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PubMed1997

The ammonia monooxygenase structural gene amoA as a functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations.

The naturally occurring genetic heterogeneity of autotrophic ammonia-oxidizing populations belonging to the beta subclass of the Proteobacteria was studied by using a newly developed PCR-based assay targeting a partial stretch of the gene which encodes the active-site polypeptide of ammonia monooxygenase (amoA). The PCR yielded a specific 491-bp fragment with all of the nitrifiers tested, but not with the homologous stretch of the particulate methane monooxygenase, a key enzyme of methane-oxidizing bacteria. The assay also specifically detected amoA in DNA extracted from various aquatic and terrestrial environments. The resulting PCR products retrieved from rice roots, activated sludge, a freshwater sample, and an enrichment culture were used for the generation of amoA gene libraries. No false positives were detected in a set of 47 randomly selected clone sequences that were analyzed further. The majority of the environmental sequences retrieved from rice roots and activated sludge grouped within the phylogenetic radiation defined by cultured strains of the genera Nitrosomonas and Nitrosospira. The comparative analysis identified members of both of these genera in activated sludge; however, only Nitrosospira-like sequences with very similar amino acid patterns were found on rice roots. Further differentiation of these molecular isolates was clearly possible on the nucleic acid level due to the accumulation of synonymous mutations, suggesting that several closely related but distinct Nitrosospira-like populations are the main colonizers of the rhizosphere of rice. Each of the amoA gene libraries obtained from the freshwater sample and the enrichment culture was dominated by a novel lineage that shared a branch with the Nitrosospira cluster but could not be assigned to any of the known pure cultures. Our data suggest that amoA represents a very powerful molecular tool for analyzing indigenous ammonia-oxidizing communities due to (i) its specificity, (ii) its fine-scale resolution of closely related populations, and (iii) the fact that a functional trait rather than a phylogenetic trait is detected.

Amino Acid Sequence↗

PubMed1996

Molecular diversity of soil and marine 16S rRNA gene sequences related to beta-subgroup ammonia-oxidizing bacteria.

We have conducted a preliminary phylogenetic survey of ammonia-oxidizing beta-proteobacteria, using 16S rRNA gene libraries prepared by selective PCR and DNA from acid and neutral soils and polluted and nonpolluted marine sediments. Enrichment cultures were established from samples and analyzed by PCR. Analysis of 111 partial sequences of c. 300 bases revealed that the environmental sequences formed seven clusters, four of which are novel, within the phylogenetic radiation defined by cultured autotrophic ammonia oxidizers. Longer sequences from 13 cluster representatives support their phylogenetic positions relative to cultured taxa. These data suggest that known taxa may not be representative of the ammonia-oxidizing beta-proteobacteria in our samples. Our data provide further evidence that molecular and culture-based enrichment methods can select for different community members. Most enrichments contained novel Nitrosomonas-like sequences whereas novel Nitrosospira-like sequences were more common from gene libraries of soils and marine sediments. This is the first evidence for the occurrence of Nitrosospira-like strains in marine samples. Clear differences between the sequences of soil and marine sediment libraries were detected. Comparison of 16S rRNA sequences from polluted and nonpolluted sediments provided no strong evidence that the community composition was determined by the degree of pollution. Soil clone sequences fell into four clusters, each containing sequences from acid and neutral soils in varying proportions. Our data suggest that some related strains may be present in both samples, but further work is needed to resolve whether there is selection due to pH for particular sequence types.

Ammonia↗

PubMed1988

Cloning of the adenylate cyclase genetic determinant of Bordetella pertussis and its expression in Escherichia coli and B. pertussis.

A recombinant plasmid, pRMB1, identified from a gene library of B. pertussis, restored adenylate cyclase (AC) and haemolysin (HLY) activities to B. pertussis BP348 (a Tn5-insertion mutant deficient in both these activities). B. pertussis BP348 was considerably less virulent than wild type strains of B. pertussis when 3-week-old mice were challenged intranasally; possession of pRMB1 restored virulence. Neither AC nor HLY activities were expressed in E. coli harbouring pRMB1. However, expression of calmodulin-responsive AC was obtained in E. coli when restriction fragments of pRMB1 were subcloned into other vectors; expression depended on the lac and tac promoters of these vectors. The enzyme was not readily solubilized from urea extracts of E. coli and required sonication for efficient release. One plasmid conferred a specific AC enzymic activity to E. coli which was greater than that for wild type B. pertussis strains. Unlike extracts of B. pertussis, extracts from E. coli expressing enzymic AC activity, did not elevate cAMP levels in S49 lymphoma cells. A second plasmid, pRMB2, was identified from the gene library, which contained a trans-acting regulatory determinant required for expression of AC, HLY and other virulence-associated factors in B. pertussis.

Adenylyl Cyclases↗

PubMed2004

Cloning and expression of clt genes encoding milk-clotting proteases from Myxococcus xanthus 422.

The screening of a gene library of the milk-clotting strain Myxococcus xanthus 422 constructed in Escherichia coli allowed the description of eight positive clones containing 26 open reading frames. Only three of them (cltA, cltB, and cltC) encoded proteins that exhibited intracellular milk-clotting ability in E. coli, Saccharomyces cerevisiae, and Pichia pastoris expression systems.

Aspartic Acid Endopeptidases↗

PubMed1987

Molecular cloning and physical mapping of the genome of insect iridescent virus type 6: further evidence for circular permutation of the viral genome.

A defined and complete gene library of the Chilo iridescent virus (CIV) genome was established. The CIV DNA was cleaved with restriction endonucleases EcoRI, NcoI, SphI, and BamHI or double digested with BamHI/SalI and the resulting DNA fragments were inserted into the corresponding sites of the bacterial vectors pACYC184, pKm2, pL-ES-C3, and pAT153 using T4 DNA ligase. All cloned fragments were identified by digestion of the recombinant plasmids with different restriction enzymes and checked by hybridization of recombinant plasmid to viral DNA. This analysis revealed that sequences representing 100% of the viral genome were cloned into the EcoRI site of pACYC184. Although the CIV genome is linear, all 32 EcoRI fragments have been cloned directly. This suggests that the CIV genome is circularly permuted. In addition, NcoI(72%), SphI(40.7%), BamHI (11.6%), and BamHI/SalI(39.7%) DNA fragments of the viral genome were inserted into the corresponding sites of pKm2, pL-ES-C3, and pAT153, respectively. The physical map of the viral genome was constructed using the established gene library for restriction enzymes ApaI, BamHI, EcoRI, NcoI, SalI, and SmaI. Although the CIV genome is linear, this analysis revealed that the restriction maps of the viral genome are circular. This finding supports the hypothesis that the CIV genome is circularly permuted.

Cloning, Molecular↗

PubMed1990

Characterization and cloning of the gerC locus of Bacillus subtilis 168.

A Bacillus subtilis gerC spore germination mutant demonstrating a temperature-sensitive response to L-alanine as germinant has been characterized in detail. The gerC58 mutation is 50% cotransformed with aroB in the gene order gerC-aroB-trpC. The mutation is responsible for a severe growth defect which is manifest at all growth temperatures and is most extreme on rich media. A second, unlinked, mutation in the original strain suppressed this growth defect, but spores of the suppressed strain failed to germinate in alanine at 42 degrees C. As this germination defect is dependent on the presence of the gerC58 allele, it is likely to be the direct result of a mutant gerC protein. The gerC gene therefore appears to have a role in both spore germination and vegetative cell growth. A gene library of BclI-digested B. subtilis chromosomal DNA was constructed in phage vector phi 105J27. A derivative containing the gerC region was obtained by complementation of the growth defect of an unsuppressed gerC58 strain. This phage contained a 6.3 kb insert of bacterial DNA, which is above the reported packaging limit of the phage. It failed to form visible plaques, although it could be handled as a prophage and sufficient phage particles be isolated to allow characterization of the insert. A deletion derivative generated in vitro and carrying only 2.9 kb of insert DNA also complemented the gerC defect. This gerC locus is the second locus to be implicated in alanine-stimulated germination. The first, gerA, is a developmentally controlled operon whose gene products are present only in the spore. This study of gerC, in contrast, reveals a role in spore germination for a normally essential vegetative protein.

Alanine↗

PubMedDate not supplied

[Isolation of individual chromosomes and their use in studying the human and animal genome].

This mini-review considers the main achievements in the field of human and animal chromosome separation: technical problems of flow-karyotyping and flow-sorting, the types of cell lines and chromosome source used, different protocols of chromosome suspension preparation, fluorochromes for chromosome staining, efficiency of chromosome sorting and purity of the fractions. The use of fractions for creating chromosome-specific human gene libraries and mapping cloned genes and DNA fragment are also considered.

Animals↗

PubMed1996

A novel and efficient route for the isolation of antibodies that recognise T cell receptor V alpha(s).

Studies of the T cell repertoire have been hindered by the lack of antibodies that recognise V region families, particularly for V alpha regions. In this report, single chain Fv (scFv) fragments have been isolated that recognise both recombinant V alpha(s) and native V alpha(s) on the surface of T cells. Mice have been immunised with purified soluble T cell receptors (TCRs) and antibody heavy and light chain variable domain (VH and VL, respectively) genes isolated from splenocytes using the polymerase chain reaction (PCR). The VH and VL genes have been assembled as scFv gene libraries and a bacteriophage display system used to isolate scFvs that recognise a soluble V alpha. Five scFvs have been purified and characterised in detail using enzyme-linked immunosorbent assays (ELISAs) and flow cytometry. Three of these five scFvs recognise native V alpha(s) on the surface of T cell hybridomas. This method therefore offers a rapid route to the generation of scFvs that recognise native TCRs and can readily be extended to the production of anti-human TCR antibodies for use in therapy and diagnosis.

Amino Acid Sequence↗

PubMed2005

Archaeal diversity along a soil salinity gradient prone to disturbance.

We employed a cultivation-independent approach to examine archaeal diversity along a transient soil salinity gradient at Salt Spring in British Columbia, Canada that is routinely eroded due to heavy, recurrent rainfall. Archaeal 16S rRNA gene libraries were created using DNA extracted from three soil samples collected along this gradient. Statistical comparisons indicated similar archaeal richness across sites but, a significant shift in archaeal community composition along the salinity gradient. Seven distinct phylogenetic groups were represented in soil libraries. Haloarchaea were the most commonly sampled group. Other 16S rRNA sequences were related to uncultured Euryarchaeota and Crenarchaeota or halophilic methanogens. Haloarchaeal diversity was remarkably high in soil of elevated salinity compared with previously characterized haloarchaeal communities. Salt Spring haloarchaea were not closely related to known low-salt adapted/tolerant species, suggesting they may be frequently faced with local mortality as a result of frequent declines in soil salinity. We speculate that ecosystem disturbance -- in the form of salinity fluctuations -- is one mechanism for maintaining a diverse community of haloarchaea at Salt Spring.

Crenarchaeota↗

PubMed1992

Cloning, sequencing, and expression of the fadD gene of Escherichia coli encoding acyl coenzyme A synthetase.

In the enteric bacterium, Escherichia coli, acyl coenzyme A synthetase (fatty acid:CoA ligase (AMP-forming) EC 6.2.1.3) activates exogenous long-chain fatty acids concomitant with their transport across the inner membrane into metabolically active CoA thioesters. These compounds serve as substrates for acyl-CoA dehydrogenase in the first step in the process of beta-oxidation. The acyl-CoA synthetase structural gene, fadD, has been identified on clone 6D1 of the Kohara E. coli gene library and by a process of subcloning and complementation analyses shown to be contained on a 2.2-kilobase NcoI-ClaI fragment of genomic DNA. The polypeptide encoded within this DNA fragment was identified following T7 RNA polymerase-dependent induction and estimated to be M(r) = 62,000 using SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of acyl-CoA synthetase was determined by automated sequencing to be Met-Lys-Lys-Val-Trp-Leu-Asn-Arg-Tyr-Pro. Sequence analysis of the 2.2-kilobase NcoI-ClaI fragment revealed a single open reading frame encoding these amino acids as the first 10 residues of a protein with a molecular weight of 62,028. The initiation codon for methionine was TTG. Primer extension of total in vivo mRNA from two fadD-specific oligonucleotides defined the transcriptional start at an adenine residue 60 base pairs upstream from the predicted translational start site. Two FadR operator sites of the fadD gene were identified at positions -13 to -29 (OD1) and positions -99 to -115 (OD2) by DNase I footprinting. Comparisons of the predicted amino acid sequence of the E. coli acyl-CoA synthetase to the deduced amino acid sequences of the rat and yeast acyl-CoA synthetases and the firefly luciferase demonstrated that these enzymes shared a significant degree of similarity. Based on the similar reaction mechanisms of these four enzymes, this similarity may define a region required for the same function.

Amino Acid Sequence↗

PubMed2006

Nitrous oxide reductase genes (nosZ) of denitrifying microbial populations in soil and the earthworm gut are phylogenetically similar.

Earthworms emit nitrous oxide (N2O) and dinitrogen (N2). It has been hypothesized that the in situ conditions of the earthworm gut activates ingested soil denitrifiers during gut passage and leads to these in vivo emissions (M. A. Horn, A. Schramm, and H. L. Drake, Appl. Environ. Microbiol. 69:1662-1669, 2003). This hypothesis implies that the denitrifiers in the earthworm gut are not endemic to the gut but rather are regular members of the soil denitrifier population. To test this hypothesis, the denitrifier populations of gut and soil from three different sites were comparatively assessed by sequence analysis of nosZ, the gene for the terminal enzyme in denitrification, N2O reductase. A total of 182 and 180 nosZ sequences were retrieved from gut and soil, respectively; coverage of gene libraries was 79 to 100%. Many of the nosZ sequences were heretofore unknown, clustered with known soil-derived sequences, or were related to N2O reductases of the genera Bradyrhizobium, Brucella, Dechloromonas, Flavobacterium, Pseudomonas, Ralstonia, and Sinorhizobium. Although the numbers of estimators for genotype richness of sequence data from the gut were higher than those of soil, only one gut-derived nosZ sequence did not group phylogenetically with any of the soil-derived nosZ sequences. Thus, the phylogenies of nosZ from gut and soil were not dissimilar, indicating that gut denitrifiers are soil derived.

Animals↗

PubMed2006

Diversity of microorganisms in Fe-As-rich acid mine drainage waters of Carnoulès, France.

The acid waters (pH 2.7 to 3.4) originating from the Carnoulès mine tailings contain high concentrations of dissolved arsenic (80 to 350 mg.liter(-1)), iron (750 to 2,700 mg.liter(-1)), and sulfate (2,000 to 7,500 mg.liter(-1)). During the first 30 m of downflow in Reigous creek issuing from the mine tailings, 20 to 60% of the dissolved arsenic is removed by coprecipitation with Fe(III). The microbial communities along the creek have been characterized using terminal-restriction fragment length polymorphism (T-RFLP) and 16S rRNA gene library analyses. The results indicate a low bacterial diversity in comparison with unpolluted water. Eighty percent of the sequences obtained are related to sequences from uncultured, newly described organisms or recently associated with acid mine drainage. As expected owing to the water chemistry, the sequences recovered are mainly related to bacteria involved in the geochemical Fe and S cycles. Among them, sequences related to uncultured TrefC4 affiliated with Gallionella ferruginea, a neutrophilic Fe-oxidizing bacterium, are dominant. The description of the bacterial community structure and its dynamics lead to a better understanding of the natural remediation processes occurring at this site.

Arsenic↗

PubMed1994

An "instant gene bank" method for heterologous gene cloning: complementation of two Aspergillus nidulans mutants with Gaeumannomyces graminis DNA.

We present a novel technique for gene cloning by complementation of mutations in Aspergillus nidulans with DNA from a heterologous organism, Gaeumannomyces graminis. This technique bypasses the time-consuming and difficult construction of gene libraries, making it both rapid and simple. The method relies on recombination between a fungal replicating vector pHELP1 and linear G. graminis genomic DNA during co-transformation. We were able to complement two out of seven A. nidulans mutants tested and to rescue transforming DNA from both in Escherichia coli. Complementation of the A. nidulans argB mutation resulted from integration of 8-10 kb segments of G. graminis DNA into pHELP1. The complementation of the A. nidulans pyrG mutation resulted from a complex rearrangement. Complementing DNA was shown to originate from G. graminis, and was capable of retransforming the original mutants to give the expected phenotype.

Aspergillus nidulans↗

PubMed1989

Cloning of the gene for cell-surface protein antigen A from Streptococcus sobrinus (serotype d).

A gene library of Strep. sobrinus B13N (serotype d) chromosomal DNA was constructed in Escherichia coli, with the bacteriophage vector lambda L47.1. A recombinant phage, lambda MDSM49, containing a 15.5 kb DNA insert, directed the expression of a 210 kDa antigenic protein. The recombinant 210 kDa protein was shown by Western blot analysis to be identical with cell-surface protein antigen A (spaA) from a serotype g strain. However, the restriction patterns of a subclone plasmid, pMD51, from lambda MDSM49 differed from those of serotype g strain. The cell-surface protein antigen I/II from serotype c Streptococcus mutans is a potential immunogen for vaccination against dental caries and corresponds to the spaA from serotype d and g strains. A recombinant clone, pDM51, will be a useful tool for serological and molecular biological studies. The recombinant spaA provides useful material for assessment of its diagnostic and immunogenic potential.

Antigens, Bacterial↗

PubMed2002

A male-specific (cysteine-rich) protein of Oesophagostomum dentatum (Strongylida) with structural characteristics of a serine protease inhibitor containing two trypsin inhibitor-like domains.

A cDNA was isolated from an adult male Oesophagostomum dentatum gene library by screening with a male-specific, partial expressed sequence tag (EST) probe identified previously using a differential display technique. The full-length cDNA of 642 bp included 5' and 3' untranslated regions of 44 and 121 nucleotides, respectively, and encoded a predicted protein with a putative 18 amino acid signal sequence and a mature polypeptide of 14.7 kDa comprising approximately 15% cysteine residues. The amino acid sequence showed similarity with a number of proteins from Caenorhabditis elegans, parasitic nematodes, insects and amphibia, all of which contain a trypsin inhibitor-like cysteine-rich domain. A 3-dimensional structure model constructed for the O. dentatum protein (designated OdmCRP) inferred that it is composed of 2 domains, each with 5 disulfide bonds, which are indicative of the Ascaris family of serine protease inhibitors. These findings indicate that OdmCRP, with 2 structural domains relating to functionally active sites, is a new member of this inhibitor family.

Amino Acid Sequence↗

PubMed2000

estA, a gene coding for a cell-bound esterase from Paenibacillus sp. BP-23, is a new member of the bacterial subclass of type B carboxylesterases.

Screening of a gene library from Paenibacillus sp. BP-23 generated in Escherichia coli led to identification of a clone that directed the production of lipolytic activity. From the sequencing data, we found an open reading frame encoding a protein of 485 amino acids with an estimated molecular mass of 53 kDa and a pI of 5.1. Absence of a signal peptide indicated that it was a cell-bound protein. Sequence analysis showed that the protein contained the signature G-XI-S-X2-G included in most serine-esterases and lipases. The cloned protein showed high homology with enzymes belonging to the bacterial subclass of type B carboxylesterases. The enzyme had a significant preference for esters of short-chain fatty acids and showed the kinetics behaviour of a true esterase. Maximum activity was found at pH 7.5 and 37 degrees C, although the enzyme was active in the pH range 6.0- 9.0 and at temperatures up to 45 degrees C. As expected for a serine-esterase, activity was inhibited by phenylmethylsulphonyl fluoride.

Amino Acid Sequence↗

PubMed2005

Diversity of bacteria growing in natural mineral water after bottling.

Bacterial growth occurs in noncarbonated natural mineral waters a few days after filling and storage at room temperature, a phenomenon known for more than 40 years. Using the full-cycle rRNA approach, we monitored the development of the planktonic bacterial community in a noncarbonated natural mineral water after bottling. Seven 16S rRNA gene libraries, comprising 108 clones in total, were constructed from water samples taken at various days after bottling and from two different bottle sizes. Sequence analyses identified 11 operational taxonomic units (OTUs), all but one affiliated with the betaproteobacterial order Burkholderiales (6 OTUs) or the class Alphaproteobacteria (4 OTUs). Fluorescence in situ hybridization (FISH) was applied in combination with DAPI (4',6'-diamidino-2-phenylindole) staining, viability staining, and microscopic counting to quantitatively monitor changes in bacterial community composition. A growth curve similar to that of a bacterium grown in a batch culture was recorded. In contrast to the current perception that Gammaproteobacteria are the most important bacterial components of natural mineral water in bottles, Betaproteobacteria dominated the growing bacterial community and accounted for 80 to 98% of all bacteria detected by FISH in the late-exponential and stationary-growth phases. Using previously published and newly designed genus-specific probes, members of the betaproteobacterial genera Hydrogenophaga, Aquabacterium, and Polaromonas were found to constitute a significant proportion of the bacterial flora (21 to 86% of all bacteria detected by FISH). For the first time, key genera responsible for bacterial growth in a natural mineral water were identified by applying molecular cultivation-independent techniques.

Bacteria↗

PubMed2002

Two non-complementing genes encoding enzymatically active methylenetetrahydrofolate reductases control methionine requirement in fission yeast Schizosaccharomyces pombe.

By transforming two methionine auxotrophic mutants from fission yeast Schizosaccharomyces pombe with a wild-type gene library, we defined two genes, met9 and met11, which both encode a methylenetetrahydrofolate reductase. The genes cannot complement each other. We detected single transcripts for both. In vitro measurements of enzymatic activities showed that the met11-encoded enzyme was responsible for only 15-20% of the total methylenetetrahydrofolate reductase activity. A strain in which gene met9 was disrupted required significantly more methionine for full growth and efficient mating and sporulation than the strain disrupted for gene met11. The in vitro and in vivo data thus indicated that met9 was the major expressed gene. Our results are in accordance with the assumption that the two methylenetetrahydrofolate reductases generate the methyl groups necessary for methionine synthetase to convert homocysteine to methionine, and suggest that expression of the two genes is an important parameter in the control of methionine biosynthesis.

Blotting, Northern↗