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A constitutively active mutant of histidine kinase sensor EvgS was found to confer multi-drug resistance (MDR) to an acrA-deficient Escherichia coil, indicating the relationship between the two-component system EvgAS and the expression of the MDR system. The observed MDR also depended on an outer-membrane channel, TolC. Microarray and S1 mapping assays indicated that, in the presence of this constitutive mutant ErgS, the level of transcription increased for some MDR genes, including the drug efflux genes emrKY, yhiUV, acrAB, mdfA and tolC. Transcription in vitro of emrK increased by the addition of phosphorylated EvgA. Transcription activation of tolC by the activated EvgS was, however, dependent on both EvgAS and PhoPQ ([Mg.sup.2+]-responsive two-component system), in agreement with the presence of the binding site (PhoP box) for the regulator PhoP in the tolC promoter region. Transcription in vitro of yhiUV also appears to require an as-yet-unidentified additional transcriptional factor besides EvgA. Taken together we propose that the expression of the MDR system is under a complex regulatory network, including the phosphorylated EvgA serving as the master regulator.

Results show that integration of insertion elements IS1 or IS2 into the acrEF operon activates the operon, which suppresses the solvent-hypersensitive phenotype due to acrB mutation in Escherichia coli. Furthermore, in the activated cells, a combination of proteins mediate solvent efflux instead of the conventional AcrAB-TolC efflux pump.

Results suggest that the AcrAB-TolC efflux pump extrudes solvents in the range of 3.4 to 6.0 of log P(sub)OW and at higher than 4.4, the solvents are retarded. Data indicate that octane, heptane, and n-hexane are preferred substrates for the efflux system.

The extracellular cholesterol oxidase of Pseudomonas sp. strain ST-200 was purified to investigate the role of this activity in ST-200 strain-mediated cholesterol oxidation in an organic solvent. The oxidase effectively oxidized cholesterol in the organic solvents but was not as effective in oxidizing cholesterol in the assay solution. It is speculated that oxidase had a mediating effect in the early stages of the cholesterol conversion reactions in the ST-200 culture.

Research was conducted to analyze the outer membrane proteins of various organic-solvent tolerant Escherichia coli mutants. Results indicate that mutants with improved tolerance levels were characterized by high levels of the inner membrane protein AcrA and the outer membrane protein TolC. The tolC gene, which is characterized by a mar-rob-box sequence, was found to be important in maintaining E. coli's organic solvent tolerance.

A 28 kDa Escherichia coli protein associated with the inner membrane was induced strongly in K-12 cells grown in the presence of a hydrophobic organic solvent. The solvent suppressed the growth of E. coli. A partial amino acid sequence revealed that this protein was the phage-shock protein PspA. Findings indicated that E. coli cells are subject to strong stress in the presence of organic solvents.

Microbiological research shows that genetically-modified bacteria with raised tolerance to organic solvents have an increased tolerance to water. Cell surface studies using mutants of the bacteria Escherichia coli K-12 demonstrate reduced hydrophobicity caused by raised lipopolysaccharide content. Variations to the organic solvent-tolerant microorganisms's lipopolysaccharide, OmpF and peptidoglycan-binding lipoprotein are thought to be responsible for increased organic solvent tolerance.

An innovative technique has been developed to measure the cytoplasmic pH of the facultative alkaliphilic strain Bacillus lentus C-125 using a pH-sensitive fluorescent probe. The technique is used to demonstrate that culture pH affects cytoplasmic pH activity. The fluorescent probe 2',7'-bis-(carboxyethyl)-5 (and-6-)-carboxyfluorescein was used to identify cytoplasmic pH changes under a range of pH environments.

Overexpression of the soxS or robA gene have been found to induce in several Escherichia coli strains the acquisition of higher organic solvent tolerance and also enhanced resistance to a number of antibiotics. The marRAB genes were isolated from a Kohara lambda phage clone and cyclohexane-tolerant mutant strain OST3408. A substitution of serine for arginine at position 73 was observed in the coding region of marR of OST3408 and named marR08. marR08 was found to be responsible for the cyclohexane-tolerant phenotype.

The anions and acidic components of the cell wall in the alkaliphile Bacillus lentus C-125 and its mutants increase with an increase in the pH of the culture medium. High pH increases the synthesis of the acidic teichuronic acid and teichuronopeptide (TUP). The growth and increase in anionic content of the TUP-deficient mutants is less than the wild-type cells. The cell walls are thicker at higher pH. An increase in negative charge of the cell wall decreases the pH by restricting the hydrogen ions to the cell wall.

Standard techniques help purify the extracellular 28- and 42-kDa beta-1,3-glucanases produced by Bacillus circulans IAM1165. A comparative study of the characteristics of these enzymes with previously isolated 91-kDa enzymes reveals that the 42-kDa enzyme is the most effective in lysing fungal cell walls. The 28- and 42-kDa beta-1, 3-glucanases exhibit similar enzymatic characteristics and are the members of endo type 1,3-beta-D-glucan glucanohydrolases. At 60 degrees Celsius these enzymes exhibit optimum activity.

An interaction between lysozyme and facultative alkaliphile Bacillus sp. C-125 facilitates the preparation of protoplasts. The presence of polyethylene glycol enables the efficient fusion of the protoplasts and regeneration of parental protoplasts from fusants occurs on solid medium having two antibiotics, nalidixic acid and streptomycin. Regeneration is faster on non-selective medium than on selective medium. Protoplast fusion permits recombination in the alkaliphile Bacillus sp. C-125 and enables the pooling of the different parental chromosomes.

Use of Southern hybridization technique with an NH2-terminal oligonucleotide probe to study the alpha-amylase gene of Natronococcus sp. strain Ah-36 reveals the presence of a 43-amino acid signal peptide. The amylase gene in Haloferax volcanii is expressed. The Natronococcus amylase signal peptide shares sequence identity with a halophilic archaeon protease, halolysin.

Pseudomonas sp. strain ST-200 dehydrogenates and oxygenates cholesterol in a two-phase water-organic solvent system to reduce cholesterol content in the system. The oxygenation induces the formation of two major compounds, (6)beta-hydroxycholest-4-en-3-one and cholest-4-ene-3,6-dione, and three minor compounds. The ST-200 strain uses a ketone or hydroxyl group to oxidize the C-3 and C-6 cholesterol positions.

Bacillus sp. strain 41M-1 is an alkaliphilic soil bacterium which produces large amounts of multiple xylanases extracellularly. The predominant xylanase from this bacterium was purified and characterized. The results showed that the xylanase, designated xylanase J, is an endoxylanase with a molecular mass of 36-kDa and is most active at a pH of 9.0 and temperature of 50 degrees celsius. Partial sequence analysis showed high N-terminal similarity to xylanases of Bacillus pumilus and Clostridium acetobutylicum.

Bacillus circulans IAM1165 is a fungus that produces cellulytic enzymes. These extracellular enzymes, beta-1,3-glucanases, produce two enzymes that have different chromatographic behaviors and molecular masses.One of these enzymes was isolated from the culture supernatant and partially characterized in this study. It was detected by immunoblotting method and purified by hydrophobic chromatography which showed that the enzyme was an anionic protein. This 87-kilodalton endo-beta-1,3-glucanase randomly cleaved with laminarin as a substrate at an optimum temperature of 70 degrees Centigrade.

The organic solvent tolerance of Escherichia coli was measured under conditions in which OmpF levels have been controlled. Genetically engineered E. coli mutants have previously been reported to tolerate high levels of organic solvents. It was speculated that the organic solvent molecules could pass through the OmpF porin protein. However, the results indicate that this hypothesis is not true. The solvent tolerance of E. coli has been found not to be dependent upon OmpF levels in the membrane.

Cloning and analysis of the ostA gene from Escherichia coli reveal that it defines the level of n-hexane tolerance in E. coli. The conversion of a JA300-based n-hexane-sensitive strain, OST4251, to an n-hexane-tolerant phenotype by using an ostA gene-derived DNA indicates that the gene complements the n-hexane-sensitive OST4251 phenotype. The ostA gene cloned from the Hex(r) strain JA300 exhibits dominant characteristics while that present on the OST4251 chromosome exhibits recessive characteristics.

Research has been conducted on Escherichia coli resistance to n-hexane. The authors suggest that the gene product, an 87 kDa minor protein associated with outer membrane, reduces n-hexane influx.