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PubMed2006

Technical note: Improved method for rapid DNA extraction of mastitis pathogens directly from milk.

Efficient control against bovine mastitis requires sensitive, rapid, and specific tests to detect and identify the main bacteria that cause heavy losses to the dairy industry. Molecular detection of pathogenic microorganisms is based on DNA amplification of the target pathogen. Therefore, efficient extraction of DNA from pathogenic bacteria is a major step. In this study, we aimed to develop a specific, sensitive, and rapid method to extract DNA directly from the main gram-positive bacteria known to cause bovine mastitis (Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus uberis) found in milk samples. The DNA extraction method is based on the lysing and nuclease-inactivating properties of the chaotropic agent, guanidinium thiocyanate, together with the nucleic acid-binding properties of the silica particles. An efficient protocol consisting of 6 basic steps (3 of which were done twice) was developed and applied directly to milk samples. Absence of PCR inhibitors and DNA quality were evaluated by PCR amplification of the species-specific DNA sequences of the target bacteria. The level of sensitivity achieved in our experiments is applicable to milk sample analysis without sample enrichment.

Animals↗
PubMed1997

From oligonucleotide shapes to genomic SELEX: novel biological regulatory loops.

The SELEX method and oligonucleotide combinatorial chemistry discovery process yields high-affinity/high-specificity ligands for virtually any molecular target. Typically, the enormous starting libraries used in the SELEX process contain 10(14)-10(15) sequences. We now ask if the smaller sequences, complexity of extant organisms, and evolutionary history provide useful interactions between oligonucleotides and at least some unexpected targets. That is, do organisms contain a robust "linkage map" between their oligonucleotides and proteins and/or small molecules that enriches life?

Base Sequence↗
PubMed1992

Rapid generation of region-specific genomic clones by chromosome microdissection: isolation of DNA from a region frequently deleted in malignant melanoma.

Malignant melanoma is frequently characterized by the deletion of the long arm of chromosome 6 (usually encompassing 6q16-q21). In an effort to saturate this region with DNA markers, microdissection and molecular cloning of DNA from banded human metaphases recent development of a novel chromosome microdissection scheme that omits microchemical manipulation of DNA. Microdissection was targeted on band 6q21. Direct PCR amplification of dissected DNA was first used as a probe in chromosomal in situ hybridization of normal metaphases to confirm the specificity of material excised for cloning. A genomic library of 20,000 clones, which is highly enriched for sequences encompassing 6q21, was then constructed. Clones from this library have been mapped against a human-rodent somatic cell hybrid mapping panel that divides chromosome 6 into seven regions, confirming the localization of probes within the target region. Direct PCR amplification of DNA excised by microdissection greatly simplifies and facilitates this chromosome band-specific cloning strategy. The isolation of microclones from this region of chromosome 6 should assist in establishing a physical map of the melanoma deletion region.

Base Sequence↗
PubMed1996

Visualization of viral candidate cDNAs in infectious brain fractions from Creutzfeldt-Jakob disease by representational difference analysis.

Creutzfeldt-Jakob Disease (CJD), a neurodegenerative and dementing disease of later life, is caused by a viruslike entity that is incompletely characterized. As in scrapie, all more purified infectious brain preparations contain nucleic acids. However, it has not been possible to visualize unique bands that may derive from a viral genome. We here used a subtractive strategy known as representational difference analysis (RDA) to uncover such sequences. To reduce the complexity of starting target nucleic acids, sucrose gradients were first used to select nuclease resistant particles with a defined 120S size. In CJD this single 120S gradient peak is highly enriched for infectivity, and contains reduced amounts of PrP (Proc. Natl. Acad. Sci. 92, 5124-8, 1995). Parallel 120S fractions from uninfected brain were made to generate subtractor sequences. 120S particles were lysed in GdnSCN, and ng amounts of released RNA were purified for random-primed cDNA synthesis. To capture representative fragments of 100-500 bp, cDNAs were cleaved with Mbo I for adaptor ligation and amplification. In the first experiment with moderate RDA selection, it was possible to visualize clones from CJD cDNA that did not hybridize to control cDNA. In the second experiment, more exhaustive subtractions yielded a discrete set of CJD derived gel bands. Competitive hybridization showed a subset of these bands were not present in either the control 120S cDNA or in the hamster genome. This represents the first demonstration of apparently CJD-specific nucleic acid bands in more purified infectious preparations. Although exhaustive cloning, sequencing and correlative titration studies need to be done, it is encouraging that most of the viral candidates selected thus far have no significant homology with any previously described sequence in the database.

Animals↗
PubMed2025

Comprehensive benchmarking of somatic structural variant detection at ultra-low allele fractions.

Postzygotic mosaicism gives rise to somatic structural variants (SVs) at ultra-low variant allele fractions (VAFs), which pose challenges for detection due to the high-coverage sequencing required and noise introduced by sequencing artifacts. Although somatic SV detection has been extensively studied in cancer, these studies are not directly applicable to the study of tissue mosaicism, as they rely on matched normals, target higher VAF ranges, and are enriched for different types of SVs. We present comprehensive benchmark data and best practices for non-cancer somatic SV detection. We created a synthetic mosaic sample by combining six HapMap individuals at varying proportions, generating allele fractions as low as 0.25%. This sample was sequenced to ~2,300x total coverage using Illumina, PacBio, and Nanopore technologies across multiple sequencing centers. A high-confidence benchmark SV set containing over 21,000 pseudo-somatic insertions and deletions ≥50bp was derived from haplotype-resolved assemblies. We evaluated 12 SV discovery pipelines and identified caller-specific strengths and sequencing platform-specific shortcomings. We find that short read-based approaches show reduced recall for insertions and repeat-associated SVs, whereas long-read sequencing achieves high accuracy throughout the genome, increasing linearly with coverage. The best algorithm's sensitivity exceeded 80% for VAFs ≥4% and 15% for VAFs of 0.5-1% with 60x coverage. The publicly available benchmarking data and comparative analysis of current methods provide a foundation for robust discovery of SV mosaicism in non-cancer tissues..

Journal Article↗
PubMed2001

Selection of phage-display library peptides recognizing ethanol targets on proteins.

There is a forthcoming link between chronic alcohol consumption and proteins covalently modified by ethanol metabolites and their antibodies. To identify sensitive probes of protein-ethanol conjugates, we screened for the ethanol-altered protein domains with a phage-display combinatorial peptide library. In principle, recognition of the epitopes by the library peptides occurs through protein-protein interactions. A general screening, M13-based library with 10(9) random sequences of linear heptameric peptides was used. The peptides were displayed in five copies each, as fusion proteins with phage's minor coat protein III. They were located on one end of the surface of the phage particles. The targets were a model protein, streptavidin, and protein-ethanol conjugates (hydroxyethyl radical- or acetaldehyde-modified bovine serum albumin). They were either immobilized on a surface by direct coating or affinity captured on floating beads. An enriched library of phages with the tightest peptide binders for each target was selected and amplified in a multiple-cycle biopanning in vitro procedure. Binders were characterized by DNA sequencing of the corresponding phages and by counter-screening with positive and negative targets in either an enzyme-linked immunosorbent assay or plaque assay. We obtained the HPQ motif for streptavidin and two unique subsets of peptides that recognized each ethanol target with a selectivity of two orders of magnitude above the carrier protein and controls. The application of biopanning processes, coupled with phage-display peptide libraries on biological fluids and tissues, could provide a systematic mapping of protein--ethanol conjugates and supply a means for early diagnosis and prognosis of chronic alcohol consumption in human beings.

Amino Acid Sequence↗
PubMed2002

Identification of active methylotroph populations in an acidic forest soil by stable-isotope probing.

Stable-isotope probing (SIP) is a culture-independent technique that enables the isolation of DNA from micro-organisms that are actively involved in a specific metabolic process. In this study, SIP was used to characterize the active methylotroph populations in forest soil (pH 3.5) microcosms that were exposed to (13)CH(3)OH or (13)CH(4). Distinct (13)C-labelled DNA ((13)C-DNA) fractions were resolved from total community DNA by CsCl density-gradient centrifugation. Analysis of 16S rDNA sequences amplified from the (13)C-DNA revealed that bacteria related to the genera Methylocella, Methylocapsa, Methylocystis and Rhodoblastus had assimilated the (13)C-labelled substrates, which suggested that moderately acidophilic methylotroph populations were active in the microcosms. Enrichments targeted towards the active proteobacterial CH(3)OH utilizers were successful, although none of these bacteria were isolated into pure culture. A parallel analysis of genes encoding the key enzymes methanol dehydrogenase and particulate methane monooxygenase reflected the 16S rDNA analysis, but unexpectedly revealed sequences related to the ammonia monooxygenase of ammonia-oxidizing bacteria (AOB) from the beta-subclass of the PROTEOBACTERIA: Analysis of AOB-selective 16S rDNA amplification products identified Nitrosomonas and Nitrosospira sequences in the (13)C-DNA fractions, suggesting certain AOB assimilated a significant proportion of (13)CO(2), possibly through a close physical and/or nutritional association with the active methylotrophs. Other sequences retrieved from the (13)C-DNA were related to the 16S rDNA sequences of members of the Acidobacterium division, the beta-Proteobacteria and the order Cytophagales, which implicated these bacteria in the assimilation of reduced one-carbon compounds or in the assimilation of the by-products of methylotrophic carbon metabolism. Results from the (13)CH(3)OH and (13)CH(4) SIP experiments thus provide a rational basis for further investigations into the ecology of methylotroph populations in situ.

Ammonia↗
PubMed1993

Ex vivo regulation of specific gene expression by nanomolar concentration of double-stranded dumbbell oligonucleotides.

Inhibition of specific transcriptional regulatory proteins is a new approach to control gene expression. Transcriptional activity of DNA-binding proteins can be inhibited by the use of double-stranded (ds) oligodeoxynucleotides that compete for the binding to their specific target sequences in promoters and enhancers. As a model, we used phosphodiester dumbbell oligonucleotides containing a binding site for the liver-enriched transcription factor HNF-1 (Hepatocyte Nuclear Factor 1). Binding affinity of HNF-1 to dumbbell oligonucleotides was the same as that to ds oligonucleotides, as determined by gel retardation assays. HNF-1 dumbbells specifically inhibited in vitro transcription driven by the albumin promoter by more than 90%. HNF-1-dependent activation of a CAT reporter plasmid was specifically inhibited when the HNF-1 dumbbell oligonucleotide was added at nM concentration to transiently transfected C33 cells. On the contrary, HNF-1 ds oligonucleotides, which displayed the same activity as the dumbbell oligonucleotides in the in vitro assays, were no more effective in the ex vivo experiments. These results might reflect the increased stability of the circular dumbbell oligonucleotides towards cellular nuclease degradation, as shown in vitro with nucleolytic enzymes. Dumbbell oligonucleotides containing unmodified phosphodiester bonds may efficiently compete for binding of specific transcription factors within cells, then providing a potential therapeutic tool to control disease-causing genes.

Albumins↗
PubMed2006

The malarial host-targeting signal is conserved in the Irish potato famine pathogen.

Animal and plant eukaryotic pathogens, such as the human malaria parasite Plasmodium falciparum and the potato late blight agent Phytophthora infestans, are widely divergent eukaryotic microbes. Yet they both produce secretory virulence and pathogenic proteins that alter host cell functions. In P. falciparum, export of parasite proteins to the host erythrocyte is mediated by leader sequences shown to contain a host-targeting (HT) motif centered on an RxLx (E, D, or Q) core: this motif appears to signify a major pathogenic export pathway with hundreds of putative effectors. Here we show that a secretory protein of P. infestans, which is perceived by plant disease resistance proteins and induces hypersensitive plant cell death, contains a leader sequence that is equivalent to the Plasmodium HT-leader in its ability to export fusion of green fluorescent protein (GFP) from the P. falciparum parasite to the host erythrocyte. This export is dependent on an RxLR sequence conserved in P. infestans leaders, as well as in leaders of all ten secretory oomycete proteins shown to function inside plant cells. The RxLR motif is also detected in hundreds of secretory proteins of P. infestans, Phytophthora sojae, and Phytophthora ramorum and has high value in predicting host-targeted leaders. A consensus motif further reveals E/D residues enriched within approximately 25 amino acids downstream of the RxLR, which are also needed for export. Together the data suggest that in these plant pathogenic oomycetes, a consensus HT motif may reside in an extended sequence of approximately 25-30 amino acids, rather than in a short linear sequence. Evidence is presented that although the consensus is much shorter in P. falciparum, information sufficient for vacuolar export is contained in a region of approximately 30 amino acids, which includes sequences flanking the HT core. Finally, positional conservation between Phytophthora RxLR and P. falciparum RxLx (E, D, Q) is consistent with the idea that the context of their presentation is constrained. These studies provide the first evidence to our knowledge that eukaryotic microbes share equivalent pathogenic HT signals and thus conserved mechanisms to access host cells across plant and animal kingdoms that may present unique targets for prophylaxis across divergent pathogens.

Amino Acid Motifs↗
PubMed2004

[Construction and identification on enriched microsatellite library from yak genome].

We constructed the first microsatellite-enriched library of yak according to the strong affinity between biotin and streptavidin. The method included ligation of 300 approximately 1 000 bp enzyme-digested fragments and adaptors, affinity capture of microsatellite repeat using biotinylated oligoprobe ((CA)12, (CCG) 8, (CAG)8, (TTTC) 8) attached to streptavidin-coated magnetic beads, PCR amplification using the 21-mer adaptor oligonucleotide as primer to obtain double-stranded targeted fragments, religated into pMD18-T vector and transformed to DH5alpha. The results of sequencing showed that 37 of 48 readable sequences contained microsatellites indicating a high degree of microsatellite enrichment. The new polymorphic microsatellite markers we have identified and characterized will contribute to the yak genetic linkage mapping, molecular evolution and phylogenetic studies, marker assistant selection and QTLs location of yak main economic traits.

Animals↗
PubMed2002

SETDB1: a novel KAP-1-associated histone H3, lysine 9-specific methyltransferase that contributes to HP1-mediated silencing of euchromatic genes by KRAB zinc-finger proteins.

Posttranslational modification of histones has emerged as a key regulatory signal in eukaryotic gene expression. Recent genetic and biochemical studies link H3-lysine 9 (H3-K9) methylation to HP1-mediated heterochromatin formation and gene silencing. However, the mechanisms that target and coordinate these activities to specific genes is poorly understood. Here we report that the KAP-1 corepressor for the KRAB-ZFP superfamily of transcriptional silencers binds to SETDB1, a novel SET domain protein with histone H3-K9-specific methyltransferase activity. Although acetylation and phosphorylation of the H3 N-terminal tail profoundly affect the efficiency of H3-K9 methylation by SETDB1, we found that methylation of H3-K4 does not affect SETDB1-mediated methylation of H3-K9. In vitro methylation of the N-terminal tail of histone H3 by SETDB1 is sufficient to enhance the binding of HP1 proteins, which requires both an intact chromodomain and chromoshadow domain. Indirect immunofluoresence staining of interphase nuclei localized SETDB1 predominantly in euchromatic regions that overlap with HP1 staining in nonpericentromeric regions of chromatin. Moreover, KAP-1, SETDB1, H3-MeK9, and HP1 are enriched at promoter sequences of a euchromatic gene silenced by the KRAB-KAP-1 repression system. Thus, KAP-1 is a molecular scaffold that is targeted by KRAB-ZFPs to specific loci and coordinates both histone methylation and the deposition of HP1 proteins to silence gene expression.

Animals↗
PubMed2026

MLL4 protects cardiomyocytes against ischemia-reperfusion injury through STAT3-mediated mitochondrial function.

Myocardial ischemia-reperfusion injury (MIRI) is an inevitable pathophysiological response during the revascularization process following myocardial ischemia. Despite its clinical significance, effective targeted therapies for MIRI remain an unmet medical need. Mixed-lineage leukemia 4 (MLL4), a member of the SET family of histone methyltransferases, exhibits particular methyltransferase action toward histone H3 lysine 4 (H3K4). This study establishes a protective role for MLL4 in MIRI pathogenesis. Utilizing cardiomyocyte-specific Mll4 knockout mice and an in vivo ischemia-reperfusion (I/R) model induced by left anterior descending coronary artery ligation, we observed significant upregulation of MLL4 expression in cardiac tissue following I/R. Genetic ablation of Mll4 in cardiomyocytes markedly exacerbated both acute and chronic phases of MIRI. In vitro, Mll4 knockdown in neonatal rat cardiomyocytes (NRCMs) amplified mitochondrial dysfunction and apoptosis under hypoxia/reoxygenation (H/R) conditions. Integrated analysis of Cleavage Under Targets and Tagmentation sequencing (CUT&Tag-seq) and RNA sequencing (RNA-seq) revealed that Mll4 deficiency induces a pronounced reduction in H3K4 monomethylation (H3K4me1) and histone H3 lysine 27 acetylation (H3K27ac) enrichment at the Stat3 genomic locus. Mechanistically, MLL4 functions as a transcriptional activator of Stat3 by depositing H3K4me1 and H3K27ac, thereby facilitating STAT3 transcription. This regulatory cascade ultimately governs STAT3-dependent mitochondrial homeostasis. Collectively, these findings identify MLL4 as a critical epigenetic regulator of MIRI and suggest its therapeutic targeting may offer a promising strategy for mitigating reperfusion injury.

Animals↗
PubMed2006

Assays for cytokines using aptamers.

Aptamers are short nucleic acid sequences that are used as ligands to bind their targets with high affinity. They are generated via the combinatorial chemistry procedure systematic evolution of ligands by exponential enrichment (SELEX). Aptamers have shown much promise towards detection of a variety of protein targets, including cytokines. Specifically, for the determination of cytokines and growth factors, several assays making use of aptamers have been developed, including aptamer-based enzyme-linked immunosorbent assays, antibody-linked oligonucleotide assay, fluorescence (anisotropy and resonance energy transfer) assays, and proximity ligation assays. In this article, the concept of aptamer selection using SELEX and the assay formats using aptamers for the detection of cytokines are discussed.

Animals↗
PubMed1999

The cleaved presequence is not required for import of subunit 6 of the cytochrome bc1 complex into yeast mitochondria or assembly into the complex.

Subunit 6 of the yeast cytochrome bc1 complex contains a 25 amino acid presequence that is not present in the mature form of the protein in the bc1 complex. The presequence of subunit 6 is atypical of presequences responsible for targeting proteins to mitochondria. Whereas mitochondrial targeting sequences rarely contain acidic residues and typically contain basic residues that can potentially form an amphiphilic structure, the presequence of subunit 6 contains only one basic amino acid and is enriched in acidic amino acids. If the 25 amino acid presequence is deleted, subunit 6 is imported into mitochondria and assembled into the cytochrome bc1 complex and the activity of the bc1 complex is identical to that from a wild-type yeast strain. However, if the C-terminal 45 amino acids are truncated from the protein, subunit 6 is not present in the mitochondria and the activity of the bc1 complex is diminished by half, identical to that of the bc1 complex from a yeast strain in which the QCR6 gene is deleted. These results indicate that the presequence of subunit 6 is not required for targeting to mitochondria or assembly of the subunit into the bc1 complex and that information necessary for targeting and import into mitochondria may be present in the C-terminus of the protein.

Amino Acid Sequence↗
PubMed2026

Genome-guided isolation and characterization of a novel bacteriophage infecting Escherichia coli reveal a putative new genus.

We have isolated and characterized a novel bacteriophage termed Jab, with lytic activity against multidrug-resistant clinical isolates of Escherichia coli. Phage Jab was identified from liquid manure by means of metagenome sequencing of a phage community enrichment using an E. coli clinical isolate ECH07 as host. The initial enrichment was composed of four phages, of which phage Jab represented only a minute fraction (less than 1%). Jab isolation strategy comprised a targeted approach using iterative replication rounds while equipping ECH07 with resistance against the numerically dominant phages coupled with a subsequent host switch to E. coli BL21. Whole-genome sequence analysis revealed only a remote evolutionary distance to known phages within the subfamily Vequintavirinae. The dsDNA genome of phage Jab comprises 142,100 bp (GC content 40.09%) and encodes 264 proteins and five transfer RNAs (tRNAs). No lysogeny-associated proteins were detected, suggesting an obligate lytic lifestyle. In silico genome analysis revealed the presence of at least four putative depolymerases. The closest homology of phage Jab is with members of the new genus Septuagintavirus with around 34% nucleotide identity. VIRIDIC and network analyses strongly suggest that phage Jab belongs to a putative novel genus. The host range of phage Jab is likely restricted to E. coli, displaying a moderately narrow host range (i.e., productive lysis in 8 out of 27 isolates tested). Notably, transmission electron microscopy (TEM) revealed the occurrence of conspicuous unique spherical structures attached at the end of the tail fibers when propagated on BL21 but not when propagated on ECH07. Although their function remains enigmatic, the possible role of those structures as a bacterial (vesicle-based) defense mechanism warrants further investigation.

Escherichia coli↗
PubMed2005

RNA sequence and secondary structure participate in high-affinity CsrA-RNA interaction.

The global Csr regulatory system controls bacterial gene expression post-transcriptionally. CsrA of Escherichia coli is an RNA binding protein that plays a central role in repressing several stationary phase processes and activating certain exponential phase functions. CsrA regulates translation initiation of several genes by binding to the mRNA leaders and blocking ribosome binding. CsrB and CsrC are noncoding regulatory RNAs that are capable of sequestering CsrA and antagonizing its activity. Each of the known target transcripts contains multiple CsrA binding sites, although considerable sequence variation exists among these RNA targets, with GGA being the most highly conserved element. High-affinity RNA ligands containing single CsrA binding sites were identified from a combinatorial library using systematic evolution of ligands by exponential enrichment (SELEX). The SELEX-derived consensus was determined as RUACARGGAUGU, with the ACA and GGA motifs being 100% conserved and the GU sequence being present in all but one ligand. The majority (51/55) of the RNAs contained GGA in the loop of a hairpin within the most stable predicted structure, an arrangement similar to several natural CsrA binding sites. Strikingly, the identity of several nucleotides that were predicted to form base pairs in each stem were 100% conserved, suggesting that primary sequence information was embedded within the base-paired region. The affinity of CsrA for several selected ligands was measured using quantitative gel mobility shift assays. A mutational analysis of one selected ligand confirmed that the conserved ACA, GGA, and GU residues were critical for CsrA binding and that RNA secondary structure participates in CsrA-RNA recognition.

Bacterial Proteins↗
PubMed1992

Cyclic amplification and selection of targets for multicomponent complexes: myogenin interacts with factors recognizing binding sites for basic helix-loop-helix, nuclear factor 1, myocyte-specific enhancer-binding factor 2, and COMP1 factor.

Myogenin is one of four muscle-specific basic helix-loop-helix regulatory factors involved in controlling myogenesis. We here describe various protein complexes that increase the affinity of myogenin for DNA. We mixed an oligonucleotide containing a degenerate center large enough to accommodate multiple binding sites with crude myotube nuclear extracts and used cyclic amplification and selection of targets with an antimyogenin monoclonal antibody to isolate protein-DNA complexes. Since each cycle of selection results in the enrichment for the sequences with the highest affinity, we isolated multicomponent sites in which myogenin binding was increased by its interaction with other DNA binding proteins. Myogenin interacts with members of the nuclear factor 1 family, the muscle-specific factor myocyte-specific enhancer-binding factor 2, and another factor, COMP1 (cooperates with myogenic proteins 1), that binds to the sequence TGATTGAC. Myogenin also exhibits cooperative binding with other proteins that recognize CANNTG motifs, and various constraints on spacing and orientation were observed. The application of this approach to other transcription factors should not only help identify the different functions of myogenin versus other members of the muscle basic helix-loop-helix regulatory family but also help define the general combinatorial mechanisms involved in eukaryotic gene regulation.

Base Sequence↗
PubMed2005

Molecular characterization of the nonphotosynthetic partner bacterium in the consortium "Chlorochromatium aggregatum".

Phototrophic consortia represent valuable model systems for the study of signal transduction and coevolution between different bacteria. The phototrophic consortium "Chlorochromatium aggregatum" consists of a colorless central rod-shaped bacterium surrounded by about 20 green-pigmented epibionts. Although the epibiont was identified as a member of the green sulfur bacteria, and recently isolated and characterized in pure culture, the central colorless bacterium has been identified as a member of the beta-Proteobacteria but so far could not be characterized further. In the present study, "C. aggregatum" was enriched chemotactically, and the 16S rRNA gene sequence of the central bacterium was elucidated. Based on the sequence information, fluorescence in situ hybridization probes targeting four different regions of the 16S rRNA were designed and shown to hybridize exclusively to cells of the central bacterium. Phylogenetic analyses of the 1,437-bp-long sequence revealed that the central bacterium of "C. aggregatum" represents a so far isolated phylogenetic lineage related to Rhodoferax spp., Polaromonas vacuolata, and Variovorax paradoxus within the family Comamonadaceae. The majority of relatives of this lineage are not yet cultured and were found in low-temperature aquatic environments or aquatic environments containing xenobiotica or hydrocarbons. In CsCl-bisbenzimidazole equilibrium density gradients, genomic DNA of the central bacterium of "Chlorochromatium aggregatum" formed a distinct band which could be detected by quantitative PCR using specific primers. Using this method, the G+C content of the central bacterium was determined to be 55.6 mol%.

Base Composition↗