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PubMed2001

Complexity and diversity of Klebsiella pneumoniae strains with extended-spectrum beta-lactamases isolated in 1994 and 1996 at a teaching hospital in Durban, South Africa.

beta-Lactamase production was investigated in cultures of 25 Klebsiella pneumoniae isolates isolated at a hospital in Durban, South Africa, in 1994 and 1996. Twenty of these isolates gave ceftazidime MIC/ceftazidime plus clavulanate MIC ratios of >/=8, implying production of extended-spectrum beta-lactamases (ESBLs), and DNA sequencing identified an ESBL gene (bla(TEM-53)) in a further two isolates. Pulsed-field gel electrophoresis (PFGE) defined 4 distinct strains among the 12 isolates collected in 1994 and 9 distinct strains among the 13 isolates collected in 1996. In three cases, multiple isolates from single patients varied in their PFGE profiles and antibiograms, implying mixed colonization or infection. Isoelectric focusing and DNA hybridization found both TEM and SHV enzymes and their genes in all 25 isolates. Many isolates had multiple identical or different beta-lactamase gene variants, with at least 84 bla(SHV) and bla(TEM) gene copies among the 25 organisms. Sequencing identified the genes for the SHV-1, -2, and -5 enzymes and for four new SHV types (SHV-19, -20, -21, and -22). These new SHV variants had novel mutations remote from sites known to affect catalytic activity. Sequencing also found the genes for TEM-1, TEM-53, and one novel type, TEM-63. All the isolates had multiple and diverse plasmids. These complex and diverse patterns of ESBL production and strain epidemiology are far removed from the concept of an ESBL outbreak and suggest a situation in which ESBL production has become endemic and in which evolution is generating a wide range of enzyme combinations. This complexity and diversity complicates patient management and the design of antibiotic use policies.

Amino Acid Substitution↗

PubMed2006

Complete sequence analysis of novel plasmids from emetic and periodontal Bacillus cereus isolates reveals a common evolutionary history among the B. cereus-group plasmids, including Bacillus anthracis pXO1.

The plasmids of the members of the Bacillus cereus sensu lato group of organisms are essential in defining the phenotypic traits associated with pathogenesis and ecology. For example, Bacillus anthracis contains two plasmids, pXO1 and pXO2, encoding toxin production and encapsulation, respectively, that define this species pathogenic potential, whereas the presence of a Bt toxin-encoding plasmid defines Bacillus thuringiensis isolates. In this study the plasmids from B. cereus isolates that produce emetic toxin or are linked to periodontal disease were sequenced and analyzed. Two periodontal isolates examined contained almost identical approximately 272-kb plasmids, named pPER272. The emetic toxin-producing isolate contained one approximately 270-kb plasmid, named pCER270, encoding the cereulide biosynthesis gene cluster. Comparative sequence analyses of these B. cereus plasmids revealed a high degree of sequence similarity to the B. anthracis pXO1 plasmid, especially in a putative replication region. These plasmids form a newly defined group of pXO1-like plasmids. However, these novel plasmids do not contain the pXO1 pathogenicity island, which in each instance is replaced by plasmid specific DNA. Plasmids pCER270 and pPER272 share regions that are not found in any other pXO1-like plasmids. Evolutionary studies suggest that these plasmids are more closely related to each other than to other identified B. cereus plasmids. Screening of a population of B. cereus group isolates revealed that pXO1-like plasmids are more often found in association with clinical isolates. This study demonstrates that the pXO1-like plasmids may define pathogenic B. cereus isolates in the same way that pXO1 and pXO2 define the B. anthracis species.

Amino Acid Sequence↗

PubMed1989

A group II intron in the Neurospora mitochondrial coI gene: nucleotide sequence and implications for splicing and molecular evolution.

The temperature-sensitive Neurospora nuclear mutant cyt18-1 is deficient in splicing many Group I mitochondrial introns when grown at its non-permissive temperature; however, splicing of intron 1 in the coI gene of the Adiopodoume (formerly called North Africa) strain is unaffected (R.A. Collins and A.M. Lambowitz, J. Mol. Biol. 184: 413-428, 1985). Here we show that coI intron 1 is a typical Group II intron, the only one identified to date in Neurospora. The differential effect of the cyt18-1 mutation suggests that splicing of certain introns could be regulated independently of others by nuclear-encoded proteins. The intron contains a long open reading frame (ORF) resembling that of the Neurospora Mauriceville mitochondrial plasmid. The intron and plasmid ORFs share unusual features of codon usage that suggest both evolved outside of the Neurospora mitochondrial genetic system.

Amino Acid Sequence↗

PubMed2003

Differences in the processing of DNA ends in Arabidopsis thaliana and tobacco: possible implications for genome evolution.

Surprising species-specific differences in non-homologous end-joining (NHEJ) of genomic double-strand breaks (DSBs) have been reported for the two dicotyledonous plants Arabidopsis thaliana and Nicotiana tabacum. In Arabidopsis deletions were, on average, larger than in tobacco and not associated with insertions. To establish the molecular basis of the phenomenon we analysed the fate of free DNA ends in both plant species by biolistic transformation of leaf tissue with linearized plasmid molecules. Southern blotting indicated that, irrespective of the nature of the ends (blunt, 5' or 3' overhangs), linearized full-length DNA molecules were, on average, more stable in tobacco than in Arabidopsis. The relative expression of a beta-glucuronidase gene encoded by the plasmid was similar in both plant species when the break was distant from the marker gene. However, if a DSB was introduced between the promoter and the open reading frame of the marker, transient expression was halved in Arabidopsis as compared to tobacco. These results indicate that free DNA ends are more stable in tobacco than in Arabidopsis, either due to lower DNA exonuclease activity or due to a better protection of DNA break ends or both. Exonucleolytic degradation of DNA ends might be a driving force in the evolution of genome size as the Arabidopsis genome is more than twenty times smaller than the tobacco genome.

Arabidopsis↗

PubMed2005

Mobile genetic elements: the agents of open source evolution.

Horizontal genomics is a new field in prokaryotic biology that is focused on the analysis of DNA sequences in prokaryotic chromosomes that seem to have originated from other prokaryotes or eukaryotes. However, it is equally important to understand the agents that effect DNA movement: plasmids, bacteriophages and transposons. Although these agents occur in all prokaryotes, comprehensive genomics of the prokaryotic mobile gene pool or 'mobilome' lags behind other genomics initiatives owing to challenges that are distinct from cellular chromosomal analysis. Recent work shows promise of improved mobile genetic element (MGE) genomics and consequent opportunities to take advantage - and avoid the dangers - of these 'natural genetic engineers'. This review describes MGEs, their properties that are important in horizontal gene transfer, and current opportunities to advance MGE genomics.

Conjugation, Genetic↗

PubMed2005

The evolution of flea-borne transmission in Yersinia pestis.

Transmission by fleabite is a recent evolutionary adaptation that distinguishes Yersinia pestis, the agent of plague, from Yersinia pseudotuberculosis and all other enteric bacteria. The very close genetic relationship between Y. pestis and Y. pseudotuberculosis indicates that just a few discrete genetic changes were sufficient to give rise to flea-borne transmission. Y. pestis exhibits a distinct infection phenotype in its flea vector, and a transmissible infection depends on genes that are specifically required in the flea, but not the mammal. Transmission factors identified to date suggest that the rapid evolutionary transition of Y. pestis to flea-borne transmission within the last 1,500 to 20,000 years involved at least three steps: acquisition of the two Y. pestis-specific plasmids by horizontal gene transfer; and recruitment of endogenous chromosomal genes for new functions. Perhaps reflective of the recent adaptation, transmission of Y. pestis by fleas is inefficient, and this likely imposed selective pressure favoring the evolution of increased virulence in this pathogen.

Animals↗

PubMed1995

Understanding cellular responses to toxic agents: a model for mechanism-choice in bacterial metal resistance.

Bacterial resistances to metals are heterogeneous in both their genetic and biochemical bases. Metal resistance may be chromosomally-, plasmid- or transposon-encoded, and one or more genes may be involved: at the biochemical level at least six different mechanisms are responsible for resistance. Various types of resistance mechanisms can occur singly or in combination and for a particular metal different mechanisms of resistance can occur in the same species. To understand better the diverse responses of bacteria to metal ion challenge we have constructed a qualitative model for the selection of metal resistance in bacteria. How a bacterium becomes resistant to a particular metal depends on the number and location of cellular components sensitive to the specific metal ion. Other important selective factors include the nature of the uptake systems for the metal, the role and interactions of the metal in the normal metabolism of the cell and the availability of plasmid (or transposon) encoded resistance mechanisms. The selection model presented is based on the interaction of these factors and allows predictions to be made about the evolution of metal resistance in bacterial populations. It also allows prediction of the genetic basis and of mechanisms of resistance which are in substantial agreement with those in well-documented populations. The interaction of, and selection for resistance to, toxic substances in addition to metals, such as antibiotics and toxic analogues, involve similar principles to those concerning metals. Potentially, models for selection of resistance to any substance can be derived using this approach.

Bacteria↗

PubMed1988

The sequence specificity of homeodomain-DNA interaction.

The Drosophila developmental gene, engrailed, encodes a sequence-specific DNA binding activity. Using deletion constructs expressed as fusion proteins in E. coli, we localized this activity to the conserved homeodomain (HD). The binding site consensus, TCAATTAAAT, is found in clusters in the engrailed regulatory region. Weak binding of the En HD to one copy of a synthetic consensus is enhanced by adjacent copies. The distantly related HD encoded by fushi tarazu binds to the same sites as the En HD, but differs in its preference for related sites. Both HDs bind a second type of sequence, a repeat of TAA. The similarity in sequence specificity of En and Ftz HDs suggests that, within families of DNA binding proteins, close relatives will exhibit similar specificities. Competition among related regulatory proteins might govern which protein occupies a given binding site and consequently determine the ultimate effect of cis-acting regulatory sites.

Allosteric Regulation↗

PubMed1991

Functional differences between yeast and human TFIID are localized to the highly conserved region.

TFIID, the general transcription factor that binds TATA promoter elements, is highly conserved throughout the eukaryotic kingdom. TFIIDs from different organisms contain C-terminal core domains that are at least 80% identical and display similar biochemical properties. Despite these similarities, yeast cells containing human TFIID instead of the endogenous yeast protein grow extremely poorly. Surprisingly, this functional distinction reflects differences in the core domains, not the divergent N-terminal regions. The N-terminal region is unimportant for the essential function(s) of yeast TFIID because expression of the core domain permits efficient cell growth. Analysis of yeast-human hybrid TFIIDs indicates that several regions within the conserved core account for the phenotypic difference, with some regions being more important than others. This species specificity might reflect differences in DNA-binding properties and/or interactions with activator proteins or other components of the RNA polymerase II transcription machinery.

Base Sequence↗

PubMed1985

Novel structure of a human U6 snRNA pseudogene.

A genomic DNA library containing human placental DNA cloned into phage lambda Charon 4A was screened for snRNA U6 genes. In vitro 32P-labeled U6 snRNA isolated from HeLa cells was used as a hybridization probe. A positive clone containing a 4.6-kb EcoRI fragment of human chromosomal DNA was recloned into the EcoRI site of pBR325 and mapped by restriction endonuclease digestion. Restriction fragments containing U6 RNA sequences were identified by hybridization with isolated U6[32P]RNA. The sequence analysis revealed a novel structure of a U6 RNA pseudogene, bearing two 17-nucleotide(nt)-long direct repeats of genuine U6 RNA sequences arranged in a head-to-tail fashion within the 5' part of the molecule. Hypothetical models as to how this type of snRNA U6 pseudogene might have been generated during evolution of the human genome are presented. When compared to mammalian U6 RNA sequences the pseudogene accounts for a 77% overall sequence homology and contains the authentic 5'- and 3'-ends of the U6 RNA.

Bacteriophage lambda↗

PubMed2005

Translational effects of differential codon usage among intragenic domains of new genes in Drosophila.

Evolved codon usages often pose a technical challenge over the expressing of eukaryotic genes in microbial systems because of changed translational machinery. In the present study, we investigated the translational effects of intragenic differential codon usage on the expression of the new Drosophila gene, jingwei (jgw), a chimera derived from two unrelated parental genes: Ymp and Adh. We found that jgw possesses a strong intragenic differential usage of synonymous codons, i.e. the Adh-derived C-domain has a significantly higher codon bias than that of the Ymp-derived N-domain (P=0.0023 by t-test). Additional evolutionary analysis revealed the heterogeneous distribution of rare codons, implicating its role in gene regulation and protein translation. The in vitro expression of jgw further demonstrated that the heterogeneous distribution of rare codons has played a role in regulating gene expression, particularly, affecting the quality of protein translation.

Alcohol Dehydrogenase↗

PubMed2006

Dual selection of a genetic switch by a single selection marker.

Forward engineering of synthetic genetic circuits in living cells is expected to deliver various applications in biotechnology and medicine and to provide valuable insights into the design principles of natural gene networks. However, lack of biochemical data and complexity of biological environment complicate rational design of such circuits based on quantitative simulation. Previously, we have shown that directed evolution can complement our weakness in designing genetic circuits by screening or selecting functional circuits from a large pool of nonfunctional ones. Here we describe a dual selection strategy that allows selection of both ON and OFF states of genetic circuits using tetA as a single selection marker. We also describe a successful demonstration of a genetic switch selection from a 2000-fold excess background of nonfunctional switches in three rounds of iterative selection. The dual selection system is more robust than the previously reported selection system employing three genes, with no observed false positive mutants during the simulated selections.

Computer Simulation↗

PubMed2006

Characterisation of KLUA-9, a beta-lactamase from extended-spectrum cephalosporin-susceptible Kluyvera ascorbata, and genetic organisation of bla(KLUA-9).

This study characterised the genetic environment of the chromosomally encoded bla(KLUA-9) gene from a clinical Kluyvera ascorbata isolate and performed a kinetic characterisation of KLUA-9. Purified KLUA-9 showed the highest catalytic efficacies towards benzylpenicillin, ampicillin, piperacillin, first-generation cephalosporins, cefuroxime and cefoperazone; like other 'cefotaximases', it showed a much higher rate of hydrolysis of cefotaxime than ceftazidime, whilst dicloxacillin, cefoxitin and imipenem behaved as poor substrates. A 9kb insert from K. ascorbata was cloned (Escherichia coli KK68C1) and sequenced. bla(KLUA-9) and its 266bp upstream flanking region (almost identical to the integron-associated bla(CTX-M-2)) are preceded by an aspat variant, a ypdABC-like operon and two open reading frames with unknown functions. Unlike ISCR1-associated bla(CTX-M-2) genes, we failed to detect the putative orf513 recombination sites. Instead, we were able to localise the 5bp target sites for insertion of ISEcp1B, suggesting that this element could be responsible for future (or still undetected) mobilisation of bla(KLUA-9) to more efficiently transferred elements.

Base Sequence↗

PubMed2004

Amplification-mutagenesis--how growth under selection contributes to the origin of genetic diversity and explains the phenomenon of adaptive mutation.

The behavior of a particular bacterial genetic system has been interpreted as evidence that selective stress induces general mutagenesis or even preferentially directs mutations to sites that improve growth (adaptive mutation). It has been proposed that changes in mutability are a programmed response to stress in non-growing cells. In contrast, the amplification-mutagenesis model suggests that stress has no direct effect on the mutation rate and that mutations arise in cells growing under strong selection. In this model, stress serves only as a selective pressure that favors cells with multiple copies of a growth-limiting gene. Mutations are made more probable because more target copies are added to the selection plate-more cells with more mutational targets per cell. The amplification-mutagenesis process involves standard genetic events and therefore should apply to all biological systems. Idiosyncrasies of the particular system described here accelerate this process, allowing an evolutionary series of events to be completed in only a few days.

Adaptation, Biological↗

PubMed2005

Interaction of heterochromatin protein 2 with HP1 defines a novel HP1-binding domain.

Heterochromatin Protein 2 (HP2) is a nonhistone chromosomal protein from Drosophila melanogaster localized principally in the pericentric heterochromatin, telomeres, and fourth chromosome, all regions associated with HP1. Mutations in HP2 can suppress position effect variegation, indicating a role in gene silencing and heterochromatin formation [Shaffer, C. D. et al. (2002) Proc. Natl. Acad. Sci.U.S.A. 99, 14332-14337]. In vitro coimmunoprecipitation experiments with various peptides from HP2 have identified a single HP1-binding domain. Conserved domains in HP2, including those within the HP1-binding region, have been identified by recovering and sequencing Su(var)2-HP2 from D. willistoni and D. virilis, as well as examining available sequence data from D. pseudoobscura. A PxVxL motif, shown to be an HP1-binding domain in many HP1-interacting proteins, is observed but is not well-conserved in location and sequence and does not mediate HP2 binding to HP1. The sole HP1-binding domain is composed of two conserved regions of 12 and 16 amino acids separated by 19 amino acids. Site-directed mutagenesis within the two conserved regions has shown that the 16 amino acid domain is critical for HP1 binding. This constitutes a novel domain for HP1 interaction, providing a critical link for heterochromatin formation in Drosophila.

Amino Acid Motifs↗

PubMed1990

Escape of DNA from mitochondria to the nucleus in Saccharomyces cerevisiae.

The migration of genetic information from ancestral prokaryotic endosymbionts into eukaryotic nuclei is thought to have had an important role in the evolution of mitochondria and chloroplasts. Here we describe an assay for the detection of movement of DNA between mitochondria and the nucleus in yeast. Because recombinant plasmid DNA replicates after transformation into mitochondria of yeast strains lacking endogenous mitochondrial DNA we were able to propagate the nuclear genetic marker URA3 in mitochondria. As expected, the wild-type URA3 gene in mitochondria failed to complement the uracil auxotrophy (Ura-) caused by a nuclear ura3 mutation. But selection of Ura+ prototrophs from a Ura- strain carrying URA3 on a plasmid in its mitochondria enabled us to detect plasmid movement to the nucleus. Conversely, as the plasmid used also contained the mitochondrial gene COX2 required for respiratory growth, we were able to set up corresponding selections to detect migration of DNA from the nucleus to the mitochondria. Our results show that, in yeast, DNA escapes from mitochondria and appears in the nucleus at a surprisingly high frequency (approximately 2 x 10(-5) per cell per generation). But the rate at which DNA makes the journey in the opposite direction--nucleus to mitochondria--is apparently at least 100,000 times less.

Cell Nucleus↗

PubMed2003

Impact of large chromosomal inversions on the adaptation and evolution of Pseudomonas aeruginosa chronically colonizing cystic fibrosis lungs.

Pseudomonas aeruginosa chronically colonizing the lungs of cystic fibrosis (CF) patients undergoes fast evolution leading to clonal divergence. More than half of the genotypes of P. aeruginosa clone C isolates exclusively from CF lung infection exhibit large chromosomal inversions (LCIs). To analyse the impact of LCIs, as a novel mechanism of bacterial adaptation, the underlying molecular mechanism was examined. Analysis of inversion breakpoints suggested an IS6100-induced coupled insertion-inversion mechanism. A selective advantage was created by insertion of IS6100 into wbpM, pilB and mutS which leads to common CF phenotypes such as O-antigen and type IV pili deficiency and hypermutability. Speciation in bacteria is accompanied by LCIs. Therefore adaptation by LCIs that allows persistence of P. aeruginosa in the CF lung and species diversification in that new ecological niche can serve as a model for bacterial genome evolution.

Adaptation, Physiological↗

PubMed1985

Molecular cloning and nucleotide sequence of the alpha and beta subunits of allophycocyanin from the cyanelle genome of Cyanophora paradoxa.

The genes for the alpha- and beta-subunit apoproteins of allophycocyanin (AP) were isolated from the cyanelle genome of Cyanophora paradoxa and subjected to nucleotide sequence analysis. The AP beta-subunit apoprotein gene was localized to a 7.8-kilobase-pair Pst I restriction fragment from cyanelle DNA by hybridization with a tetradecameric oligonucleotide probe. Sequence analysis using that oligonucleotide and its complement as primers for the dideoxy chain-termination sequencing method confirmed the presence of both AP alpha- and beta-subunit genes on this restriction fragment. Additional oligonucleotide primers were synthesized as sequencing progressed and were used to determine rapidly the nucleotide sequence of a 1336-base-pair region of this cloned fragment. This strategy allowed the sequencing to be completed without a detailed restriction map and without extensive and time-consuming subcloning. The sequenced region contains two open reading frames whose deduced amino acid sequences are 81-85% homologous to cyanobacterial and red algal AP subunits whose amino acid sequences have been determined. The two open reading frames are in the same orientation and are separated by 39 base pairs. AP alpha is 5' to AP beta and both coding sequences are preceded by a polypurine, Shine-Dalgarno-type sequence. Sequences upstream from AP alpha closely resemble the Escherichia coli consensus promoter sequences and also show considerable homology to promoter sequences for several chloroplast-encoded psbA genes. A 56-base-pair palindromic sequence downstream from the AP beta gene could play a role in the termination of transcription or translation. The allophycocyanin apoprotein subunit genes are located on the large single-copy region of the cyanelle genome.

Amino Acid Sequence↗