The Use of Suppression Subtractive Hybridization to Identify Strain-Specific Sequences in Bacterial Genomes

Comparisons of bacterial genomes demonstrate that even strains of one species may strikingly differ in gene set. Strain-specific genes are of considerable interest, as they may be responsible for distinguishing features, such as virulence or drug resistance, of the strain and may be employed as markers in epidemiological or evolutionary studies. Suppression subtractive hybridization (SSH) was shown to be suitable for generating a set of DNA fragments differing between two closely related bacterial strains. More than 95% DNA fragments selected by SSH proved to be specific for Staphylococcus aureus strains ZW compared with strain 29213.     

Isolation and characterization of transposon Tn5-induced symbiotic mutants of Rhizobium loti

Rhizobium loti NZP2037 and NZP2213, each cured of its single large indigenous plasmid, formed effective nodules on Lotus spp., suggesting that the symbiotic genes are carried on the chromosome of these strains. By using pSUP1011 as a vector for introducing transposon Tn5 into R. loti NZP2037, symbiotic mutants blocked in hair curling (Hac), nodule initiation (Noi), bacterial release (Bar), and nitrogen fixation (Nif/Cof) on Lotus pedunculatus were isolated. Cosmids complementing the Hac, Noi, and Bar mutants were isolated from a pLAFR1 gene library of NZP2037 DNA by in planta complementation and found to contain EcoRI fragments of identical sizes to those into which Tn5 had inserted in the mutants. The cosmids that complemented the mutants of these phenotypic classes did not share common fragments, nor did cosmids that complemented four mutants within the Noi class, suggesting that these symbiotically important regions are not tightly linked on the R. loti chromosome.     

Cellular mechanisms of pH tolerance in Rhizobium loti

Seven strains of Rhizobium loti were tested for acid tolerance in yeast-extract mannitol (YEM) broth at pH values ranging from 4.0 to 8.0. The strains that grew at pH 4.0 showed the slowest generation time when grown at pH above 7.0 and also produced the most acid. The acid tolerance was related to the composition and structure of the membrane. pH influenced protein expression in acid-tolerant strains growing at pH 4.0 or 7.0. Acid tolerant strains showed one membrane protein of 49.5 kDa and three soluble proteins of 66.0, 58.0 and 44.0kDa; their expression increased when the cells grew at pH 4.0. It is suggested that acid tolerance in Rhizobium loti involves constitutive mechanisms, such as permeability of the outer membrane together with adaptive responses, including the state of bacterial growth and concomitant changes in protein expression.     

Detection of Viral DNA Sequences in Adenovirus-Transformed Cells by In Situ Hybridization

Cytological preparations of cells transformed by members of three groups of human adenoviruses, adenovirus 12, 7, and 2, were annealed with radioactive complementary RNA (cRNA) (4 x 10(7) to 4.5 x 10(7) dpm/mug) prepared by copying viral DNA with the Escherichia coli DNA-directed RNA polymerase. These in situ hybridizations detected adenovirus-specific DNA sequences in interphase nuclei when transformed cells were annealed with homologous viral cRNA, but not with heterologous viral cRNA. The highest autoradiographic grain counts were found over adenovirus 7-transformed cell nuclei, next over adenovirus 12-, and the lowest over adenovirus 2-transformed cell nuclei. This is the same order as found by reassociation kinetic measurements (K. Fujinaga and M. Green, unpublished data).     

Isolation and characterization of a Rhizobium loti gene required for effective nodulation of Lotus pedunculatus

A Rhizobium loti gene required for effective invasion of the host Lotus pedunculatus has been identified by transposon Tn5 mutagenesis. Cosmids that complemented a previously isolated mutation (239) at this invasion (inv) locus were identified by in planta complementation and used to construct a physical map of the gene region. The insertion site of Tn5 in PN239 was mapped to a 7.5-kb EcoRI fragment, which complemented the mutation when subcloned into pLAFR1. Further Tn5 mutagenesis of the 7.5-kb fragment was carried out in Escherichia coli using bacteriophage lambda 467, and the mutations homogenotized into R. loti NZP2037. Three additional Fix- mutations were isolated, and these were found to map adjacent to the position of the original mutation in strain PN239. All the other Tn5 insertions isolated in the 7.5-kb fragment gave a Fix+ phenotype on L. pedunculatus. Electron microscopic examination of the L. pedunculatus nodules induced by the isolated Fix- mutants showed that bacteria were either blocked in release from the infection threads or were unable to undergo normal bacteroid development. The inv locus as defined by the Tn5 insertions was sequenced, and a single open-reading frame (ORF) of 576 bp, corresponding to a polypeptide of 21.3 kDa, was identified. The position and orientation of this ORF were consistent with those of the isolated Tn5 Fix- insertions.     

Identification of DNA Sequences Specific for Vibrio vulnificus Biotype 2 Strains by Suppression Subtractive Hybridization

Vibrio vulnificus can be divided into three biotypes, and only biotype 2, which is further divided into serovars, contains eel-virulent strains. We compared the genomic DNA of a biotype 2 serovar E isolate (tester) with the genomic DNAs of three biotype 1 strains by suppression subtractive hybridization and then tested the distribution of the tester-specific DNA sequences in a wide collection of bacterial strains. In this way we identified three plasmid-borne DNA sequences that were specific for biotype 2 strains irrespective of the serovar and three chromosomal DNA sequences that were specific for serovar E biotype 2 strains. These sequences have potential for use in the diagnosis of eel vibriosis caused by V. vulnificus and in the detection of biotype 2 serovar E strains.     

Characterization of Rhizobium loti strains from the Salado River Basin

Thirty indigenous rhizobia strains, isolated from Lotus tenuis in the area of Chascomús and other regions of the Salado River Basin (Argentina), were characterized based on generation time, acid production, carbon utilization, protein profile, and molecular characterization by restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes amplified by the polymerase chain reaction (PCR). The results indicated that native rhizobia isolates from the Chascomús area are predominantly fast and intermediate-growers. The unclassified rhizobia examined by PCR-RFLP were found to be closely related to the reference strains of validly described Rhizobium species.     

Localized Changes in Flavonoid Biosynthesis in Roots of Lotus pedunculatus after Infection by Rhizobium loti

Two-dimensional paper chromatography in four solvent systems, high-sensitivity spray reagents, and UV absorption spectroscopy were used to separate and characterize flavonoids and isoflavonoids in roots and root nodules of 20-d-old Lotus pedunculatus Cav. Seedlings were grown either under sterile conditions or after inoculation with Fix⁺ or Fix⁻ strains of Rhizobium loti. Flavonoids rather than isoflavonoids predominated in all tissues. Flavonoid profiles in sterile and denodulated root tissues were remarkably similar, both qualitatively and quantitatively. At least 14 partially purified flavonoid aglycones and conjugates were found in root extracts; denodulated root tissues contained no compounds that were not also present in sterile roots. Fix⁺ rhizobia were responsible for major postinfection shifts in plant flavonoid biosynthesis at the sites of nodule morphogenesis. Polymeric flavolans were absent from Fix⁺ nodules but present in all root tissues and in Fix⁻ nodules. Catechin was detected only in Fix⁺ nodules.     

Structural identification of the iipo-chitin oligosaccharide nodulation signals of Rhizobium loti

Rhizobium loti is a fast-growing Rhizobium species that has been described as a microsymbiont of plants of the genus Lotus. Nodulation studies show that Lotus plants are nodulated by R loti, but not by most other Rhizobium strains, indicating that R. loti produces specific lipo-chitin oligosaccharides (LCOs) which are necessary for the nodulation of Lotus plants. The LCOs produced by five different Rhizobium ioti strains have been purified and were shown to be N-acetylglucosamine pentasaccharides of which the non-reducing residue is N-methylated and N-acylated with c/s-vaccenic acid (C18:1) or stearic acid (C18:O) and carries a carbamoyl group. In one R. loti strain, NZP2037, an additional carbamoyl group is present on the non-reducing terminal residue. The major class of LCO molecules is substituted on the reducing terminal residue with 4-O-acetylfucose. Addition of LCOs to the roots of Lotus plants results in abundant distortion, swelling and branching of the root hairs, whereas spot inoculation leads to the formation of nodule primordia.     

Biotransformation of the Pentahydroxy Flavone Quercetin by Rhizobium loti and Bradyrhizobium Strains (Lotus)

Lotus rhizobia catabolized quercetin in an arabinose-based medium via a novel form of C-ring cleavage, yielding phloroglucinol and protocatechuic acid. Conservation of the A and B rings of the flavone suggests that a chalcone could be formed as a transient intermediate.     

Chemical characterization of two lipopolysaccharide species isolated from Rhizobium loti NZP2213

Phenol-water extraction of Rhizobium loti NZP2213 cells allowed a simultaneous isolation of two structurally different lipopolysaccharides, from the aqueous (LPS-W) and phenol (LPS-P) phase that differed in their sodium doexycholate-PAGE pattern and composition. LPS-W showed a profile indicating an R-type LPS; LPS-P had a cluster of poorly resolved bands in the high-molecular-weight region. LPS-P contained large amounts of 6-deoxy-l-talose (6dTal), and a small amount of 2-O-methyl-6-deoxy-talose (molar ratio 30:1), both of which were completely absent in LPS-W. Methylation analysis gave only one major product, 2,4-di-O-methyl-6dTal, indicating that the O-chain is composed of a homopolymer of 1,3-linked 6dTal, having the methylated 6dTal (2-O-me-6dTal) probably localized at the non-reducing end of the O-chain. This homopolymeric O-chain was additionally O-acetylated, as evidenced by GC-MS and by ¹³C NMR analysis. The lipid A moieties of both LPS-W and LPS-P showed almost identical composition, with six, different 3-OH fatty acids and with two, so far not described, long-chain 4-oxo-fatty acids, all being amide-linked, and with 27-OH-28:0 as the main ester-linked fatty acid. Lipid A was of the lipid A_DAG-type, i.e., having a (phosphorylated) 2,3-diamino-2,3-dideoxy-d-glucose-containing lipid A backbone. Lipid A_DAG is widespread among species of the -2 group of Proteobacteria, but has so far not been encountered in any other rhizobial or agrobacterial species.     

Conserved Symbiotic Plasmid DNA Sequences in the Multireplicon Pangenomic Structure of Rhizobium etli

Strains of the same bacterial species often show considerable genomic variation. To examine the extent of such variation in Rhizobium etli, the complete genome sequence of R. etli CIAT652 and the partial genomic sequences of six additional R. etli strains having different geographical origins were determined. The sequences were compared with each other and with the previously reported genome sequence of R. etli CFN42. DNA sequences common to all strains constituted the greater part of these genomes and were localized in both the chromosome and large plasmids. About 700 to 1,000 kb of DNA that did not match sequences of the complete genomes of strains CIAT652 and CFN42 was unique to each R. etli strain. These sequences were distributed throughout the chromosome as individual genes or chromosomal islands and in plasmids, and they encoded accessory functions, such as transport of sugars and amino acids, or secondary metabolism; they also included mobile elements and hypothetical genes. Sequences corresponding to symbiotic plasmids showed high levels of nucleotide identity (about 98 to 99%), whereas chromosomal sequences and the sequences with matches to other plasmids showed lower levels of identity (on average, about 90 to 95%). We concluded that R. etli has a pangenomic structure with a core genome composed of both chromosomal and plasmid sequences, including a highly conserved symbiotic plasmid, despite the overall genomic divergence.It is becoming clear that bacterial genomes of strains of the same species vary widely both in size and in gene composition (39). An unexpected degree of genomic diversity has been found by comparing whole genomes (39). For instance, in Escherichia coli strains, differences of up to 1,400 kb account for some strain-specific pathogenic traits (5, 56). The extent of intraspecies genome diversity varies in different bacterial lineages. Some species have a wide range of variation; these species include E. coli (42), Streptococcus agalactiae (53), and Haloquadratum walsbyi (34). Other bacteria display only limited gene content diversity; an example is Ureaplasma urealyticum (1, 54). Tettelin and colleagues have suggested that bacterial species can be characterized by the presence of a pangenome consisting of a core genome containing genes present in all strains and a dispensable genome consisting of partially shared and strain-specific genes (53, 54). This concept is rooted in the earlier ideas of Reanney (43) and Campbell (7) concerning the structure of bacterial populations, and it indicates both that there is a pool of accessory genetic information in bacterial species and that strains of the same or even different species can obtain this information by horizontal transfer mechanisms (7, 43).Genome size and diversity are related to bacterial lifestyle. Small genomes are typical of strict pathogens such as Rickettsia prowazekii (2) and endosymbionts such as Buchnera aphidicola (44a). In contrast, free-living bacteria, such as Pseudomonas syringae and Streptomyces coelicolor, have large genomes (4, 6). The bacteria with the largest genomes are common inhabitants of heterogeneous environments, such as soil, where energy sources are limited but diverse (32). An increase in genome size is attributable mainly to expansion of functions such as secondary metabolism, transport of metabolites, and gene regulation. All these features are common to the nitrogen-fixing symbiotic bacteria of legumes, which are collectively known as rhizobia, and their close relative the plant pathogen Agrobacterium. The genomes of such bacterial species have diverse architectures with circular chromosomes that are different sizes or linear chromosomes, like that in Agrobacterium species, and the organisms contain variable numbers of large plasmids (31, 49). Comparative genomic studies have highlighted the conservation of gene content and order among the chromosomes of some species of rhizobia (22, 23, 25, 40). Furthermore, Guerrero and colleagues (25) observed that most essential genes occur in syntenic arrangements and display a higher level of sequence identity than nonsyntenic genes. In contrast, plasmids, including symbiotic plasmids and symbiotic chromosomal islands (like those in Mesorhizobium loti and Bradyrhizobium japonicum) are poorly conserved in terms of both gene content and gene order (21). It is not clear what evolutionary advantage, if any, is provided by multipartite genomes, but some authors have speculated that such genomes may allow further accumulation of genes independent of the chromosome. Recently, Slater and coworkers (46) proposed a model for the origin of secondary chromosomes. Their idea is based on the notion of intragenomic gene transfers that might occur from primary chromosomes to ancestral plasmids of the repABC type. Observations of conservation of clusters of genes in secondary chromosomes or in large plasmids that retain synteny with respect to the main chromosome support this hypothesis (46).We have been studying Rhizobium etli as a multipartite genome model species (23). This organism is a free-living soil bacterium that is able to form nodules and fix nitrogen in the roots of bean plants. The genome of R. etli is partitioned into several replicons, a circular chromosome, and several large plasmids. In the reference strain R. etli CFN42, the genome is composed of a circular chromosome consisting of about 4,381 kb and 6 large plasmids whose total size is 2,148 kb (23). A 371-kb plasmid, termed pSym or the symbiotic plasmid, contains most of the genes required for symbiosis (21). Previous studies have described the high level of genetic diversity among geographically different R. etli isolates (41). The strains are also variable with respect to the number and size of plasmids. Nevertheless, there has been no direct measurement of diversity at the genomic level, nor have comparative studies of shared and particular genomic features of R. etli strains been reported. Therefore, to assess the degrees of genomic difference and genomic similarity in R. etli, we obtained the complete genomic sequence of an additional R. etli strain and partial genomic sequences of six other R. etli strains isolated worldwide. Our results support the concept of a pangenomic structure at the multireplicon level and show that a highly conserved symbiotic plasmid is present in divergent R. etli isolates.     

Reliability of the RNA-DNA Filter Hybridization for the Detection of Oncornavirus-Specific DNA Sequences

Denatured DNA from leukemic myeloblasts or uninfected chicken embryos, immobilized on nitrocellulose filters, was hybridized to a vast excess of [(3)H]70S RNA from purified avian myeloblastosis virus. The viral RNA was eluted from the RNA-DNA hybrids, purified, and then rehybridized in solution to an excess of either leukemic or normal chicken embryonic DNA. This study revealed that all the slow and the fast hybridizing viral RNA sequences detectable by liquid hybridization in DNA excess had hybridized to the filter bound DNA. Both techniques also gave similar values for the number of 28S ribosomal RNA genes contained in a chicken cell genome: 210 by the liquid hybridization procedure and 218 by the filter hybridization technique. Therefore, filter hybridization can accurately detect DNA sequences present in relatively few numbers in the genome of higher organisms.     

Biosynthesis and degradation of nodule-specific Rhizobium loti compounds in Lotus nodules.

Two nodule-specific Rhizobium loti compounds were identified in Lotus tenuis and Lotus pedunculatus nodules induced by strain NZP2037. One, a silver nitrate-positive cation called rhizolotine, has been characterized as the riboside of a novel alpha-hydroxyimino acid containing a 1,4,5,6-tetrahydropyrimidine ring (G. J. Shaw, R. D. Wilson, G. A. Lane, L. D. Kennedy, D. B. Scott, and G. J. Gainsford, J. Chem. Soc. Chem. Commun., p. 180-181, 1986), and the other, yellow-1, stains yellow with ninhydrin. Both compounds were degraded by R. loti NZP2037 but not by strains of Rhizobium meliloti, Rhizobium trifolii, or Agrobacterium tumefaciens. Under the conditions tested neither compound was able to serve as a sole source of C or N for growth of R. loti NZP2037. Rhizolotine and yellow-1 were found in nodules from a range of different legumes inoculated with NZP2037, suggesting that the Rhizobium and not the host plant determines their synthesis. Neither compound was found in nodulelike structures of L. pedunculatus induced by transposon Tn5-induced noninfectious (Inf-) mutants of NZP2037 or in similar structures induced by a transconjugant of NZP2037 containing the symbiotic (Sym) cointegrate plasmid pPN1 of R. trifolii. Both compounds were also absent in the ineffective nodules induced by the bacterial-release-negative (Bar-) mutant, strain PN239. However, both compounds were present in nodules induced by the fixation-negative (Fix-) mutant PN235 and in Fix+ nodules formed by a plasmid-cured derivative of NZP2037. These results would suggest that infection and bacterial release from the infection thread are necessary for nodule (symbiotic) synthesis of these compounds.     

Isolation and Partial Characterization of DNA in Capsular Preparations of Rhizobium trifolii

The capsular polysaccharides from thymidine-(methyl-³H) labeled cultures of Rhizobium trifolii; strain 162S7 (Nitragin Co.) were centrifuged from bacterial cells and collected by ethanol precipitation. Following the addition of unlabeled carrier nucleic acids, labeled DNA, termed cap-DNA, was isolated from the capsular polysaccharides. Cap-DNA absorbed maximally at H-260 nm and was DNase sensitive. Approximately 11 μg of ³H-cap-DNA were consistently isolated per liter of 48 h cultures. Cap-DNA production was generally synchronized with the synthesis of the capsular polysaccharide and bacterial growth, attaining maximum recoverable amounts in 48 h cultures. By five days of culture growth, significant decreases in the amount of recoverable cap-DNA were noted. The presence of label in the cap-DNA demonstrated that the cap-DNA originated via de novo synthesis by the Rhizobium cells rather than from an anomalous source. The cap-DNA and intracellular Rhizobium DNA had similar buoyant densities of p= 1.719, indicating that cap-DNA arose specifically from the intracellular DNA. In 48-h cultures the specific activity of the cap-DNA was about one-third that of the intracellular DNA. This implies intracellular DNA was released during early growth with a relatively low specific activity which diluted the isotopic label of DNA released later. The evidence suggests lysogeny was the principal release mechanism.     

Nitrate reductase and nitrite reductase activity in free-living cells and bacteroids of Rhizobium loti

Cells of Rhizobium loti strains T1 and U226 cultured in defined medium with glutamate as the only nitrogen source and bacteroids isolated from root nodules of Lotus corniculatus, L. pedunculatus and L. tenuis did not show constitutive (non-nitrate induced) nitrate reductase activity (NRA). In contrast, nitrite reductase activity (NiRA) was present in both free-living cells and bacteroids of either strain T1 or U226. Constitutive NRA and NiRA were detected in the cytosol fraction from nodules of all three symbioses examined. An induced NRA was expressed in bacteroids after a 10 h incubation in the presence of nitrate.     

The requirement for exopolysaccharide precedes the requirement for flavolan-binding polysaccharide in nodulation of Leucaena leucocephala by Rhizobium loti

Rhizobium loti strain PN4115 (NZP2213 str-1) ineffectively nodulates Leucaena leucocephala, i.e., strain PN4115 induces nodulation (Nod⁺) and is able to invade these nodules (Inv⁺), but fails to fix nitrogen (Fix^–). Strain PN4115 does not synthesize a flavolan-binding polysaccharide (FBP), which is synthesized by the fully effective (Nod⁺Inv⁺Fix⁺) R. loti strain PN184 (NZP2037 str-1). The FBP may offer protection from prodelphinidin-rich flavolans synthesized by Lc. leucocephala. In this work, we show that exopolysaccharide (EPS)-negative mutants derived from strain PN4115 have a more severe ineffective phenotype (Nod⁺Inv^–Fix^–) on Lc. leucocephala than strain PN4115. This suggests that EPS from strain PN4115 is functional during invasion of Lc. leucocephala and that the requirement for EPS precedes the requirement for FBP. Received: 8 October 1996 / Accepted: 11 December 1996