Sequence Analysis of a DNA Fragment from Buchnera aphidicola (Aphid Endosymbiont) Containing the Genes dapD-htrA-ilvI-ilvH-ftsL-ftsI-murE

Buchnera aphidicola is a prokaryotic endosymbiont of the aphid Schizaphis graminum. One of the endosymbiont's functions is the synthesis of branched-chain amino acids. A 9.7-kilobase B. aphidicola chromosomal DNA fragment was cloned and sequenced and found to contain genes encoding acetohydroxy acid synthase (ilvIH), the first enzyme of the parallel pathway of isoleucine and valine biosynthesis. Previously we have detected ilvC and ilvD, encoding the two other enzymes of this pathway. In addition the DNA fragment contained genes for cell division (ftsL, ftsI), murein biosynthesis (murE), lysine biosynthesis (dapD) and a periplasmic protease (htrA). In these properties B. aphidicola resembles free-living bacteria. Received: 25 April 1998 / Accepted: 28 April 1998     

Ribosomal Protein S1(RpsA) of Buchnera aphidicola,the Endosymbiont of Aphids: Characterization of the Gene and Detection of the Product

Buchnera aphidicola is a prokaryotic endosymbiont found in specialized cells of the aphid Schizaphis graminum. Many of the previously cloned B. aphidicola genes are preceded by a poor ribosome-binding site. Ribosomal protein S1 (RpsA) allows the translation of messenger RNAs that lack or have a poor ribosome binding site. We have cloned and sequenced a 4.5-kilobase (kb) B. aphidicola DNA fragment containing four open reading frames corresponding to aroA–rpsA–himD–tpiA. The deduced amino acid sequence of B. aphidicola RpsA was 75% identical to that of the Escherichia coli protein. The major difference was in the number of basic amino acids, which were present in higher numbers in B. aphidicola RpsA. Antiserum to E. coli RpsA was prepared and used to detect B. aphidicola RpsA in cell-free extracts of aphids. During the first 12 days of aphid growth there is a slight decrease in the amount of RpsA per unit of aphid weight. The three additional genes found on the 4.5-kb DNA fragment encoded for proteins involved in aromatic amino acid biosynthesis (aroA), DNA bending (himD), and carbohydrate metabolism (tpiA). The presence of these genes in B. aphidicola is additional evidence of its similarity to free-living bacteria.     

Aromatic amino acid biosynthesis in Buchnera aphidicola (Endosymbiont of aphids): Cloning and sequencing of a DNA fragment containing aroH-thrS-infC-rpmI-rplT

A 4.5-kilobase DNA fragment from Buchnera aphidicola, the endosymbiont of the aphid Schizaphis graminum, was cloned and sequenced. On the basis of homology to Escherichia coli, the following genes were found in the order listed: aroH-thrS-infC-rpmI-rplT. AroH corresponds to the E. coli tryptophan-inhibited 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase. Evidence was presented indicating that this is the sole gene for DAHP synthase in the B. aphidicola genome. This enzyme initiates the complex branched pathway leading to aromatic amino acid biosynthesis. The presence of aroH is consistent with past observations indicating that aphid endosymbionts are able to synthesize tryptophan for the aphid host. thrS, infC, rpmI, and rplT correspond to genes for threonine tRNA synthase, initiation factor-3, and large ribosome subunit proteins L35 and L20, respectively. Sequence comparisons indicate some differences and similarities between E. coli and B. aphidicola with respect to the possible regulation of synthesis of these proteins.     

Characterization of ftsZ, the Cell Division Gene of Buchnera aphidicola (Endosymbiont of Aphids) and Detection of the Product

Buchnera aphidicola, the endosymbiont of the aphid Schizaphis graminum, contains the gene ftsZ, which codes for a protein involved in the initiation of septum formation during cell division. With immunological techniques, this protein has been detected in cell-free extracts of the endosymbiont. Nucleotide sequence determination of a 6.4-kilobase B. aphidicola DNA fragment has indicated that, as in E. coli, ftsZ is adjacent to genes coding for other cell division proteins as well as genes involved in murein synthesis (murC–ddlB–ftsA–ftsZ). Although B. aphidicola ftsZ is expressed in E. coli, it cannot complement E. coli ftsZ mutants. High levels of B. aphidicola FtsZ results in the formation of long filamentous E. coli cells, suggesting that this protein interferes with cell division. The presence of FtsZ indicates that in this, as well as in many other previously described properties, B. aphidicola resembles free-living bacteria. Received: 22 July 1997 / Accepted: 28 July 1997     

Endosymbionts (Buchnera) from the Aphids Schizaphis graminum and Diuraphis noxia Have Different Copy Numbers of the Plasmid Containing the Leucine Biosynthetic Genes

The prokaryotic endosymbiont (Buchnera) of the aphid Schizaphis graminum contains 24 copies of a plasmid that has genes encoding enzymes of the leucine biosynthetic pathway while the endosymbiont of the related aphid Diuraphis noxia has only one copy of this plasmid. These results, in conjunction with similar results for the trpEG-containing plasmids, suggest that D. noxia has a reduced demand for endosymbiont-derived essential amino acids. Received: 11 September 1997 / Accepted: 23 September 1997     

Sequence Analysis of a 34.7-kb DNA Segment from the Genome of Buchnera aphidicola (Endosymbiont of Aphids) Containing groEL, dnaA, the atp operon, gidA, and rho

Buchnera aphidicola is a prokaryotic endosymbiont of the aphid Schizaphis graminum. From past and present nucleotide sequence analyses of the B. aphidicola genome, we have assembled a 34.7-kilobase (kb) DNA segment. This segment contains genes coding for 32 open reading frames (ORFs), which corresponded to 89.9% of the DNA. All of these ORFs could be identified with homologous regions of the Escherichia coli genome. The order of the genes with established functions was groELS–trmE–rnpA–rpmH–dnaA–dnaN–gyrB–atpCDGAHFEB–gidA–fdx–hscA– hscB–nifS–ilvDC–rep–trxA–rho. The order of genes in small DNA fragments was conserved in both B. aphidicola and E. coli. Most of these fragments were in approximately the same region of the E. coli genome. The latter organism, however, contained many additional inserted genes within and between the fragments. The results of the B. aphidicola genome analyses indicate that the endosymbiont has many properties of free-living bacteria. Received: 15 August 1997 / Accepted: 29 August 1997     

Levels of Buchnera aphidicola Chaperonin GroEL During Growth of the Aphid Schizaphis graminum

Buchnera aphidicola is the prokaryotic, intracellular symbiont found in the aphid Schizaphis graminum. Using an immunological approach, we have quantitated the amount of the B. aphidicola chaperonin, GroEL, present in aphid cell-free extracts during the growth cycle of S. graminum at 23°C. Our results indicate that the increase in GroEL approximately follows the increase in aphid weight and endosymbiont number for the first 12 days after birth of the aphid. A 9-day-old aphid contains 1.6 × 10⁵ molecules of GroEL per μm³ of cell volume. This number is similar to that found in Escherichia coli growing at 46°C, close to its maximal growth temperature, and a condition at which there is a major increase in the levels of chaperonins and other stress proteins. It is estimated that at 23°C, 10% of the B. aphidicola protein is GroEL. When S. graminum grown at 23°C was shifted to 33°C for 1 day and subsequently to 23°C, there was no change in the level of GroEL or the rate of growth. It is possible that the high level of GroEL in the endosymbiont masked an increase in the protein owing to a heat shock response.     

Buchnera aphidicola (Aphid-Endosymbiont) glyceraldehyde-3-phosphate dehydrogenase: Molecular cloning and sequence analysis

Buchnera aphidicola is an endosymbiont of the aphid Schizaphis graminum. A 3.9-kb B. aphidicola DNA fragment was sequenced and found to contain two open reading frames (ORFs). The deduced amino acid sequence of one of the ORFs had an 85% identity to Escherichia coli glyceraldehyde-3-phosphate dehydrogenase (Gap). Both of these proteins have a higher similarity to eukaryotic than to prokaryotic Gaps. The second ORF could not be readily identified. The sequence of the putative product indicated that it was a member of the family of ATP-binding, membrane-associated proteins. The highest amino acid identity (36%) was with E. coli FtsE, a protein involved in cell division.     

News & Notes: Nucleotide Sequence of a DNA Fragment from Buchnera aphidicola (Aphid Endosymbiont) Containing the Genes aspS–trxB–serS–serC–aroA–rpsA–himD–tpiA

Buchnera aphidicola is an endosymbiont of aphids. The nucleotide sequence of an 11.5-kilobase DNA fragment from this prokaryotic organism was determined. Eight open reading frames were found coding for putative proteins involved in protein synthesis, serine and aromatic amino acid biosynthesis, as well as thioredoxin and carbohydrate metabolism. These results indicate that B. aphidicola has many genetic properties of free-living bacteria. Received: 31 December 1996 / Accepted: 6 January 1997     

Growth Kinetics of the Endosymbiont Buchnera aphidicola in the Aphid Schizaphis graminum

The aphid Schizaphis graminum is dependent on its prokaryotic endosymbiont, Buchnera aphidicola. As a means of determining B. aphidicola numbers during the growth cycle of the aphid we have used the quantitative PCR to measure the number of copies of rrs (the gene coding for 16S rRNA, which is present as one copy in the B. aphidicola genome). In addition we have measured the aphid wet weight and the DNA and protein content. The results indicate an approximately parallel (23- to 31-fold) increase of these properties during the period of aphid growth. A 1-day-old aphid (24 μg [wet weight]) has 0.2 × 10⁶ copies of rrs, while a 9-day-old aphid (497 μg [wet weight]) has 5.6 × 10⁶ copies. The coupling of endosymbiont and aphid growth is consistent with the requirement of the endosymbiont for growth and reproduction of the aphid.     

The striking case of tryptophan provision in the cedar aphid Cinara cedri

Buchnera aphidicola BCc has lost its symbiotic role as the tryptophan supplier to the aphid Cinara cedri. We report the presence of a plasmid in this endosymbiont that contains the trpEG genes. The remaining genes for the pathway (trpDCBA) are located on the chromosome of the secondary endosymbiont “Candidatus Serratia symbiotica.” Thus, we propose that a symbiotic consortium is necessary to provide tryptophan.     

News & Notes: Buchnera Plasmid-Associated trpEG Probably Originated from a Chromosomal Location Between hsIU and fpr

Buchnera are prokaryotic endosymbionts found in most aphids. One of their functions is the synthesis of the essential amino acid tryptophan for the aphid host. In Buchnera from some aphids that have a long development time, trpEG, which encodes the first enzyme of the tryptophan biosynthetic pathway (anthranilate synthase), is found as one copy on the endosymbiont chromosome and is located between hsIU and fpr. In Buchnera from Schizaphis graminum, which has a short development time, trpEG is amplified on plasmids. We have cloned and sequenced a 4.1-kb DNA fragment from Buchnera of S. graminum and have found the gene order hsIU-ibp-fpr-yjeA-kdtB. The proximity of hsIU and fpr is consistent with the excision, in an endosymbiont ancestor, of trpEG from a location between these two genes, with the excision either followed or preceded by acquisition of ibp. Received: 5 December 1998 / Accepted: 10 December 1998     

Temperature effect on the growth of <Emphasis Type="Italic">Buchnera</Emphasis> endosymbiont in <Emphasis Type="Italic">Aphis craccivora</Emphasis> (Hemiptera: Aphididae)

Effect of temperature on the growth of the primary endosymbiont Buchnera aphidicola in the cowpea aphid Aphis craccivora was studied by measuring quantitatively the copy number of 16S rDNA of this endosymbiont. A 1.5 kb segment of eubacterial 16S rDNA amplified by PCR from total DNA of Aphis craccivora was confirmed by RFLP analysis and sequence BLAST as that of Buchnera aphidicola. No secondary endosymbiont was detected in the aphid population studied. The relative levels of Buchnera ratio, quantified by real-time PCR, were higher in old nymphs than in young ones at temperatures between 10–30˚C, and this age-dependent difference was more pronounced at lower temperatures. Throughout the entire reproductive stage of Aphis craccivora, the relative levels of Buchnera ratio were higher at 10–25˚C than at 30˚C and 35˚C. A close relationship was found between these levels and the net reproductive rate (R ₀ ) of aphid, which was suppressed not only at 35˚C but also at 10˚C. The decoupling of Aphis craccivora and Buchnera response at low temperatures suggests that the cowpea aphid was more sensitive to low temperatures, while Buchnera was more sensitive to high temperatures.     

Aspects of energy-yielding metabolism in the aphid,Schizaphis graminum,and its endosymbiont: Detection of gene fragments potentially coding for the ATP synthaseβ-subunit and glyceraldehyde-3-phosphate dehydrogenase

Specialized cells within the aphid,Schizaphis graminum, contain intracellular, vesicleenclosed eubacterial endosymbionts (Buchnera aphidicola). Using oligonucleotide probes derived from conserved sequences of the ATP synthase -subunit and glyceraldehyde-3-phosphate dehydrogenase, and the polymerase chain reaction (PCR), we have amplified, cloned, and sequenced three DNA fragments. Amino acid sequence similarity indicated that two of these fragments corresponded to endosymbiont and host genes potentially coding for the -subunit of ATP synthase. The host gene fragment contained two putative introns. The third DNA fragment corresponded to a portion of a gene coding for a glyceraldehyde-3-phosphate dehydrogenase that was highly related to one of the enzymes fromEscherichia coli (GapA). These results indicate thatB. aphidicola may have an ATP synthase and consequently could synthesize ATP from a proton motive force generated within the intracellular vesicles of host cells containing the endosymbionts. The detection of a gene fragment coding for a protein similar to glyceraldehyde-3-phosphate dehydrogenase suggests the presence of this glycolytic enzyme in the endosymbiont and its involvement in energy-yielding metabolism.     

A substrate ambiguous enzyme facilitates genome reduction in an intracellular symbiont

Background

Genome evolution in intracellular microbial symbionts is characterized by gene loss, generating some of the smallest and most gene-poor genomes known. As a result of gene loss these genomes commonly contain metabolic pathways that are fragmented relative to their free-living relatives. The evolutionary retention of fragmented metabolic pathways in the gene-poor genomes of endosymbionts suggests that they are functional. However, it is not always clear how they maintain functionality. To date, the fragmented metabolic pathways of endosymbionts have been shown to maintain functionality through complementation by host genes, complementation by genes of another endosymbiont and complementation by genes in host genomes that have been horizontally acquired from a microbial source that is not the endosymbiont. Here, we demonstrate a fourth mechanism.

Results

We investigate the evolutionary retention of a fragmented pathway for the essential nutrient pantothenate (vitamin B5) in the pea aphid, Acyrthosiphon pisum endosymbiosis with Buchnera aphidicola. Using quantitative analysis of gene expression we present evidence for complementation of the Buchnera pantothenate biosynthesis pathway by host genes. Further, using complementation assays in an Escherichia coli mutant we demonstrate functional replacement of a pantothenate biosynthesis enzyme, 2-dehydropantoate 2-reductase (E.C. 1.1.1.169), by an endosymbiont gene, ilvC, encoding a substrate ambiguous enzyme.

Conclusions

Earlier studies have speculated that missing enzyme steps in fragmented endosymbiont metabolic pathways are completed by adaptable endosymbiont enzymes from other pathways. Here, we experimentally demonstrate completion of a fragmented endosymbiont vitamin biosynthesis pathway by recruitment of a substrate ambiguous enzyme from another pathway. In addition, this work extends host/symbiont metabolic collaboration in the aphid/Buchnera symbiosis from amino acid metabolism to include vitamin biosynthesis.

     

News & Notes: Buchnera aphidicola (Endosymbiont of Aphids) Contains nuoC(D) Genes That Encode Subunits of NADH Dehydrogenase

A two-kilobase DNA fragment from Buchnera aphidicola, the endosymbiont of aphids, was cloned and sequenced. One open reading frame was detected, coding for a putative protein of 600 amino acids. The N-terminal portion of this protein corresponded to NuoC, while the C-terminal portion corresponded to NuoD. These proteins are constituents of the membrane-associated NADH dehydrogenase. Our results suggest that these two proteins are fused in Buchnera aphidicola, a result consistent with their previously postulated spatial association. Received: 28 January 1997 / Accepted: 12 February 1997     

Genetic Characterization of Plasmids Containing Genes Encoding Enzymes of Leucine Biosynthesis in Endosymbionts (Buchnera) of Aphids

The prokaryotic endosymbionts (Buchnera) of aphids are known to provision their hosts with amino acids that are limiting in the aphid diet. Buchnera from the aphids Schizaphis graminum and Diuraphis noxia have plasmids containing leuABCD, genes that encode enzymes of the leucine biosynthetic pathway, as well as genes encoding proteins probably involved in plasmid replication (repA1 and repA2) and an open reading frame (ORF1) of unknown function. The newly reported plasmids closely resemble a plasmid previously described in Buchnera of the aphid Rhopalosiphum padi [Bracho AM, Martínez-Torres D, Moya A, Latorre A (1995) J Mol Evol 41:67–73]. Nucleotide sequence comparisons indicate conserved regions which may correspond to an origin of replication and two promoters, as well as inverted repeats, one of which resembles a rho-independent terminator. Phylogenetic analyses based on amino acid sequences of leu gene products and ORF1 resulted in trees identical to those obtained from endosymbiont chromosomal genes and the plasmid-borne trpEG. These results are consistent with a single evolutionary origin of the leuABCD-containing plasmid in a common ancestor of Aphididae and the lack of plasmid exchange between endosymbionts of different aphid species. Trees for ORF1 and repA (based on both nucleotides and amino acids) are used to examine the basis for leu plasmid differences between Buchnera of Thelaxes suberi and Aphididae. The most plausible explanation is that a single transfer of the leu genes to an ancestral replicon was followed by rearrangements. The related replicon in Buchnera of Pemphigidae, which lacks leuABCD, appears to represent the ancestral condition, implying that the plasmid location of the leu genes arose after the Pemphigidae diverged from other aphid families. This conclusion parallels previously published observations for the unrelated trpEG plasmid, which is present in Aphididae and absent in Pemphigidae. Recruitment of amino acid biosynthetic genes to plasmids has been ongoing in Buchnera lineages after the infection of aphid hosts. Received: 9 March 1998 / Accepted: 18 May 1998.     

Discovery and molecular characterization of a plasmid localized in Buchnera sp. bacterial endosymbiont of the aphid Rhopalosiphum padi

We have identified and completely sequenced a novel plasmid isolated from the aphid Rhopalosiphum padi. Evidence which suggests that the plasmid occurs localized within the bacterial endosymbionts is presented. The plasmid contains the four genes that constitute the entire leucine operon. This fact makes it really unique since most plasmids are dispensable and lack genes that encode essential anabolic functions. Four more phloem-feeding aphid species also seem to contain homologous plasmids.Although further work is necessary, we hypothesize that this plasmid has appeared during the evolution of the symbiotic association between the aphid and the bacterial endosymbiont. The fact that this plasmid contains the entire leucine operon can be related to physiological evidence showing that the aphid host's diet of plant phloem is deficient in essential amino acids.