Strain TR-1 Research Articles

Activity assessment of tomato endophytic bacteria bioactive compounds for the postharvest biocontrol of Botrytis cinerea

• Healthy tomato plants harbor diverse endophytic bacteria colonizing mainly leaves. • 36 % of the strains produce antifungal volatiles against Botrytis cinerea . • Antagonistic bacteria belong to 9 Bacillus and 2 Enterobacter species. • Enterobacter volatile and Bacillus soluble compounds reduced B. cinerea infection. • 3-Methylbutan-1-ol reduces postharvest decay of B. cinerea on tomato fruit. Botrytis cinerea is one of the most important postharvest fungal pathogens causing significant losses in fresh fruits, vegetables and ornamentals. Synthetic fungicides are primarily used to control postharvest decay loss, but due to their hazardous use the recent trend is shifting toward safer and more eco-friendly alternatives. The use of antagonistic microorganisms is becoming popular throughout the world. This study investigated the antifungal, the plant growth promoting activities and the identity of volatile organic compounds (VOCs) produced by tomato-derived endophytic bacteria strains. The capacity of selected strains to prevent postharvest B. cinerea infection on tomato fruit through VOCs and soluble compounds was also studied. A collection of 50 bacterial strains was established from different organs of tomato plants sampled from six localities in Cape Bon region (Tunisia). Despite the small geographical scale, complexity and abundance of endophytic communities varied greatly according to the site of origin. Healthy tomato plants harbor diverse endophytic bacteria of Bacillus and Enterobacter genera colonizing mainly leaves with a significant enrichment with Bacillus strains. The in vitro dual culture assays showed that 36 % of the endophytic bacterial strains produce antifungal VOCs against B. cinerea . To our knowledge, this is the first report of VOCs antifungal activity produced by B. nakamurai, B. pseudomycoides, B. proteolyticus, B. thuringiensis, E. asburiae and E. cloacae against B. cinerea . About 14 % of bacterial strains produce VOCs with in vitro specific promoting effects on tomato seedling length or biomass production. The five selected antagonistic endophytic bacterial strains produced a core set of seven VOCs along with different strain-specific and known antifungal VOCs such as 3-Methylbutan-1-ol, sulfur-containing compounds, 2-Heptanone and Dodecanal. Tomato fruit bio-protection assay showed that the Enterobacter strain TR1 produces the most protective VOCs against B. cinerea infection with 3-Methylbutan-1-ol as a major volatile compound which totally suppressed B. cinerea growth and infection on tomato fruit at 0.442 mL L −1 headspace, whereas the Bacillus strains showed better protection against fungal infection when applied as vegetative cells on tomato fruit. These results support the use of the selected strains as potential biocontrol agents to reduce postharvest decay of B. cinerea , as well as 3-Methylbutan-1-ol as promising antifungal volatile to apply during postharvest commercialization of tomato fruit.

Halophilic mechanism of the enzymatic function of a moderately halophilic dihydrofolate reductase from Haloarcula japonica strain TR-1.

Dihydrofolate (DHF) reductase coded by a plasmid of the extremely halophilic archaeon Haloarcula japonica strain TR-1 (HjDHFR P1) shows moderate halophilicity on enzymatic activity at pH 6.0, although there is no significant effect of NaCl on its secondary structure. To elucidate the salt-activation and -inactivation mechanisms of this enzyme, we investigated the effects of pH and salt concentration, deuterium isotope effect, steady-state kinetics, and rapid-phase ligand-binding kinetics. Enzyme activity was increased eightfold by the addition of 500mM NaCl at pH 6.0, fourfold by 250mM at pH 8.0, and became independent of salt concentration at pH 10.0. Full isotope effects observed at pH 10.0 under 0-1000mM NaCl indicated that the rate of hydride transfer, which was the rate-determining step at the basic pH region, was independent of salt concentration. Conversely, rapid-phase ligand-binding experiments showed that the amplitude of the DHF-binding reaction increased and the tetrahydrofolate (THF)-releasing rate decreased with increasing NaCl concentration. These results suggested that the salt-activation mechanism of HjDHFR P1 is via the population change of the anion-unbound and anion-bound conformers, which are binding-incompetent and -competent conformations for DHF, respectively, while that of salt inactivation is via deceleration of the THF-releasing rate, which is the rate-determining step at the neutral pH region.

Genome Sequence of Desulfurella amilsii Strain TR1 and Comparative Genomics of Desulfurellaceae Family.

The acidotolerant sulfur reducer Desulfurella amilsii was isolated from sediments of Tinto River, an extremely acidic environment. Its ability to grow in a broad range of pH and to tolerate certain heavy metals offers potential for metal recovery processes. Here we report its high-quality draft genome sequence and compare it to the available genome sequences of other members of Desulfurellaceae family: D. acetivorans. D. multipotens, Hippea maritima. H. alviniae, H. medeae, and H. jasoniae. For most species, pairwise comparisons for average nucleotide identity (ANI) and in silico DNA–DNA hybridization (DDH) revealed ANI values from 67.5 to 80% and DDH values from 12.9 to 24.2%. D. acetivorans and D. multipotens, however, surpassed the estimated thresholds of species definition for both DDH (98.6%) and ANI (88.1%). Therefore, they should be merged to a single species. Comparative analysis of Desulfurellaceae genomes revealed different gene content for sulfur respiration between Desulfurella and Hippea species. Sulfur reductase is only encoded in D. amilsii, in which it is suggested to play a role in sulfur respiration, especially at low pH. Polysulfide reductase is only encoded in Hippea species; it is likely that this genus uses polysulfide as electron acceptor. Genes encoding thiosulfate reductase are present in all the genomes, but dissimilatory sulfite reductase is only present in Desulfurella species. Thus, thiosulfate respiration via sulfite is only likely in this genus. Although sulfur disproportionation occurs in Desulfurella species, the molecular mechanism behind this process is not yet understood, hampering a genome prediction. The metabolism of acetate in Desulfurella species can occur via the acetyl-CoA synthetase or via acetate kinase in combination with phosphate acetyltransferase, while in Hippea species, it might occur via the acetate kinase. Large differences in gene sets involved in resistance to acidic conditions were not detected among the genomes. Therefore, the regulation of those genes, or a mechanism not yet known, might be responsible for the unique ability of D. amilsii. This is the first report on comparative genomics of sulfur-reducing bacteria, which is valuable to give insight into this poorly understood metabolism, but of great potential for biotechnological purposes and of environmental significance.

Sulfur Reduction in Acid Rock Drainage Environments.

Microbiological suitability of acidophilic sulfur reduction for metal recovery was explored by enriching sulfur reducers from acidic sediments at low pH (from 2 to 5) with hydrogen, glycerol, methanol and acetate as electron donors at 30 °C. The highest levels of sulfide in the enrichments were detected at pH 3 with hydrogen and pH 4 with acetate. Cloning and sequencing of the 16S rRNA gene showed dominance of the deltaproteobacterial sulfur-reducing genus Desulfurella in all the enrichments and subsequently an acidophilic strain (TR1) was isolated. Strain TR1 grew at a broad range of pH (3-7) and temperature (20-50 °C) and showed good metal tolerance (Pb(2+), Zn(2+), Cu(2+), Ni(2+)), especially for Ni(2+) and Pb(2+), with maximal tolerated concentrations of 0.09 and 0.03 mM, respectively. Different sources of sulfur were tested in the enrichments, from which biosulfur showed fastest growth (doubling time of 1.9 days), followed by colloidal, chemical and sublimated sulfur (doubling times of 2.2, 2.5, and 3.6 days, respectively). Strain TR1's physiological traits make it a good candidate to cope with low pH and high metal concentration in biotechnological processes for treatment of metal-laden acidic streams at low and moderately high temperature.

Effects of salt on the structure, stability, and function of a halophilic dihydrofolate reductase from a hyperhalophilic archaeon, Haloarcula japonica strain TR-1.

The effects of salt on the structure, stability, and enzymatic function of a novel dihydrofolate reductase (HjDHFR P1) from a hyperhalophilic archaeon, Haloarcula japonica strain TR-1 living in a Japanese saltern, were studied using ultraviolet absorption, circular dichroism (CD), and fluorescence spectroscopy. HjDHFR P1 had a partial structure at pH 8.0 in the absence of NaCl, and the addition of NaCl (0-500 mM concentration) induced significant structural formation to HjDHFR P1. The addition of NADPH, which is a coenzyme for its catalytic reaction, and lowering the pH from 8 to 6 also induced the same CD change, indicating the formation of the NADPH-binding site in HjDHFR P1. The NaCl dependence of thermal and urea-induced unfolding measurements suggested that protein stability increased depending on NaCl concentration regardless of structural formation, and HjDHFR P1 achieved the same stability as Escherichia coli DHFR at 750 mM NaCl. Halophilic characteristics were also observed for enzymatic function, although its structure had already formed under the conditions that enzymatic activity was measured at due to the presence of NADPH. These results suggest that the halophilic mechanism on structural stability and function was caused by factors other than structural formation, which are suggested to be the contributions of preferential interactions between the protein and salt ions and the specific binding of salt ions.

Gene cloning, expression and partial characterization of cell division protein FtsZ1 from extremely halophilic archaeon Haloarcula japonica strain TR-1

The gene encoding a cell division protein FtsZ1 was cloned from an extremely halophilic archaeon, Haloarcula japonica strain TR-1. Nucleotide sequencing analysis of the ftsZ1 gene revealed that the structural gene consisted of an open reading frame of 1,158 nucleotides encoding 386 amino acids. Transcription of the ftsZ1 gene in Ha. japonica was confirmed by RT-PCR. A modified ftsZ1 gene was inserted into the shuttle vector pWL102 and used to transform Ha. japonica. The recombinant FtsZ1 was produced as a fusion with hexahistidine-tag in Ha. japonica host cells and purified. Purified recombinant FtsZ1 exhibited GTP-dependent polymerization activity and GTP-hydrolyzing activity in the presence of high concentrations of KCl.

Molecular cloning of transducer genes hjtA and hjtC from extremely halophilic archaeon Haloarcula japonica

The genes encoding putative taxis transducers hjtA and hjtC were cloned from triangular disc-shaped extremely halophilic archaeon Haloarcula japonica strain TR-1. Nucleotide sequencing analysis revealed that HjtA and HjtC consisted of 438 and 630 amino acids, respectively. HjtA and HjtC have the highly conserved sequence which is commonly found in signaling domains of bacterial and archaeal transducers.

Isolation of crtI homolog from extremely halophilic archaeon Haloarcula japonica strain TR-1

Halobacterium sp. strain NRC-1 possesses phytoene dehydrogenase genes (crtIs). A crtI2 homolog was cloned from Haloarcula japonica strain TR-1. Nucleotide sequencing analysis of the possible crtI2 revealed that the structural gene consisted of an open reading frame of 1,521 nucleotides encoding 507 amino acids. Transcription of crtI2 in Ha. japonica was confirmed by RT-PCR.

Gene cloning of ftsZ1 homolog from extremely halophilic archaeon Haloarcula japonica.

The gene encoding FtsZ1 was cloned from triangular disc-shaped extremely halophilic archaeon Haloarcula japonica strain TR-1. Nucleotide sequencing analysis of the possible ftsZ1 gene revealed that the structural gene consisted of an open reading frame of 1,038 nucleotides encoding 346 amino acids. Transcription of the ftsZ1 gene in Ha. japonica was confirmed by RT-PCR.

A brp homolog of Haloarcula japonica: gene cloning and transcriptional analysis.

The gene encoding a homolog of Halobacterium salinarum bacterioopsin-related protein (Brp) was cloned from Haloarcula japonica strain TR-1. Nucleotide sequencing analysis of the possible brp gene revealed that the structural gene consisted of an open reading frame of 1,062 nucleotides encoding 354 amino acids. Transcription of the brp homolog in Ha. japonica was confirmed by RT-PCR.

Gene clusters encoding ATP synthase of Haloarcula japonica strain TR-1.

The genes encoding I, K, E, C, F, A, B and D subunits of A0A1 ATP synthase were cloned from Haloarcula japonica strain TR-1. The genes were split into two clusters. Northern analysis revealed that the larger gene cluster would be transcribed as a single mRNA.

Soil bacteria Pseudomonas putida and Alcaligenes xylosoxidans subsp. denitrificans inactivate triclosan in liquid and solid substrates.

Triclosan is a broad-spectrum antimicrobial agent that has been incorporated into many household and medical products. Bacteria with high levels of triclosan resistance were isolated from compost, water, and soil samples. Two of these bacteria, Pseudomonas putida TriRY and Alcaligenes xylosoxidans subsp. denitrificans TR1, were able to use triclosan as a sole carbon source and clear particulate triclosan from agar. A decrease in triclosan concentration was measured by HPLC within 6 h of inoculation with strain TriRY and 24 h with strain TR1. Bioassays demonstrated that triclosan was inactivated in liquid cultures and/or embedded in plastic by the growth of strain TriRY and strain TR1, permitting the growth of triclosan-sensitive bacteria.

Soil bacteria Pseudomonas putida and Alcaligenes xylosoxidans subsp. denitrificans inactivate triclosan in liquid and solid substrates

Triclosan is a broad-spectrum antimicrobial agent that has been incorporated into many household and medical products. Bacteria with high levels of triclosan resistance were isolated from compost, water, and soil samples. Two of these bacteria, Pseudomonas putida TriRY and Alcaligenes xylosoxidans subsp. denitrificans TR1, were able to use triclosan as a sole carbon source and clear particulate triclosan from agar. A decrease in triclosan concentration was measured by HPLC within 6 h of inoculation with strain TriRY and 24 h with strain TR1. Bioassays demonstrated that triclosan was inactivated in liquid cultures and/or embedded in plastic by the growth of strain TriRY and strain TR1, permitting the growth of triclosan-sensitive bacteria.

Cloning and expression of the ferredoxin gene from extremely halophilic archaeon Haloarculajaponica strain TR-1.

The gene encoding a ferredoxin (Fd) from Haloarculajaponica strain TR-1 was cloned and sequenced. Sequence analysis of the cloned Ha. japonica Fd gene revealed that the structural gene consisted of an open reading frame of 387 nucleotides encoding 129 amino acids. The deduced amino acid sequence of Ha. japonica Fd showed 84 to 98% identity with corresponding sequences in other extremely halophilic archaea. The Ha. japonica Fd gene was inserted into the shuttle vector pWL102 and used to transform Ha. japonica. Ha. japonica Fd could then be produced as a fusion with His-Tag (6xHis) in Ha. japonica host cells. The absorption and ESR spectra of the Fd/His Tag fusion protein revealed the presence of a [2Fe-2S] cluster which is characteristic of native Ha. japonica Fd.

The gene encoding a novel halorhodopsin-like protein of extremely halophilic archaeon Haloarcula japonica strain TR-1.

The gene encoding the halorhodopsin (hR)-like protein was cloned and sequenced from Haloarcula japonica strain TR-1. The structural gene consisted of an open reading frame of 828 nucleotides encoding 276 amino acids. The deduced amino acid sequence of the Ha. japonica hR-like protein showed the highest homology to those of cruxhalorhodopsins. Furthermore, we have demonstrated that the Ha. japonica chR gene is transcriptionally induced by light.

A novel bacteriorhodopsin-like protein from Haloarcula japonica strain TR-1: gene cloning, sequencing, and transcript analysis

A novel bacteriorhodopsin-like protein from Haloarcula japonica strain TR-1: gene cloning, sequencing, and transcript analysis

A novel bacteriorhodopsin-like protein from Haloarcula japonica strain TR-1: gene cloning, sequencing, and transcript analysis.

The gene encoding a novel bacteriorhodopsin (bR)-like protein from Haloarcula japonica strain TR-1 was cloned and sequenced. The nucleotide sequence of the gene contained an open reading frame that corresponded to a protein of 250 amino acids. The deduced amino acid sequence of Ha. japonica bR-like protein exhibited the highest homology to those of cruxrhodopsins (cRs) produced by members of the genus Haloarcula, suggesting that the bR-like protein of Ha. japonica belonged to the cR subfamily. The hydropathy analysis of Ha. japonica bR-like protein (cR) revealed that the Ha. japonica cR had a transmembrane heptahelical structure similar to that of bR. Furthermore, transcription of the cR gene in Ha. japonica was confirmed by the reverse transcription-polymerase chain reaction method.

Molecular cloning of A1-ATPase gene from extremely halophilic archaeon Haloarcula japonica strain TR-1.

The genes encoding A1-ATPase A- and B-subunits were cloned from Haloarcula japonica strain TR-1. Nucleotide sequencing analysis of the A1-ATPase gene revealed that the A- and B-subunits consisted of 586 and 473 amino acids, respectively. The deduced amino acid sequences of the A- and B-subunits of Ha. japonica showed high identities with those of Halobacterium salinarum and Haloferax volcanii. The consensus ATP-binding motif was found in the A-subunit.

Transcriptional regulation of cruxrhodopsin gene from extremely halophilic archaeon Haloarcula japonica strain TR-1.

Transcription of the cruxrhodopsin (cR) gene in extremely halophilic archaeon Haloarcula japonica strain TR-1 was investigated using Northern analysis to quantify message level. In the cell cultures growing in the dark, cR transcript level remained very low. In contrast, exposure of the cell cultures to light stimulated transcription of the cR gene. In addition, cR gene transcription was also induced when Ha. japonica was grown under high oxygen tension and then shifted to low oxygen tension in the dark. These results suggested that transcription of the cR gene is regulated by high light intensity and low oxygen tension.

Purification and characterization of a ferredoxin from Haloarcula japonica strain TR-1, and cloning of its gene

Purification and characterization of a ferredoxin from Haloarcula japonica strain TR-1, and cloning of its gene