Identification of facultatively alkaliphilic Bacillus sp. strain YN-2000 and its fatty acid composition and cell-surface aspects depending on culture pH

Facultatively alkaliphilic Bacillus sp. strain YN-2000 was isolated from an indigo ball. Although the strain has been extensively investigated as a representative strain of alkaliphilic bacillus, its taxonomic position is not yet known. Morphological, biochemical, and physiological characteristics and chemotaxonomic properties indicated that the strain was closely related to Bacillus cohnii; this was confirmed by the high homology of the 16S rRNA sequence and the construction of a phylogenetic tree on the basis of the 16S rRNA sequence and DNA–DNA relatedness data. Strain YN-2000 contained a larger amount of unsaturated fatty acids compared with Bacillus subtilis and the obligate alkaliphile, Bacillus alcalophilus, regardless of its culture pH. When the cells were grown at pH 10, the unsaturated fatty acid content and anteiso-/iso-branched fatty acid ratio became lower than those at pH 7. This result suggests that membrane fluidity decreases when the cells are grown at pH 10 compared to those of pH 7. In the cells of strain YN-2000 grown at pH 10, the cell-surface aspect was rougher, the cell shape was longer, and the cell-surface layer was thicker compared with those of the cells grown at pH 7. The cell-surface structural change might be related to adaptation to an alkaline environment. Received: April 6, 2000 / Accepted: May 8, 2000     

Leaching of copper ore of the Udokanskoe deposit at low temperatures by an association of acidophilic chemolithotrophic microorganisms

Pure cultures of indigenous microorganisms Acidithiobacillus ferrooxidans strain TFUd, Leptospirillum ferrooxidans strain LUd, and Sulfobacillus thermotolerans strain SUd have been isolated from the oxidation zone of sulfide copper ore of the Udokanskoe deposit. Regimes of bacterial-chemical leaching of ore have been studied over a temperature range from −10 to +20°C. Effects of pH, temperature, and the presence of microorganisms on the extraction of copper have been shown. Bacterial leaching has been detected only at positive values of temperature, and has been much more active at +20 than at +4°C. The process of leaching was more active when the ore contained more hydrophilic and oxidized minerals. The possibility of copper ore leaching of the Udokanskoe deposit using sulfuric acid with pH 0.4 at negative values of temperature and applying acidophilic chemolithotrophic microorganisms at positive values of temperature and low pH values was shown.     

Response of Rhizobium leguminosarum bv phaseoli to acidity

Soil acidity constraints grain legume production in tropical soils, both limiting Rhizobium survival and reducing nodulation. Strains of rhizobia with greater tolerance to hydrogen-ion concentration have been identified, but the basis for strain differences in pH tolerance has yet to be determined. In this study, strains of Rhizobium leguminosarum by phaseoli which differed in their tolerance to acidity were exposed to acid pH, then cell levels of potassium and calcium determined, and specific ‘acid-shock’ proteins identified. Lowering the external pH to 4.6–4.7 resulted in an immediate efflux of calcium from the cell of both acid tolerant and sensitive bean strains. Change in cell potassium levels on exposure to acidity varied with the strain. Strain UMR 1899 and an acid-sensitive mutant derived from it maintained high cytoplasmic potassium at acid pH, whereas an acid-sensitive strain UMR 1632 underwent a marked decline in cell potassium at pH 4.6. Exposure of these strains to pH 4.5 in the presence of [³⁵S]-labeled methionine enhanced production of a number of proteins, while synthesis of other proteins at this pH was significantly reduced. Differences in banding pattern were also evident between UMR1899 and the Tn5-induced pH-sensitive mutant UMR5005 derived from it, and between cells grown in the presence and absence of calcium and phosphorus.     

Transport kinetics of maltotriose in strains ofSaccharomyces

Summary Maltotriose transport was studied in two brewer's yeast strains, an ale strain 3001 and a lager strain 3021, using laboratory-synthesized¹⁴C-maltotriose. The maltotriose transport systems preferred a lower pH (pH 4.3) to a higher pH (pH 6.6). Two maltotriose transport affinity systems have been indentified. The high affinity system hasK _m values of 1.3 mM for strain 3021 and 1.4 mM for strain 3001. The low affinity competitively inhibited by maltose and glucose withK _i values of 58 mM and 177 mM. respectively, for strain 3021, and 55 mM and 147 mM, respectively, for strain 3001. Cells grown in maltotriose and maltose had higher maltotriose and maltose transport rates, and cells grown in glucose had lower maltortriose and maltose transport rates. Early-logarithmic phase cells transported glucose faster than either maltose or maltotriose. Cells harvested later in the growth phase had increased maltotriose and maltose transport activity. Neither strain exhibited significant differences with respect to maltose and maltotriose transport activity.     

Application of31P nuclear magnetic resonance spectroscopy to investigate plasmid effects onEscherichia coli metabolism

Summary Glucose metabolism inE. coli strain HB101, as a plasmid-free cell and as a host to two plasmids of different copy numbers, has been characterized using³¹P NMR. While the low-copy-number strain was found to behave very similarly to the plasmid-free strain, dramatically different behavior was exhibited by the high-copy-number strain. This strain maintained a nearly constant intracellular pH after addition of glucose to a starved suspension while intracellular pH of the other strains dropped considerably. The inorganic phosphate level in the high-copy-number strain was substantially higher than in the other strains, and the NTP level was much lower. Glycolytic rates of all three strains, however, were nearly identical. The trend in glycolytic rate strongly suggests that transport of glucose into the cell is the rate-limiting step under these conditions.     

Tindallia magadii gen. nov., sp. nov.: An Alkaliphilic Anaerobic Ammonifier from Soda Lake Deposits

Strain Z-7934, an alkaliphilic, obligately anaerobic, fermentative, asporogenous bacterium with Gram-positive cell wall structure, was isolated from soda deposits in Lake Magadi, Kenya. The organism ferments only a few amino acids, preferentially arginine and ornithine, with production of acetate, propionate, and ammonia. It is a true alkaliphile, with pH range for growth ranging from 7.5 to 10.5 (optimum pH 8.5), and growth is dependent on the presence of sodium ions. The G+C content of the genomic DNA is 37.6 mol%. 16S rDNA sequence analysis of strain Z-7934 shows that it belongs phylogenetically to cluster XI of the low G+C Gram-positive bacteria. On the basis of its distinct phylogenetic position and unique physiological properties, we propose a new genus and new species, Tindallia magadii, for this strain. The type strain is Z-7934^T (=DSM 10318). Received: 5 January 1998 / Accepted: 5 February 1998     

Characterization and molecular cloning of thermostable alpha-amylase from Streptomyces sp.To1

Summary A new thermophilic Streptomyces sp. TO1, isolated from Tunisian soil, produced a thermostable alpha-amylase and pullulanase. The gene encoding for the alpha-amylase activity was cloned into the multicopy cloning plasmid pLM1 using S. lividans ZX1 as host strain. The ZX1 / pLM1 strain has the same activity than the initial TO1 strain and about 25 fold higher activity than the ZX1 strain. This alpha-amylase has an optimum of pH and temperature at 6 and 70 °C respectively.     

Reiterated DNA sequences in a mutant strain of Streptomyces glaucescens and cloning of the sequence in Escherichia coli

Summary Streptomyces glaucescens exhibits a high degree of genetic instability. A sequence of 7.2 kb has been found which is present in a few tandemly repeated copies in the wild type strain GLA 0 and is amplified to ca. 500 copies per genome in the mutant strain GLA 1204. This sequence was cloned in Escherichia coli using pBR325 as vector.     

Sequence of the gene for a high-alkaline mannanase from an alkaliphilic Bacillus sp. strain JAMB-750, its expression in Bacillus subtilis and characterization of the recombinant enzyme

A novel alkaline mannanase Man26A has been found in the culture of an alkaliphilic Bacillus sp. strain JAMB-750 and the optimal pH for the mannanase activity of the enzyme was around pH 10 (J Biol Macromol 4: 67–74, 2004). This optimal pH is the highest among those of the mannanases reported to date. The gene man26A coding the enzyme was cloned from the genomic DNA of strain JAMB-750 and sequenced. It encodes a protein of 997 amino acids including a signal peptide. The N-terminal half (Glu27–Val486) of the enzyme exhibited moderate similarities to other mannanases belonging to glycoside hydrolase family 26, such as the enzymes from Cellvibrio japonicus (37% identity), Cellulomonas fimi (33% identity), and Bacillus sp. strain AM-001 (28% identity). The C-terminal half was found to contain four domains. The first, second, third, and fourth domains exhibited similarities to the carbohydrate-binding module, the mannan-binding module, the Homo sapiens collagen type IX alpha I chain, and the membrane anchor region of Gram-positive surface proteins, respectively. Its recombinant mannanase was produced extracellularly using Bacillus subtilis as the host. The optimal pH for the mannanase activity of the recombinant enzyme was around pH 10. The enzyme was very resistant to surfactants, for example, SDS up to 2.0% (w/v).     

Enhanced production of aureofuscin by over-expression of AURJ3M, positive regulator of aureofuscin biosynthesis in Streptomyces aureofuscus

Aims: The production of aureofuscin is very low in the wild‐type strain. We attempt to increase the production of aureofuscin by over‐expression of a controlling gene in the wild‐type strain. Methods and Results: The aurj3M gene was PCR‐amplified from Streptomyces aureofuscus SYAU0709, ligated into vector pMD19 and sequenced. The predicted translation of the 579‐bp cloned fragment was 97% similar to pimM from Streptomyces natalensis, which has an N‐terminal PAS domain and a LuxR‐type C‐terminal helix–turn–helix. Recombinant bacterial strains were constructed by transforming SYAU0709 with an expression plasmid (pBJJ3M) that contained aurj3M, thereby increasing the number of aurj3M gene copies. Conclusions: Bioassays for the antibiotic compound aureofuscin indicated that the recombinant bacteria had greater antifungal activity than the wild‐type strain. Specifically, the recombinant strain produced approx. 600% more aureofuscin, as quantified by high‐performance liquid chromatography analysis. Significance and Impact of the Study: To our knowledge, this approach has not been attempted in S. aureofuscus before and few genes in the aureofuscin pathway have been cloned and characterized.     

Isolation of a New Root Growth-stimulating Substance from a Fungus

In the last few decades, enzymatic production of 3,4-dihydroxyphenyl-L-alanine (L-dopa) using tyrosine phenol-lyase (Tpl) has been industrialized. This method has an intrinsic problem of tyrosine contamination because Tpl is synthesized under tyrosine-induced conditions. Herein, we constructed a hyper-L-dopa-producing strain by exploiting a mutant TyrR, an activator of tpl. The highest productivity was obtained for the strain grown under non-induced conditions. It was 30-fold higher than that obtained for tyrosine-induced wild-type cells.     

The adaptive acid response in Mesorhizobium sp.

Mesorhizobium huakuii strain LL56 and Mesorhizobium sp. strain LL22, which nodulate Lotus glaber, developed an adaptive acid response during exponential growth upon exposure to sublethal acid conditions. The adaptive acid response was found to be dependent on the sublethal pH and the strain intrinsic acid tolerance: the lowest adaptation pH was 4.0 for strain LL56 and 5.7 for strain LL22, and the lowest pH values tolerated after adaptation were 3.0 and 4.0, respectively. Both complex and minimal medium allowed the development of the adaptive acid response, although in complex medium this response was more effective. Three low molecular weight polypeptides (LMWPs) showed increased expression in strain LL56 during the adaptation to pH 4.0. However, the adaptive acid tolerance was only partially dependent on de novo protein synthesis, and constitutive systems may play a significant role on the acid tolerance of Mesorhizobium huakuii strain LL56.     

Protease Production by Alternaria tenuissima

Five strains of A. tenuissima showed wide variation in ability to produce extracellular proteases and the amount of protease produced by a given strain varied greatly with the medium used. Strain E-34 produced at least three proteases, with pH optima for casein digestion of pH 2.8, 6.6 and 9.5 respectively. The effect of the constituents of a liver-glucose medium on production of pH 9.5 protease by strain E-34 was investigated. Yields of pH 9.5 protease of up to 1.4 enzyme units per 1 have been obtained using this strain.     

Viability and ATP content of conidia of sorbic acid-sensitive and-resistant strains of Penicillium roqueforti after exposure to sorbic acid

Summary Conidia from two strains of Penicillium roqueforti, one sensitive and one resistant to inhibition by sorbic acid, were tested to determine how the chemical affected viability and ATP content of the spores. The minimum inhibitory concentration was less than 1,000 ppm for the sensitive strain and 3,000 ppm for the resistant strain. Exposing conidia to 6,000 ppm sorbic acid caused complete loss of viability in 1 day by those of the sensitive strain and in 4 days by those of the resistant strain. Exposure of conidia to sorbate solutions caused a rapid initial decrease in ATP content during the first few hours, followed by a more gradual decrease over the next 48–72 h. The same general trend was observed for both strains, but the resistant strain recovered some of the lost ATP following the rapid initial decrease. Results suggest that increased viability in the resistant strain may result from maintainance of ionic balance and an internal pH high enough to reduce the effectiveness of sorbic acid.     

Growth in acidic media increases production of phosphatidylinositol-specific phospholipase C byStaphylococcus aureus

Production of phosphatidylinositol-specific phospholipase C (PIPLC), an exoenzyme of some strains ofStaphylococcus aureus, has been epidemiologically associated with virulence. To investigate the elaboration ofS. aureus PIPLC, we have evaluated in vitro conditions that maximize production of enzymatically active PIPLC. PIPLC activity was assessed by measuring the release of³H-inositol-phosphate from the substrate³H-phosphatidylinositol. Lowering the pH ofS. aureus cultures from 7.0 to 5.4 progressively increased the yield of PIPLC. The final yield of PIPLC was at least five-fold greater when the initial culture pH was 5.4 compared with 7.0. Low pH enhanced PIPLC activity recovered from twoS. aureus strains capable of high PIPLC production, but not from a strain producing little PIPLC. At both pH 5.0 and 7.4, PIPLC production peaked during mid- to late-logarithmic phase. We conclude that an acidic starting pH of culture media increases the yield of PIPLC activity elaborated during active growth ofS. aureus.     

Tropical Drosophila pandora carry Wolbachia infections causing cytoplasmic incompatibility or male killing

Wolbachia infections have been described in several Drosophila species, but relatively few have been assessed for phenotypic effects. Cytoplasmic incompatibility (CI) is the most common phenotypic effect that has been detected, while some infections cause male killing or feminization, and many Wolbachia infections have few host effects. Here, we describe two new infections in a recently described species, Drosophila pandora, one of which causes near‐complete CI and near‐perfect maternal transmission (the “CI” strain). The other infection is a male killer (the “MK” strain), which we confirm by observing reinitiation of male production following tetracycline treatment. No incompatibility was detected in crosses between CI strain males and MK strain females, and rare MK males do not cause CI. Molecular analyses indicate that the CI and MK infections are distantly related and the CI infection is closely related to the wRi infection of Drosophila simulans. Two population surveys indicate that all individuals are infected with Wolbachia, but the MK infection is uncommon. Given patterns of incompatibility among the strains, the infection dynamics is expected to be governed by the relative fitness of the females, suggesting that the CI infection should have a higher fitness. This was evidenced by changes in infection frequencies and sex ratios in population cages initiated at different starting frequencies of the infections.     

Isolation and characterization of an isoproturon mineralizing <Emphasis Type="Italic">Sphingomonas</Emphasis> sp. strain SH from a French agricultural soil

The phenylurea herbicide isoproturon, 3-(4-isopropylphenyl)-1,1-dimethylurea (IPU), was found to be rapidly mineralized in an agricultural soil in France that had been periodically exposed to IPU. Enrichment cultures from samples of this soil isolated a bacterial strain able to mineralize IPU. 16S rRNA sequence analysis showed that this strain belonged to the phylogeny of the genus Sphingomonas (96% similarity with Sphingomonas sp. JEM-14, AB219361) and was designated Sphingomonas sp. strain SH. From this strain, a partial sequence of a 1,2-dioxygenase (catA) gene coding for an enzyme degrading catechol putatively formed during IPU mineralization was amplified. Phylogenetic analysis revealed that the catA sequence was related to Sphingomonas spp. and showed a lack of congruence between the catA and 16S rRNA based phylogenies, implying horizontal gene transfer of the catA gene cluster between soil microbiota. The IPU degrading ability of strain SH was strongly influenced by pH with maximum degradation taking place at pH 7.5. SH was only able to mineralize IPU and its known metabolites including 4-isopropylaniline and it could not degrade other structurally related phenylurea herbicides such as diuron, linuron, monolinuron and chlorotoluron or their aniline derivatives. These observations suggest that the catabolic abilities of the strain SH are highly specific to the metabolism of IPU.     

The lysins of bacteriophages infecting lactic acid bacteria

This short review highlights the complete absence of literature on lysins of bacteriophages infecting species like S. salivarius subsp. thermophilus, Pediococcus and Leuconostoc species, L. helveticus, L. acidophilus, L. plantarum and L. brevis, which are also widely used in the dairy industry. The lysins described share some similar biochemical characteristics: optimal pH and temperature, site of hydrolysis inside the peptidoglycan, and some activators and inhibitors. The cloning of the genes encoding these lysins only began in the last few years and four of them have been completely sequenced. In the future, these lysin genes could be interestingly compared to the host autolysin(s) gene(s). By contrast, the passage of phage lysins through the cytoplasmic membrane of the host cell in order to reach the peptidoglycan (via a signal sequence or the presence of a holin) seems not to be clearly resolved. The presence of a second open-reading frame upstream from the gene of the lysin, enabling a putative holin to be encoded, has already been suggested. No doubt our ever increasing knowledge about bacteriophage genome organization will help to elucidate this question. Meanwhile the obtention of a Lactococcus strain with an autolytic phenotype, using a bacteriophage lysin gene, as well as the successful use of purified PL1 lysin to obtain protoplasts of L. casei encourage us to continue to explore the field of bacteriophage lysins.