Expression of<Emphasis Type="Italic"> FoxE</Emphasis> and<Emphasis Type="Italic"> FoxQ</Emphasis> genes in the endostyle of<Emphasis Type="Italic"> Ciona intestinalis</Emphasis>

We have analyzed the expression patterns of two Fox genes, FoxE and FoxQ, in the ascidian Ciona intestinalis. Expression of Ci-FoxE was specific to the endostyle of adults, being prominent in the thyroid-equivalent region of zone 7. Ci-FoxQ was expressed in several endodermal organs of adult ascidians, such as the endostyle, branchial sac and esophagus. In the endostyle, the pattern of Ci-FoxQ expression was similar to that of CiTTF-1, being prominent in the thyroid-equivalent regions of zones 7 and 8. Therefore, these Fox genes may perform thyroid-equivalent functions in the ascidian endostyle.Edited by N. Satoh     

Genetic organization of the putative salbostatin biosynthetic gene cluster including the 2-<Emphasis Type="Italic">epi</Emphasis>-5-<Emphasis Type="Italic">epi</Emphasis>-valiolone synthase gene in <Emphasis Type="Italic">Streptomyces albus</Emphasis> ATCC 21838

In this paper, we provide the first report of utilizing recombinant fungal whole cells in enzymatic biodiesel production. Aspergillus oryzae, transformed with a heterologous lipase-encoding gene from Fusarium heterosporum, produced fully processed and active forms of recombinant F. heterosporum lipase (FHL). Cell immobilization within porous biomass support particles enabled the convenient usage of FHL-producing A. oryzae as a whole-cell biocatalyst for lipase-catalyzed methanolysis. The addition of 5% water to the reaction mixture was effective in both preventing the lipase inactivation by methanol and facilitating the acyl migration in partial glycerides, resulting in the final methyl ester content of 94% even in the tenth batch cycle. A comparative study showed that FHL-producing A. oryzae attained a higher final methyl ester content and higher lipase stability than Rhizopus oryzae, the previously developed whole-cell biocatalyst. Although both FHL and R. oryzae lipase exhibit 1,3-regiospecificity towards triglyceride, R. oryzae accumulated a much higher amount of sn−2 isomers of partial glycerides, whereas FHL-producing A. oryzae maintained a low level of the sn−2 isomers. This is probably because FHL efficiently facilitates the acyl migration from the sn−2 to the sn−1(3) position in partial glycerides. These findings indicate that the newly developed FHL-producing A. oryzae is an effective whole-cell biocatalyst for enzymatic biodiesel production.     

Existence of<Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> genomic fragment hybridizing with the<Emphasis Type="Italic">nifM</Emphasis> gene of<Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

A novel finding that genomic restriction fragments of symbiotic nitrogen fixer S. meliloti hybridized with nifM gene probe of the free-living diazotroph Klebsiella pneumoniae is reported. When SmaI endonuclease was used to digest S. meliloti DNA, a unique hybridizing band was obtained.     

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.     

Targeted disruption of<Emphasis Type="Italic">Streptomyces globisporus lndF</Emphasis> and<Emphasis Type="Italic">lndL</Emphasis> cyclase genes involved in landomycin E biosynthesis

Streptomyces globisporus strains with knockouts in lndF and lndL genes, previously identified as possibly encoding cyclases governing cyclization of the nascent oligoketide ('polyketide') chain during the biosynthesis of the antitumor angucycline landomycin E, were prepared. On combining the results of sequence analysis and HPLC of extracts from mutant strains, lndL was suggested to control the first cyclization-aromatization event and lndF to be responsible for the 3rd-4th ring formation.     

Organization of<Emphasis Type="Italic"> Iroquois</Emphasis> genes in fish

In mammals, a total of six iroquois (Irx) genes exist, which are organized into two clusters. Here we report on the organization of all iroquois genes present in fish, using zebrafish (Danio rerio) and pufferfish (Fugu rubripes and Tetraodon nigroviridis) as examples. A total of 10 Irx genes were found in pufferfish, and 11 in zebrafish; all but one of these genes are organized into clusters (four clusters plus one isolated gene locus). The extra fish clusters result from chromosome duplication in the fish lineage, after its divergence from tetrapod vertebrates. Two of the four fish clusters are highly conserved to the ones in mammals, with regard to similarity of genes and cluster architecture. Irx genes within the other two clusters have diverged in sequence and cluster organization, suggesting functional divergence. These results will allow us to use the zebrafish system for functional and comparative studies of iroquois genes in vertebrate development.Electronic Supplementary Material Supplementary material is available in the online version of this article at Edited by D. Tautz     

Evaluation of<Emphasis Type="Italic">in Vitro</Emphasis> antifungal activity of<Emphasis Type="Italic">N</Emphasis>-Benzylsalicylamide derivatives

A series of 65 derivatives of N-benzylsalicylamide was tested against eight potentially human pathogenic fungi by microdilution broth method modified according to M27-A standard. The majority of these compounds showed only weak in vitro antifungal activity. The most significant effect was observed against filamentous fungi Trichophyton mentagrophytes, Absidia corymbifera, and Aspergillus fumigatus while yeasts, in general, were less susceptible. N-(4'-Chlorobenzyl) salicylamides, N-(3',4'-dichlorobenzyl)-salicylamides, and partially N-benzylsalicylamides exhibited relatively high in vitro antifungal activity. The most efficient derivatives had MIC < or = 7.8 mumol/L against T. mentagrophytes. Regression analysis suggested an indirect relationship between MIC values and lipophilicity (log P).     

The importance of<Emphasis Type="Italic"> porE</Emphasis> and<Emphasis Type="Italic"> porF</Emphasis> in the anabolic pyruvate oxidoreductase of<Emphasis Type="Italic"> Methanococcus maripaludi</Emphasis>s

The operon of the anabolic pyruvate oxidoreductase (POR) of Methanococcus maripaludis encodes two genes (porEF) whose functions are unknown. Because these genes possess sequence similarity to polyferredoxins, they may be electron carriers to the POR. To elucidate whether the methanococcal POR requires PorEF for activity, a deletion mutant, strain JJ150, lacking porEF was constructed. Compared to the wild-type strain JJ1, the mutant grew more slowly in minimal medium and minimal plus acetate medium, and pyruvate-dependent methanogenesis was inhibited. In contrast, the methyl-viologen-dependent pyruvate-oxidation activity of POR, carbon monoxide dehydrogenase, and hydrogenase activities of the mutant were similar to those of the wild-type. Upon genetic complementation of the mutant with porEF in the methanococcal shuttle vector pMEV2+porEF, growth in minimal medium and pyruvate-dependent methanogenesis were restored to wild-type levels. Complementation with porE alone restored methanogenesis from pyruvate but not growth in minimal medium. Complementation with porF alone partially restored growth but not methanogenesis from pyruvate. Although the specific roles of porE and porF have not been determined, these results suggest that PorEF play important roles in the anabolic POR in vivo even though they are not required for the dye-dependent activity.Abbreviations CODH/ACS Carbon monoxide dehydrogenase/acetyl-CoA synthase - POR Pyruvate oxidoreductase     

Complementary expression of<Emphasis Type="Italic"> AP-2</Emphasis> and<Emphasis Type="Italic"> AP-2rep</Emphasis> in ectodermal derivatives of<Emphasis Type="Italic"> Xenopus</Emphasis> embryos

In an attempt to define the pattern of developmental expression of AP-2rep and AP-2 in Xenopus embryos, we cloned a Xenopus AP-2rep cDNA. The AP-2rep message was localized in the organizer region at the gastrula stage whereas AP-2 was expressed ventro-laterally in the animal hemisphere. Later, AP-2rep was expressed in the entire neural tissue at the neurula stage while AP-2 was predominantly expressed in the cranial neural crest areas. The endogenous expression of AP-2 in the neural crest area was diminished by ectopic injection of AP-2rep RNA, suggesting a role for AP-2rep in the differentiation of neural tissues by restricting the expression of AP-2 in the Xenopus embryo.     

Structural and transcriptional analysis of<Emphasis Type="Italic"> S</Emphasis>-locus F-box genes in<Emphasis Type="Italic"> Antirrhinum</Emphasis>

A class of ribonucleases termed S-RNases, which control the pistil expression of self-incompatibility, represents the only known functional products encoded by the S locus in species from the Solanaceae, Scrophulariaceae and Rosaceae. Previously, we identified a pollen-specific F-box gene, AhSLF (S locus F-box)-S₂, very similar to S₂-RNase in Antirrhinum, a member of the Scrophulariaceae. In addition, AhSLF-S₂ also detected the presence of its homologous DNA fragments. To identify these fragments, we constructed two genomic DNA libraries from Antirrhinum self-incompatible lines carrying alleles S₁S₅ and S₂S₄, respectively, using a transformation-competent artificial chromosome (TAC) vector. With AhSLF-S₂-specific primers, TAC clones containing both AhSLF-S₂ and its homologs were subsequently identified (S₂TAC, S₅TACa, S₄TAC, and S₁TACa). DNA blot hybridization, sequencing and segregation analyses revealed that they are organized as single allelic copies (AhSLF-S₂, -S₁, -S₄ and -S₅) tightly linked to the S-RNases. Furthermore, clusters of F-box genes similar to AhSLF-S₂ were identified. In total, three F-box genes (AhSLF-S₂, -S₂A and -S₂C) in S₂TAC (51 kb), three (AhSLF-S₄, -S₄A and -S₄D) in S₄TAC (75 kb), two (AhSLF-S₅ and -S₅A) in S₅TACa (55 kb), and two (AhSLF-S₁ and -S₁E) in S₁TACa (71 kb), respectively, were identified. Paralogous copies of these genes show 38–54% identity, with allelic copies sharing 90% amino acid identity. Among these genes, three (AhSLF-S₂C, -S₄D and -S₁E) were specifically expressed in pollen, similar to AhSLF-S₂, implying that they likely play important roles in pollen, whereas three AhSLF-SA alleles showed no detectable expression. In addition, several types of retroelements and transposons were identified in the sequenced regions, revealing some detailed information on the structural diversity of the S locus region. Taken together, these results indicate that both single allelic and tandemly duplicated genes are associated with the S locus in Antirrhinum. The implications of these findings in evolution and possible roles of allelic AhSLF-S genes in the self-incompatible reaction are discussed in species like Antirrhinum.Sequence data from this article have been deposited with the EMBL/GenBank databases under accession numbers AJ300474, AJ515534, AJ515536 and AJ515535     

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.     

New isolates and physiological properties of the<Emphasis Type="Italic"> Aquificales</Emphasis> and description of<Emphasis Type="Italic"> Thermocrinis albus</Emphasis> sp. nov.

The ecology of the Aquificales was studied using a combination of phylogenetic and cultivation approaches. Enrichment cultures were prepared from low-salt and marine samples of geothermally and volcanically heated environments of the United States (Yellowstone National Park), Russia (Kamchatka), Italy, Germany, Djibouti, Iceland, and Africa (Lake Tanganyika). Isolation of single cells using the selected cell cultivation technique resulted in 15 different pure cultures. Comparisons of their 16S rRNA gene sequences showed that most of the isolates were new representatives of the major lineages of the Aquificaceae, represented by the genera Aquifex, Thermocrinis, Hydrogenobaculum, and Hydrogenobacter. Isolate HI 11/12, which was obtained from whitish streamers in the Hveragerthi area of Iceland, represents a separate branch within the Aquificaceae. The organism grew at salinities up to 0.7% NaCl and at temperatures up to 89 degrees C. Depending on the culture conditions, the organisms occurred as single motile rods, as aggregates, or as long filaments that formed whitish streamer-like cell masses. The novel isolate grew chemolithoautotrophically with hydrogen, sulfur, or thiosulfate as the electron donor under microaerophilic conditions. It represents a second species within the order Thermocrinis, which we name Thermocrinis albus HI 11/12 (DSM 14484, JCM 11386).     

Granaticins and their biosynthetic gene cluster from <Emphasis Type="Italic">Streptomyces vietnamensis</Emphasis>: evidence of horizontal gene transfer

Streptomyces vietnamensis, a recently designated species isolated from tropical forest soil, was found to be a new granaticin producer. The granaticin biosynthetic gene cluster (gra) and flanking genes from S. vietnamensis were cloned and sequenced by a sequential cloning strategy. All biosynthetic genes were found as expected. The high overall homology of the gra cluster from S. vietnamensis to that of Streptomyces violaceoruber Tü22 indicated a recent common ancestor of the two clusters. However, a flanking gene orf35 was missing from the gra cluster of S. vietnamensis, and high frequency of insertions and deletions of short fragment (shorter than 63 bp) were observed throughout the sequenced region compared to that of S. violaceoruber Tü22. These revealed a rapid evolution of the gra cluster and suggested that small insertions and deletions might be one of the basic evolution mechanisms for streptomycete genomes. The phylogenetic incongruence between 16S rDNA and the gra cluster and the scattered distribution of the granaticin producers within Streptomyces implicated horizontal gene transfer (HGT) being involved in the gra cluster dispersion. The remnants of orf35 found in S. vietnamensis present a scenario on how the antibiotic gene clusters evolved after HGT. The contemporary gra cluster residing in S. vietnamensis could be interpreted as a combination of HGT and highly variable vertical transmission.     

Diversity of<Emphasis Type="Italic">fliC</Emphasis> gene in commensal<Emphasis Type="Italic">Escherichia coli</Emphasis> derived from various mammals

Relations between the diversity of the fliC gene conditioning flagellum protein in E. coli and the source of the strain origin are presented. The fliC genes have been identified and characterized in commensal E. coli derived from 10 healthy animal species living in Zoo Safari Park (Poland). The fliC gene was found in 150 strains by the PCR method. The amplifiedfliC products revealed single bands within the range 1.26-2.16 kbp. Forty restriction patterns (classed by restriction analysis with the use of RsaI (PCR-RFLP RsaI; R-types) were determined. The neighbor-joining method was employed to illustrate the distribution of the kinds of R-types. There are 3-8 various R-types of a diversified frequency of occurrence in strains. Application of PCR-RFLP RsaI permitted the identification of alleles of fliC genes characteristic for E. coli and the estimation of their diversity among the animal species. The transmission ways of E. coli fliC+ between organisms of different species were determined and confirmed the role of transmission and horizontal gene transfer in the generation of the allelic diversity of fliC gene in natural E. coli populations.