Regulation of transcription in a reduced bacterial genome: nutrient-provisioning genes of the obligate symbiont Buchnera aphidicola.
ABSTRACT: Buchnera aphidicola, the obligate symbiont of aphids, has an extremely reduced genome, of which about 10% is devoted to the biosynthesis of essential amino acids needed by its hosts. Most regulatory genes for these pathways are absent, raising the question of whether and how transcription of these genes responds to the major shifts in dietary amino acid content encountered by aphids. Using full-genome microarrays for B. aphidicola of the host Schizaphis graminum, we examined transcriptome responses to changes in dietary amino acid content and then verified behavior of individual transcripts using quantitative reverse transcriptase PCR. The only gene showing a consistent and substantial (>twofold) response was metE, which underlies methionine biosynthesis and which is the only amino acid biosynthetic gene retaining its ancestral regulator (metR). In another aphid host, Acyrthosiphon pisum, B. aphidicola has no functional metR and shows no response in metE transcript levels to changes in amino acid concentrations. Thus, the only substantial transcriptional response involves the one gene for which an ancestral regulator is retained. This result parallels that from a previous study on heat stress, in which only the few genes retaining the global heat shock promoter showed responses in transcript abundance. The irreversible losses of transcriptional regulators constrain ability to alter gene expression in the context of environmental fluctuations affecting the symbiotic partners.
Project description:A 8,392-nucleotide-long DNA fragment from Buchnera aphidicola (endosymbiont of the aphid Schizaphis graminum) contained five genes of the tryptophan biosynthetic pathway [trpDC(F)BA] which code for enzymes converting anthranilate to tryptophan. These genes are probably arranged as a single transcription unit. Downstream of the trp genes were ORF-V, ORF-VI, and P14, three open reading frames which in Escherichia coli are also found downstream of the trp operon. Upstream of the B. aphidicola trp genes were two unidentified open reading frames, one of which potentially codes for a membrane-spanning protein with a leader sequence. Evidence for the presence of trpB in the endosymbionts of eight additional species of aphids and two species of whiteflies was obtained. These results as well as those of A. E. Douglas and W. A. Prosser (J. Insect Physiol. 38:565-568, 1992) suggest that aphid endosymbionts are capable of synthesizing tryptophan, which is required by the aphid host.
Project description:The symbiotic association between aphids (Homoptera) and Buchnera aphidicola (Gammaproteobacteria) started about 100 to 200 million years ago. As a consequence of this relationship, the bacterial genome has undergone a prominent size reduction. The downsize genome process starts when the bacterium enters the host and will probably end with its extinction and replacement by another healthier bacterium or with the establishment of metabolic complementation between two or more bacteria. Nowadays, several complete genomes of Buchnera aphidicola from four different aphid species (Acyrthosiphon pisum, Schizaphis graminum, Baizongia pistacea, and Cinara cedri) have been fully sequenced. C. cedri belongs to the subfamily Lachninae and harbors two coprimary bacteria that fulfill the metabolic needs of the whole consortium: B. aphidicola with the smallest genome reported so far and "Candidatus Serratia symbiotica." In addition, Cinara tujafilina, another member of the subfamily Lachninae, closely related to C. cedri, also harbors "Ca. Serratia symbiotica" but with a different phylogenetic status than the one from C. cedri. In this study, we present the complete genome sequence of B. aphidicola from C. tujafilina and the phylogenetic analysis and comparative genomics with the other Buchnera genomes. Furthermore, the gene repertoire of the last common ancestor has been inferred, and the evolutionary history of the metabolic losses that occurred in the different lineages has been analyzed. Although stochastic gene loss plays a role in the genome reduction process, it is also clear that metabolism, as a functional constraint, is also a powerful evolutionary force in insect endosymbionts.
Project description:An analysis of the impact of infection by Buchnera aphidicola APS (isolated from Acyrthosiphon pisum strain LL01) on gene expression of S2 cells. All comparisons are made against a pool of RNA from S2 cells not exposed to B. aphidicola. B. aphidicola freshly isolated from the aphids, and data are collected at 1, 6 and 24 hours after exposure of S2 cells to the B. aphidicola preparation. Keywords: time course, Buchnera aphidicola APS, aphids, Drosophila melanogaster S2 cells Overall design: All comparisons are made against a pool of RNA from S2 cells not exposed to B. aphidicola. B. aphidicola freshly isolated from the aphids, and data are collected at 1, 6 and 24 hours after exposure of S2 cells to the B. aphidicola preparation.
Project description:Aphids, important agricultural pests, can grow and reproduce thanks to their intimate symbiosis with the ?-proteobacterium Buchnera aphidicola that furnishes them with essential amino acids lacking in their phloem sap diet. To study how B. aphidicola, with its reduced genome containing very few transcriptional regulators, responds to variations in the metabolic requirements of its host, we concentrated on the leucine metabolic pathway. We show that leucine is a limiting factor for aphid growth and it displays a stimulatory feeding effect. Our metabolic analyses demonstrate that symbiotic aphids are able to respond to leucine starvation or excess by modulating the neosynthesis of this amino acid. At a molecular level, this response involves an early important transcriptional regulation (after 12 h of treatment) followed by a moderate change in the pLeu plasmid copy number. Both responses are no longer apparent after 7 days of treatment. These experimental data are discussed in the light of a re-annotation of the pLeu plasmid regulatory elements. Taken together, our data show that the response of B. aphidicola to the leucine demand of its host is multimodal and dynamically regulated, providing new insights concerning the genetic regulation capabilities of this bacterium in relation to its symbiotic functions.
Project description: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.
Project description:The best studied insect-symbiont system is that of aphids and their primary bacterial endosymbiont Buchnera aphidicola. Buchnera inhabits specialized host cells called bacteriocytes, provides nutrients to the aphid and has co-speciated with its aphid hosts for the past 150 million years. We have used a single microarray to examine gene expression in the pea aphid, Acyrthosiphon pisum, and its resident Buchnera. Very little is known of gene expression in aphids, few studies have examined gene expression in Buchnera, and no study has examined simultaneously the expression profiles of a host and its symbiont. Expression profiling of aphids, in studies such as this, will be critical for assigning newly discovered A. pisum genes to functional roles. In particular, because aphids possess many genes that are absent from Drosophila and other holometabolous insect taxa, aphid genome annotation efforts cannot rely entirely on homology to the best-studied insect systems. Development of this dual-genome array represents a first attempt to characterize gene expression in this emerging model system.We chose to examine heat shock response because it has been well characterized both in Buchnera and in other insect species. Our results from the Buchnera of A. pisum show responses for the same gene set as an earlier study of heat shock response in Buchnera for the host aphid Schizaphis graminum. Additionally, analyses of aphid transcripts showed the expected response for homologs of known heat shock genes as well as responses for several genes with unknown functional roles.We examined gene expression under heat shock of an insect and its bacterial symbiont in a single assay using a dual-genome microarray. Further, our results indicate that microarrays are a useful tool for inferring functional roles of genes in A. pisum and other insects and suggest that the pea aphid genome may contain many gene paralogs that are differentially regulated.
Project description:A plasmid (pRSE562) containing the metE and metR genes of Escherichia coli was used to study the expression of these genes and the role of the MetR protein in regulating metE expression. DNA sequence analysis of the 236-base-pair region separating these genes showed the presence of seven putative met boxes. When this plasmid was used to transform either wild-type E. coli, metE mutant, or metR mutant, MetE enzyme activity increased 5- to 7-fold over wild-type levels. The metR gene was subcloned from pRSE562, and this plasmid, pMRIII, relieved the methionine auxotrophy of a metR mutant after transformation. The metR gene was also cloned into a vector containing the lambda PL promoter, and the MetR protein was overexpressed and purified to near homogeneity. This protein, when added to an in vitro DNA-dependent protein synthesis system in which the MetE and/or MetR proteins were synthesized, caused a large increase in the expression of the metE gene but a decrease in the expression of the metR gene. The in vitro expression of both genes was inhibited by the MetJ protein and S-adenosylmethionine in the presence or absence of MetR protein. These results provide evidence that the product of the metR gene is a trans-activator of the expression of the metE gene and that the expression of the metR gene is under autogenous regulation and is repressed by the MetJ protein.
Project description:Intracellular symbiosis is very common in the insect world. For the aphid Cinara cedri, we have identified by electron microscopy three symbiotic bacteria that can be characterized by their different sizes, morphologies, and electrodensities. PCR amplification and sequencing of the 16S ribosomal DNA (rDNA) genes showed that, in addition to harboring Buchnera aphidicola, the primary endosymbiont of aphids, C. cedri harbors a secondary symbiont (S symbiont) that was previously found to be associated with aphids (PASS, or R type) and an alpha-proteobacterium that belongs to the Wolbachia genus. Using in situ hybridization with specific bacterial probes designed for symbiont 16S rDNA sequences, we have shown that Wolbachia was represented by only a few minute bacteria surrounding the S symbionts. Moreover, the observed B. aphidicola and the S symbionts had similar sizes and were housed in separate specific bacterial cells, the bacteriocytes. Interestingly, in contrast to the case for all aphids examined thus far, the S symbionts were shown to occupy a similarly sized or even larger bacteriocyte space than B. aphidicola. These findings, along with the facts that C. cedri harbors the B. aphidicola strain with the smallest bacterial genome and that the S symbionts infect all Cinara spp. analyzed so far, suggest the possibility of bacterial replacement in these species.
Project description:Buchnera aphidicola is an obligate symbiotic bacterium that sustains the physiology of aphids by complementing their exclusive phloem sap diet. In this study, we reappraised the transport function of different Buchnera strains, from the aphids Acyrthosiphon pisum, Schizaphis graminum, Baizongia pistaciae and Cinara cedri, using the re-annotation of their transmembrane proteins coupled with an exploration of their metabolic networks. Although metabolic analyses revealed high interdependencies between the host and the bacteria, we demonstrate here that transport in Buchnera is assured by low transporter diversity, when compared to free-living bacteria, being mostly based on a few general transporters, some of which probably have lost their substrate specificity. Moreover, in the four strains studied, an astonishing lack of inner-membrane importers was observed. In Buchnera, the transport function has been shaped by the distinct selective constraints occurring in the Aphididae lineages. Buchnera from A. pisum and S. graminum have a three-membraned system and similar sets of transporters corresponding to most compound classes. Transmission electronic microscopic observations and confocal microscopic analysis of intracellular pH fields revealed that Buchnera does not show any of the typical structures and properties observed in integrated organelles. Buchnera from B. pistaciae seem to possess a unique double membrane system and has, accordingly, lost all of its outer-membrane integral proteins. Lastly, Buchnera from C. cedri revealed an extremely poor repertoire of transporters, with almost no ATP-driven active transport left, despite the clear persistence of the ancestral three-membraned system.
Project description:An analysis of the impact of infection by Buchnera aphidicola APS (isolated from Acyrthosiphon pisum strain LL01) on gene expression of S2 cells. All comparisons are made against a pool of RNA from S2 cells not exposed to B. aphidicola. B. aphidicola freshly isolated from the aphids, and data are collected at 1, 6 and 24 hours after exposure of S2 cells to the B. aphidicola preparation. Keywords: time course, Buchnera aphidicola APS, aphids, Drosophila melanogaster S2 cells All comparisons are made against a pool of RNA from S2 cells not exposed to B. aphidicola. B. aphidicola freshly isolated from the aphids, and data are collected at 1, 6 and 24 hours after exposure of S2 cells to the B. aphidicola preparation.