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 (ΔcycHJKL) 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.     

The cycHJKL genes of Rhizobium meliloti involved in cytochrome c biogenesis are required for “respiratory” nitrate reduction ex planta and for nitrogen fixation during symbiosis

We report the genetic and biochemical analysis of Rhizobium meliloti mutants defective in symbiotic nitrogen fixation (Fix^?) and “respiratory” nitrate reduction (Rnr^?). The mutations were mapped close to the ade-1 and cys-46 chromosomal markers and the mutated locus proved to be identical to the previously described fix-14 locus. By directed Tn5 mutagenesis, a 4.5 kb segment of the chromosome was delimited in which all mutations resulted in Rnr^? and Fix^? phenotypes. Nucleotide sequence analysis of this region revealed the presence of four open reading frames coding for integral membrane and membrane-anchored proteins. Biochemical analysis of the mutants showed that the four proteins were necessary for the biogenesis of all cellular c-type cytochromes. In agreement with the nomenclature proposed for rhizobial genes involved in the formation of c-type cytochromes, the four genes were designated cycH, cycJ, cycK, and cycL, respectively. The predicted protein product of cycH exhibited a high degree of similarity to the Bradyrhizobium japonicum counterpart, while CycK and CycL shared more than 50% amino acid sequence identity with the Rhodobacter capsulatus Ccll and Cc12 proteins, respectively. cycJ encodes a novel membrane anchored protein of 150 amino acids. We suggest that this gene cluster codes for (parts of) a multi-subunit cytochrome c haem lyase. Moreover, our results indicate that in R. meliloti c-type cytochromes are required for respiratory nitrate reduction ex planta, as well as for symbiotic nitrogen fixation in root nodules.     

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.     

Characterization of the cycHJKL genes involved in cytochrome c biogenesis and symbiotic nitrogen fixation in Rhizobium leguminosarum.

Mutants of Rhizobium leguminosarum bv. viciae unable to respire via the cytochrome aa3 pathway were identified by the inability to oxidize N,N'-dimethyl-p-phenylenediamine. Two mutants which were complemented by cosmid pIJ1942 from an R. leguminosarum clone bank were identified. Although pea nodules induced by these mutants contained many bacteroids, no symbiotic nitrogen fixation was detected. Heme staining of cellular proteins revealed that all cytochrome c-type heme proteins were absent. These mutants lacked spectroscopically detectable cytochrome c, but cytochromes aa3 and d were present, the latter at a higher-than-normal level. DNA sequence analysis of complementing plasmids revealed four apparently cotranscribed open reading frames (cycH, cycJ, cycK, and cycL). CycH, CycJ, CycK, and CycL are homologous to Bradyrhizobium japonicum and Rhizobium meliloti proteins thought to be involved in the attachment of heme to cytochrome c apoproteins; CycK and CycL are also homologous to the Rhodobacter capsulatus ccl1 and ccl2 gene products and the Escherichia coli nrfE and nrfF gene products involved in the assembly of c-type cytochromes. The absence of cytochrome c heme proteins in these R. leguminosarum mutants is consistent with the view that the cycHJKL operon could be involved in the attachment of heme to apocytochrome c.     

Formation of several bacterial c-type cytochromes requires a novel membrane-anchored protein that faces the periplasm

We report here the discovery of a novel bacterial gene (cycH) whose product is involved in the biogenesis of most of the cellular cytochromes c. The cycH gene was detected in the course of characterizing a cytochrome oxidase-deficient Bradyrhizobium japonicum Tn5 mutant (strain CO×3) in which the transposon insertion disrupted cycH. Ali of the c-type cytochromes detectable in aerobically grown B. Japonicum wild-type cells were absent in the C0X3 mutant, with the exception of cytochrome c₁. A secondary phenotypic effect was the spectroscopic absence of the aa₃-type cytochrome c oxidase. The nucleotide sequence of the cloned wild-type cycH gene predicted a membrane-bound 369-amino-acid protein with an M_r of 39727. Results from studies on its membrane topology suggested that approximately 110 N-terminal amino acids are involved in anchoring the protein in the membrane, whereas the remaining two-thirds of the protein are exposed to the periplasm. We postulate that the CycH protein plays an essential role in an as yet unidentified periplasmic step in the biogenesis of holocytochromes c, except that of cytochrome c₁.     

The Rhizobium etli FixL protein differs in structure from other known FixL proteins

The central heme-binding domain in the FixL proteins of Sinorhizobium meliloti, Bradyrhizobium japonicum, Rhizobium leguminosarum biovar viciae and Azorhizobium caulinodans, is highly conserved. The similarity with the corresponding domain in the Rhizobium etli FixL protein is considerably less. This observation prompted us to analyze the heme-binding capacities of the R. etli FixL protein. The R. etlifixL gene was overexpressed in Escherichia coli. In the presence of S. meliloti FixJ, the overexpressed R. etli FixL protein was able to enhance FixJ-mediated activation of an S. meliloti pnifA-lacZ fusion, indicating that the R.?etli FixL protein possesses an active conformation in E. coli. Subsequently, using a non-denaturing gel assay for heme, we analyzed the heme-binding capacity of the R.?etli FixL protein expressed in E. coli, taking the S.?meliloti FixL protein as a positive control. The R. etli FixL protein expressed in E. coli does not contain a heme group, in contrast to the S. meliloti FixL protein. Therefore we conclude that the R. etli FixL is a non-heme protein in the nif regulatory cascade.     

Conservation of Plasmid-Encoded Traits among Bean-Nodulating Rhizobium Species

Rhizobium etli type strain CFN42 contains six plasmids. We analyzed the distribution of genetic markers from some of these plasmids in bean-nodulating strains belonging to different species (Rhizobium etli, Rhizobium gallicum, Rhizobium giardinii, Rhizobium leguminosarum, and Sinorhizobium fredii). Our results indicate that independent of geographic origin, R. etli strains usually share not only the pSym plasmid but also other plasmids containing symbiosis-related genes, with a similar organization. In contrast, strains belonging to other bean-nodulating species seem to have acquired only the pSym plasmid from R. etli.     

Rhizobium ruizarguesonis sp. nov., isolated from nodules of Pisum sativum L

Four strains, coded as UPM1132, UPM1133^T, UPM1134 and UPM1135, and isolated from nodules of Pisum sativum plants grown on Ni-rich soils were characterised through a polyphasic taxonomy approach. Their 16S rRNA gene sequences were identical and showed 100% similarity with their closest phylogenetic neighbors, the species included in the ‘R. leguminosarum group’: R. laguerreae FB206^T, R. leguminosarum USDA 2370^T, R. anhuiense CCBAU 23252^T, R. sophoreae CCBAU 03386^T, R. acidisoli FH13^T and R. hidalgonense FH14^T, and 99.6% sequence similarity with R. esperanzae CNPSo 668^T. The analysis of combined housekeeping genes recA, atpD and glnII sequences showed similarities of 92-95% with the closest relatives. Whole genome average nucleotide identity (ANI) values were 97.5-99.7% ANIb similarity among the four strains, and less than 92.4% with closely related species, while digital DNA-DNA hybridization average values (dDDH) were 82-85% within our strains and 34-52% with closely related species. Major fatty acids in strain UPM1133^T were C18:1 ω7c / C18:1 ω6c in summed feature 8, C14:0 3OH/ C16:1 iso I in summed feature 2 and C18:0. Colonies were small to medium, pearl-white coloured in YMA at 28 °C and growth was observed in the ranges 8-34 °C, pH 5.5-7.5 and 0-0.7% (w/v) NaCl. The DNA G + C content was 60.8 mol %. The combined genotypic, phenotypic and chemotaxonomic data support the classification of strains UPM1132, UPM1133^T, UPM1134 and UPM1135 into a novel species of Rhizobium, for which the name Rhizobium ruizarguesonis sp. nov. is proposed. The type strain is UPM1133^T (=CECT 9542^T = LMG 30526^T).     

The new class 11 transposon Tn163 is plasmid-borne in two unrelated Rhizobium leguminosarum biovar viciae strains

Tn163 is a transposable element identified in Rhizobium leguminosarum bv. viciae by its high insertion rate into positive selection vectors. The 4.6 kb element was found in only one further R. leguminosarum bv. viciae strain out of 70 strains investigated. Both unrelated R. leguminosarum bv. viciae strains contained one copy of the transposable element, which was localized in plasmids native to these strains. DNA sequence analysis revealed three large open reading frames (ORFs) and 38 bp terminal inverted repeats. ORF1 encodes a putative protein of 990 amino acids displaying strong homologies to transposases of class 11 transposons. ORF2, transcribed in the opposite direction, codes for a protein of 213 amino acids which is highly homologous to DNA invertases and resolvases of class II transposons. Homology of ORF1 and ORF2 and the genetic structure of the element indicate that Tn163 can be classified as a class II transposon. It is the first example of a native transposon in the genus Rhizobium. ORF3, which was found not to be involved in the transposition process, encodes a putative protein (256 amino acids) of unknown function. During transposition Tn163 produced direct repeats of 5 bp, which is typical for transposons of the Tn3 family. However, one out of the ten insertion sites sequenced showed a 6 by duplication of the target DNA; all duplicated sequences were A/T rich. Insertion of Tn163 into the sacB gene revealed two hot spots. Chromosomes of different R. leguminosarum bv. viciae strains were found to be highly refractory to the insertion of Tn163.     

Near-full length sequencing of 16S rDNA and RFLP indicates that Rhizobium etli is the dominant species nodulating Egyptian winter Berseem clover (Trifolium alexandrinum L.)

Egyptian winter Berseem clover (EWBC) is one of the main important forage legume crops in Egypt that is used for animal feeding in winter and it occupies about 2.5 million feddans (Feddan = 4200 m²) in winter agricultural rotation systems. Forty-eight rhizobial isolates that nodulated this legume host from different geographical regions within Egypt were isolated. RFLP analyses of 16S rDNA (1.5 kb) and whole ribosomal DNA (5 kb), the sequencing of 16S rDNA, and the sequencing of nodC, nifH and house keeping genes were used to identify these isolates. The RFLP analysis of 16S rDNA (1.5 kb) among 15 representative strains with three enzymes generated two genotypes. The largest genotype was similar to Rhizobium etli CFN42T (93.33%) except for strain 902 that failed to re-nodulate EWBC. RFLP analysis of complete ribosomal DNA (5 kb) produced five genotypes. The majority of tested strains shared the genotype with R. etli CFN42T (53.33%). Only one strain (1002) shared the genotype with Rhizobium leguminosarum sv. trifolii 3023. The other four strains were comprised of two unique genotypes. Phylogenetic analysis of 16S rDNA sequences revealed that seven representative strains could be divided into two genetic clusters sharing the ancestral clad with R. etli CFN42T. A phylogenetic tree based on nodC gene sequence confirmed that all the examined strains shared the genetic lineage with R. leguminosarum sv. trifolii WSM1325. The phylogenetic trees of house keeping genes are supported strongly the identification of majority of strains as a novel symbiovar of R. etli with new lineages.     

Sol–gel immobilization of haloalkane dehalogenase from Bradyrhizobium japonicum for the remediation 1,2-dibromoethane

Haloalkane dehalogenases catalyze the hydrolytic cleavage of carbon–halogen bonds in a broad range of environmental pollutants such as aliphatic mono-, di-, and polyhalogenated alkanes. From the biotechnology point of view haloalkane dehalogenases attract attention because of many potential uses for the bioremendation of soil, water and air. In the present study, different Rhizobium strains (Sinorhizobium meliloti 1021, Rhizobium leguminosarum bv. trifolii, Mesorhizobium loti MAFF, Bradyrhizobium japonicum usda 110) were screened for their ability to produce stable and active 1,2-dibromoethane-degrading dehalogenase. The results showed that B. japonicum produces the most potent dehalogenase. This enzyme was cloned, expressed in Escherichia coli BL21(DE3), purified and was entrapped in tetraethylorthosilicate derived sol–gel. The tetraethylorthosilicate sol–gel entrapped haloalkane dehalogenases exhibited higher storage and operational stability at 4 °C and 25 °C, compared to the free enzyme. Kinetic analysis of the entrapped enzyme using 1,2-dibromoethane showed that substrate turnover was limited by partitioning effects or diffusion through the sol–gel matrix. The biocatalyst was used in a packed bed bioreactor for the biodegradation of 1,2-DBE. Under selected conditions the sol–gel entrapped dehalogenase was able to hydrolyze 91.8% of the loaded 1,2-DBE, within 16.7 h. The results of the present study suggest that the use of HLD biocatalysis may provide a ‘green chemistry’ tool for sustainable remediation of 1,2-DBE.     

The Rhizobium etli FixL protein differs in structure from other known FixL proteins

The central heme-binding domain in the FixL proteins of Sinorhizobium meliloti, Bradyrhizobium japonicum, Rhizobium leguminosarum biovar viciae and Azorhizobium caulinodans, is highly conserved. The similarity with the corresponding domain in the Rhizobium etli FixL protein is considerably less. This observation prompted us to analyze the heme-binding capacities of the R. etli FixL protein. The R. etlifixL gene was overexpressed in Escherichia coli. In the presence of S. meliloti FixJ, the overexpressed R. etli FixL protein was able to enhance FixJ-mediated activation of an S. meliloti pnifA-lacZ fusion, indicating that the R.␣etli FixL protein possesses an active conformation in E. coli. Subsequently, using a non-denaturing gel assay for heme, we analyzed the heme-binding capacity of the R.␣etli FixL protein expressed in E. coli, taking the S.␣meliloti FixL protein as a positive control. The R. etli FixL protein expressed in E. coli does not contain a heme group, in contrast to the S. meliloti FixL protein. Therefore we conclude that the R. etli FixL is a non-heme protein in the nif regulatory cascade. Received: 22 August 1997 / Accepted: 20 October 1997     

Phenotypic and genetic diversity of Moroccan rhizobia isolated from Vicia faba and study of genes that are likely to be involved in their osmotolerance

Rhizobia are symbiotic nitrogen-fixing bacteria in root nodules of legumes. In Morocco, faba bean (Vicia faba L.), which is the main legume crop cultivated in the country, is often grown in marginal soils of arid and semi-arid regions. This study examines the phenotypic diversity of rhizobia nodulating V. faba isolated from different regions in Morocco for tolerance to some abiotic stresses. A total of 106 rhizobia strains isolated from nodules were identified at the species level by analysing 16S rDNA. Additionally, for selected strains recA, otsA, kup and nodA fragments were sequenced. 102 isolates are likely to belong to Rhizobium leguminosarum or R. laguerreae and 4 isolates to Ensifer meliloti. All strains tolerating salt concentrations of 428 or 342 mM NaCl as well as 127 or 99 mM Na2SO4 were highly resistant to alkaline conditions (pH 10) and high temperature (44 °C). Three strains: RhOF4 and RhOF53 (both are salt-tolerant) and RhOF6 (salt-sensitive) were selected to compare the influence of different levels of salt stress induced by NaCl on growth and on trehalose and potassium accumulation. We find a direct correlation between the trehalose contents of the rhizobial strains and their osmotolerance.     

Shigella flexneri type-specific antigen V: cloning,sequencing and characterization of the glucosyl transferase gene of temperate bacteriophage SfV

With lysogeny by bacteriophage SfV, Shigella flexneri serotype Y is converted to serotype 5a. The glucosyl transferase gene (gtr) from bacteriophage SfV of S. flexneri, involved in serotype-specific conversion, was cloned and characterized. The DNA sequence of a 3.7 kb EcoRI–BamHI fragment of bacteriophage SfV which includes the gtr gene was determined. This gene, encoding a polypeptide of 417 aa with 47.67 kDa molecular mass, caused partial serotype conversion of S. flexneri from serotype Y to type V antigen as demonstrated by Western blotting and the sensitivity of the hybrid strain to phage Sf6. The deduced protein of the partially sequenced open reading frame upstream of the gtr showed similarity to various glycosyl transferases of other bacteria. Orf3, separated from the gtr by a non-coding region and transcribed convergently, codes for a 167 aa (18.8 kDa) protein found to have homology with tail fibre genes of phage lambda and P2.     

Genome-wide analysis of long,exact DNA repeats in rhizobia

The rhizobia are a group of bacteria widely studied for their capacity to form intimate symbiotic relationships with leguminous plants. However, they are also interesting for containing a remarkable abundance of repetitive genetic elements, such as long DNA repeats. In this study we deeply analyzed long, exact DNA repeats in five representative rhizobial genomes; Rhizobium etli, Rhizobium leguminosarum, Bradyrhizobium japonicum, Sinorhizobium meliloti and Mesorhizobium loti. The results suggest that a huge proportion of repeats can be located in either plasmid or chromosome replicons, except in B. japonicum, which lacks plasmids, but contains the largest number, and longest repeat elements of the genomes analyzed here. Interestingly, we detected a slight correlation between the density of repeats (either number or length) and genome size. As expected, the highest percentage of DNA repeats code for mobile genetic elements, including insertion sequences, recombinases, and transposases. Some repeats corresponded to non-coding or intergenic regions, while in genomes like that of R. etli, a significant percentage of large repeats, mainly located in plasmids, were strongly associated with symbiotic and nitrogen fixation activities. In conclusion, our analysis shows that rhizobial genomes contain a high density of long DNA repeats, which might facilitate recombination events and genome rearrangements, functioning in adaption and persistence during saprophytic or symbiotic life.     

The synthesis of cholestane and furostan saponin analogues and the determination of sapogenin's absolute configuration at C-22

A facile and efficient way for the synthesis of cholestane and furostan saponin analogues was established and adopted for the first time. Following this strategy, starting from diosgenin, three novel cholestane saponin analogues: (22S,25R)-3β,22,26-trihydroxy-cholest-5-ene-16-one 22-O-[O-α-l-rhamnopyranosyl-(1 → 2)-β-d-glucopyranoside] 11, (25R)-3β,16β,26-trihydroxy-cholest-5-ene-22-one 16-O-[O-α-l-rhamnopyranosyl-(1 → 2)-α-d-glucopyranoside] 14 and (25R)-3β,16β,26-trihydroxy-cholest-5-ene-22-one 16-O-[O-α-l-rhamnopyranosyl-(1 → 2)-β-d-glucopyranoside] 17, three novel furostan saponin analogues: (22S,25R)-furost-5-ene-3β,22,26-triol 22-O-(α-d-glucopyranoside) 23, (22R,25R)-furost-5-ene-3β,22,26-triol 22-O-(α-d-glucopyranoside) 24 and (22S,25R)-furost-5-ene-3β,22,26-triol 22-O-[O-α-l-rhamnopyranosyl-(1 → 2)-α-d-glucopyranoside] 26, were synthesized ultimately. The structures of all the synthesized analogues were confirmed by spectroscopic methods. The S-chirality at C-22 of cholestane was confirmed by Mosher's method. The absolute configuration at C-22 of furostan saponin analogues was distinguished by conformational analysis combined with the NMR spectroscopy. The cytotoxicities of the synthetic analogues toward four types of tumor cells were shown also.     

Effects of combining releases of non-viable host eggs with insecticide application on Riptortus pedestris population and its egg parasitoids

Our previous study demonstrated that the release of refrigerated non-viable eggs of Riptortus pedestris (Fabricius) (Hemiptera: Alydidae) enhanced parasitism rates in soybean fields but did not result in the reduction of R. pedestris populations. This study was further conducted using an open-cage exclusion design in a soybean field in order to evaluate the compatibility of combining releases of non-viable host eggs with a single pre-harvest application of insecticide for the control of R. pedestris. Refrigerated eggs of R. pedestris were released twice in treatment plots, and fresh (< 1 day old) eggs of R. pedestris were deployed in all experimental arenas, every 6 days, for host resource and measurement of field parasitism. The releases of host eggs did not reduce the number of R. pedestris in any life stage except the adult stage on two sampling dates. However, parasitism by Gryon japonicum (Ashmead) (Hymenoptera: Scelionidae) was higher in treated plots (9–25%) than in the control plots (1–9%). Statistical significant reduction was not found in the pest population, but parasitism rates significantly increased. Pesticide application did not reduce the bug population but did affect the parasitoids population. Pest management tactics, using both artificially deployed host eggs and insecticide, are discussed.     

A rapid,simple and sensitive loop-mediated isothermal amplification method to detect Anaplasma bovis in sheep and goats samples

A loop-mediated isothermal amplification (LAMP) technique has been widely used in detecting the nucleic acid of various pathogenic bacteria. In this study, a set of four LAMP primers was designed to specifically test Anaplasma bovis. The LAMP assay was performed at 62 °C for 60 min in a water bath. The specificity was confirmed by amplifying A. bovis isolate, while no cross reaction was observed with other five pathogens (Anaplasma bovis, Anaplasma phagocytophilum, Theileria luwenshuni, Babesia motasi and Schistosoma japonicum). The sensitivity of LAMP was 5 × 10⁰ copies/μL, 100 times more than that of conventional PCR (5 × 10² copies/μL). Of 120 blood DNA extracted from sheep and goats field samples, 81 (67.5%), 22 (18.3%) and 43 (35.8%) were positively detected by LAMP, conventional PCR and nested PCR, respectively. The findings indicated that the developed LAMP assay is a new convenient tool for rapid and cost-effective detection of A. bovis.