Alkaliphiles: some applications of their products for biotechnology.

The term "alkaliphile" is used for microorganisms that grow optimally or very well at pH values above 9 but cannot grow or grow only slowly at the near-neutral pH value of 6.5. Alkaliphiles include prokaryotes, eukaryotes, and archaea. Many different taxa are represented among the alkaliphiles, and some of these have been proposed as new taxa. Alkaliphiles can be isolated from normal environments such as garden soil, although viable counts of alkaliphiles are higher in samples from alkaline environments. The cell surface may play a key role in keeping the intracellular pH value in the range between 7 and 8.5, allowing alkaliphiles to thrive in alkaline environments, although adaptation mechanisms have not yet been clarified. Alkaliphiles have made a great impact in industrial applications. Biological detergents contain alkaline enzymes, such as alkaline cellulases and/or alkaline proteases, that have been produced from alkaliphiles. The current proportion of total world enzyme production destined for the laundry detergent market exceeds 60%. Another important application is the industrial production of cyclodextrin by alkaline cyclomaltodextrin glucanotransferase. This enzyme has reduced the production cost and paved the way for cyclodextrin use in large quantities in foodstuffs, chemicals, and pharmaceuticals. It has also been reported that alkali-treated wood pulp could be biologically bleached by xylanases produced by alkaliphiles. Other applications of various aspects of alkaliphiles are also discussed.

Tryptophan permease gene TAT2 confers high-pressure growth in Saccharomyces cerevisiae.

Hydrostatic pressure in the range of 15 to 25 MPa was found to cause arrest of the cell cycle in G(1) phase in an exponentially growing culture of Saccharomyces cerevisiae, whereas a pressure of 50 MPa did not. We found that a plasmid carrying the TAT2 gene, which encodes a high-affinity tryptophan permease, enabled the cells to grow under conditions of pressure in the range of 15 to 25 MPa. Additionally, cells expressing the Tat2 protein at high levels became endowed with the ability to grow under low-temperature conditions at 10 or 15 degrees C as well as at high pressure. Hydrostatic pressure significantly inhibited tryptophan uptake into the cells, and the Tat2 protein level was down-regulated by high pressure. The activation volume associated with tryptophan uptake was found to be a large positive value, 46.2 +/- 3.85 ml/mol, indicating that there was a net volume increase in a rate-limiting step in tryptophan import. The results showing cell cycle arrest in G(1) phase and down-regulation of the Tat2 protein seem to be similar to those observed upon treatment of cells with the immunosuppressive drug rapamycin. Although rapamycin treatment elicited the rapid dephosphorylation of Npr1 and induction of Gap1 expression, hydrostatic pressure did not affect the phosphorylation state of Npr1 and it decreased the level of Gap1 protein, suggesting that the pressure-sensing pathway may be independent of Npr1 function. Here we describe high-pressure sensing in yeast in comparison with the TOR-signaling pathway and discuss an important factor involved in adaptation of organisms to high-pressure environments.

Barophiles: deep-sea microorganisms adapted to an extreme environment.

The deep-sea environment is characterized by high pressure and low temperature but in the vicinity of hydrothermal vents regions of extremely high temperature exist. Deep-sea microorganisms have specially adapted features that enable them to live and grow in this extreme environment. Recent research on the physiology and molecular biology of deep-sea barophilic bacteria has identified pressure-regulated operons and shown that microbial growth is influenced by the relationship between temperature and pressure in the deep-sea environment.

Complete genome sequence of the alkaliphilic bacterium Bacillus halodurans and genomic sequence comparison with Bacillus subtilis.

The 4 202 353 bp genome of the alkaliphilic bacterium Bacillus halodurans C-125 contains 4066 predicted protein coding sequences (CDSs), 2141 (52.7%) of which have functional assignments, 1182 (29%) of which are conserved CDSs with unknown function and 743 (18. 3%) of which have no match to any protein database. Among the total CDSs, 8.8% match sequences of proteins found only in Bacillus subtilis and 66.7% are widely conserved in comparison with the proteins of various organisms, including B.subtilis. The B. halodurans genome contains 112 transposase genes, indicating that transposases have played an important evolutionary role in horizontal gene transfer and also in internal genetic rearrangement in the genome. Strain C-125 lacks some of the necessary genes for competence, such as comS, srfA and rapC, supporting the fact that competence has not been demonstrated experimentally in C-125. There is no paralog of tupA, encoding teichuronopeptide, which contributes to alkaliphily, in the C-125 genome and an ortholog of tupA cannot be found in the B.subtilis genome. Out of 11 sigma factors which belong to the extracytoplasmic function family, 10 are unique to B. halodurans, suggesting that they may have a role in the special mechanism of adaptation to an alkaline environment.

Pressure-regulated metabolism in microorganisms.

There has been a renewal of interest in the survival strategies employed by deep-sea, high-pressure-adapted (piezophilic) microorganisms as well as in the effects of high pressure on mesophilic, 1-atmosphere-pressure-adapted microorganisms. This is partly the result of a greater appreciation of the adaptations of microorganisms to life in extreme environments and partly the result of the development of new techniques for examining physiological and molecular processes as a function of pressure.

Production of 1, 3-beta-glucanase by Bacillus No. 221. An alkalophilic microorganism.

A 1, 3-beta-glucanase of Bacillus No. 221 has been extensively purified by a DEAE-cellulose column followed by a Sephadex G-75 gel filtration, and crystallized in ammonium sulfate solution. The crystalline enzyme is homogenous on the basis of polyacrylamide gel electrophoresis, sedimentation in ultracentrifuge (3.2 S), Ampholine electrofocusing (pI=4.1) and dodecylsulfate-polyacrylamide gel electrophoresis (Mr=36 000). The enzyme has an optimum pH for enzyme action at 8.5 which is higher than those of other 1, 3-beta-glucanases so far reported. The enzyme is very thermostable; about 90% of activity remains after being heated at 70 degrees C for 10 min, and no effect of Ca-2's obversed. The enzyme does not hydrolyse laminaritriose, but hydrolyses laminaritetraose, and yields glucose and laminaritriose. The enzyme splits laminaran at random and yields glucose, laminaribiose, laminaritriose and higher oligosaccharides. From these results, this enzyme is a type of endo-1, 3-beta-glucanase.

Purification and characterization of extracellular polyamine oxidase produced by Penicillium sp. no. PO-1.

An extracellular polyamine oxidase produced by Penicillium sp. No. PO-1 was completely purified using the chromatofocusing method with a very high yield (93%) of the activity. The enzyme was composed of two identical subunits (Mr 64 000) and contained FAD. The optimal pH for activity was approx. 4.0. The enzyme oxidized spermidine and spermine. Km and Vmax values for spermidine were respectively 8.2 microM and 16.4 mumol H2O2/mg protein per min. Corresponding values for spermine were 5.3 microM and 13.3 mumol H2O2/mg protein per min. The enzyme attacked the secondary amino group of spermidine and spermine, and produced putrescine, 3-aminopropionaldehyde and H2O2. The enzyme activity was completely inhibited by phenylhydrazine. However, sulfhydryl reagents showed no effect on the activity. It is expected that the enzyme will be useful in determining the amount of polyamine in body fluids.

Purification and properties of NAD+-dependent maltose dehydrogenase produced by alkalophilic Corynebacterium sp. No. 93-1.

NAD+-dependent maltose dehydrogenase was purified about 250-fold from the cell free extract of an alkalophilic Corynebacterium sp. No. 93-1. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis, sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and ultracentrifugation. The molecular weight of the enzyme was determined to be 40 000 +/- 2000 by gel filtration and SDS-polyacrylamide gel electrophoresis. The enzyme appeared to be a single peptide chain. The isoelectric point was pH 4.50. The optimal pH was 10.2. The enzyme was stable over the range of pH 6 to 10. NAD+-dependent maltose dehydrogenase showed very wide substrate specificity on monosaccharides, disaccharides and trisaccharides. Among these substrates, maltose was the most reactive. Also, the enzyme showed oxidative activity on maltotetraose and maltopentaose. The Km values at pH 10 were 2.1 mM for maltose and 0.15 mM for NAD+. It was conjectured that the primary product of this reaction was maltono-delta-lactone and its was hydrolyzed non-enzymatically to maltobionic acid. p-Chloromercuribenzoic acid, Hg2+ and Ag2+ completely inhibited the activity, and HADH also showed competitive inhibition on the activity.

Molecular cloning of the gene encoding RNA polymerase alpha subunit from deep-sea barophilic bacterium.

We have cloned the gene encoding RNA polymerase alpha subunit from a gene library of deep-sea barophilic bacterium strain DB6705. The clone contains the genes for ribosomal protein S4, RNA polymerase subunit alpha and ribosomal protein L17 in this order. The alpha gene has 328 amino acids and a molecular mass of 36 100 Da with 86.9% identity to Escherichia coli alpha gene. Differences between the two sequences were mainly in the N-terminal portion of the alpha subunit, which is involved in the assembly of the core RNA polymerase; while the 87 C-terminal residues, which form a region involved in contact with some positive regulators and rrnB P1 promoter region called UP-element, were identical in the both strain. Plasmid encoding the alpha subunit with an N-terminal hexahistidine tag was constructed. Using the plasmid, the recombinant fusion alpha subunit was overexpressed and successfully purified to near homogeneity.

Purification and some properties of alkaline pullulanase from a strain of bacillus no. 202-1, an alkalophilic microorganism.

Pullulanase (pullulan 6-glucanohydrolase EC 3.2.1.41) was purified about 290-fold from the culture fluid of Bacillus No. 202-1 by DEAE-cellulose adsorption, acetone fractionation, (NH4) 2SO4 precipitation and DEAE--cellulose column chromatography followed by Sephadex G-200 molecular sieve chromatography. The enzyme gave a single band of protein by disc polyacrylamide gel electrophoresis. The molecular weight was estimated as 92 000 by sodium dodecyl sulfate gel electrophoresis. The isolectric point was lower than pH 2.5. The optimum pH for enzyme action was about 8.5-9.0. The action of the enzyme on amylopectin and glycogen resulted in increase in the iodine coloration of 85% and 70%, respectively. The enzyme completely hydrolyzed 1,6-alpha-glucosidic linkages in amylopectin, glycogen and pullulan.

Oxidation of acetylpolyamines by extracellular polyamine oxidase produced by Penicillium sp. No. PO-1.

The oxidation of acetylpolyamines by an extracellular polyamine oxidase of Penicillium sp. No. PO-1 was investigated. The optimal pH value for oxidation of acetylpolyamines was 6.0. The purified enzyme oxidized spermidine, spermine, N1-acetylspermidine, N8-acetylspermidine, N1,8-diacetylspermidine, N1-acetylspermine and N1,12-diacetylspermine. The relative velocities for oxidation of acetylpolyamines were lower than those of spermidine and spermine. The Km values for oxidation of acetylpolyamines were higher than those of spermidine and spermine. The enzyme split N1-acetylspermidine and N8-acetylspermidine at the same position of the linkage as in spermidine oxidation. N1-Acetylspermine was changed to N1-acetylspermidine. This oxidation mechanism was different from that of rat liver polyamine oxidase. N1-Acetylspermine inhibited the oxidation of spermine. Putrescine, N8-acetylspermidine and N1,12-diacetylspermine also inhibited the N1-acetylspermidine oxidation by the enzyme.

Cloning and characterization of the gene encoding RNA polymerase sigma factor sigma(54) of deep-sea piezophilic Shewanella violacea.

We have recently reported that a sigma(54)-like factor recognizes a DNA element, designated as region A, upstream of a pressure-regulated operon in piezophilic Shewanella violacea strain DSS12 (Nakasone et al., FEMS Microbiology Lett. 176 (1999) 351-356). In this study, we isolated and characterized the rpoN gene of this piezophilic bacterium. The rpoN gene was found to encode a putative protein consisting of 492 amino acid residues with a predicted molecular mass of 55359 Da. Significant homology was evident comparing the rpoN sequence of S. violacea with that of Escherichia coli (62.8% identity), Vibrio anguillarum (61.7% identity) and Pseudomonas putida (57.0% identity). The DNA-binding domain at the C-terminus of sigma(54) is well conserved in the case of the S. violacea rpoN gene product and the helix-turn-helix motif and the RpoN box are also present. In addition, the conserved glutamine-rich domain is present at the N-terminus. sigma(54) in S. violacea was expressed at a relatively constant level under various growth conditions as determined by both primer extension and Western blotting analyses. By means of a recombinant plasmid, a hexahistidine-tagged derivative of the sigma(54) from strain DSS12 was overexpressed in Escherichia coli and purified to near homogeneity. An electrophoretic mobility shift assay demonstrated that the purified sigma(54) protein specifically recognizes region A in the above-mentioned pressure-regulated operon.

Genetic diversity of archaea in deep-sea hydrothermal vent environments.

Molecular phylogenetic analysis of naturally occurring archaeal communities in deep-sea hydrothermal vent environments was carried out by PCR-mediated small subunit rRNA gene (SSU rDNA) sequencing. As determined through partial sequencing of rDNA clones amplified with archaea-specific primers, the archaeal populations in deep-sea hydrothermal vent environments showed a great genetic diversity, and most members of these populations appeared to be uncultivated and unidentified organisms. In the phylogenetic analysis, a number of rDNA sequences obtained from deep-sea hydrothermal vents were placed in deep lineages of the crenarchaeotic phylum prior to the divergence of cultivated thermophilic members of the crenarchaeota or between thermophilic members of the euryarchaeota and members of the methanogen-halophile clade. Whole cell in situ hybridization analysis suggested that some microorganisms of novel phylotypes predicted by molecular phylogenetic analysis were likely present in deep-sea hydrothermal vent environments. These findings expand our view of the genetic diversity of archaea in deep-sea hydrothermal vent environments and of the phylogenetic organization of archaea.

The biotechnological potential of piezophiles.

Microorganisms that prefer high-pressure conditions are termed piezophiles (previously termed barophiles). The molecular basis of piezophily is now being investigated extensively focusing on aspects of gene regulation and the function of certain proteins in deep-sea isolates. Little attention has been paid, however, to the potential biotechnological applications of piezophiles compared with other extremophiles. Based on the fundamental knowledge available, we will try to answer the following questions: How can we exploit the biotechnological potential of piezophiles? What can be understood by the application of high-pressure in biological systems?

Isolating and characterizing deep-sea marine microorganisms.

We have isolated several microorganisms that are adapted to living in the extremes of the deep-sea environment. They include barophilic bacteria, which are able to grow at high hydrostatic pressure, but that are unable to grow at atmospheric pressure, and organic-solvent-tolerant bacteria, which are able to grow in the presence of toxic organic solvents such as toluene or benzene. In this review, we describe how to isolate such extremophiles, and we outline the characteristics of several strains that have been recovered from the deep-sea environment.

The third cellulase of alkalophilic Bacillus sp. strain N-4: evolutionary relationships within the cel gene family.

The third cellulase gene (celC) of Bacillus sp. strain N-4 was cloned in plasmid pBR322 and was located within a 5.5-kb HindIII fragment. The cellulase encoded by this fragment had an Mr of about 100,000 and showed optimum activity around pH 9. These properties were different from those of the enzymes encoded by the celA and celB genes of the same organism. The amino acid sequence deduced from the nucleotide sequence was found to be highly homologous to the CEL-F enzyme from Bacillus sp. strain No. 1139 [Fukumori et al., J. Gen. Microbiol. 132 (1986) 2329-2335]. An evolutionary relationship observed among the four cellulases of alkalophilic Bacillus strains and that of Bacillus subtilis endoglucanase suggested that ancestral genes for alkaline and neutral cellulases diverged early in the evolution of these enzymes.

Production of mitogen-free immune interferon and T-cell growth factor by human peripheral blood lymphocytes induced with biotin-labeled staphylococcal enterotoxin A.

A method for the facile removal of mitogens or inducers of lymphokine production from cell culture medium of stimulated cells is described. The technique is based on the covalent attachment of biotin to mitogen or inducer and the removal of the biotinylated products from stimulated cell culture medium using immobilized avidin. Using this procedure, biotin-labeled staphylococcal enterotoxin A (SEA-B) was shown not to differ significantly from unmodified SEA in its capacity to stimulate mitogenesis and induce production of immune interferon (IFN) and T-cell growth factor (TCGF) in cultures of human mononuclear cells from peripheral blood. SEA-B was also shown not to differ from SEA in its binding to SEA antibodies. Results of mitogenicity studies and competitive radioimmune assay (RIA) measurements indicate that SEA-B is essentially completely removed from stimulated cell culture medium by absorption with avidin coupled to Sepharose 4B.

High pressure influences on gene and protein expression.

Elevated hydrostatic pressure can influence gene and protein expression in both 1 atmosphere-adapted and high pressure-adapted microorganisms. Here we review experiments documenting these effects and describe their significance towards understanding the molecular bases of life in deep-sea high pressure environments.

Successful repair of double-outlet right ventricle with bilateral conus, 1-transposition of great arteries (S,D,L), and subpulmonary ventricular septal defect.

Surgical correction was carried out successfully in a severely cyanotic 3-year-old Japanese girl who had a very rare type of double-outlet right ventricle. The malformation was associated with bilateral conus, 1-transposition of the great arteries, and subpulmonary ventricular septal defect without significant pulmonary stenosis in situs solitus. A large amount of subaortic conal musculature which separated the aortic valve from the subpulmonary ventricular septal defect was removed, as was the anterior rim of the ventricular septal defect. A tunnel, constructed with a woven Teflon prosthesis, was inserted in such a manner as to direct blood from the left ventricle through the defect and out to the aorta. The pulmonary outflow tract was reconstructed with a Teflon patch lined with pericardium. The patient's postoperative recovery was uneventful, and she was doing well 3 months postoperatively. To our knowledge, no identical case with a similar type of surgical correction has previously been reported.

Lung pathology in infants with severe pulmonary hypertension and cardiac disease.

Lung biopsy specimens were taken from 39 infants younger than 12 months of age with congenital heart disease and severe pulmonary hypertension (Pp/Ps greater than or equal to 0.75) accompanied by respiratory distress. The pathological change in lung specimens and clinical courses were compared. These 39 infants underwent surgical treatment of patent ductus arteriosus (PDA), seven patients; ventricular septal defect (VSD), 13 patients; and complex heart anomaly, 19 patients. The common pathological findings of the lung specimens taken from these infants were lymphoid cellular infiltration and thickening of the alveolar septum, which we have called "septitis" in the present study. In most cases pulmonary vascular obstructive change was within Grade 2 of the Health-Edwards criteria. Septitis was classified into three categories: mild, moderate, and severe. Only three of the 19 infants with severe septitis survived postoperatively, whereas 10 of the 12 infants with moderate septitis and all eight with mild septitis could be successfully weaned. The cause of septitis remains unidentified. We have found the patient's age and pulmonary hypertension to be closely related to the grade of septitis in this study. Septitis plays a much more important role than pulmonary vascular obstructive change in the prognosis of pulmonary hypertensive heart disease in early infancy.