Presence of Two Different Active nirS Nitrite Reductase Genes in a Denitrifying Thauera sp. from a High-Nitrate-Removal-Rate Reactor

The nirS nitrite reductase genes were studied in two strains (strains 27 and 28) isolated from two denitrifying reactors and characterized as Thauera according to their 16S rRNA gene sequences. Strain 28 contains a single nirS sequence, which is related to the nirS of Thauera mechernichensis, and strain 27 contains two nirS sequences; one is similar to the nirS sequence from Thauera mechernichensis (gene 2), but the second one (gene 8) is from a separate clade with nirS from Pseudomonas stutzeri, Azoarcus species, Alcaligenes faecalis, and other Thauera species. Both genes were expressed, but gene 8 was constitutively expressed while gene 2 was positively regulated by nitrate.     

九龙江河口区nirS型反硝化细菌多样性及系统发育学分析

【目的】结合16S rRNA基因克隆文库和nirS基因克隆文库的分析,揭示九龙江河口区nirS型反硝化细菌多样性。【方法】选取九龙江河口区一富营养化采样点,分别采集水样及沉积物样品,进行理化因子的测定并提取细菌总DNA。以水样DNA构建16S rRNA基因克隆文库,以沉积物DNA构建nirS基因克隆文库,分析微生物群落结构的多样性并构建系统发育树。【结果】从16S rRNA基因克隆文库中获得86条有效序列,按97%的序列相似性划分为53个OTU,分别属于Proteobacteria门、Planctomycetes门、Bacteroidetes门、Actinobacteria门、Firmicutes门和Chloroflexi门。其中属于Proteobacteria门OTU的克隆子占克隆数的62.9%,是最优势的类群,分属于Alphaproteobacteria、Betaproteobacteria、Gammaproteobacteria和Deltaproteobacteria纲等。从nirS基因克隆文库中获得190条有效序列,翻译为氨基酸序列后,按82%的序列相似性划分为60个OTU,并定位到属的水平。其中Proteobacteria门是最优势的类群,占文库克隆子总数的71.6%,包括Alphaproteobacteria纲(5.8%)、Betaproteobacteria纲(49.0%)和Gammaproteobacteria纲(16.9%)。nirS基因克隆文库中丰度最高的OTU与GenBank中的一株可培养反硝化菌Thauera sp. R-26906具有100%的序列相似性。【结论】九龙江河口区的微生物以及亚硝酸盐还原酶基因(nirS)具有丰富的多样性。大部分NirS序列在GenBank中的最相似序列来源于河口、海湾等相似的环境。     

Analysis of nitrite reductase (nirK and nirS) genes and cultivation reveal depauperate community of denitrifying bacteria in the Black Sea suboxic zone

Chemical profiles of the Black Sea suboxic zone show a distribution of nitrogen species which is traditionally associated with denitrification, i.e. a secondary nitrite maximum associated with nitrate depletion and a N(2) gas peak. To better understand the distribution and diversity of the denitrifier community in the Black Sea suboxic zone, we combined a cultivation approach with cloning and sequencing of PCR-amplified nitrite reductase (nirS and nirK) genes. The Black Sea suboxic zone appears to harbour a homogeneous community of denitrifiers. For nirK, over 94% of the sequences fell into only three distinct phylogenetic clusters, and for nirS, a single closely related sequence type accounted for 91% of the sequences retrieved. Both nirS and nirK genes showed a dramatic shift in community composition at the bottom of the suboxic zone, but overall, nirK-based community composition showed much greater variation across depths compared with the highly uniform distribution of nirS sequences throughout the suboxic zone. The dominant nirK and nirS sequences differed at the amino acid level by at least 17% and 8%, respectively, from their nearest database matches. Denitrifying isolates recovered from the suboxic zone shared 97% 16S rRNA gene sequence similarity with Marinobacter maritimus. Analysis of the recently discovered nirS gene from the anammox bacterium Candidatus'Kuenenia stuttgartiensis' revealed that mismatches with commonly used primers may have prevented the previous detection of this divergent sequence.     

Characterization of a Bacteroides mobilizable transposon, NBU2, which carries a functional lincomycin resistance gene

The mobilizable Bacteroides element NBU2 (11 kbp) was found originally in two Bacteroides clinical isolates, Bacteroides fragilis ERL and B. thetaiotaomicron DOT. At first, NBU2 appeared to be very similar to another mobilizable Bacteroides element, NBU1, in a 2.5-kbp internal region, but further examination of the full DNA sequence of NBU2 now reveals that the region of near identity between NBU1 and NBU2 is limited to this small region and that, outside this region, there is little sequence similarity between the two elements. The integrase gene of NBU2, intN2, was located at one end of the element. This gene was necessary and sufficient for the integration of NBU2. The integrase of NBU2 has the conserved amino acids (R-H-R-Y) in the C-terminal end that are found in members of the lambda family of site-specific integrases. This was also the only region in which the NBU1 and NBU2 integrases shared any similarity (28% amino acid sequence identity and 49% sequence similarity). Integration of NBU2 was site specific in Bacteroides species. Integration occurred in two primary sites in B. thetaiotaomicron. Both of these sites were located in the 3' end of a serine-tRNA gene NBU2 also integrated in Escherichia coli, but integration was much less site specific than in B. thetaiotaomicron. Analysis of the sequence of NBU2 revealed two potential antibiotic resistance genes. The amino acid sequences of the putative proteins encoded by these genes had similarity to resistances found in gram-positive bacteria. Only one of these genes was expressed in B. thetaiotaomicron, the homolog of linA, a lincomycin resistance gene from Staphylococcus aureus. To determine how widespread elements related to NBU1 and NBU2 are in Bacteroides species, we screened 291 Bacteroides strains. Elements with some sequence similarity to NBU2 and NBU1 were widespread in Bacteroides strains, and the presence of linA(N) in Bacteroides strains was highly correlated with the presence of NBU2, suggesting that NBU2 has been responsible for the spread of this gene among Bacteroides strains. Our results suggest that the NBU-related elements form a large and heterogeneous family, whose members have similar integration mechanisms but have different target sites and differ in whether they carry resistance genes.     

The incidence of nirS and nirK and their genetic heterogeneity in cultivated denitrifiers

Gene sequence analysis of nirS and nirK, both encoding nitrite reductases, was performed on cultivated denitrifiers to assess their incidence in different bacterial taxa and their taxonomical value. Almost half of the 227 investigated denitrifying strains did not render an nir amplicon with any of five previously described primers. NirK and nirS were found to be prevalent in Alphaproteobacteria and Betaproteobacteria, respectively, nirK was detected in the Firmicutes and Bacteroidetes and nirS and nirK with equal frequency in the Gammaproteobacteria. These observations deviated from the hitherto reported incidence of nir genes in bacterial taxa. NirS gene phylogeny was congruent with the 16S rRNA gene phylogeny on family or genus level, although some strains did group within clusters of other bacterial classes. Phylogenetic nirK gene sequence analysis was incongruent with the 16S rRNA gene phylogeny. NirK sequences were also found to be significantly more similar to nirK sequences from the same habitat than to nirK sequences retrieved from highly related taxa. This study supports the hypothesis that horizontal gene transfer events of denitrification genes have occurred and underlines that denitrification genes should not be linked with organism diversity of denitrifiers in cultivation-independent studies.     

Mouse histone H2A and H2B genes: four functional genes and a pseudogene undergoing gene conversion with a closely linked functional gene.

The sequence of five mouse histone genes, two H2a and three H2b genes on chromosome 13 has been determined. The three H2b genes all code for different proteins, each differing in two amino acids from the others. The H2b specific elements present 5' to H2b genes from other species are present in all three mouse H2b genes. All three H2b genes are expressed in the same relative amounts in three different mouse cell lines and fetal mice. The H2b gene with the H2b specific sequence closest to the TATAA sequence is expressed in the highest amount. One of the H2a genes lacks the first 9 amino acids, the promoter region, the last 3 amino acids and contains an altered 3' end sequence. Despite these multiple defects, there is only one nucleotide change between the two H2a genes from codon 9 to 126. This indicates that a recent gene conversion has occurred between these two genes. The similarity of the nucleotide sequences in the coding regions of mouse histone genes is probably due to gene conversion events targeted precisely at the coding region.     

Two genes encoding ''minor'' legumin polypeptides in pea (Pisum sativum L.). Characterization and complete sequence of the LegJ gene.

A genomic clone from pea (Pisum sativum L.) contains all of one gene encoding a 'minor' (B-type) legumin polypeptide, and most of a second very similar gene. The two genes, designated LegJ and LegK, are arranged in tandem, separated by approx. 6 kb. A complete sequence of gene LegJ and its flanking sequences is given, with as much of the sequence of gene LegK as is present on the genomic clone. Hybridization of 3' flanking sequence probes to seed mRNA, and sequence comparisons with cDNA species, suggested that gene LegJ, and probably gene LegK, was expressed. The partial amino acid sequences of 'minor' legumin alpha- and beta-polypeptides were used to confirm the identity of these genes. The transciption start in gene LegJ was mapped. The 5' flanking sequence of gene LegJ contains a sequence conserved in legumin genes from pea and other species, which is likely to have functional significance in control of gene expression. Sequence comparisons with legumin genes and cDNA species from Vicia faba and soya bean show that separation of legumin genes into A- and B-type subfamilies occurred before separation of the Viciae and Glycinae tribes.     

Nitrite reductase genes (nirK and nirS) as functional markers to investigate diversity of denitrifying bacteria in pacific northwest marine sediment communities

Genetic heterogeneity of denitrifying bacteria in sediment samples from Puget Sound and two sites on the Washington continental margin was studied by PCR approaches amplifying nirK and nirS genes. These structurally different but functionally equivalent single-copy genes coding for nitrite reductases, a key enzyme of the denitrification process, were used as a molecular marker for denitrifying bacteria. nirS sequences could be amplified from samples of both sampling sites, whereas nirK sequences were detected only in samples from the Washington margin. To assess the underlying nir gene structure, PCR products of both genes were cloned and screened by restriction fragment length polymorphism (RFLP). Rarefraction analysis revealed a high level of diversity especially for nirS clones from Puget Sound and a slightly lower level of diversity for nirK and nirS clones from the Washington margin. One group dominated within nirK clones, but no dominance and only a few redundant clones were seen between sediment samples for nirS clones in both habitats. Hybridization and sequencing confirmed that all but one of the 228 putative nirS clones were nirS with levels of nucleotide identities as low as 45.3%. Phylogenetic analysis grouped nirS clones into three distinct subclusters within the nirS gene tree which corresponded to the two habitats from which they were obtained. These sequences had little relationship to any strain with known nirS sequences or to isolates (mostly close relatives of Pseudomonas stutzeri) from the Washington margin sediment samples. nirK clones were more closely related to each other than were the nirS clones, with 78.6% and higher nucleotide identities; clones showing only weak hybridization signals were not related to known nirK sequences. All nirK clones were also grouped into a distinct cluster which could not be placed with any strain with known nirK sequences. These findings show a very high diversity of nir sequences within small samples and that these novel nir clusters, some very divergent from known sequences, are not known in cultivated denitrifiers.     

Two gene families clustered in a small region of the Drosophila genome

Three Drosophila genes that are clustered within 8 X 10(3) bases of DNA at the chromosomal region 44D have been identified and mapped, and the gene cluster entirely sequenced. The three genes are 55 to 60% homologous in DNA sequence. One gene contains an intron in its 5'-proximal protein coding sequence while the other two have none at this position; similarly, another gene has an intron in its 3'-proximal protein coding sequence which is not found in the other genes. All three genes are abundantly expressed together in Drosophila first, second, and early third instar larval stages and in adults, but they are not abundantly expressed in either embryonic, late third instar larval, or pupal stages. This gene family lies 11 X 10(3) bases away from another cluster containing four Drosophila larval cuticle protein genes plus a pseudogene. The cuticle genes are all abundantly expressed throughout third instar larval development. Thus, at least seven protein-coding genes and one pseudogene lie within 27 X 10(3) bases of DNA. Moreover, two small gene families can lie adjacent on a chromosome and exhibit different patterns of developmental regulation, even though individual genes within each clustered family are co-ordinately expressed.     

Diversity in the Major Polysaccharide Antigen of Acinetobacter Baumannii Assessed by DNA Sequencing,and Development of a Molecular Serotyping Scheme

We have sequenced the gene clusters for type strains of the Acinetobacter baumannii serotyping scheme developed in the 1990s, and used the sequences to better understand diversity in surface polysaccharides of the genus. We obtained genome sequences for 27 available serovar type strains, and identified 25 polysaccharide gene cluster sequences. There are structures for 12 of these polysaccharides, and in general the genes present are appropriate to the structure where known. This greatly facilitates interpretation. We also find 53 different glycosyltransferase genes, and for 7 strains can provisionally allocate specific genes to all linkages. We identified primers that will distinguish the 25 sequence forms by PCR or microarray, or alternatively the genes can be used to determine serotype by “molecular serology”. We applied the latter to 190 Acinetobacter genome-derived gene-clusters, and found 76 that have one of the 25 gene-cluster forms. We also found novel gene clusters and added 52 new gene-cluster sequence forms with different wzy genes and different gene contents. Altogether, the strains that have one of the original 25 sequence forms include 98 A. baumannii (24 from our strains) and 5 A. nosocomialis (3 from our strains), whereas 32 genomes from 12 species other than A. baumannii or A. nosocomialis, all have new sequence forms. One of the 25 serovar type sequences is found to be in European clone I (EC I), 2 are in EC II but none in EC III. The public genome strains add an additional 52 new sequence forms, and also bring the number found in EC I to 5, in EC II to 9 and in EC III to 2.     

Relationship of human variable region heavy chain germ-line genes to genes encoding anti-DNA autoantibodies

In order to identify the V region genes encoding systemic lupus erythematosus (SLE)-derived anti-DNA autoantibodies, we have determined the nucleotide sequence of heavy chain mRNA from several DNA-binding immunoglobulins secreted by human hybridomas. We used the technique of cDNA primer extension for determining sequences of the VH, D, and JH gene segments of anti-DNA autoantibodies from three different primary hybridoma growths from an SLE patient and one hybridoma from a leprosy patient. Immunoglobulins from two of the SLE hybridomas expressed the same idiotype, Id-16/6, which is also expressed on immunoglobulins in sera of patients with active SLE. Their mRNA sequences showed complete homology to each other in the V, D, and J genes and more than 99% homology to the VH26 germ-line gene sequence, a member of the human VHIII gene family. The VH mRNA sequence of the third SLE hybridoma, 21/28, which was idiotypically unrelated to the other two, was 93% homologous to a different VH germ-line gene sequence, HA2, a member of the human VHI gene family. The fourth anti-DNA-producing hybridoma, 8E10, was derived from a leprosy patient of different ethnic origin than the SLE patient. It was idiotypically related to 21/28 and expressed a VH segment gene identical to that of 21/28. Hybridomas 21/28 and 8E10 shared sequence homology with the VH26 anti-DNA antibodies in the first complementarity-determining region. In addition, 21/28 shared sequence homology with the Id-16/6+ group in the region encoded by the D and J gene segments. Our findings indicate that some SLE autoantibodies are encoded by unmodified or scarcely modified VH germ-line genes that are conserved in the human population and identify two distinct VH germ-line genes that can encode segments of anti-DNA immunoglobulins.     

Diversity of nitrite reductase (nirK and nirS) gene fragments in forested upland and wetland soils

The genetic heterogeneity of nitrite reductase gene (nirK and nirS) fragments from denitrifying prokaryotes in forested upland and marsh soil was investigated using molecular methods. nirK gene fragments could be amplified from both soils, whereas nirS gene fragments could be amplified only from the marsh soil. PCR products were cloned and screened by restriction fragment length polymorphism (RFLP), and representative fragments were sequenced. The diversity of nirK clones was lower than the diversity of nirS clones. Among the 54 distinct nirK RFLP patterns identified in the two soils, only one pattern was found in both soils and in each soil two dominant groups comprised >35% of all clones. No dominance and few redundant patterns were seen among the nirS clones. Phylogenetic analysis of deduced amino acids grouped the nirK sequences into five major clusters, with one cluster encompassing most marsh clones and all upland clones. Only a few of the nirK clone sequences branched with those of known denitrifying bacteria. The nirS clones formed two major clusters with several subclusters, but all nirS clones showed less than 80% identity to nirS sequences from known denitrifying bacteria. Overall, the data indicated that the denitrifying communities in the two soils have many members and that the soils have a high richness of different nir genes, especially of the nirS gene, most of which have not yet been found in cultivated denitrifiers.     

Isolation, characterization, and distribution of denitrifying toluene degraders from a variety of habitats.

Enrichments capable of toluene degradation under O2-free denitrifying conditions were established with diverse inocula including agricultural soils, compost, aquifer material, and contaminated soil samples from different geographic regions of the world. Successful enrichment was strongly dependent on the initial use of relatively low toluene concentrations, typically 5 ppm. From the enrichments showing positive activity for toluene degradation, 10 bacterial isolates were obtained. Fingerprints generated by PCR-amplified DNA, with repetitive extragenic palindromic sequence primers, showed that eight of these isolates were different. Under aerobic conditions, all eight isolates degraded toluene, five degraded ethylbenzene, three consumed benzene, and one degraded chlorobenzene, meta-Xylene was the only other substrate used anaerobically and was used by only one isolate. All isolates were motile gram-negative rods, produced N2 from denitrification, and did not hydrolyze starch. All strains but one fixed nitrogen as judged by ethylene production from acetylene, but only four strains hybridized to the nifHDK genes. All strains appeared to have heme nitrite reductase since their DNA hybridized to the heme (nirS) but not to the Cu (nirU) genes. Five strains hybridized to a toluene ortho-hydroxylase catabolic probe, and two of those also hybridized to a toluene meta-hydroxylase probe. Partial sequences of the 16S rRNA genes of all isolates showed substantial similarity to 16S rRNA sequences of Azoarcus sp. Physiological, morphological, fatty acid, and 16S rRNA analyses indicated that these strains were closely related to each other and that they belong to the genus Azoarcus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Organization of the gene for gelatin-binding protein (GBP28)

GBP28 is a novel human plasma gelatin-binding protein that is encoded by apM1 mRNA, expressed specifically in adipose tissue. Three overlapping clones (two lambda clones and one BAC clone) containing the human plasma gelatin-binding protein (GBP28) gene were isolated and characterized. The GBP28 gene spans 16kb and is composed of three exons from 18bp to 4277bp in size with consensus splice sites. The sizes of the two introns were 0.8 and 12kb, respectively. The gene's regulatory sequences contain putative promoter elements, but no typical TATA box.The third exon of this gene contains a long 3'-untranslated sequence containing three Alu repeats. The exon-intron organization of this gene was very similar to that of obese gene, encoding leptin. We also report the chromosome mapping of this gene by fluorescence in situ hybridization (FISH) using a genomic DNA fragment as a probe. The GBP28 gene was located on human chromosome 3q27. The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession numbers ABO12163, ABO12164 or ABO12165.