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.     

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     

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.     

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.     

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     

Partial Sequence of the Mitochondrial Genome of Littorina saxatilis: Relevance to Gastropod Phylogenetics

A 8022 base pair fragment from the mitochondrial DNA of the prosobranch gastropod Littorina saxatilis has been sequenced and shown to contain the complete genes for 12 transfer RNAs and five protein genes (CoII, ATPase 6, ATPase 8, ND1, ND6), two partial protein genes (CoI and cyt b), and two ribosomal RNAs (small and large subunits). The order of these constituent genes differs from those of other molluscan mitochondrial gene arrangements. Only a single rearrangement involving a block of protein coding genes and three tRNA translocations are necessary to produce identical gene orders between L. saxatilis and K. tunicata. However, only one gene boundary is shared between the L. saxatilis gene order and that of the pulmonate gastropod Cepaea nemoralis. This extends the observation that there is little conservation of mitochrondrial gene order amongst the Mollusca and suggests that radical mitochondrial DNA gene rearrangement has occurred on the branch leading to the pulmonates. Received: 4 June 1998 / Accepted: 20 August 1998     

Genetic aspects of aromatic amino acid biosynthesis in Lactococcus lactic

Polymerase chain reaction (PCR) primers designed from a multiple alignment of predicted amino acid sequences from bacterial aroA genes were used to amplify a fragment of Lactococcus lactis DNA. An 8 kb fragment was then cloned from a lambda library and the DNA sequence of a 4.4 kb region determined. This region was found to contain the genes tyrA, aroA, aroK, and pheA, which are involved in aromatic amino acid biosynthesis and folate metabolism. TyrA has been shown to be secreted and AroK also has a signal sequence, suggesting that these proteins have a secondary function, possibly in the transport of amino acids. The aroA gene from L. lactis has been shown to complement an E. coli mutant strain deficient in this gene. The arrangement of genes involved in aromatic amino acid biosynthesis in L. lactis appears to differ from that in other organisms.The nucleotide sequence data reported in this paper have been submitted to the EMBL, GenBank, and DDBJ Nucleotide Sequence Databanks and have been assigned the accession number X78413     

Buchnera aphidicola,the endosymbiont of aphids,contains genes for four ribosomal RNA proteins,initiation factor-3, and the α-subunit of RNA polymerase

Buchnera aphidicola is a prokaryotic endosymbiont of the aphidSchizaphis graminum. With the polymerase chain reaction (PCR) and oligonucleotide primers to conserved regions, two DNA fragments of the endosymbiont -operon and L20 operon were amplified, cloned intoEscherichia coli, and their sequences were determined. The results indicated that the organization of the endosymbiont genes on these fragments was identical with that of the corresponding operons ofE. coli. The 1032 base pair (bp) fragment of the -operon contained the genes for small ribosomal subunit proteins S11 and S4, followed by the gene for the -subunit of RNA polymerase (-RNAP). The 702-bp fragment of the L20-operon contained the genes for initiation factor-3 (IF3) and large ribosomal subunit proteins L35 and L20. As in other prokaryotes, the genes of the -operon and the L20-operon were present as single copies in the genome ofB. aphidicola. Comparisons of the amino acid sequences of these proteins were consistent with the previously established close relationship betweenB. aphidicola andE. coli and a distant relationship to species ofBacillus.     

Cloning, Sequencing, and Function of sanF: A Gene Involved in Nikkomycin Biosynthesis of Streptomyces ansochromogenes

A 111-bp DNA fragment related to nikkomycin biosynthesis of Streptomyces ansochromogenes 7100 was obtained with the method of reverse genetics. Then, a 2.2-kb DNA fragment was cloned from the DNA library of S. ansochromogenes 7100 by using the 111-bp fragment as a probe. Sequence analysis showed that the fragment contains one complete open reading frame (ORF) that encodes a 219-amino acid (aa) protein, and this gene was designated sanF (GenBank Accession No. AF223971). The function of the sanF gene was studied by a strategy of gene disruption, and the resulting sanF mutants lost the ability to synthesize biologically active nikkomycin, indicating that sanF is essential for nikkomycin biosynthesis. Received: 17 April 2000 / Accepted: 23 May 2000     

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     

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 transketolase mutant of Corynebacterium glutamicum

A transketolase mutant was first isolated from Corynebacterium glutamicum, an organism of industrial importance. The mutant strain exhibited an absolute requirement for shikimic acid or the aromatic amino acids and vitamins for growth, and also failed to grow on ribose or gluconic acid as sole carbon source, even with the aromatic supplement. All of these defective properties were fully restored in spontaneous revertants, indicating the existence of a single transketolase in C. glutamicum that was indispensable both for aromatic biosynthesis and for utilization of these carbohydrates in vivo. The transketolase mutant accumulated ribulose extracellularly when cultivated in glucose medium with shikimic acid, but no ribose was detected. Received: 10 April 1998 / Received revision: 26 May 1998 / Accepted: 14 June 1998     

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     

Sequence analysis of an aphid endosymbiont DNA fragment containingrpoB (β-subunit of RNA polymerase) and portions ofrplL andrpoC

The aphidSchizaphis graminum is dependent on an association with a prokaryotic endosymbiont (Buchnera aphidicola). The nucleotide (nt) sequence of a 5040 base pair (bp) DNA fragment ofB. aphidicola, homologous to therplL-rpoB-rpoC portion of theEscherichia coli -operon, was determined. The DNA coded for the terminal 35 amino acids of RplL (large ribosomal subunit protein L7/L12), the complete RpoB (-subunit of RNA polymerase), and the first 209 amino acids of RpoC (-subunit of RNA polymerase). The deduced sequences ofB. aphidicola RplL, RpoB, and RpoC were 71, 84, and 91% identical, respectively, to the homologous proteins ofE. coli. The sequences of two portions of the intergenic region betweenrplL andrpoB were nearly identical in bothB. aphidicola andE. coli. One sequence constituted an inverted repeat that could be an RNase III-messenger RNA processing site; the other sequence preceded RpoB. A compilation of the codon usage for RpoB, RpoC, and otherB. apidicola proteins indicated a major preference for A or T in the first and third positions, a result consistent with the low guanine plus cytosine (G+C) content of the DNA of this organism.     

A divergent non-classical class I gene conserved in salmonids

 Complementary DNA for two class I genes of the rainbow trout, Oncorhynchus mykiss, were characterized. MhcOnmy-UBA*01 is similar to Onmy-UA-C32 and the classical major histocompatibility complex class I genes of other fish species, whereas Onmy-UAA*01 is divergent from all class I genes so far characterized. Onmy-UAA*01 is expressed at lower levels than Onmy-UBA*01. Although Onmy-UAA*01 exhibits restriction fragment length polymorphism on Southern blotting, the encoded protein is highly conserved. Two allotypes, which differ only by substitution at amino acid position 223 of the α₃ domain, have been defined. Onmy-UAA*01 has an exon-intron organization like other class I genes and contains a Tc1-like transposon element in intron III. Orthologues of Onmy-UAA*01 have been characterized in four other species of salmonid. Between four species of Oncorhynchus, UAA*01 proteins differ by only 2–6 amino acids, whereas comparison of Oncorhynchus with Salmo trutta (brown trout) reveals 14–16 amino acid differences. The Onmy-UAA*01 gene has properties indicative of a particularly divergent non-classical class I gene. Received: 22 September 1998 / Revised: 24 November 1998     

Genetic aspects of aromatic amino acid biosynthesis in Lactococcus lactic

Polymerase chain reaction (PCR) primers designed from a multiple alignment of predicted amino acid sequences from bacterial aroA genes were used to amplify a fragment of Lactococcus lactis DNA. An 8 kb fragment was then cloned from a lambda library and the DNA sequence of a 4.4 kb region determined. This region was found to contain the genes tyrA, aroA, aroK, and pheA, which are involved in aromatic amino acid biosynthesis and folate metabolism. TyrA has been shown to be secreted and AroK also has a signal sequence, suggesting that these proteins have a secondary function, possibly in the transport of amino acids. The aroA gene from L. lactis has been shown to complement an E. coli mutant strain deficient in this gene. The arrangement of genes involved in aromatic amino acid biosynthesis in L. lactis appears to differ from that in other organisms.     

Thiamin biosynthesis in prokaryotes

Twelve genes involved in thiamin biosynthesis in prokaryotes have been identified and overexpressed. Of these, six are required for the thiazole biosynthesis (thiFSGH, thiI, and dxs), one is involved in the pyrimidine biosynthesis (thiC), one is required for the linking of the thiazole and the pyrimidine (thiE), and four are kinase genes (thiD, thiM, thiL, and pdxK). The specific reactions catalyzed by ThiEF, Dxs, ThiDM, ThiL, and PdxK have been reconstituted in vitro and ThiS thiocarboxylate has been identified as the sulfur source. The X-ray structures of thiamin phosphate synthase and 5-hydroxyethyl-4-methylthiazole kinase have been completed. The genes coding for the thiamin transport system (thiBPQ) have also been identified. Remaining problems include the cloning and characterization of thiK (thiamin kinase) and the gene(s) involved in the regulation of thiamin biosynthesis. The specific reactions catalyzed by ThiC (pyrimidine formation), and ThiGH and ThiI (thiazole formation) have not yet been identified. Received: 23 August 1998 / Accepted: 16 January 1999