The Xylella fastidosa RTX operons: evidence for the evolution of protein mosaics through novel genetic exchanges.

BACKGROUND: Xylella fastidiosa (Xf) is a gram negative bacterium inhabiting the plant vascular system. In most species this bacterium lives as a benign symbiote, but in several agriculturally important plants (e.g. coffee, citrus, grapevine) Xf is pathogenic. Xf has four loci encoding homologues to hemolysin RTX proteins, virulence factors involved in a wide range of plant pathogen interactions. RESULTS: We show that all four genes are expressed during pathogenesis in grapevine. The sequences from these four genes have a complex repetitive structure. At the C-termini, sequence diversity between strains is what would be expected from orthologous genes. However, within strains there is no N-terminal homology, indicating these loci encode RTXs of different functions and/or specificities. More striking is that many of the orthologous loci between strains share this extreme variation at the N-termini. Thus these RTX orthologues are most easily visualized as fusions between the orthologous C-termini and different N-termini. Further, the four genes are found in operons having a peculiar structure with an extensively duplicated module encoding a small protein with homology to the N-terminal region of the full length RTX. Surprisingly, some of these small peptides are most similar not to their corresponding full length RTX, but to the N-termini of RTXs from other Xf strains, and even other remotely related species. CONCLUSIONS: These results demonstrate that these genes are expressed in planta during pathogenesis. Their structure suggests extensive evolutionary restructuring through horizontal gene transfers and heterologous recombination mechanisms. The sum of the evidence suggests these repetitive modules are a novel kind of mobile genetic element.

Evolutionary implications of horizontal gene transfer.

The flow of genes between different species represents a form of genetic variation whose implications have not been fully appreciated. Here I examine some key findings on the extent of horizontal gene transfer (HGT) revealed by comparative genome analysis and their theoretical implications. In theoretical terms, HGT affects ideas pertaining to the tree of life, the notion of a last universal common ancestor, and the biological unities, as well as the rules of taxonomic nomenclature. This review discusses the emergence of the eukaryotic cell and the occurrence of HGT among metazoan phyla involving both transposable elements and structural genes for normal housekeeping functions. I also discuss the bacterial pangenome, which provides an important case study on the permeability of species boundaries. An interesting observation about bdelloid rotifers and their reversion to asexual reproduction as it pertains to HGT is included.

The gut mucosal viral reservoir in HIV-infected patients is not the major source of rebound plasma viremia following interruption of highly active antiretroviral therapy.

Interruption of suppressive highly active antiretroviral therapy (HAART) in HIV-infected patients leads to increased HIV replication and viral rebound in peripheral blood. Effects of therapy interruption on gut-associated lymphoid tissue (GALT) have not been well investigated. We evaluated longitudinal changes in viral replication and emergence of viral variants in the context of T cell homeostasis and gene expression in GALT of three HIV-positive patients who initiated HAART during primary HIV infection but opted to interrupt therapy thereafter. Longitudinal viral sequence analysis revealed that a stable proviral reservoir was established in GALT during primary HIV infection that persisted through early HAART and post-therapy interruption. Proviral variants in GALT and peripheral blood mononuclear cells (PBMCs) displayed low levels of genomic diversity at all times. A rapid increase in viral loads with a modest decline of CD4(+) T cells in peripheral blood was observed, while gut mucosal CD4(+) T cell loss was severe following HAART interruption. This was accompanied by increased mucosal gene expression regulating interferon (IFN)-mediated antiviral responses and immune activation, a profile similar to those found in HAART-naive HIV-infected patients. Sequence analysis of rebound virus suggested that GALT was not the major contributor to the postinterruption plasma viremia nor were GALT HIV reservoirs rapidly replaced by HIV rebound variants. Our data suggest an early establishment and persistence of viral reservoirs in GALT with minimal diversity. Early detection of and therapy for HIV infection may be beneficial in controlling viral evolution and limiting establishment of diverse viral reservoirs in the mucosal compartment.

Evolution of nef variants in gut associated lymphoid tissue of rhesus macaques during primary simian immunodeficiency virus infection.

We utilized the simian immunodeficiency virus model of AIDS to examine evolution of nef gene in gut-associated lymphoid tissue (GALT) during primary and early asymptomatic stages of infection. Macaques were infected with a cloned virus, SIVmac239/nef-stop harboring a premature stop codon in the nef gene. Restoration of the nef open reading frame occurred in GALT early at 3 days post-infection. Analysis of nef sequences by phylogenetic tools showed that evolution of nef was neutral thereafter, as evidenced by the ratio of synonymous to nonsynonymous substitutions, a star pattern in unrooted trees and distribution of amino acid replacements fitting a simple Poisson process. Two regions encoding for a nuclear localization signal and a CTL epitope were conserved. Thus, GALT was a site for strong positive selection of functional nef during initial stages of infection. However, evolution of the nef gene thereafter was neutral during early asymptomatic stage of infection.

Transcription profile of Helicobacter pylori in the human stomach reflects its physiology in vivo.

BACKGROUND: Little is known about levels of expression of Helicobacter pylori genes in the human host. We therefore developed a quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) assay to measure transcript profiles of H. pylori in the human stomach. METHODS: In vivo expression of 16 genes on the cag pathogenicity island and of 18 putative virulence genes was quantitated by isolation of total RNA directly from infected human gastric mucosa. The results were compared with in vitro expression determined from H. pylori cells grown in culture. RESULTS: The highest levels of expression were found for cag1 and cag25 and for genes, such as urease and catalase, that may be important for bacterial homeostasis in the relatively hostile environment of the gastric mucosa. Transcript abundance, relative to 16S rRNA, was lower in vivo than in vitro, which suggests that H. pylori cells are in stationary phase in the gastric environment. This was particularly apparent for cagA. Since CagA is arguably of unique importance, in terms of interaction with the host, tight control of its in vivo expression might be particularly important. CONCLUSIONS: qRT-PCR is a powerful tool to measure gene expression in human or animal tissue that contains minute amounts of microbial mRNA, and the results reflect on the physiology of the pathogen in its natural host.

Modification of Helicobacter pylori outer membrane protein expression during experimental infection of rhesus macaques.

Clinical isolates of Helicobacter pylori show marked diversity, which may derive from genomic changes that occur during the often lifelong association of the bacterium with its human host. We used the rhesus macaque model, together with DNA microarrays, to examine genomic changes in H. pylori that occur early during experimental infection. Microarray analysis showed that H. pylori recovered from challenged macaques had deleted babA, a member of a large family of paralogous outer membrane proteins (OMPs) that mediates attachment of H. pylori to the Lewis B blood group antigen on gastric epithelium. In some cases the babA gene was replaced by babB, an uncharacterized OMP that is closely related to babA. In other cases the babA gene was present but was not expressed because of alteration in dinucleotide CT repeats in the 5' coding region. In either case, strains lacking babA did not adhere to Lewis B, which is expressed on macaque gastric epithelium. Absence of babA and duplication of babB was also seen in H. pylori isolates derived from human clinical samples, suggesting that this gene conversion event is not unique to experimentally infected rhesus monkeys. These results demonstrate in real time with a relevant animal model that H. pylori regulates OMP expression in vivo by using both antigenic variation and phase variation. We suggest that changes in babA and babB after experimental infection of macaques represent a dynamic response in the H. pylori outer membrane that facilitates adherence to the gastric epithelium and promotes chronic infection.

Rates of ribosomal RNA evolution are uniquely accelerated in eukaryotes.

A novel procedure for testing the relative rates of evolution is described. The procedure, the distance-matrix rate test, consists of creating a graph that displays two complete distance matrices for two different genes derived from the same group of species, an approach made practical by numerous whole genomic sequences. The results in this paper show that the molecular clock of ribosomal RNA from Eukaryotes is uniquely accelerated and highly variable while those of Archaea and Bacteria are not. This idiosyncratic eukaryotic rRNA evolution is not observed with four different protein genes. The distance matrix rate test consists of plotting the distance of one gene (from two different species) against the distance of a second gene (from the same pair of species) in the form of a simple X-Y plot. Because it is not possible to compute variances (or co-variances in this case) that can be meaningfully compared to expectations from a Poisson process, the test does not permit calculations of an index of dispersion. In place of this, equations are given for the 95% confidence limits expected for a Poisson process. The test was applied to the proteins rpsl1 and rp114, as one example, and to rps11 and ssu rRNA as a second example. In addition, the cytochrome c and cytochrome c oxidase evolution from a larger group of Eukaryotes are compared to each other and that of the ssu rRNA. This graphical test shows that the evolution of the four proteins and the archael and bacterial ssu rRNA's are consistent with a Poisson process since last common ancestor. The distance-matrix rate test that is introduced in this study needs to make no assumptions regarding evolutionary rates, divergence times, or phylogenetic relationships.

Recent emergence of the modern genetic code: a proposal.

This article proposes that the genetic code was not fully formed before the divergence of life into three kingdoms. Rather, at least arginine and tryptophan evolved after the diversification of archaea, bacteria and eukaryotes, and were spread by horizontal gene transfer. Evidence for this hypothesis is based on data suggesting that enzymes for biosynthesis of arginine and tryptophan, and for arginine tRNA ligase, have shorter divergence times than the underlying lineages. Also, many of these genes display "star" phylogenies. This proposal is an extension of the idea that the genetic code was unified because of the evolutionary pressure from horizontal gene transfer. These considerations further undermine the need to postulate the existence of a "last common ancestor"; a simpler model would be that multiple lineages gave rise to life today.

On the occurrence of horizontal gene transfer among an arbitrarily chosen group of 26 genes.

The deduced amino acid sequences from 1200 Haemophilus influenzae genes was compared to a data set that contained the orfs from yeast, two different Archaea and the Gram+ and Gramminus sign bacteria, Bacillus subtilis and Escherichia coli. The results of the comparison yielded a 26 orthologous gene set that had at least one representative from each of the four groups. A four taxa phylogenetic relationship for these 26 genes was determined. The statistical significance of each minimal tree was tested against the two alternative four taxa trees. The result was that four genes significantly supported the (Archaea, Eukaryota) (Gram+, Gramminus sign) topology, two genes supported the one where Gramminus sign and Eukaryota form a clade, and one gene supported the tree where Gram+ and Eukaryota define one clade. The remaining genes do not uniquely support any phylogeny, thereby collapsing the two central nodes into a single node. These are referred to as star phylogenies. I offer a new suggestion for the mechanism that gave rise to the star phylogenies. Namely, these are genes that are younger than the underlying lineages that currently harbor them. This hypothesis is examined with two proteins that display the star phylogeny; namely onithine transcarbamylase and tryptophan synthetase. It is shown, using the distance matrix rate test, that the rate of evolution of these two proteins is comparable to a control gene when rates are determined by comparing closely related species. This implies that the genes under comparison experience comparable functional constraint. However, when the genes from remotely related species are compared, a plateau is encountered. Since we see no unusual levels of functional constraint this plateau cannot be attributed to the divergence of the protein having reached saturation. The simplest explanation is that the genes displaying the star phylogenies were introduced after Archaea, Eukaryota, and Bacteria had diverged from one another. They presumably spread through life by horizontal gene transfer.