Electroporation of Bradyrhizobium japonicum

Summary Electroporation offers a fast, efficient and reproducible way to introduce DNA into bacteria. We have successfully used this technique to transform two commercially important strains of Bradyrhizobium japonicum, the nitrogen-fixing soybean symbiont. Initially, electroporation conditions were optimized using plasmid DNA which had been prepared from the same B. japonicum strain into which the{imDNA was to b}e transformed. Efficiencies of 10⁵-10⁶ transformants/g DNA were obtained for strains USDA 110 and 61A152 with ready-to-use frozen cells. Successful electroporation of B. japonicum with plasmid DNA prepared from Escherichia coli varied with the E. coli strain from which the plasmid was purified. The highest transformation efficiencies (10⁴ transformants/g DNA) were obtained using DNA prepared from a dcm ^– dam ^– strain of E. coli. This suggests that routine isolation of DNA from an E. coli strain incapable of DNA modification should help in increasing transformation efficiencies for other strains of bacteria where DNA restriction appears to be a significant obstacle to successful transformation. We have also monitored the rate of spontaneous mutation in electroporated cells and saw no significant difference in the frequency of streptomycin resistance for electroporated cells compared to control cells.     

The cycHJKL gene cluster plays an essential role in the biogenesis of c-type cytochromes in Bradyrhizobium japonicum

We present an extended genetic analysis of the previously identified cycH locus in Bradyrhizobium japonicum. Three new open reading frames found in an operon-like structure immediately adjacent to the 3 end of cycH were termed cycJ, cycK and cycL. A deletion mutant (cycHJKL) and biochemical analysis of its phenotype showed that the genes of the cluster are essential for the biogenesis of cellular c-type cytochromes. Mutations in discrete regions of each of the genes were also constructed and shown to affect anaerobic respiration with nitrate and the ability to elicit an effective symbiosis with soybean, both phenotypes being a consequence of defects in cytochrome c formation. The CycK and CycL proteins share up to 53% identity in amino acid sequence with the Rhodobacter capsulatus Ccll and Cc12 proteins, respectively, which have been shown previously to be essential for cytochrome c biogenesis, where-as cycJ codes for a novel protein of 169 amino acids with an M_r of 17857. Localisation studies revealed that CycJ is located in the periplasmic space; it is probably anchored to the cytoplasmic membrane via an N-terminal hydrophobic domain. Based on several considerations discussed here, we suggest that the proteins encoded by the cycHJKL-cluster may be part of a cytochrome c-haem lyase complex whose active site faces the periplasm.     

Identification and inter-relationship analysis of Bradyrhizobium japonicum strains by restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD)

Genomic DNA of 13 Bradyrhizobium japonicum strains was prepared and analysed by restriction fragment length polymorphism (RFLP) with nif and nod probes, and by random amplified polymorphic DNA (RAPD) with 11 primers of arbitrary nucleotide sequence. Polymorphism was observed in both analyses. The RFLP and RAPD banding patterns of different strains were used to calculate genetic divergence and to construct phylogenetic trees, allowing studies on the relationships between the strains. RFLP with nif and nod probes permitted the separation of the strains into two divergent groups, whereas RAPD separated them into four main groups. RAPD allowed closely related strains to be distinguished.     

Genomic fingerprinting of Bradyrhizobium japonicum isolates by RAPD and rep-PCR

Genetic diversity of indigenous Bradyrhizobium japonicum population in Croatia was studied by using different PCR-based fingerprinting methods. Characteristic DNA profiles for 20 B. japonicum field isolates and two reference strains were obtained using random primers (RAPD) and two sets of repetitive primers (REP- and ERIC-PCR). In comparison with the REP, the ERIC primer set generates fingerprints of lower complexity, but still several strain-specific bands were detected. Different B. japonicum isolates could be more efficiently distinguished by using combined results from REP- and ERIC-PCR. The most polymorphic bands were observed after amplification with four different RAPD primers. Both methods, RAPD and rep-PCR, resulted in identical grouping of the strains. Cluster analysis, irrespective of the fingerprinting method used, revealed that all the isolates could be divided into three major groups. Within the major groups, the degree of relative similarity between B. japonicum isolates was dependent upon the method used. Our results indicate that both RAPD and rep-PCR fingerprinting can effectively distinguish different B. japonicum strains. RAPD fingerprinting proved to be slightly more discriminatory than rep-PCR.     

Identification of nifE-, nifN- and nifS-like genes in Bradyrhizobium japonicum

Summary The 17 kb region between the Bradyrhizobium japonicum nitrogenase genes (nifDK and nifH) was investigated for the presence of further nif or fix genes by site-directed insertion or deletion/replacement mutagenesis and interspecies hybridization. Mutant strains were tested for their ability to reduce acetylene in free-living, microaerobic culture (Nif phenotype) and in soybean root nodules (Fix phenotype). The presence of a gene, previously identified by hybridization with the Klebsiella pneumoniae nifB gene, was proved by isolation of a nifB insertion mutant which was completely Nif^- and Fix^-. Three other regions were found to be homologous to the K. pneumoniae genes nifE, nifN, and nifS, NifE and nifN insertion mutants were completely Nif^-/Fix^- whereas nifS mutants were leaky with 30% residual Fix activity. Taken together, the data show that the B. japonicum genome harbours a cluster of closely adjacent genes which are directly concerned with nitrogenase function.     

Regulation of nod gene expression in Bradyrhizobium japonicum

Summary The best inducers of nod:: lacZ translational fusions in Bradyrhizobium japonicum are isoflavones, primarily genistein and daidzein. Upstream of the nodABC genes in B. japonicum is a novel gene, nodY, which is coregulated with nodABC. Measurements of the activity of lacZ fusions to the nodD gene of B. japonicum show that this gene is inducible by soybean seed extract and selected flavonoid chemicals. The induction of the nodY ABC and nodD operons appears to require a functional nodD gene, indicating that the nodD gene product controls its own synthesis as well as other nod genes.     

Mutational analysis of the Bradyrhizobium japonicum common nod genes and further nod box-linked genomic DNA regions

Summary By insertional and deletional marker replacement mutagenesis the common nod region of Bradyrhizobium japonicum was examined for the presence of additional, essential nodulation genes. An open reading frame located in the 800 bp large intergenic region between nodD1 and nodA did not appear to be essential for nodulation of soybean. Furthermore, a strain with a deletion of the nodI- and nodJ-like genes downstream of nodC had a Nod⁺ phenotype. A mutant with a 1.7 kb deletion immediately downstream of nodD1 considerably delayed the onset of nodulation. This region carried a second copy of nodD (nodD2). A nodD1-nodD2 double mutant had a similar phenotype to the nodD2 mutant. Using a 22-mer oligonucleotide probe partially identical to the nod box sequence, a total of six hybridizing regions were identified in B. japonicum genomic DNA and isolated from a cosmid library. Sequencing of the hybridizing regions revealed that at least three of them represented true nod box sequences whereas the others showed considerable deviations from the consensus sequence. One of the three nod box sequences was the one known to be associated with nodA, whereas the other two were located 60 to 70 kb away from nif cluster I. A deletion of one of these two sequences plus adjacent DNA material mmutant 308) led to a reduced nodulation on Vigna radiata but not on soybean. Thus, this region is probably involved in the determination of host specificity.Dedicated to Prof. Giorgio Semenza on the occasion of his 60th birthday     

Oligonucleotide fingerprinting and RAPD analysis of Achillea species: Characterization and long-term monitoring of micropropagated clones

Two different DNA fingerprinting techniques were applied to a set of Achillea samples (Asteraceae), comprising ten taxa of the medicinally important A. millefolium group and six related species. Field-grown as well as in vitro-micropropagated plants were individually screened for abundance and polymorphism of target sequences recognized by oligonucleotide fingerprinting with 13 different microsatellite-complementary probes. While most probes revealed a high level of intra- and interspecific variability, fingerprints proved to be somatically stable in vegetatively propagated plant material. Analysis of the same samples by polymerase chain reaction with arbitrary 10-mer primers yielded less polymorphic patterns. Because of its higher discriminatory ability, oligonucleotide fingerprinting offers itself as the method of choice for the identification and discrimination of A. asplenifolia and A. roseoalba clones, as well as for monitoring their stability during micropropagation.Abbreviations BPA N-benzyl-9-[2-tetrahydropyranyl]-adenine - PCR polymerase chain reaction - RAPD random amplified polymorphic DNA - RFLP restriction fragment length polymorphism - TAE buffer 40 mM Tris acetate, 20 mM sodium acetate, 1 mM EDTA, pH 7.8Part of the Ph.D. Thesis     

In Bradyrhizobium japonicum the common nodulation genes, nodABC, are linked to nifA and fixA

Summary Using cloned Rhizobium phaseoli nodulation (nod) genes as hybridization probes homologous restriction fragments were detected in the genome of the slow-growing soybean symbiont, Bradyrhizobium japonicum strain 110. These fragments were isolated from a cosmid library, and were shown to lie 10 kilobasepairs (kb) upstream from the nifA and fixA genes. Specific nod probes from Rhizobium leguminosarum were used to identify nodA-, nodB-, and nodC-like sequences clustered within a 4.5 kb PstI fragment. A mutant was constructed in which the kanamycin resistance gene from Tn5 was inserted into the nodA homologous B. japonicum region. This insertion was precisely located, by DNA sequencing, to near the middle of the nodA gene. B. japonicum mutants carrying this insertion were completely nodulation deficient (Nod^-).     

Comparison of 16S rDNA analysis and rep-PCR genomic fingerprinting for molecular identification of Yersinia pseudotuberculosis

16S rDNA sequence analysis and repetitive element sequence-based PCR (rep-PCR) genomic fingerprinting were evaluated on 11 type strains of the genus Yersinia and 17 recognized serotype strains of Y. pseudotuberculosis to investigate their genetic relatedness and to establish the value of techniques for the identification of Y. pseudotuberculosis. A phylogenetic tree constructed from 16S rDNA sequences showed that the type strains of Yersinia species formed distinct clusters with the exception of Y. pestis and Y. pseudotuberculosis. Moreover, Y. pestis NCTC 5923^T was found to be closely related to Y. pseudotuberculosis serotypes 1b, 3, and 7. Dendrograms generated from REP-PCR, and ERIC-PCR data revealed that members of the genus Yersinia differed from each other with the degree of similarity 62% and 58%, respectively. However, the BOX-PCR results showed that Y. pestis 5923^T clustered with the Y. pseudotuberculosis group with a degree of similarity 74%. According to these findings, 16S rDNA sequence analysis was unable to reliably discriminate Y. pseudotuberculosis from Y. pestis. However, REP-PCR and especially ERIC-PCR provided an effective means of differentiating between members of the taxa. This revised version was published online in June 2006 with corrections to the Cover Date.     

DNA fingerprinting of isolates of Streptococcus mutans by pulsed-field gel electrophoresis

Forty isolates and five standard laboratory strains, representing serotypes c, e and f of Streptococcus mutans were analyzed by pulsed-field gel electrophoresis (PFGE) after digestion of the genomic DNA with BssH II. The digestion patterns of standard laboratory strains were characteristic of serotypes c, e and f. Serotypes c and f generated diagnostic DNA fragments of approximately 145 kbp and of approximately 130-175 kbp in length, respectively. Serotype e generated a ladder of at least 14 fragments of 15-155 kbp in length. The digestion patterns of isolates were essentially similar to those of the standard laboratory strains. The patterns of almost all isolates obtained from a single individual were identical, but patterns of a few different types were also observed among isolates obtained from two individuals. Digestion with BssH II revealed differences among isolates obtained from different individuals. We used differences in banding patterns among isolates to construct a dendrogram. The dendrogram included two major clusters, one that consisted of isolates of serotypes c and f, and an other that consisted of isolates of serotype e. Our results indicate that BssH II is a useful enzyme for distinguishing among isolates of S. mutans and that digestion patterns obtained by PFGE can be used for chromosomal DNA fingerprinting.     

Molecular characterization of Macrophomina phaseolina and Fusarium species by a single primer RAPD technique

Charcoal root rot and wilt, are two economically important diseases of many crop plants in North and South America, Asia and Africa and some parts of Europe. Genetic variation in 43 isolates of Macrophomina phaseolina and 22 isolates of Fusarium species, collected from geographically distinct regions over a range of hosts, was studied using random amplified polymorphic DNA (RAPD) markers. Initially, 210 arbitrary nucleotide (10-mer) primers were tested for amplification of genomic DNA of one M. phaseolina isolate, 70 primers amplified the genomic DNA of M. phaseolina. One primer OPA-13 (5'-CAGCACCCAC-3') produced fingerprint profiles, which clearly distinguished between the different isolates of M. phaseolina. UPGMA analysis classified these isolates into five major groups. By primer OPA-13, 22 isolates of pathogenic and non-pathogenic Fusarium species of different formae-speciales and races, were also distinguished from M. phaseolina. This marker is useful for distinguishing between these two important plant pathogens irrespective of hosts, virulence spectrum and races. This is the first report of reliable diagnosis of two soilborne pathogens (root/collar rot and wilt causing pathogens) at the level of isolates, formae-speciales and races by a single primer RAPD procedure with uniform PCR conditions.     

Antioxidant defense system responses and role of nitrate reductase in the redox balance maintenance in Bradyrhizobium japonicum strains exposed to cadmium

In this work, we evaluated the effects of cadmium (Cd) on the antioxidant defense system responses and the role of nitrate reductase (NR) in the redox balance maintenance in Bradyrhizobium japonicum strains. For that, B. japonicum USDA110 and its NR defective mutant strain (GRPA1) were used. Results showed that the addition of 10 μM Cd did not modify the aerobic growth of the wild type strain while the mutant strain was strongly affected. Anaerobic growth revealed that only the parental strain was able to grow under this condition. Cd reduced drastically the NR activity in B. japonicum USDA110 and increased lipid peroxide content in both strains. Cd decreased reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio in B. japonicum USDA110 although, a significant increased was observed in the mutant GRPA1. GSH-related enzymes were induced by Cd, being more evident the increase in the mutant strain. This different behavior observed between strains suggests that NR enzyme plays an important role in the redox balance maintenance in B. japonicum USDA 110 exposed to Cd.     

Combination of ARDRA and RAPD genotyping techniques in identification of Acinetobacter spp. genomic species

A total of 10 non-repetitive multi-drug-resistant Acinetobacter strains were collected. With reference to A. calcoaceticus (ATCC23055), A. baumannii (ATCC19606), A. lwoffii (ATCC17986), and A. junii (NCTC5866),DNA fingerprint technique, amplified ribosomal DNA restriction analysis (ARDRA), and random amplified polymorphism DNA (RAPD) were carried out to identify the genomic species of Acinetobacter spp. The distances between them were calculated by the unweighted pair group method with arithmetic (UPGMA). Genotypes of Acinetobacter spp. were effectively classified and an A. junii together with nine A. baumannii isolates was genomically identified. The combination of ARDRA and RAPD DNA-fingerprint technique shows high complementarity, and could be a useful tool in Acinetobacter genomic species identification. __________ Translated from Microbiology, 2007, 34(2): 303–306 [译自:微生物学通报]     

Soybean response to nodulation by rhizobitoxine-producing bradyrhizobia as influenced by nitrate application

Foliar chlorosis of soybean (Glycine max [L.] Merr.) resulting from nodulation by rhizobitoxine-producing (RT⁺) strains of Bradyrhizobium japonicum is commonly less severe in the field than under greenhouse conditions. Differences in nutritional conditions between the field and greenhouse may contribute to this phenomenon. In particular, field-grown plants obtain some N from soil sources, whereas in the greenhouse soybean is often grown in low-N rooting media to emphasize symbiotic responses. Therefore, we examined the effect of NO₃ ^- on the expression of RT-induced symptoms. Soybean plants inoculated with RT⁺ bradyrhizobia were grown for 42 days in horticultural vermiculite receiving nutrient solution amended with 0.0, 2.5, or 7.5 mM KNO₃. Foliar chlorosis decreased with increasing NO₃ ^- application whereas nodule mass per plant was generally increased by NO₃ ^- application. Total amounts of nodular RT remained constant or increased with NO₃ ^- application, but nodular concentrations of RT decreased. Chlorosis severity was negatively correlated with shoot dry weight, chlorophyll concentration, and total shoot N content. It was concluded that application of NO₃ ^- can reduce the negative effects of RT production on the host plant. This suggests that any NO₃ ^- present in field soils may serve to limit chlorosis development in soybeans.Abbreviations RT rhizobitoxine - RT⁺ rhizobitoxine-producing - Lb leghemoglobin Published as Miscellaneous Paper No. 1429 of the Delaware Agricultural Experiment Station.     

Electrochemical assay of the nitrate and nitrite reductase activities of Rhizobium japonicum

This work describes an electrochemical method for the determination of the nitrate and nitrite reductase activities of Rhizobium japonicum. The advantage of the method lies in the use of whole cells for the analysis and we earlier developed this protocol for the assay of NO. The results obtained are comparable to the spectrophotometric Griess assay. As the method is based on electrochemical reduction, the commonly interfering biological components like ascorbic acid, uric acid, dopamine, etc., will not interfere with the analysis. This method can be extended to the fabrication of biosensors for nitrate and nitrite using the same principle.     

Visualization of bioluminescence as a marker of gene expression in rhizobium-infected soybean root nodules

The linked structural genes lux A and lux B, encoding bacterial luciferase of a marine bacterium Vibrio harveyi, were fused with the nitrogenase nifD promoter from Bradyrhizobium japonicum and with the P1 promoter of pBR322. Both fusions were integrated into the B. japonicum chromosome by site-specific recombination. Soybean roots infected with the two types of rhizobium transconjugants formed nitrogen-fixing nodules that produced bright blue-green light. Cells containing the P1 promoter/lux AB fusion resulted in continuously expressed bioluminescence in both free-living rhizobium and in nodule bacteriods. However, when under control of the nifD promoter, luciferase activity was found only in introgen-fixing nodules. Light emission from bacteroids allowed us to visualize and to photograph nodules expressing this marker gene fusion in vivo at various levels of resolution, including within single, living plant cells. Localization of host cells containing nitrogen-fixing bacteroids within nodule tissue was accomplished using low-light video microscopy aided by realtime image processing techniques developed specifically to enhance extreme low-level luminescent images.     

RAPD cluster analysis and chlorate sensitivity of some Indian isolates of Macrophomina phaseolina from sorghum and their relationships with pathogenicity

Charcoal rot caused by Macrophomina phaseolina is an economically important disease in sorghum grown during the post rainy season in India. Variations in random amplified polymorphic DNA (RAPD) polymorphisms, chlorate sensitivity and pathogenicity were studied among sorghum isolates of M. phaseolina collected from different parts of India. RAPD data based on 14 random primers of Kit A and C (OPA and OPC) on 20 isolates showed a high degree of polymorphism (98.1%) in different isolates. UPGMA dendrogram on RAPD data produced 7 clusters at the level of 37% similarity. Isolates from the same locations showed a tendency to group closer, substantiating closer genetic relatedness. Sorghum infecting Macrophomina isolates showed a mixed response for sensitivity to potassium chlorate (120 mM). Chlorate-resistant isolates were predominant (>65% of the isolates) over sensitive isolates. Chlorate-sensitive isolates were found to be genetically closer among them than the resistant ones. For the first time it was shown that chlorate sensitivity in Macrophomina had some relations with charcoal rot severity in sorghum.