Surfeit locus gene homologs are widely distributed in invertebrate genomes.

The mouse Surfeit locus contains six sequence-unrelated genes (Surf-1 to -6) arranged in the tightest gene cluster so far described for mammals. The organization and juxtaposition of five of the Surfeit genes (Surf-1 to -5) are conserved between mammals and birds, and this may reflect a functional or regulatory requirement for the gene clustering. We have undertaken an evolutionary study to determine whether the Surfeit genes are conserved and clustered in invertebrate genomes. Drosophila melanogaster and Caenorhabditis elegans homologs of the mouse Surf-4 gene, which encodes an integral membrane protein associated with the endoplasmic reticulum, have been isolated. The amino acid sequences of the Drosophila and C. elegans homologs are highly conserved in comparison with the mouse Surf-4 protein. In particular, a dilysine motif implicated in endoplasmic reticulum localization of the mouse protein is conserved in the invertebrate homologs. We show that the Drosophila Surf-4 gene, which is transcribed from a TATA-less promoter, is not closely associated with other Drosophila Surfeit gene homologs but rather is located upstream from sequences encoding a homolog of a yeast seryl-tRNA synthetase protein. There are at least two closely linked Surf-3/rpL7a genes or highly polymorphic alleles of a single Surf-3/rpL7a gene in the C. elegans genome. The chromosomal locations of the C. elegans Surf-1, Surf-3/rpL7a, and Surf-4 genes have been determined. In D. melanogaster the Surf-3/rpL7a, Surf-4, and Surf-5 gene homologs and in C. elegans the Surf-1, Surf-3/rpL7a, Surf-4, and Surf-5 gene homologs are located on completely different chromosomes, suggesting that any requirement for the tight clustering of the genes in the Surfeit locus is restricted to vertebrate lineages.     

Mutation discovery in bacterial genomes: metronidazole resistance in Helicobacter pylori

We developed a microarray hybridization-based method, 'comparative genome sequencing' (CGS), to find mutations in bacterial genomes and used it to study metronidazole resistance in H. pylori. CGS identified mutations in several genes, most likely affecting metronidazole activation, and produced no false positives in analysis of three megabases. We conclude that CGS identifies mutations in bacterial genomes efficiently, should enrich understanding of systems biology and genome evolution, and help track pathogens during outbreaks.     

A tale of two genomes: contrasting patterns of phylogeographic structure in a widely distributed bat

One of the most widely distributed bats in the New World, the big brown bat (Eptesicus fuscus) exhibits well-documented geographic variation in morphology and life history traits, suggesting the potential for significant phylogeographic structure as well as adaptive differentiation among populations. In a pattern broadly consistent with morphologically defined subspecies, we found deeply divergent mitochondrial lineages restricted to different geographic regions. In contrast, sequence data from two nuclear loci suggest a general lack of regional genetic structure except for peripheral populations in the Caribbean and Mexico/South America. Coalescent analyses suggest that the striking difference in population structure between genomes cannot be attributed solely to different rates of lineage sorting, but is likely due to male-mediated gene flow homogenizing nuclear genetic diversity across most of the continental range. Despite this ongoing gene flow, selection has apparently been effective in producing and maintaining adaptive differentiation among populations, while strong female site fidelity, maintained over the course of millions of years, has produced remarkably deep divergence among geographically isolated matrilines. Our results highlight the importance of evaluating multiple genetic markers for a more complete understanding of population structure and history.     

Phosphoenolpyruvate:glycose phosphotransferase system in species of Vibrio, a widely distributed marine bacterial genus.

The genus Vibrio is one of the most common and widely distributed groups of marine bacteria. Studies on the physiology of marine Vibrio species were initiated by examining 15 species for the bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS). All species tested contained a PTS analogous to the glucose-specific (IIGlc) system in enteric bacteria. Crude extracts of the cells showed immunological cross-reactivity with antibodies to enzyme I, HPr, and IIIGlc from Salmonella typhimurium when assayed by the rocket-line method. Toluene-permeabilized cells of 11 species were tested and were active in phosphorylating methyl alpha-D-glucoside with phosphoenolpyruvate but not ATP as the phosphoryl donor. Membranes from 10 species were assayed, and they phosphorylated methyl alpha-D-glucoside when supplemented with a phospho-IIIGlc-generating system composed of homogeneous proteins from enteric bacteria. Toluene-permeabilized cells and membranes of seven species were assayed, as were phosphorylated fructose and 2-deoxyglucose. IIIGlc was isolated from Vibrio fluvialis and was active in phosphorylating methyl alpha-D-glucoside when supplemented with a phospho-HPr-generating system composed of homogeneous proteins from Escherichia coli and membranes from either E. coli or V. fluvialis. These results show that the bacterial PTS is widely distributed in the marine environment and that it is likely to have a significant role in marine bacterial physiology and in the marine ecosystem.     

The generation of variation in bacterial genomes

In the context of a general overview of molecular mechanisms of microbial evolution, several genetic systems known to either promote or restrain the generation of genetic variations are discussed. Particular attention is given to functions involved in DNA rearrangements and DNA acquisition. Sporadic actions by a variety of such systems influencing genetic stability in either way result in a level of genetic plasticity which is tolerable to the overall wealth of microbial populations but which allows for evolutionary change needed for a steady adaptation to variable selective forces. Although these evolutionarily relevant biological functions are encoded by the genome of each individual, their actions are exerted to some degree randomly in rare individuals and are therefore seemingly nondeterministic and become manifest at the population level.     

The evolution of domain-content in bacterial genomes

Background  

Across all sequenced bacterial genomes, the number of domains n _c in different functional categories c scales as a power-law in the total number of domains n, i.e. , with exponents α _c that vary across functional categories. Here we investigate the implications of these scaling laws for the evolution of domain-content in bacterial genomes and derive the simplest evolutionary model consistent with these scaling laws.     

The Neolithic revolution of bacterial genomes

Current human activities undoubtedly impact natural ecosystems. However, the influence of Homo sapiens on living organisms must have also occurred in the past. Certain genomic characteristics of prokaryotes can be used to study the impact of ancient human activities on microorganisms. By analyzing DNA sequence similarity features of transposable elements, dramatic genomic changes have been identified in bacteria that are associated with large and stable human communities, agriculture and animal domestication: three features unequivocally linked to the Neolithic revolution. It is hypothesized that bacteria specialized in human-associated niches underwent an intense transformation after the social and demographic changes that took place with the first Neolithic settlements. These genomic changes are absent in related species that are not specialized in humans.     

An epitope of elongation factor Tu is widely distributed within the bacterial and archaeal domains.

A monoclonal antibody (MAb), MAb 900, which detects a 43-kDa protein present on Escherichia coli was found. Subsequently, more than 90 organisms, belonging to either the bacterial, archaeal, or eucaryal domain, were tested for reactivity to this MAb. Of the bacterial and archaeal domains, almost all species proved to be positive, whereas all organisms from the eucaryal domain gave negative results. The 43-kDa protein was purified by affinity chromatography and subsequently analyzed by microsequencing methods. Two peptide sequences which showed a high degree of homology (> 99%) to the prokaryotic elongation factor Tu (EF-Tu) were obtained. Western blot (immunoblot) analysis using both purified EF-Tu and EF-Tu domains confirmed that the unknown protein was EF-Tu. The panbacterial distribution of EF-Tu, which is present in large amounts in every prokaryotic cell, renders this protein a good candidate for a diagnostic approach. In consequence, we have used the anti-EF-Tu MAb 900 to design both a dot blot assay and an enzyme-linked immunosorbent assay. From either blood culture, urine, or gall-bladder fluid, bacterial contamination could be detected. The sensitivity of these tests is currently 10(4) bacteria per ml.     

The discovery of mRNA interferases: implication in bacterial physiology and application to biotechnology

Escherichia coli contains a large number of suicide or toxin genes, whose expression leads to cell growth arrest and eventual cell death. This raises intriguing questions as to why E. coli contains so many toxin genes and what are their roles in bacterial physiology. Among these, MazF has been shown to be a sequence-specific endoribonuclease, which cleaves mRNAs at ACA sequences to completely inhibit protein synthesis. MazF is therefore called mRNA interferase. A number of other mRNA interferases with different cleavage specificities have been discovered not only in E. coli, but also in other bacteria including Mycobacterium tuberculosis. Induction of MazF in the cell leads to cellular dormancy termed quasi-dormancy. In spite of complete cell growth inhibition, cells in the quasi-dormant state are fully capable of energy metabolism, amino acids and nucleic acids biosynthesis and RNA and protein synthesis. The quasi-dormancy may be implicated in cell survival under stress conditions and may play a major role in pathogenicity of M. tuberculosis. The quasi-dormant cells provide an intriguing novel biotechnological system producing only a protein of interest in a high yield. MazF causing Bak-dependent programmed cell death in mammalian cells may be used as a tool for gene therapy against cancer and AIDS. The discovery of a novel way to interfere with mRNA function by mRNA interferases opens a wide variety of avenues in basic as well as applied and clinical sciences.     

The source of laterally transferred genes in bacterial genomes

Background  

Laterally transferred genes have often been identified on the basis of compositional features that distinguish them from ancestral genes in the genome. These genes are usually A+T-rich, arguing either that there is a bias towards acquiring genes from donor organisms having low G+C contents or that genes acquired from organisms of similar genomic base compositions go undetected in these analyses.     

The discovery of highly potent CGRP receptor antagonists

Rational modification of a previously identified spirohydantoin lead structure has identified a series of potent spiroazaoxindole CGRP receptor antagonists. The azaoxindole was found to be a general replacement for the hydantoin that consistently improved in vitro potency. The combination of the indanylspiroazaoxindole and optimized benzimidazolinones led to highly potent antagonists (e.g., 25, CGRP K_i = 40 pM). The closely related compound 27 demonstrated good oral bioavailability in dog and rhesus.     

The discovery of potent cRaf1 kinase inhibitors

A series of benzylidene-1H-indol-2-one (oxindole) derivatives was synthesized and evaluated as cRaf-1 kinase inhibitors. The key features of the molecules were the donor/acceptor motif common to kinase inhibitors and a critical acidic phenol flanked by two substitutions. Diverse 5-position substitutions provided compounds with low nanomolar kinase enzyme inhibition and inhibited the intracellular MAPK pathway.