Rikizo Aono

Entry into and Release of Solvents by Escherichia coli in an Organic-Aqueous Two-Liquid-Phase System and Substrate Specificity of the AcrAB-TolC Solvent-Extruding Pump

Growth of Escherichia coli is inhibited upon exposure to a large volume of a harmful solvent, and there is an inverse correlation between the degree of inhibition and the log P(OW) of the solvent, where P(OW) is the partition coefficient measured for the partition equilibrium established between the n-octanol and water phases. The AcrAB-TolC efflux pump system is involved in maintaining intrinsic solvent resistance. We inspected the solvent resistance of delta acrAB and/or delta tolC mutants in the presence of a large volume of solvent. Both mutants were hypersensitive to weakly harmful solvents, such as nonane (log P(OW) = 5.5). The delta tolC mutant was more sensitive to nonane than the delta acrAB mutant. The solvent entered the E. coli cells rapidly. Entry of solvents with a log P(OW) higher than 4.4 was retarded in the parent cells, and the intracellular levels of these solvents were maintained at low levels. The delta tolC mutant accumulated n-nonane or decane (log P(OW) = 6. 0) more abundantly than the parent or the delta acrAB mutant. The AcrAB-TolC complex likely extrudes solvents with a log P(OW) in the range of 3.4 to 6.0 through a first-order reaction. The most favorable substrates for the efflux system were considered to be octane, heptane, and n-hexane.

Suppression of hypersensitivity of Escherichia coli acrB mutant to organic solvents by integrational activation of the acrEF operon with the IS1 or IS2 element.

The AcrAB-TolC efflux pump plays an intrinsic role in resistance to hydrophobic solvents in Escherichia coli. E. coli OST5500 is hypersensitive to solvents due to inactivation of the acrB gene by insertion of IS30. Suppressor mutants showing high solvent resistance were isolated from OST5500. These mutants produced high levels of AcrE and AcrF proteins, which were not produced in OST5500, and in each mutant an insertion sequence (IS1 or IS2) was found integrated upstream of the acrEF operon, coding for the two proteins. The suppressor mutants lost solvent resistance on inactivation of the acrEF operon. The solvent hypersensitivity of OST5500 was suppressed by introduction of the acrEF operon with IS1 or IS2 integrated upstream but not by introduction of the operon lacking the integrated IS. It was concluded that IS integration activated acrEF, resulting in functional complementation of the acrB mutation. The acrB mutation was also complemented by a plasmid containing acrF or acrEF under the control of Plac. The wild-type tolC gene was found to be essential for complementation of the acrB mutation by acrEF. Thus, it is concluded that in these cells a combination of the proteins AcrA, AcrF, and TolC or the proteins AcrE, AcrF, and TolC is functional in solvent efflux instead of the AcrAB-TolC efflux pump.

Appearance of a stress-response protein, phage-shock protein A, in Escherichia coli exposed to hydrophobic organic solvents.

A 28 kDa protein associated with the inner membrane was induced strongly in Escherichia coli K-12 cells grown in the presence of a hydrophobic organic solvent, n-hexane or cyclooctane. These organic solvents suppressed the growth (growth rate and yield) of E. coli. A partial amino acid sequence showed that this protein was the phage-shock protein PspA. PspA is known to be induced in E. coli cells under extreme stress conditions. The results suggest that E. coli cells are subject to strong stress in the presence of organic solvents. Introduction of a multi-copy plasmid vector carrying the psp operon into E. coli improved the survival frequency of cells exposed suddenly to n-hexane but not the growth rate of cells growing in the presence of n-hexane.

Thermophilic Alkaline Xylanase from Newly Isolated Alkaliphilic and Thermophilic Bacillus sp. Strain TAR-1

Alkaliphilic and thermophilic Bacillus sp. strain TAR-1, isolated from soil, produced a xylanase extracellularly. The xylanase was most active over a pH range of 5.0 to 9.5 at 50°C. Optimum temperatures of the crude xylanase preparation were 75°C at pH 7.0 and 70°C at pH 9.0. Zymogram analyses of the culture supernatant showed that the molecular mass of the xylanase was 40 kDa and the isoelectric point was pH 4.1. The predominant products of xylan hydrolysate were xylobiose, xylotriose, and higher oligosaccharides, indicating that the enzyme was an endoxylanase. Production of the thermophilic alkaline xylanase was induced by xylan and xylose, but was repressed in the presence of glucose.

Natronococcus amylolyticus sp. nov., a Haloalkaliphilic Archaeon

The alpha-amylase-producing haloalkaliphilic archaeon Natronococcus sp. strain Ah-36T (T = type strain) was isolated previously from a Kenyan soda lake, Lake Magadi. Most cells of strain Ah-36T occurred in irregular clusters, and the colonies were orange-red. The polar lipids of this organism were composed of C20, C20 and C20, C25 derivatives of phosphatidylglycerol and phosphatidylglycerophosphate. Phosphatidylglycero-(cyclo-) phosphate, which is characteristic of Natronococcus occultus, was not detected. The complete nucleotide sequence of the 16S rRNA gene revealed that the closest relative of strain Ah-36T is N. occultus ATCC43101T (level of similarity, 96.4%), an extremely halophilic archaeon. However, strain Ah-36T did not exhibit a significant level of DNA homology to N. occultus ATCC43101T, which represents the only previously described species in the genus Natronococcus. We describe a new species for strain Ah-36T, for which we propose the name Natronococcus amylolyticus.

Cloning and sequencing of the gene encoding the cell surface glycoprotein of Haloarcula japonica strain TR-1.

Abstract The triangular disk-shaped halophilic archaeon Haloarcula japonica strain TR-1 has a glycoprotein on its cell surface. The complete gene encoding the cell surface glycoprotein (CSG) was cloned and sequenced. The gene has an open reading frame of 2586 bp, and a potential archaeal promoter sequence approximately 150 bp upstream of the ATG initiation codon. The mature CSG is composed of 828 amino acids and is preceded by a signal sequence of 34 amino acid residues. A hydropathy analysis showed a hydrophobic stretch at the C-terminus, that probably serves as a transmembrane domain. The amino acid sequence of the Ha. japonica CSG showed 52.1% and 43.2% identities to those from the Halobacterium halobium and Haloferax volcanii CSGs, respectively. Five potential N-glycosylation sites were found in the mature Ha. japonica CSG, sites that were distinctly different from those in Hb. halobium and Hf. volcanii. The Ha. japonica CSG gene was expressed in Escherichia coli.

Isolation of Paenibacillus illinoisensis That Produces Cyclodextrin Glucanotransferase Resistant to Organic Solvents

A bacterium that secreted cyclodextrin glucanotransferase (CGTase) in a medium overlaid with n-hexane was isolated and identified as Paenibacillus illinoisensis strain ST-12 K. The CGTase of the strain was purified from the culture supernatant. The molecular mass was 70 kDa. The enzyme was stable at pH 6 to 10 and active at pH 5.0 to 8.0. The optimum temperature at pH 7.0 was 65°C in the presence of 5 mM CaCl2. The enzyme produced mainly β-cyclodextrin. The total yield of α-, β-, and γ- cyclodextrins was increased 1.4-fold by the addition of ethanol. In particular, the yield of β-cyclodextrins in the presence of 10% (vol/vol) ethanol was 1.6-fold that without ethanol. The CGTase was stable and active in the presence of large amounts of various organic solvents.

Biodegradation of indole at high concentration by persolvent fermentation with Pseudomonas sp. ST-200

AbstractPseudomonas sp. strain ST-200 grew on indole as a sole carbon source. The minimal inhibitory concentration of indole was 0.3 mg/ml for ST-200. However, ST-200 grew in a persolvent fermentation system containing a large amount of indole (a medium containing 20% by vol. diphenylmethane and 4 mg/ml indole), because most of the indole was partitioned in the organic solvent layer. When the organism was grown in the medium containing indole at 1 mg/ml in the presence of diphenylmethane, more than 98% of the indole was consumed after 48 h. Isatic acid (0.4 mg/ml) and isatin (0.03 mg/ml) were produced as the metabolites in the aqueous medium layer.

A high cell wall negative charge is necessary for the growth of the alkaliphile Bacillus lentus C-125 at elevated pH

Summary: The structural components in cell walls of three mutants of a facultative alkaliphile, Bacillus lentus C-125, defective in certain cell-wall components were characterized in detail. The cell walls of the wild-type C-125 were thick and increased in thickness when grown at high pH. Electron microscopy showed that triple layers developed when the bacteria were grown in an alkaline environment. In contrast, cell walls of teichuronopeptide (TUP)-defective mutants consisted of a single layer. For both the wild-type and mutants, the cell-wall concentrations of the acidic structural polymers teichuronic acid and TUP increased with respect to peptidoglycan as culture pH increased. For all four strains, the anion content of their cell walls was the greatest at high pH. The cell-wall density of the negatively charged compounds (uronic acids plus L-glutamic acid) was calculated as about 3 and 9 equivalents (I cell wall region)−1 for C-125 cells grown at pH 7 and 10, respectively. At high pH, the specific growth rates of the two TUP-defective mutants were much lower than those of the wild-type. It is concluded that increased levels of acidic polymers in the cell walls of alkaliphilic bacteria may be a necessary adaptation for growth at elevated pH.

Chemical Composition of Cell Walls of Alkalophilic Strains of Bacillus

SUMMARY: Cell walls of 10 alkalophilic Bacillus strains were prepared by inactivation of autolytic enzymes with SDS, disruption with a sonic oscillator, trypsin digestion and washing with SDS. The walls were composed of peptidoglycan and acidic compounds. The peptidoglycan was similar in composition to that of B. subtilis. After hydrolysis, the acidic compounds detected were glucuronic acid, glutamic acid and aspartic acid. The strains tested could be divided into three groups as follows. Group 1, the glucuronic acid and hexosamine contents were high; no growth was observed at pH 7.0; Na+ was essential for their growth. Group 2, large amounts of glutamic acid, aspartic acid and glucuronic acid were detected. Growth at higher pH values increased the content of acidic compounds; growth was observed at pH 7.0; Na+ was essential for their growth. Group 3, no remarkable difference was detected in the chemical components in comparison with B. subtilis; growth was observed in the presence of Na+ or K+ at pH 7.0 and 10.2.