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Genetically engineered poly-\gamma-glutamate producer from bacillus subtilis isw1214

Biosci. Biotechnol. Biochem., 70 (7), 1794–1797, 2006 Genetically Engineered Poly--glutamate Producerfrom Bacillus subtilis ISW1214 Makoto ASHIUCHI,y Kazuya SHIMANOUCHI, Terumi HORIUCHI,Tohru KAMEI, and Haruo MISONO Department of Bioresources Science, Kochi University, Nankoku, Kochi 783-8502, Japan Received February 13, 2006; Accepted April 4, 2006; Online Publication, July 23, 2006 The pgsBCA-gene disruptant from Bacillus subtilis a domestic strain very useful in gene engineering,13) ISW1214, i.e., MA41, does not produce poly--gluta- which harbors no plasmid DNA.14) It, however, has mate (PGA). We newly constructed an MA41 recombi- remained obscure about whether the ISW1214 strain nant bearing the plasmid-borne PGA synthetic system, cannot produce PGA as well as the 168 strain.11) Then in which PGA production was strictly controlled by the we examined the polymer productivity of B. subtilis use of xylose. Unlike the parent strain, ISW1214, the ISW1214. Growing cells (wet weight, 0.4 g) of B. sub- genetically engineered strain produced abundant PGA tilis ISW1214 were first inoculated into the following in both L-glutamate-rich and D-glutamate-rich media.
three media (50 ml): a standard (S) medium consistingof 5% sucrose, 0.5% MgSO .
0.42% Na2HPO4, 0.05% NaCl, a Murashige-Skoog vitamin solution (PhytoTechnology Laboratories, Shaw-nee Mission, KS.), and two essential amino acids (L- Poly--glutamate (PGA) is the most promising bio- leucine and L-methionine, each 0.5 mg mlÀ1); and the LS polymer in industry, the environment, and pharmaceut- and DS media, in which excess L- and D-glutamate icals.1) It has been assumed that Bacillus licheniformis (50 mg mlÀ1, viz., 340 mM) were further added to the S produces poly--D-glutamate (D-PGA) from L-gluta- medium, respectively. Cells were incubated in each mate alone in a thiotemplate-dependent multi-enzyme- medium for 5 d at 30 C and centrifuged at 12;000 Â g like fashion,2) which involves a unidirectional isomer- for 30 min at 4 C. PGA was prepared from the ization process3) of L- to D-glutamyl residues in a PGA supernatant according to the usual procedures1) and chain. In contrast, the extracellular polymer from purified by anion-exchange chromatography.15) Purified Bacillus subtilis is actually a mixture of multi-anionic PGA was determined by the method described previ- copolymers in which D- and L-glutamyl residues are ously.15) B. subtilis ISW1214 produced a large amount randomly aligned,1) as in the case of poly--DL-gluta- of PGA in the LS medium (about 4.4 mg mlÀ1) and only mate (DL-PGA). Although two distinct mechanisms for a slight amount of PGA in the DS medium (about the biosynthesis of DL-PGA have been proposed,1) it 0.3 mg mlÀ1), but did not produce PGA in the S medium was difficult to assess whether, in the polymer produc- with no glutamate. PGA productivity of ISW1214, tion by B. subtilis cells, it is formed from L-glutamate however, was not observed in the presence of a lower alone,4,5) as does B. licheniformis, or whether D-gluta- concentration of L-glutamate (e.g., 2, 3, or 5 mM).4) mate also serves as a substrate6) according to an amide B. subtilis ISW1214 is thus similar to naturally occur- ligase7,8)-like manner.9,10) Intricate regulation of PGA ring PGA over-producers of B. subtilis, such as B. sub- production in B. subtilis11,12) causes a difficulty in the tilis (natto) and B. subtilis subsp. chungkookjang,1) in interpretation of this issue. In this study, to simplify the the requirement of a high concentration of L-glutamate regulation of PGA production and construct a more convenient PGA producer, genetic alterations were Recent research indicates that the pgsBCA genes preformed to B. subtilis ISW1214, which is a tetracy- encode the sole machinery for PGA synthesis in cline-susceptible derivative from the 1012 strain of B. subtilis,9,16) viz., the PGA synthetase complex, in B. subtilis R13,14) and also the leucine/methionine which the PgsB component shows structural features seen commonly in the amide ligase family6) but the other Similarly to B. subtilis 168,11) B. subtilis ISW1214 is two components (PgsC and PgsA)16) do not encompass y To whom correspondence should be addressed. Fax: +81-888-64-5200; E-mail: ashiuchi@cc.kochi-u.ac.jpAbbreviations: PGA, poly--glutamate; Cm, chloramphenicol; LA-PCR, long amplification-polymerase chain reaction; Tc, tetracycline; SDS– PAGE, SDS–polyacrylamide gel electrophoresis any structural motifs that involve thiotemplate-depend- SpeI
SmaI
KpnI
SmaI
BamHI KpnI
SphI
BglII
ent biopolymer syntheses.3,17) In fact, a naturally occur-ring PGA over-producer, B. subtilis subsp. chungkook- B
jang, completely lost the polymer productivity due to thepgsBCA-gene disruption,9) and the constructed mutant C
was named MA11. To ascertain the similarity between A
B. subtilis ISW1214 and B. subtilis subsp. chungkook-jang in PGA production, we constructed the pgsBCA- pWPGBC1:
B
C
gene disruptant of B. subtilis ISW1214 as follows: First,the pKPSd plasmid9) carrying the chloramphenicol pWPGBA1:
B
A
(Cm)-resistance gene, which is useful for pgsBCA-genedisruption of various B. subtilis strains, was applied.
pWPGCA1:
C
A
Then chromosomal alteration was verified by a geneticstrategy involving the amplification of the target region B
C
A
by LA-PCR and the sequencing of the DNA fragmentusing the PPGS-U (50-TCATAGTGATTCTATATACT- B
C
A
GATGAAT-30) and PPGS-D (50-TTTGAATATGTTA-AGAGACTTTTTAAT-30) primers, as described previ-ously.9) The pgsBCA-gene disruptant obtained was xylR
PxylA
named MA41. It acquired Cm-resistance but lost PGAproductivity. The phenotype of the MA41 mutant was (pWH1520)
thus consistent with that of the MA11 mutant ofB. subtilis subsp. chungkookjang.9) tet
It appears likely that the pgsB, -C, and -A genes are at least indispensable for transformation of E. coli into a PGA producer,1,16,18) but a recent study of PGA PxylA, D-xylose-inducible promoter; xylR, D-xylose-dependent production by B. subtilis cells suggests that both the repressor; tet, tetracycline-resistance gene; open arrows B, the pgsBgene; open arrows C, the pgsC gene; open arrows A, the pgsA gene; ywsC (corresponding to pgsB) and ywtA (pgsC) gene short black bars, the designed ribosome-binding sequence. Among products are indeed essential, but that the ywtB (pgsA) these, only the pWPGO1 vector bears the original pgsBCA gene- gene product is dispensable.18) We expected that a containing sequence of B. subtilis ISW1214. These pgs genes cloned genetic complementation test of MA41 about PGA in B. subtilis cells are overexpressed in the presence of D-xylose and productivity would give a clue to this puzzle. Vectors to control pgs-gene expression were constructed as fol-lows: First, DNA fragments containing the pgsB, -C, and-A genes of B. subtilis ISW1214 were amplified by the medium, and named the LX and DX media, respective- PCR method with the PPGSB-NF2 and PPGSB-CR ly. PGA productivities of these pgs recombinants were primers, the PPGSC-NF and PPGSC-CR primers, and then examined, and we found that only the MA41/ the PPGSA-NF and PPGSA-CR2 primers,9) respective- pWPGS1 and MA41/pWPGO1 recombinants, which ly. The pgsB, -C, and -A gene-containing fragments are complemented by all the pgsB, -C, and -A genes, were verified with an automatic DNA sequencer, and produced high-molecular-mass PGA in the presence of then introduced into the multi-cloning site of Bacillus D-xylose and L-arabinose (Fig. 2 top, lanes 7 and 8). In expression vector pWH1520 carrying the tetracycline contrast, the MA41/pWPGBC1 recombinant (lacking (Tc)-resistance gene (MoBiTec, Go¨ttingen, Germany).
the pgsA gene) did not produce the extracellular polymer Figure 1 shows the structures of eight pgs vectors thus (lane 4). These results indicate that all the pgsB, -C, and developed: pWPGB1, pWPGC1, pWPGA1, pWPGBC1, -A gene products are indispensable for B. subtilis PGA pWPGBA1, pWPGCA1, pWPGS1, and pWPGO1. The production, at least in the use of the plasmid-borne PGA MA41 mutant was transformed with these pgs vectors synthetic system. In addition, the genetically engineered by the competence method.6) Recombinants were PGA producers never produce the polymer in the screened on a plate of Luria-Bertani medium19) with absence of D-xylose and L-arabinose (Fig. 2, bottom).
appropriate antibiotics (e.g., Tc, 10 mg mlÀ1; Cm, 5 mg Development of the plasmid-borne PGA synthetic mlÀ1). The recombinant harboring the pWPGS1 vector system for B. subtilis thus allowed a strict control in was tentatively abbreviated to MA41/pWPGS1, and the initiation of the polymer production. We further other recombinants constructed were also named in the observed that the genetically engineered strains, MA41/ same way. For pgs-gene induction, we newly prepared pWPGS1 and MA41/pWPGO1, produced abundant a modified (X) medium, in which D-xylose (5%) and PGA even in the DX medium (data not shown), unlike L-arabinose (1%) that activates the xylose uptake of the parent strain ISW1214. In fact, the former and latter B. subtilis cells20) were substituted for sucrose of the S strains accumulated PGA at about 8.2 and 3.8 mg mlÀ1 medium. Excess L- and D-glutamate were added to the X in the LX medium and at 9.0 and 5.5 mg mlÀ1 in the DX Kimura, K., Tran, L.-S., and Itoh, Y., Roles and M 1 2 3 4 5 6 7 8 9
regulation of the glutamate racemase isogenes, racEand yrpC, in Bacillus subtilis. Microbiology, 150, 2911– Itoh, Y., Molecular regulation and genetic instability of -polyglutamic acid production in Bacillus subtilis(natto). Bioscience and Industry (in Japanese), 57, Kubota, H., Matsunobu, T., Uotani, K., Takebe, H., Satoh, A., Tanaka, T., and Taniguchi, M., Production ofpoly(-glutamic acid) by Bacillus subtilis F-2-01. Biosci.
SDS–PAGE of PGAs Produced by the pgs Recombinants Biotechnol. Biochem., 57, 1212–1213 (1993).
Eveland, S. S., Pompliano, D. L., and Anderson, M. S., PGA was visualized on the gel by methylene-blue staining.1,10,16) Conditionally lethal Escherichia coli murein mutants Smear bands correspond to PGAs accumulated in 10 ml of the culturefiltrates of B. subtilis MA41/pWPGS1 (lane 7) and 50 ml of those of contain point defects that map tp regions conserved among murein and folyl poly--glutamate ligases: pWPGA1 (lane 3), MA41/pWPGBC1 (lane 4), MA41/pWPBA1 Identification of a ligase superfamily. Biochemistry, 36, (lane 5), MA41/pWPGCA1 (lane 6), MA41/pWPGO1 (lane 8), and MA41/pWH1520 (the negative control, lane 9), which were Vaganay, S., Tanner, M. E., van Heijenoort, J., and prepared after incubation for 5 d at 30 C in the LX medium (top) Blanot, D., Study of the reaction mechanism of the D- and the LS medium (bottom). Tetracycline (10 mg mlÀ1) was glutamic acid-adding enzyme from Escherichia coli.
essentially added to the media for the PGA-production test (5 d of Microb. Drug Resist., 2, 51–54 (1996).
Ashiuchi, M., Nawa, C., Kamei, T., Song, J.-J., Hong,S.-P., Sung, M.-H., Soda, K., and Misono, H., Physio-logical and biochemical characteristics of poly--gluta- medium, respectively. Accordingly, the polymer pro- mate synthetase complex of Bacillus subtilis. Eur. J.
ductivity of MA41/pWPGS1 is usually higher than that of MA41/pWPGO1. This benefit is probably brought by Ashiuchi, M., Shimanouchi, K., Nakamura, H., Kamei,T., Soda, K., Park, C., Sung, M.-H., and Misono, H., a simple modification in the gene structure, namely that Enzymatic synthesis of high-molecular-mass poly-- the typical ribosome-binding sequence is designed at the glutamate and regulation of its stereochemistry. Appl.
immediate upstream of each pgs gene on the pWPGS1 Environ. Microbiol., 70, 4249–4255 (2004).
vector so as to increase the translation levels of the pgsC Urushibata, Y., Tokuyama, S., and Tahara, Y., Differ- and -A genes besides that of the pgsB gene (Fig. 1).
ence in transcription levels of cap genes for -poly- Nevertheless, neither engineered strain produced PGA in glutamic acid production between Bacillus subtilis the presence of a lower concentration of glutamate (e.g., IFO16449 and Marburg 168. J. Biosci. Bioeng., 93, 5 mM).4) The result indicates that excess glutamate is required as the polymer substrate in B. subtilis.
Stanley, N. R., and Lazazzera, B. A., Defining the To our knowledge, this is the first example of genetic differences between wild and domestic strains of effective PGA production in the presence of Bacillus subtilis that affect poly--DL-glutamic acid production and biofilm formation. Mol. Microbiol., 57, mate. It appears likely that the genetic strategy con- structed in this study not only serves to deepen under- Yoshimoto, T., Oyama, H., Honda, T., Tone, H., standing of PGA biosynthesis, but also is applicable in Takeshita, T., Kamiyama, T., and Tsuru, D., Cloning mass-production of the useful biopolymer, because and expression of subtilisin amylosacchariticus gene.
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Park, C., Choi, J.-C., Choi, Y.-H., Nakamura, H.,Shimanouchi, K., Horiuchi, T., Misono, H., Sewaki, T., Ashiuchi, M., and Misono, H., Poly--glutamic acid. In Soda, K., Ashiuchi, M., and Misono, H., Synthesis ‘‘Biopolymers’’ Vol. 7, eds. Fahnestock, S. R., and of super-high-molecular-weight poly--glutamate from Steinbu¨chel, A., Wiley-VCH, Weinheim, pp. 123–174 Bacillus subtilis subsp. chungkookjang. J. Mol. Cat. B: Troy, F. A., Chemistry and biosynthesis of the poly(-D- Ashiuchi, M., Soda, K., and Misono, H., A poly-- glutamyl) capsule in Bacillus licheniformis. I. Properties glutamate synthetic system of Bacillus subtilis IFO of the membrane-mediated biosynthesis reaction. J. Biol.
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Thrash, I. & Derry, J.F. (1999) The nature and modelling of piospheres: a review. Koedoe 42 (2): 73-94. Pretoria. ISSN 0075-6458. REVIEW OF LITERATURE ON THE NATURE AND MODELLING OF PIOSPHERES I. Thrash and J.F. Derry A piosphere is an ecological system of interactions between a watering point, its surrounding vegetation and the grazing animal. In the simplest case of an isolated

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