Agrobacterium Tumefaciens Ti Plasmid Research Articles

Archaeal DNA-import apparatus is homologous to bacterial conjugation machinery

Conjugation is a major mechanism of horizontal gene transfer promoting the spread of antibiotic resistance among human pathogens. It involves establishing a junction between a donor and a recipient cell via an extracellular appendage known as the mating pilus. In bacteria, the conjugation machinery is encoded by plasmids or transposons and typically mediates the transfer of cognate mobile genetic elements. Much less is known about conjugation in archaea. Here, we determine atomic structures by cryo-electron microscopy of three conjugative pili, two from hyperthermophilic archaea (Aeropyrum pernix and Pyrobaculum calidifontis) and one encoded by the Ti plasmid of the bacterium Agrobacterium tumefaciens, and show that the archaeal pili are homologous to bacterial mating pili. However, the archaeal conjugation machinery, known as Ced, has been ‘domesticated’, that is, the genes for the conjugation machinery are encoded on the chromosome rather than on mobile genetic elements, and mediates the transfer of cellular DNA.

Domesticated conjugation machinery promotes DNA exchange in hyperthermophilic archaea.

Conjugation, a major mechanism of horizontal gene transfer promoting the spread of antibiotic resistance among human pathogens, involves establishing a junction between a donor and a recipient cell. The mating pilus plays a prominent role in this process. In bacteria, the conjugation machinery is encoded by plasmids or transposons and typically mediates the transfer of cognate mobile genetic elements. Much less has been known about conjugation in archaea, with no homologs of bacterial mating pilins having been identified in sequence searches. Here, using cryo-electron microscopy, we determined high-resolution structures of three conjugative pili, two from hyperthermophilic archaea, Aeropyrum pernix and Pyrobaculum calidifontis, and one encoded by the Ti plasmid of the bacterium Agrobacterium tumefaciens. We show that the archaeal pili are homologous to bacterial mating pili and display intricate pilin-lipid interaction networks. Unlike in bacteria, the conjugation machinery in hyperthermophilic archaea has been domesticated and mediates the transfer of cellular DNA. We suggest that the constitutive expression of the pili may be due to the chronic damage of DNA at high temperatures that is constantly being repaired using homologous recombination.

A Survey of Genetically Modified Foods and Feed in Namibian Supermarkets

The current study was carried out to survey if genetically modified organisms (GMOs) are present in feeds and foods found in local supermarkets in Namibia. Little is known of the presence and prevalence of GMOs in the local supermarkets or information available on the labelling provided on some. The aim was to prove the absence or existence of GMOs in the foods and feeds by means of molecular extraction and screening procedures. Samples purposefully selected were nine (6 food and 3 chicken feed samples). DNA was extracted from them, and they were screened by means of polymerase chain reaction (PCR) for the presence of the Cauliflower mosaic virus (CaMV) 35S promoter and the terminator of Agrobacterium tumefaciens Ti plasmid nopaline synthase gene (NOS) that are commonly found in transgene constructs. Screening results indicated that GMOs were present in the 3 chicken feed samples, absent in 1 food sample, present in 3 food samples and the other 2 food samples’ results were inconclusive. These results and survey information reveal the extent of GMOs in the food chain in supermarkets in Namibia and highlight the need for regulatory bodies in the food and feed industry to protect consumers from being misled by absence of properly informative labels on genetic modifications.

Features of Expression of Foreign Genes in Complex Insertions in Transgenic Tobacco Plants with a Mosaic Pattern of nptII Gene Expression

The features of a mosaic pattern of the expression of the nptII selective gene, which provides plant resistance to kanamycin antibiotic, and the target gene of Serratia marcescens secretory endonuclease under control of the bidirectional MAS promoter of the mannopine synthase gene of Agrobacterium tumefaciens Ti plasmid in epiallelic lines of transgenic tobacco plants (Nu5 and Nu6) were studied. Both genes are part of a complex insertion represented by two full-size T-DNA copies and one truncated copy located between them in the reverse orientation. Transgenic tobacco lines contrastingly differ in the phenotypic expression of the nptII gene (low frequency of mosaics in the Nu5 line and high in Nu6). It was established that a decrease in the level of expression of the selective gene occurs when transgenic plants pass from hemi- to homozygous state, and it is most pronounced for the Nu6 epiallele. It was demonstrated that synthesis of aberrant sense and antisense transcripts in the region of a truncated T-DNA copy occurs in the transgenic lines. Namely these transcripts can act as triggers and trigger the inactivation of the selective nptII gene expression.

The Composite 259-kb Plasmid of Martelella mediterranea DSM 17316T–A Natural Replicon with Functional RepABC Modules from Rhodobacteraceae and Rhizobiaceae

A multipartite genome organization with a chromosome and many extrachromosomal replicons (ECRs) is characteristic for Alphaproteobacteria. The best investigated ECRs of terrestrial rhizobia are the symbiotic plasmids for legume root nodulation and the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens. RepABC plasmids represent the most abundant alphaproteobacterial replicon type. The currently known homologous replication modules of rhizobia and Rhodobacteraceae are phylogenetically distinct. In this study, we surveyed type-strain genomes from the One Thousand Microbial Genomes (KMG-I) project and identified a roseobacter-specific RepABC-type operon in the draft genome of the marine rhizobium Martelella mediterranea DSM 17316T. PacBio genome sequencing demonstrated the presence of three circular ECRs with sizes of 593, 259, and 170-kb. The rhodobacteral RepABC module is located together with a rhizobial equivalent on the intermediate sized plasmid pMM259, which likely originated in the fusion of a pre-existing rhizobial ECR with a conjugated roseobacter plasmid. Further evidence for horizontal gene transfer (HGT) is given by the presence of a roseobacter-specific type IV secretion system on the 259-kb plasmid and the rhodobacteracean origin of 62% of the genes on this plasmid. Functionality tests documented that the genuine rhizobial RepABC module from the Martelella 259-kb plasmid is only maintained in A. tumefaciens C58 (Rhizobiaceae) but not in Phaeobacter inhibens DSM 17395 (Rhodobacteraceae). Unexpectedly, the roseobacter-like replication system is functional and stably maintained in both host strains, thus providing evidence for a broader host range than previously proposed. In conclusion, pMM259 is the first example of a natural plasmid that likely mediates genetic exchange between roseobacters and rhizobia.

Estandarización de un protocolo para detección de OGMs: evaluación de la presencia de OGMs en granos de soya colectados en diferentes centros de acopio de Ecuador

La soya es un cultivo cuya producción nacional en el Ecuador no es suficiente para suplir la demanda interna. Por esta razón, se recurre a la importación de semilla de soya para siembra y soya en grano para consumo, desde países donde el uso de soya transgénica ha sido aprobado. Como consecuencia, existe la probabilidad del ingreso de este cultivo a territorio ecuatoriano. En esta investigación, se estandarizó una metodología para la detección y cuantificación de soya genéticamente modificada con tecnología SYBR Green. Los primers utilizados para la amplificación corresponden a secuencias específicas de los dos elementos recombinantes más representativos en soya genéticamente modificada, el promotor 35S del Virus del Mosaico de la Coliflor (P35S) y el terminador NOS del plásmido Ti de Agrobacterium tumefaciens (TNOS), generando amplicones de 200pb y de 69pb respectivamente. El análisis cuantitativo se realizó a partir de la generación de una curva estándar en base a la amplificación usando los primers mencionados en material de referencia certificado con concentraciones de 0.01%, 0.1%, 1% y 10% de soya transgénica del evento GTS 40-3-2. El límite de detección se estableció en 0.01%, mientras que el límite de cuantificación fue establecido en 0.1%. En 2 de las 26 muestras de soya analizadas se encontró más del 0.1% de OGM (organismo genéticamente modificado) (muestra 22 con 0.2% y la muestra 23 con 14%). Además se detectó presencia adventicia de soya transgénica en 8 de las muestras estudiadas. Debido a las regulaciones de OGMs que existen en el Ecuador, este tipo de análisis es importante para confirmar la presencia de OGMs en el territorio ecuatoriano y evidencia la necesidad de contar con personal capacitado e instituciones especializadas en la detección y análisis de organismos genéticamente modificados.

Review: Plant Binary Vectors of Ti Plasmid in <i>Agrobacterium tumefaciens</i> with a Broad Host-Range Replicon of pRK2, pRi, pSa or pVS1

This review chronicles the development of the plant binary vectors of Ti plasmid in Agrobacterium tumefaciens during the last 30 years. A binary vector strategy was designed in 1983 to separate the T-DNA region in a small plasmid from the virulence genes in avirulent T-DNA-less Ti plasmid. The small plant vectors with the T-DNA region have been simply now called binary Ti vectors. A binary Ti vector consist of a broad host-range replicon for propagation in A. tumeraciens, an antibiotic resistance gene for bacterial selection and the T-DNA region that would be transferred to the plant genome via the bacterial virulence machinery. The T-DNA region delimited by the right and left border sequences contains an antibiotic resistance gene for plant selection, reporter gene, and/or any genes of interest. The ColEI replicon was also added to the plasmid backbone to enhance the propagation in Escherichia coli. A general trend in the binary vector development has been to increase the plasmid stability during a long co-cultivation period of A. tumefaciens with the target host plant tissues. A second trend is to understand the molecular mechanism of broad host-range replication, and to use it to reduce the size of plasmid for ease in cloning and for higher plasmid yield in E. coli. The broad host-range replicon of VS1 was shown to be a choice of replicon over those of pRK2, pRi and pSA because of the superior stability and of small well-defined replicon. Newly developed plant binary vectors pLSU has the small size of plasmid backbone (4566 bp) consisting of VS1 replicon (2654 bp), ColE1 replicon (715 bp), a bacterial kanamycin (999 bp) or tetracycline resistance gene, and the T-DNA region (152 bp).

Inducible expression of the gene of Zinnia elegans coding for extracellular ribonuclease in Nicotiana tabacum plants

The gene of Zinnia elegans L. coding for S-like extracellular ribonuclease (ZRNase II) was used to produce transgenic tobacco plants with an increased ribonuclease activity. The protein-coding part of ZRNase II included the signal peptide sequence so the transgenic protein was located extracellularly. The cDNA of ZRNase II was cloned under the control of 2′-promoter of the mannopine synthase (MAS 2′) gene from Ti-plasmid of Agrobacterium tumefaciens. It was shown that the resultant transgenic plants had an increased ribonuclease activity of the crude extracts and the induction of MAS 2′ promoter by wounding additionally increased the activity. The plants of two transforming lines characterized by different ribonuclease activities were used to analyze the transgene influence on plant resistance to tobacco mosaic virus. The plants demonstrated either absence of disease symptoms or a significant delay in their appearance, depending on the virus content in the inoculum and ribonuclease activity.

RepC protein of the octopine‐type Ti plasmid binds to the probable origin of replication within repC and functions only in cis

Vegetative replication and partitioning of many plasmids and some chromosomes of alphaproteobacteria are directed by their repABC operons. RepA and RepB proteins direct the partitioning of replicons to daughter cells, while RepC proteins are replication initiators, although they do not resemble any characterized replication initiation protein. Here we show that the replication origin of an Agrobacterium tumefaciens Ti plasmid resides fully within its repC gene. Purified RepC bound to a site within repC with moderate affinity, high specificity and with twofold cooperativity. The binding site was localized to an AT-rich region that contains a large number of GANTC sites, which have been implicated in replication regulation in related organisms. A fragment of RepC containing residues 26-158 was sufficient to bind DNA, although with limited sequence specificity. This portion of RepC is predicted to have structural homology to members of the MarR family of transcription factors. Overexpression of RepC in A. tumefaciens caused large increases in copy number in cis but did not change the copy number of plasmids containing the same oriV sequence in trans, confirming other observations that RepC functions only in cis.

High level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector

We constructed a novel autonomously replicating gene expression shuttle vector, with the aim of developing a system for transiently expressing proteins at levels useful for commercial production of vaccines and other proteins in plants. The vector, pRIC, is based on the mild strain of the geminivirus Bean yellow dwarf virus (BeYDV-m) and is replicationally released into plant cells from a recombinant Agrobacterium tumefaciens Ti plasmid. pRIC differs from most other geminivirus-based vectors in that the BeYDV replication-associated elements were included in cis rather than from a co-transfected plasmid, while the BeYDV capsid protein (CP) and movement protein (MP) genes were replaced by an antigen encoding transgene expression cassette derived from the non-replicating A. tumefaciens vector, pTRAc. We tested vector efficacy in Nicotiana benthamiana by comparing transient cytoplasmic expression between pRIC and pTRAc constructs encoding either enhanced green fluorescent protein (EGFP) or the subunit vaccine antigens, human papillomavirus subtype 16 (HPV-16) major CP L1 and human immunodeficiency virus subtype C p24 antigen. The pRIC constructs were amplified in planta by up to two orders of magnitude by replication, while 50% more HPV-16 L1 and three- to seven-fold more EGFP and HIV-1 p24 were expressed from pRIC than from pTRAc. Vector replication was shown to be correlated with increased protein expression. We anticipate that this new high-yielding plant expression vector will contribute towards the development of a viable plant production platform for vaccine candidates and other pharmaceuticals.

Identification of amino acid residues of the pheromone‐binding domain of the transcription factor TraR that are required for positive control

Genes required for replication and for conjugal transfer of the Agrobacterium tumefaciens Ti plasmid are regulated by the quorum sensing transcription factor TraR, whose N-terminal domain binds to the pheromone 3-oxo-octanoylhomoserine lactone (OOHL) and whose C-terminal domain binds to specific DNA sequences called tra boxes. Here, we constructed 117 mutants, altering 103 surface-exposed amino acid residues of the TraR N-terminal domain. Each mutant was tested for activation of the traI promoter, where TraR binds to a site centred 45 nucleotides upstream of the transcription start site, and of the traM promoter, where TraR binds a site centred 66 nucleotides upstream. Alteration of 18 residues blocked activity at the traI promoter. Of these, alteration at three positions impaired TraR abundance or DNA binding, leaving 15 residues that are specifically needed for positive control. Of these 15 residues, nine also blocked or reduced activity at the traM promoter, while six had no effect. Amino acid residues required for activation of both promoters probably contact the C-terminal domain of the RNA polymerase alpha subunit, while residues required only for traI promoter activation may contact another RNA polymerase component.

Terminal proteins of Streptomyces chromosome can target DNA into eukaryotic nuclei

Streptomyces species are highly abundant soil bacteria that possess linear chromosomes (and linear plasmids). The 5′ ends of these molecules are covalently bound by terminal proteins (TPs), that are important for integrity and replication of the telomeres. There are at least two types of TPs, both of which contain a DNA-binding domain and a classical eukaryotic nuclear localization signal (NLS). Here we show that the NLS motifs on these TPs are highly efficient in targeting the proteins along with covalently bound plasmid DNA into the nuclei of human cells. The TP-mediated nuclear targeting resembles the inter-kingdom gene transfer mediated by Ti plasmids of Agrobacterium tumefaciens, in which a piece of the Ti plasmid DNA is targeted to the plant nuclei by a covalently bound NLS-containing protein. The discovery of the nuclear localization functions of the Streptomyces TPs not only suggests possible inter-kingdom gene exchanges between Streptomyces and eukaryotes in soil but also provides a novel strategy for gene delivery in humans and other eukaryotes.

Molecular biology and plant breeding

The use of modern molecular biological techniques has played a significant role in elucidating various aspects of the molecular organization of plant genes. Most of the information gained concerns characters and phenomena that are of relevance to plant breeding. It has been shown at the molecular level that cereal and legume seed storage proteins are controlled by complex multigenic nuclear loci. The analysis of movable nuclear genes, exemplified by the Ds-Ac transposable elements in maize, has given insight into the nature of these elements and the genetic changes that occur at their insertion and deletion sites. The characterization of the heterogeneous mitochondrial DNA and the uniform chloroplast DNA has helped to elucidate the molecular basis of cytoplasmic male sterility, RuBP carboxylase enzyme synthesis and herbicide resistance, In N2 fixation, the emphasis has been put on the molecular dissection of N2-fixing genes in bacteria and the interactions between symbiotic and host DNAs. It has been possible to develop vectors of Agrobacterium tumefaciens Ti-plasmids which lack oncogenes for DNA transfer. Cauliflower and gemini viruses as well as liposomes are also potential systems for DNA transport to protoplasts which can be regenerated into plants. Furthermore, it has been shown that certain molecular biological techniques are useful for screening purposes in genetic analysis and plant breeding work.

Localization of the plasmid-encoded proteins TraI and MobA in eukaryotic cells

Conjugation mediates gene transfer not only between bacterial species but also from bacteria to yeast, plant, and animal cells. DNA transferred by conjugative plasmids from bacteria to eukaryotes must traverse subcellular membranes in the recipient before the transferred genes can be expressed and inherited. This process is most likely facilitated by putative DNA pilot proteins such as VirD2 of the Agrobacterium tumefaciens Ti plasmid. Here, we test this model as a general feature of trans-kingdom conjugation using the DNA-relaxases TraI and MobA of the IncP and IncQ groups. TraI localized unambiguously and uniformly to the nuclei of both yeast and human cells, whereas MobA displayed a range of subcellular localization patterns. The tendency to localize to the nucleus was not correlated with predicted nuclear localization sequence motifs in either protein, suggesting a lack of stringent requirements for nuclear localizing potential in pilot proteins mediating conjugative DNA transfer to eukaryotes. Further, our results indicate that nuclear localization ability may be more commonly associated with conjugative pilot proteins than previously recognized.

Oncogene 6b from Agrobacterium tumefaciens Induces Abaxial Cell Division at Late Stages of Leaf Development and Modifies Vascular Development in Petioles

The 6b gene in the T-DNA region of the Ti plasmids of Agrobacterium tumefaciens and A. vitis is able to generate shooty calli in phytohormone-free culture of leaf sections of tobacco transformed with 6b. In the present study, we report characteristic morphological abnormalities of the leaves of transgenic tobacco and Arabidopsis that express 6b from pTiAKE10 (AK-6b), and altered expression of genes related to cell division and meristem formation in the transgenic plants. Cotyledons and leaves of both transgenic tobacco and Arabidopsis exhibited various abnormalities including upward curling of leaf blades, and transgenic tobacco leaves produced leaf-like outgrowths from the abaxial side. Transcripts of some class 1 KNOX homeobox genes, which are thought to be related to meristem functions, and cell cycle regulating genes were ectopically accumulated in mature leaves. M phase-specific genes were also ectopically expressed at the abaxial sides of mature leaves. These results suggest that the AK-6b gene stimulates the cellular potential for division and meristematic functions preferentially in the abaxial side of leaves and that the leaf phenotypes generated by AK-6b are at least in part due to such biased cell division during polar development of leaves. The results of the present experiments with a fusion gene between the AK-6b gene and the glucocorticoid receptor gene showed that nuclear import of the AK-6b protein was essential for upward curling of leaves and hormone-free callus formation, suggesting a role for AK-6b in nuclear events.

A small antisense RNA downregulates expression of an essential replicase protein of an Agrobacterium tumefaciens Ti plasmid

Tumour-inducing (Ti) plasmids of Agrobacterium tumefaciens replicate via the products of the repABC genes, which are highly conserved among plasmids of the alpha-Proteobacteria. RepA and RepB direct stable partitioning of daughter plasmids, while the RepC directs replicative DNA synthesis. We have identified a new gene (repE) within the repB-repC intergenic region of an octopine-type Ti plasmid. This gene encodes a small, non-translated RNA that is transcribed in the direction opposite to the repABC mRNA. Increased expression of repE blocked plasmid replication of a repABC-dependent miniplasmid, while decreased repE expression increased plasmid copy number. Direct RNA measurements and repC-lacZ fusions demonstrated that RepE inhibits the expression of RepC at the transcriptional level and possibly also at the translational level. Based on our experimental results and an RNA folding algorithm, we predict that RepE binding to the repABC mRNA would promote termination of the repABC transcript near the start codon of repC. Sequence analysis suggests that this phenomenon may be widespread among plasmids of this family.

Identification of amino acid residues of the Agrobacterium tumefaciens quorum‐sensing regulator TraR that are critical for positive control of transcription

The LuxR-type quorum-sensing transcription factor TraR regulates replication and conjugal transfer of the tumour-inducing (Ti) plasmid in the plant pathogen Agrobacterium tumefaciens. TraR is a two-domain protein with an N-terminal domain that binds to the quorum-sensing signal N-3-oxooctanoyl- l-homoserine lactone (OOHL) and a C-terminal domain that binds to specific DNA sequences called tra boxes. TraR-OOHL complexes form homodimers that activate transcription of at least seven promoters on the Ti plasmid. At five promoters, a tra box overlaps the binding site of core RNA polymerase (class II promoters), while in the other two promoters, this site is located farther upstream (class I promoters). In this study, we performed saturating point mutagenesis of the surface residues of the TraR C-terminal domain. Each mutant was tested for proteolytic stability and transcription activity in vivo, and for DNA binding activity in vitro. Mutants of TraR with single substitutions at positions W184, V187, K189, E193Q, V197 and D217 have wild-type levels of accumulation and DNA binding, but are defective in transcription of both types of promoters. These residues constitute a patch on the surface of the DNA-binding domain. We propose that this patch is an activating region that recruits RNA polymerase to TraR-dependent promoters through direct contact. As residues of this patch are critical for activation at both a class I and a class II promoter, we predict that these residues may contact the C-terminal domain of the RNA polymerase alpha-subunit.

Agrobacterium proteins VirD2 and VirE2 mediate precise integration of synthetic T-DNA complexes in mammalian cells.

Agrobacterium tumefaciens-mediated plant transformation, a unique example of interkingdom gene transfer, has been widely adopted for the generation of transgenic plants. In vitro synthesized transferred DNA (T-DNA) complexes comprising single-stranded DNA and Agrobacterium virulence proteins VirD2 and VirE2, essential for plant transformation, were used to stably transfect HeLa cells. Both proteins positively influenced efficiency and precision of transgene integration by increasing overall transformation rates and by promoting full-length single-copy integration events. These findings demonstrate that the virulence proteins are sufficient for the integration of a T-DNA into a eukaryotic genome in the absence of other bacterial or plant factors. Synthetic T-DNA complexes are therefore unique protein:DNA delivery vectors with potential applications in the field of mammalian transgenesis.

Crystallization and preliminary X-ray diffraction studies of the quorum-sensing regulator TraM from Agrobacterium tumefaciens.

TraM is a 11.4 kDa protein involved in the control of the conjugal transfer of Agrobacterium tumefaciens Ti plasmids by quorum-sensing. TraM was overexpressed and purified from Escherichia coli. This protein binds to the transcriptional regulator TraR, abolishing its function. Size-exclusion chromatography and dynamic light scattering show that the recombinant protein has an apparent molecular weight of 30 kDa in solution. Crystals have been obtained of both native and selenomethionine-substituted TraM by the vapour-diffusion method. Crystals diffract to 1.67 A and belong to the space group P2(1)2(1)2, with unit-cell parameters a = 76.43, b = 47.09, c = 47.46 A and two molecules in the asymmetric unit. A two-wavelength MAD data set for the selenomethionine-substituted form has been collected to a resolution of 2.0 A. The selenium substructure (five out of six possible sites) has been solved using direct methods.

Complete nucleotide sequence of the circular megaplasmid pHCG3 of Oligotropha carboxidovorans: function in the chemolithoautotrophic utilization of CO, H 2 and CO 2

Oligotropha carboxidovorans harbors the low-copy-number, circular, 133,058-bp DNA megaplasmid pHCG3, which is essential in the chemolithoautotrophic utilization of CO (carboxidotrophy), H 2 (hydrogenotrophy) and CO 2 under aerobic conditions. The complete nucleotide sequence of pHCG3 revealed 125 open reading frames. Of these, 95 were identified as putative structural genes. The plasmid carries the four gene clusters cox (14.54 kb, 12 genes), cbb (13.33 kb, 13 genes), hox (23.35 kb, 19 genes plus one ORF) and tra/trb (25.01 kb, 22 genes plus 2 ORFs), which assemble the functions required for the utilization of CO, CO 2 or H 2, and the conjugal transfer of the plasmid, respectively. The gene clusters cox, cbb and hox form a 51.2-kb chemolithoautotrophy module. The tra/trb cluster on the plasmid pHCG3 of O. carboxidovorans has a similar architecture as the Ti-plasmid of Agrobacterium tumefaciens. The tra/trb cluster is separated from the chemolithoautotrophy module by two regions (25.2 and 29.6 kb) with miscellaneous or mostly unknown functions. These regions carry a number of single genes coding for replication and stabilization of pHCG3 as well as the components of a putative system of global regulation of plasmid replication in O. carboxidovorans. An oriV encodes the replication proteins RepABC. Sequence comparisons of pHCG3-encoded genes suggest that major genetic exchange between O. carboxidovorans and the proteobacteria has occurred.