Resazurin reducing time as an indicator of Bradyrhizobium viable cell count

A method based on the use of resazurin (RSZ) is described to determine the number of viable Bradyrhizobium japonicum cells in culture medium. The observation of RSZ reduction can be done spectrophotometrically or visually. B. japonicum strains behaved differently when the reducing time was considered. This methodology can be used to determine the number of viable cells during the liquid culture stage of inoculant production.     

Use of the mitochondria toxicity assay for quantifying the viable cell density of microencapsulated jurkat cells

The mitochondria toxicity assay (MTT assay) is an established method for monitoring cell viability based on mitochondrial activity. Here the MTT assay is proposed for the in situ quantification of the living cell density of microencapsulated Jurkat cells. Three systems were used to encapsulate the cells, namely a membrane consisting of an interpenetrating polyelectrolyte network of sodium cellulose sulphate/poly(diallyldimethylammonium chloride) (NaCS/PDADMAC), a calcium alginate hydrogel covered with poly(L ‐lysine) (Ca‐alg‐PLL), and a novel calcium alginate‐poly(ethylene glycol) hybrid material (Ca‐alg‐PEG). MTT results were correlated to data obtained by the trypan blue exclusion assay after release of the cells from the NaCS/PDADMAC and Ca‐alg‐PLL capsules, while a resazurin‐based assay was used for comparison in case of the Ca‐alg‐PEG material. Analysis by MTT assay allows quick and reliable determination of viable cell densities of encapsulated cells independent of the capsule material. The assay is highly reproducible with inter‐assay relative standard deviations below 10%. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:986–993, 2013     

Degradation of catechin by Bradyrhizobium japonicum

Rhizobia utilize phenolic substances as sole carbonsource. Bradyrhizobium japonicum utilizescatechin, a unit of condensed tannin as carbonsource. To establish the degradative pathway ofcatechin, the products of catechin degradation wereisolated by paper chromatography and TLC andidentified by HPLC, UV, IR and NMR spectra. B.japonicum cleaves catechin through catechinoxygenase. Phloroglucinolcarboxylic acid andprotocatechuic acid were identified as the initialproducts of degradation. Phloroglucinolcarboxylicacid is further decarboxylated to phloroglucinolwhich is dehydroxylated to resorcinol. Resorcinolis hydroxylated to hydroxyquinol. Protocatechuicacid and hydroxyquinol undergo intradiol cleavagethrough protocatechuate 3,4-dioxygenase andhydroxyquinol 1,2-dioxygenase to form-carboxy cis, cis-muconic acidand maleylacetate respectively. The enzymes ofcatechin degradative pathway are inducible. Estimation of all the enzymes involved in thecatabolism of catechin reveals the existence of acatechin degradative pathway in B. japonicum.     

Transfer and maintenance of IncP and IncW group plasmids into and between extra-slow-growing Bradyrhizobium japonicum strains

Abstract IncP group plasmid pRL180 was conjugally transferred from Agrobacterium tumefaciens LBA928 into extra-slow-growing (ESG) Bradyrhizobium japonicum strains and between ESG strains, RJ17W and RJ12S. pRL180 was integrated into the chromosome of RJ12S, RJ17W and RJ19FY. ESG strains efficiently transferred pRL180 into Escherichia coli at about a 3 × 10⁻⁵ frequency. IncW group plasmid pTY97 was transferred in intergeneric matings from E. coli into ESG strains at a high frequency of 2.5 × 10⁻³; between RJ17W and RJ12S transfer was about 5.6 × 10⁻⁴. pTY97 was maintained as an R' plasmid in RJ12S. The R' plasmid was resolved upon transfer into E. coli C where only pTY97 was autonomously replicated.     

Chemotaxis and growth of Bradyrhizobium japonicum in the presence of fine-dispersed silica

The chemotactic properties of the soybean nodule bacterium Bradyrhizobium japonicum were studied in the presence of synthetic fine-dispersed materials. It was shown that fine-dispersed silica (FDS) and its variety modified with aluminum oxide (MFDS) reduce bacterial chemotaxis to glucose. In addition, FDS increases the irregular motility of B. japonicum, and MFDS decreases it. This is in agreement with the effect of the materials on the rate of nodule bacterium growth.     

Lack of O‐polysaccharide enhances biofilm formation by Bradyrhizobium japonicum

Aims: To reveal the effects of the O‐polysaccharide antigen of Bradyrhizobium japonicum LPS on biofilm formation and motility. Methods and Results: Wild type and O‐antigen‐deficient mutant strains of B. japonicum were tested for biofilm formation on polyvinyl chloride (PVC) surfaces and motility on semi‐solid (0·3%) agar media. After 7 days of incubation, the amount of biofilms formed by the mutant was c. 3·5‐fold greater than that of the wild type. Unlike biofilm formation, the motility assay revealed that the mutant strain was less motile than the wild type. Conclusions: This study shows enhanced biofilm formation and decreased motility by the O‐antigen‐deficient mutant, suggesting that the lack of the O‐polysaccharide of the rhizobial LPS is associated with biofilm‐forming ability and movement. Significance and Impact of the Study: LPS plays an important role in both pathogenic and beneficial bacteria. It has also been reported that LPS deficiency negatively affects biofilm formation. However, our results demonstrate that the O‐antigen‐deficient mutant enhances biofilm formation, presumably through a significant increase in hydrophobicity. It is notable that the hydrophobicity of cell walls might be a key regulator in controlling biofilm development in B. japonicum.     

Rapid and efficient selection of recombinant site-directed mutants of Bradyrhizobium japonicum by colony hybridization

Abstract Due to the high incidence of spontaneous antibiotic resistance and slow growth of Bradyrhizobium japonicum strains, screening for site-directed mutants is cumbersome and time-consuming. A rapid method for selection of recombinant site-directed mutants of B. japonicum was developed. A kanamycin (Km) and a spectinomycin (Sp) cassette were each used to replace DNA fragments in the chromosome by homologous recombination. The primary new features of this method involve a simple plate selection for the antibiotic (Km or Sp) resistant mutants, then colony streaking, and lysis for DNA hybridization on a nitrocellulose filter enabling direct identification of the recombinant site-directed mutants. This method has permitted us to quickly and easily identify a large number of positive recombinant mutants from a large number of indicidual colonies. The procedure eliminates the need to first isolate genomic DNA from each mutant for Southern hybridization. All of the tested site-directed mutants from this method were confirmed to exhibit the expected mutant phenotype.     

Increase of Bradyrhizobium japonicum numbers in soils and enhanced nodulation of soybean (Glycine max (L) merr.) using granular inoculants amended with nutrients

Abstract: Mineral microgranules, amended with nutrients and inoculated with either peat or liquid Bradyrhizobium japonicum inoculants, increased the growth and recovery of the bacterium during laboratory incubation in unsterilized soil. Increases in the range of 1 log unit per g or ml inoculant used were observed in different soil types. B. japonicum showed better survival with nutrient-amended granules than in unamended ones, in soil undergoing desiccation. In a growth chamber experiment, the number of nodules per plant were significantly increased by nutrient-amendment of the granules, but only under suboptimal conditions for nodulation. Nutrient-amended granules significantly enhanced early nodulation of soybean and increased N content of the grain at harvest in four field trials. All these effects were obtained using an average of 10 kg granules amended with 1.14 kg glycerol and 0.16 kg sodium glutamate per hectare. The possible use of nutrient-amended granules to improve efficacy and reliability of microbial inoculation is discussed.     

Complete genomic sequence of nitrogen-fixing symbiotic bacterium Bradyrhizobium japonicum USDA110.

The complete nucleotide sequence of the genome of a symbiotic bacterium Bradyrhizobium japonicum USDA110 was determined. The genome of B. japonicum was a single circular chromosome 9,105,828 bp in length with an average GC content of 64.1%. No plasmid was detected. The chromosome comprises 8317 potential protein-coding genes, one set of rRNA genes and 50 tRNA genes. Fifty-two percent of the potential protein genes showed sequence similarity to genes of known function and 30% to hypothetical genes. The remaining 18% had no apparent similarity to reported genes. Thirty-four percent of the B. japonicum genes showed significant sequence similarity to those of both Mesorhizobium loti and Sinorhizobium meliloti, while 23% were unique to this species. A presumptive symbiosis island 681 kb in length, which includes a 410-kb symbiotic region previously reported by G?ttfert et al., was identified. Six hundred fifty-five putative protein-coding genes were assigned in this region, and the functions of 301 genes, including those related to symbiotic nitrogen fixation and DNA transmission, were deduced. A total of 167 genes for transposases/104 copies of insertion sequences were identified in the genome. It was remarkable that 100 out of 167 transposase genes are located in the presumptive symbiotic island. DNA segments of 4 to 97 kb inserted into tRNA genes were found at 14 locations in the genome, which generates partial duplication of the target tRNA genes. These observations suggest plasticity of the B. japonicum genome, which is probably due to complex genome rearrangements such as horizontal transfer and insertion of various DNA elements, and to homologous recombination.     

An isothermal absorptiometric assay for viable microbes using the redox color indicator 2,6-dichlorophenolindophenol

A simple and rapid isothermal absorptiometric assay for detection of viable microbes using the redox color indicator 2,6-dichlorophenolindophenol (DCPIP) was studied. The absorbance of DCPIP decreased at 600 nm because of a redox reaction occurring between DCPIP and the surface membrane of viable microbes and was inversely proportional to the viable cell density. The redox reaction was found not only with bacteria, but also with yeast and a mixture of bacteria and yeast. In this assay, the influence of light scattering and absorption caused by microbial cells and coexisting substances in the sample was excluded by a time difference method. The assay required only 10 min for one incubation mixture, and highly repeatable results from three consecutive measurements were obtained by isothermal incubation for specific times at 30 °C using a thermostable three-cuvette-stir system. Thus, the cell density of microbial cell suspensions or growth medium was successfully determined, and a practical lower detection limit for food inspection was obtained at 10⁴–10⁶ cfu/ml. Single-cell effects on DCPIP reduction were evaluated and compared between species. Consequently, this assay is expected to be a useful tool for the rapid measurement of viable microbes as a preliminary assay for the Hazard Analysis Critical Control Point program.     

Increased divalent metal ion uptake associated with hydrogenase constitutive phenotypes of Bradyrhizobium japonicum

Hydrogenase-constitutive (Hup^c) mutants of Bradyrhizobium japonicum were previously shown to accumulate more nickel than the wild-type strain. In a 2 h period Hup^c strains JH101 and JH103 also accumulated 2- to 3-fold more Mg²⁺, Zn²⁺ and Cu²⁺, and about 4-fold more Co²⁺ and Mn²⁺ than the wild-type strain JH. Init uptake rates (first 10 min) by the Hup^c strains were also greater for all the metals. The mutation in the Hup^c strains affecting a trans-acting regulator of the hup structural genes appears to have also amplified a metal uptake/accumulation process common to many divalent metal ions. From efflux experiments (suspension of cells in metal-free medium after metal accumulation) to determine the degree of dissociation of each metal with the cells it was concluded that Zn²⁺, like Ni²⁺, was rapidly and tightly cell-associated. In contrast, about 50% of the accumulated Cu²⁺ and about 30% of the Mn²⁺ was effluxed within 2 h by both the Hup^c and wild-type strains. Cobalt was more tightly cell-associated than Mn²⁺ or Cu²⁺, as the strains effluxed about 26% of the previously accumulated metal in 2 h. Even after accounting for effluxed metal, the Hup^c strains retained more of each metal than the wild-type. The increased metal accumulation by Hup^c strains could not be accounted for solely at the level of transport, as known metabolic inhibitors (carbonyl cyanide m-chlorophenylhydrazone and nigericin) of nickel transport partially inhibited (1 h) accumulation of only some (magnesium, zinc and copper) of the other metals. Hydrogenase-derepressed wild-type cells exhibited slightly higher (22–27% more) 2 h accumulation capacity for some of the metals (nickel, zinc and copper) than did non-derepressed cells, but not to the 2- to 4-fold greater level observed for Hup^c strains compared with the wild-type. The Hup^c strains JH101 and JH103 do not synthesize more capsular/cell wall carbohydrate than the wild-type strain.     

A comparative proteomic evaluation of culture grown vs nodule isolated Bradyrhizobium japonicum

Total protein extract of Bradyrhizobium japonicum cultivated in HM media were resolved by 2-D PAGE using narrow range IPG strips. More than 1200 proteins were detected, of which nearly 500 proteins were analysed by MALDI-TOF and 310 spots were tentatively identified. The present study describes at the proteome level a significant number of metabolic pathways related to important cellular events in free-living B. japonicum. A comparative analysis of proteomes of free-living and nodule residing bacteria revealed major differences and similarities between the two states. Proteins related to fatty acid, nucleic acid and cell surface synthesis were significantly higher in cultured cells. Nitrogen metabolism was more pronounced in bacteroids whereas carbon metabolism was similar in both states. Relative percentage of proteins related to global functions like protein synthesis, maturation & degradation and membrane transporters were similar in both forms, however, different proteins provided these functions in the two states.     

Global protein expression pattern of Bradyrhizobium japonicum bacteroids: a prelude to functional proteomics

As a prelude to using functional proteomics towards understanding the process of symbiotic nitrogen fixation between the legume soybean and the soil bacteria Bradyrhizobium japonicum, we examined the total protein expression pattern of the nodule bacteria, often referred to as bacteroids. A partial proteome map was constructed by separating the total bacteroid proteins using high-resolution 2-DE. Of the several hundred protein spots analyzed using PMF, 180 spots were tentatively identified by searching the available database for B. japonicum, (http://www.kazusa.or.jp/index.html). The data showed that the bacteroid expressed a dominant and elaborate protein network for nitrogen and carbon metabolism, which is closely dependent on the plant supplied metabolites, and seems aptly supported by a selective group of bacteroid transporter proteins. However, they seem to lack a defined fatty acid and nucleic acid metabolism. Interestingly, the proteins related to protein synthesis, scaffolding and degradation were among the most predominant spots of the bacteroid proteome. In addition, several proteins, which showed fairly good expression, were identified to be involved with cellular detoxification, stress regulation and signaling communication components. This preliminary proteomic data matches very well with several biochemical and genetic reports, and clearly shows the inter-connection between several metabolic pathways that meet the needs of the bacteroid. It is expected that in the future this will allow us to develop testable hypotheses about the roles of several of these proteins in context to the metabolic pathway connections and metabolite fluxes.     

Getting more from cell size distributions: Establishing more accurate biovolumes by estimating viable cell populations

Current approaches for cell size distribution modeling are attempting to describe the behavior of the entire distribution with respect to time. Although some advances have been made in this area, the modeling process requires a large number of culture‐specific parameters and an a priori assumption of the distribution nature (Poisson, Gaussian, etc.). In this work, we propose a deconvolution of the distribution into size ranges and an iterative regression process with respect to a single culture variable, such as viability. Following this approach, two example applications are outlined using data collected with a Coulter Counter Multisizer. In the first, traditional biovolume measurements are corrected to account for the noneven distribution of nonviable cells. These corrections amount to an average increase of 7–65% in the calculated biovolume from 24 to 72 h postinfection and are expected to aid in the development of a new basis for nutrient consumption postinfection. In the second example, viability is predicted from the cell size distribution using both linear and exponential regressions. Differences between predicted and measured viabilities were found to be normally distributed with means of 0.4% and 0% as well as standard deviations of 7.6% and 8.1% for linear and exponential regression, respectively. Although only viability relationships were tested, our approach yielded significant results for both applications, allowing the possibility for further development. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Regulation of nitrogenase activity in Bradyrhizobium japonicum/soybean symbiosis by plant N status as determined by shoot C:N ratio

Regulation of nitrogenase is not sufficiently understood to engineer symbioses that achieve a high N₂ fixation rate under high levels of soil N. In the present hydroponic growth chamber study we evaluated the hypothesis that nitrogenase activity and the extent of its inhibition by NO₃⁻ may be related to both N and carbohydrate levels in plant tissues. A wide range of C:N ratios in various plant tissues (8.5 to 41.0, 1.9 to 3.7, and 0.8 to 1.8, respectively, in shoots, roots, and nodules) was generated through a combination of light and CO₂ levels, using two soybean genotypes differing in C and N acquisition rates. For both genotypes, N concentration in shoots was negatively correlated to nitrogenase activity and positively correlated to the extent of nitrogenase inhibition by NO₃⁻. Furthermore, nitrogenase activity was positively correlated to total nonstructural carbohydrates (TNC) and C:N ratio in shoot and nodules for both genotypes. Nitrogenase inhibition by NO₃⁻ was negatively correlated to TNC and C:N ratio in shoots, but not in nodules for both genotypes. At the onset of nitrogenase inhibition by NO₃⁻, C:N ratio declined in shoots but not in nodules. These results indicate that both C and N levels in plant tissues are involved in regulation of nitrogenase activity. We suggest that the level of nitrogenase activity may be determined by (1) N needs (as determined by shoot C:N) and (2) availability of carbohydrates in nodules. Modulation of the nitrogenase activity may occur through sensing changes in plant N, i.e. changes in shoot C:N ratio, possibly through some phloem translocatable compound(s).     

Comparison of MTT and ATP-based assays for the measurement of viable cell number

Cell viability assays are widely used to assess the effect of chemotherapeutic drugs and other agents on cell lines and have shown promise for the prediction of tumour chemosensitivity. In this study we have compared two viability assays using Daudi and CCRF-CEM cell lines over a range of 1500–100,000 cells/well of a microplate. The ATP assay was able to detect the lower limit of 1563 cells/well with luminescence values at least 100× background readings, while the MTT assay could not detect less than 25,000 cells/well above background readings. The ATP assay also showed better reproducibility and sensitivity when cells were grown in microtitre plates over several days, and is particularly useful for the measurement of viability with low cell numbers.