Chromosomal stasis versus plasmid plasticity in aphid endosymbiont Buchnera aphidicola

The study of three genomes of the aphid endosymbiont Buchnera aphidicola has revealed an extraordinary stasis: conservation of gene order and genetic composition of the chromosome, while the chromosome size and number of genes has reduced. The reduction in genome size appears to be ongoing since some lineages we now know to have even smaller chromosomes than the first B. aphidicola analysed. The current sequencing by our group of one of these smaller genomes with an estimated size of 450 kb, and its comparison with the other three available genomes provide insights into the nature of processes involved in shrinkage. We discuss whether B. aphidicola might be driven to extinction and be replaced by secondary aphid endosymbionts. In some lineages, genes encoding key enzymes in the pathways leading to tryptophan and leucine biosynthesis (trpEG and leuABCD, respectively) are located on plasmids, rather than the main chromosome. In contrast to the stasis of the main chromosome, plasmid genes have frequently been transferred to the main chromosome and undergone other gene rearrangements. We propose a two-step scenario to explain these contrasting modes of evolution. Essential genes may have escaped regulation by moving to plasmids in a moving B. aphidicola ancestor. B. aphidicola became polyploidy at a given stage of its evolution and plasmid genes have been transferred to the main chromosome through several independent events.     

Low and homogeneous copy number of plasmid-borne symbiont genes affecting host nutrition in Buchnera aphidicola of the aphid Uroleucon ambrosiae

The bacterial endosymbiont of aphids, Buchnera aphidicola, often provides amino acids to its hosts. Plasmid amplification of leucine (leuABCD) and tryptophan (trpEG) biosynthesis genes may be a mechanism by which some Buchnera over-produce these nutrients. We used quantitative polymerase chain reaction to assess the leuABCD/trpEG copy variability within Uroleucon ambrosiae, an aphid with a wide diet breadth and range. Both leuABCD and trpEG abundances are: (i) similar for aphids across 15 populations, and (ii) low compared to Buchnera from other aphid species (particularly trpEG). Consequently, the plasmid location of trpEG combined with Buchnera's chromosomal polyploidy may functionally limit, rather than increase, tryptophan production within Uroleucon ambrosiae.     

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.     

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.     

Molecular characterization of the leucine cluster in Buchnera sp. strain PSY, a primary endosymbiont of the aphid Pemphigus spyrothecae

Buchnera strains from most aphid subfamilies studied to date have been found to carry the leucine gene cluster (leuA, -B, -C, and -D) on a plasmid, an organization unique among bacteria. Here, however, we demonstrate a classical chromosomal location of the cluster in Buchnera sp. strain PSY from the aphid Pemphigus spyrothecae (subfamily Pemphiginae). The genes that flank leuABCD in Buchnera sp. strain PSY appear to be adjacent in the genome of Buchnera sp. strain APS, a strain carrying a leucine plasmid. We propose that the presence of a leucine plasmid predates the diversification of symbiotic Buchnera and that the chromosomal location observed in Buchnera sp. strain PSY arose by a transfer of the leucine genes from a plasmid to the chromosome.     

Development and application of molecular tools in the study of IncN-related plasmids from lakewater sediments

Homology to IncN, P, Q and W inc regions was investigated amongst 114 Hg(2+)-resistant or antibiotic-resistant bacteria isolated from lakewater sediments. No hybridisation signals were found with Inc P, Q and W probes, and only one plasmid, pLV1402, hybridised to the IncN probe. PCR primers designed to conserved regions in the replicon of the IncN plasmid pCU1 and the related beta replicon from pGSH500 were used to amplify a 978-bp fragment from pLV1402, with sequence analysis showing a close relationship (99.2% identity) between their replication genes. A 387-bp region from the pLV1402 rep gene was used to re-screen the isolates and identified another related plasmid, pLV1403. A 3.7-kb probe containing the alpha replicon from pGSH500 hybridised to both pLV1402 and pLV1403, suggesting that both are multi-replicon plasmids. The PCR primers and probes described will be useful in future studies of plasmid diversity.     

Dispersion of a gene that codifies fosfomycin resistance among plasmids from enterobacteriaceae isolated from sewage

Abstract The presence of plasmid encoded resistance to fostomycin among enterobacteriaceae isolated from sewage was studied. These plasmids found were classified into 7 varieties according to their original host, size, resistance determinants, restriction pattern and incompatibility group. All of them were related to the Inc M group, indicating a probable common origin from an ancestral replicon. The bacteria carrying these plasmids modified fosfomycin as did those carrying fosfomycin resistant (Fo^r) hospital plasmids. The plasmids showed positive hybridization with a 1-kb DNA fragment which carried a Fo^r-determinant from a hospital plasmid.     

Characterization of cp18, a naturally truncated member of the cp32 family of Borrelia burgdorferi plasmids.

We have mapped the genes encoding the antigenic lipoproteins OspE and OspF to an approximately 18-kb circular plasmid in Borrelia burgdorferi N40. Sequencing and restriction mapping have revealed that this plasmid, cp18, is homologous to an 18-kb region of the cp32 circular plasmids found in the Lyme disease spirochetes. Our data show that cp18 may have arisen from an ancestral cp32 plasmid by deletion of a 14-kb region of DNA, indicating that a significant portion of the cp32 plasmid is not essential in cis for plasmid maintenance. These findings suggest that a relatively small recombinant plasmid capable of being stably maintained in B. burgdorferi could be constructed from a cp32 plasmid.     

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.     

Plasmid-Encoded Anthranilate Synthase (TrpEG) in Buchnera aphidicola from Aphids of the Family Pemphigidae

Buchnera aphidicola is an obligate intracellular symbiont of aphids. One of its proposed functions is the synthesis of essential amino acids, nutrients required by aphids but deficient in their diet of plant phloem sap. The genetic organization of the tryptophan pathway in Buchnera from proliferous aphids of the family Aphididae has previously been shown to reflect a capacity to overproduce this essential amino acid (C.-Y. Lai, L. Baumann, and P. Baumann, Proc. Natl. Acad. Sci. USA 91:3819–3823, 1994). This involved amplification of the genes for the first enzyme in the pathway, anthranilate synthase (TrpEG), on a low-copy-number plasmid. Here we report on the finding and molecular characterization of TrpEG-encoding plasmids in Buchnera from aphids of the distantly related family Pemphigidae. Buchnera from Tetraneura caerulescens contained a 3.0-kb plasmid (pBTc2) that carried a single copy of trpEG and resembled trpEG plasmids of Buchnera from the Aphididae. The second plasmid (pBPs2), isolated from Buchnera of Pemphigus spyrothecae, contained a different replicon. It consisted of a putative origin of replication containing iterons and an open reading frame, designated repAC, which showed a high similarity to the gene encoding the replication initiation protein RepA of the RepA/C replicon from the broad-host-range IncA/C group of plasmids. The plasmid population was heterogeneous with respect to the number of tandem repeats of a 1.8-kb unit carrying repAC₁, trpG, and remnants of trpE. The two principal forms consisted of either five or six copies of this repeat and a single-copy region carrying repAC₂, the putative origin of replication, and trpE. The unexpected finding of elements of the RepA/C replicon in previously characterized trpEG plasmids from Buchnera of the Aphididae suggests that a replacement of replicons has occurred during the evolution of these plasmids, which may point to a common ancestry for all Buchnera trpEG amplifications.     

Identification of contemporary plasmid virulence genes in ancestral isolates of Salmonella enteritidis and Salmonella typhimurium

Six epidemiologically distinct ancestral strains of Salmonella enteritidis and 5 of S. typhimurium from the pre-antibiotic era were examined for plasmid content, and for presence of plasmid genes implicated in mouse-virulence. Five sizes of plasmid were detected in S. enteritidis varying from 1 to 60 MDa. Two sizes of plasmid were found in S. typhimurium, 28 and 60 MDa. Plasmids of the same size were not common to both serovars. The HindIII restriction patterns of 3 of the ancestral S. enteritidis plasmids were identical to the modern 38 MDa plasmid, while all contained identical bands of 3.5, 2.7 and 1.9 kb. All the 60-MDa S. typhimurium plasmids, ancestral and contemporary, had an identical restriction pattern. Three different sized S. enteritidis plasmids and one size S. typhimurium plasmid contained a 3.5-kb DNA fragment carrying the virulence locus VirA. The VirB virulence locus was located on a 2.7-kb DNA fragment in S. enteritidis and on a 2.5-kb fragment in S. typhimurium. Both loci were precisely conserved between the ancestral strains and the modern representatives of both serovars.     

Replication Mechanism and Sequence Analysis of the Replicon of pAW63, a Conjugative Plasmid from Bacillus thuringiensis

A 5.8-kb fragment of the large conjugative plasmid pAW63 from Bacillus thuringiensis subsp. kurstaki HD73 containing all the information for autonomous replication was cloned and sequenced. By deletion analysis, the pAW63 replicon was reduced to a 4.1-kb fragment harboring four open reading frames (ORFs). Rep63A (513 amino acids [aa]), encoded by the largest ORF, displayed strong similarity (40% identity) to the replication proteins from plasmids pAMbeta1, pIP501, and pSM19035, indicating that the pAW63 replicon belongs to the pAMbeta1 family of gram-positive theta-replicating plasmids. This was confirmed by the facts that no single-stranded DNA replication intermediates could be detected and that replication was found to be dependent on host-gene-encoded DNA polymerase I. An 85-bp region downstream of Rep63A was also shown to have strong similarity to the origins of replication of pAMbeta1 and pIP501, and it is suggested that this region contains the bona fide pAW63 ori. The protein encoded by the second large ORF, Rep63B (308 aa), was shown to display similarity to RepB (34% identity over 281 aa) and PrgP (32% identity over 310 aa), involved in copy control of the Enterococcus faecalis plasmids pAD1 and pCF10, respectively. No significant similarity to known proteins or DNA sequences could be detected for the two smallest ORFs. However, the location, size, hydrophilicity, and orientation of ORF6 (107 codons) were analogous to those features of the putative genes repC and prgO, which encode stability functions on plasmids pAD1 and pCF10, respectively. The cloned replicon of plasmid pAW63 was stably maintained in Bacillus subtilis and B. thuringiensis and displayed incompatibility with the native pAW63. Hybridization experiments using the cloned replicon as a probe showed that pAW63 has similarity to large plasmids from other B. thuringiensis subsp. kurstaki strains and to a strain of B. thuringiensis subsp. alesti.     

Characterization and properties of a novel plasmid vector for Bacillus thuringiensis displaying compatibility with host plasmids.

A novel plasmid vector, composed of a 1.7-kb Bacillus thuringiensis (B.t.) replicon, a multiple cloning site, and an erythromycin-resistance marker gene from Bacillus subtilis, was constructed for use in B.t. Unlike other vectors which have been reported to be acceptable for B.t., this new B.t. vector was stably maintained in the absence of Er and did not displace host plasmids, some of which carry crystal protein-encoding genes (cry genes). The compatibility of this B.t. vector with native plasmids is highly desirable when introducing new cry genes into a wild-type B.t. strain. When a cryIIIA gene of B.t. tenebrionis was cloned in this vector and introduced into B.t. kurstaki (kur) HD119, cryIIIA was highly expressed without affecting the level of expression of native cry genes. The stability of this vector and its compatibility with native B.t. plasmids were achieved by subcloning only nucleotide sequences required for the vector to replicate in B.t. The origin of replication was first cloned on a 9.6-kb Bg/II fragment from a 75-kb plasmid of B.t. kur HD73 and then localized to a 2.4-kb region within the 9.6-kb fragment. Sequencing of the 2.4-kb region revealed the presence of an open reading frame (ORF), encoding a putative 312-amino acid (aa) protein. The deduced aa sequence of the ORF showed no homology to any published aa sequences. Deletion analysis indicated that the B.t. vector required at least the ORF and up to 300 bp surrounding the ORF, in order to replicate.     

Characterization of a small cryptic plasmid from Salmonella enteritidis that affects the growth of Escherichia coli

Abstract We examined the plasmid content of 25 clinical isolates of Salmonella enteritidis , and detected the presence of small plasmids (3–5.3 kb) in 9 of them, alone, or in addition to the large, so-called virulence plasmid. A 5.3-kb plasmid isolated as unique extrachromosomal DNA from a strain responsible for a high-mortality outbreak was characterized by restriction mapping and cloning. The plasmid replicon was localized in a 1.7-kb fragment, that hybridized with three of the small plasmids detected in S. enteritidis , and with another small plasmid from Salmonella typhimurium . A strain of Escherichia coli carrying this plasmid, or a cloned 3.7-kb Pvu II restriction fragment, showed a slower growth rate, especially in minimal medium, as well as a noticeable increase in DNA methyltransferase activity.     

Application of the resident plasmid integration technique to construct a strain of Streptococcus godronii able to express the Bacillus circulans cycloisomaltooligosaccharide glucanotransferase gene,and secrete its active gene product

A novel transformation technique, resident plasmid integration, for the cloning of foreign DNA in oral streptococci was described recently (T. Shiroza and H. K. Kuramitsu, Plasmid, 1995, 34, 85–95). This technique is based on the integration of linearized foreign genes by recombination-proficient bacteria onto a resident plasmid, if an appropriate selection marker is flanked by the same anchor sites present in the resident plasmid. Since the transforming vehicles for this system included a pUC-derived replication origin, the high level expression in Escherichia coli cells hindered the cloning of certain genes. In the present study, new plasmids were constructed, two resident plasmids, four integration plasmids, and four cloning plasmids, all of which possess the medium-copy number replication origin, p15A ori, isolated from pACYC177. The resident plasmids consisted of the following three components: the p15A ori (0.65-kb BglII fragment), the pVA380-1 basic replicon functional in mutans streptococci (2.5-kb BamHI fragment), and either an erythromycin resistance or a spectinomycin resistance gene (0.9- or 1.1-kb BamHI fragment, respectively). Most of the basic replicon of pVA380-1, except for the 3′-portion of the 0.2-kb region, in the resident plasmid was replaced with a kanamycin resistance gene to construct the four integration plasmids. Therefore, the upstream and downstream anchor sites for the double cross-over event in this new system were 0.65-kb p15A ori and the 0.2-kb portion of the 3′-end of pVA380-1 replicon, respectively. This system was used to clone the gene coding for cycloisomaltooligosaccharide glucanotransferase which produces cycloisomaltooligosaccharide, a potent inhibitor of oral streptococcal glucosyltransferase, isolated from Bacillus circulans chromosome, into Streptococcus gordonii, and its gene product was successfully secreted into the culture media. Plasmids described here should be useful tools for introducing heterologous DNA into resident plasmids following integration in oral streptococci.     

Presence of 16S rRNA genes in multiple replicons in Azospirillum brasilense

Rhizosphere and endophytic Azospirillum brasilense isolates recovered from sugarcane plants and the reference strains Sp7 and Cd were analyzed for plasmid occurrence. All of the 26 A. brasilense isolates analyzed harbored from five to eight replicons. Several strains contained small plasmids from 45 to 70 kb, but all of the isolates harbored other plasmids ranging from 100 to 290 kb and two megareplicons of approximately 1700 and over 1800 kb. Most of the strains contained a replicon with a size of either 570 or 630 kb, and another large 910- or 980-kb replicon. The 1700-kb megareplicon and some others around 600 kb strongly hybridized to 16S rDNA genes, while the 910- or 980-kb replicons hybridized only slightly. This suggests that the A. brasilense genome is composed of multiple minichromosomes instead of a single circular chromosome. The apparent genome complexity of A. brasilense deserves to eventually be resolved by complete genome sequencing.