A new bacteriocinogenic activity: Megacin BII encoded by plasmid pSE 203 in strains of Bacillus megaterium

Mesophilic strains producing a new bacteriocin: Megacin BII, have been isolated from strains of Bacillus megaterium. Facultatively thermophilic strains producing Megacin BI were less sensitive to this new activity than nonproducing mesophiles and strains producing Megacin BII were also also more resistant to Megacin BI. Strains producing Megacin BII contained a large plasmid of 36·10⁶:pSE 203. This plasmid was introduced into non-megacinogenic acceptor strains by protoplast transformation, they then became megacin producers and immune to Megacin BII. Plasmid pSE 203 has been mapped with endonucleases. No similarity to the Megacin A plasmids pBM 309 [Rostás et al. (1980) and pBM 113 (von Tersch and Carlton (1983b)] was evident.Abbreviations CFU colony forming units - CCO covalently closed circular - OC open circular - LIN linear - EB ethidium bromide - Meg megacin - Tris tris (hydroxyl methyl) amino methane - Ot obligately thermophilic - Ft facultatively thermophilic - Sm streptomycin - Trim trimethoprim - Rif rifampicin - Thy thymine - Tc tetracycline     

Obligately and facultatively autotrophic,sulfur- and hydrogen-oxidizing thermophilic bacteria isolated from hot composts

A variety of autotrophic, sulfur- and hydrogen-oxidizing thermophilic bacteria were isolated from thermogenic composts at temperatures of 60–80° C. All were penicillin G sensitive, which proves that they belong to the Bacteria domain. The obligately autotrophic, non-spore-forming strains were gram-negative rods growing at 60–80°C, with an optimum at 70–75°C, but only under microaerophilic conditions (5 kPa oxygen). These strains had similar DNA G+C content (34.7–37.6 mol%) and showed a high DNA:DNA homology (70–87%) with Hydrogenobacter strains isolated from geothermal areas. The facultatively autotrophic strains isolated from hot composts were gram-variable rods that formed spherical and terminal endospores, except for one strain. The strains grew at 55–75° C, with an optimum at 65–70° C. These bacteria were able to grow heterotrophically, or autotrophically with hydrogen; however, they oxidized thiosulfate under mixotrophic growth conditions (e.g. pyruvate or hydrogen plus thiosulfate). These strains had similar DNA G+C content (60–64 mol%) to and high DNA:DNA homology (> 75%) with the reference strain of Bacillus schlegelii. This is the first report of thermogenic composts as habitats of thermophilic sulfur- and hydrogen-oxidizing bacteria, which to date have been known only from geothermal manifestations. This contrasts with the generally held belief that thermogenic composts at temperatures above 60° C support only a very low diversity of obligatory heterotrophic thermophiles related to Bacillus stearothermophilus. Received: 20 July 1995 / Accepted: 25 September 1995     

Iron Demand by Thermophilic and Mesophilic Bacteria Isolated from an Antarctic Geothermal Soil

The thermophilic bacterial strain MP4 assigned to a new species, likely of the genus Alicyclobacillus, was isolated from geothermal soils on the NW slope of Mount Melbourne, Antarctica. These soils have high iron concentrations and the strain MP4 requires iron additions for growth. Four mesophilic bacterial strains Paenibacillus validus MP5, MP8, and MP10, and P. apiarius MP7, isolated from the same site, need iron supply for growth depending on the medium. Growth temperature of thermophilic strain ranges from 42 to 70 °C, and that one of mesophiles from 25 to 44 °C. Thermophilic and mesophilic strains shared microenvironments with temperature of 42–44 °C and showed optima of pH values ranging from 5.5 to 6.0. The thermophilic strain MP4 reached values of 10⁶ CFU ml⁻¹ in aqueous soil extract from the NW slope of Mt. Melbourne, and 10⁵ CFU ml⁻¹ in water extracts from other geothermal Antarctic areas (Mt. Rittmann and Cryptogam Ridge). Growth of thermophilic bacteria in aqueous extracts of the NW slope of Mount Melbourne soils caused a reduction of 50% of soluble iron content, which was recovered in bacterial biomass. These results suggest a possible involvement of the thermophilic strain MP4 in iron bioavailability in these geothermal soils.     

Thermophilic Bacilli growing with carbon monoxide

Four strains of obligately thermophilic Bacilli capable of growing with carbon monoxide as a sole carbon and energy source were isolated from settling ponds of a sugar factory. Most of them could be identified as strains of Bacillus schlegelii on the basis of cell wall composition, DNA homology menaquinone and DNA base content. Growth with CO was very fast (t _d =3 h) and was optimal at 65°C. No growth occurred below 50°C. As with the mesophilic carboxydotrophs, hydrogen plus carbon dioxide could also serve as autotrophic substrates. Growth of the isolates with CO depended on the presence of molybdenum in the growth medium. This suggested CO oxidase in the newly isolated Bacilli being a molybdenum hydroxylase similar to the enzymes from the mesophilic carboxydotrophs. Some data characterizing the CO-oxidizing activity in extracts of the thermophilic isolates are also provided.This paper is respectively dedicated to Professor Dr. H. G. Schlegel on the occasion of his 60th birthday     

Fractionation of sulfur isotopes during dissimilatory reduction of sulfate by a thermophilic gram-negative bacterium at 60 °C

Sulfur isotope (³⁴S/³²S) fractionation during reduction of dissolved sulfate was investigated with a growing batch culture of a thermophilic, gram-negative, sulfate-reducing bacterium (strain MT-96) at 60 °C. The completely oxidizing strain was isolated from geothermally heated sediments of a shallow-water hydrothermal vent in the Mediterranean Sea. The hydrogen sulfide produced in the experiments was enriched in ³²S by approximately 19‰ as compared to sulfate, which indicates that stable isotope discrimination by this thermophile is within the range found previously for mesophilic sulfate-reducing bacteria, and only slightly higher than that observed for the thermophilic gram-positive Desulfotomaculum nigrificans. Received: 1 December 1998 / Accepted: 25 May 1999     

The Phylogenetic Diversity of Aerobic Organotrophic Bacteria from the Dagang High-Temperature Oil Field

The distribution and species diversity of aerobic organotrophic bacteria in the Dagang high-temperature oil field (China), which is exploited with water-flooding, have been studied. Twenty-two strains of the most characteristic thermophilic and mesophilic aerobic organotrophic bacteria have been isolated from the oil stratum. It has been found that, in a laboratory, the mesophilic and thermophilic isolates grow in the temperature, pH, and salinity ranges characteristic of the injection well near-bottom zones or of the oil stratum, respectively, and assimilate a wide range of hydrocarbons, fatty acids, lower alcohols, and crude oil, thus exhibiting adaptation to the environment. Using comparative phylogenetic 16S rRNA analysis, the taxonomic affiliation of the isolates has been established. The aerobic microbial community includes gram-positive bacteria with a high and low G+C content of DNA, and γ and β subclasses of Proteobacteria. The thermophilic bacteria belong to the genera Geobacillus and Thermoactinomyces, and the mesophilic strains belong to the genera Bacillus, Micrococcus, Cellulomonas, Pseudomonas, and Acinetobacter. The microbial community of the oil stratum is dominated by known species of the genus Geobacillus (G. subterraneus, G. stearothermophilus, and G. thermoglucosidasius) and a novel species “Geobacillus jurassicus.” A number of novel thermophilic oil-oxidizing bacilli have been isolated.__________Translated from Mikrobiologiya, Vol. 74, No. 3, 2005, pp. 401–409.Original Russian Text Copyright © 2005 by Nazina, Sokolova, Shestakova, Grigoryan, Mikhailova, Babich, Lysenko, Tourova, Poltaraus, Qingxian Feng, Fangtian Ni, Belyaev.     

Temperature Effects on Kinetic Parameters and Substrate Affinity of Cel7A Cellobiohydrolases

We measured hydrolytic rates of four purified cellulases in small increments of temperature (10–50 °C) and substrate loads (0–100 g/liter) and analyzed the data by a steady state kinetic model that accounts for the processive mechanism. We used wild type cellobiohydrolases (Cel7A) from mesophilic Hypocrea jecorina and thermophilic Rasamsonia emersonii and two variants of these enzymes designed to elucidate the role of the carbohydrate binding module (CBM). We consistently found that the maximal rate increased strongly with temperature, whereas the affinity for the insoluble substrate decreased, and as a result, the effect of temperature depended strongly on the substrate load. Thus, temperature had little or no effect on the hydrolytic rate in dilute substrate suspensions, whereas strong temperature activation (Q₁₀ values up to 2.6) was observed at saturating substrate loads. The CBM had a dual effect on the activity. On one hand, it diminished the tendency of heat-induced desorption, but on the other hand, it had a pronounced negative effect on the maximal rate, which was 2-fold larger in variants without CBM throughout the investigated temperature range. We conclude that although the CBM is beneficial for affinity it slows down the catalytic process. Cel7A from the thermophilic organism was moderately more activated by temperature than the mesophilic analog. This is in accord with general theories on enzyme temperature adaptation and possibly relevant information for the selection of technical cellulases.     

Production of a thermostable ATPase by the facultative thermophile,Bacillus coagulans

The properties of the ATPase in the facultative thermophile, Bacillus coagulans, grown at thermophilic or mesophilic temperatures were similar. Arrhenius plots did not show discontinuities indicative of thermoadaptation. Magnesium stimulation of the enzyme was dependant on the assay temperature but independant of the growth temperature. The ATPase in cells grown at 35°C or 55°C was equally thermostable at 65°C. In contrast, the ATPase from the mesophile, Bacillus megaterium (T _max=42°C) was completely inactivated at 55°C in 5 min.     

Isolation and characterization of two novel ethanol-tolerant facultative-anaerobic thermophilic bacteria strains from waste compost

In a search for potential ethanologens, waste compost was screened for ethanol-tolerant thermophilic microorganisms. Two thermophilic bacterial strains, M5EXG and M10EXG, with tolerance of 5 and 10% (v/v) ethanol, respectively, were isolated. Both isolates are facultative anaerobic, non-spore forming, non-motile, catalase-positive, oxidase-negative, Gram-negative rods that are capable of utilizing a range of carbon sources including arabinose, galactose, mannose, glucose and xylose and produce low amounts of ethanol, acetate and lactate. Growth of both isolates was observed in fully defined minimal media within the temperature range 50–80°C and pH 6.0–8.0. Phylogenetic analysis of the 16S rDNA sequences revealed that both isolates clustered with members of subgroup 5 of the genus Bacillus. G+C contents and DNA–DNA relatedness of M5EXG and M10EXG revealed that they are strains belonging to Geobacillus thermoglucosidasius. However, physiological and biochemical differences were evident when isolates M5EXG and M10EXG were compared with G. thermoglucosidasius type strain (DSM 2542^T). The new thermophilic, ethanol-tolerant strains of G. thermoglucosidasius may be candidates for ethanol production at elevated temperatures.     

The new alkaliphilic bacteriochlorophyll <Emphasis Type="Italic">a</Emphasis>-containing bacterium <Emphasis Type="Italic">Roseinatronobacter monicus</Emphasis> sp. nov. from the hypersaline Soda Mono Lake (California,United States)

Two strains of pink-colored aerobic bacteriochlorophyll a-containing bacteria were isolated from aerobic (strain ROS 10) and anaerobic (strain ROS 35) zones of the water column of Mono Lake (California, United States). Cells of the bacteria were nonmotile oval gram-negative rods multiplying by binary fission by means of a constriction. No intracellular membranes were detected. Polyphosphates and poly-β-hydroxybutyric acid were the storage compounds. Pigments were represented by bacteriochlorophyll a and carotenoids of the spheroidene series. The strains were obligately aerobic, mesophilic (temperature optimum of 25–30°C), alkaliphilic (pH optimum of 8.5–9.5), and moderately halophilic (optimal NaCl concentration of 40 g/l). They were obligately heterotrophic and grew aerobically in the dark and in the light. Respiration was inhibited by light at wavelengths corresponding to the absorption of the cellular pigments. The substrate utilization spectra were strain-specific. In the course of organotrophic growth, the bacteria could oxidize thiosulfate to sulfate; sulfide and polysulfide could also be oxidized. The DNA G+C content was 59.4 mol % in strain ROS 10 and 59 mol % in strain ROS 35. In their phenotypic properties, the new strains were close but not identical to the alkaliphilic bacterium Roseinatronobacter thiooxidans. The distinctions in the nucleotide sequences of the 16S rRNA genes (2%) and low DNA-DNA hybridization level with Rna. thiooxidans (22–25%) allow the new strains to be assigned to a new species of the genus Roseinatronobacter, Roseinatronobacter monicus sp. nov. with the type strain ROS 35^T (=UNIQEM U-251^T = VKM B-2404^T).     

Isolation of thermophilic mutants of Bacillus subtilis and Bacillus pumilus and transformation of the thermophilic trait to mesophilic strains

Thermophilic mutants were isolated from mesophilic Bacillus subtilis and Bacillus pumilus by plating large numbers of cells and incubating them for several days at a temperature about 10 degrees C above the upper growth temperature limit for the parent mesophiles. Under these conditions we found thermophilic mutant strains that were able to grow at temperatures between 50 degrees C and 70 degrees C at a frequency of less than 10(-10). The persistence of auxotrophic and antibiotic resistance markers in the thermophilic mutants confirmed their mesophilic origin. Transformation of genetic markers between thermophilic mutants and mesophilic parents was demonstrated at frequencies of 10(-3) to 10(-2) for single markers and about 10(-7) for two unlinked markers. With the same procedure we were able to transfer the thermophilic trait from the mutant strains of Bacillus to the mesophilic parental strains at a frequency of about 10(-7), suggesting that the thermophilic trait is a phenotypic consequence of mutations in two unlinked genes.     

Biochemical and physiological characteristics of Xenorhabdus species,symbiotically associated with entomopathogenic nematodes including Steinernema kushidai and their pathogenicity against Spodoptera litura (Lepidoptera: Noctuidae)

The symbiotic bacterium strain, SK-1 isolated from Steinernema kushidai, a new species of entomopathogenic nematode, was compared with other strains of Xenorhabdus species. Like other Xenorhabdus nematophilus strains, this new strain is gram-negative, facultatively anaerobic, peritrichously flagellated rod and negative for catalase and nitrate reduction. It can be distinguished from the other Xenorhabdus spp. by differences in reactions to phenylalanine deaminase, no acid production from myo-inositol and utilizations of inosine, dl-malate, formate and methanol. Intra-haemocoelic injection of actual cells or liquid culture supernatant into sixth instar larvae of Spodoptera litura for either Phase I or II variants were not pathogenic. Other strains of X. nematophilus showed pathogenicity for whole cell injections. The supernatants of strain D-1 and ATCC 19061, which are symbionts of Steinernema carpocapsae were pathogenic, however pathogenicity decreased and then terminated by increases in temperature.     

Physiological Properties and Phylogenetic Affiliations of Anaerobic Bacteria Isolated from Roots of Rice Plants Cultivated on a Paddy Field

Anaerobic bacteria associated with roots of rice plants cultivated on a paddy field were isolated, and their physiological properties and phylogenetic affiliations were investigated. The roots harbored culturable populations of anaerobic microorganisms at 10⁷ levels of viable counts (CFU/g dry roots), and the isolates were thought to represent numerically abundant populations of culturable anaerobic microorganisms present on the roots. Among 18 strains isolated in pure culture, five strains were obligately anaerobic and others were facultatively anaerobic. Eight strains including four obligately anaerobic strains were selected for further study. Of eight strains, seven strains were saccharolytic, and one strain was a non-saccharolytic sulfate-reducer. Glucose was fermented into ethanol and/or acetate by the saccharolytic strains, lactate, succinate or H₂ was also produced by some strains. Four facultatively anaerobic strains were saccharolytic and grew with the fermentative metabolism even under the oxic condition. Three facultatively anaerobic strains and one obligately anaerobic strain exhibited the Fe(III)-reducing ability. The comparative analysis of 16S rRNA gene sequences indicated that the sequence of any strain did not completely match to the sequences available in the database. The seven saccharolytic strains represented diverse phylogenetic groups: the classes ‘Alphaproteobacteria’ (two strains) and ‘Gammaproteobacteria’ (one strain), the family Bacteroidaceae (one strain), the orderActinomycetales (two strains), and the family Clostridiaceae. The sulfate-reducing strain was a close relative ofDesulfovibriodesulfuricans . At least five strains were considered to represent novel species. In particular, two strains were considered to represent novel lines of descent at the family level within the order ‘Rhizobiales’. These results suggested that phylogenetically different bacteria with a common physiological trait as the saccharolytic fermentative acidogen formed numerically most abundant populations of culturable anaerobes in the microbial community on rice roots.     

Atrazine degradation by aerobic microorganisms isolated from the rhizosphere of sweet flag (Acorus calamus L.)

In presented study the capability of microorganisms isolated from the rhizosphere of sweet flag (Acorus calamus) to the atrazine degradation was assessed. Following isolation of the microorganisms counts of psychrophilic bacteria, mesophilic bacteria and fungi were determined. Isolated microorganisms were screened in terms of their ability to decompose a triazine herbicide, atrazine. Our results demonstrate that within the rhizosphere of sweet flag there were 3.8 × 10⁷ cfu of psychrophilic bacteria, 1.8 × 10⁷ cfu of mesophilic bacteria, and 6 × 10⁵ cfu of fungi per 1 g of dry root mass. These microorganisms were represented by more than 20 different strains, and at the first step these strains were grown for 5 days in the presence of atrazine at a concentration of 5 mg/l. In terms of the effect of this trial culture, the bacteria reduced the level of atrazine by an average of about 2–20%, but the average level of reduction by fungi was in the range 18–60%. The most active strains involved in atrazine reduction were then selected and identified. These strains were classified as Stenotrophomonas maltophilia, Bacillus licheniformis, Bacillus megaterium, Rahnella aquatilis (three strains), Umbelopsis isabellina, Volutella ciliata and Botrytis cinerea. Culturing of the microorganisms for a longer time resulted in high atrazine degradation level. The highest degradation level was observed at atrazine concentrations of 5 mg/l for S. maltophilia (83.5% after 15 days of culture) and for Botrytis sp. (82% after 21 days of culture). Our results indicate that microorganisms of the sweet flag rhizosphere can play an important role in the bioremediation of atrazine-contaminated sites.     

Analysis of the Heat-Shock Response Displayed by Two Chaetomium Species Originating from Different Thermal Environments

Three features of the heat shock response, reorganization of protein expression, intracellular accumulation of trehalose, and alteration in unsaturation degree of fatty acids were investigated in the thermophilic fungus Chaetomium thermophile and compared to the response displayed by a closely related mesophilic species, C. brasiliense. Thermophilic heat shock response paralleled the mesophilic response in many respects like (i) the temperature difference observed between normothermia and the upper limit of translational activity, (ii) the transient nature of the heat shock response at the level of protein expression including both the induction of heat shock proteins (HSPs) as well as the repression of housekeeping proteins, (iii) the presence of representatives of high-molecular-weight HSPs families, (iv) intracellular accumulation of trehalose, and finally (v) modifications in fatty acid composition. On the other hand, a great variability between the two organisms was observed for the proteins expressed during stress, in particular a protein of the HSP60 family that was only observed in C. thermophile. This peptide was also present constitutively at normal temperature and may thus fulfil thermophilic functions. It is shown that accumulation of trehalose does not play a part in thermophily but is only a stress response. C. thermophile contains less polyunsaturated fatty acids at normal temperature than C. brasiliense, a fact that can be directly related to thermophily. When subjected to heat stress, both organisms tended to accumulate shorter and less unsaturated fatty acids.     

Growth characteristics of selected thermophilic strains of cyanobacteria using crossed gradients of temperature and light

Growth requirements of 10 possibly thermophilic strains of cyanobacteria were compared under a wide spectrum of light and temperature conditions (7–80Wm^?2, 12–40°C). The strains were isolated from different localities: six of thermal springs in Slovakia (4 from Pie??any, 2 from Sklené Teplice), one from thermal waters in Rupite, Bulgaria, 2 strains from a hypersaline lake Chott-el-Djerid, Tunisia, and one strain from the tropical island of Cebu, Philippines. Although the crossed gradient unit allowed only sub-optimal temperature range with respect to thermophile definition, i.e. optimum temperature above 45°C, there were difference among the strains. The most thermophilic and high-light tolerant strain was Synechococcus bigranulatus strain Lukavsky 2005/66, with a peak above 45°C; the second was Chroococcidiopsis thermalis strain Hindák 2008/9, and Isocystis sp. strain Hindák 2006/1. The temperature requirements of other strains were similar; the least thermophilic were both Slovakian strains of the genus Hapalosiphon. Growth was not limited below 80 W m^?2, except for H. fontinalis strain Hindák 2008/3, which prefers lower irradiance. Hapalosiphon delicatulus strain Hindák 2007/20, isolated from a bark tree on the tropical island of Cebu was rather mesophilic than thermophilic and shade-preferring. The CCA revealed that the ecologically similar strains originated from the same or nearby localities. There were no significant correlations between temperature optima in culture and in nature. Bulgarian and Tunisian strains preferred higher irradiances.     

Comparative Arrhenius plots of enzyme activity from penicillium

Comparison of the Arrhenius plots of three enzymes, formyltetrahydrofolate synthetase, glutathione reductase (GSSGR) and chorismate mutase (CM) from a thermophilic (Penicillium duponti) and a mesophilic (Penicillium chrysogenum) fungus reveals a fairly consistent pattern. In general, those enzymes extracted from mesophiles had lower activation energies than similar enzymes extracted from thermophiles. One enzyme studied, mesophilic glutathione reductase, exhibited a break in its Arrhenius plot. The allosteric enzyme studied showed slightly different sensitivities in the thermophilic versus the mesophilic extracts.     

Nucleoside deoxyribosyltransferase and inosine phosphorylase activity in lactic acid bacteria

Phosphate-independent nucleoside deoxyribosyltransferase activity was detected in Aerococcus viridans, Leuconostoc mesenteroides, two species of Pediococcus, eight obligately homofermentative and three obligately heterofermentative lactobacilli. In cellfree extracts of one strain of Lactococcus, one strain of Streptococcus and three strains of facultatively heterofermentative lactobacilli no phosphate-independent nucleoside deoxyribosyltransferase could be detected and inosine nucleoside phosphorylase was the only enzyme catalysing pentosyl transfer to purines. A classification based on the presence or absence of these enzymes separates Streptococcus and Lactococcus from the remaining genera which is in agreement with studies using anti-aldolase sera and 16S rRNA oligonucleotide catalogues.Abbreviations NCIMB National Collection of Industrial and Marine bacteria - NCDO National Collection of Dairy organisms - MRS deMan Rogosa Sharpe - Pipes Piperazine-N,N-bis[2-ethanesulphonic acid]     

Using a strategy based on the concept of convergent evolution to identify residue substitutions responsible for thermal adaptation

Factors that are related to thermostability of proteins have been extensively studied in recent years, especially by comparing thermophiles and mesophiles. However, most of them are global characters. It is still not clear how to identify specific residues or fragments which may be more relevant to protein thermostability. Moreover, some of the differences among the thermophiles and mesophiles may be due to phylogenetic differences instead of thermal adaptation. To resolve these problems, I adopted a strategy to identify residue substitutions evolved convergently in thermophiles or mesophiles. These residues may therefore be responsible for thermal adaptation. Four classes of genomes were utilized in this study, including thermophilic archaea, mesophilic archaea, thermophilic bacteria, and mesophilic bacteria. For most clusters of orthologous groups (COGs) with sequences from all of these four classes of genomes, I can identify specific residues or fragments that may potentially be responsible for thermal adaptation. Functional or structural constraints (represented as sequence conservation) were suggested to have higher impact on thermal adaptation than secondary structure or solvent accessibility does. I further compared thermophilic archaea and mesophilic bacteria, and found that the most diverged fragments may not necessarily correspond to the thermostability-determining ones. The usual approach to compare thermophiles and mesophiles without considering phylogenetic relationships may roughly identify sequence features contributing to thermostability; however, to specifically identify residue substitutions responsible for thermal adaptation, one should take sequence evolution into consideration.