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PubMed2005

Characterisation of the nitrile hydratase gene clusters of Rhodococcus erythropolis strains AJ270 and AJ300 and Microbacterium sp. AJ115 indicates horizontal gene transfer and reveals an insertion of IS1166.

The nitrile metabolising strains AJ270, AJ300 and AJ115 were isolated from the same location. The strains have very similar nitrile metabolising profiles. Sequencing of the 16S rRNA gene indicates that strains AJ270 and AJ300 are novel strains of Rhodococcus erythropolis while strain AJ115 is a novel Microbacterium strain very closely related to Microbacterium oxydans and Microbacterium liquefaciens. Analysis of the structure of the nitrile hydratase/amidase gene clusters in the three strains indicates that this region is identical in these strains and that this structure is different to other nitrile hydratase/amidase gene clusters. The major difference seen is the insertion of a complete copy of the insertion sequence IS1166 in the nhr2 gene. This copy of IS1166 generates a 10 bp direct duplication at the point of insertion and has one ORF encoding a protein of 434 amino acids, with 98% homology to the transposase of IS666 from Mycobacterium avium. A gene oxd, encoding aldoxime dehydratase is found upstream of the nitrile hydratase gene cluster and an open reading frame encoding a protein with homology to GlnQ type ABC transporters is found downstream of the nitrile hydratase/amidase genes. The identity of the nitrile hydratase/amidase gene clusters in the three strains suggests horizontal gene transfer of this region. Analysis of the strains for both linear and circular plasmids indicates that both are present in the strains but hybridisation studies indicate that the nitrile hydratase/amidase gene cluster is chromosomally located. The nitrile hydratase/amidase enzymes of strain AJ270 are inducible with acetonitrile or acetamide. Interestingly although a number of Fe-type nitrile hydratases have been shown to be photosensitive, the enzyme from strain AJ270 is not.

ATP-Binding Cassette Transporters↗

PubMed2003

Evolutionary implications of microbial genome tetranucleotide frequency biases.

We compared nucleotide usage pattern conservation for related prokaryotes by examining the representation of DNA tetranucleotide combinations in 27 representative microbial genomes. For each of the organisms studied, tetranucleotide usage departures from expectations (TUD) were shared between related organisms using both Markov chain analysis and a zero-order Markov method. Individual strains, multiple chromosomes, plasmids, and bacteriophages share TUDs within a species. TUDs varied between coding and noncoding DNA. Grouping prokaryotes based on TUD profiles resulted in relationships with important differences from those based on 16S rRNA phylogenies, which may reflect unequal rates of evolution of nucleotide usage patterns following divergence of particular organisms from a common ancestor. By both symmetrical tree distance and likelihood analysis, phylogenetic trees based on TUD profiles demonstrate a level of congruence with 16S rRNA trees similar to that of both RpoA and RecA trees. Congruence of these trees indicates that there exists phylogenetic signal in TUD patterns, most prominent in coding region DNA. Because relationships demonstrated in TUD-based analyses utilize whole genomes, they should be considered complementary to phylogenies based on single genetic elements, such as 16S rRNA.

Chromosome Mapping↗

PubMed1992

Behavior in agricultural soils of a recombinant Pseudomonas bacterium that simultaneously degrades alkyl- and haloaromatics.

Pseudomonas sp. FR1 (pFRC20P) is a recombinant bacterium able simultaneously to degrade alkyl- and haloaromatics due to the xylXYZL and xylS genes from the TOL plasmid borne on the bacterial chromosome, and to the gene encoding for a gamma-lactone isomerase from Alcaligenes eutrophus on pFRC20P. The survival of this strain in sterile soils was shown to depend on the physicochemical properties of the soil. The recombinant information was stable in bacteria introduced in soils and conferred selective advantage to the host bacterium when the soils were amended with low amounts of p-methylbenzoate. However, relatively high amounts of this chemical significantly reduced survival of the bacterium. Survival was more seriously affected by chlorobenzoates than by methylbenzoates. Evolution of 14CO2 from p-methyl-14C-benzoate in soils confirmed that Pseudomonas sp. FR1 (pFRC20P) functions in a complex system in accordance with its design. Survival of Pseudomonas sp. FR1 (pFRC20P) was better at 4 degrees C and 25 degrees C than at 37 degrees C. On plates, the pFRC20P plasmid was mobilized by helper plasmids to P. putida at a frequency of about 10(-6) transconjugants per recipient. However, no transfer of recombinant DNA integrated on the chromosome was found, nor was lateral transfer of the recombinant DNA borne by Pseudomonas sp. FR1 (pFRC20P) detected in soils.

Benzoates↗

PubMed2002

Typing of Yersinia pestis isolates from the state of Ceará, Brazil.

AIMS: To investigate whether modifications in Yersinia pestis isolates from three plague foci from the state of Ceará, Brazil, had occurred over the years as a consequence of genetic adaptation to the environment. METHODS AND RESULTS: The isolates were studied with respect to susceptibility to antimicrobial drugs, plasmid and protein profiling, pigmentation on Congo red-agar plates, and the presence of some pathogenicity genes using PCR. Most of the expected virulence markers were detected in the cultures examined. There was no evidence of any alteration that could be associated with their origin (patients, rodents and fleas) or period of isolation (1971-1997). CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Phenotypic or genotypic changes were not detected in the cultures examined. However, the results obtained will serve as a reference to follow the evolution of Y. pestis in these foci.

Animals↗

PubMed2006

Probing genomic diversity and evolution of Escherichia coli O157 by single nucleotide polymorphisms.

Infections by Shiga toxin-producing Escherichia coli O157:H7 (STEC O157) are the predominant cause of bloody diarrhea and hemolytic uremic syndrome in the United States. In silico comparison of the two complete STEC O157 genomes (Sakai and EDL933) revealed a strikingly high level of sequence identity in orthologous protein-coding genes, limiting the use of nucleotide sequences to study the evolution and epidemiology of this bacterial pathogen. To systematically examine single nucleotide polymorphisms (SNPs) at a genome scale, we designed comparative genome sequencing microarrays and analyzed 1199 chromosomal genes (a total of 1,167,948 bp) and 92,721 bp of the large virulence plasmid (pO157) of eleven outbreak-associated STEC O157 strains. We discovered 906 SNPs in 523 chromosomal genes and observed a high level of DNA polymorphisms among the pO157 plasmids. Based on a uniform rate of synonymous substitution for Escherichia coli and Salmonella enterica (4.7x10(-9) per site per year), we estimate that the most recent common ancestor of the contemporary beta-glucuronidase-negative, non-sorbitolfermenting STEC O157 strains existed ca. 40 thousand years ago. The phylogeny of the STEC O157 strains based on the informative synonymous SNPs was compared to the maximum parsimony trees inferred from pulsed-field gel electrophoresis and multilocus variable numbers of tandem repeats analysis. The topological discrepancies indicate that, in contrast to the synonymous mutations, parts of STEC O157 genomes have evolved through different mechanisms with highly variable divergence rates. The SNP loci reported here will provide useful genetic markers for developing high-throughput methods for fine-resolution genotyping of STEC O157. Functional characterization of nucleotide polymorphisms should shed new insights on the evolution, epidemiology, and pathogenesis of STEC O157 and related pathogens.

Chromosome Mapping↗

PubMed1997

Generation of large libraries of random mutants in Bacillus subtilis by PCR-based plasmid multimerization.

We describe a PCR-based method for the generation of plasmid multimers that can be directly transformed into Bacillus subtilis with very high efficiency. This technique is particularly useful for the generation of large libraries of randomly mutagenized genes, which are required for the optimization of enzymes by directed evolution. We subjected the gene coding for the protease subtilisin to six consecutive rounds of PCR at three different levels of mutagenicity. The resulting 18 populations were cloned using our PCR multimerization protocol, and the mutation frequencies were determined by DNA sequencing. The resulting data demonstrate that the mutation frequency during PCR can be controlled by adding varying concentrations of manganese chloride to the reaction mixture. We observed a bias in the type of base pair changes with A and T being mutated much more frequently than C and G. We determined the fraction of active clones in all populations and found that its natural logarithm is proportional to the average mutation frequency of the populations. These data reveal that a fraction of about 0.27 of all possible mutations leads to the inactivation of the subtilisin gene, which provides a measure for its structural plasticity.

Bacillus subtilis↗

PubMed2001

Identification and characterization of variable-number tandem repeats in the Yersinia pestis genome.

Yersinia pestis, the infamous plague-causing pathogen, appears to have emerged in relatively recent history. Evidence of this fact comes from several studies that document a lack of nucleotide diversity in the Y. pestis genome. In contrast, we report that variable-number tandem repeat (VNTR) sequences are common in the Y. pestis genome and occur frequently in gene coding regions. Larger tandem repeat arrays, most useful for phylogenetic analysis, are present at an average of 2.18 arrays per 10 kbp and are distributed evenly throughout the genome and the two virulence plasmids, pCD1 and pMT1. We examined allelic diversity at 42 chromosomal VNTR loci in 24 selected isolates (12 globally distributed and 12 from Siskiyou County, Calif.). Vast differences in diversity were observed among the 42 VNTR loci, ranging from 2 to 11 alleles. We found that the maximum copy number of repeats in an array was highly correlated with diversity (R = 0.86). VNTR-based phylogenetic analysis of the 24 strains successfully grouped isolates from biovar orientalis and most of the antiqua and mediaevalis strains. Hence, multiple-locus VNTR analysis (MLVA) appears capable of both distinguishing closely related strains and successfully classifying more distant relationships. Harnessing the power of MLVA to establish standardized databases will enable researchers to better understand plague ecology and evolution around the world.

Animals↗

PubMed1997

Lamprey fructose-1,6-bisphosphate aldolase: characterization of the muscle-type and non-muscle-type isozymes.

To study evolutionary aspects of fructose-1,6-bisphosphate (Fru-1,6-P2) aldolase during deuterostomian evolution, we have purified and characterized aldolases from the muscle and liver of lamprey (Entosphenus japonicus). Aldolase from the skeletal muscle and liver was identified to be the muscle-type isozyme and the non-muscle-type isozyme that was encoded by cDNAs M8 and L3, respectively, as described previously (Zhang, R., Yatsuki, H., Kusakabe, T., Iwabe, Miyata, T., Imai, T., Yoshida, M., and Hori, K., J. Biochem. 117, 545-553, 1995). The muscle-type isozyme has properties similar to vertebrate aldolase A, while the non-muscle-type isozyme shows a similarity to bacterial class I aldolase and vertebrate aldolase C but not to aldolase B, the liver-type aldolase, in terms of kinetic parameters: the Kcat values toward Fru-1,6-P2 and Fru-1-P, the Fru-1,6-P2/Fru-1-P activity ratio, and the Km values toward these substrates. The two enzymes have tetrameric forms with a molecular mass of approximately 160,000 and have similar pH optimum. The muscle-type and non-muscle-type isozymes from the tissues show different electrophoretic mobility; the muscle-type isozyme moves much faster than the non-muscle-type isozyme toward anodic side. The recombinant muscle-type and non-muscle-type aldolases gave similar characteristics as those from the tissues. The results presented in this paper, together with the data presented in the previous paper, strongly suggest that in lamprey it is possible to have two types of aldolase isozymes rather than one or three isozymes.

Amino Acid Sequence↗

PubMed2002

Cardiac-specific activity of an Nkx2-5 enhancer requires an evolutionarily conserved Smad binding site.

Heart formation in vertebrates and fruit flies requires signaling by bone morphogenetic proteins (BMPs) to cardiogenic mesodermal precursor cells. The vertebrate homeobox gene Nkx2-5 and its Drosophila ortholog, tinman, are the earliest known markers for the cardiac lineage. Transcriptional activation of tinman expression in the cardiac lineage is dependent on a mesoderm-specific enhancer that binds Smad proteins, which activate transcription in response to BMP signaling, and Tinman, which maintains its own expression through an autoregulatory loop. Here, we show that an evolutionarily conserved, cardiac-specific enhancer of the mouse Nkx2-5 gene contains multiple Smad binding sites, as well as a binding site for Nkx2-5. A single Smad site is required for enhancer activity at early and late stages of heart development in vivo, whereas the Nkx2-5 site is not required for enhancer activity. These findings demonstrate that Nkx2-5, like tinman, is a direct target for transcriptional activation by Smad proteins; however, the independence of this Nkx2-5 enhancer of Nkx2-5 binding suggests a fundamental difference in the transcriptional circuitry for activation of Nkx2-5 and tinman expression during cardiogenesis in vertebrates and fruit flies.

5' Untranslated Regions↗

PubMed1989

Structure of the multigene family of MAL loci in Saccharomyces.

Multigene families are a ubiquitous feature of eukaryotes; however, their presence in Saccharomyces is more limited. The MAL multigene family is comprised of five unlinked loci, MAL1, MAL2, MAL3, MAL4 and MAL6, any one of which is sufficient for yeast to metabolize maltose. A cloned MAL6 locus was used as a probe to facilitate the cloning of the other four functional loci as well as two partially active alleles of MAL1. Each locus could be characterized as a cluster of three genes, MALR (regulatory), MALT (maltose transport or permease) and MALS (structural or maltase), encoded by a total of about 7 kb of DNA; however, homologous sequences at each locus extend beyond the coding regions. Our results indicate that there is extensive homology among the MAL loci, especially within their maltase genes. The greatest sequence diversity occurs in their regulatory gene regions. Southern cross analyses of the cloned MAL loci indicate a single duplication of the MAL6R-homologous sequences upstream of the MAL6R gene as well as an extensive duplication of more than 10 kb at the MAL3 locus. The large repeat at the MAL3 locus results in the presence of four copies of MAL3R-homologous sequences and two copies of MAL3T-homologous sequences at that locus. Two naturally occurring inactive alleles of MAL1 show a deletion or divergence of their MALR sequences. The significance of these repeats in the evolution of the MAL multigene family is discussed.

Biological Evolution↗

PubMed1994

Functional and molecular analysis of liver arginase promoter sequences from man and Macaca fascicularis.

Functional and DNA binding analyses were used to investigate transcriptional regulation of liver arginase, a mammalian urea cycle enzyme with marked tissue specificity. Reporter constructs containing the proximal 111 bp of the gene from man and Macaca fascicularis showed over sixfold background activity in HepG2 hepatoma cells, which express significant levels of liver arginase, and 12-fold background activity in minimally expressing HEK cells. Longer constructs, active in both cell lines, showed greater activity in the liver cell line. The constructs showed no activity in arginase-negative NIH 3T3 fibroblasts. A 54-bp dyad insert present in the human sequence and absent in M. fascicularis did not affect function. DNA binding analyses localized multiple liver-specific complexes as well as complexes shared among cell types. Little binding was evident in fibroblast extracts. Despite liver-specific binding, there was no evidence of a strong liver-specific enhancer. HEK and NIH 3T3 nuclear extracts showed strikingly different patterns of DNA binding. These studies demonstrate that molecular regulation of liver arginase transcription is complex and that control mechanisms differ among tissue types.

3T3 Cells↗

PubMed1992

The integron In1 in plasmid R46 includes two copies of the oxa2 gene cassette.

The sequence of the insert region of the integron In1 found in the IncN plasmid R46 was completed. The insert region is 2929 bases long and includes four gene cassettes, two of which are identical copies of the oxa2 gene cassette flanking an aadA1 cassette. The fourth cassette encodes an open reading frame orfD. From comparison of these data with published maps and sequences it is argued that the integrons found in the IncN plasmids pCU1 and R1767 and in the transposon Tn2410 are closely related to In1 from R46. Both site-specific gene insertion and recA-dependent recombination are likely to have contributed to the evolution of these integrons.

Amino Acid Sequence↗

PubMed2004

Molecular evolution of the ATPase subunit of three archaeal sugar ABC transporters.

The sequence of genes encoding extracellular amylopullulanase and putative maltodextrin ATP binding cassette (ABC) transporter of a hyperthermophilic archaeon, Thermococcus litoralis, was determined. The mdxK gene, which encodes an ATPase subunit of the putative maltodextrin ABC transporter, has extraordinarily high similarity with the malK gene, which encodes an ATPase subunit of trehalose/maltose ABC transporter of the same organism. DNA sequence comparison revealed that the malK gene was generated through the duplication of the mdxK gene before lateral gene transfer of the mal gene cluster from T. litoralis to Pyrococcus furiosus.

ATP-Binding Cassette Transporters↗

PubMed1998

LTR and tat variability of HIV-1 isolates from patients with divergent rates of disease progression.

The genetic heterogeneity and transcription activity of the human immunodeficiency virus type 1 (HIV-1) LTR region and tat gene have been examined. Comparison involved the relevant genomic regions of viruses isolated from twenty long-term survivors and from ten typical progressors. No significant differences were observed in mutation frequencies among the two groups, although there was a significant higher proportion of synonymous substitutions in the tat gene of viruses from typical progressors. Four LTR sequences showed an insertion of 20-31 residues at the junction between the LTR Nef-coding and the LTR noncoding region. Neither these insertions nor other genetic changes found in these sequences affected the LTR transcription function, as measured in transient expression assays using transfection of both established cell lines and peripheral blood lymphocytes with plasmid DNA. The results did not allow the association of structural or functional alterations in LTR or tat with a degree of disease progression. The results reinforce the concepts of complexity of HIV-1 evolution in infected individuals, and the multifactorial nature of progression to AIDS.

Adult↗

PubMed2003

A despecialization step underlying evolution of a family of serine proteases.

In the trypsin superfamily of serine proteases, non-trypsin-like primary specificities have arisen in only two monophyletic descendent subbranches. We have recreated an ancestor to one of these subbranches (granzyme) using phylogenetic inference, gene synthesis, and protein expression. This ancestor has two unusual properties. First, it has broad primary specificity encompassing the entire repertoire of novel primary specificities found in its descendents. Second, unlike extant members that have narrow primary specificities, the ancestor exhibits tolerance to mutational changes in primary specificity-conferring residues-that is, structural plasticity. Molecular modeling and mutagenesis studies indicate that these unusual properties are due to a particularly wide substrate binding pocket. These two crucial properties of the ancestor not only distinguish it from its extant descendents but also from the trypsin-like proteases that preceded it. This indicates that a despecialization step, characterized by broad specificity and structural plasticity, underlies evolution of new primary specificities in this protease superfamily.

Amino Acid Sequence↗

PubMed1994

Emergence of a replicating species from an in vitro RNA evolution reaction.

The technique of self-sustained sequence replication allows isothermal amplification of DNA and RNA molecules in vitro. This method relies on the activities of a reverse transcriptase and a DNA-dependent RNA polymerase to amplify specific nucleic acid sequences. We have modified this protocol to allow selective amplification of RNAs that catalyze a particular chemical reaction. During an in vitro RNA evolution experiment employing this modified system, a unique class of "selfish" RNAs emerged and replicated to the exclusion of the intended RNAs. Members of this class of selfish molecules, termed RNA Z, amplify efficiently despite their inability to catalyze the target chemical reaction. Their amplification requires the action of both reverse transcriptase and RNA polymerase and involves the synthesis of both DNA and RNA replication intermediates. The proposed amplification mechanism for RNA Z involves the formation of a DNA hairpin that functions as a template for transcription by RNA polymerase. This arrangement links the two strands of the DNA, resulting in the production of RNA transcripts that contain an embedded RNA polymerase promoter sequence.

Avian Myeloblastosis Virus↗

PubMed2000

CyaG, a novel cyanobacterial adenylyl cyclase and a possible ancestor of mammalian guanylyl cyclases.

A novel gene encoding an adenylyl cyclase, designated cyaG, was identified in the filamentous cyanobacterium Spirulina platensis. The predicted amino acid sequence of the C-terminal region of cyaG was similar to the catalytic domains of Class III adenylyl and guanylyl cyclases. The N-terminal region next to the catalytic domain of CyaG was similar to the dimerization domain, which is highly conserved among guanylyl cyclases. As a whole, CyaG is more closely related to guanylyl cyclases than to adenylyl cyclases in its primary structure. The catalytic domain of CyaG was expressed in Escherichia coli and partially purified. CyaG showed adenylyl cyclase (but not guanylyl cyclase) activity. By site-directed mutagenesis of three amino acid residues (Lys(533), Ile(603), and Asp(605)) within the purine ring recognition site of CyaG to Glu, Arg, and Cys, respectively, CyaG was transformed to a guanylyl cyclase that produced cGMP instead of cAMP. Thus having properties of both cyclases, CyaG may therefore represent a critical position in the evolution of Class III adenylyl and guanylyl cyclases.

Adenylyl Cyclases↗

PubMed1996

Tracking the evolutionary origin of the methicillin resistance gene: cloning and sequencing of a homologue of mecA from a methicillin susceptible strain of Staphylococcus sciuri.

The gene mecA, a central genetic determinant of methicillin resistance in Staphylococcus aureus and coagulase-negative staphylococci of human origin, has an unknown extra species origin in these human pathogens. After screening isolates representing over 15 different species within the genus Staphylococcus we could identify only one--S. sciuri--in which each of over 150 independent isolates showed positive hybridization with a mecA-specific DNA probe isolated from a methicillin-resistant strain of S. aureus, (MRSA). Yet, the majority of these isolates showed no resistance to penicillin or methicillin. The mecA gene homologue was cloned and sequenced from a S. sciuri strain and the sequence of mecA was compared to that of the mecA of prototype strains of methicillin resistant S. aureus and S. epidermidis. Similarly to mecA of MRSA, the mecA homologue of S. sciuri was composed of a putative transglycosylase and a transpeptidase domain the latter showing all the conserved motifs typical of the active sites of the penicillin binding domain of transpeptidases. Overall similarity between the deduced amino acid sequences of mecA of MRSA and the mecA homologue of S. sciuri was 88%. On the other hand, comparison of the transpeptidase domain of the S. sciuri mecA to the corresponding domain alone of the MRSA mecA showed a similarity of 96% and an identity of 91%, while comparison of the putative transglycosylase domains of the two bacteria showed only a 80% similarity and 68% identity of amino acid sequences. Our data suggests that mecA of methicillin-resistant strains of staphylococci pathogenic to Man originated within the genus of Staphylococcus from an evolutionary relative of the mecA homologue that we have identified in S. sciuri and which may perform a normal physiological function in this bacterium unrelated to beta-lactam resistance.

Animals↗