Strain RG1 Research Articles

Rhizobacteria of Bali With Obvious Growth-Promoting Properties on Corn (Zea mays L.)

Corn productivity in Indonesia is still relatively low compared with other countries. Therefore, it is necessary to increase the productivity of corn by using rhizobacteria, which have multiple traits. This study was conducted to obtain indigenous rhizobacteria of Bali that have multiple traits, can produce indoleacetic acid (IAA), fix nitrogen from atmosphere, produce siderophores, colonize roots, increase seed germination, and promote the growth of corn. Isolation of rhizobacteria was carried out from the rhizosphere of plants belonging to the Gramineae family that grows in Bali Island, Indonesia. Six isolates, namely, Sr3, Tb9, Rg1, Rg23, Al27, and Jg8, could produce IAA, fix nitrogen from the atmosphere, produce siderophores, and increased germination rate and vigor index of corn seedling. Among them, three isolates, namely, Rg1, Sr3, and Jg8, significantly (p <0.05) increased the germination rate of corn seeds, increased vigor index, increased root dry weight and shoot dry weight of corn at the age of 7 days, and were able to colonize corn roots. Compared with the control, the rhizobacteria treatment increased the germination of corn seeds ranging from 5.04 to 13.05%. Based on the analysis of the 16S rRNA gene, it was found that these rhizobacteria species were Glutamicibacter nicotianae strain Rg1 (accession number OM349119), Brevibacillus invocatus strain Sr3 (accession number OM327515), and Micrococcus luteus strain Jg8 (accession number OM362349). Under a greenhouse condition, all the three isolates significantly (p <0.05) increased nutrient uptake, the leaf chlorophyll content, net assimilation rate, and crop growth rate of corn when compared with control. These results suggested that these isolates of rhizobacteria obviously promoted the growth of corn and can be developed as biostimulant to promote the growth and increase the corn yield in Bali, Indonesia.

Enterobacter asburiae and Pantoea ananatis Causing Rice Bacterial Blight in China.

Rice bacterial blight is a devastating bacterial disease threatening rice yield all over the world and Xanthomonas oryzae pv. oryzae is traditionally believed to be the pathogen. In recent years, we have received diseased rice samples with symptoms of blighted leaves from Sichuan and Guangdong provinces, China. Pathogen isolation and classification identified two different enterobacteria as the causal agents, namely Enterobacter asburiae and Pantoea ananatis. Among them, E. asburiae was isolated from samples of both provinces, and P. ananatis was only isolated from the Sichuan samples. Different from rice foot rot pathogen Dickeya zeae EC1 and rice bacterial blight pathogen X. oryzae pv. oryzae PXO99A, strains SC1, RG1, and SC7 produced rare cell wall degrading enzymes (CWDEs) but more extrapolysaccharides (EPS). E. asburiae strains SC1 and RG1 produced bacteriostatic substances while P. ananatis strain SC7 produced none. Pathogenicity tests indicated that all of them infected monocotyledonous rice and banana seedlings, but not dicotyledonous potato, radish, or cabbage. Moreover, strain RG1 was most virulent, while strains SC1 and SC7 were similarly virulent on rice leaves, even though strain SC1 propagated significantly faster in rice leaf tissues than strain SC7. This study firstly discovered E. asburiae as a new pathogen of rice bacterial blight, and in some cases, P. ananatis could be a companion pathogen. Analysis on production of virulence factors suggested that both pathogens probably employ a different mechanism to infect hosts other than using cell wall degrading enzymes to break through host cell walls.

Silencing of transcription factor encoding gene StTCP23 by small RNAs derived from the virulence modulating region of potato spindle tuber viroid is associated with symptom development in potato.

Viroids are small, non-protein-coding RNAs which can induce disease symptoms in a variety of plant species. Potato (Solanum tuberosum L.) is the natural host of Potato spindle tuber viroid (PSTVd) where infection results in stunting, distortion of leaves and tubers and yield loss. Replication of PSTVd is accompanied by the accumulation of viroid-derived small RNAs (sRNAs) proposed to play a central role in disease symptom development. Here we report that PSTVd sRNAs direct RNA silencing in potato against StTCP23, a member of the TCP (teosinte branched1/Cycloidea/Proliferating cell factor) transcription factor family genes that play an important role in plant growth and development as well as hormonal regulation, especially in responses to gibberellic acid (GA). The StTCP23 transcript has 21-nucleotide sequence complementarity in its 3ʹ untranslated region with the virulence-modulating region (VMR) of PSTVd strain RG1, and was downregulated in PSTVd-infected potato plants. Analysis using 3ʹ RNA ligase-mediated rapid amplification of cDNA ends (3ʹ RLM RACE) confirmed cleavage of StTCP23 transcript at the expected sites within the complementarity with VMR-derived sRNAs. Expression of these VMR sRNA sequences as artificial miRNAs (amiRNAs) in transgenic potato plants resulted in phenotypes reminiscent of PSTVd-RG1-infected plants. Furthermore, the severity of the phenotypes displayed was correlated with the level of amiRNA accumulation and the degree of amiRNA-directed down-regulation of StTCP23. In addition, virus-induced gene silencing (VIGS) of StTCP23 in potato also resulted in PSTVd-like phenotypes. Consistent with the function of TCP family genes, amiRNA lines in which StTCP23 expression was silenced showed a decrease in GA levels as well as alterations to the expression of GA biosynthesis and signaling genes previously implicated in tuber development. Application of GA to the amiRNA plants minimized the PSTVd-like phenotypes. Taken together, our results indicate that sRNAs derived from the VMR of PSTVd-RG1 direct silencing of StTCP23 expression, thereby disrupting the signaling pathways regulating GA metabolism and leading to plant stunting and formation of small and spindle-shaped tubers.

Investigation of conformational changes of levansucrase isolated from Acetobacter nitrogenifigens strain RG1 by mercuric and cadmium ion

Levansucrase is a secretary enzyme of Acetobacter nitrogenifigens strain RG1. The enzyme shows enhanced activity in the presence of Hg2+ in spite of being inhibited by other heavy metal ion Cd2+. In this study the structural characterization of levansucrase in native state as well as in the presence of Hg2+ and Cd2+ by CD spectroscopy is done. The secondary structures of the native enzyme and the enzyme treated with Hg2+ and Cd2+ on comparison by their CD spectra revealed that their spectra showed no significant difference indicating that both Hg2+ as well as Cd2+ had no effect on the overall secondary structure of the protein. The respective CD spectra on analysis revealed that they have almost identical percentage of secondary structural elements. The interaction of levansucrase with Hg2+ as well as Cd2+ was studied further by tryptophan fluorescence spectroscopy which on analysis revealed static quenching indicating protein-heavy metal complex formation. A blue shift in the tryptophan fluorescence spectra of Hg2+ treated protein indicated that the tryptophan residues have moved to a more hydrophobic environment in the protein away from aqueous phase. The mechanism of interaction of enzyme with mercury and cadmium was determined from their tryptophan fluorescence spectra.

Phylogenetic analysis and characterization of an alkane-degrading yeast strain isolated from oil-polluted soil

The isolation and phylogenetic analysis of new yeast strains represent an important basis for the development of practical applications in biotechnology, industry, and bioremediation. The yeast strain RG1 was isolated from oil-polluted soil and characterized using morphological observations and the API 20C AUX system. The amplified ITS1-5.8S rDNA-ITS2 region had 653 bp and the restriction profiles obtained with Cfo I, Dde I, Hae III, Hinf I, and Hpa II endonucleases showed high similarities with those from Rhodotorula glutinis reference strains. The phylogenetic tree based on the Cfo I patterns confirmed the classification of the strain RG1 as belonging to the R. glutinis species. The studies performed during the present work indicate the existence of different mechanisms of n-alkane assimilation and biodegradation in the RG1 cells and their correlation with the production of biosurfactants. An evaluation of the emulsifying activity revealed that biosurfactant-mediated assimilation of n-alkanes decreases in the order n-decane > n-tetradecane > n-hexadecane. Biodegradation and emulsification assays using culture media supplemented with 1% n-alkanes as the sole carbon source showed that n-tetradecane represented the optimal hydrocarbon substrate for the growth of the strain RG1 over 14 days and for obtaining biosurfactants at stable rates.

Mercuric Ion Stabilizes Levansucrase Secreted by Acetobacter nitrogenifigens Strain RG1T

The purification and characterization of an extracellular levansucrase enzyme produced by novel nitrogen-fixer Acetobacter nitrogenifigens strain RG1(T) is described. Culture conditions were optimized for maximum levansucrase production. Levansucrase purified to homogeneity by tenfold purification has a molecular weight of 65 kDa, contained four cysteine residues, polymerized raffinose and was stable for 21 days at pH 6.0 when stored at 4 °C or -20 °C but was vulnerable to DTT and β-mercaptoethanol. Interestingly, this enzyme showed enhanced hydrolytic and polymerization activity in the presence of mercuric ion which, to our knowledge, is the first report for any levansucrase enzyme characterized so far. Evidences obtained from Native PAGE, tryptophan fluorescence study and activity measurements at different temperatures and in the presence of thiol modifying agents, show that mercuric ion stabilizes the enzyme. Levan, synthesized by the enzyme, has a molecular weight of 7,080 kDa and was shown to be a homopolymer of fructose.

Comparison of the removal of ethanethiol in twin-biotrickling filters inoculated with strain RG-1 and B350 mixed microorganisms

This study aims to compare the biological degradation performance of ethanethiol using strain RG-1 and B350 commercial mixed microorganisms, which were inoculated and immobilized on ceramic particles in twin-biotrickling filter columns. The parameters affecting the removal efficiency, such as empty bed residence time (EBRT) and inlet concentration, were investigated in detail. When EBRT ranged from 332 to 66 s at a fixed inlet concentration of 1.05 mg L −1, the total removal efficiencies for RG-1 and B350 both decreased from 100% to 70.90% and 47.20%, respectively. The maximum elimination capacities for RG-1 and B350 were 38.36 (removal efficiency = 89.20%) and 25.82 g m −3 h −1 (removal efficiency = 57.10%), respectively, at an EBRT of 83 s. The variation of the inlet concentration at a fixed EBRT of 110 s did not change the removal efficiencies which remained at 100% for RG-1 and B350 at concentrations of less than 1.05 and 0.64 mg L −1, respectively. The maximum elimination capacities were 39.93 (removal efficiency = 60.30%) and 30.34 g m −3 h −1 (removal efficiency = 46.20%) for RG-1 and B350, respectively, at an inlet concentration of 2.03 mg L −1. Sulfate was the main metabolic product of sulfur in ethanethiol. Based the results, strain RG-1 would be a better choice than strain B350 for the biodegradation of ethanethiol.

Biodegradation of ethanethiol in aqueous medium by a new Lysinibacillus sphaericus strain RG-1 isolated from activated sludge

In the present study, a new bacterial strain isolated from activated sludge has been identified as Lysinibacillus sphaericus based on its morphology, physiochemical properties, and the results of 16S ribosomal RNA (rRNA) gene sequence analysis. This new bacterial strain uses ethanethiol as both carbon source and energy source. The key factors for controlling the degradation efficiency of ethanethiol by this strain were found to be initial ethanethiol concentration, temperature, and pH value of solutions. Under the optimized conditions, as well as 4mgl(-1) ethanethiol, 30°C, and pH=7.0, almost 100% ethanethiol can be degraded within 96h and sulfate as a final product was detected in aqueous medium. The degradation reaction of ethanethiol over this newly isolated strain can be described by pseudo first-order equation in which the maximum degradation rate constant K and the minimum half-life were respectively calculated to be 0.0308h(-1) and 22.5h under the optimal conditions.

RamB, a novel transcriptional regulator of genes involved in acetate metabolism of Corynebacterium glutamicum.

The adaptation of Corynebacterium glutamicum to acetate as a carbon and energy source involves transcriptional regulation of the pta-ack operon coding for the acetate-activating enzymes phosphotransacetylase and acetate kinase and of the aceA and aceB genes coding for the glyoxylate cycle enzymes isocitrate lyase and malate synthase, respectively. Deletion and mutation analysis of the respective promoter regions led to the identification of highly conserved 13-bp motifs (AA/GAACTTTGCAAA) as cis-regulatory elements for expression of the pta-ack operon and the aceA and aceB genes. By use of DNA affinity chromatography, a 53-kDa protein specifically binding to the promoter/operator region of the pta-ack operon was purified. Mass spectrometry and peptide mass fingerprinting identified the protein as a putative transcriptional regulator (which was designated RamB). Purified His-tagged RamB protein was shown to bind specifically to both the pta-ack and the aceA/aceB promoter/operator regions. Directed deletion of the ramB gene in the genome of C. glutamicum resulted in mutant strain RG1. Whereas the wild type of C. glutamicum showed high-level specific activities of acetate kinase, phosphotransacetylase, isocitrate lyase, and malate synthase when grown on acetate and low-level specific activities when grown on glucose as sole carbon and energy sources, mutant RG1 showed high-level specific activities with all four enzymes irrespective of the substrate. Comparative transcriptional cat fusion experiments revealed that this deregulation takes place at the level of transcription. The results indicate that RamB is a negative transcriptional regulator of genes involved in acetate metabolism of C. glutamicum.

Molecular and functional characterization of the kstD2 gene of Rhodococcus erythropolis SQ1 encoding a second 3-ketosteroid Δ1-dehydrogenase isoenzyme b bThe GenBank accession number for the sequence reported in this paper is AY078169

Previously, Rhodococcus erythropolis SQ1 kstD, encoding ketosteroid Delta(1)-dehydrogenase (KSTD1) was characterized. Surprisingly, a kstD gene deletion mutant (strain RG1) grew normally on steroids. UV mutagenesis of strain RG1 allowed isolation of strains (e.g. strain RG1-UV29) unable to perform the Delta(1)-dehydrogenation of 4-androstene-3,17-dione (AD) and 9alpha-hydroxy-4-androstene-3,17-dione (9OHAD). Functional complementation of strain RG1-UV29 with total genomic DNA of strain RG1 resulted in identification of a 1698 nt ORF (kstD2) showing clear similarity (35% identity at amino acid sequence level) with KSTD1. Expression of kstD2 in Escherichia coli resulted in high KSTD2 activity levels. Single gene deletion mutants of either kstD (strain RG1) or kstD2 (strain RG7) appeared unaffected in growth on the steroid substrates AD, 1,4-androstadiene-3,17-dione and 9OHAD. Strain RG7, but not strain RG1, showed temporary accumulation of 9OHAD during AD conversion. A kstD kstD2 double deletion mutant (strain RG8) was constructed. Strain RG8 was unable to grow on steroid substrates, had undetectable steroid Delta(1)-dehydrogenation activity and efficiently converted AD into 9OHAD. Strain SQ1 thus employs two KSTD isoenzymes in steroid catabolism. Analysis of two null mutants in KSTD2 showed that the semi-conserved Ser325 and the highly conserved Thr503 play a role in KSTD enzyme activity.

Unmarked gene deletion mutagenesis of kstD, encoding 3-ketosteroid Δ1-dehydrogenase, in Rhodococcus erythropolis SQ1 using sacB as counter-selectable marker

This paper reports the first method for the construction of unmarked gene deletion mutants in the genus Rhodococcus. Unmarked deletion of the kstD gene, encoding 3-ketosteroid Δ1-dehydrogenase (KSTD1) in Rhodococcus erythropolis SQ1, was achieved using the sacB counter-selection system. Conjugative mobilization of the mutagenic plasmid from Escherichia coli S17-1 to R. erythropolis strain SQ1 was used to avoid its random genomic integration. The kstD gene deletion mutant, designated strain RG1, still possessed about 10% of the KSTD enzyme activity of wild-type and was not affected in its ability to grow on the steroid substrates 4-androstene-3,17-dione (AD) and 9α-hydroxy-4-androstene-3,17-dione (9OHAD). Biochemical evidence subsequently was obtained for the presence of a second KSTD enzyme (KSTD2) in R. erythropolis SQ1. UV mutants of strain RG1 unable to grow on AD were isolated. One of these mutants, strain RG1-UV29, had lost all KSTD enzyme activity and was also unable to grow on 9OHAD. It stoichiometrically converted AD into 9OHAD in concentrations as high as 20 g l−1. The two KSTD enzymes apparently both function in AD and 9OHAD catabolism. These isoenzymes have been inactivated in strain RG1 (KSTD1 negative) and strain RG1-UV29 (KSTD1 and KSTD2 negative), respectively.

Unmarked gene deletion mutagenesis of kstD, encoding 3-ketosteroid Δ 1-dehydrogenase, in Rhodococcus erythropolis SQ1 using sacB as counter-selectable marker

This paper reports the first method for the construction of unmarked gene deletion mutants in the genus Rhodococcus. Unmarked deletion of the kstD gene, encoding 3-ketosteroid Δ 1-dehydrogenase (KSTD1) in Rhodococcus erythropolis SQ1, was achieved using the sacB counter-selection system. Conjugative mobilization of the mutagenic plasmid from Escherichia coli S17-1 to R. erythropolis strain SQ1 was used to avoid its random genomic integration. The kstD gene deletion mutant, designated strain RG1, still possessed about 10% of the KSTD enzyme activity of wild-type and was not affected in its ability to grow on the steroid substrates 4-androstene-3,17-dione (AD) and 9α-hydroxy-4-androstene-3,17-dione (9OHAD). Biochemical evidence subsequently was obtained for the presence of a second KSTD enzyme (KSTD2) in R. erythropolis SQ1. UV mutants of strain RG1 unable to grow on AD were isolated. One of these mutants, strain RG1-UV29, had lost all KSTD enzyme activity and was also unable to grow on 9OHAD. It stoichiometrically converted AD into 9OHAD in concentrations as high as 20 g l −1. The two KSTD enzymes apparently both function in AD and 9OHAD catabolism. These isoenzymes have been inactivated in strain RG1 (KSTD1 negative) and strain RG1-UV29 (KSTD1 and KSTD2 negative), respectively.

Rhodosporidium Banno: dose-effect relations, mutagenic efficiency, and spectrum of mutants in the induction of auxotrophic mutants by ultraviolet light and N-methyl-N'-nitro-N-nitrosoguanidine

The kinetics, efficiency, and specificity of induction of forward mutations to auxotrophy by ultraviolet light (UV) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was examined in stationary phase cells of Rhodosporidium (Rhodotorula) wild strain Rg1. In comparison to the spontaneous level the frequency of auxotrophic mutants was increased more than 1000 times by both mutagens, however, the mutagenic efficiency of MNNG was higher than that of UV. We found that the forward mutation rate is a linear function of the applicated UV and MNNG doses in the range to 600 J m-2 or 25 mM X min, respectively. The 35 studied biosynthetic pathways to amino acids, purines, pyrimidines, and vitamins are genetically blocked at different frequencies, but there is not any significant difference between UV and MNNG induced frequencies of mutants with a specific requirement. However, in difference to the approximately equal distribution of the MNNG-induced nic mutants among the genetic blocks of the tryptophan-nicotinamide pathway, UV-induced nic mutants occurred with a higher frequency in the genes of the tryptophan pyrrolase and the 3-hydroxykynureninase than in the genes of the other enzymes of the pathway.