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PubMed2005

Large-scale genetic variation of the symbiosis-required megaplasmid pSymA revealed by comparative genomic analysis of Sinorhizobium meliloti natural strains.

BACKGROUND: Sinorhizobium meliloti is a soil bacterium that forms nitrogen-fixing nodules on the roots of leguminous plants such as alfalfa (Medicago sativa). This species occupies different ecological niches, being present as a free-living soil bacterium and as a symbiont of plant root nodules. The genome of the type strain Rm 1021 contains one chromosome and two megaplasmids for a total genome size of 6 Mb. We applied comparative genomic hybridisation (CGH) on an oligonucleotide microarrays to estimate genetic variation at the genomic level in four natural strains, two isolated from Italian agricultural soil and two from desert soil in the Aral Sea region. RESULTS: From 4.6 to 5.7 percent of the genes showed a pattern of hybridisation concordant with deletion, nucleotide divergence or ORF duplication when compared to the type strain Rm 1021. A large number of these polymorphisms were confirmed by sequencing and Southern blot. A statistically significant fraction of these variable genes was found on the pSymA megaplasmid and grouped in clusters. These variable genes were found to be mainly transposases or genes with unknown function. CONCLUSION: The obtained results allow to conclude that the symbiosis-required megaplasmid pSymA can be considered the major hot-spot for intra-specific differentiation in S. meliloti.

Blotting, Southern↗

PubMed2000

High-resolution physical map of the pSymb megaplasmid and comparison of the three replicons of Sinorhizobium meliloti strain 1021.

A high-resolution physical map of the larger megaplasmid (pSymb) of Sinorhizobium meliloti strain 1021 has been constructed by using BAC libraries and an original two-step PCR screening method. This method, previously used to map both the chromosome and the smaller megaplasmid (pSyma), allowed us to position over the genome a total of 842 markers with an average density of one marker every 8.3 kb. In addition, we used BLASTX and PRODOM analysis to predict a function for a number of STSs. This work led to the discovery of several interesting loci and to a comparison of the genetic information carried by each replicon. The two main results emerging from this study are (i) a biased distribution of housekeeping genes, mainly detected on chromosome, and (ii) the presence of an unexpected number of transporters, mainly belonging to the ABC superfamily. These are broadly distributed across the whole genome, but particularly found on pSymb.

ATP-Binding Cassette Transporters↗

PubMed1984

Visualization and exact molecular weight determination of a Rhizobium meliloti megaplasmid.

The entire DNA of Rhizobium meliloti MVII /1 cells was isolated preparatively by gentle lysis and sucrose gradient centrifugation. Fractions of the sucrose gradient were investigated by electron microscopy. We found intact megaplasmids in supercoiled and relaxed form and total chromosomes. The length of the megaplasmid could be measured, which allowed an exact determination of the molecular weight. We found a length of 0.48 +/- 0.019 mm equivalent to a molecular weight of about 1000 X 10(6).

Chromosomes, Bacterial↗

PubMed2003

Complete nucleotide sequence of pHG1: a Ralstonia eutropha H16 megaplasmid encoding key enzymes of H(2)-based ithoautotrophy and anaerobiosis.

The self-transmissible megaplasmid pHG1 carries essential genetic information for the facultatively lithoautotrophic and facultatively anaerobic lifestyles of its host, the Gram-negative soil bacterium Ralstonia eutropha H16. We have determined the complete nucleotide sequence of pHG1. This megaplasmid is 452,156 bp in size and carries 429 potential genes. Groups of functionally related genes form loose clusters flanked by mobile elements. The largest functional group consists of lithoautotrophy-related genes. These include a set of 41 genes for the biosynthesis of the three previously identified hydrogenases and of a fourth, novel hydrogenase. Another large cluster carries the genetic information for denitrification. In addition to a dissimilatory nitrate reductase, both specific and global regulators were identified. Also located in the denitrification region is a set of genes for cytochrome c biosynthesis. Determinants for several enzymes involved in the mineralization of aromatic compounds were found. The genes for conjugative plasmid transfer predict that R.eutropha forms two types of pili. One of them is related to the type IV pili of pathogenic enterobacteria. pHG1 also carries an extensive "junkyard" region encompassing 17 remnants of mobile elements and 22 partial or intact genes for phage-type integrase. Among the mobile elements is a novel member of the IS5 family, in which the transposase gene is interrupted by a group II intron.

Anaerobiosis↗

PubMed2003

Megaplasmids in Gram-negative, moderately halophilic bacteria.

We have discovered that many Halomonas species harbour large extrachromosomal DNA elements. Using currently available protocols it is technically very difficult to identify large plasmids in bacteria, and even more so when they are coated in mucous polysaccharide. We used culture conditions suitable for both halophilic and halophilic exopolysaccharide-producing bacteria and applied a modified gel electrophoresis method to locate and visualise the megaplasmids. Almost all the species of Halomonas studied harbour two plasmids of about 70 kb and 600 kb and some species carry other smaller extrachromosomal DNA elements. The common presence of these megaplasmids may well be related to the survival strategies of the bacteria in their special surroundings.

DNA, Bacterial↗

PubMed1997

Megaplasmid and chromosomal loci for the PHB degradation pathway in Rhizobium (Sinorhizobium) meliloti.

Chromosomal and megaplasmid loci that affect the poly-3-hydroxybutyrate (PHB) degradation pathway in Rhizobium meliloti were identified. A clone that restores the ability of certain R. meliloti mutants with defined deletions in megaplasmid pRmeSU47b to use 3-hydroxybutyrate or acetoacetate as the sole carbon source was isolated from a cosmid library of R. meliloti genomic DNA. Tn5 insertion mutagenesis, followed by merodiploid complementation analysis, demonstrated that the locus consists of at least four transcriptional units, bhbA-D. We also identified loci involved in 3-hydroxybutyrate and/or acetoacetate utilization by screening for mutants that had lost the ability to use 3-hydroxybutyrate as the sole carbon source while retaining the ability to use acetate (thus ensuring an intact glyoxylate cycle and gluconeogenic pathway). These mutants fell into four classes, as determined by replicon mobilization experiments and genetic linkage in phage transduction; one class corresponded to the bhb locus on pRmeSU47b, two classes mapped to different regions on the chromosome and the fourth, bdhA, represented by a single mutant, mapped to another pRmeSU47b locus, near bacA. The bdhA mutant is deficient in 3-hydroxybutrate dehydrogenase activity.

Acetoacetates↗

PubMed1994

Halobacterial megaplasmids are negatively supercoiled.

Several covalently closed circular halobacterial megaplasmids (up to more than 500 kb) from different strains of Haloferax mediterranei, have been resolved by orthogonal-field alternating gel electrophoresis (OFAGE). These molecules seem to be negatively supercoiled in vivo, as deduced from the effect of intercalating agents affecting their topology and, therefore, their electrophoretic mobility. It has also been demonstrated that the topoisomerase II inhibitor novobiocin affects the native topological state of halobacterial megaplasmids impeding their migration in OFAGE under standard conditions for resolution of large supercoiled molecules.

DNA, Superhelical↗

PubMed2006

Identification of a megaplasmid centromere reveals genetic structural diversity within the repABC family of basic replicons.

The basic replication unit of many plasmids and second chromosomes in the alpha-proteobacteria consists of a repABC locus that encodes the trans- and cis-acting components required for both semiautonomous replication and replicon maintenance in a cell population. In terms of physical genetic organization and at the nucleotide sequence level, repABC loci are well conserved across various genera. As with all repABC-type replicons that have been genetically characterized, the 1.4 Mb pSymA and 1.7 Mb pSymB megaplasmids from the plant endosymbiont Sinorhizobium meliloti encode strong incompatibility (inc) determinants. We have identified a novel inc sequence upstream of the repA2 gene in pSymA that is not present on pSymB and not reported in other repABC plasmids that have been characterized. This region, in concert with the repA and repB genes, stabilizes a test plasmid indicating that it constitutes a partitioning (par) system for the megaplasmid. Purified RepB binds to this sequence and binding may be enhanced by RepA. We have isolated 19 point mutations that eliminate incompatibility, reduce RepB binding or the stabilization phenotype associated with this sequence and all of these map to a 16-nucleotide palindromic sequence centred 330 bp upstream of the repA2 gene. An additional five near-perfect repeats of this palindrome are located further upstream of the repA2 gene and we show that they share some conservation with known RepB binding sites in different locations on other repABC plasmids and to two sequences found on the tumour inducing plasmid of Agrobacterium tumefaciens. These additional palindromes also bind RepB but one of them does not display obvious incompatibility effects. A heterogenic distribution of par sequences demonstrates unexpected diversity in the structural genetic organization of repABC loci, despite their obvious levels of similarity.

Bacterial Proteins↗

PubMed2001

A physical map of the megaplasmid pHG1, one of three genomic replicons in Ralstonia eutropha H16.

We have used pulsed field gel electrophoresis and megabase DNA techniques to investigate the basic genomic organization of Ralstonia eutropha H16, and to construct a physical map of its indigenous megaplasmid pHG1. This Gram-negative, soil-dwelling bacterium is a facultative chemolithoautotroph and a denitrifier. In the absence of organic substrates it can grow on H2 as its sole energy source and CO2 as its sole source of carbon. Under anaerobic conditions it can utilize nitrate as a terminal electron acceptor, whereby dinitrogen is released. Essential genetic determinants of the enzyme systems responsible for these metabolic processes are linked to the 0.44-Mb conjugative megaplasmid pHG1. Aside from pHG1, the genome of R. eutropha H16 is comprised of two circular chromosomes measuring 4.1 and 2.9 Mb, adding up to a total genome size of 7.1 Mb. An estimated five copies of rDNA are distributed on the two chromosomes. A macrorestriction map of pHG1 was derived for the endonucleases DraI and XbaI. Hybridization studies showed that genes for anaerobic metabolism are located on all three genomic replicons.

Anaerobiosis↗

PubMed1990

Conjugal Transfer of Megaplasmid 2 between Rhizobium meliloti Strains in Alfalfa Nodules.

A DNA fragment containing the RP4 mob function, as well as the gentamicin and spectinomycin resistance genes, was inserted by gene replacement onto the megaplasmid 2 (pM2) of Rhizobium meliloti 0540 (Inf EPS), resulting in PG101 (Inf EPS). The self-transfer of pM2 and the mobilization of pM2 by plasmid RP4-4 were investigated during conjugation between PG101 and R. meliloti 2526 (Nod). In filter conjugations, pM2 was readily mobilized by RP4-4. In addition to this, the self-transfer of one megaplasmid (pM) was detected at a frequency of 3 x 10. Bacteria isolated from the nodules of alfalfa and coinoculated with strains PG101 and 2526 showed that pM2 was mobilized at a frequency of approximately 7 x 10. Bacterial cell numbers were too low in the nodules for detection of the self-transfer of pM2 to occur. No pM2 transfer was detected in the inoculum. A comparison of the transfer frequencies for the various conjugation conditions revealed that pM2 transfer occurred as frequently in the nodules as in filter conjugations. These results indicate that the nodule creates conditions for gene transfer that are comparable to optimal laboratory conditions.

Journal Article↗

PubMed1999

A megaplasmid-borne anaerobic ribonucleotide reductase in Alcaligenes eutrophus H16.

The conjugative 450-kb megaplasmid pHG1 is essential for the anaerobic growth of Alcaligenes eutrophus H16 in the presence of nitrate as the terminal electron acceptor. We identified two megaplasmid-borne genes (nrdD and nrdG) which are indispensable under these conditions. Sequence alignment identified significant similarity of the 76.2-kDa gene product NrdD and the 30.9-kDa gene product NrdG with anaerobic class III ribonucleotide reductases and their corresponding activases. Deletion of nrdD and nrdG in A. eutrophus abolished anaerobic growth and led to the formation of nondividing filamentous cells, a typical feature of bacteria whose DNA synthesis is blocked. Enzyme activity of NrdD-like ribonucleotide reductases is dependent on a stable radical at a glycine residue in a conserved C-terminal motif. A mutant of A. eutrophus with a G650A exchange in NrdD showed the DNA-deficient phenotype as the deletion strain, suggesting that G650 forms the glycyl radical. Analysis of transcriptional and translational fusions indicate that nrdD and nrdG are cotranscribed and that the translation efficiency of nrdD is 40-fold higher than that of nrdG. Thus, the two proteins NrdD and NrdG are not synthesized at a stoichiometric level.

Anaerobiosis↗

PubMed1993

Why it pays for bacteria to delete disused DNA and to maintain megaplasmids.

Genetic information for disused metabolic systems is easily lost. This can be understood on the basis of a mathematic model for the description of the growth of individual microbial cells. The essential features of the model are that the uptake of nutrients is proportional to the surface area of the cell and maintenance costs are proportional to the volume. This explains why disused genes are easily lost. Growth of the individual cell continues until DNA replication of the genome is completed. The consequence is that cells with a large genome size are bigger at the moment of cell division. This results in a less favourable surface to volume ratio, which has a negative influence on the population growth rate. This means that cells with a smaller genome size will have a selective advantage. In cells in which the total DNA is divided over several units which replicate simultaneously (e.g. a cell with more chromosomes or a cell with one chromosome and one or more megaplasmids) a high surface to volume ratio can be maintained. The great metabolic differences between phylogenetically strongly related bacteria are due to loss of genetic information for complex metabolic characters in order to maintain a small genome size and a favourable surface to volume ratio.

Bacteria↗

PubMed1984

Isolation and characterization of megaplasmid DNA from lithoautotrophic bacteria.

A method is described for the preparative isolation of megaplasmids ranging in size from 340 to 700 kb. These plasmids were isolated from chemolithoautotrophic bacteria including the species Alcaligenes, Pseudomonas, and Paracoccus. The procedure was based on alkaline sodium dodecyl sulfate lysis of the cells, followed by heat treatment, salt precipitation, several phenol extractions, dialysis steps, and proteinase and RNase treatment. The various parameters were evaluated and controlled. Hydrogen-oxidizing-ability (Hox) encoding plasmids were compared by EcoRI restriction enzyme analysis. pHG plasmids from Alcaligenes eutrophus wild-type strains appeared to be closely related; plasmids derived from the type strain TF93 and from A. hydrogenophilus exhibited major differences in restriction sites. Two cryptic plasmids harbored by Pseudomonas facilis and Paracoccus denitrificans showed scarcely detectable similarity to the plasmid species of Alcaligenes.

Alcaligenes↗

PubMed2003

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.

Carbon Dioxide↗

PubMed2005

Cloning and characterization of a region responsible for the maintenance of megaplasmid pTAV3 of Paracoccus versutus UW1.

Using cointegrate formation, we constructed a basic replicon of the megaplasmid/mini-chromosome pTAV3 of Paracoccus versutus UW1. It is composed of two adjacent modules, responsible for plasmid replication (rep) and partitioning (par). Functional analysis of the par region identified a determinant of incompatibility (inc2), whose presence is crucial for proper partitioning (the partitioning site). Database searches revealed that the only known replicon with significant homology to that of pTAV3 is encoded by the chromosome cII of Rhodobacter sphaeroides 2.4.1. Incompatibility studies showed that closely related basic replicons are also encoded by megaplasmids (above 400 kb) harbored by four strains of P. pantotrophus. Basic replicons of the pTAV3-type are able to maintain large bacterial genomes, therefore they appear to be good candidates for the construction of vectors specific for Alphaproteobacteria.

Base Sequence↗

PubMed1995

Detection, isolation, and stability of megaplasmid-encoded chloroaromatic herbicide-degrading genes within Pseudomonas species.

Dicamba is used as a model system for microbial degradation of chloroaromatic benzoic acids. The detection, isolation, and stability of a megaplasmid within a Pseudomonas sp. is described as the first step in optimizing the growth of this microorganism and other microorganisms similar to it. A large plasmid, pDK1, consisting of approximately 250 kb, was purified from dicamba-degrading Pseudomonas sp. PXM. This plasmid was purified by the method of Allen (personal communication, 1994), which is a modified version of several that have been attempted for the isolation of large plasmids (Lee and Rasheed, 1990). The restriction analysis of this plasmid (pDK1) from PXM. revealed many distinctive bands on agarose gel electrophoresis. Based on the preliminary restriction enzyme analysis, the estimated size of this plasmid is 250 kb, which could make it one of the largest procaryotic plasmids encoding for chloroaromatic degrading enzymes. Allen's methodology results in very high purity and reproducibility compared to the other methods used in this study. As described in this work, the method of Kado and Liu (1981) is easier to perform and results in a more reproducible plasmid preparation than the method of Casse et al. (1979). Casse's protocol requires the use of a highly alkaline SDS solution (pH 12.45) in order to eliminate the chromosomal DNA. However, only incomplete removal of the chromosomal DNA results. Compared to the Casse et al. protocol, the Kado and Liu protocol requires the use of a highly alkaline solution (pH 12.6) and a high temperature (55-65 degrees C) to eliminate the chromosomal DNA. This results in a nearly complete removal of the chromosomal DNA. The high temperature treatment also quickly eliminates the RNA. Another advantage of the protocol of Kado and Liu over the protocol of Casse et al. is that the former uses phenol-chloroform extraction while the latter uses only phenol extraction. The phenol-chloroform extraction step denatures the DNA along with the proteins. In addition to this, the phenol-chloroform mixture minimizes the formation of a brown oxidation pigment that usually occurs with phenol extraction alone. Finally, the time needed to complete the Kado and Liu protocol is much shorter (2 hr) than the time needed to complete the Casse protocol (8 hr). As described previously, a highly purified plasmid preparation with minimal chromosomal DNA was prepared by following the suggestions of L. Allen.(ABSTRACT TRUNCATED AT 400 WORDS)

Biodegradation, Environmental↗

PubMed1999

Sequence analysis, characterization and CO-specific transcription of the cox gene cluster on the megaplasmid pHCG3 of Oligotropha carboxidovorans.

Sequence, transcriptional, mutational and physiological analyses indicate that the carbon monoxide (CO) dehydrogenase of Oligotropha carboxidovorans is an integral and unique part of an elaborate CO oxidizing system. It is encoded by the 14.5kb gene cluster coxBCMSLDEFGHIK residing on the 128kb megaplasmid pHCG3. The CO dehydrogenase structural genes coxMSL are flanked by nine accessory genes arranged as the cox gene cluster. The cox genes are specifically and coordinately transcribed under chemolithoautotrophic conditions in the presence of CO as carbon and energy source. With the exception of CoxB and CoxK, all deduced products of the cox genes of O. carboxidovorans have counterparts in so far uncharacterized gene clusters of Pseudomonas thermocarboxydovorans, Hydrogenophaga pseudoflava, Bradyrhizobium japonicum, and Mycobacterium tuberculosis. Transposon mutagenesis suggests a function of CoxH and CoxI in the interaction of CO dehydrogenase with the cytoplasmic membrane. The specific functions of the other accessory Cox proteins are difficult to envisage right now, as the polypeptides do not show significant homologies with functionally characterized proteins in the databases. In addition to the clustered cox genes, mutational analyses have identified the genes lon, cycH and orfX which reside on the plasmid pHCG3. The Lon protease, the CycH protein and the unknown orfX gene product have essential functions in the utilization of CO.

Aldehyde Oxidoreductases↗

PubMed2001

The complete sequence of the 1,683-kb pSymB megaplasmid from the N2-fixing endosymbiont Sinorhizobium meliloti.

Analysis of the 1,683,333-nt sequence of the pSymB megaplasmid from the symbiotic N(2)-fixing bacterium Sinorhizobium meliloti revealed that the replicon has a high gene density with a total of 1,570 protein-coding regions, with few insertion elements and regions duplicated elsewhere in the genome. The only copies of an essential arg-tRNA gene and the minCDE genes are located on pSymB. Almost 20% of the pSymB sequence carries genes encoding solute uptake systems, most of which were of the ATP-binding cassette family. Many previously unsuspected genes involved in polysaccharide biosynthesis were identified and these, together with the two known distinct exopolysaccharide synthesis gene clusters, show that 14% of the pSymB sequence is dedicated to polysaccharide synthesis. Other recognizable gene clusters include many involved in catabolic activities such as protocatechuate utilization and phosphonate degradation. The functions of these genes are consistent with the notion that pSymB plays a major role in the saprophytic competence of the bacteria in the soil environment.

Bacterial Proteins↗