Construction of physical map and mapping of chromosomal virulence genes of the biovar 3 Agrobacterium (Rhizobium vitis) strain K-Ag-1

Most plant pathogenic Agrobacterium strains have been classified into three biovars, "biovar 1 (A. tumefaciens; Rhizobium radiobacter), biovar 2 (A. rhizogenes; R. rhizogenes) and biovar 3 (A. vitis; R. vitis)". The bacteria possess diverse types of genomic organization depending on the biovar. Previous genomic physical maps indicated difference in location of rDNA and chromosomally-coded virulence genes between biovar 1 and 2 genomes. In order to understand biovar 3 genome and its evolution in relation to the biovar 1, 2 and 3 genomes, we constructed physical map of a pathogenic biovar 3 strain K-Ag-1 in this study. Its genome consisted of two circular chromosomes (3.6 and 1.1 Mbp in length), and three plasmids (560, 230 and 70 kbp). Gene mapping based on the physical map showed presence of two rDNA loci in the larger chromosome and at least one rDNA locus in the smaller chromosome. Six chromosomal virulence genes, namely chvA, chvD, chvE, glgP, exoC and ros were found in the larger chromosome and not in the smaller chromosome. The location of rDNA loci is similar with that of biovar 1 genome, whereas the location of chromosomal virulence genes is similar with that of biovar 2 genome despite of the closer 16S-rRNA based phylogenetic relation of biovar 3 with biovar 1 than with biovar 2. Genomic PFGE RFLP analysis revealed that the K-Ag-1 strain, which was isolated on a kiwifruit plant in Japan, has the closest intra-species relation with two strains isolated from grapevine plants in Japan among eight biovar 3 strains examined. This datum suggests that the line of the strain is a major one in biovar 3 in Japan. Evolution of the genome of the strain is discussed based on the data.     

Genome analysis of Agrobacterium tumefaciens: linkage map and genetic features of the left region of the linear chromosome

In addition to a unique tumor-inducing (Ti) plasmid, the plant pathogenic bacterium Agrobacterium tumefaciens has an unconventional chromosomal organization. Our previous studies on A. tumefaciens MAFF301001 revealed that it possesses a 2 Mb linear and a 2.8 Mb circular chromosome plus a 206.479 kbp Ti plasmid (pTi-SAKURA). In this study, a linkage map for the left half of its linear chromosome covering a 900 kbp region was constructed and the number of potential genes existing in the region was estimated. The linkage map consists of 31 BAC and 8 lambda phage recombinants without any gaps. It confirmed the size and all the structural landmarks indicated in the corresponding region of our previously constructed physical map for the linear chromosome. Sequencing analysis of the end-regions of each linking clone led to the identification of 6 genes and another 27 potential genes or ORFs, including genes and/or gene clusters responsible for homologous recombination (ruvB), trehalose/maltose sugar transport (thuR, thuG) and alanine catabolism (dadR). Two virulence-related gene homologues (attK and celB), previously reported in the circular chromosome of a different strain of A. tumefaciens were found in this region. These findings will provide a ready-to-use linkage map for further functional analysis of the linear chromosome.     

Physical and genetic map of the Pseudomonas fluorescens SBW25 chromosome

Pseudomonas fluorescens is a saprophytic bacterium commonly isolated from soil, water, and the surfaces and tissues of plants and animals. The species has important applications in biotechnology because it can enhance plant growth and protect crops against disease. A complete physical map of the 6.63 Mbp P. fluorescens SBW25 chromosome was constructed using data obtained from combinations of one- and two-dimensional electrophoresis of completely or partially digested chromosomal DNA with end labelling. In total, 139 restriction sites (15 Pac I, 53 Spe I, 71 Xba I) were placed on the physical map and complete maps of the circular chromosome were obtained for both Pac I and Spe I; only Xba I fragments linking Spe I fragments were positioned. The average resolution of restriction sites was 48 kbp. A genetic map was derived from the physical map by Southern hybridization and 31 genes were positioned including oriC , rDNA operons ( rnnA–E ), recA gacA and pyvD     

Physical and genetic mapping of the Rhodobacter sphaeroides 2.4.1 genome: presence of two unique circular chromosomes.

A macrorestriction map representing the complete physical map of the Rhodobacter sphaeroides 2.4.1 chromosomes has been constructed by ordering the chromosomal DNA fragments from total genomic DNA digested with the restriction endonucleases AseI, SpeI, DraI, and SnaBI. Junction fragments and multiple restriction endonuclease digestions of the chromosomal DNAs derived from wild-type and various mutant strains, in conjunction with Southern hybridization analysis, have been used to order all of the chromosomal DNA fragments. Our results indicate that R. sphaeroides 2.4.1 carries two different circular chromosomes of 3,046 +/- 95 and 914 +/- 17 kilobases (kb). Both chromosome I (3,046 kb) and chromosome II (914 kb) contain rRNA cistrons. It appears that only a single copy of the rRNA genes is contained on chromosome I (rrnA) and that two copies are present on chromosome II (rrnB, rrnC). Additionally, genes for glyceraldehyde 3-phosphate dehydrogenase (gapB) and delta-aminolevulinic acid synthase (hemT) are found on chromosome II. In each instance, there appears to be a second copy of each of these genes on chromosome I, but the extent of the DNA homology is very low. Genes giving rise to enzymes involved in CO2 fixation and linked to the gene encoding the form I enzyme (i.e., the form I region) are on chromosome I, whereas those genes representing the form II region are on chromosome II. The complete physical and partial genetic maps for each chromosome are presented.     

Presence of one linear and one circular chromosome in the Agrobacterium tumefaciens C58 genome.

Analysis of the entire Agrobacterium tumefaciens C58 genome by pulsed-field gel electrophoresis (PFGE) reveals four replicons: two large molecules of 3,000 and 2,100 kb, the 450-kb cryptic plasmid, and the 200-kb Ti plasmid. Digestion by PacI or SwaI generated 12 or 14 fragments, respectively. The two megabase-sized replicons, used as probes, hybridize with different restriction fragments, showing that these replicons are two independent genetic entities. A 16S rRNA probe and genes encoding functions essential to the metabolism of the organism were found to hybridize with both replicons, suggesting their chromosomal nature. In PFGE, megabase-sized circular DNA does not enter the gel. The 2.1-Mb chromosome always generated an intense band, while the 3-Mb band was barely visible. After linearization of the DNA by X-irradiation, the intensity of the 3-Mb band increased while that of the 2.1-Mb remained constant. This suggests that the 3-Mb chromosome is circular and that the 2.1-Mb chromosome is linear. To confirm this hypothesis, genomic DNA, trapped in an agarose plug, was first submitted to PFGE to remove any linear DNA present. The plug was then recovered, and the remaining DNA was digested with either PacI or SwaI and then separated by PFGE. The fragments corresponding to the small chromosome were found to be absent, while those corresponding to the circular replicon remained, further proof of the linear nature of the 2.1-Mb chromosome.     

Isolation and characterization of a new chromosomal virulence gene of Agrobacterium tumefaciens.

A mutant (strain B119) of Agrobacterium tumefaciens with a chromosomal mutation was isolated by transposon (Tn5) mutagenesis. The mutant exhibited growth rates on L agar and minimal medium (AB) plates similar to those of the parent strain (strain A208 harboring a nopaline-type Ti plasmid). The mutant was avirulent on all host plants tested: Daucus carota, Cucumis sativus, and Kalanchoe diagremontiana. The mutant was not impaired in attachment ability to carrot cells. The mutant had one insertion of Tn5 in its chromosome. The avirulent phenotype of B119 was shown to be due to the Tn5 insertion in the chromosome by the marker exchange technique. A wild-type target chromosomal segment (3.0 kb) which included the site of mutation was cloned and sequenced. Two open reading frames, ORF-1 (468 bp) and ORF-2 (995 bp), were identified in the 3.0-kb DNA segment. Tn5 was inserted in the middle of ORF-2 (acvB gene). Introduction of the acvB gene into the mutant B119 strain complemented the avirulent phenotype of the strain. Homology search found no genes homologous to acvB, although it had some similarity to the open reading frame downstream of the virA gene on the Ti plasmid. Thus, the acvB gene identified in this study seems to be a new chromosomal virulence gene of A. tumefaciens.     

Isolation and genetic analysis of an Agrobacterium tumefaciens avirulent mutant with a chromosomal mutation produced by transposon mutagenesis.

A transposon 5 (Tn5) insertion was introduced into the genome of A. tumefaciens (A-208 strain harboring a nopaline type Ti-plasmid) using a conjugative pJB4JI plasmid containing Tn5. Five thousand transconjugants were assayed for virulence on carrot (Daucus carota L.) disks; 54 isolates were avirulent or very attenuated. The cellular localization (plasmid or chromosome) of the Tn5 insertion in those isolates were identified by Southern hybridization analysis. An avirulent mutant (B-90 strain) with the Tn5 insertion in the chromosome was selected and characterized. The mutant had the same growth rate as that of the parent strain in L-broth. The mutant and the parent strain had similar attachment ability to carrot root cells. Tn5 was inserted into one site of the chromosome. The wild-type target chromosomal region (1281 base pairs) was cloned and sequenced. An open reading frame (ORF) consisting of 395 base pairs was identified. The wild-type DNA fragment (1.6 kb) containing the ORF introduced into B-90 strain complemented the avirulent phenotype of the strain. A soluble protein was predicted from the ORF. The Tn5 was inserted near the 3'-terminal of the ORF. Homology search of this ORF found no significant homology to known genes and proteins. Thus, the ORF identified in this paper seems to be a new chromosomal virulence gene of A. tumefaciens.     

Ti plasmid and chromosomal ornithine catabolism genes of Agrobacterium tumefaciens C58.

The pTiC58 plasmid noc genes of Agrobacterium tumefaciens C58 code for nopaline oxidase (nocC), nopaline permease (nocP), the inducible periplasmic protein n1 (nocB), and a function(s) required for ornithine catabolism (nocA). In addition, strains C58 and Ach-5 of A. tumefaciens have chromosomal ornithine catabolism genes. The chromosomal orc gene codes for ornithine dehydrogenase. Strain C58 is normally orc, but orc+ mutants can be selected. We have characterized both chromosomal orc and pTiC58 nocA plasmid genes. Complementation of most chromosomal orc mutants by pTiC58 restored growth on both nopaline and L-ornithine but did not restore ornithine dehydrogenase activity. We conclude that ornithine is an intermediate of nopaline degradation and that the Ti plasmid and chromosome both code for ornithine-degradative enzymes. A model for nopaline catabolism is presented.     

Plasmid loss and changes within the chromosomal DNA of Streptomyces reticuli.

The sporulating wild-type strain of Streptomyces reticuli, which produces a melanin pigment and the macrolide leucomycin, contains plasmid DNA of 48 to 49 megadaltons. Plasmidless variants had an altered secondary metabolism and a changed antibiotic resistance pattern. By using a new colony hybridization technique developed for streptomycetes, it could be shown that plasmidless variants could be transformed with the wild-type plasmid DNA, which, however, is quickly lost from regenerated mycelium. In contrast to the wild-type strain, the plasmidless variants contain amplified nucleotide sequences within the chromosomal DNA. The number and size of these sequences vary with the strain tested. Hybridization studies revealed that the reiterated sequences are neither amplified ribosomal nor plasmid genes, but are present in small concentrations within the wild-type chromosome. Some of them share extensive homologies with each other and are located at different positions within the chromosome. It is assumed that alterations in secondary metabolism are due to changes within both the chromosomal and the extrachromosomal DNAs of S. reticuli.     

A novel plasmid-transcribed regulatory sRNA,QfsR, controls chromosomal polycistronic gene expression in Agrobacterium fabrum

Plasmids are mobile DNAs that can adjust host cell functions for their own amplification and dissemination. We identified Quorum sensing flagella small RNA regulator (QfsR), a small RNA, transcribed from the virulence tumour-inducing (Ti) plasmid in the phytopathogen Agrobacterium fabrum. QfsR is widely conserved throughout RepABC plasmids carried by Rhizobiaceae. Target prediction, expression analysis and site-direct mutagenesis experiments showed that QfsR directly pairs within polycistronic mRNAs transcribed from chromosome (genes involved in flagella apparatus) and Ti plasmid (genes involved in conjugative transfer). QfsR leads to a coordinated expression of whole polycistronic mRNA molecules. Whereas a lack of QfsR represses motility, its overproduction increases the quorum sensing signal accumulation and the Ti plasmid conjugative transfer. Based on these observations, we propose QfsR as a hub connecting regulatory networks of motility and plasmid conjugative transfer. To our knowledge, QfsR is the first example of a plasmid-encoded sRNA that controls chromosomal polycistronic gene expression.     

Physical map of the linear chromosome of Streptomyces hygroscopicus 10-22 deduced by analysis of overlapping large chromosomal deletions

The chromosomal DNA of Streptomyces hygroscopicus 10-22, a derivative of strain 5102-6, was digested with several restriction endonucleases and analyzed by pulsed-field gel electrophoresis (PFGE). Digestions with AseI gave 11 fragments with a total length of ca. 7.36 Mb. The AseI sites were mapped by analysis of overlapping chromosomal deletions in different mutants and confirmed by Southern hybridizations using partially digested genome fragments and linking cosmids as probes. PFGE analysis of DNA with and without proteinase K treatment, together with the hybridization results, suggested a linear organization with terminal proteins and large terminal inverted repeats. Some deletion mutants had circular chromosomes.     

Activation of Agrobacterium tumefaciens vir gene expression generates multiple single-stranded T-strand molecules from the pTiA6 T-region: requirement for 5'' virD gene products.

Agrobacterium tumefaciens transfers its Ti-plasmid T-DNA to plant cells. This process is initiated by plant-induced activation of the Ti-plasmid virulence loci, resulting in the generation of single stranded (ss) cleavages of the Ti-plasmid T-DNA border sequences (border nicks) and ss linear unipolar T-DNA molecules (T-strands). A single T-strand is produced from the two-border T-region of the pGV3850 nopaline plasmid. In this paper the induced molecular events for the complex T-region of the pTiA6 octopine plasmid are analyzed. This T-region carries four T-DNA borders delimiting three T-DNA elements (TR, TC and TL). Induction of pTiA6 generates cleavages independently at its border repeats, and six distinct T-strand species corresponding to TR, TR/TC, TR/TC/TL, TC, TC/TL and TL. These T-strand molecules are linear and correspond to the bottom strand of the pTiA6 T-region. Thus, borders can function for both initiation and termination of T-strand synthesis. We propose that the different pTiA6 T-strands are independently generated, and that the distribution of border nicks within the parental T-region determines which T-strand is produced. To identify genes involved in T-strand production, pTiA6 virulence (vir) and chromosomal virulence (chv) mutant strains were analyzed. VirA and VirG, the vir regulatory loci are required. Furthermore, the two 5' cistrons of virD are required for both border nicks and T-strands, suggesting that these genes encode the border endonuclease, and that T-strand production is dependent on border nicks. That no mutants are defective for T-strands alone suggests that functions encoded outside of vir and chv might mediate some of the later reactions of T-strand synthesis.     

Characterization of conjugal transfer functions of Agrobacterium tumefaciens Ti plasmid pTiC58.

Physical characterization of 13 transposon Tn5 insertions within the agrocinopine-independent, transfer-constitutive Ti plasmid pTiC58Trac identified three separate loci essential for conjugation of this nopaline/agrocinopine A + B-type Ti plasmid. Complementation analysis with relevant subcloned DNAs indicated that the three physically separated blocks of conjugal genes constitute distinct complementation groups. Two independent Tn5 insertions within the wild-type, agrocinopine-dependent, repressed pTiC58 plasmid resulted in constitutive expression of conjugal transfer. These two insertions were physically indistinguishable and could not be complemented in trans. However, the Trac phenotype resulted when the Tn5-mutated fragment cointegrated into the wild-type Ti plasmid. While the spontaneous Trac mutant Ti plasmids were also derepressed for agrocinopine catabolism, those generated by Tn5 insertions remained inducible, indicating that this apparent cis-acting site is different from that affected in the spontaneous mutants. No chromosomal Tn5 insertion mutations were obtained that affected conjugal transfer. An octopine-type Ti plasmid, resident in different Agrobacterium tumefaciens chvB mutants, transferred at normal frequencies, demonstrating that this virulence locus affecting plant cell binding is not required for Ti plasmid conjugation. None of our conjugal mutants limited tumor development on Kalanchoe diagremontiana. Known lesions in pTiC58 vir loci had no effect on conjugal transfer of this Ti plasmid. These results show that pTiC58 Ti plasmid conjugal transfer occurs by functions independent of those required for transfer of DNA to plant cells.     

Characterization and chromosomal distribution of satellite DNA sequences of the water buffalo (Bubalus bubalis).

Satellite DNA sequences were isolated from the water buffalo (Bubalus bubalis) after digestion with two restriction endonucleases, BamHI and StuI. These satellite DNAs of the water buffalo were classified into two types by sequence analysis: one had an approximately 1,400 bp tandem repeat unit with 79% similarity to the bovine satellite I DNA; the other had an approximately 700 bp tandem repeat unit with 81% similarity to the bovine satellite II DNA. The chromosomal distribution of the satellite DNAs were examined in the river-type and the swamp-type buffaloes with direct R-banding fluorescence in situ hybridization. Both the buffalo satellite DNAs were localized to the centromeric regions of all chromosomes in the two types of buffaloes. The hybridization signals with the buffalo satellite I DNA on the acrocentric autosomes and X chromosome were much stronger than that on the biarmed autosomes and Y chromosome, which corresponded to the distribution of C-band-positive centromeric heterochromatin. This centromere-specific satellite DNA also existed in the interstitial region of the long arm of chromosome 1 of the swamp-type buffalo, which was the junction of the telomere-centromere tandem fusion that divided the karyotype in the two types of buffaloes. The intensity of the hybridization signals with buffalo satellite II DNA was almost the same over all the chromosomes, including the Y chromosome, and no additional hybridization signal was found in noncentromeric sites.     

Identification of a chromosomal tra-like region in Agrobacterium tumefaciens

The virD4 gene is one of the virulence genes present on the pTiC58 plasmid of Agrobacterium tumefaciens. Unexpectedly, we found that a pTi-free A. tumefaciens strain carried a protein of similar size to the plasmid-encoded VirD4 protein which reacted with VirD4-specific antibodies. This suggested that this strain may contain a homologue of the VirD4 protein. A chromosomal fragment encoding a protein of similar sequence to VirD4 was isolated and a 7.8 kilobase region surrounding the gene encoding this putative homologue was sequenced. This region contained four open reading frames, encoding putative proteins similar to proteins of known bacterial transfer and conjugation systems, viz., orf1 encoded a putative homologue of the TraA protein of the Rhizobium symbiosis plasmid pNGR234 and the TraA protein encoded by pTiC58 from A. tumefaciens plasmid pTiC58, orf3 encoded a protein very similar to the MobC protein encoded by the IncQ plasmid RSF1010 of E. coli and to MobS encoded by pTF1 from Thiobacillus ferrooxidans, whereas the predicted product of orf4 displayed similarity to the TraG protein encoded by the IncPalpha plasmid RP4 of E. coli, TraG and VirD4 encoded by A. tumefaciens plasmid pTiC58. The product of orf2 showed no significant similarity to any known protein. Preliminary assays with two orf4 mutants suggested that the product of this orf is involved in DNA transfer. The 7.8 kb chromosomal fragment seems to be closely related to the tra region of different conjugative plasmids and appears to be confined to Agrobacterium species, raising the question of the role of a chromosomal tra-like region during evolution.     

The nir, nor, and nos denitrification genes are dispersed over the Bradyrhizobium japonicum chromosome

Cleavage of genomic DNA from Bradyrhizobium japonicum strain 3I1b110 by the restriction enzymes PmeI, PacI, and SwaI has been used together with pulsed-field gel electrophoresis and Southern hybridization to locate the nirK, norCBQD, and nosRZDFYLX denitrification genes on the chromosomal map of B. japonicum strain 110spc4. Mutant strains GRK13, GRC131, and GRZ25 were obtained by insertion of plasmid pUC4-KIXX-aphII-PSP, which carries recognition sites for the enzymes PacI, PmeI and SwaI, into the B. japonicum 3I1b110 nirK, norC and nosZ genes, respectively. Restriction of strain 3I1b110 genomic DNA with PacI, PmeI and SwaI yielded three, five and nine fragments, respectively. Pulsed-field gel electrophoresis of restricted mutant DNAs resulted in an altered fragment pattern that allowed determination of the position of the selected genes. Complementary mapping data were obtained by hybridization using digoxigenin-labeled B. japonicum 3I1b110 nirK, norBQD and nosZD as gene probes. The nirK, norCBQD and nosRZDFYLX genes were located close to the groEL(2), cycH and cycVWX genes, respectively, on the strain 110spc4 genetic map. In contrast to other denitrifiers, B. japonicum 3I1b110 denitrification genes were dispersed over the entire chromosome.     

Development of a physical map for three Frankia strains and a partial genetic map for Frankia EuI1c

Physical maps for Frankia strains CcI3, EAN1pec and EuI1c chromosomes were constructed by the use of macrorestriction analysis and pulsed-field gel electrophoresis (PFGE). The restriction enzymes Ase I, Pme I, Swa I and Ssp I were used to cut the Frankia chromosome into a limited number of large fragments and for double digestions. The genomes sizes, as determined by the addition of the estimated fragment sizes, were 5430 ± 35 kb, 9101 ± 109 kb and 8105 ± 842 kb for strains CcI3, EAN1pec and EuI1c, respectively. A complete physical map was achieved by the analysis of PFGE for the single and double digestions and by two-dimensional PFGE to determine doublets and overlapping fragments. For strain EuI1c, a partial genetic map was also constructed by positioning the 16S rRNA, gln II, gln A and hbo O genes on the physical map. PFGE analysis of DNA with and without proteinase K treatment together with the other results suggested a circular genome.     

DNA homology of surface protein antigen A gene in mutans streptococci

1. A recombinant plasmid, pYA724, containing an 8.45 kb DNA fragment encoding surface protein antigen A (spaA) from Streptococcus sobrinus 6715 was used to examine the DNA homology of the spaA gene with chromosomal DNA of various mutans streptococci strains. 2. Restriction endonuclease BamHI-digested pYA724 DNA was radio-labeled by nick-translation, and a DNA-DNA hybridization experiment was carried out. pYA724 DNA hybridized with chromosomal DNA of serotypes a, c, d, e, f and g strains, but not with b by dot DNA-hybridization and Southern blot DNA hybridization. 3. Chromosomal DNAs were isolated from several serotype c Streptococcus mutans strains, digested with BamHI, and analyzed by Southern blot DNA hybridization. pYA724 DNA hybridized with different sizes and numbers of BamHI-digested DNA fragments of the chromosomal DNAs. 4. These data indicated that all mutans streptococci strains except serotype b have DNA homologous with the spaA gene, although within the same serotype strain the spaA gene has a diversity of arrangement within the chromosome.