Streptomyces rimosus aminoglycoside-3'-phosphotransferase VIII: comparisons with aminoglycoside-3'-phosphotransferases of aminoglycoside-producing strains with eukaryotic protein kinases

The nucleotide sequence was established for the aphVIII aminoglycoside phosphotransferase gene of an oxytetracycline-producing Streptomyces rimosus strain. The gene is 804 bp in size and possibly codes for APHVIII of 267 residues. Heterologous expression of aphVIII was studied in Escherichia coli and Chlamydomonas reinhardtii. The deduced APHVIII sequence was compared with known sequences of aminoglycoside phosphotransferases of aminoglycoside-producing actinomycete strains and of eukaryotic protein kinases. A local homology of 38 residues was found between APHVIII and actinomycete serine-threonine protein kinases in the conserved region possibly involved in ATP binding. APHVIII differed from aminoglycoside 3'-phosphotransferases of aminoglycoside-producing actinomycete strains and of clinical isolates, and can be classed to a separate group.     

Dependence of aminoglycoside 3′-phosphotransferase VIII activity on serine/threonine protein kinases in <Emphasis Type="Italic">Streptomyces rimosus</Emphasis>

In Streptomyces rimosus, selection for resistance to the aminoglycoside antibiotic kanamycin triggers the normally silent aminoglycoside 3-phosphotransferase VIII gene (aphVIII). The expression of APHVIII is accompanied by amplification of the chromosomal DNA fragment containing the aphVIII gene. Earlier, S. rimosus aphVIII gene was isolated and sequenced. Using in vitro labeling and immunoprecipitation with anti-APHVIII antibodies, we have demonstrated that endogenous protein kinases (PKs) in extracts of S. rimosus strain S683 actively phosphorylate two serine residues in the APHVIII molecule. The amount of phosphate incorporated into APHVIII in the presence of Ca²⁺ is 1.84-fold greater than that without Ca²⁺. Analysis of ingel autophosphorylation and phosphorylation of the substrate incorporated into the gel matrix has shown that modification of APHVIII is catalyzed by two serine/threonine PKs (74 kDa and 55 kDa). The activity of 55-kDa PK is dependent on Ca²⁺ and calmodulin. The specific kanamycin phosphotransferase activity of exhaustively phosphorylated APHVIII is 3.72 times higher than that of the unmodified enzyme. It is proposed that the above PKs may be involved in the regulation of kanamycin resistance in S. rimosus.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 255–263.Original Russian Text Copyright © 2005 by Elizarov, Sergienko, Sizova, Danilenko.     

Identification of phosphorylation sites in aminoglycoside phosphotransferase VIII from Streptomyces rimosus

We demonstrate for the first time the role of phosphorylation in the regulation of activities of enzymes responsible for inactivation of aminoglycoside antibiotics. The aminoglycoside phosphotransferase VIII (APHVIII) from the actinobacterial strain Streptomyces rimosus ATCC 10970 is an enzyme regulated by protein kinases. Two serine residues in APHVIII are shown to be phosphorylated by protein kinases from extracts of the kanamycin-resistant strain S. rimosus 683 (a derivative of strain ATCC 10970). Using site-directed mutagenesis and molecular modeling, we have identified the Ser146 residue in the activation loop of the enzyme as the key site for Ca²⁺-dependent phosphorylation of APHVIII. Comparison of the kanamycin kinase activities of the unphosphorylated and phosphorylated forms of the initial and mutant APHVIII shows that the Ser146 modification leads to a 6–7-fold increase in the kanamycin kinase activity of APHVIII. Thus, Ser146 in the activation loop of APHVIII is crucial for the enzyme activity. The resistance of bacterial cells to kanamycin increases proportionally. From the practical viewpoint, our results increase prospects for creation of highly effective test systems for selecting inhibitors of human and bacterial serine/threonine protein kinases based on APHVIII constructs and corresponding human and bacterial serine/threonine protein kinases.     

Dependence of aminoglycoside 3'-phosphotransferase VIII activity on protein serine/threonine kinases in Streptomyces rimosus

In Streptomyces rimosus, selection with aminoglycoside kanamycin triggers "silent" aminoglycoside 3'-phosphotransferase (aph) VIII gene. Expression of aphVIII was accompanied by amplification of a chromosomal DNA fragment, which contained aphVIII. Earlier, S. rimosus aphVIII gene was isolated, sequenced, and deduced APHVIII protein sequence was reported. Using in vitro labeling and immunoprecipitation with anti-APHVIII antibody, we demonstrate that one of the abundant proteins phosphorylated by endogenous protein kinases (PKs) in extracts of S. rimosus strain S683 is APHVIII. Phosphoamino acid assay has shown phosphorylation of two seryl residues in APH molecule. The amount of phosphate incorporated into APHVIII in the presence of Ca2+ was 1.84-fold as much as that detected without Ca2+. As shown by in the gel self-phosphorylation and in the substrate-containing gel phosphorylation analyses, two serine PKs with molecular masses of 74 kDa and 55 kDa were active against APHVIII. The 55-kDa PK showed a clear Ca2+ and calmodulin dependency in activity. The specific kanamycin phosphotransferase activity of exhaustedly phosphorylated APHVIII was 3.72-fold as much as that detected in the preparation of nonphosphorylated enzyme. These results suggest involvement of PKs under study in the modulation of APHVIII aminoglycoside phosphorylating activity and in the generation of kanamycin resistance in S. rimosus.     

Ca2+-dependent modulation of antibiotic resistance in Streptomyces lividans 66 and Streptomyces coelicolor A3(2)

The level of resistance to antibiotics of various chemical structure in actinobacteria of the genus Streptomyces is shown to be regulated by Ca²⁺ ions. The inhibitors of Ca²⁺/calmodulin and Ca²⁺/phospholipid-dependent serine/threonine protein kinases (STPK) are found to reduce antibiotic resistance of actinobacteria. The effect of Ca²⁺-dependent phosphorylation on the activity of the enzymatic aminoglycoside phosphotransferase system protecting actinobacteria from aminoglycoside antibiotics was studied. It is shown that inhibitors of Ca²⁺/calmodulin and Ca²⁺/phospholipid-dependent STPK reduced the Ca²⁺-induced kanamycin resistance in Streptomyces lividans cells transformed by a hybrid plasmid which contained the aminoglycoside phosphotransferase VIII (APHVIII) gene. In S. coelicolor A3(2) cells, the protein kinase PK25 responsible for APHVIII phosphorylation in vitro was identified. It is suggested that STPK play a major role in the regulation of antibiotic resistance in actinobacteria.     

Isolation and characterization of a <Emphasis Type="Italic">Paramecium</Emphasis> cDNA clone encoding a putative serine/threonine protein kinase

Mitogen-activated protein (MAP) kinases, a closely related family of protein kinases, are involved in cell cycle regulation and differentiation in yeast and human cells. They have not been documented in ciliates. We used PCR to amplify DNA sequences of a ciliated protozoan—Paramecium caudatum—using primers corresponding to amino acid sequences that are common to MAP kinases. We isolated and sequenced one putative MAP kinase-like serine/threonine kinase cDNA from P. caudatum. This cDNA, called pcstk1 (Paramecium caudatum Serine/Threonine Kinase 1) shared approximately 35% amino acid identity with MAP kinases from yeast. MAP kinases are activated by phosphorylation of specific threonine and tyrosine residues. These two amino acid residues are conserved in the PCSTK1 sequence at positions Thr 159 and Tyr 161. The PSTAIRE motif, which is characteristic of the CDK2 gene family, cannot be found in ORF of PCSTK1. The highest homology score was to human STK9, which contains MAP type kinase domains. Comparisons of expression level have shown that pcstk1 is expressed equally in cells at different stages (sexual and asexual). We discussed the possibility, as in other organisms, that a family of MAP kinase genes exists in P. caudatum.     

Characterization of the autophosphorylating kinase,PkaF, in <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> A3(2) M130

Streptomyces coelicolor, the model species for morphologically complex actinomycete bacteria, has unique characteristics such as morphological and physiological differentiation, which are controlled by various factors and several protein kinases. From the whole genomic sequence of S. coelicolor A3(2), 44 putative serine/threonine (Ser/Thr) protein kinases were identified, and the pkaF gene was chosen as the best-conserved protein for typical Ser/Thr protein kinases. pkaF encodes a 667-amino acid protein with a predicted N-terminal Ser/Thr kinase domain and four repeated C-terminal penicillin-binding domains and Ser/Thr kinase-associated (PASTA) domains. Based on PCR, a pkaF gene was cloned and heterologously expressed. PkaF expressed in Escherichia coli had the bigger molecular size than the expected value (75 kDa) and was further purified by Ni²⁺-NTA agarose affinity column chromatography to homogeneity. The purified PkaF was autophosphorylated through the transfer of the γ-phosphate group of ATP. The extent of phosphorylation was proportional to the amount of PkaF, and the phospho-PkaF was dephosphorylated by the addition of the cell lysate of S. coelicolor A3(2). Although no change was observed in the pkaF disruptant, overexpression of pkaF induced severe repression of morphogenesis and actinorhodin production, but not undecylprodigiosin production, implying that PkaF specifically regulates morphogenesis and actinorhodin production in S. coelicolor.     

Cloning and partial characterization of the putative rifamycin biosynthetic gene cluster from the actinomycete Amycolatopsis mediterranei DSM 46095

The actinomycete Amycolatopsis mediterranei produces the commercially and medically important polyketide antibiotic rifamycin, which is widely used against mycobacterial infections. The rifamycin biosynthetic (rif) gene cluster has been isolated, cloned and characterized from A. mediterranei S699 and A. mediterranei LBGA 3136. However, there are several other strains of A. mediterranei which also produce rifamycins. In order to detect the variability in the rif gene cluster among these strains, several strains were screened by PCR amplification using oligonucleotide primers based on the published DNA sequence of the rif gene cluster and by using dEBS II (second component of deoxy-erythronolide biosynthase gene) as a gene probe. Out of eight strains of A. mediterranei selected for the study, seven of them showed the expected amplification of the DNA fragments whereas the amplified DNA pattern was different in strain A. mediterranei DSM 46095. This strain also showed striking differences in the banding pattern obtained after hybridization of its genomic DNA against the dEBS II probe. Initial cloning and characterization of the 4-kb DNA fragment from the strain DSM 46095, representing a part of the putative rifamycin biosynthetic cluster, revealed nearly 10% and 8% differences in the DNA and amino acid sequence, respectively, as compared to that of A. mediterranei S699 and A. mediterranei LBGA 3136. The entire rif gene cluster was later cloned on two cosmids from A. mediterranei DSM 46095. Based on the partial sequence analysis of the cluster and sequence comparison with the published sequence, it was deduced that among eight strains of A. mediterranei, only A. mediterranei DSM 46095 carries a novel rifamycin biosynthetic gene cluster.     

Nucleotidylation of Aminoglycoside Antibiotics by Bacillus brevis

Biochemical transformations of aminoglycoside antibiotics by Bacillus species were examined. Among 39 strains of 8 Bacillus species, 4 strains of B. brevis were found to inactivate several aminoglycoside antibiotics: neamine, xylostasin, butirosin A and kanamycin A.

In the presence of Mg⁺² and ATP, the cell-free extracts of B. brevis IFO 12334 catalyzed the transformation of xylostasin to its inactive form. The structure of this inactivated xylostasin was determined to be 4′-O-monoadenylylxylostain from the ¹³C-NMR spectra, and from biochemical and spectroscopic studies.     

Molecular cloning and analysis of a cDNA coding for nucleoside diphosphate kinase from ryegrass

A full-length cDNA, LpNDPK, encoding ryegrass nucleoside diphosphate kinase (EC 2.7.4.6) has been cloned and sequenced. The nucleotide sequence of the clone contains an open reading frame of 450 nucleotides encoding a protein of 150 amino acid residues with a calculated molecular mass of 16.5 kDa and a P_i of 6.62. The LpNDPK encoded protein possesses substantial homology with nucleoside diphosphate kinases (NDPKs) isolated and cloned form other sources; the highest identity (86 percnt;) was observed with NDPK from sugarcane (Saccharum officinarum). Amino acid comparisons with other NDPKs show that the presented ryegrass NDPK sequence also contains several motifs and specific residues crucial for catalytic activity which are highly conserved among other NDPKs. RT-PCR expression analysis using primers covering the coding region of LpNDPK revealed that the ryegrass NDPK gene is equally expressed in stem, leaf, and flower tissue.     

Molecular Cloning and Nucleotide Sequencing of a Novel Aminoglycoside 6′-N-Acetyltransferase Gene from an R-Plasmid of Salmonella typhimurium S24 Isolated in Taiwan

A conjugative aminoglycoside resistance plasmid pST2 has been isolated from Escherichia coli K-12 14R525, which was mated with a clinical isolate of Salmonella typhimurium S24. A novel resistance gene of aminoglycoside 6′-N-acetyltransferase[AAC(6′)] was cloned from plasmid pST2 on a 1,393 kilobase (kb) of Sphl-SalI fragment into vector pACYC184 and pUC18. This novel A AC (6′) gene in plasmid pST2 acetylated kanamycin, amikacin, dibekacin, tobramycin, gentamicin, netilmicin, and sisomicin. The complete nucleotide sequence of the novel AAC(6′) gene and its neighboring sequences were also determined. Minicell experiments detected only one protein of 24.7 kilodaltons (kDa) translated from an open reading frame of the 618 base pairs (bp) gene.     

地黄RgCDPK基因的克隆与表达分析

钙依赖型蛋白激酶(calcium-dependent protein kinases, CDPKs)是高等植物细胞中重要的钙离子信号受体,在植物抵御逆境胁迫过程中发挥着重要作用。该研究以地黄为材料,设计特异引物,克隆地黄RgCDPK基因全长序列,并使用在线软件进行生物信息学分析,采用荧光定量PCR技术进行组织特异性分析。结果表明:(1)克隆得到的地黄CDPK基因长度为1 770 bp,编码589个氨基酸;(2)多序列比对和结构分析显示,该蛋白含有钙依赖蛋白激酶典型结构域丝氨酸/苏氨酸蛋白激酶区及EF-手性区。系统进化分析表明其与拟南芥 AtCDPK28 的同源关系最近,因此命名为RgCDPK(Genbank登录号为MT024235);(3)组织特异性分析得出RgCDPK在地黄叶中表达量最高。该研究成功克隆出地黄CDPK基因,且发现该基因在不同组织中的表达存在差异,为以后深入研究CDPK在地黄连作障碍等生物及非生物胁迫中的分子机制提供理论基础。     

Isolation of Endophytic Actinomycetes From Roots and Leaves of Banana (Musa Acuminata) Plants and Their Activities Against Fusarium oxysporumf. sp. cubense

Two hundred and forty-two actinomycete strains were isolated from the interior of leaves and roots of healthy and wilting banana plants. Most of them were streptomycetes, Streptomyces griseorubiginosus-like strains were the most frequently isolated strains. Community analysis demonstrated increased actinomycete diversity in wilting leaves compared to that in healthy leaves, similar actinomycete communities were found in wilting and healthy roots. Screening of the isolates for antagonistic activity against Fusarium oxysporumf. sp. cubenserevealed that the proportion of antagonistic streptomycetes in healthy roots was higher than that in wilting roots (P < 0.01), but no difference was found between antagonistic strains isolated from healthy and wilting leaves. The potential biological control of Panama disease of banana by endophytic streptomycetes, especially Streptomyces griseorubiginosus-like strains was discussed.     

Cloning,characterization and expression of the extracellular lipase gene from <Emphasis Type="Italic">Aureobasidium </Emphasis><Emphasis Type="Italic">pullulans</Emphasis> HN2-3 isolated from sea saltern

The extracellular lipase structural gene was isolated from cDNA of Aureobasidium pullulans HN2-3 by using SMART^TM RACE cDNA amplification kit. The gene had an open reading frame of 1245 bp long encoding a lipase. The coding region of the gene was interrupted by only one intron (55 bp). It encodes 414 amino acid residues of a protein with a putative signal peptide of 26 amino acids. The protein sequence deduced from the extracellular lipase structural gene contained the lipase consensus sequence (G-X-S-X-G) and three conserved putative N-glycosylation sites. According to the phylogenetic tree of the lipases, the lipase from A. pullulans was closely related to that from Aspergillus fumigatus (XP_750543) and Neosartorya fischeri (XP_001257768) and the identities were 50% and 52%, respectively. The mature peptide encoding cDNA was subcloned into pET-24a (+) expression vector. The recombinant plasmid was expressed in Escherichia coli BL21(DE3). The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 47 kDa was found. Enzyme activity assay verified the recombinant protein as a lipase. A maximum activity of 0.96 U/mg was obtained from cellular extract of E. coli BL21(DE3) harboring pET-24a(+)LIP1. Optimal pH and temperature of the crude recombinant lipase were 8.0 and 35 °C, respectively and the crude recombinant lipase had the highest hydrolytic activity towards peanut oil.     

Cloning and expression of <Emphasis Type="SmallCaps">d</Emphasis> -lactonohydrolase cDNA from <Emphasis Type="Italic">Fusarium moniliforme</Emphasis> in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A d -lactonohydrolase gene of about 1.1 kb was cloned from Fusarium moniliforme. The ORF sequence predicted a protein of 382 amino acids with a molecular mass of about 40 kDa. An expression plasmid carrying the gene under the control of the triose phosphate isomerase gene promotor was introduced into Saccharomyces cerevisiae, and the d -lactonohydrolase gene was successfully expressed in the recombinant strains.Revisions requested 10 September 2004; Revisions received 15 October 2004The nucleotide sequence data reported in this paper has been assigned accession number AY728018 in the GeneBank database.     

A new approach to the production of the recombinant SOD protein by methylotrophic <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The gene for the copper, zinc–superoxide dismutase (SOD) from the yeast Saccharomyces cerevisiae was cloned, characterized, and overexpressed in the methylotrophic Pichia pastoris. The sod gene sequence obtained is 465 bp and encodes 154 amino acid residues. The sod gene sequence was cloned into the pPIC9K vector, yielding pAB22. The linearized pAB22 DNA, digested with restriction enzyme SacI, was transformed into the genome of the GS115 strain of yeast P. pastoris. The overexpressed SOD protein was shown to have immunologically biological activity and to be enzymatically active. The SOD protein was purified from the cultured yeast by ammonium sulfate precipitation and diethylaminoethyl–cellulose column chromatography. This relatively simple purification method produced a single band on analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which indicated that the SOD protein obtained attained to higher purity and specific activity.     

Characterization of the <Emphasis Type="Italic">AXDH</Emphasis> gene and the encoded xylitol dehydrogenase from the dimorphic yeast <Emphasis Type="Italic">Arxula adeninivorans</Emphasis>

The xylitol dehydrogenase-encoding Arxula adeninivorans AXDH gene was isolated and characterized. The gene includes a coding sequence of 1107 bp encoding a putative 368 amino acid protein of 40.3 kDa. The identity of the gene was confirmed by a high degree of homology of the derived amino acid sequence to that of xylitol dehydrogenases from different sources. The gene activity was regulated by carbon source. In media supplemented with xylitol, D-sorbitol and D-xylose induction of the AXDH gene and intracellular accumulation of the encoded xylitol dehydrogenase was observed. This activation pattern was confirmed by analysis of AXDH promoter – GFP gene fusions. The enzyme characteristics were analysed from isolates of native strains as well as from those of recombinant strains expressing the AXDH gene under control of the strong A. adeninivorans-derived TEF1 promoter. For both proteins, a molecular mass of ca. 80 kDa was determined corresponding to a dimeric structure, an optimum pH at 7.5 and a temperature optimum at 35 °C. The enzyme oxidizes polyols like xylitol and D-sorbitol whereas the reduction reaction is preferred when providing D-xylulose, D-ribulose and L-sorbose as substrates. Enzyme activity exclusively depends on NAD⁺ or NADH as coenzymes.     

Nit-3, the structural gene of nitrate reductase in Neurospora crassa: nucleotide sequence and regulation of mRNA synthesis and turnover

Summary The nit-3 gene of the filamentous fungus Neurospora crassa encodes the enzyme nitrate reductase, which catalyzes the first reductive step in the highly regulated nitrate assimilatory pathway. The nucleotide sequence of nit-3 was determined and translates to a protein of 982 amino acid residues with a molecular weight of approximately 108 kDa. Comparison of the deduced nit-3 protein sequence with the nitrate reductase protein sequences of other fungi and higher plants revealed that a significant amount of homology exists, particularly within the three cofactor-binding domains for molybdenum, heme and FAD. The synthesis and turnover of the nit-3 mRNA were also examined and found to occur rapidly and efficiently under changing metabolic conditions.