| 1. |
Lobo S,
Florova G,
Reynolds KA,
( 2001 ) A Streptomyces collinus thiolase with novel acetyl-CoA:acyl carrier protein transacylase activity. PMID : 11570897 : DOI : 10.1021/bi011325a Abstract >>
Acetyl-CoA:acyl carrier protein (ACP) transacylase (ACT) activity has been demonstrated for the 3-ketoacyl-ACP synthase III (KASIII) which initiates fatty acid biosynthesis in the type II dissociable fatty acid synthases of plants and bacteria. Several lines of evidence have indicated the possibility of ACT activity being associated with proteins other than KASIII. Using a crude extract of Streptomyces collinus, we have resolved from KASIII an additional protein with ACT activity and subsequently purified it 85-fold in five chromatographic steps. The 45 kDa protein was shown by gel filtration to have a molecular mass of 185 +/- 35 kDa, consistent with a homotetrameric structure for the native enzyme. The corresponding gene (fadA) was cloned and sequenced and shown to encode a protein with amino acid sequence homology to type II thiolases. The fadA was expressed in Escherichia coli, and the resulting recombinant FadA enzyme purified by metal chelate chromatography was shown to have both ACT and thiolase activities. Kinetic studies revealed that in an ACT assay FadA had a substrate specificity for a two-carbon acetyl-CoA substrate (K(m) 8.7 +/- 1.4 microM) but was able to use ACPs from both type II fatty acid and polyketide synthases (Streptomyces glaucescens FabC ACP, K(m) 10.7 +/- 1.4 microM; E. coli FabC ACP, K(m) 8.8 +/- 2 microM; FrenN ACP, K(m) 44 +/- 12 microM). In the thiolase assay kinetic analyses revealed similar K(m) values for binding of substrates acetoacetyl-CoA (K(m) 9.8 +/- 0.8 microM) and CoA (K(m) 10.9 +/- 1.8 microM). A Cys92Ser mutant of FadA possessed virtually unchanged K(m) values for acetoacetyl-CoA and CoA but had a greater than 99% decrease in k(cat) for the thiolase activity. No detectable ACT activity was observed for the Cys92Ser mutant, demonstrating that both activities are associated with FadA and likely involve formation of the same covalent acetyl-S-Cys enzyme intermediate. An ACT activity with ACP has not previously been observed for thiolases and in the case of the S. collinus FadA is significantly lower (k(cat) 3 min(-1)) than the thiolase activity of FadA (k(cat) 2170 min(-1)). The ACT activity of FadA is comparable to the KAS activity and significantly higher than the ACT activity, reported for a streptomycete KASIII.
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2. |
Mikulík K,
Man P,
Halada P,
( 2001 ) Characterization of the rplB gene from Streptomyces collinus and its protein product by mass spectrometry. PMID : 11478805 : DOI : 10.1006/bbrc.2001.5336 Abstract >>
Ribosomal protein L2 is the largest protein components of 50S subunits. The protein is implicated in peptidyl transferase activity and binds to functionally important domains of 23S rRNA. The rplB gene, which codes for ribosomal protein L2 was cloned from Streptomyces collinus. The gene rplB was isolated from BamHI fragment (3.0 kb) of chromosomal DNA possessing two partial and four complete ORF's in the order from 5' to 3': rplC, rplD, rplW, rplB, rpsS, and rplV. The gene organization corresponds to the S10 operon. Gene rplB (834 bp) encodes a polypeptide chain of 278 amino acids. The molecular mass calculated from genomic structure is 30.5 kDa and pI 11.87. Protein L2 is rich in positively charged amino acids (Arg 36, Lys 20, and His 11). N-terminal domain possesses topology similar to the oligonucleotide/oligosaccharide binding OB folds. The availability of genome sequence makes it possible to identify L2 protein by mass spectrometry, moreover it facilitates the characterization of its potential posttranslational modifications. To confirm the protein sequence derived from the rplB gene the tryptic peptides of L2 were analyzed by mass spectrometric techniques. The obtained data matched exactly with the results of DNA sequencing.
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3. |
Cropp TA,
Patton SM,
( 2000 ) A novel delta(3),delta(2)-enoyl-CoA isomerase involved in the biosynthesis of the cyclohexanecarboxylic acid-derived moiety of the polyketide ansatrienin A. PMID : 10858310 : DOI : 10.1021/bi0005714 Abstract >>
The side chain of the antifungal polyketide ansatrienin A produced by Streptomyces collinus contains a cyclohexanecarboxylic acid (CHC) derived moiety. This CHC in the coenzyme A activated form (CHC-CoA) is derived from shikimic acid via a pathway in which the penultimate step is the isomerization of 2-cyclohexenylcarbonyl-CoA to 1-cyclohexenylcarbonyl-CoA. We have purified a 28 kDa 2-cyclohexenylcarbonyl-CoA isomerase (ChcB) from S. collinus and cloned and sequenced the corresponding chcB gene. The predicted amino acid sequence of ChcB showed moderate sequence identity to members of the hydratase/isomerase superfamily of enzymes. The recombinant ChcB was overexpressed in Escherichia coli and purified to homogeneity using metal chelate chromatography. Kinetic analysis demonstrated that recombinant ChcB had wide substrate specificity and could catalyze a double bond isomerization using 2-cyclohexenylcarbonyl-CoA (K(m) 116 +/- 68 microM, k(cat)()()3.7 +/- 1.0 min(-)(1)), trans-3-hexenyl-CoA (K(m) 39 +/- 10 microM, k(cat)()()12.8 +/- 1 min(-)(1)), and vinylacetyl-CoA (K(m) 156 +/- 34 microM, k(cat)()()29 +/- 3 min(-)(1)) as substrates. ChcB activity in cell extracts of S. collinus SP1, an insertionally disrupted chcB mutant, was shown to decrease by more than 99% (as compared to the wild-type strain) using all three of these substrates. The S. collinus SP1 strain, unlike the wild-type strain, could not produce omega-cyclohexyl fatty acids but was still able to grow efficiently on methyl oleate as a sole carbon source. These observations demonstrate that the S. collinus ChcB is required for catalyzing the isomerization of 2-cyclohexenylcarbonyl-CoA to 1-cyclohexenylcarbonyl-CoA during CHC-CoA biosynthesis but not for degradation of unsaturated fatty acids. The chcB gene does not appear to be associated with the ansatrienin biosynthetic gene cluster, which has previously been shown to contain at least one gene known to be essential for CHC-CoA biosynthesis. This finding represents a notable exception to the general rule regarding the clustering of polyketide biosynthetic pathway genes.
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4. |
Cropp TA,
Wilson DJ,
Reynolds KA,
( 2000 ) Identification of a cyclohexylcarbonyl CoA biosynthetic gene cluster and application in the production of doramectin. PMID : 10973220 : DOI : 10.1038/79479 Abstract >>
The side chain of the antifungal antibiotic ansatrienin A from Streptomyces collinus contains a cyclohexanecarboxylic acid (CHC)-derived moiety. This moiety is also observed in trace amounts of omega-cyclohexyl fatty acids (typically less than 1% of total fatty acids) produced by S. collinus. Coenzyme A-activated CHC (CHC-CoA) is derived from shikimic acid through a reductive pathway involving a minimum of nine catalytic steps. Five putative CHC-CoA biosynthetic genes in the ansatrienin biosynthetic gene cluster of S. collinus have been identified. Plasmid-based heterologous expression of these five genes in Streptomyces avermitilis or Streptomyces lividans allows for production of significant amounts of omega-cyclohexyl fatty acids (as high as 49% of total fatty acids). In the absence of the plasmid these organisms are dependent on exogenously supplied CHC for omega-cyclohexyl fatty acid production. Doramectin is a commercial antiparasitic avermectin analog produced by fermenting a bkd mutant of S. avermitilis in the presence of CHC. Introduction of the S. collinus CHC-CoA biosynthetic gene cassette into this organism resulted in an engineered strain able to produce doramectin without CHC supplementation. The CHC-CoA biosynthetic gene cluster represents an important genetic tool for precursor-directed biosynthesis of doramectin and has potential for directed biosynthesis in other important polyketide-producing organisms.
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5. |
Zhulanova E,
Krátký M,
Mikulík K,
( 2000 ) Isolation and characterization of dcw cluster from Streptomyces collinus producing kirromycin. PMID : 10679194 : DOI : 10.1006/bbrc.2000.2127 Abstract >>
A 4.5-kb BamHI fragment of chromosomal DNA of Streptomyces collinus containing gene ftsZ was cloned and sequenced. Upstream of ftsZ are localized genes ftsQ, murG, and ftsW, and downstream is yfiH. Gene ftsA is not adjacent to ftsZ or other genes of the cloned fragment. Protein FtsZ was isolated and characterized with respect to its binding to GTP and GTPase activity. The binding of GTP to FtsZ was Ca(2+) or Mg(2+) dependent with an optimum at 10 mM. The rate of GTP hydrolysis by FtsZ was stimulated by KCl. The presence of Ca(2+) (3-5 mM) resulted in a significant increase of GTPase activity. Higher concentrations of Ca(2+) than 5 mM had an inhibitory effect on GTPase activity. These results indicate that divalent ions (Ca(2+) or Mg(2+)) can be involved in regulation of GTP binding and hydrolysis of FtsZ. The maximum level of FtsZ was detected in aerial mycelium when spiral loops and sporulation septa were formed. FtsZ is degraded after finishing sporulation septa.
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6. |
Leistner E,
Reynolds KA,
Floss HG,
Breuer M,
Chen S,
Müller R,
Hardt B,
( 1999 ) Biosynthesis of ansatrienin (mycotrienin) and naphthomycin. Identification and analysis of two separate biosynthetic gene clusters in Streptomyces collinus T? 1892. PMID : 10103039 : DOI : 10.1046/j.1432-1327.1999.00244.x Abstract >>
The polyketide chains of the two ansamycin antibiotics, ansatrienin (mycotrienin) and naphthomycin produced by Streptomyces collinus are assembled using 3-amino-5-hydroxybenzoic acid (AHBA) as a starter unit. The gene encoding AHBA synthase, an enzyme which catalyzes the final step of AHBA biosynthesis in the recently discovered aminoshikimate pathway, has been used to identify two separate antibiotic biosynthetic gene clusters in S. collinus. In one of these clusters, analysis of approximately 20 kb of contiguous sequence has revealed both a cluster of six genes presumed to play a role in the AHBA pathway and the beginning of a polyketide synthase (PKS) gene containing an acyl ACP ligase domain. This domain is likely responsible for loading AHBA onto the PKS. This gene cluster also contains chcA, encoding the enzyme 1-cyclohexenylcarbonyl CoA reductase, which is essential for the biosynthesis of the cyclohexanecarboxylic acid moiety of ansatrienin from shikimic acid, and a peptide synthetase. This gene cluster thus seems to control the biosynthesis of ansatrienin, which contains a side chain of N-cyclohexanecarbonyl-d-alanine esterified to the macrocyclic lactam backbone. In the putative naphthomycin biosynthetic gene cluster approximately 13 kb of contiguous sequence has revealed a second set of the genes required for AHBA biosynthesis. In addition the end of a polyketide synthase and a gene putatively involved in termination of the chain extension process, formation of an intramolecular amide bond between the AHBA nitrogen and the carboxyl group of the fully extended polyketide chain, have been identified. Thus, despite commonality in biosynthesis, the ansatrienin and naphthomycin biosynthetic gene clusters show clear organizational differences and carry separate sets of genes for AHBA biosynthesis.
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7. |
Kim BJ,
Kim CJ,
Chun J,
Koh YH,
Lee SH,
Hyun JW,
Cha CY,
Kook YH,
( 2004 ) Phylogenetic analysis of the genera Streptomyces and Kitasatospora based on partial RNA polymerase beta-subunit gene (rpoB) sequences. PMID : 15023980 : DOI : 10.1099/ijs.0.02941-0 Abstract >>
The RNA polymerase beta-subunit genes (rpoB) of 67 Streptomyces strains, representing 57 species, five Kitasatospora strains and Micromonospora echinospora KCTC 9549 were partially sequenced using a pair of rpoB PCR primers. Among the streptomycetes, 99.7-100 % similarity within the same species and 90.2-99.3 % similarity at the interspecific level were observed by analysis of the determined rpoB sequences. The topology of the phylogenetic tree based on rpoB sequences was similar to that of 16S rDNA. The five Kitasatospora strains formed a stable monophyletic clade and a sister group to the clade comprising all Streptomyces species. Although there were several discrepancies in the details, considerable agreement was found between the results of rpoB analysis and those of numerical phenetic classification. This study demonstrates that analysis of rpoB can be used as an alternative genetic method in parallel to conventional taxonomic methods, including numerical phenetic and 16S rDNA analyses, for the phylogenetic analyses of the genera Streptomyces and Kitasatospora.
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8. |
Gueneau de Novoa P,
Williams KP,
( 2004 ) The tmRNA website: reductive evolution of tmRNA in plastids and other endosymbionts. PMID : 14681369 : DOI : 10.1093/nar/gkh102 PMC : PMC308836 Abstract >>
tmRNA combines tRNA- and mRNA-like properties and ameliorates problems arising from stalled ribosomes. Research on the mechanism, structure and biology of tmRNA is served by the tmRNA website (http://www.indiana.edu/~ tmrna), a collection of sequences, alignments, secondary structures and other information. Because many of these sequences are not in GenBank, a BLAST server has been added; another new feature is an abbreviated alignment for the tRNA-like domain only. Many tmRNA sequences from plastids have been added, five found in public sequence data and another 10 generated by direct sequencing; detection in early-branching members of the green plastid lineage brings coverage to all three primary plastid lineages. The new sequences include the shortest known tmRNA sequence. While bacterial tmRNAs usually have a lone pseudoknot upstream of the mRNA segment and a string of three or four pseudoknots downstream, plastid tmRNAs collectively show loss of pseudoknots at both postions. The pseudoknot-string region is also too short to contain the usual pseudoknot number in another new entry, the tmRNA sequence from a bacterial endosymbiont of insect cells, Tremblaya princeps. Pseudoknots may optimize tmRNA function in free-living bacteria, yet become dispensible when the endosymbiotic lifestyle relaxes selective pressure for fast growth.
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9. |
Weber T,
Laiple KJ,
Pross EK,
Textor A,
Grond S,
Welzel K,
Pelzer S,
Vente A,
Wohlleben W,
( 2008 ) Molecular analysis of the kirromycin biosynthetic gene cluster revealed beta-alanine as precursor of the pyridone moiety. PMID : 18291322 : DOI : 10.1016/j.chembiol.2007.12.009 Abstract >>
Kirromycin is a complex linear polyketide that acts as a protein biosynthesis inhibitor by binding to the bacterial elongation factor Tu. The kirromycin biosynthetic gene cluster was isolated from the producer, Streptomyces collinus T? 365, and confirmed by targeted disruption of essential biosynthesis genes. Kirromycin is synthesized by a large hybrid polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) encoded by the genes kirAI-kirAVI. This complex involves some very unusual features, including the absence of internal acyltransferase (AT) domains in KirAI-KirAV, multiple split-ups of PKS modules on separate genes, and swapping in the domain organization. Interestingly, one PKS enzyme, KirAVI, contains internal AT domains. Based on in silico analysis, a route to pyridone formation involving PKS and NRPS steps was postulated. This hypothesis was experimentally proven by feeding studies with [U-13C3(15)N]beta-alanine and NMR and MS analyses of the isolated pure kirromycin.
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10. |
( 1997 ) A novel alternate anaplerotic pathway to the glyoxylate cycle in streptomycetes. PMID : 9260959 : DOI : 10.1128/jb.179.16.5157-5164.1997 PMC : PMC179375 Abstract >>
ccr encoding crotonyl coenzyme A (CoA) reductase (CCR), which catalyzes the conversion of crotonyl-CoA to butyryl-CoA in the presence of NADPH, was previously cloned from Streptomyces collinus. We now report that a complete open reading frame, designated meaA, is located downstream from ccr. The predicted gene product showed 35% identity with methylmalonyl-CoA mutases from various sources. In addition, the predicted amino acid sequences of S. collinus ccr and meaA exhibit strong similarity to that of adhA (43% identity), a putative alcohol dehydrogenase gene, and meaA (62% identity) of Methylobacterium extorquens, respectively. Both adhA and meaA are involved in the assimilation of C1 and C2 compounds in an unknown pathway in the isocitrate lyase (ICL)-negative Methylobacterium. We have demonstrated that S. collinus can grow with acetate as its sole carbon source even though there is no detectable ICL, suggesting that in this organism ccr and meaA may also be involved in a pathway for the assimilation of C2 compounds. Previous studies with streptomycetes provided a precedent for a pathway that initiates with the condensation of two acetyl-CoA molecules to form butyryl-CoA, which is then transformed to succinyl-CoA with two separate CoB12-mediated rearrangements and a series of oxidations. The biological functions of ccr and meaA in this process were investigated by gene disruption. A ccr-blocked mutant showed no detectable crotonyl-CoA reductase activity and, compared to the wild-type strain, exhibited dramatically reduced growth when acetate was the sole carbon source. An meaA-blocked mutant also exhibited reduced growth on acetate. However, both methylmalonyl-CoA mutase and isobutyryl-CoA mutase, which catalyze the two CoB12-dependent rearrangements in this proposed pathway, were shown to be present in the meaA-blocked mutant. These results suggested that both ccr and meaA are involved in a novel pathway for the growth of S. collinus when acetate is its sole carbon source.
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11. |
( 1997 ) Protein kinase associated with ribosomes phosphorylates ribosomal proteins of Streptomyces collinus. PMID : 9299515 : DOI : 10.1006/bbrc.1997.7297 Abstract >>
Protein kinase activity associated with ribosomes of a kirromycin-producing strain of Streptomyces collinus was detected. The enzyme utilizes [gamma-32P]ATP to phosphorylate proteins, yielding acid-stable phosphoamino acids. Two-dimensional electrophoresis of proteins from a crude ribosomal fraction revealed 17 phosphoproteins. Eleven of the phosphoproteins exhibited electrophoretic mobility identical to that of S. collinus ribosomal proteins S3, S4, S12, S13, S14, S18, L2, L7, L16, L17, and L23. Protein L2 was identified by microsequencing of internal peptide fragments. Immunodetection with monoclonal antibodies indicated that the ribosomal proteins are phosphorylated on serine and threonine residues. Phosphorylation of ribosomal proteins led to the reduction of activity of ribosomes in the translation of poly(U). These results provide the first evidence of phosphorylation of ribosomal proteins in bacteriophage-uninfected cells of eubacteria.
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12. |
( 1996 ) Cloning and characterization of the gene encoding 1-cyclohexenylcarbonyl coenzyme A reductase from Streptomyces collinus. PMID : 8955309 : DOI : 10.1128/jb.178.23.6873-6881.1996 PMC : PMC178588 Abstract >>
We report the cloning of the gene encoding the 1-cyclohexenylcarbonyl coenzyme A reductase (ChcA) of Streptomyces collinus, an enzyme putatively involved in the final reduction step in the formation of the cyclohexyl moiety of ansatrienin from shikimic acid. The cloned gene, with a proposed designation of chcA, encodes an 843-bp open reading frame which predicts a primary translation product of 280 amino acids and a calculated molecular mass of 29.7 kDa. Highly significant sequence similiarity extending along almost the entire length of the protein was observed with members of the short-chain alcohol dehydrogenase superfamily. The S. collinus chcA gene was overexpressed in Escherichia coli by using a bacteriophage T7 transient expression system, and a protein with a specific ChcA activity was detected. The E. coli-produced ChcA protein was purified and shown to have similar steady-state kinetics and electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels as the enoyl-coenzyme A reductase protein prepared from S. collinus. The enzyme demonstrated the ability to catalyze, in vitro, three of the reductive steps involved in the formation of cyclohexanecarboxylic acid. An S. collinus chcA mutant, constructed by deletion of a genomic region comprising the 5' end of chcA, lost the ChcA activity and the ability to synthesize either cyclohexanecarboxylic acid or ansatrienin. These results suggest that chcA encodes the ChcA that is involved in catalyzing multiple reductive steps in the pathway that provides the cyclohexanecarboxylic acid from shikimic acid.
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13. |
( 1995 ) Purification of crotonyl-CoA reductase from Streptomyces collinus and cloning, sequencing and expression of the corresponding gene in Escherichia coli. PMID : 8521864 : DOI : 10.1111/j.1432-1033.1995.954_3.x Abstract >>
A crotonyl-CoA reductase (EC 1.3.1.38, acyl-CoA:NADP+ trans-2-oxidoreductase) catalyzing the conversion of crotonyl-CoA to butyryl-CoA has been purified and characterized from Streptomyces collinus. This enzyme, a dimer with subunits of identical mass (48 kDa), exhibits a Km = 18 microM for crotonyl-CoA and 15 microM for NADPH. The enzyme was unable to catalyze the reduction of any other enoyl-CoA thioesters or to utilize NADH as an electron donor. A highly effective inhibition by straight-chain fatty acids (Ki = 9.5 microM for palmitoyl-CoA) compared with branched-chain fatty acids (Ki > 400 microM for isopalmitoyl-CoA) was observed. All of these properties are consistent with a proposed role of the enzyme in providing butyryl-CoA as a starter unit for straight-chain fatty acid biosynthesis. The crotonyl-CoA reductase gene was cloned in Escherichia coli. This gene, with a proposed designation of ccr, is encoded in a 1344-bp open reading frame which predicts a primary translation product of 448 amino acids with a calculated molecular mass of 49.4 kDa. Several dispersed regions of highly significant sequence similarity were noted between the deduced amino acid sequence and various alcohol dehydrogenases and fatty acid synthases, including one region that contains a putative NADPH binding site. The ccr gene product was expressed in E. coli and the induced crotonyl-CoA reductase was purified tenfold and shown to have similar steady-state kinetics and electrophoretic mobility on sodium dodecyl sulfate/polyacrylamide to the native protein.
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14. |
Mikulík K,
Zhulanova E,
( 1995 ) Sequencing of the tuf1 gene and the phosphorylation pattern of EF-Tu1 during development and differentiation in Streptomyces collinus producing kirromycin. PMID : 7646499 : DOI : 10.1006/bbrc.1995.2153 Abstract >>
We have cloned and sequenced the tuf1 gene from a kirromycin-producing strain of Streptomyces collinus. The gene encodes a polypeptide of 396 amino acid residues with a molecular weight of 43,849. The protein shows 97% identity with EF-Tu1 of S. coelicolor and is sensitive to kirromycin. EF-Tu-dependent translation of poly(U) was reduced to 50% in the presence of 0.25 microM kirromycin. Using high resolution two-dimensional electrophoresis and specific immunodetection with monoclonal antibodies we found that the EF-Tu1 is phosphorylated on threonine and that serine is the second phosphate-accepting amino acid. EF-Tu1 phosphorylated on threonine and serine residues was detected among the S150 supernatant proteins of vegetative cells, aerial mycelium and spores. The level of phosphorylated EF-Tu1 varied during the growth and differentiation.
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15. |
( 1998 ) Characterization of ftsZ gene and its protein product from Streptomyces collinus producing kirromycin. PMID : 9712736 : DOI : 10.1006/bbrc.1998.9193 Abstract >>
The FtsZ protein is required for septation and conversion of aerial mycelium into chains of streptomycete spores. We have cloned and sequenced the ftsZ gene from Streptomyces collinus. The ftsZ of S. collinus is not in juxtaposition with ftsA and IpxC as in Escherichia coli or Bacillus subtilis. The gene encodes a polypeptide of 402 amino acid residues with a molecular mass of 41.3 kDa. N-terminus shares a high level of sequence similarity with FtsZ of S. coelicolor and S. griseus, respectively. C-terminal part is variable both in length and sequence. The purified protein binds GTP. Using polyclonal antisera against FtsZ, we have found that the protein is expressed at the beginning of germination of spores and is present in vegetative cells and aerial mycelium, but not in spores.
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