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     

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     

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.     

The process of genome shrinkage in the obligate symbiont Buchnera aphidicola

Background  

Very small genomes have evolved repeatedly in eubacterial lineages that have adopted obligate associations with eukaryotic hosts. Complete genome sequences have revealed that small genomes retain very different gene sets, raising the question of how final genome content is determined. To examine the process of genome reduction, the tiny genome of the endosymbiont Buchnera aphidicola was compared to the larger ancestral genome, reconstructed on the basis of the phylogenetic distribution of gene orthologs among fully sequenced relatives of Escherichia coli and Buchnera.     

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     

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.     

蚜虫初级内共生菌Buchnera aphidicola研究进展

动物和细菌的内共生关系一直是生物学关注的热点,相关研究不但对于了解动物宿主的生长、繁殖等有重要意义,也有助于探讨生命起源和进化等生命现象。蚜虫类昆虫体内存在一类专性的胞内共生菌Buchnera,它对于蚜虫营养代谢和正常发育至关重要,被称为蚜虫的初级内共生菌。由于两者间具有专性共生关系,使其成为内共生关系研究的理想模型。本文将从Buchnera的基本特征、Buchnera与蚜虫进化关系、Buchnera在共生关系中的作用及Buchnera基因组学等方面对Buchnera研究现状进行综述,并对未来的研究热点进行展望。     

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 evolutionary fate of nonfunctional DNA in the bacterial endosymbiont Buchnera aphidicola

Reduction of the genome size in endosymbiotic bacteria is the main feature linked to the adaptation to a host-associated lifestyle. We have analyzed the fate of the nonfunctional DNA in Buchnera aphidicola, the primary endosymbiont of aphids. At least 164 gene losses took place during the recent evolution of three B. aphidicola strains, symbionts of the aphids Acyrthosiphon pisum (BAp), Schizaphis graminum (BSg), and Baizongia pistacia (BBp). A typical pattern starts with the inactivation of a gene, which produces a pseudogene, and is followed by the progressive loss of its DNA. Our results show that during the period from the separation of the Aphidinae and Pemphiginae lineages (86-164 MYA) to the divergence of BAp and BSg (50-70 MYA) the half-life of a pseudogene was 23.9 Myr. For the remaining periods of evolution, the ranges of values obtained for this parameter are of the same order of magnitude. These results have revealed that a gene inactivated during B. aphidicola evolution requires 40-60 Myr to become almost completely disintegrated. Moreover, we have shown a positive correlation between the decrease in the GC content and the DNA loss for these nonfunctional DNA regions. When gene losses are classified, based on the detection of a pseudogene or otherwise of an absent gene in the modern B. aphidicola genomes, we have observed a drastic reduction of DNA length in the latter versus the former relative to the functional gene. Finally, we have also detected a slight reduction in size of the intergenic regions in the three B. aphidicola strains, when they are compared with the size of the close relative Escherichia coli.     

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.     

Evolution of the leucine gene cluster in Buchnera aphidicola: insights from chromosomal versions of the cluster

In Buchnera aphidicola strains associated with the aphid subfamilies Thelaxinae, Lachninae, Pterocommatinae, and Aphidinae, the four leucine genes (leuA, -B, -C, and -D) are located on a plasmid. However, these genes are located on the main chromosome in B. aphidicola strains associated with the subfamilies Pemphiginae and Chaitophorinae. The sequence of the chromosomal fragment containing the leucine cluster and flanking genes has different positions in the chromosome in B. aphidicola strains associated with three tribes of the subfamily Pemphiginae and one tribe of the subfamily Chaitophorinae. Due to the extreme gene order conservation of the B. aphidicola genomes, the variability in the position of the leucine cluster in the chromosome may be interpreted as resulting from independent insertions from an ancestral plasmid-borne leucine gene. These findings do not support a chromosomal origin for the leucine genes in the ancestral B. aphidicola and do support a back transfer evolutionary scenario from a plasmid to the main chromosome.     

Interactions of Chaperonin with a Weakly Active Anthranilate Synthase from the Aphid Endosymbiont <Emphasis Type="Italic">Buchnera aphidicola</Emphasis>

The endosymbiotic bacterium Buchnera provides its aphid host with essential amino acids. Buchnera is typical of intracellular symbiotic and parasitic microorganisms in having a small effective population size, which is believed to accelerate genetic drift and reduce the stability of gene products. It is hypothesized that Buchnera mitigates protein instability with an increased production of the chaperonins GroESL. In this paper, we report the expression and functional analysis of trpE, a plasmid-borne fast-evolving gene encoding the tryptophan biosynthesis enzyme anthranilate synthase. We overcame the problem of low enzyme stability by using an anthranilate synthase-deficient mutant of E. coli as the expression host and the method of genetic complementation for detection of the enzyme activity. We showed that the Buchnera anthranilate synthase was only weakly active at the temperature of 26°C but became inactive at the higher temperatures of 32°C and 37°C and that the coexpression with chaperonin genes groESL of E. coli enhanced the function of the Buchnera enzyme. These findings are consistent with the proposed role of groESL in the Buchnera–aphid symbiosis.     

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.     

Isolation and Characterization of the Azospirillum brasilense trpE(G) Gene,Encoding Anthranilate Synthase

The Azospirillum brasilense trpE gene has been isolated by DNA hybridization and by genetic complementation of an Escherichia coli trpE deletion mutant. DNA sequence analysis of a 3.1-kb PstI restriction fragment of A. brasilense revealed the presence of an open reading frame encoding a putative TrpE(G) fusion protein. Previously an A. brasilense clone containing trpGDC was identified (Zimmer et al. Mol Gen Genet 229:41–51, 1991). It can, therefore, be concluded that A. brasilense contains two trpG genes. A putative leader peptide is found upstream of trpE(G), containing three consecutive tryptophan residues. Putative terminator and anti-terminator loops have also been identified. The LLESX₁₀S motif, which is responsible for feedback inhibition by tryptophan in other TrpE proteins, is absent in the A. brasilense TrpE(G) fused protein. Received: 29 May 1996 / Accepted: 5 July 1996     

Genome reduction of the aphid endosymbiont Buchnera aphidicola in a recent evolutionary time scale

Genome reduction, a typical feature of symbiotic bacteria, was analyzed in the last stages of evolution of Buchnera aphidicola, the primary aphid endosymbiont, in two neutrally evolving regions: the pseudogene cmk and an intergenic region. These two regions were examined in endosymbionts from several lineages of their aphid host Rhopalosiphum padi, and different species of the same genus, whose divergence times ranged from 0.62 to 19.51 million years. Estimates of nucleotide substitution rates were between 4.3 and 6.7x10(-9) substitution/site/year, with G or C nucleotides being substituted around four times more frequently than A or T. Two different types of indel events were detected, of which many were small (1-10 nt) but one was large (about 200 nucleotides).With respect to the large one and considering the proportion and size of the deletions and insertions, the reduction rate was 1.3x10(-8) lost nucleotides/site/year. We propose a stepwise scenario for the last stages of evolution in B. aphidicola: together with a very slow and gradual degradation, considerable indels would punctually emerge. The only restriction to large deletion fixation is that the lost fragment does not contain essential genes.