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The microsatellites of Escherichia coli: rapidly evolving repetitive DNAs in a non-pathogenic prokaryote.

We have demonstrated hypervariability of native short-motif repeats (microsatellites) in Escherichia coli. Twenty-five of the longest microsatellites in the E. coli genome were identified. These were analysed for length variability among 22 wild-type (non-mutator) isolates from the E. coli collection of reference (ECOR). Non-coding mononucleotide repeats are consistently polymorphic among these genetically diverse E. coli. Length differences in variable microsatellites allowed all E. coli strains examined to be uniquely differentiated. Phylogenetic analysis of the variable repeats shows ubiquitous homoplasy at the level of divergence represented by the sample set, suggesting that these markers are hypermutable and should prove valuable for the discrimination of closely related strains that are not otherwise genetically differentiable. Genomic analyses suggest that similar markers are also likely to be found in all other prokaryotes.

Biological Evolution↗

A molecular phylogeny of enteric bacteria and implications for a bacterial species concept.

A molecular phylogeny for seven taxa of enteric bacteria (Citrobacter freundii, Enterobacter cloacae, Escherichia coli, Hafnia alvei, Klebsiella oxytoca, Klebsiella pneumoniae, and Serratia plymuthica) was made from multiple isolates per taxa taken from a collection of environmental enteric bacteria. Sequences from five housekeeping genes (gapA, groEL, gyrA, ompA, and pgi) and the 16S rRNA gene were used to infer individual gene trees and were concatenated to infer a composite molecular phylogeny for the species. The isolates from each taxa formed tight species clusters in the individual gene trees, suggesting the existence of 'genotypic' clusters that correspond to traditional species designations. These sequence data and the resulting gene trees and consensus tree provide the first data set with which to assess the utility of the recently proposed core genome hypothesis (CGH). The CGH provides a genetically based approach to applying the biological species concept to bacteria.

DNA, Bacterial↗

Cohn's Crenothrix is a filamentous methane oxidizer with an unusual methane monooxygenase.

135 years ago Ferdinand Cohn, the founder of bacteriology, microscopically observed a conspicuous filamentous bacterium with a complex life cycle and described it as Crenothrix polyspora. This uncultured bacterium is infamous for mass developments in drinking water systems, but its phylogeny and physiology remained unknown. We show that C. polyspora is a gammaproteobacterium closely related to methanotrophs and capable of oxidizing methane. We discovered that C. polyspora encodes a phylogenetically very unusual particulate methane monooxygenase whose expression is strongly increased in the presence of methane. Our findings demonstrate a previously unrecognized complexity of the evolutionary history and cell biology of methane-oxidizing bacteria.

Bacterial Proteins↗

How to become a uropathogen: comparative genomic analysis of extraintestinal pathogenic Escherichia coli strains.

Uropathogenic Escherichia coli (UPEC) strain 536 (O6:K15:H31) is one of the model organisms of extraintestinal pathogenic E. coli (ExPEC). To analyze this strain's genetic basis of urovirulence, we sequenced the entire genome and compared the data with the genome sequence of UPEC strain CFT073 (O6:K2:H1) and to the available genomes of nonpathogenic E. coli strain MG1655 (K-12) and enterohemorrhagic E. coli. The genome of strain 536 is approximately 292 kb smaller than that of strain CFT073. Genomic differences between both UPEC are mainly restricted to large pathogenicity islands, parts of which are unique to strain 536 or CFT073. Genome comparison underlines that repeated insertions and deletions in certain parts of the genome contribute to genome evolution. Furthermore, 427 and 432 genes are only present in strain 536 or in both UPEC, respectively. The majority of the latter genes is encoded within smaller horizontally acquired DNA regions scattered all over the genome. Several of these genes are involved in increasing the pathogens' fitness and adaptability. Analysis of virulence-associated traits expressed in the two UPEC O6 strains, together with genome comparison, demonstrate the marked genetic and phenotypic variability among UPEC. The ability to accumulate and express a variety of virulence-associated genes distinguishes ExPEC from many commensals and forms the basis for the individual virulence potential of ExPEC. Accordingly, instead of a common virulence mechanism, different ways exist among ExPEC to cause disease.

Biological Evolution↗

Aminoglycoside-modifying enzyme of an antibiotic-producing bacterium acts as a determinant of antibiotic resistance in Escherichia coli.

Bacillus circulans NRRL B-3312, a nonpathogenic bacterium that produces the aminoglycoside antibiotic butirosin, is known to contain an aminoglycoside phosphotransferase that is similar to the neomycin phosphotransferases of clinically isolated antibiotic-resistant bacteria. Purified DNAs from B. circulans and the plasmid ColE1-ApR were digested with EcoRI endonuclease and the resulting fragments covalently joined with polynucleotide ligase. The recombined DNA was used to transform E. coli and ampicillin-neomycin resistant colonies were selected. Analysis of several clones indicated that neomycin resistance in the E. coli transformants was due to the presence of the B. circulans phosphotransferase gene. This observation is consistent with the notion that anitbiotic-modifying enzymes from antibiotic-producing organisms may be the sources of antibiotic resistance in plasmid-containing bacteria.

Bacillus↗

Adaptive evolution that requires multiple spontaneous mutations: mutations involving base substitutions.

A previous study has demonstrated that adaptive missense mutations occur in the trp operon of Escherichia coli. In this study it is shown that, under conditions of intense selection, a strain carrying missense mutations in both trpA and trpB reverts to Trp+ 10(8) times more frequently than would be expected if the two mutations were the result of independent events. Comparison of the single mutation rates with the double mutation rate and information obtained by sequencing DNA from double revertants show that neither our classical understanding of spontaneous mutation processes nor extant models for adaptive mutations can account for all of the observations. Despite a current lack of mechanistic understanding, it is clear that adaptive mutations can permit advantageous phenotypes that require multiple mutations to arise and that they appear enormously more frequently than would be expected.

Biological Evolution↗

An intron within the 16S ribosomal RNA gene of the archaeon Pyrobaculum aerophilum.

The 16S rRNA genes of Pyrobaculum aerophilum and Pyrobaculum islandicum were amplified by the polymerase chain reaction, and the resulting products were sequenced directly. The two organisms are closely related by this measure (over 98% similar). However, they differ in that the (lone) 16S rRNA gene of Pyrobaculum aerophilum contains a 713-bp intron not seen in the corresponding gene of Pyrobaculum islandicum. To our knowledge, this is the only intron so far reported in the small subunit rRNA gene of a prokaryote. Upon excision the intron is circularized. A secondary structure model of the intron-containing rRNA suggests a splicing mechanism of the same type as that invoked for the tRNA introns of the Archaea and Eucarya and 23S rRNAs of the Archaea. The intron contains an open reading frame whose protein translation shows no certain homology with any known protein sequence.

Amino Acid Sequence↗

Nucleotide polymorphism in colicin E1 and Ia plasmids from natural isolates of Escherichia coli.

We examined DNA sequence polymorphism for the colicin gene clusters of seven ColE1 and six ColIa plasmids obtained from natural isolates of Escherichia coli. These gene clusters harbor levels of nucleotide diversity ranging from 0.006 (ColIa) to 0.054 (ColE1). This level of diversity is similar to that observed for chromosomally encoded E. coli genes. However, the variance associated with these estimates is severalfold higher for the plasmid-encoded genes. This increased variance may be due to the differing plasmid population sizes. The pattern of colicin gene cluster polymorphism suggests that the two colicins are evolving in different fashions. ColE1 accumulates polymorphism at an elevated rate in the central domain of the colicin protein, while ColIa polymorphism is distributed evenly along the gene cluster. Comparison of the patterns of divergence between colicin and related proteins of ColIa and Ib and patterns of polymorphism within ColIa suggest that this gene cluster is not evolving in a neutral fashion. These data lend support to the hypothesis that colicin gene clusters may evolve under the influence of diversifying selection.

Bacteriocin Plasmids↗

Which bacterium is the ancestor of the animal mitochondrial genome?

We present considerable data supporting the hypothesis that a Sulfolobus- or Mycoplasma-like endosymbiont, rather than an alpha-proteobacterium, is the ancestor of animal mitochondrial genomes. This hypothesis is based on pronounced similarities in oligonucleotide relative abundance extremes common to animal mtDNA, Sulfolobus, and Mycoplasma capricolum and pronounced discrepancies of these relative abundance values with respect to alpha-proteobacteria. In addition, genomic dinucleotide relative abundance measures place Sulfolobus and M. capricolum among the closest to animal mitochondrial genomes, whereas the classical eubacteria, especially the alpha-proteobacteria, are at excessive distances. There are also considerable molecular and cellular phenotypic analogies among mtDNA, Sulfolobus, and M. capricolum.

Base Composition↗

Identification of Mycobacterium tuberculosis DNA in a pre-Columbian Peruvian mummy.

The existence of tuberculosis in the pre-Columbian Americas is controversial because the morphology of the lesion is not specific, the organism is culturally nonviable in ancient tissues, and nonpathogenic soil mycobacteria can contaminate buried bodies. We report the recovery of DNA unique to Mycobacterium tuberculosis from a lung lesion of a spontaneously mummified, 1000-year-old adult female body in southern Peru. This provides the most specific evidence possible for the pre-Columbian presence of human tuberculosis in the New World.

Adult↗

Amplification of trpEG: adaptation of Buchnera aphidicola to an endosymbiotic association with aphids.

Survival of aphids is dependent on an association with a prokaryotic endosymbiont (Buchnera aphidicola) found in specialized cells within the aphid body cavity. Recent nutritional and physiological studies have indicated that one of the functions of the endosymbionts is the synthesis of tryptophan [Douglas, A. E. & Prosser, W. A. (1992) J. Insect Physiol. 38, 565-568]. B. aphidicola resembles in many of its properties free-living prokaryotes. An adaptation to an endosymbiosis involving the overproduction of tryptophan would necessitate alterations that modify the effect of regulatory systems that in free-living organisms function to reduce enzyme activity under conditions of excess tryptophan. We have cloned and sequenced the genes for B. aphidicola trpEG encoding anthranilate synthase, the first enzyme of the tryptophan biosynthetic pathway, which in free-living bacteria is feedback-inhibited by tryptophan. Amino acid sequence comparisons indicate that the B. aphidicola enzyme has all of the key residues involved in allosteric feedback inhibition. Evidence is presented indicating that trpEG is present as four tandem repeats on a circular plasmid. Relative to B. aphidicola trpDC(F)BA (the chromosomal genes coding for the remaining enzymes of the tryptophan biosynthetic pathway) trpEG is amplified 14- to 15-fold. These findings suggest that the effect of inhibition by accumulated tryptophan may be overcome by overproduction of anthranilate synthase. Our results demonstrate the acquisition of a new property (gene amplification) as an adaptation to an endosymbiotic association in which B. aphidicola overproduces tryptophan for the aphid host.

Amino Acid Sequence↗

cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-associated virulence factors.

cagA, a gene that codes for an immunodominant antigen, is present only in Helicobacter pylori strains that are associated with severe forms of gastroduodenal disease (type I strains). We found that the genetic locus that contains cagA (cag) is part of a 40-kb DNA insertion that likely was acquired horizontally and integrated into the chromosomal glutamate racemase gene. This pathogenicity island is flanked by direct repeats of 31 bp. In some strains, cag is split into a right segment (cagI) and a left segment (cagII) by a novel insertion sequence (IS605). In a minority of H. pylori strains, cagI and cagII are separated by an intervening chromosomal sequence. Nucleotide sequencing of the 23,508 base pairs that form the cagI region and the extreme 3' end of the cagII region reveals the presence of 19 ORFs that code for proteins predicted to be mostly membrane associated with one gene (cagE), which is similar to the toxin-secretion gene of Bordetella pertussis, ptlC, and the transport systems required for plasmid transfer, including the virB4 gene of Agrobacterium tumefaciens. Transposon inactivation of several of the cagI genes abolishes induction of IL-8 expression in gastric epithelial cell lines. Thus, we believe the cag region may encode a novel H. pylori secretion system for the export of virulence determinants.

Antigens, Bacterial↗

Molecular keys to speciation: DNA polymorphism and the control of genetic exchange in enterobacteria.

Speciation involves the establishment of genetic barriers between closely related organisms. The extent of genetic recombination is a key determinant and a measure of genetic isolation. The results reported here reveal that genetic barriers can be established, eliminated, or modified by manipulating two systems which control genetic recombination, SOS and mismatch repair. The extent of genetic isolation between enterobacteria is a simple mathematical function of DNA sequence divergence. The function does not depend on hybrid DNA stability, but rather on the number of blocks of sequences identical in the two mating partners and sufficiently large to allow the initiation of recombination. Further, there is no obvious discontinuity in the function that could be used to define a level of divergence for distinguishing species.

Biological Evolution↗

Unequal fidelity of leading strand and lagging strand DNA replication on the Escherichia coli chromosome.

We have investigated the question whether during chromosomal DNA replication in Escherichia coli the two DNA strands may be replicated with differential accuracy. This possibility of differential replication fidelity arises from the distinct modes of replication in the two strands, one strand (the leading strand) being synthesized continuously, the other (the lagging strand) discontinuously in the form of short Okazaki fragments. We have constructed a series of lacZ strains in which the lac operon is inserted into the bacterial chromosome in the two possible orientations with regard to the chromosomal replication origin oriC. Measurement of lac reversion frequencies for the two orientations, under conditions in which mutations reflect replication errors, revealed distinct differences in mutability between the two orientations. As gene inversion causes a switching of leading and lagging strands, these findings indicate that leading and lagging strand replication have differential fidelity. Analysis of the possible mispairs underlying each specific base pair substitution suggests that the lagging strand replication on the E. coli chromosome may be more accurate than leading strand replication.

Alleles↗

Evolution of rhizobia by acquisition of a 500-kb symbiosis island that integrates into a phe-tRNA gene.

Nodulation and nitrogen fixation genes of Mesorhizobium loti are encoded on the chromosome of the bacterium. Nevertheless, there is strong evidence that these genes can be transferred from an inoculant strain to nonsymbiotic mesorhizobia in the field environment. Here we report that the chromosomal symbiotic element of M. loti strain ICMP3153 is transmissible in laboratory matings to at least three genomic species of nonsymbiotic mesorhizobia. The element is 500 kb in size, integrates into a phe-tRNA gene, and encodes an integrase of the phage P4 family just within its left end. The entire phe-tRNA gene is reconstructed at the left end of the element upon integration, whereas the 3' 17 nucleotides of the tRNA gene are present as a direct repeat at the right end. We termed the element a symbiosis island on the basis of its many similarities to pathogenicity islands. It may represent a class of genetic element that contributes to microbial evolution by acquisition.

Amino Acid Sequence↗

Protein import into mitochondria: origins and functions today (review).

Mitochondria are organelles derived from alpha-proteobacteria over the course of one to two billion years. Mitochondria from the major eukaryotic lineages display some variation in functions and coding capacity but sequence analysis demonstrates them to be derived from a single common ancestral endosymbiont. The loss of assorted functions, the transfer of genes to the nucleus, and the acquisition of various 'eukaryotic' proteins have resulted in an organelle that contains approximately 1000 different proteins, with most of these proteins imported into the organelle across one or two membranes. A single translocase in the outer membrane and two translocases in the inner membrane mediate protein import. Comparative sequence analysis and functional complementation experiments suggest some components of the import pathways to be directly derived from the eubacterial endosymbiont's own proteins, and some to have arisen 'de novo' at the earliest stages of 'mitochondrification' of the endosymbiont. A third class of components appears lineage-specific, suggesting they were incorporated into the process of protein import long after mitochondria was established as an organelle and after the divergence of the various eukaryotic lineages. Protein sorting pathways inherited from the endosymbiont have been co-opted and play roles in intraorganelle protein sorting after import. The import apparatus of animals and fungi show significant similarity to one another, but vary considerably to the plant apparatus. Increasing complexity in the eukaryotic lineage, i.e., from single celled to multi-cellular life forms, has been accompanied by an expansion in genes encoding each component, resulting in small gene families encoding many components. The functional differences in these gene families remain to be elucidated, but point to a mosaic import apparatus that can be regulated by a variety of signals.

Bacterial Physiological Phenomena↗

What we have learned about prokaryotes from structural genomics.

Five years ago systematic determination and theoretical analysis of all protein structures encoded in model prokaryotic organisms was proposed as a powerful way to obtain new insights into protein function and the variety of protein folds. What has been the pay-off from studying structures in genomic context? Have we learned anything new about protein structure? Can we now predict protein function better? In this contribution, I summarize the status of large-scale structure determination projects on prokaryotes and provide an overview of the main results obtained from experimental and theoretical studies in this dynamic research field.

Bacterial Proteins↗

Multilocus sequence typing of hospital-associated Enterococcus faecium from Brazil reveals their unique evolutionary history.

We studied the genetic relationships between vancomycin-susceptible (n = 11) and -resistant Enterococcus faecium (VRE, n = 20) recovered from Brazil using a multilocus sequence typing (MLST) scheme. Grouping of allelic profiles revealed six clusters of related sequence types (STs) that differ in no more than two of the seven alleles. Of these, one cluster harbored 16 of the 20 isolates recovered during the first VRE outbreak in Brazil. The ampicillin and gentamicin resistance profiles were stable in the isolates that clustered within the groups I-III. Comparison with the allelic profiles of 139 E. faecium from different geographical regions and origins found in the international database http://www.mlst.net revealed that the Brazilian outbreak clone did not cluster in the previously named complex-17. This genetic complex contains hospital epidemic and clinical isolates recovered from different countries and continents. Twenty two of the 31 Brazilian isolates, including the VRE outbreak clone, clustered apart from the E. faecium isolates from the database, suggesting that these Brazilian isolates have a distinct evolutionary history.

Alleles↗