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PubMed1990

Evolutionary relationship of some stalked and budding bacteria (genera Caulobacter, "Hyphobacter", Hyphomonas and Hyphomicrobium) as studied by the new integral taxonomical method.

A new approach was developed for the determination of taxonomic and evolutional relationships among four genera of oligotrophic bacteria. The main idea of this approach is the algorithmized integrative analysis of the morphological and physiological specificity of these bacteria, their 5S rRNA sequences, fatty acid and lipid composition of their membranes, as well as their sensitivity to a large variety of antibiotics. It was shown that the genera Caulobacter and Hyphomonas are closely related to each other, but they are both distant from Hyphomicrobium species. The new genus, "Hyphobacter", is placed between Caulobacter and Hyphomonas. Taxonomic heterogeneity was found to exist within the genera Caulobacter and Hyphomicrobium. Evolutional pathways from Caulobacter to Hyphomicrobium are proposed on the basis of the present data. No correlations were found between the cell morphology of the organisms and their geno- and chemotaxonomy.

Anti-Bacterial Agents↗
PubMed2003

Motifs in Schizosaccharomyces pombe ars3002 important for replication origin activity in Saccharomyces cerevisiae.

Ars3002 is an efficient single-copy replication origin in the fission yeast, Schizosaccharomyces pombe. In a previous study, we tested the effects of consecutive approximately 50-bp deletions throughout ars3002 on the replication efficiency of those origins in S. pombe. Here we report the results of our use of the same approximately 50-bp deletions to test the hypothesis that some of the cis-acting sequences important for replication origin activity in fission yeast might be conserved in the evolutionarily distant budding yeast, Saccharomyces cerevisiae. We found that in most cases there was no correlation between the effects of particular mutations in S. pombe and in S. cerevisiae. We conclude that it is unlikely that any of the cis-acting sequences recognised by homologous replication proteins is conserved between these two yeast species.

Base Sequence↗
PubMed2003

The yersiniae--a model genus to study the rapid evolution of bacterial pathogens.

Yersinia pestis, the causative agent of plague, seems to have evolved from a gastrointestinal pathogen, Yersinia pseudotuberculosis, in just 1,500-20,000 years--an 'eye blink' in evolutionary time. The third pathogenic Yersinia, Yersinia enterocolitica, also causes gastroenteritis but is distantly related to Y. pestis and Y. pseudotuberculosis. Why do the two closely related species cause remarkably different diseases, whereas the distantly related enteropathogens cause similar symptoms? The recent availability of whole-genome sequences and information on the biology of the pathogenic yersiniae have shed light on this paradox, and revealed ways in which new, highly virulent pathogens can evolve.

Animals↗
PubMed1997

Genetic definition of a protein-splicing domain: functional mini-inteins support structure predictions and a model for intein evolution.

Inteins are protein-splicing elements, most of which contain conserved sequence blocks that define a family of homing endonucleases. Like group I introns that encode such endonucleases, inteins are mobile genetic elements. Recent crystallography and computer modeling studies suggest that inteins consist of two structural domains that correspond to the endonuclease and the protein-splicing elements. To determine whether the bipartite structure of inteins is mirrored by the functional independence of the protein-splicing domain, the entire endonuclease component was deleted from the Mycobacterium tuberculosis recA intein. Guided by computer modeling studies, and taking advantage of genetic systems designed to monitor intein function, the 440-aa Mtu recA intein was reduced to a functional mini-intein of 137 aa. The accuracy of splicing of several mini-inteins was verified. This work not only substantiates structure predictions for intein function but also supports the hypothesis that, like group I introns, mobile inteins arose by an endonuclease gene invading a sequence encoding a small, functional splicing element.

Amino Acid Sequence↗
PubMed1998

Evolution of an Escherichia coli protein with increased resistance to oxidative stress.

L-1,2-Propanediol:NAD+ 1-oxidoreductase of Escherichia coli is encoded by the fucO gene, a member of the regulon specifying dissimilation of L-fucose. The enzyme normally functions during fermentative growth to regenerate NAD from NADH by reducing the metabolic intermediate L-lactaldehyde to propanediol which is excreted. During aerobic growth L-lactaldehyde is converted to L-lactate and thence to the central metabolite pyruvate. The wasteful excretion of propanediol is minimized by oxidative inactivation of the oxidoreductase, an Fe2+-dependent enzyme which is subject to metal-catalyzed oxidation (MCO). Mutants acquiring the ability to grow aerobically on propanediol as sole carbon and energy source can be readily selected. These mutants express the fucO gene constitutively, as a result of an IS5 insertion in the promoter region. In this study we show that continued selection for aerobic growth on propanediol resulted in mutations in the oxidoreductase conferring increased resistance to MCO. In two independent mutants, the resistance of the protein was respectively conferred by an Ile7 --> Leu and a Leu8 --> Val substitution near the NAD-binding consensus amino acid sequence. A site-directed mutant protein with both substitutions showed an MCO resistance greater than either mutant protein with a single amino acid change.

Alcohol Oxidoreductases↗
PubMed1998

The complete DNA sequence and analysis of the large virulence plasmid of Escherichia coli O157:H7.

The complete DNA sequence of pO157, the large virulence plasmid of EHEC strain O157:H7 EDL 933, is presented. The 92 kb F-like plasmid is composed of segments of putative virulence genes in a framework of replication and maintenance regions, with seven insertion sequence elements, located mostly at the boundaries of the virulence segments. One hundred open reading frames (ORFs) were identified, of which 19 were previously sequenced potential virulence genes. Forty-two ORFs were sufficiently similar to known proteins for suggested functions to be assigned, and 22 had no convincing similarity with any known proteins. Of the newly identified genes, an unusually large ORF of 3169 amino acids has a putative cytotoxin active site shared with the large clostridial toxin (LCT) family and proteins such as ToxA and B of Clostridium difficile . A conserved motif was detected that links the large ORF and the LCT proteins with the OCH1 family of glycosyltransferases. In the complete sequence, the mosaic form can be observed at the levels of base composition, codon usage and gene organization. Insights were obtained from patterns of DNA composition as well as the pathogenic and 'housekeeping' gene segments. Evolutionary trees built from shared plasmid maintenance genes show that even these genes have heterogeneous origins.

Amino Acid Sequence↗
PubMed2000

cis and trans factors affecting Mos1 mariner evolution and transposition in vitro, and its potential for functional genomics.

Mos1 and other mariner / Tc1 transposons move horizon-tally during evolution, and when transplanted into heterologous species can transpose in organisms ranging from prokaryotes to protozoans and vertebrates. To further develop the Drosophila Mos1 mariner system as a genetic tool and to probe mechanisms affecting the regulation of transposition activity, we developed an in vitro system for Mos1 transposition using purified transposase and selectable Mos1 derivatives. Transposition frequencies of nearly 10(-3)/target DNA molecule were obtained, and insertions occurred at TA dinucleotides with little other sequence specificity. Mos1 elements containing only the 28 bp terminal inverted repeats were inactive in vitro, while elements containing a few additional internal bases were fully active, establishing the minimal cis -acting requirements for transposition. With increasing transposase the transposition frequency increased to a plateau value, in contrast to the predictions of the protein over-expression inhibition model and to that found recently with a reconstructed Himar1 transposase. This difference between the 'natural' Mos1 and 'reconstructed' Himar1 transposases suggests an evolutionary path for down-regulation of mariner transposition following its introduction into a naïve population. The establishment of the cis and trans requirements for optimal mariner transposition in vitro provides key data for the creation of vectors for in vitro mutagenesis, and will facilitate the development of in vivo systems for mariner transposition.

Animals↗
PubMed2006

Evolution of variants of yeast site-specific recombinase Flp that utilize native genomic sequences as recombination target sites.

As a tool in directed genome manipulations, site-specific recombination is a double-edged sword. Exquisite specificity, while highly desirable, makes it imperative that the target site be first inserted at the desired genomic locale before it can be manipulated. We describe a combination of computational and experimental strategies, based on the tyrosine recombinase Flp and its target site FRT, to overcome this impediment. We document the systematic evolution of Flp variants that can utilize, in a bacterial assay, two sites from the human interleukin 10 gene, IL10, as recombination substrates. Recombination competence on an end target site is acquired via chimeric sites containing mixed sequences from FRT and the genomic locus. This is the first time that a tyrosine site-specific recombinase has been coaxed successfully to perform DNA exchange within naturally occurring sequences derived from a foreign genomic context. We demonstrate the ability of an Flp variant to mediate integration of a reporter cassette in Escherichia coli via recombination at one of the IL10-derived sites.

Base Sequence↗
PubMed2005

Directed evolution of Pseudomonas aeruginosa lipase for improved amide-hydrolyzing activity.

A lipase from Pseudomonas aeruginosa was subjected to directed molecular evolution for increased amide-hydrolyzing (amidase) activity. A single round of random mutagenesis followed by screening for hydrolytic activity for oleoyl 2-naphthylamide as compared with that for oleoyl 2-naphthyl ester identified five mutants with 1.7-2.0-fold increased relative amidase activities. Three mutational sites (F207S, A213D and F265L) were found to affect the amidase/esterase activity ratios. The combination of these mutations further improved the amidase activity. Active-site titration using a fluorescent phosphonic acid ester allowed the molecular activities for the amide and the ester to be determined for each mutant without purification of the lipase. A double mutant F207S/A213D gave the highest molecular activity of 1.1 min(-1) for the amide, corresponding to a 2-fold increase compared with that of the wild-type lipase. A structural model of the lipase indicated that the mutations occurred at the sites near the surface and remote from the catalytic triad, but close to the calcium binding site. This study is a first step towards understanding why lipases do not hydrolyze amides despite the similarities to serine proteases in the active site structure and the reaction mechanism and towards the preparation of a general acyl transfer catalyst for the biotransformation of amides.

Amides↗
PubMed2001

The social evolution of bacterial pathogenesis.

Many of the genes responsible for the virulence of bacterial pathogens are carried by mobile genetic elements that can be transferred horizontally between different bacterial lineages. Horizontal transfer of virulence-factor genes has played a profound role in the evolution of bacterial pathogens, but it is poorly understood why these genes are so often mobile. Here, I present a hypothetical selective mechanism maintaining virulence-factor genes on horizontally transmissible genetic elements. For virulence factors that are secreted extracellularly, selection within hosts may favour mutant 'cheater' strains of the pathogen that do not produce the virulence factor themselves but still benefit from factors produced by other members of the pathogen population within a host. Using simple mathematical models, I show that if this occurs then selection for infectious transmission between hosts favours pathogen strains that can reintroduce functional copies of virulence-factor genes into cheaters via horizontal transfer, forcing them to produce the virulence factor. Horizontal gene transfer is thus a novel mechanism for the evolution of cooperation. I discuss predictions of this hypothesis that can be tested empirically and its implications for the evolution of pathogen virulence.

Bacteria↗
PubMed2003

Limitations on geminivirus genome size imposed by plasmodesmata and virus-encoded movement protein: insights into DNA trafficking.

Animals and plants evolved systems to permit non-cell-autonomous trafficking of RNA, whereas DNA plays a cell-autonomous role. In plants, plasmodesmata serve as the conduit for this phenomenon, and viruses have evolved to use this pathway for the spread of infectious nucleic acids. In this study, a plant DNA virus was used to explore the constraints imposed on the movement of DNA through this endogenous RNA trafficking pathway. The combined properties of the geminivirus-encoded movement protein and plasmodesmata were shown to impose a strict limitation on the size of the viral genome at the level of cell-to-cell movement. Size-increased viral genome components underwent homologous and nonhomologous recombination to overcome this strict limitation. Our results provide insights into the genetic mechanisms that underlie viral evolution and provide a likely explanation for why relatively few types of plant DNA viruses have evolved: they would have had to overcome the constraints imposed by an endogenous system operating to ensure that DNA acts in a cell-autonomous manner.

Cloning, Molecular↗
PubMed1999

Structure and function of a conserved DNA region coding for tartrate utilization in Agrobacterium vitis.

Three tartrate utilization regions from Agrobacterium vitis strains involved in host specificity have been compared, to clearly define the borders of these regions and eventually identify specific sequences that could provide a mechanism of duplication of this region. A 10.8-kb conserved DNA fragment called the TAR element, found in different genetic contexts, was defined. A comparison of the two tartrate dehydrogenase genes (ttuC and ttuC') in each of the three TAR elements suggests that these genes co-evolve.

Alcohol Oxidoreductases↗
PubMed2004

Long-term experimental evolution in Escherichia coli. XII. DNA topology as a key target of selection.

The genetic bases of adaptation are being investigated in 12 populations of Escherichia coli, founded from a common ancestor and serially propagated for 20,000 generations, during which time they achieved substantial fitness gains. Each day, populations alternated between active growth and nutrient exhaustion. DNA supercoiling in bacteria is influenced by nutritional state, and DNA topology helps coordinate the overall pattern of gene expression in response to environmental changes. We therefore examined whether the genetic controls over supercoiling might have changed during the evolution experiment. Parallel changes in topology occurred in most populations, with the level of DNA supercoiling increasing, usually in the first 2000 generations. Two mutations in the topA and fis genes that control supercoiling were discovered in a population that served as the focus for further investigation. Moving the mutations, alone and in combination, into the ancestral background had an additive effect on supercoiling, and together they reproduced the net change in DNA topology observed in this population. Moreover, both mutations were beneficial in competition experiments. Clonal interference involving other beneficial DNA topology mutations was also detected. These findings define a new class of fitness-enhancing mutations and indicate that the control of DNA supercoiling can be a key target of selection in evolving bacterial populations.

Adaptation, Physiological↗
PubMed1996

Adaptation of a conjugal transfer system for the export of pathogenic macromolecules.

Conjugal transfer of bacterial plasmids requires a pore through which DNA can traverse the envelopes of the donor and recipient cells. Recent studies indicate that these pores, which are composed of approximately ten proteins, are evolutionarily related to the transport systems required for the transfer of oncogenic T-DNA from Agrobacterium tumefaciens to plant cells and for toxin secretion from Bordetella pertussis.

Bacterial Proteins↗