Identification of the genus Geobacillus using genus-specific primers, based on the 16S-23S rRNA gene internal transcribed spacer

The aim of this study was to develop an easy and accurate technique for the identification of the genus Geobacillus. For this purpose, Geobacillus genus-specific primers GEOBAC (GEOBAC-F and GEOBAC-R) based on the 16S-23S rRNA gene internal transcribed spacer (ITS) region sequences have been designed. In total, 52 sequences from three species of the genus Geobacillus (Geobacillus stearothermophilus, Geobacillus kaustophilus and Geobacillus lituanicus) were examined for the design of these primers. Analysis of the sequences revealed three highly conservative regions common to these species: 5' and 3' end regions of 16S-23S rRNA gene ITSs and box A. Some sequences possessed two additional conservative regions - genes of tRNA(Ile) and tRNA(Ala). These particular sequences were chosen for the construction of the primers. The designed primers targeted the gene of tRNA(Ile) and the 3' end region of ITSs. This technique was validated with both the reference strains of the genus Geobacillus and the thermophilic aerobic endospore-forming environmental isolates. Different Geobacillus species could be grouped according to the number and size of GEOBAC-PCR products and identified on the basis of the AluI and TaqI restriction analysis of these products.     

Three novel halotolerant and thermophilic Geobacillus strains from shallow marine vents

During a polyphasic taxonomic analysis performed on isolates from shallow marine hydrothermal vents of Eolian Islands (Italy), three thermophilic, halotolerant bacilli, designated as strain 1bw, strain 5-2 and strain 10-1, could not be affiliated to any described species. Physiological and biochemical characteristics, membrane lipids composition, mol % G+C content, and phylogenetic relationships determined on the basis of the 16S rRNA gene sequence analysis, placed these strains within the genus Geobacillus. The three strains were only moderately related to species of Geobacillus and their relatives, members of Saccharococcus. Determination of the relatedness among each other at a higher taxonomic level by DNA-DNA reassociation experiments demonstrated the three isolates to represent three different novel Geobacillus genomospecies. The taxonomic novelty of these three marine strains was substantiated by their physiological properties and by fatty acid patterns that did not match closely those of any Geobacillus type strain. These three novel strains could be of interest to biotechnology because of their ability to produce exopolysaccharides and to adhere on polystirene, characteristics undescribed so far for other Geobacillus species. They are also able to utilise hydrocarbons such as gas oil, kerosene and mineral lubricating oil. Strain 5-2 is tolerant to zinc.     

Characterization of Ferroplasma isolates and Ferroplasma acidarmanus sp. nov., extreme acidophiles from acid mine drainage and industrial bioleaching environments

Three recently isolated extremely acidophilic archaeal strains have been shown to be phylogenetically similar to Ferroplasma acidiphilum Y(T) by 16S rRNA gene sequencing. All four Ferroplasma isolates were capable of growing chemoorganotrophically on yeast extract or a range of sugars and chemomixotrophically on ferrous iron and yeast extract or sugars, and isolate "Ferroplasma acidarmanus" Fer1(T) required much higher levels of organic carbon. All four isolates were facultative anaerobes, coupling chemoorganotrophic growth on yeast extract to the reduction of ferric iron. The temperature optima for the four isolates were between 35 and 42 degrees C and the pH optima were 1.0 to 1.7, and "F. acidarmanus" Fer1(T) was capable of growing at pH 0. The optimum yeast extract concentration for "F. acidarmanus" Fer1(T) was higher than that for the other three isolates. Phenotypic results suggested that isolate "F. acidarmanus" Fer1(T) is of a different species than the other three strains, and 16S rRNA sequence data, DNA-DNA similarity values, and two-dimensional polyacrylamide gel electrophoresis protein profiles clearly showed that strains DR1, MT17, and Y(T) group as a single species. "F. acidarmanus" Fer1(T) groups separately, and we propose the new species "F. acidarmanus" Fer1(T) sp. nov.     

Ecotypes of planktonic actinobacteria with identical 16S rRNA genes adapted to thermal niches in temperate, subtropical, and tropical freshwater habitats

Seven strains with identical 16S rRNA genes affiliated with the Luna2 cluster (Actinobacteria) were isolated from six freshwater habitats located in temperate (Austria and Australia), subtropical (People's Republic of China), and tropical (Uganda) climatic zones. The isolates had sequence differences at zero to five positions in a 2,310-nucleotide fragment of the ribosomal operon, including part of the intergenic spacer upstream of the 16S rRNA gene, the complete 16S rRNA gene, the complete 16S-23S internal transcribed spacer (ITS1), and a short part of the 23S rRNA gene. Most of the few sequence differences found were located in the internal transcribed spacer sequences. Two isolates obtained from habitats in Asia and Europe, as well as two isolates obtained from different habitats in the People's Republic of China, had identical sequences for the entire fragment sequenced. In spite of minimal sequence differences in the part of the ribosomal operon investigated, the strains exhibited significant differences in their temperature response curves (with one exception), as well as pronounced differences in their temperature optima (25.0 to 35.6 degrees C). The observed differences in temperature adaptation were generally in accordance with the thermal conditions in the habitats where the strains were isolated. Strains obtained from temperate zone habitats had the lowest temperature optima, strains from subtropical habitats had intermediate temperature optima, and a strain from a tropical habitat had the highest temperature optimum. Based on the observed temperature responses, we concluded that the strains investigated are well adapted to the thermal conditions in their home habitats. Consequently, these closely related strains represent different ecotypes adapted to different thermal niches.     

Engineering of a multifunctional hemicellulase

To engineer a multifunctional xylan-degrading enzyme, a chimera was created by fusing the xylanase domain of the Clostridium thermocellum xylanase (xynZ) and a dual functional arabinofuranosidase/xylosidase (DeAFc; from a compost starter mixture) through a flexible peptide linker. The xylanase domain of xynZ possesses previously unreported endoglucanase activity. The chimera, possessing the activities of xylanase, endoglucanase, arabinofuranosidase and xylosidase, was expressed in E. coli and purified. The chimera closely resembled the parental enzymes in pH, temperature optima and kinetics, and was more active than the parental enzyme mixture in the hydrolysis of natural xylans and corn stover. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Application of recA and rpoB sequence analysis on phylogeny and molecular identification of Geobacillus species

Aims:  Some Geobacillus species have highly similar 16S rRNA gene sequences, making 16S rDNA sequence analysis-based identification problematic. To overcome this limitation, recA and rpoB sequence analysis was evaluated as an alternative for distinguishing Geobacillus species.
Methods and Results:  The phylogram of 16S rRNA gene sequences inferred from the neighbour-joining method showed that nine clusters of Geobacillus species were characterized with bootstrap values >90%. The recA and rpoB sequences of 10 reference strains in clusters V, VIb and VIc were amplified and sequenced using consensus primers. Alignment of recA sequences in clusters V, VIb and VIc revealed three types of recA genes, consistent with the putative amino acid sequences and in vivo recA splicing analysis. The phylogram constructed from rpoB sequences showed more divergence than that constructed from 16S rRNA gene sequences.
Conclusions:  recA and rpoB sequence analysis differentiated closely-related Geobacillus species and provided direct evidence for reclassifying some species dubiously categorized as Geobacilli . Additionally, this study revealed three types of recA genes in the different Geobacillus species.
Significance and Impact of the Study:  This study highlights the advantage of recA and rpoB sequence analysis to supplement 16S rRNA gene sequence analysis for efficient and convenient determination of Geobacillus species.     

The phylogenetic diversity of aerobic organotrophic bacteria from the Dagan high-temperature oil field

The distribution and species diversity of aerobic organotrophic bacteria in the Dagan high-temperature oil field (China), which is exploited via 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 gamma and beta 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.     

Application of rpoB sequence similarity analysis, REP-PCR and BOX-PCR for the differentiation of species within the genus Geobacillus

Aim:  To investigate the applicability of rpoB gene, which encodes the β subunit of RNA polymerase, to be used as an alternative to 16S rRNA for sequence similarity analysis in the thermophilic genus Geobacillus. Rapid and reproducible repetitive extragenic palindromic fingerprinting techniques (REP‐ and BOX‐polymerase chain reaction) were also used. Methods and Results:  rpoB DNA (458 bp) were amplified from 21 Geobacillus‐ and Bacillus type strains, producing different BOX‐ and REP‐PCR profiles, in addition to 11 thermophilic isolates of Geobacillus and Bacillus species from a Santorini volcano habitat. The sequences and the phylogenetic tree of rpoB were compared with those obtained from 16S rRNA gene analysis. The results demonstrated between 90–100% (16S rRNA) and 74–100% (rpoB) similarity among examined bacteria. Conclusion:  BOX‐ and REP‐PCR can be applied for molecular typing within Geobacillus genus. rpoB sequence similarity analysis permits a more accurate discrimination of the species within the Geobacillus genus than the more commonly used 16S rRNA. Significance and Impact of the Study:  The obtained results suggested that rpoB sequence similarity analysis is a powerful tool for discrimination between species within the ecologically and industrially important strains of Geobacillus genus.     

Geobacillus toebii subsp. decanicus subsp. nov., a hydrocarbon-degrading, heavy metal resistant bacterium from hot compost

A thermophilic, spore-forming bacterial strain L1(T) was isolated from hot compost "Pomigliano Environment" s.p.a., Pomigliano, Naples, Italy. The strain was identified by using a polyphasic taxonomic approach. L1(T) resulted in an aerobic, gram-positive, rod-shaped, thermophilic with an optimum growth temperature of 68 degrees C chemorganotrophic bacterium which grew on hydrocarbons as unique carbon and energy sources and was resistant to heavy metals. The G+C DNA content was 43.5 mol%. Phylogenetic analysis of 16S rRNA gene sequence and Random Amplified Polymorphic DNA-PCR (RAPD-PCR) analysis of L1(T) and related strains showed that it forms within Geobacillus toebii, a separate cluster in the Geobacillus genus. The composition of cellular fatty acids analyses by Gas-Mass Spectroscopy differed from that typical for the genus Geobacillus in that it is lacking in iso-C15 fatty acid, while iso-C16 and iso-C17 were predominant. Isolates grew on a rich complex medium at temperatures between 55-75 degrees C and presented a doubling time (t(d)) of 2 h and 6 h using complex media and hydrocarbon media, respectively. Among hydrocarbons tested, n-decane (2%) was the more effective to support the growth (1 g/L of wet cells). The microorganism showed resistance to heavy metal tested during the growth. Furthermore, intracellular alpha-galactosidase and alpha-glucosidase enzymatic activities were detectable in the L1(T) strain. Based on phenotypic, phylogenetic, fatty acid analysis and results from DNA-DNA hybridization, we propose assigning a novel subspecies of Geobacillus toebii, to be named Geobacillus toebii subsp. decanicus subsp. nov., with the type strain L1(T) (=DSM 17041=ATCC BAA 1004).     

Gamma-proteobacteria Aquicella lusitana gen. nov., sp. nov., and Aquicella siphonis sp. nov. infect protozoa and require activated charcoal for growth in laboratory media

Several isolates, belonging to two new species of the same novel genus of gamma-proteobacteria, were recovered from drilled well (borehole) and spa water at S?o Gemil in central Portugal. These organisms are phylogenetically most closely related to the strictly intracellular uncultured species of the genus Rickettsiella, which cause disease in arthropods, and to the facultatively intracellular species of the genus Legionella, some of which cause Legionnaires' disease and Pontiac fever. The S?o Gemil strains grew only on media containing charcoal, as is also true of the species of the genus LEGIONELLA: Unlike the vast majority of Legionella isolates, the new isolates did not require L-cysteine or ferric pyrophosphate for growth but like the legionellae had an absolute requirement for alpha-ketoglutarate. Strains SGT-39(T) and SGT-56 grew consistently between 30 and 43 degrees C, while strains SGT-108(T) and SGT-109 grew between 30 and 40 degrees C. The pH ranges for growth of these organisms were surprisingly narrow: strains SGT-39(T) and SGT-56 grew between pH 6.3 and 7.3, while strains SGT-108(T) and SGT-109 grew between pH 6.3 and 7.0. Both organisms proliferated in the amoeba Hartmannella vermiformis but did not grow in U937 human cells. Based on 16S rRNA gene sequence analysis and physiological, biochemical, and chemical analysis we describe two new species of one novel genus; one species is represented by strain SGT-39(T), for which we propose the name Aquicella lusitana, while strain SGT-108(T) represents a second species of the same genus, for which we propose the name Aquicella siphonis.     

Geobacillus jurassicus sp. nov., a new thermophilic bacterium isolated from a high-temperature petroleum reservoir, and the validation of the Geobacillus species

Four thermophilic, spore-forming bacterial strains, DS1(T), DS2, 46 and 49, were isolated from the high-temperature Dagang oilfield, located in China. The strains were identified by using the polyphasic taxonomy approach. These were aerobic, gram-positive, rod-shaped, moderately thermophilic (with an optimum growth temperature of 60-65 degrees C), chemoorganotrophic bacteria capable of growing on various sugars, carboxylic acids and crude oil. Two strains, DS1(T) and DS2, were capable of growing on individual saturated hydrocarbons. The G + C content of the DNA of strains DS1(T) and DS2 was 54.5 and 53.8 mol%, respectively. The phylogenetic analysis of the 16S rDNA of strains DS1(T) and DS2 showed that they form a separate cluster within the genus Geobacillus. The cellular fatty acids of the isolates were dominated by iso-15:0, iso-16:0 and iso-17:0 acids, which are the typical fatty acids of bacteria from the genus Geobacillus. The DNA-DNA hybridization study and the comparative analysis of the morphological and chemotaxonomic characteristics of strains DS1(T) and DS2 showed that they differ from the previously described Geobacillus species and belong to a new species, which was called Geobacillus jurassicus. DS1(T) (=VKM B2301(T), = DSM 15726(T)) is the type strain of this species. According to both DNA-DNA reassociation studies and 16S rDNA sequence analysis, two other strains, 46 and 49, were assigned to the species G. stearothermophilus. In this paper, we provide evidence that the new combinations G. stearothermophilus, G. thermoleovorans, G. kaustophilus, G. thermoglucosidasius and G. thermodenitrificans may be considered to be valid.     

Relatedness of Thermomyces lanuginosus strains producing a thermostable xylanase

Properties of an endo-beta-xylanase produced by a locally isolated Thermomyces lanuginosus strain SSBP was compared to seven other T. lanuginosus strains isolated from different geographical regions. Strain SSBP produced the highest xylanase activity of 59600 nkat ml(-1) when cultivated on corn cobs (maize) medium, whereas the seven other strains produced xylanase activities ranging from 6000 to 32000 nkat ml(-1). No cellulase activity was produced by the strains. Despite the variability in the production of xylanase, little difference in the other characteristics of the strains could be found. The optimal temperature and pH for xylanase production by the strains was either 40 or 50 degrees C and between pH 6 and 7, respectively. Optimal xylanase activity of the strains was observed at 70 degrees C and at pH 6 or 6.5. Culture supernatant analysis by SDS-PAGE and isoelectric focusing PAGE of all strains revealed the presence of a single 24.7 kDa and pI 3.9 xylanase. Phylogenetic analysis by PCR amplification and sequencing of the internal transcribed spacer of nuclear rRNA repeat units and 5.8S rDNA revealed no strain diversity. However, random amplified polymorphic DNA analysis pointed to greater diversity and with one primer (5'-GCCCGACGCG-3'), a relationship was established between xylanase levels and the RAPD pattern.     

Identification of Thermophilic Bacterial Strains Producing Thermotolerant Hydrolytic Enzymes from Manure Compost

Ten thermophilic bacterial strains were isolated from manure compost. Phylogenetic analysis based on 16S rRNA genes and biochemical characterization allowed identification of four different species belonging to four genera: Geobacillus thermodenitrificans, Bacillus smithii, Ureibacillus suwonensis and Aneurinibacillus thermoaerophilus. PCR-RFLP profiles of the 16S-ITS-23S rRNA region allowed us to distinguish two subgroups among the G. thermodenitrificans isolates. Isolates were screened for thermotolerant hydrolytic activities (60–65°C). Thermotolerant lipolytic activities were detected for G. thermodenitrificans, A. thermoaerophilus and B. smithii. Thermotolerant protease, α-amylase and xylanase activities were also observed in the G. thermodenitrificans group. These species represent a source of potential novel thermostable enzymes for industrial applications.     

Polyphasic characterization of benthic, moderately halophilic, moderately thermophilic cyanobacteria with very thin trichomes and the proposal of Halomicronema excentricum gen. nov., sp. nov.

A new genus of moderately halophilic, moderately halotolerant and moderately thermophilic cyanobacteria with very thin trichomes is described. The four strains included in this genus were isolated from benthic microbial mats in a man-made hypersaline pond. Trichomes were around 1 microm thick, with small constrictions at the cross-walls and diffluent colorless sheaths. Thylakoids were parallel to the cell wall, but thylakoids and nucleoid were often excentrically arranged within the cytoplasm with respect to the main trichome axis. Strains grew at between 3.2 and 12-15% (w/v) salinity with optima between 3.2 and 12%. They showed lower temperature limits around 20 degrees C and upper limits between 45 and 50 degrees C, with optima between 28 and 45-50 degrees C. Carotenoid and mycosporine amino-acid complements were identical among strains. Phylogenetic analyses based on 16S rRNA gene sequence showed that all strains were closely related (99% or higher similarity) and distantly related to other cyanobacteria (91% or lower similarity). We propose the new genus and species Halomicronema excentricum for these strains. The type strain is TFEP1.     

Characterization of psychrotolerant heterotrophic bacteria from Finnish Lapland

A total of 331 aerobic heterotrophic bacterial strains were isolated from various ecosystems of Finnish Lapland (68-69 degrees N) including forest soil, arctic alpine-tundra soil, stream water, lake and mire sediments, lichen and snow algae. Whole cell fatty acid and 16S rRNA gene sequence analysis and microscopy indicated that the isolates were dominated by Gram-negative bacteria, while only 20 Gram-positive strains were isolated. Based on 16S rRNA gene sequences the isolates were members of alpha-, beta-, gamma-Proteobacteria, Gram-positives with low G+C content, Actinobacteria and the Cytophaga/Flexibacter/Bacteroides group. More than one-third of the isolates could be tentatively identified as Pseudomonas spp. which were particularly abundant in the alpine-tundra soils where they represented 60% of all isolates. Other frequently isolated Gram-negative taxa were Burkholderia sp., Collimonas sp., Pedobacter sp., Janthinobacter sp., Duganella sp., Dyella sp. and Sphingomonas sp. Growth temperature ranges and hydrolytic enzyme activities of selected ca.100 strains were screened. The strains were psychrotolerant growing generally at temperatures ranging from 0 to 30 degrees C, as 82% of the isolates grew at 0 degrees C while only 7% grew at 35 degrees C. Protease and lipase activities at 5 degrees C were detected in more than half of the strains while approximately 20% of the strains possessed amylase and/or cellulase activities.     

九香虫(椿象)体内可培养细菌的多样性分析

【目的】认识药用昆虫九香虫(Aspongopus chinesis Dallas)成虫体内可培养细菌资源多样性。【方法】运用纯培养法、反转录重复因子扩增(BOXA1R-PCR)分析技术、16S r RNA基因测序和系统发育分析对样品中可培养细菌进行多样性研究,测定了分离菌株的抗菌特性、吲哚乙酸(IAA)含量和产淀粉酶活性等指标。【结果】通过6种不同培养基共分离得到52株菌落特征不同的细菌菌株。基于菌落特征和BOXA1R-PCR图谱选取12株代表菌株用于16S r RNA基因序列测定。16S r RNA基因序列系统发育分析显示,52株菌株分属于芽孢杆菌属(Bacillus)、假单胞菌属(Pseudomonas)、寡养单胞菌属(Stenotrophomonas)和伯克霍尔德氏菌属(Burkholderia)4个属,其中芽孢杆菌属(Bacillus)为优势菌属。分离到的52株细菌有44株(占总分离菌株的84.6%)表现出对供试病原菌具有较好的抑制作用,高达94.2%的分离菌株能产IAA,有43株(占总分离菌株的82.7%)表现出淀粉酶活性。【结论】九香虫内细菌种群较为多样,具有潜在应用价值。     

Thiobacillus sajanensis sp. nov., a new obligately autotrophic sulfur-oxidizing bacterium isolated from Khoito-Gol hydrogen-sulfide springs, Buryatia

Four strains of rod-shaped gram-negative sulfur-oxidizing bacteria were isolated from Khoito-Gol hydrogen-sulfide springs in the eastern Sayan Mountains (Buryatia). The cells of the new isolates were motile by means of a single polar flagellum. The strains were obligately chemolithoautotrophic aerobes that oxidized thiosulfate (with the production of sulfur and sulfates) and hydrogen sulfide. They grew in a pH range of 6.8-9.5, with an optimum at pH 9.3 and in a temperature range of 5-39 degrees C, with an optimum at 28-32 degrees C. The cells contained ubiquinone Q-8. The DNA G+C content of the new strains was 62.3-64.2 mol %. According to the results of analysis of their 16S rRNA genes, the isolates belong to the genus Thiobacillus within the subclass Betaproteobacteria. However, the similarity level of nucleotide sequences of the 16S rRNA genes was insufficient to assign the isolates to known species of this genus. The affiliation to the genus Thiobacillus was confirmed by DNA-DNA hybridization of the isolates with the type strain of the type species of the genus Thiobacillus, T. thioparus DSM 505T (= ATCC 8158T). Despite the phenotypic similarity, the hybridization level was as low as 21-29%. In addition, considerable differences were revealed in the structure of the genes encoding RuBPC, the key enzyme of autotrophic CO2 assimilation, between the known Thiobacillus species and the new isolates. Based on molecular-biological features and certain phenotypic distinctions, the new isolates were assigned to a new Thiobacillus species, T. sajanensis sp. nov., with the type strain 4HGT (= VKM B-2365T).     

Identification of enzyme-producing thermophilic bacilli isolated from marine vents of Aeolian Islands (Italy)

Enzyme-producing thermophilic bacilli were isolated from different thermal sites located in and around Aeolian Islands (Italy) and characterised by both molecular and culture-based methods. Spore-forming bacteria with optimal growth from 45 to 70 °C were isolated from submarine vents and a geothermal well of Aeolian Islands (Italy). Eighteen selected strains were screened for extracellular enzyme presence by using nine substrates: Tween 20, Tween 80, tributyrin, soluble starch, xylan, dextran, carragenan, gelatine and casein. Sixteen isolates were able to grow at pH 9. The isolates were differentiated on the basis of restriction pattern of their amplified 16S rDNA (ARDRA) prior to 16S rRNA gene sequence analysis. On the basis of the most complete sequencing results strain V3 was identified asGeobacillus thermodenitrificans, most of isolates (10/14) was similar at high level (≥95%) to different reference strains of the speciesBacillus licheniformis. The remaining isolates, exhibiting sequence similarity below 95%, may represent novel species of the genusBacillus.     

Fiber-degrading systems of different strains of the genus Fibrobacter

The S85 type strain of Fibrobacter succinogenes, a major ruminal fibrolytic species, was isolated 49 years ago from a bovine rumen and has been used since then as a model for extensive studies. To assess the validity of this model, we compared the cellulase- and xylanase-degrading activities of several other F. succinogenes strains originating from different ruminants, including recently isolated strains, and looked for the presence of 10 glycoside hydrolase genes previously identified in S85. The NR9 F. intestinalis type strain, representative of the second species of the genus, was also included in this study. DNA-DNA hybridization and 16S rRNA gene sequencing first classified the strains and provided the phylogenetic positions of isolates of both species. Cellulase and xylanase activity analyses revealed similar activity profiles for all F. succinogenes strains. However, the F(E) strain, phylogenetically close to S85, presented a poor xylanolytic system and weak specific activities. Furthermore, the HM2 strain, genetically distant from the other F. succinogenes isolates, displayed a larger cellulolytic profile on zymograms and higher cellulolytic specific activity. F. intestinalis NR9 presented a higher cellulolytic specific activity and a stronger extracellular xylanolytic activity. Almost all glycoside hydrolase genes studied were found in the F. succinogenes isolates by PCR, except in the HM2 strain, and few of them were detected in F. intestinalis NR9. As expected, the fibrolytic genes of strains of the genus Fibrobacter as well as the cellulase and xylanase activities are better conserved in closely related phylogenetic isolates.     

产耐热木聚糖酶细菌的分离鉴定及酶易错PCR致突变条件优化

从福建省永泰县温泉采集样品中筛选到1株产耐热木聚糖酶嗜热菌株TC-W7,并获得该木聚糖酶基因。在此基础上,采用易错PCR技术在木聚糖酶基因中引入突变,研究Mg2＋浓度、Mn2＋浓度、dTTP/dCTP浓度等条件对突变率的影响。通过形态特征、生理生化试验及16S rRNA序列相似性比对分析,初步鉴定菌株TC-W7为土壤芽胞杆菌（Geobacillus）,菌株TC-W7在最适温度75℃和 pH 8.2条件下,其木聚糖酶活力为215.83 U/mL,Triton X-100和DDT能显著增强该酶的活性。在 Mg2＋浓度为20μmol/L,Mn2＋浓度为0.80μmol/L,dTTP/dCTP浓度为0.30 mmol/L的致突变条件下,碱基突变率为0.98%。 Geobacillus sp. TC-W7产木聚糖酶具有较好的耐热和耐碱等工业应用特性,对该酶易错PCR致突变条件优化结果,可用于后续木聚糖酶的耐热定向进化。