Takahisa Tajima

Identification of Chemotaxis Sensory Proteins for Amino Acids in Pseudomonas fluorescens Pf0-1 and Their Involvement in Chemotaxis to Tomato Root Exudate and Root Colonization

Pseudomonas fluorescens Pf0-1 showed positive chemotactic responses toward 20 commonly-occurring l-amino acids. Genomic analysis revealed that P. fluorescens Pf0-1 possesses three genes (Pfl01_0124, Pfl01_0354, and Pfl01_4431) homologous to the Pseudomonas aeruginosa PAO1 pctA gene, which has been identified as a chemotaxis sensory protein for amino acids. When Pf01_4431, Pfl01_0124, and Pfl01_0354 were introduced into the pctA pctB pctC triple mutant of P. aeruginosa PAO1, a mutant defective in chemotaxis to amino acids, its transformants showed chemotactic responses to 18, 16, and one amino acid, respectively. This result suggests that Pf01_4431, Pfl01_0124, and Pfl01_0354 are chemotaxis sensory proteins for amino acids and their genes were designated ctaA, ctaB, and ctaC, respectively. The ctaA ctaB ctaC triple mutant of P. fluorescens Pf0-1 showed only weak responses to Cys and Pro but no responses to the other 18 amino acids, indicating that CtaA, CtaB, and CtaC are major chemotaxis sensory proteins in P. fluorescens Pf0-1. Tomato root colonization by P. fluorescens strains was analyzed by gnotobiotic competitive root colonization assay. It was found that ctaA ctaB ctaC mutant was less competitive than the wild-type strain, suggesting that chemotaxis to amino acids, major components of root exudate, has an important role in root colonization by P. fluorescens Pf0-1. The ctaA ctaB ctaC triple mutant was more competitive than the cheA mutant of P. fluorescens Pf0-1, which is non-chemotactic, but motile. This result suggests that chemoattractants other than amino acids are also involved in root colonization by P. fluorescens Pf0-1.

Development of butanol-tolerant Bacillus subtilis strain GRSW2-B1 as a potential bioproduction host

As alternative microbial hosts for butanol production with organic-solvent tolerant trait are in high demands, a butanol-tolerant bacterium, Bacillus subtilis GRSW2-B1, was thus isolated. Its tolerance covered a range of organic solvents at high concentration (5%v/v), with remarkable tolerance in particular to butanol and alcohol groups. It was susceptible for butanol acclimatization, which resulted in significant tolerance improvement. It has versatility for application in a variety of fermentation process because it has superior tolerance when cells were exposed to butanol either as high-density, late-exponential grown cells (up to 5%v/v) or under growing conditions (up to 2.25%v/v). Genetic transformation procedure was optimized, yielding the highest efficiency at 5.17 × 103 colony forming unit (μg DNA)-1. Gene expression could be effectively driven by several promoters with different levels, where as the highest expression was observed with a xylose promoter. The constructed vector was stably maintained in the transformants, in the presence or absence of butanol stress. Adverse effect of efflux-mediated tetracycline resistance determinant (TetL) to bacterial organic-solvent tolerance property was unexpectedly observed and thus discussed. Overall results indicate that B. subtilis GRSW2-B1 has potential to be engineered and further established as a genetic host for bioproduction of butanol.

Evaluation of marine sediments as microbial sources for methane production from brown algae under high salinity

Various marine sediments were evaluated as promising microbial sources for methane fermentation of Saccharina japonica, a brown alga, at seawater salinity. All marine sediments tested produced mainly acetate among volatile fatty acids. One marine sediment completely converted the produced volatile fatty acids to methane in a short period. Archaeal community analysis revealed that acetoclastic methanogens belonging to the Methanosarcina genus dominated after cultivation. Measurement of the specific conversion rate at each step of methane production under saline conditions demonstrated that the marine sediments had higher conversion rates of butyrate and acetate than mesophilic methanogenic granules. These results clearly show that marine sediments can be used as microbial sources for methane production from algae under high-salt conditions without dilution.

Identification of Pseudomonas fluorescens chemotaxis sensory proteins for malate, succinate, and fumarate, and their involvement in root colonization.

Pseudomonas fluorescens Pf0-1 exhibited chemotactic responses to l-malate, succinate, and fumarate. We constructed a plasmid library of 37 methyl-accepting chemotaxis protein (MCP) genes of P. fluorescens Pf0-1. To identify a MCP for l-malate, the plasmid library was screened using the PA2652 mutant of Pseudomonas aeruginosa PAO1, a mutant defective in chemotaxis to l-malate. The introduction of Pfl01_0728 and Pfl01_3768 genes restored the ability of the PA2652 mutant to respond to l-malate. The Pfl01_0728 and Pfl01_3768 double mutant of P. fluorescens Pf0-1 showed no response to l-malate or succinate, while the Pfl01_0728 single mutant did not respond to fumarate. These results indicated that Pfl01_0728 and Pfl01_3768 were the major MCPs for l-malate and succinate, and Pfl01_0728 was also a major MCP for fumarate. The Pfl01_0728 and Pfl01_3768 double mutant unexpectedly exhibited stronger responses toward the tomato root exudate and amino acids such as proline, asparagine, methionine, and phenylalanine than those of the wild-type strain. The ctaA, ctaB, ctaC (genes of the major MCPs for amino acids), Pfl01_0728, and Pfl01_3768 quintuple mutant of P. fluorescens Pf0-1 was less competitive than the ctaA ctaB ctaC triple mutant in competitive root colonization, suggesting that chemotaxis to l-malate, succinate, and/or fumarate was involved in tomato root colonization by P. fluorescens Pf0-1.

Identification of CtpL as a chromosomally-encoded chemoreceptor for 4-chloroaniline and catechol in Pseudomonas aeruginosa PAO1

ABSTRACT Bacterial chemotaxis influences the ability of bacteria to survive and thrive in most environments, including polluted ones. Despite numerous reports of the phenotypic characterization of chemotactic bacteria, only a few molecular details of chemoreceptors for aromatic pollutants have been described. In this study, the molecular basis of chemotaxis toward an environmentally toxic chlorinated aromatic pollutant, 4-chloroaniline (4CA), was evaluated. Among the three Pseudomonas spp. tested, Pseudomonas aeruginosa PAO1 exhibited positive chemotaxis both to the nonmetabolizable 4CA, where 4-chloroacetanilide was formed as a dead-end transformation product, and to the metabolizable catechol. Molecular analysis of all 26 mutants with a disrupted methyl-accepting chemotaxis gene revealed that CtpL, a chromosomally encoded chemoreceptor, was responsible for the positive chemotactic response toward 4CA. Since CtpL has previously been described to be a major chemoreceptor for inorganic phosphate at low concentrations in PAO1, this report describes a fortuitous ability of CtpL to function toward aromatic pollutants. In addition, its regulation not only was dependent on the presence of the chemoattractant inducer but also was regulated by conditions of phosphate starvation. These results expand the range of known chemotactic transducers and their function in the environmental bacterium PAO1.

Identification of the mcpA and mcpM Genes, Encoding Methyl-Accepting Proteins Involved in Amino Acid and l-Malate Chemotaxis, and Involvement of McpM-Mediated Chemotaxis in Plant Infection by Ralstonia pseudosolanacearum (Formerly Ralstonia solanacearum Phylotypes I and III)

ABSTRACT Sequence analysis has revealed the presence of 22 putative methyl-accepting chemotaxis protein (mcp) genes in the Ralstonia pseudosolanacearum GMI1000 genome. PCR analysis and DNA sequencing showed that the highly motile R. pseudosolanacearum strain Ps29 possesses homologs of all 22 R. pseudosolanacearum GMI1000 mcp genes. We constructed a complete collection of single mcp gene deletion mutants of R. pseudosolanacearum Ps29 by unmarked gene deletion. Screening of the mutant collection revealed that R. pseudosolanacearum Ps29 mutants of RSp0507 and RSc0606 homologs were defective in chemotaxis to l-malate and amino acids, respectively. RSp0507 and RSc0606 homologs were designated mcpM and mcpA. While wild-type R. pseudosolanacearum strain Ps29 displayed attraction to 16 amino acids, the mcpA mutant showed no response to 12 of these amino acids and decreased responses to 4 amino acids. We constructed mcpA and mcpM deletion mutants of highly virulent R. pseudosolanacearum strain MAFF106611 to investigate the contribution of chemotaxis to l-malate and amino acids to tomato plant infection. Neither single mutant exhibited altered virulence for tomato plants when tested by root dip inoculation assays. In contrast, the mcpM mutant (but not the mcpA mutant) was significantly less infectious than the wild type when tested by a sand soak inoculation assay, which requires bacteria to locate and invade host roots from sand. Thus, McpM-mediated chemotaxis, possibly reflecting chemotaxis to l-malate, facilitates R. pseudosolanacearum motility to tomato roots in sand.

Chemotaxis of the nematode Caenorhabditis elegans toward cycloheximide and quinine hydrochloride.

We investigated the chemotaxis of the nematode Caenorhabditis elegans toward cycloheximide, denatonium benzoate, 6-n-propyl-2-thiouracil, and quinine hydrochloride. Interestingly, C. elegans was strongly attracted to cycloheximide, while avoiding quinine hydrochloride. This is the first report thus far to describe the chemotactic responses of C. elegans toward bitter tastants for humans.

Improved methane production from brown algae under high salinity by fed-batch acclimation

Here, a methanogenic microbial community was developed from marine sediments to have improved methane productivity from brown algae under high salinity. Fed-batch cultivation was conducted by adding dry seaweed at 1wt% total solid (TS) based on the liquid weight of the NaCl-containing sediment per round of cultivation. The methane production rate and level of salinity increased 8-fold and 1.6-fold, respectively, at the 10th round of cultivation. Moreover, the rate of methane production remained high, even at the 10th round of cultivation, with accumulation of salts derived from 10wt% TS of seaweed. The salinity of the 10th-round culture was equivalent to 5% NaCl. The improved methane production was attributed to enhanced acetoclastic methanogenesis because acetate became rapidly converted to methane during cultivation. The family Fusobacteriaceae and the genus Methanosaeta, the acetoclastic methanogen, predominated in bacteria and archaea, respectively, after the cultivation.

Enhancement of (R)-1,3-butanediol production by engineered Escherichia coli using a bioreactor system with strict regulation of overall oxygen transfer coefficient and pH

(R)-1,3-butanediol ((R)-1,3-BD) is an important substrate for the synthesis of industrial chemicals. Despite its large demand, a bioprocess for the efficient production of 1,3-BD from renewable resources has not been developed. We previously reported the construction of recombinant Escherichia coli that could efficiently produce (R)-1,3-BD from glucose. In this study, the fermentation conditions were optimized to further improve 1,3-BD production by the recombinant strain. A batch fermentation was performed with an optimized overall oxygen transfer coefficient (82.3 h−1) and pH (5.5); the 1,3-BD concentration reached 98.5 mM after 36 h with high-yield (0.444 mol (mol glucose)−1) and a high maximum production rate (3.63 mM h−1). In addition, a fed-batch fermentation enabled the recombinant strain to produce 174.8 mM 1,3-BD after 96 h cultivation with a yield of 0.372 mol (mol glucose)−1, a maximum production rate of 3.90 mM h−1, and a 98.6% enantiomeric excess (% ee) of (R)-1,3-BD. Graphical Abstract E. coli engineered to synthesize (R)-1,3-butanediol from glucose provides the possibility to produce industrial chemicals from renewable resources.

Degradation of chloroanilines by toluene dioxygenase from Pseudomonas putida T57

In this study, we investigated the ability of Pseudomonas putida toluene dioxygenase to oxidize chloroanilines. Toluene-induced P. putida T57 cells degraded 4-chloroaniline (4CA) more rapidly than toluene-non-induced cells, suggesting that toluene dioxygenase pathway was involved in 4CA degradation. Escherichia coli harboring P. putida T57 genes encoding toluene dioxygenase complex (todC1C2BA) showed 4CA degradation activity, demonstrating that toluene dioxygenase oxidizes 4CA. Thin-layer chromatography (TLC) and mass spectrometry (MS) analyses identified 4-chlorocatechol and 2-amino-5-chlorophenol as reaction products, suggesting that toluene dioxygenase catalyzes both 1,2- and 2,3-dioxygenation of 4CA. A plasmid containing the entire tod operon (todC1C2BADE) was introduced to P. putida T57 to enhance its ability to degrade 4CA. Resulting P. putida T57 (pHK-C1C2BADE) showed 250-fold higher 4CA degradation activity than P. putida T57 parental strain. P. putida T57 (pHK-C1C2BADE) degraded 2-chloroaniline (2CA), 3-chloroaniline (3CA), and 3,4-dichloroaniline (34DCA) as well as 4CA, but not 3,5-dichloroaniline (35DCA). The order of the degradation rate was: 4CA > 3CA > 2CA > 34DCA.