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Acidobacteria form a coherent but highly diverse group within the bacterial domain: evidence from environmental genomics.

Acidobacteria have been established as a novel phylum of Bacteria that is consistently detected in many different habitats around the globe by 16S rDNA-based molecular surveys. The phylogenetic diversity, ubiquity and abundance of this group, particularly in soil habitats, suggest an important ecological role and extensive metabolic versatility. However, the genetic and physiological information about Acidobacteria is scarce. In order to gain insight into genome structure, evolution and diversity of these microorganisms we have initiated an environmental genomic approach by constructing large insert libraries directly from DNA of a calcerous grassland soil. Genomic fragments of Acidobacteria were identified with specific 16S rDNA probes and sequence analyses of six independently identified clones were performed, representing in total more than 210,000 bp. The 16S rRNA genes of the genomic fragments differed between 2.3% and 19.9% and were placed into two different subgroups of Acidobacteria (groups III and V). Although partial co-linearity was found between genomic fragments, the gene content around the rRNA operons was generally not conserved. Phylogenetic reconstructions with orthologues that were encoded on two of the six genomic fragments (PurF, PurL, PurB and formamidopyrimidine-DNA glycosylase) confirmed the coherence of the acidobacterial phylum. One genomic fragment harboured a cluster of eight genes which was syntenic and highly homologous to genomic regions in Rhodopseudomonas palustris and Bradyrhizobium japonicum, including a conserved two-component system. Phylogenetic analysis of the putative response regulator confirmed that this similarity between Rhizobiales and Acidobacteria might be due to a horizontal gene transfer. In total, our data give first insight into the genome content and diversity of the ubiquitously distributed but poorly characterized phylum of Acidobacteria. Furthermore they support the phylogenetic inferences made from 16S rRNA gene libraries, suggesting that Acidobacteria form a broad group in the same sense and with a similar diversity as that of many well-studied bacterial phyla.

Bacteria↗

Novel predominant archaeal and bacterial groups revealed by molecular analysis of an anaerobic sludge digester.

A culture-independent molecular phylogenetic approach was used to study prokaryotic diversity in an anaerobic sludge digester. Two 16S rRNA gene libraries were constructed using total genomic DNA, and amplified by polymerase chain reaction (PCR) using primers specific for archaeal or bacterial domains. Phylogenetic analysis of 246 and 579 almost full-length 16S rRNA genes for Archaea and Bacteria, respectively, was performed using the ARB software package. Phylogenetic groups affiliated with the Archaea belong to Euryarchaeota and Crenarchaeota. Interestingly, we detected a novel monophyletic group of 164 clones representing 66.6% of the archaeal library. Culture enrichment and probe hybridization show that this group grows better under formate or H2-CO2. Within the bacterial library 95.6% of the operational taxonomic units (OTUs) represent novel putative phylotypes never described before, and affiliated with eight divisions. The Bacteroidetes phylum is the most abundant and diversified phylogenetic group representing 38.8% of the OTUs, followed by the gram-positives (27.7%) and the Proteobacteria (21.3%). Sequences affiliated with phylogenetic divisions represented by few cultivated representatives such as the Chloroflexi, Synergistes, Thermotogales or candidate divisions such as OP9 and OP8 are represented by <5% of the total OTUs. A comprehensive set of 15 16S and 23S rRNA-targeted oligonucleotide hybridization probes was used to quantify these major groups by dot blot hybridization within 12 digester samples. In contrast to the clone library, Firmicutes and Actinobacteria together accounted for 21.8 +/- 14.9% representing the most abundant phyla. They were surprisingly followed by the Chloroflexi representing 20.2 +/- 4.6% of the total 16S rRNA. The Proteobacteria and the Bacteroidetes group accounted for 14.4 +/- 4.9% and 14.5 +/- 4.3%, respectively, WWE1, a novel lineage, accounted for 11.9 +/- 3.1% while Planctomycetes and Synergistes represented <2% each. Using the novel set of probes we extended the coverage of bacterial populations from 52% to 85.3% of the total rRNA within the digester samples.

Anaerobiosis↗

An 'instant gene bank' method for gene cloning by mutant complementation.

We describe a new method of gene cloning by complementation of mutant alleles which obviates the need for construction of a gene library in a plasmid vector in vitro and its amplification in Escherichia coli. The method involves simultaneous transformation of mutant strains of the fungus Aspergillus nidulans with (i) fragmented chromosomal DNA from a donor species and (ii) DNA of a plasmid without a selectable marker gene, but with a fungal origin of DNA replication ('helper plasmid'). Transformant colonies appear as the result of the joining of chromosomal DNA fragments carrying the wild-type copies of the mutant allele with the helper plasmid. Joining may occur either by ligation (if the helper plasmid is in linear form) or recombination (if it is cccDNA). This event occurs with high efficiency in vivo, and generates an autonomously replicating plasmid cointegrate. Transformants containing Penicillium chrysogenum genomic DNA complementing A. nidulans niaD, nirA and argB mutations have been obtained. While some of these cointegrates were evidently rearranged or consisted only of unaltered replicating plasmid, in other cases plasmids could be recovered into E. coli and were subsequently shown to contain the selected gene. The utility of this "instant gene bank" technique is demonstrated here by the molecular cloning of the P. canescens trpC gene.

Aspergillus nidulans↗

A method for construction of long randomized open reading frames and polypeptides.

A method is presented for construction of randomized open reading frame sequences (ORFs) and gene libraries containing them. The building blocks for the ORFs were 75 bp long DNA fragments generated by cloning sequences from a single synthetic oligonucleotide preparation by bridge mutagenesis. The fragments had the property that, regardless of their orientation in the ligated product, the ORF of the construct was maintained. The heterogeneity of the ORFs resulted from the random ligation of 2000 different DNA fragments. The randomized ORFs were cloned downstream from the lac promoter in a multicopy plasmid in Escherichia coli. To test the method, a library of 10(6) clones was constructed.

Amino Acid Sequence↗

A molecular microbial ecology approach to studying hemodialysis water and fluid.

Bacteria, or bacteria-derived products, might be responsible for deleterious effects in hemodialysis patients. Most microorganisms in hemodialysis water, including potential pathogens, are difficult to isolate and might subsist in a 'viable but not culturable' state or may need specific culture media. A molecular culture-independent approach based on the analysis of the 16S rDNA obtained from total DNA has been used to better know the diversity of bacteria inhabiting hemodialysis water and fluid, and to address the possible health effects associated with their presence. Four clone libraries from 16S rDNA (274 clones) were analyzed to characterize the species or groups of bacteria present, to assess their distribution in the water circuit, and to compare the results with those previously obtained in culture-dependent analysis. One hundred and ninety-seven clones of four gene libraries were analyzed by sequencing, and were identified phylogenetically. Clones affiliated to the Alphaproteobacteria group led the diversity. The presence in several samples of Alpha-4-proteobacteria, recognized as sphingolipids producers, was to be noted. The most abundant clones were affiliated to the Betaproteobacteria branch, closely related to the genus Herbaspirillum. As known, Alphaproteobacteria and Betaproteobacteria genomes might present a manifest excess in CpG sequences and most of them show a lipopolysaccharide-rich outer membrane, both described as inducers of innate immunity responses. Another abundant group, belonging to the Cyanobacteria class, a possible source of cyanotoxins, was not related to any previously cultured bacterium. Possible risk implications for hemodialysis patients of the bacterial community detected are discussed.

Alphaproteobacteria↗

Genetic analysis of the phosphinothricin-tripeptide biosynthetic pathway of Streptomyces viridochromogenes Tü494.

Streptomyces viridochromogenes Tü494 produces the antibiotic phosphinothricyl-alanyl-alanine (Ptt). Ptt-non-producing mutants were isolated following N-methyl-N'-nitro-N-nitrosoguanidine (NTG) or UV light treatment of spore suspensions. In co-synthesis and bioconversion experiments the mutational blocks in the biosynthetic pathway were localized. The mutant NTG1 was analysed in detail. This mutant acts as a secretor for all other mutants. From bioconversion experiments with presumptive precursors circumstantial evidence was obtained that NTG1 is mutated in a gene involved in the alanylation of N-acetyl-demethyl-phosphinothricin. Using a cosmid gene library the DNA region complementing the defective gene of mutant NTG1 was isolated on a 4-kb BamHI fragment. Subcloning experiments showed that a 3-kb BglII/BamHI fragment is sufficient for complementation of mutant NTG1.

Anti-Bacterial Agents↗

Are data from different gene expression microarray platforms comparable?

Many commercial and custom-made microarray formats are routinely used for large-scale gene expression surveys. Here, we sought to determine the level of concordance between microarray platforms by analyzing breast cancer cell lines with in situ synthesized oligonucleotide arrays (Affymetrix HG-U95v2), commercial cDNA microarrays (Agilent Human 1 cDNA), and custom-made cDNA microarrays from a sequence-validated 13K cDNA library. Gene expression data from the commercial platforms showed good correlations across the experiments (r = 0.78-0.86), whereas the correlations between the custom-made and either of the two commercial platforms were lower (r = 0.62-0.76). Discrepant findings were due to clone errors on the custom-made microarrays, old annotations, or unknown causes. Even within platform, there can be several ways to analyze data that may influence the correlation between platforms. Our results indicate that combining data from different microarray platforms is not straightforward. Variability of the data represents a challenge for developing future diagnostic applications of microarrays.

Cell Line, Tumor↗

Isolation of novel tRNA(Ala) mutants by library selection in a tRNA(Ala) knockout strain.

The relationship between tRNA structure and function has been widely investigated by site-directed mutagenesis. This method has been a very useful tool to reveal the critical bases in tRNAs that are important for recognition and aminoacylation, but has been limited by the large number of possible base combinations in tRNA molecules. We have devised a new method that uses tRNA knockout cells for selection of functional tRNAs from a mutant tRNA gene library to overcome this limitation. To explore the mechanism of tRNA(Ala) recognition, the bases of the acceptor-stem region were randomized and active mutants were selected in a tRNA(Ala) knockout strain. Mutants of tRNA(Ala) having diverse sequence combinations in the acceptor-stem region and a broad range of functional activity to support knockout cell growth were isolated. The mutant tRNAs selected by the method included molecules containing novel base substitutions as well as extensively altered base combinations that would not be readily generated by rationally designed site-directed mutagenesis. Our results emphasize the importance of the acceptor stem as a structural unit in which some nucleotides may carry more weight than others, but in summation every nucleotide contributes to the interaction with the enzyme.

Amino Acyl-tRNA Synthetases↗

Isolation and genetic characterization of pGKL killer-insensitive mutants (iki) from Saccharomyces cerevisiae.

The linear double stranded DNA plasmid pGKL1 encodes the yeast killer toxin complex (Gunge et al., 1981) of which the killing mechanism is not understood. We isolated and characterized eight mutants in Saccharomyces cerevisiae that were insensitive to both the intracellularly expressed 28-kDa killer subunit and the native killer toxin complex. These mutations (iki1 through iki5) were all recessive, and classified into five complementation groups. The iki2 mutation was mapped to a position near the centromere on chromosome XIII. We developed a novel screening system to isolate the DNA fragments complementing the iki mutations from a Saccharomyces gene library, and isolated three DNA fragments that complement the iki1, iki3, and iki4 mutations, respectively.

Cloning, Molecular↗

The isolation of specific genes from the basidiomycete Schizophyllum commune.

We have developed a routine way to isolate genes directly from the basidiomycete fungus, Schizophyllum commune. Plasmid DNA from a genomic gene library was used to isolate five specific genes by complementation of Schizophyllum mutations via transformation. The mutant strains were deficient in the ability to synthesize either adenine (ade2 and ade5), uracil (ura1, encoding orotidine-5'-phosphate decarboxylase; OMPdecase), tryptophan (trp1, encoding indole-3-glycerol phosphate synthetase; IGPS) or para aminobenzoic acid (pab1). In each case, Southern analysis revealed that transformation to prototrophy was concomitant with the integration of vector sequence into the genome of the S. commune mutant. Total DNA from transformants was restricted, religated, and used to transform E. coli. Ampicillin resistant plasmids were recovered from E. coli and tested for their ability to transform the corresponding mutant of S. commune. Plasmids complementing the ade2, ade5, pab1, trp1, and ura1 mutations were recovered.

Basidiomycota↗

Thermotoga maritima AglA, an extremely thermostable NAD+-, Mn2+-, and thiol-dependent alpha-glucosidase.

The gene for the alpha-glucosidase AglA of the hyperthermophilic bacterium Thermotoga maritima MSB8, which was identified by phenotypic screening of a T. maritima gene library, is located within a cluster of genes involved in the hydrolysis of starch and maltodextrins and the uptake of maltooligosaccharides. According to its primary structure as deduced from the nucleotide sequence of the gene, AglA belongs to family 4 of glycosyl hydrolases. The enzyme was recombinantly expressed in Escherichia coli, purified, and characterized. The T. maritima alpha-glucosidase has the unusual property of requiring NAD+ and Mn2+ for activity. Co2+ and Ni2+ also activated AglA, albeit less efficiently than Mn2+. T. maritima AglA represents the first example of a maltodextrin-degrading alpha-glucosidase with NAD+ and Mn2+ requirement. In addition, AglA activity depended on reducing conditions. This third requirement was met by the addition of dithiothreitol (DTT) or beta-mercaptoethanol to the assay. Using gel permeation chromatography, T. maritima AglA behaved as a dimer (two identical 55-kDa subunits), irrespective of metal depletion or metal addition, and irrespective of the presence or absence of NAD+ or DTT. The enzyme hydrolyzes maltose and other small maltooligosaccharides but is inactive against the polymeric substrate starch. AglA is not specific with respect to the configuration at the C-4 position of its substrates because glycosidic derivatives of D-galactose are also hydrolyzed. In the presence of all cofactors, maximum activity was recorded at pH 7.5 and 90 degrees C (4-min assay). AglA is the most thermoactive and the most thermostable member of glycosyl hydrolase family 4. When incubated at 50 degrees C and 70 degrees C, the recombinant enzyme suffered partial inactivation during the first hours of incubation, but thereafter the residual activity did not drop below about 50% and 20% of the initial value, respectively, within a period of 48 h.

Cations, Divalent↗

Isolation and structure of the gene for the progesterone-inducible protein uteroglobin.

Uteroglobin is a small steroid-binding protein that is differentially regulated by steroid hormones in several tissues of the rabbit. In endometrium, the levels of uteroglobin mRNA increase after progesterone administration due to an enhanced rate of transcription of the uteroglobin gene. As a prerequisite for understanding the molecular mechanisms that modulate uteroglobin gene expression, we have isolated and characterized the uteroglobin gene. We first synthesized, cloned, and sequenced a uteroglobin cDNA that was used to screen a rabbit gene library and to show that the uteroglobin gene is not reiterated in the rabbit genome. We obtained three recombinant phages containing uteroglobin gene sequences and covering 35 kilobases of the rabbit genome. The uteroglobin gene is 3 kilobases long and is composed of three short exons separated by a long and a short intron. The complete coding sequence, the short intron, part of the large intron, and the flanking sequences have been subjected to sequence analysis. The salient features of the nucleotide sequence, including the absence of a canonical "T-A-T-A box," are discussed. A possible relationship is considered between the exon-intron structure of the gene and the known structure and function of uteroglobin.

Amino Acid Sequence↗

Nucleotide sequence of the human parathyroid hormone gene.

From a lambda phage gene library we have isolated phage containing the gene encoding human preproparathyroid hormone. The phage were isolated by using both the plaque-hybridization technique and the in vivo recombination-selection technique. The human preproparathyroid hormone gene contains two intervening sequences that separate the gene into a 5' noncoding domain, a "prepro" sequence domain, and a domain containing the parathyroid hormone sequence and the 3' noncoding region. The gene is approximately 4,200 base pairs long. Restriction endonuclease analysis of human leukocyte DNA shows that the haploid human genome contains one copy of the preproparathyroid hormone gene. A 14-base-pair sequence of alternating purines and pyrimidines that has the potential of adopting the Z-DNA conformation lies 134 base pairs upstream from the presumed site of initiation of transcription.

Amino Acid Sequence↗

Degradation signals for ubiquitin system proteolysis in Saccharomyces cerevisiae.

Combinations of different ubiquitin-conjugating (Ubc) enzymes and other factors constitute subsidiary pathways of the ubiquitin system, each of which ubiquitinates a specific subset of proteins. There is evidence that certain sequence elements or structural motifs of target proteins are degradation signals which mark them for ubiquitination by a particular branch of the ubiquitin system and for subsequent degradation. Our aim was to devise a way of searching systematically for degradation signals and to determine to which ubiquitin system subpathways they direct the proteins. We have constructed two reporter gene libraries based on the lacZ or URA3 genes which, in Saccharomyces cerevisiae, express fusion proteins with a wide variety of C-terminal extensions. From these, we have isolated clones producing unstable fusion proteins which are stabilized in various ubc mutants. Among these are 10 clones whose products are stabilized in ubc6, ubc7 or ubc6ubc7 double mutants. The C-terminal extensions of these clones, which vary in length from 16 to 50 amino acid residues, are presumed to contain degradation signals channeling proteins for degradation via the UBC6 and/or UBC7 subpathways of the ubiquitin system. Some of these C-terminal tails share similar sequence motifs, and a feature common to almost all of these sequences is a highly hydrophobic region such as is usually located inside globular proteins or inserted into membranes.

Amino Acid Sequence↗

Identification of the gene encoding the major capsid protein of fish lymphocystis disease virus.

The gene encoding the major capsid protein (MCP) of fish lymphocystis disease virus (flounder isolate; FLCDV-f) has been identified by PCR using oligonucleotide primers corresponding to different regions of the MCP of Tipula iridescent virus (TIV), iridescent virus 22 (IV22) and Chilo iridescent virus (CIV). DNA fragments of 0.4 kbp, 0.5 kbp and 0.27 kbp in size were amplified using oligonucleotide primers corresponding to amino acids (aa) 146 to 153 (primer 1) and 274 to 268 (primer 6), or aa 146 to 153 (primer 1) and 313 to 304 (primer 8), or aa 304 to 312 (primer 7) and 385 to 381 (primer 9) of the MCP of TIV, respectively. The PCR products were used as hybridization probes for screening the gene library of FLCDV-f. The MCP gene of FLCDV-f(1377 bp; 459 aa; 51.4K) was identified within the DNA sequence of the EcoRI FLCDV-f DNA fragment C (11.2 kbp; 0.611 to 0.718 map units). A high degree of aa sequence identity/similarity was detected between the MCP of FLCDV-f and TIV (50.3%/33.8%), IV22 (49.1%/34.2%). CIV (53%/29.5%) and African swine fever virus (16%/38.1%).

Amino Acid Sequence↗

Molecular cloning and sequencing of a non-haem bromoperoxidase gene from Streptomyces aureofaciens ATCC 10762.

A bromoperoxidase gene (bpoT), recently cloned from Streptomyces aureofaciens Tü24, was used as a probe in Southern blot hybridization of total DNA from S. aureofaciens ATCC 10762. A single SstI fragment of 5.4 kb was detected, which was cloned via an enriched gene library into Escherichia coli. The functional bromoperoxidase gene was located on a 2.1 kb BamHI-HindIII fragment by subcloning into S. lividans TK64, using the multicopy plasmid pIJ486. The enzyme was overproduced in S. lividans TK64 (up to 30,000 times compared to S. aureofaciens ATCC 10762) and showed the same electrophoretic and immunological properties as the bromoperoxidase BPO-A2 purified from S. aureofaciens ATCC 10762. DNA sequence analysis revealed an open reading frame encoding a predicted polypeptide with the same M(r) and N-terminal amino acid sequence as the purified subunit of BPO-A2.

Amino Acid Sequence↗

ScCypB is a novel second cytosolic cyclophilin from Streptomyces chrysomallus which is phylogenetically distant from ScCypA.

A novel second streptomycete cyclophilin gene-designated sccypB-was isolated from a cosmid gene library of Streptomyces chrysomallus by using as gene probe a fragment of the previously isolated cyclophilin gene sccypA of the same organism. From its sequence the gene sccypB should encode a protein of M(r) 18868. Expression of sccypB in Escherichia coli as a hexaHis-tagged fusion protein (H6ScCypB) and enzymic characterization of the purified protein showed that, like ScCypA, ScCypB is a peptidyl-prolyl cis-trans isomerase (PPIase). The specific activity and substrate specificity of the enzyme were comparable to that of ScCypA, but it was threefold less sensitive to inhibition by cyclosporin A (CsA). In contrast to ScCypA, which is abundant and exists in free and liganded form, ScCypB was 50- to 100-fold less abundant in cytosol-derived protein fractions of S. chrysomallus or Streptomyces lividans, as revealed by Western blot analyses, suggesting a specialized function for this enzyme in the streptomycete cell. Both sccypB and sccypA were found to be present as single copies in the genome of S. chrysomallus and hybridized to a single band in chromosomal DNAs of other streptomycetes. High-level expression of sccypB as well as of sccypA cloned into the expression vector pIJ702 did not produce detectable changes in growth and morphology of S. chrysomallus and S. lividans. Calculations of similarities to known cyclophilin sequences and construction of phylogenetic trees indicated that ScCypB and ScCypA are phylogenetically distant from each other. While ScCypA is clearly related to the eukaryotic cyclophilins, the analyses show the sequence of ScCypB to be the most divergent of all cyclophilin sequences, indicating that it possibly constitutes a cluster by itself.

Amino Acid Isomerases↗

Molecular characteristics of anti-self antibody fragments against neutrophil cytoplasmic antigens from human V gene phage display libraries.

Recently it has been demonstrated that human antibody fragments with binding activities against self antigens can be isolated from repertoires of rearranged V genes from non-immunized humans. We have applied phage display technology to study the B cell repertoire for antibody activity against neutrophil cytoplasmic antigens. These antibodies may play an important role in Wegener's granulomatosis (WG) and related forms of vasculitides. Autoantibodies in patients with WG are directed against proteinase 3. The immunodominant antigen in other forms of vasculitis is myeloperoxidase, but the B cell response can also be directed against other neutrophil enzymes, e.g. lysozyme, human neutrophil elastase, lactoferrin and cathepsin G. We show here that anti-self reactivity against neutrophil cytoplasmic antigens can be detected in the rearranged V gene repertoire of healthy individuals and that the reactivity can be directed against structural related epitopes which are present on different neutrophil cytoplasmic antigens. The scFv with binding activities were sequenced and the V gene usage, the level of somatic mutations and the immunoserological characteristics of the antibody fragments are discussed. Further evidence is presented that antibody fragments consisting only of a heavy chain variable domain can recognize neutrophil cytoplasmic antigens in a specific manner. These single-domain antibody fragments were used in experiments designed to establish the relative role of the light chain variable domains in antigen binding.

Amino Acid Sequence↗