Chthoniobacter flavus gen. nov., sp. nov., the first pure-culture representative of subdivision two, Spartobacteria classis nov., of the phylum Verrucomicrobia

The phylum Verrucomicrobia is increasingly recognized as an environmentally significant group of bacteria, particularly in soil habitats. At least six subdivisions of the Verrucomicrobia are resolved by comparative analysis of 16S rRNA genes, mostly obtained directly from environmental samples. To date, only two of these subdivisions (1 and 4) have characterized pure-culture representatives. We have isolated and characterized the first known pure-culture representative of subdivision 2. Strain Ellin428 is an aerobic heterotrophic bacterium that is able to grow with many of the saccharide components of plant biomass but does not grow with amino acids or organic acids other than pyruvate. Cells are yellow, rod-shaped, nonmotile, and gram-stain negative, and they contain peptidoglycan with direct cross-linkages of the A1 gamma meso-Dpm type. The isolate grows well at 25 degrees C on a variety of standard biological media, including some used in the routine cultivation of bacteria from soil. The pH range for growth is 4.0 to 7.0. Low levels of menaquinones MK-10 and MK-11 were detected. The major cellular fatty acids are C(14:0), a-C(15:0), C(16:1 omega 7c), and/or 2OH i-C(15:0), and C(16:0). The G+C content of the genomic DNA is 61 mol%. We propose a new genus and species, Chthoniobacter flavus gen. nov., sp. nov., with isolate Ellin428 as the type strain, and a new class for the subdivision to which it belongs, Spartobacteria classis nov. Environmental sequences indicate that the class Spartobacteria is largely represented by globally distributed, abundant, and active soil bacteria.     

Biological consequences of ancient gene acquisition and duplication in the large genome of Candidatus Solibacter usitatus Ellin6076

Members of the bacterial phylum Acidobacteria are widespread in soils and sediments worldwide, and are abundant in many soils. Acidobacteria are challenging to culture in vitro, and many basic features of their biology and functional roles in the soil have not been determined. Candidatus Solibacter usitatus strain Ellin6076 has a 9.9 Mb genome that is approximately 2-5 times as large as the other sequenced Acidobacteria genomes. Bacterial genome sizes typically range from 0.5 to 10 Mb and are influenced by gene duplication, horizontal gene transfer, gene loss and other evolutionary processes. Our comparative genome analyses indicate that the Ellin6076 large genome has arisen by horizontal gene transfer via ancient bacteriophage and/or plasmid-mediated transduction, and widespread small-scale gene duplications, resulting in an increased number of paralogs. Low amino acid sequence identities among functional group members, and lack of conserved gene order and orientation in regions containing similar groups of paralogs, suggest that most of the paralogs are not the result of recent duplication events. The genome sizes of additional cultured Acidobacteria strains were estimated using pulsed-field gel electrophoresis to determine the prevalence of the large genome trait within the phylum. Members of subdivision 3 had larger genomes than those of subdivision 1, but none were as large as the Ellin6076 genome. The large genome of Ellin6076 may not be typical of the phylum, and encodes traits that could provide a selective metabolic, defensive and regulatory advantage in the soil environment.     

Preliminary X-ray diffraction analysis of a crystallizable mutant of malate dehydrogenase from the thermophile Thermus flavus

Malate dehydrogenase from mutant strain F428 of the thermophilic bacterium Thermus flavus has now been crystallized from polyethylene glycol 8000 in a form suitable for diffraction studies. The protein crystallizes in the orthorhombic P2(1)2(1)2(1) space group with unit cell dimensions a = 71.8 A, b = 88.6 A, c = 119.0 A. The asymmetric unit consists of one homodimer of molecular mass 67,000 Da. The X-ray diffraction extends beyond 1.7 A and a full data set to 1.9 A has been collected.     

Clostridium botulinum C2 toxin. Identification of the binding site for chloroquine and related compounds and influence of the binding site on properties of the C2II channel

Clostridium botulinum C2 toxin belongs to the family of binary AB type toxins that are structurally organized into distinct enzyme (A, C2I) and binding (B, C2II) components. The proteolytically activated 60-kDa C2II binding component is essential for C2I transport into target cells. It oligomerizes into heptamers and forms channels in lipid bilayer membranes. The C2II channel is cation-selective and can be blocked by chloroquine and related compounds. Residues 303-330 of C2II contain a conserved pattern of alternating hydrophobic and hydrophilic residues, which has been implicated in the formation of two amphipathic beta-strands involved in membrane insertion and channel formation. In the present study, C2II mutants created by substitution of different negatively charged amino acids by alanine-scanning mutagenesis were analyzed in artificial lipid bilayer membranes. The results suggested that most of the C2II mutants formed SDS-resistant oligomers (heptamers) similar to wild type. The mutated negatively charged amino acids did not influence channel properties with the exception of Glu(399) and Asp(426), which are probably localized in the vestibule near the channel entrance. These mutants show a dramatic decrease in their affinity for binding of chloroquine and its analogues. Similarly, F428A, which represents the Phi-clamp in anthrax protective antigen, was mutated in C2II in several other amino acids. The C2II mutants F428A, F428D, F428Y, and F428W not only showed altered chloroquine binding but also had drastically changed single channel properties. The results suggest that amino acids Glu(399), Asp(426), and Phe(428) have a major impact on the function of C2II as a binding protein for C2I delivery into target cells.     

Genome sequence of "Pedosphaera parvula" Ellin514, an aerobic Verrucomicrobial isolate from pasture soil

"Pedosphaera parvula" Ellin514 is an aerobically grown verrucomicrobial isolate from pasture soil. It is one of the few cultured representatives of subdivision 3 of the phylum Verrucomicrobia. Members of this group are widespread in terrestrial environments.     

NMR studies of interactions between C-terminal tail of Kir2.1 channel and PDZ1,2 domains of PSD95

Control of surface expression of inwardly rectifying potassium (Kir) channels is important for regulating membrane excitability. Kir2 channels have been shown to interact directly with PDZ-containing proteins in the postsynaptic density (PSD). These scaffold proteins, such as PSD95, bind to Kir2.1 channels via a PDZ-binding motif (T/S-x-Phi) in the C-terminal tail (SEI428). By utilizing a multidimensional solution NMR approach, we show that the previously unresolved structure of Kir2.1 tail (residues 372-428) is highly flexible. Using in vitro binding assays, we determined that shortening the flexible tail of Kir2.1 preceding the C-terminal region (residues 414-428) does not significantly disrupt PDZ binding. We also investigated which amino acids in the Kir2.1 tail associated with PSD95 PDZ1,2 by NMR spectroscopy, revealing that a stretch of 12 C-terminal amino acids is involved in interaction with both PDZ domains (residues 417-428). Deletion of the 11 amino acids preceding the C-terminal tail, Delta414-424, completely disrupts binding to PSD95 PDZ1,2. Therefore, the molecular interfaces formed between PDZ domains and Kir2.1 tail involve regions outside the previously identified binding motif (SEI428) and may be important for additional channel-specific interactions with associating PDZ-containing proteins.     

Cloning and expression in Escherichia coli of the gene encoding Aspergillus flavus urate oxidase.

Amino acid sequencing of peptides obtained after proteolytic hydrolysis of Aspergillus flavus urate oxidase (uricase) permitted the design of oligodeoxynucleotide probes that were used to obtain 1.2- and 5-kilobase pair DNA fragments from A. flavus cDNA and genomic libraries, respectively. The cDNA fragment contained the entire coding region for uricase, and comparison with the genomic fragment revealed the presence of two short introns in the coding region of the gene. A. flavus uricase has around 40% overall identity with uricases from higher organisms but with many conserved amino acids. Hitherto highly conserved consensus patterns found in other uricases were found to be modified in the A. flavus enzyme, notably the sequence Val-Leu-Lys-Thr-Thr-Gln-Ser near position 150, which in the filamentous fungus is uniquely modified to Val-Leu-Lys-Ser-Thr-Asn-Ser. Silent mutations were introduced by cassette mutagenesis near the 5'-extremity of the coding sequence in order to conform with Escherichia coli codon usage, and the uricase was expressed in the E. coli cytoplasm in a completely soluble, biologically active form.     

Comparative analysis of ribosomal protein L5 sequences from bacteria of the genus Thermus.

The genes for the ribosomal 5S rRNA binding protein L5 have been cloned from three extremely thermophilic eubacteria, Thermus flavus, Thermus thermophilus HB8 and Thermus aquaticus (Jahn et al, submitted). Genes for protein L5 from the three Thermus strains display 95% G/C in third positions of codons. Amino acid sequences deduced from the DNA sequence were shown to be identical for T flavus and T thermophilus, although the corresponding DNA sequences differed by two T to C transitions in the T thermophilus gene. Protein L5 sequences from T flavus and T thermophilus are 95% homologous to L5 from T aquaticus and 56.5% homologous to the corresponding E coli sequence. The lowest degrees of homology were found between the T flavus/T thermophilus L5 proteins and those of yeast L16 (27.5%), Halobacterium marismortui (34.0%) and Methanococcus vannielii (36.6%). From sequence comparison it becomes clear that thermostability of Thermus L5 proteins is achieved by an increase in hydrophobic interactions and/or by restriction of steric flexibility due to the introduction of amino acids with branched aliphatic side chains such as leucine. Alignment of the nine protein sequences equivalent to Thermus L5 proteins led to identification of a conserved internal segment, rich in acidic amino acids, which shows homology to subsequences of E coli L18 and L25. The occurrence of conserved sequence elements in 5S rRNA binding proteins and ribosomal proteins in general is discussed in terms of evolution and function.     

Molecular cloning of the pyrE gene from the extreme thermophile Thermus flavus.

Mutants of the extreme thermophile Thermus flavus in the pyrimidine biosynthetic pathway (Pyr-) were isolated by resistance to 5-fluoroorotic acid. The pyrE gene, which codes for the orotate phosphoribosyltransferase, was cloned by recombination with one of the isolated Pyr- T. flavus mutant strains. It was subcloned by complementation of an Escherichia coli pyrE mutant strain and was sequenced. The deduced polypeptide sequence extends over 183 amino acids. Several independent Pyr- mutations were mapped within the pyrE locus by recombination with fragments of the cloned gene.     

Mutation-induced reinforcement of prestin-expressing cells

The motor protein prestin in cochlear outer hair cells is a member of the solute carrier 26 family, but among the proteins of that family, only prestin can confer the cells with nonlinear capacitance (NLC) and motility. In the present study, to clarify contributions of unique amino acids of prestin, namely, Met-122, Met-225 and Thr-428, to the characteristics of prestin, mutations were introduced into those amino acids. As a result, NLC remained unchanged by both replacement of Met-122 by isoleucine and that of Thr-428 by leucine, suggesting that those amino acids were not important for the generation of NLC. Surprisingly, the replacement of Met-225 by glutamine statistically increased NLC as well as the motility of prestin-expressing cells without an increase in the amount of prestin expression in the plasma membrane. This indicates that Met-225 in prestin somehow adjusts NLC and the motility of prestin-expressing cells.     

Cloning and functional expression of the gene encoding an inhibitor against <Emphasis Type="Italic">Aspergillus flavus</Emphasis> α-amylase,a novel seed lectin from <Emphasis Type="Italic">Lablab purpureus</Emphasis> (<Emphasis Type="Italic">Dolichos lablab</Emphasis>)

Maize is one of the more important agricultural crops in the world and, under certain conditions, prone to attack from pathogenic fungi. One of these, Aspergillus flavus, produces toxic and carcinogenic metabolites, called aflatoxins, as byproducts of its infection of maize kernels. The alpha-amylase of A. flavus is known to promote aflatoxin production in the endosperm of these infected kernels, and a 36-kDa protein from the Lablab purpureus, denoted AILP, has been shown to inhibit alpha-amylase production and the growth of A. flavus. Here, we report the isolation of six full-length labAI genes encoding AILP and a detailed analysis of the activities of the encoded proteins. Each of the six labAI genes encoded sequences of 274 amino acids, with the deduced amino acid sequences showing approximately 95-99% identity. The sequences are similar to those of lectin members of a legume lectin-arcelin-alpha-amylase inhibitor family reported to function in plant resistance to insect pests. The labAI genes did not show any of the structures characteristic of conserved structures identified in alpha-amylase inhibitors to date. The recombinant proteins of labAI-1 and labAI-2 agglutinated human red blood cells and inhibited A. flavus alpha-amylase in a manner similar to that shown by AILP. These data indicate that labAI genes are a new class of lectin members in legume seeds and that their proteins have both lectin and alpha-amylase inhibitor activity. These results are a valuable contribution to our knowledge of plant-pathogen interactions and will be applicable for developing protocols aimed at controlling A. flavus infection.     

Derivation of stable zebrafish ES-like cells in feeder-free culture

Zebrafish offers an excellent opportunity to combine embryological, genetic and molecular analyses of vertebrate development in vivo. Embryonic stem (ES) cells have enormous potential to study developmental potency and differentiation in vitro and thus to complement in vivo approaches. Zebrafish ES-like cells have been produced on a feeder cell layer. Here, we report the derivation of Z428, a zebrafish ES-like cell line, from blastula embryos in feeder-free culture. Fetal bovine serum, fish serum, fish embryo extract, basic fibroblast growth factor, non-essential amino acids and 2-mercaptoethanol were found to be important for Z428 growth. After more than 120 passages and many freezing/thawing cycles over a period of 20 years, Z428 exhibits stable growth and manifests many ES cell features including an ES cell phenotype, high alkaline phosphatase activity and spontaneous differentiation in culture. Most importantly, Z428 was transplantable to blastula hosts and capable of contributing to embryonic tissues and organ systems of the three germ layers. Therefore, Z428 is a stable cell line and contains ES-like cells with pluripotency in vitro and in vivo, and a feeder layer is dispensable for ES-like cell derivation in zebrafish. The derivation and easy maintenance of zebrafish ES-like cells under feeder-free conditions provide a useful extension of the present toolbox for studying development and differentiation in the zebrafish model.     

Aspergillus infection inhibits the expression of peanut 13S-HPODE-forming seed lipoxygenases

Oxylipins recently have been implicated as signaling molecules for cross-kingdom communication in plant-pathogen interactions. Linoleic acid and its two plant lipoxygenase (LOX) oxylipin products 9- and 13-hydroperoxy fatty acids (9S- and 13S-HPODE) have been shown to have a significant effect on differentiation processes in the mycotoxigenic seed pathogens Aspergillus spp. Whereas both fatty acids promote sporulation, 9S-HPODE stimulates and 13S-HPODE inhibits mycotoxin production. Additionally, Aspergillus flavus infection of seed promotes linoleate 9-LOX expression and 9S-HPODE accumulation. Here, we describe the characterization of two peanut seed lipoxygenase alleles (PnLOX2 and PnLOX3) highly expressed in mature seed. PnLOX2 and PnLOX3 both are 13S-HPODE producers (linoleate 13-LOX) and, in contrast to previously characterized 9-LOX or mixed function LOX genes, are repressed between 5-fold and 250-fold over the course of A. flavus infection. The results of these studies suggest that 9S-HPODE and 13S-HPODE molecules act as putative susceptibility and resistance factors respectively, in Aspergillus seed-aflatoxin interactions.     

Stimulation of aflatoxin biosynthesis by lipophilic epoxides

Epoxy fatty acids added to the culture media either with the inoculum or at the end of exponential growth phase stimulated aflatoxin production by toxigenic strains of Aspergillus flavus and Aspergillus parasiticus. This effect did not appear when the unsaturated fatty acids used for the synthesis of the epoxides and the polyhydroxyacids (which can be considered to be derived from the opening of the oxirane ring) replaced the epoxides in the culture media. No significant differences were detected in the lipid fractions (diglycerides, sterols, triglycerides, free fatty acids, sterol esters) extracted from the mycelia grown in the presence of any of the fatty acid derivates.     

Characterization of a novel allergen, a major IgE-binding protein from Aspergillus flavus, as an alkaline serine protease.

Aspergillus species of fungi have been known to be one of the most prevalent aeroallergens. One important A. flavus allergen (Asp fl 1) was identified by means of immunoblotting with a serum pool of allergic patients on a two-dimensional electrophoretic gel. The cDNA coding for Asp fl 1 was cloned and sequenced. The clone encodes a full-length protein of 403 amino acid precursors of 42 kDa. After cleavage of a putative signal peptide of 21 amino acids and a prepeptide of 100 amino acids, a mature protein of 282 amino acids was obtained with a molecular mass of 33 kDa and a pI of 6.3. A degree of identity was found in a range of 27 to 84% among related allergens derived from bacteria allergen subtilisin, mold allergen Pen c 1, and virulence factor of A. fumigatus. Recombinant Asp fl 1 (rAsp fl 1) was cloned into vector pQE-30 and expressed in E. coli M15 as a histidine-tag fusion protein and purified to homogeneity. The IgE binding capacity of rAsp fl 1 was tested by immunoblotting using a serum pool of Aspergillus-allergic patients. Recombinant allergen cross-reacted strongly with IgE specific for natural Asp fl 1 and Pen c 1, indicating that common IgE epitopes may exist between allergens of A. flavus and P. citrinum.     

Cloning and molecular analysis of cDNA encoding a carboxymethylcellulase of the yeast Cryptococcus flavus.

A cDNA copy for carboxymethylcellulase (CMCase 1) of the yeast Cryptococcus flavus was cloned by screening an expression cDNA library with anti-CMCase 1 antibody. The sequence of the cDNA had an open reading frame of 1023 bp that encoded a preprotein of 341 amino acids with a molecular weight of 35,698. The putative precursor begins with a hydrophobic segment that possibly acts as a signal sequence for secretion, which is followed by a presumed prosequence and a sequence consistent with the N-terminal amino acid sequence of secreted CMCase 1. No potential N-glycosylation site was found in the sequence of putative pro-CMCase 1. Comparison of the deduced protein sequence shows that the C. flavus CMCase 1 is partially homologous to the Trichoderma reesei endoglucanase EGIII. Alignment of the cDNA copy and the chromosomal DNA showed seven putative introns of 45 to 134 bp. When introduced into E. coli, the cDNA directed the synthesis of CMCase 1 as seen by CMCase activity and Western blotting using anti-CMCase 1 antibody.     

A membrane-associated lipomannan in micrococci.

Membranes of Micrococcus lysodeikticus, Micrococcus flavus and Micrococcus sodonensis contain acidic lipomannans. Lipoteichoic acids could not be detected in these organisms, and the suggestion that they are substituted for by the lipomannans is strengthened by the chemical and physical resemblances between the two polymers. The mannans contain glycerol, ester-linked fatty acids and mono-esterified succinic acid residues, giving them both hydrophobic and charged properties. The M. lysodeikticus mannan has a chain of about 60 hexose units with two branch points, and is joined at its reducing end to the 1-position of a glycerol moiety bearing two fatty acid residues. Succinic acid on the mannan enables it to bind Mg2+ efficiently, and the polymer is firmly associated with the cytoplasmic membrane, probably by intercalation of its fatty acids with those of the membrane lipids.     

Optimization of L-malic acid production by Aspergillus flavus in a stirred fermentor

Effects of various nutritional and environmental factors on the accumulation of organic acids (mainly L-malic acid) by the filamentous fungus Aspergillus flavus were studied in a 16-L stirred fermentor. Improvement of the molar yield (moles acid produced per moles glucose consumed) of L-malic acid was obtained mainly by increasing the agitation rate (to 350 rpm) and the Fe(z+) ion concentration (to 12 mg/L) and by lowering the nitrogen (to 271 mg/L) and phosphate concentrations (to 1.5 mM) in the medium. These changes resulted in molar yields for L-malic acid and total C(4) acids (L-malic, succinic, and fumaric acids) of 128 and 155%, respectively. The high molar yields obtained (above 100%) are additional evidence for the operation of part of the reductive branch of the tricarboxylic acid cycle in L-malic acid accumulation by A. flavus. The fermentation conditions developed using the above mentioned factors and 9% CaCO(3) in the medium resulted in a high concentration (113 g/L L-malic acid from 120 g/L glucose utilized) and a high overall productivity (0.59 g/L h) of L-malic acid. These changes in acid accumulation coincide with increases in the activities of NAD(+)-malate dehydrogenase, fumarase, and citrate synthase.