streptomyces anulatus 9663
1 Department of Biotechnology, School of Biotechnology Chemical and Biomedical Engineering, Vellore Institute of Technology University (VITU), Vellore 632014, India 2 Department of Biological Chemistry, Indian Association for Cultivation of Science (IACS), Kolkata 700032, India
Orwah Saleh 1 , Bertolt Gust 1 , Hans-Peter Fiedler 2 , Lutz Heide 1,∗
E-mail address: nilanjan [email protected] (N. Kayal).
I1-Y-064
1
Pharmazeutische Biologie, Eberhard-Karls-Universität Tübingen, auf der Morgenstelle 8, 72076 Tübingen, Germany 2 Mikrobiologisches Institut, Eberhard-Karls-Universität Tübingen, auf der Morgenstelle 28, 72076 Tübingen, Germany E-mail address: [email protected] (L. Heide). Naturally occurring phenazines are primarily isolated from Pseudomonas and Streptomyces. The biological properties of this class of natural products include antibiotic, antitumor, antimalarial and antiparasitic activities (Laursen and Nielsen, 2004). Several gene clusters for phenazine biosynthesis have been isolated from Pseudomonas, but no complete cluster from Streptomyces could be isolated yet (Mavrodi et al., 2006). To study the genetic basis of the phenazine biosynthesis in Streptomyces, we isolated a phenazine biosynthetic cluster from Streptomyces anulatus 9663. A cosmid library was prepared and screened for phenazine and mevalonate biosynthetic genes by Southern blot and PCR. One cosmid was selected for total sequencing. Sequence analysis revealed 30 open reading frames, including orthologues of all seven genes involved in the biosynthesis of phenazine-1-carboxylic acid in Pseudomonas (Mavrodi et al., 2006). Furthermore, this cluster contained a set of six genes for the biosynthesis of the prenyl moiety via the mevalonate pathway, a putative gene (ppz1) for an aromatic prenyltransferase from the ABBA family (Tello et al., 2008) and a putative gene for the N-methyltransferase in endophenazine B biosynthesis (ppzM). PCR targeting was used to introduce the integration functions of phage ФC31 and the resulting cosmid, containing the entire cluster, was heterologously expressed in Streptomyces coelicolor M512. HPLC and LC–MS analysis revealed in the formation of phenazine-1-
Cationic surfactants bear anti-bacterial activity. Surfactants are usually organic compounds which are amphiphilic in nature; they contain both hydrophobic groups (their “tails”) and hydrophilic groups (their “heads”). Thus they are soluble in both organic solvents and water. Quaternary ammonium compounds are found to be quite effective against both Gram’s positive and Gram’s negative bacteria, but they are also toxic. Their toxicity is related primarily to the various biological effects of the quaternary ammonium head and its metabolism (such as oxidative dealkylation), but it is also believed that the surfactant characteristics of the molecules, particularly in liver, causes additional alterations in a number of chemical, biological and transport phenomena. The mechanism of action of cationic surfactants on bacteria is understood to be purely electrostatic interaction and physical disruption. The cationic site of the agent is able to bind to the anionic sites of the cell wall surface. With a significant lipophilic component present, it is then able to diffuse through the cell wall and bind to membrane. As a surfactant it is able to disrupt the membrane and permit the release of electrolytes and nucleic materials, leading to cell death. Amino acid (Tryptophan)based cationic surfactants having carbon lengths C14 and C16 were tested with Klebsiela aerogenes (Gram negative) and Bacillus subtilis (Gram positive) and also give rise to semi-solid materials i.e. Gels, which can in turn have antimicrobial properties and can have a variety of uses in terms of antibiotics. Mainly peptides and dipeptides have been used which contains a combination of a number of amino acids. It was found that both the surfactants have their MIC (minimum inhibitory concentration) values far below their CMC (critical micellar concentration) and MGC (minimum gelation concentration) values hence thereby showing that they have antimicrobial activity.