Directed evolution of <Emphasis Type="Italic">Streptomyces lividans</Emphasis> xylanase B toward enhanced thermal and alkaline pH stability

The thermal and alkaline pH stability of Streptomyces lividans xylanase B was improved greatly by random mutagenesis using DNA shuffling. Positive clones with improved thermal stability in an alkaline buffer were screened on a solid agar plate containing RBB-xylan (blue). Three rounds of directed evolution resulted in the best mutant enzyme 3SlxB6 with a significantly improved stability. The recombinant enzyme exhibited significant thermostability at 70°C for 360 min, while the wild-type lost 50% of its activity after only 3 min. In addition, mutant enzyme 3SlxB6 shows increased stability to treatment with pH 9.0 alkaline buffer. The K _m value of 3SlxB6 was estimated to be similar to that of wild-type enzyme; however k _cat was slightly decreased, leading to a slightly reduced value of k _cat/K _m, compared with wild-type enzyme. DNA sequence analysis revealed that eight amino acid residues were changed in 3SlxB6 and substitutions included V3A, T6S, S23A, Q24P, M31L, S33P, G65A, and N93S. The stabilizing effects of each amino acid residue were investigated by incorporating mutations individually into wild-type enzyme. Our results suggest that DNA shuffling is an effective approach for simultaneous improvement of thermal and alkaline pH stability of Streptomyces lividans xylanase B even without structural information.     

Repressed multidrug resistance genes in <Emphasis Type="Italic">Streptomyces lividans</Emphasis>

Multidrug resistance (MDR) systems are ubiquitously present in prokaryotes and eukaryotes and defend both types of organisms against toxic compounds in the environment. Four families of MDR systems have been described, each family removing a broad spectrum of compounds by a specific membrane-bound active efflux pump. In the present study, at least four MDR systems were identified genetically in the soil bacterium Streptomyces lividans. The resistance genes of three of these systems were cloned and sequenced. Two of them are accompanied by a repressor gene. These MDR gene sequences are found in most other Streptomyces species investigated. Unlike the constitutively expressed MDR genes in Escherichia coli and other gram-negative bacteria, all of the Streptomyces genes were repressed under laboratory conditions, and resistance arose by mutations in the repressor genes.Abbreviations MDR Multidrug resistance     

Efficient purification of transglutaminase from recombinant <Emphasis Type="Italic">Streptomyces platensis</Emphasis> at various scales

An efficient system for the fast and efficient purification of transglutaminase from recombinant Streptomyces platensis and expressed in Streptomyces lividans 25-2 is described. Because the purification procedure of this system is flexible, culture broth from laboratory (20 l) and pilot-plant (130 l) fermentations were used to purify the enzyme to electrophoretic homogeneity with high purity (90–95%) and yield (61–77%) within 1 or 2 days.     

Expression of 2-deoxy-<Emphasis Type="Italic">scyllo</Emphasis>-inosose synthase (<Emphasis Type="Italic">kan</Emphasis>A) from kanamycin gene cluster in <Emphasis Type="Italic">Streptomyces lividans</Emphasis>

An actinomycetes expression vector (pIBR25) was constructed and applied to express a gene from the kanamycin biosynthetic gene cluster encoding 2-deoxy-scyllo-inosose synthase (kanA) in Streptomyces lividans TK24. The expression of kanA in pIBR25 transformants reached a maximum after 72 h of culture. The plasmid pIBR25 showed better expression than pSET152, and resulted in the formation of insoluble KanA when it was expressed in Escherichia coli. This strategy thus provides a valuable tool for expressing aminoglycoside-aminocyclitols (AmAcs) biosynthetic genes in Streptomyces spp.     

Purification and Properties of an <Emphasis Type="Italic">N</Emphasis>-acetylglucosaminidase from <Emphasis Type="Italic">Streptomyces cerradoensis</Emphasis>

An N-acetylglucosaminidase produced by Streptomyces cerradoensis was partially purified giving, by SDS-PAGE analysis, two main protein bands with Mr of 58.9 and 56.4 kDa. The K_m and V_max values for the enzyme using p-nitrophenyl-β-N-acetylglucosaminide as substrate were of 0.13 mM and 1.95 U mg⁻¹ protein, respectively. The enzyme was optimally activity at pH 5.5 and at 50 °C when assayed over 10 min. Enzyme activity was strongly inhibited by Cu²⁺ and Hg²⁺ at 10 mM, and was specific to substrates containing acetamide groups such as p-nitrophenyl-β-N-acetylglucosaminide and p-nitrophenyl-β-D-N,N′-diacetylchitobiose.     

Overexpression of yeast <Emphasis Type="Italic">S</Emphasis>-adenosylmethionine synthetase <Emphasis Type="Italic">metK</Emphasis> in <Emphasis Type="Italic">Streptomyces actuosus</Emphasis> leads to increased production of nosiheptide

S-Adenosylmethionine (SAM) is synthesized via the metabolic reaction involving adenosine triphosphate and l-methionine that is catalyzed by the enzyme S-adenosyl-l-methionine synthetase (SAM-s) and encoded by the gene metK. In the present study, metK with the absence of introns from Saccharomyces cerevisiae was introduced into Streptomyces actuosus, a nosiheptide (Nsh) producer. Intracellular SAM levels were determined by high-pressure liquid chromatography. Through optimizing the nutrient content of the medium, it was shown that increased SAM production induced by the overexpression of SAM-s leads to an increase in the intracellular cysteine pool and overproduction of Nsh in S. actuosus. This investigation shows that increased SAM promotes the elevated production of the non-ribosomal thiopeptide Nsh in Streptomyces sp.     

L-asparaginase production by <Emphasis Type="Italic">Streptomyces albidoflavus</Emphasis>

Attempts were made to optimize the cultural conditions for the production of L-asparaginase by Streptomyces albidoflavus under submerged fermentations. Enhanced level of L-asparaginase was found in culture medium supplemented with maltose as carbon source. Yeast extract (2%) was served as good nitrogen source for the production of L-asparaginase. The optimum pH for enzyme production was 7.5 and temperature was 35°C. The release of L-asparaginase from the cells of S. albidoflavus was high when strain was treated with cell disrupting agents like EDTA and lysozyme. The enzyme produced by the strain was purifi ed by ammonium sulfate, Sephadex G-100 and CM-Sephadex C-50 gel fi ltration and the molecular weight was apparently determined as 112 kDa.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Thermostable malate synthase of<Emphasis Type="Italic"> Streptomyces thermovulgaris</Emphasis>

The gene, encoding malate synthase (MS), aceB, was cloned from the thermophilic bacterium Streptomyces thermovulgaris by homology-based PCR. The 1,626-bp cloned fragment encodes a protein consisting of 541 amino acids. S. thermovulgaris malate synthase (stMS) gene was over-expressed in Escherichia coli using a glutathione-S transferase (GST) fusion vector (pGEX-6P-1), purified by affinity chromatography, and subsequently cleaved from its GST fusion partner. The purified stMS was characterized and compared to a mesophilic malate synthase (scMS) from Streptomyces coelicolor. stMS exhibited higher temperature optima (40–60 °C) than those of scMS (28–37 °C). It was more thermostable and very resistant to the chemical denaturant urea. Amino acid sequence comparison of stMS with four mesophilic streptomycete MSs indicated that they share 70.9–91.4% amino acid identities, with stMS possessing slightly more charged residues (~31%) than its mesophilic counterparts (~28–29%). Seven charged residues (E85, R187, R209, H239, H364, R382 and K520) that were unique to stMS may be selectively and strategically placed to support its peculiar characteristics.     

Phylogenetic analyses of <Emphasis Type="Italic">Uromyces viciae-fabae</Emphasis> and its varieties on <Emphasis Type="Italic">Vicia</Emphasis>, <Emphasis Type="Italic">Lathyrus</Emphasis>, and <Emphasis Type="Italic">Pisum</Emphasis> in Japan

A pea rust fungus, Uromyces viciae-fabae, has been classified into two varieties, var. viciae-fabae and var. orobi, based on differences in urediniospore wall thickness and putative host specificity in Japan. In principal component analyses, morphological features of urediniospores and teliospores of 94 rust specimens from Vicia, Lathyrus, and Pisum did not show definite host-specific morphological groups. In molecular analyses, 23 Uromyces specimens from Vicia, Lathyrus, and Pisum formed a single genetic clade based on D1/D2 and ITS regions. Four isolates of U. viciae-fabae from V. cracca and V. unijuga could infect and sporulate on P. sativum. These results suggest that U. viciae-fabae populations on different host plants are not biologically differentiated into groups that can be recognized as varieties.Contribution no. 184, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

<Emphasis Type="Italic">pspA</Emphasis> overexpression in <Emphasis Type="Italic">Streptomyces lividans</Emphasis> improves both Sec- and Tat-dependent protein secretion

Streptomyces is an interesting host for the secretory production of recombinant proteins because of its innate capacity to secrete proteins at high level in the culture medium. In this report, we evaluated the importance of the phage-shock protein A (PspA) homologue on the protein secretion yield in Streptomyces lividans. The PspA protein is supposed to play a role in the maintenance of the proton motive force (PMF). As the PMF is an energy source for both Sec- and Tat-dependent secretion, we evaluated the influence of the PspA protein on both pathways by modulating the pspA expression. Results indicated that pspA overexpression can improve the Tat-dependent protein secretion as illustrated for the Tat-dependent xylanase C and enhanced green fluorescent protein (EGFP). The effect on Sec-dependent secretion was less pronounced and appeared to be protein dependent as evidenced by the increase in subtilisin inhibitor (Sti-1) secretion but the lack of increase in human tumour necrosis factor (hTNFα) secretion in a pspA-overexpressing strain.     

Overproduction of soluble recombinant transglutaminase from <Emphasis Type="Italic">Streptomyces netropsis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A novel microbial transglutaminase (TGase) from the cultural filtrate of Streptomyces netropsis BCRC 12429 (Sn) was purified. The specific activity of the purified TGase was 18.2 U/mg protein with an estimated molecular mass of 38 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The TGase gene of S. netropsis was cloned and an open reading frame of 1,242 bp encoding a protein of 413 amino acids was identified. The Sn TGase was synthesized as a precursor protein with a preproregion of 82 amino acid residues. The deduced amino acid sequence of the mature S. netropsis TGase shares 78.9–89.6% identities with TGases from Streptomyces spp. A high level of soluble Sn TGase with its N-terminal propeptide fused with thioredoxin was expressed in E. coli. A simple and efficient process was applied to convert the purified recombinant protein into an active enzyme and showed activity equivalent to the authentic mature TGase. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Diversification of the barley and wheat <Emphasis Type="Italic">blt101</Emphasis>/<Emphasis Type="Italic">wpi6</Emphasis> promoters by the <Emphasis Type="Italic">Xumet</Emphasis> element without affecting stress responsiveness

BLT101-family plasma membrane proteins are found in a wide range of organisms from bacteria to nematodes and are involved in the regulation of cellular cation concentration under stress conditions. A comparison of the promoter regions of barley blt101 and its wheat ortholog, wpi6, revealed highly conserved nucleotide sequences between both genes and a unique insertion of a Xumet element in the blt101 promoter. The Xumet insertion occurred between a putative abscisic acid-responsive element (ABRE) and the dehydration-responsive element/c-repeat (DRE/CRT) within the blt101 promoter. However, blt101 and wpi6 were induced similarly in response to ABA, drought and low temperature, suggesting that the insertion does not affect promoter functions. The Xumet insertion in the blt101/wpi6 promoter region was detected in five barley cultivars, but absent in two wheat cultivars tested, suggesting that the insertion is barley-specific. Genomic Southern blot analysis revealed a large number of Xumet sequences interspersed in the barley genome, whereas only one or very few copies are present in the wheat genome. The data suggested that an expansion in copy number of Xumet elements occurred in the barley genome through evolution.     

The <Emphasis Type="Italic">kgmB</Emphasis> gene,encoding ribosomal RNA methylase from <Emphasis Type="Italic">Streptomyces tenebrarius</Emphasis>, is autogenously regulated

The KgmB methylase (the kanamycin–gentamicin resistance methylase from Streptomyces tenebrarius) acts at G-1405 of 16S rRNA within the sequence CGUCA that is also found 6 bp in front of ribosomal binding site of the kgmB gene. The kgmBlacZ gene and operon fusions were used in order to test for translational autoregulation of kgmB gene. Overexpression of kgmB either in cis or in trans drastically decreased the level of expression of the fusion protein. However, mutagenesis eliminated any role for the CGUCA sequence in translational autoregulation. Hence, the role of second putative regulatory sequence (CGCCC) that was shown to be involved in regulation of another methylase, Sgm (sisomicin–gentamicin methylase gene from Micromonospora zionensis) was examined. It was shown that the Sgm methylase can also decrease the level of expression of the kgmBlacZ fusion protein.     

Floral development and systematic position of <Emphasis Type="Italic">Arillastrum</Emphasis>, <Emphasis Type="Italic">Allosyncarpia</Emphasis>, <Emphasis Type="Italic">Stockwellia</Emphasis> and <Emphasis Type="Italic">Eucalyptopsis</Emphasis> (Myrtaceae)

We have investigated the floral ontogeny of Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis (of the eucalypt group, Myrtaceae) using scanning electron microscopy and light microscopy. Several critical characters for establishing relationships between these genera and to the eucalypts have been determined. The absence of compound petaline primordia in Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis excludes these taxa from the eucalypt clade. Post-anthesis circumscissile abscission of the hypanthium above the ovary in Stockwellia, Eucalyptopsis and Allosyncarpia is evidence that these three taxa form a monophyletic group; undifferentiated perianth parts and elongated fusiform buds are characters that unite Stockwellia and Eucalyptopsis as sister taxa. No floral characters clearly associate Arillastrum with either the eucalypt clade or the clade of Stockwellia, Eucalyptopsis and Allosyncarpia.We gratefully acknowledge Clyde Dunlop and Bob Harwood (Northern Territory Herbarium) for collecting specimens of Allosyncarpia, and Bruce Gray (Atherton) for collecting specimens of Stockwellia. The Australian National Herbarium (CANB) kindly lent herbarium specimens of Eucalyptopsis for examination. This research was supported by a University of Melbourne Research Development Grant to Andrew Drinnan.     

<Emphasis Type="Italic">fabC</Emphasis> of <Emphasis Type="Italic">Streptomyces lydicus</Emphasis> involvement in the biosynthesis of streptolydigin

Streptolydigin, a secondary metabolite produced by Streptomyces lydicus, is a potent inhibitor of bacterial RNA polymerases. It has been suggested that streptolydigin biosynthesis is associated with polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS). Thus, there is great interest in understanding the role of fatty acid biosynthesis in the biosynthesis of streptolydigin. In this paper, we cloned a type II fatty acid synthase (FAS II) gene cluster of fabDHCF from the genome of S. lydicus and constructed the SlyfabCF-disrupted mutant. Sequence analysis showed that SlyfabDHCF is 3.7 kb in length and encodes four separated proteins with conserved motifs and active residues, as shown in the FAS II of other bacteria. The SlyfabCF disruption inhibited streptolydigin biosynthesis and retarded mycelial growth, which were likely caused by the inhibition of fatty acid synthesis. Streptolydigin was not detected in the culture of the mutant strain by liquid chromatography–mass spectrometry. Meanwhile, the streptolol moiety of streptolydigin accumulated in cultures. As encoded by fabCF, acyl carrier protein (ACP) and β-ketoacyl-ACP synthase II are required for streptolydigin biosynthesis and likely involved in the step between PKS and NRPS. Our results provide the first genetic and metabolic evidence that SlyfabCF is shared by fatty acid synthesis and antibiotic streptolydigin synthesis.     

The Histidinol Phosphate Phosphatase Involved in Histidine Biosynthetic Pathway Is Encoded by <Emphasis Type="Italic">SCO5208</Emphasis> (<Emphasis Type="Italic">hisN</Emphasis>) in <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> A3(2)

Through the screening of a Streptomyces coelicolor genomic library, carried out in a histidinol phosphate phosphatase (HolPase) deficient strain, SCO5208 was identified as the last unknown gene involved in histidine biosynthesis. SCO5208 is a phosphatase, and it can restore the growth in minimal medium in this HolPase deficient strain when cloned in a high or low copy number vector. Moreover, it shares sequence homology with other HolPases recently identified in Actinobacteria. During this work a second phosphatase, SCO2771, sharing no homologies with SCO5208 and all so far described phosphatases was identified. It can complement HolPase activity mutation only at high copy number. Sequence analysis of SCO5208 and SCO2771, amplified from the HolPase mutant strain, revealed that SCO5208 shows a mutation in a conserved amino acid, whereas SCO2771 does not show any mutation. All these results show that S. coelicolor SCO5208, recently renamed hisN, is the HolPase involved in histidine biosynthesis.