Trichloroethene reductive dehalogenase from Dehalococcoides ethenogenes: sequence of tceA and substrate range characterization

The anaerobic bacterium Dehalococcoides ethenogenes is the only known organism that can completely dechlorinate tetrachloroethene or trichloroethene (TCE) to ethene via dehalorespiration. One of two corrinoid-containing enzymes responsible for this pathway, TCE reductive dehalogenase (TCE-RDase) catalyzes the dechlorination of TCE to ethene. TCE-RDase dehalogenated 1,2-dichloroethane and 1, 2-dibromoethane to ethene at rates of 7.5 and 30 micromol/min/mg, respectively, similar to the rates for TCE, cis-dichloroethene (DCE), and 1,1-DCE. A variety of other haloalkanes and haloalkenes containing three to five carbon atoms were dehalogenated at lower rates. The gene encoding TCE-RDase, tceA, was cloned and sequenced via an inverse PCR approach. Sequence comparisons of tceA to proteins in the public databases revealed weak sequence similarity confined to the C-terminal region, which contains the eight-iron ferredoxin cluster binding motif, (CXXCXXCXXXCP)(2). Direct N-terminal sequencing of the mature enzyme indicated that the first 42 amino acids constitute a signal sequence containing the twin-arginine motif, RRXFXK, associated with the Sec-independent membrane translocation system. This information coupled with membrane localization studies indicated that TCE-RDase is located on the exterior of the cytoplasmic membrane. Like the case for the two other RDases that have been cloned and sequenced, a small open reading frame, tceB, is proposed to be involved with membrane association of TCE-RDase and is predicted to be cotranscribed with tceA.     

Cloning and sequencing of the beta-lactamase gene and surrounding DNA sequences of Citrobacter braakii,Citrobacter murliniae,Citrobacter werkmanii,Escherichia fergusonii and Enterobacter cancerogenus

To further identify the origins of plasmid-mediated cephalosporinases that are currently spreading worldwide, the chromosomal beta-lactamase genes of Citrobacter braakii, Citrobacter murliniae, Citrobacter werkmanii reference strains and of Escherichia fergusonii and Enterobacter cancerogenus clinical isolates were cloned and expressed into Escherichia coli and sequenced. These beta-lactamases had all a single pI value >8 and conferred a typical AmpC-type resistance pattern in E. coli recombinant strains. The cloned inserts obtained from genomic DNAs of each strain encoded Ambler class C beta-lactamases. The AmpC-type enzymes of C. murliniae, C. braakii and C. werkmanii shared 99%, 96% and 95% amino acid sequence identity, respectively, with chromosomal AmpC beta-lactamases from Citrobacter freundii. The AmpC-type enzyme of E. cancerogenus shared 85% amino acid sequence identity with the chromosomal AmpC beta-lactamase of Enterobacter cloacae OUDhyp and the AmpC-type enzyme of E. fergusonii shared 96% amino acid sequence identity with that of E. coli K12. The ampC genes, except for E. fergusonii, were associated with genes homologous to regulatory ampR genes of other chromosomal class C beta-lactamases that explain inducibility of beta-lactamase expression in these strains. This work provides further evidence of the molecular heterogeneity of class C beta-lactamases.     

Indications for acquisition of reductive dehalogenase genes through horizontal gene transfer by Dehalococcoides ethenogenes strain 195

The genome of Dehalococcoides ethenogenes strain 195, an anaerobic dehalorespiring bacterium, contains 18 copies of putative reductive dehalogenase genes, including the well-characterized tceA gene, whose gene product functions as the key enzyme in the environmentally important dehalorespiration process. The genome of D. ethenogenes was analyzed using a bioinformatic tool based on the frequency of oligonucleotides. The results in the form of a genomic signature revealed several local disruptions of the host signature along the genome sequence. These fractures represent DNA segments of potentially foreign origin, so-called atypical regions, which may have been acquired by an ancestor through horizontal gene transfer. Most interestingly, 15 of the 18 reductive dehalogenase genes, including the tceA gene, were found to be located in these regions, strongly indicating the foreign nature of the dehalorespiration activity. The GC content and the presence of recombinase genes within some of these regions corroborate this hypothesis. A hierarchical classification of the atypical regions containing the reductive dehalogenase genes indicated that these regions were probably acquired by several gene transfer events.     

Escherichia coli chromosomal mutations that permit direct cloning of the Bacteroides fragiiis metallo-β-lactamase gene, ccrA

The class B, metallo-beta-lactamase genes ccrA (carbapenem- and cephamycin resistance) from three Bacteroides fragilis isolates--QMCN3, QMCN4, and TAL3636--were cloned and expressed in Escherichia coli. Cloning of the genes, by selecting for ampicillin resistance, was facilitated by two classes of Escherichia coli chromosomal mutations which resulted in at least a 5-10-fold increase in metallo-beta-lactamase enzymatic activity. The observed increase in enzymatic activity is due to either increased translation of the ccrA gene or an effect on localization or stability of the protein. Comparison of the DNA sequences of the three ccrA genes revealed that their protein-coding sequences shared greater than 97% DNA sequence identity. However, the 5' upstream sequence for the TAL3636 ccrA gene was unrelated to that of the other two genes.     

Two-domain hemoglobin gene of the water flea Moina macrocopa: duplication in the ancestral Cladocera, diversification, and loss of a bridge intron

Two cDNAs encoding the two-domain hemoglobin (Hb) chains of a crustacean Cladocera, Moina macrocopa, were cloned and their nucleotide (nt) sequences were determined. The amino acid (aa) sequences of both the gene products deduced from the nt sequences consisted of 348 residues and showed 98% identity with each other. These sequences together with the NH(2)-terminal aa sequences of the Hb chains determined after separation by two-dimensional gel electrophoresis showed that the Hb chains are synthesized as a secretory precursor with a signal peptide of 17 aa residues. The aa sequences of M. macrocopa Hb chains shared the following features with those of Daphnia Hb chains. Firstly, the signal peptide is followed by an NH(2)-terminal extension containing a threonine-rich sequence that might play a role in the multimerization of subunit chains. Secondly, the identity between the aa sequences of the first and second domains is exceptionally low. These facts suggest that duplication of the cladoceran Hb gene occurred before the divergence of families Moinidae and Daphniidae. Analysis of genomic DNA showed that the M. macrocopa Hb genes consist of two large repeated regions, encoding the first and second domains of Hb chains, respectively. The intron-exon organization of the first region of the M. macrocopa Hb genes was similar to that found in the Daphnia Hb genes, having the three-exon, two-intron structure characteristic of animal Hb genes. However, the intron bridging the two regions and the most downstream intron in the second region were missing in the Moina genes, providing a new example of intron loss. The following elements in the 5' flanking region were conserved in the Moina and Daphnia genes: (1) TATAAA, a typical TATA box sequence accompanied by a downstream sequence, GAAXAGCATCAGTT (the fourth residue X was G or A in Daphnia and absent in Moina); (2) CCAAT boxes, located upstream of the TATA box; (3) the binding sites for HIF-1 and GATA-1, also located upstream of the TATA box, that may be responsible for up-regulation of the cladoceran Hb genes under hypoxia.     

Comparative genomics of the T4-Like Escherichia coli phage JS98: implications for the evolution of T4 phages

About 130 kb of sequence information was obtained from the coliphage JS98 isolated from the stool of a pediatric diarrhea patient in Bangladesh. The DNA shared up to 81% base pair identity with phage T4. The most conserved regions between JS98 and T4 were the structural genes, but their degree of conservation was not uniform. The head genes showed the highest sequence conservation, followed by the tail, baseplate, and tail fiber genes. Many tail fiber genes shared only protein sequence identity. Except for the insertion of endonuclease genes in T4 and gene 24 duplication in JS98, the structural gene maps of the two phages were colinear. The receptor-recognizing tail fiber proteins gp37 and gp38 were only distantly related to T4, but shared up to 83% amino acid identity to other T6-like phages, suggesting lateral gene transfer. A greater degree of variability was seen between JS98 and T4 over DNA replication and DNA transaction genes. While most of these genes came in the same order and shared up to 76% protein sequence identity, a few rearrangements, insertions, and replacements of genes were observed. Many putative gene insertions in the DNA replication module of T4 were flanked by intron-related endonuclease genes, suggesting mobile DNA elements. A hotspot of genome diversification was located downstream of the DNA polymerase gene 43 and the DNA binding gene 32. Comparative genomics of 100-kb genome sequence revealed that T4-like phages diversify more by the accumulation of point mutations and occasional gene duplication events than by modular exchanges.     

Positional gene enrichment analysis of gene sets for high-resolution identification of overrepresented chromosomal regions

The search for feature enrichment is a widely used method to characterize a set of genes. While several tools have been designed for nominal features such as Gene Ontology annotations or KEGG Pathways, very little has been proposed to tackle numerical features such as the chromosomal positions of genes. For instance, microarray studies typically generate gene lists that are differentially expressed in the sample subgroups under investigation, and when studying diseases caused by genome alterations, it is of great interest to delineate the chromosomal regions that are significantly enriched in these lists. In this article, we present a positional gene enrichment analysis method (PGE) for the identification of chromosomal regions that are significantly enriched in a given set of genes. The strength of our method relies on an original query optimization approach that allows to virtually consider all the possible chromosomal regions for enrichment, and on the multiple testing correction which discriminates truly enriched regions versus those that can occur by chance. We have developed a Web tool implementing this method applied to the human genome (http://www.esat.kuleuven.be/~bioiuser/pge). We validated PGE on published lists of differentially expressed genes. These analyses showed significant overrepresentation of known aberrant chromosomal regions.     

Comparative sequence analysis of the sorghum Rph region and the maize Rp1 resistance gene complex

A 268-kb chromosomal segment containing sorghum (Sorghum bicolor) genes that are orthologous to the maize (Zea mays) Rp1 disease resistance (R) gene complex was sequenced. A region of approximately 27 kb in sorghum was found to contain five Rp1 homologs, but most have structures indicating that they are not functional. In contrast, maize inbred B73 has 15 Rp1 homologs in two nearby clusters of 250 and 300 kb. As at maize Rp1, the cluster of R gene homologs is interrupted by the presence of several genes that appear to have no resistance role, but these genes were different from the ones found within the maize Rp1 complex. More than 200 kb of DNA downstream from the sorghum Rp1-orthologous R gene cluster was sequenced and found to contain many duplicated and/or truncated genes. None of the duplications currently exist as simple tandem events, suggesting that numerous rearrangements were required to generate the current genomic structure. Four truncated genes were observed, including one gene that appears to have both 5' and 3' deletions. The maize Rp1 region is also unusually enriched in truncated genes. Hence, the orthologous maize and sorghum regions share numerous structural features, but all involve events that occurred independently in each species. The data suggest that complex R gene clusters are unusually prone to frequent internal and adjacent chromosomal rearrangements of several types.     

Two plus two does not equal three: statistical tests for multiple genome comparison

Gene clusters that span three or more chromosomal regions are of increasing importance, yet statistical tests to validate such clusters are in their infancy. Current approaches either conduct several pairwise comparisons or consider only the number of genes that occur in all of the regions. In this paper, we provide statistical tests for clusters spanning exactly three regions based on genome models of typical comparative genomics problems, including analysis of conserved linkage within multiple species and identification of large-scale duplications. Our tests are the first to combine evidence from genes shared among all three regions and genes shared between pairs of regions. We show that our tests of clusters spanning three regions are more sensitive than existing approaches, and can thus be used to identify more diverged homologous regions.     

Structure and expression of the U5 snRNA gene of Arabidopsis thaliana. Conserved upstream sequence elements in plant U-RNA genes.

We have previously characterized the U2 small nuclear (sn) RNA gene family of Arabidopsis thaliana. To find out the structural features of upstream and downstream non-coding regions that are shared by different U-RNA genes in higher plants we have isolated the gene encoding a 125 nt-long U5 snRNA of Arabidopsis. Activity of the cloned gene was demonstrated in stably transformed tobacco calli and by transient expression in transfected protoplasts of Nicotiana plumbaginifolia. Southern analysis indicated that the Arabidopsis genome contains 8-9 copies of the U5 gene. Alignment of upstream non-coding regions revealed two elements conserved between all plant U-RNA genes characterized so far: the sequence RTCCCACATCG (-70/-80 region, 100% conservation) and the TATA homology around position -30. The coding regions in all genes are followed by the sequence CAN4-9AGTN (A/T)AA which may correspond to a termination and/or processing signal.     

Distinct control sites located upstream from the levansucrase gene of Bacillus subtilis

The sacR regulatory region, which modulates the expression of sacB, the structural gene for levansucrase, was separated into two parts: an upstream region which carries a constitutive promoter and a downstream region which carries a palindromic structure. Three types of fusions were constructed in which the aphA3 gene coding for kanamycin resistance of Streptococcus faecalis was placed downstream from different deleted sacR regions. Other fusions were constructed by inserting a promoter from phage SPO1 upstream from the sacB gene and part of the sacA region. A third kind of fusion was constructed in which the palindromic structure was flanked by a heterologous promoter and a heterologous structural gene. After introduction of these fusions into the chromosomal DNA of mutants affected in sacB regulation, it was possible to reveal different targets for the regulatory genes sacU, sacQ and sacS: the sacU and sacQ genes act on a region located near or just upstream from the promoter, and the sacS gene, which is involved in the induction process, acts on the palindromic structure.     

tRNA gene identity affects nuclear positioning

The three-dimensional organization of genomes is dynamic and plays a critical role in the regulation of cellular development and phenotypes. Here we use proximity-based ligation methods (i.e. chromosome conformation capture [3C] and circularized chromosome confrmation capture [4C]) to explore the spatial organization of tRNA genes and their locus-specific interactions with the ribosomal DNA. Directed replacement of one lysine and two leucine tRNA loci shows that tRNA spatial organization depends on both tRNA coding sequence identity and the surrounding chromosomal loci. These observations support a model whereby the three-dimensional, spatial organization of tRNA loci within the nucleus utilizes tRNA gene-specific signals to affect local interactions, though broader organization of chromosomal regions are determined by factors outside the tRNA genes themselves.     

The<Emphasis Type="Italic">Escherichia fergusonii iucABCD iutA</Emphasis> genes are located within a larger chromosomal region similar to pathogenicity islands

Three strains of Escherichia fergusonii (EF873, EF1496, EF939) of 50 strains tested produced the hydroxamate siderophore aerobactin. Screening of a cosmid library of the strain EF873 chromosomal DNA (in aerobactin nonproducing Escherichia coli VCS257) for aerobactin production identified iucABCD and iutA gene orthologues. The predicted IucABCD and IutA proteins showed 59-65% identity to the corresponding proteins of Shigella flexneri and E. coli. Aerobactin molecules synthesized by E. fergusonii and E. coli strains stimulated growth of aerobactin indicator strains harboring either E. coli or E. fergusonii iutA genes. In the 12 kb upstream and 17 kb downstream regions of the iuc and iut genes, 20 additional ORFs were identified. Their gene products showed homology to proteins from E. coli, S. flexneri, Klebsiella aerogenes, Pseudomonas aeruginosa and Vibrio cholerae. Probes recognizing DNA sequences from a region of more than 25 kb, which included the iucABCD and iutA genes, hybridized with chromosomal DNA of two aerobactin-producing strains (EF873 and EF939), but not with other nonproducing E. fergusonii strains tested. These data, together with the genetic organization of this region, suggest that E. fergusonii iucABCD iutA genes are a portion of a larger segment of DNA similar to pathogenicity islands of other bacteria.