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PubMed2001

Sporosarcina aquimarina sp. nov., a bacterium isolated from seawater in Korea, and transfer of Bacillus globisporus (Larkin and Stokes 1967), Bacillus psychrophilus (Nakamura 1984) and Bacillus pasteurii (Chester 1898) to the genus Sporosarcina as Sporosarcina globispora comb. nov., Sporosarcina psychrophila comb. nov. and Sporosarcina pasteurii comb. nov., and emended description of th.

A light-orange-coloured, facultatively anaerobic, rod-shaped bacterium (strain SW28T), which was isolated from seawater in Korea, was taxonomically studied by a polyphasic approach. This organism formed round terminal endospores in swollen sporangia. The peptidoglycan type is A4alpha, based on L-Lys-L-Ala-D-Asp. The predominant menaquinone is MK-7 and the major fatty acid is ante-C15:0. The G+C content of the DNA is 40 mol%. Phylogenetic analysis based on 16S rDNA sequences showed that strain SW28T falls within the radiation of a cluster comprising the rRNA group 2 bacilli and non-Bacillus-type organisms. Strain SW28T showed the highest degree of relatedness to the type strain of Sporosarcina ureae, sharing 96.8% 16S rDNA similarity. Levels of DNA-DNA relatedness between strain SW28T and S. ureae DSM 2281T and the type strains of some Bacillus species forming a coherent phylogenetic cluster are less than 12.5%. On the basis of phenotypic and chemotaxonomic characteristics, 16S rDNA sequence analysis and DNA-DNA relatedness data, it is proposed that strain SW28T should be placed in the genus Sporosarcina as a new species, Sporosarcina aquimarina sp. nov. The type strain is SW28T (= KCCM 41039T = JCM 10887T). From the results of the taxonomic re-evaluation, it is also proposed that Bacillus globisporus, Bacillus psychrophilus and Bacillus pasteurii be transferred to the genus Sporosarcina as Sporosarcina globispora, Sporosarcina psychrophila and Sporosarcina pasteurii, respectively.

Bacillaceae↗
PubMed2001

Alkaliphilus transvaalensis gen. nov., sp. nov., an extremely alkaliphilic bacterium isolated from a deep South African gold mine.

A novel extreme alkaliphile was isolated from a mine water containment dam at 3.2 km below land surface in an ultra-deep gold mine near Carletonville, South Africa. The cells of this bacterium were straight to slightly curved rods, motile by flagella and formed endospores. Growth was observed over the temperature range 20-50 degrees C (optimum 40 degrees C; 45 min doubling time) and pH range 8.5-12.5 (optimum pH 10.0). The novel isolate, one of the most alkaliphilic micro-organisms yet described, was a strictly anaerobic chemo-organotroph capable of utilizing proteinaceous substrates such as yeast extract, peptone, tryptone and casein. Elemental sulfur, thiosulfate or fumarate, when included as accessory electron acceptors, improved growth. The G+C content of genomic DNA was 36.4 mol %. Phylogenetic analysis based on the 16S rDNA sequence indicated that the isolate is a member of cluster XI within the low G+C gram-positive bacteria, but only distantly related to previously described members. On the basis of physiological and molecular properties, the isolate represents a novel species, for which the name Alkaliphilus transvaalensis gen. nov., sp. nov. is proposed (type strain SAGM1T = JCM 10712T = ATCC 700919T). The mechanism of generation of the highly alkaline microbial habitat and the possible source of the alkaliphile are discussed.

Bacillaceae↗
PubMed2001

Thermoanaerobacter tengcongensis sp. nov., a novel anaerobic, saccharolytic, thermophilic bacterium isolated from a hot spring in Tengcong, China.

A new, extremely thermophilic bacterium, designated strain MB4T, was isolated from a Chinese hot spring. The new isolate was an obligately anaerobic, rod-shaped, gram-negative, saccharolytic bacterium. Spore formation was not observed. Growth occurred at temperatures between 50 and 80 degrees C, with an optimum of around 75 degrees C; at pH values between 5.5 and 9.0, with an optimum of 7.0-7.5; and at salinities between 0 and 2.5% NaCl, with an optimum of around 0.2% NaCl. The organism utilized glucose, galactose, maltose, cellobiose, mannose, fructose, lactose, mannitol and starch. Acetate was the main end product from glucose fermentation. Thiosulfate and sulfur were reduced to hydrogen sulfide. Sulfate, sulfite and nitrate were not reduced. Growth was inhibited by hydrogen. The G+C content of the DNA was 33 mol%. Phylogenetic analyses based on the 16S rDNA sequence indicated that the isolate was a new member of the genus Thermoanaerobacter and formed a monophyletic unit within the Thermoanaerobacter cluster. Based on its phenotypic and phylogenetic characteristics, the isolate was proposed as a new species, Thermoanaerobacter tengcongensis. The type strain is MB4T (= Chinese Collection of Microorganisms AS 1.2430T = JCM 11007T).

Anaerobiosis↗
PubMed2001

Simplified technique for identification of the aerobic spore-forming bacteria by phenotype.

The use of modern research approaches of genetics, biochemistry and molecular biology has led to progress in bacterial taxonomy. Systematic study of the aerobic spore-forming bacteria has resulted in the realignment of the genus Bacillus into several new genera. In the meantime, the identification process has become more difficult for the non-specialist in Bacillus taxonomy. This paper presents a key for the simplified phenotypic identification of the mesophilic, aerobic, spore-forming bacteria belonging to the genera Bacillus, Paenibacillus, Brevibacillus, Aneurinibacillus, Geobacillus and Virgibacillus. A total of 81 species were included and 115 morphological and physiological tests were analysed for their discriminative efficiency. This key is practical for rough but quick identification of aerobic spore-forming bacteria isolated from nature. Such preliminary identification will be helpful for the selection of reference strains and methods for more precise identification using the newest techniques. The reliability of the proposed identification key was tested on 100 cultures from the Ukrainian Collection of Microorganisms. The developed identification key is represented in interactive mode on a website (http://www/imv.kiev.ua/key/).

Bacillaceae↗
PubMed2001

Facklamia miroungae sp. nov., from a juvenile southern elephant seal (Mirounga leonina).

An unusual gram-positive, catalase-negative, facultatively anaerobic, coccus-shaped organism that originated from a juvenile elephant seal was characterized by phenotypic and molecular taxonomic methods. Comparative 16S rRNA gene sequencing showed that the unknown coccus represents a new subline within the genus Facklamia. The unknown strain was readily distinguishable from all currently recognized species of the genus Facklamia (Facklamia hominis, Facklamia languida, Facklamia ignava, Facklamia sourekii and Facklamia tabacinasalis) by biochemical tests and electrophoretic analysis of whole-cell proteins. Based on phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium be classified as Facklamia miroungae sp. nov. The type strain of F. miroungae is CCUG 42728T (= CIP 106764T). F. miroungae is the first member of the genus Facklamia to be isolated from an animal other than man.

Animals↗
PubMed2001

Planomicrobium koreense gen. nov., sp. nov., a bacterium isolated from the Korean traditional fermented seafood jeotgal, and transfer of Planococcus okeanokoites (Nakagawa et al. 1996) and Planococcus mcmeekinii (Junge et al. 1998) to the genus Planomicrobium.

A bacterial strain, JG07T, isolated from the Korean traditional fermented seafood jeotgal, was subjected to a polyphasic taxonomic study. Cells of strain JG07T are cocci or short rods in the early growth phase but change to rods as the cultures age. The peptidoglycan type is A4alpha, based on L-Lys-D-Glu. The menaquinone profile is characterized by the predominance of MK-8 followed by MK-7 and MK-6. The cellular fatty acid profile contains major amounts of saturated, unsaturated and branched fatty acids. The cellular phospholipids are phosphatidylethanolamine, phosphatidylglycerol and bisphosphatidylglycerol. The G+C content of the DNA is 47 mol%. Phylogenetic analysis showed that strain JG07T forms a cluster with Planococcus okeanokoites and Planococcus mcmeekinii, and the relationship between this cluster and two other Planococcus species described previously is supported by bootstrap analysis at a confidence level of 100%. The 16S-23S internally transcribed spacer (ITS) sequence similarity and DNA-DNA relatedness values between strain JG07T and the type strains of other Planococcus species are in the range 74.6-83.2% and 10.4-20.5%, respectively. On the basis of the phenotypic and phylogenetic data and the genomic distinctiveness, strain JG07T is considered to represent a new genus and a new species, for which the name Planomicrobium koreense gen. nov., sp. nov. is proposed. It is also proposed that Planococcus okeanokoites and Planococcus mcmeekinii be transferred to the new genus Planomicrobium as Planomicrobium okeanokoites and Planomicrobium mcmeekinii, respectively.

Bacillaceae↗
PubMed2001

Proposal of the genera Anaerococcus gen. nov., Peptoniphilus gen. nov. and Gallicola gen. nov. for members of the genus Peptostreptococcus.

Members of genus Peptostreptococcus have previously been found to be distantly related to the type species, Peptostreptococcus anaerobius, on the basis of 16S rDNA sequence similarities. They were divided into three major phylogenetic groups, and their peptidoglycan structure and biochemical traits differed between groups. The reclassification of the species of these three groups into three new genera, Peptoniphilus gen. nov., Anaerococcus gen. nov. and Gallicola gen. nov., is proposed. The genus Peptoniphilus gen. nov. includes the following butyrate-producing, non-saccharolytic species that use peptone and amino acids as major energy sources: Peptoniphilus asaccharolyticus comb. nov. (type species), Peptoniphilus lacrimaris comb. nov., Peptoniphilus harei comb. nov., Peptoniphilus indolicus comb. nov. and Peptoniphilus ivorii comb. nov. The genus Anaerococcus gen. nov. contains the saccharolytic, butyrate-producing species Anaerococcus prevotii comb. nov. (type species), Anaerococcus tetradius comb. nov., Anaerococcus lactolyticus comb. nov., Anaerococcus hydrogenalis comb. nov., Anaerococcus vaginalis comb. nov. and Anaerococcus octavius sp. nov. The genus Gallicola gen. nov. contains a single species, Gallicola barnesae comb. nov.

Animals↗
PubMed2001

Thermoanaerobacter yonseiensis sp. nov., a novel extremely thermophilic, xylose-utilizing bacterium that grows at up to 85 degrees C.

A novel strictly anaerobic, extremely thermophilic, spore-forming and xylose-utilizing bacterium, designated strain KB-1TP (type and patent strain), was isolated from a geothermal hot stream at Sileri on Java island, Indonesia. The cells were rod-shaped, motile and had terminal spores. The newly isolated strain stained gram-positive and the cells occurred singly or in pairs during the exponential growth phase. The temperature optimum for growth was 75 degrees C and growth occurred in the range 50-85 degrees C. The pH range for growth was 4.5-9.0, with an optimum at pH 6.5. Strain KB-1TP grew chemo-organotrophically by fermenting a wide range of substrates such as glucose, fructose, D-xylose, lactose, maltose, sucrose, mannose, galactose, cellobiose, pullulan and soluble starch. Arabinose, xylan, cellulose, olive oil and Tween 80 were not fermented. The predominant fermentation end products after growth on glucose were lactate, acetate, ethanol, CO2 and small amounts of isovaleric acid, butyric acid, propionic acid, 1-pentanol and 2-propanol. Thiosulfate was reduced to H2S. Strain KB-1TP was sensitive to tetracycline, chloramphenicol, penicillin G, neomycin, kanamycin, vancomycin and rifampicin at concentrations of 100 microg ml(-1). No effect was observed with chloramphenicol and neomycin at concentrations of 10 microg ml(-1). This indicates that strain KB-1TP belongs to the bacterial domain. The G+C content of the DNA was 37 mol%. The comparison of the 165 rDNA sequence to that of closely related strains revealed that strain KB-1TP belonged to clostridial cluster V, showing highest sequence identities (92.7%) to members of the genus Thermoanaerobacter. Taking into account the physiological and molecular properties of the new isolate, it is proposed that strain KB-1TP should be classified as a new species of the genus Thermoanaerobacter, designated Thermoanaerobacter yonseiensis. The type strain, KB-1TP, has been deposited in the Korean Federation of Culture Collections (KFCC 11116P) as a patent strain and in the Deutsche Sammlung von Mikroorganismen und Zellkulturen as a type strain (= DSM 13777T).

Bacillaceae↗
PubMed2001

Reclassification of Saccharococcus caldoxylosilyticus as Geobacillus caldoxylosilyticus (Ahmad et al. 2000) comb. nov.

A polyphasic study was performed on five thermophilic strains belonging to the genus Bacillus, isolated from soil of different geographical areas. 16S rRNA gene sequence analysis placed these isolates in RNA group 5, with Saccharococcus caldoxylosilyticus and [Bacillus] thermoglucosidasius being the closest phylogenetic neighbours. The type species of Saccharococcus, Saccharococcus thermophilus, was only moderately related to these two species and the novel isolates. DNA-DNA hybridization studies and comparison of morphological, chemotaxonomic and phenotypic features supported the close relationship between the novel isolates and Saccharococcus caldoxylosilyticus. These data justify the reclassification of Saccharococcus caldoxylosilyticus. Following the transfer of the validly described Bacillus species of group 5 into the genus Geobacillus, the reclassification of Saccharococcus caldoxylosilyticus as Geobacillus caldoxylosilyticus comb. nov. is proposed. This species can be distinguished genomically from Geobacillus thermoglucosidasius, Geobacillus stearothermophilus, Geobacillus thermodenitrificans and Saccharococcus thermophilus by a specific PCR-RFLP assay targeting the 16S rDNA.

Bacillaceae↗
PubMed2002

PAH-degradation by Paenibacillus spp. and description of Paenibacillus naphthalenovorans sp. nov., a naphthalene-degrading bacterium from the rhizosphere of salt marsh plants.

Bacteria belonging to the genus Paenibacillus were isolated by enrichment from petroleum-hydrocarbon-contaminated sediment and salt marsh rhizosphere using either naphthalene or phenanthrene as the sole carbon source, and were characterized using phenotypic, morphological and molecular techniques. The isolates were grouped by their colony morphologies and polyaromatic hydrocarbon-degradation patterns. Phenanthrene-degrading isolates produced mottled colonies on solid media and were identified as P. validus by fatty acid methyl ester and 16S rRNA gene sequence analyses. In contrast, the naphthalene-degrading isolates with mucoid colony morphology were distantly related to Paenibacillus validus, according to fatty acid methyl ester and 16S rRNA gene sequence analyses. The predominant fatty acids of the mucoid isolates were 15:0 anteiso, 16:1omega11c, 16:0 and 17:0 anteiso, constituting, on average, 50.5, 12.0, 11.2 and 6.5% of the total, respectively. The G+C contents of their DNA ranged from 47 to 52 mol%. The 16S rDNA sequence analysis revealed the highest (< or = 94%) similarity to P. validus. In addition, phylogenetic analyses based on 16S rDNA sequences showed that the mucoid isolates formed a distinct cluster within Paenibacillus. DNA-DNA hybridization experiments showed only a 6% DNA similarity between the type strain of P. validus and mucoid strain PR-N1. On the basis of the morphological, phenotypic and molecular data, the naphthalene-degrading isolates merit classification as a new Paenibacillus species, for which the name Paenibacillus naphthalenovorans sp. nov. is proposed, with PR-N1T (= ATCC BAA-206T = DSM 14203T) as the type strain.

Bacillaceae↗
PubMed2002

Exiguobacterium undae sp. nov. and Exiguobacterium antarcticum sp. nov.

Four orange-pigmented strains from pond water (L1-L4) have been subjected to polyphasic taxonomic analyses. On the basis of ribotype analysis and Fourier-transform infrared spectroscopy, these strains form a genomically highly related group. 16S rDNA sequence analysis revealed 98.8% similarity between the 16S rDNA sequences of strains L2T and H2T, isolated previously from a microbial mat from Lake Fryxell, Antarctica. DNA-DNA reassociation values indicated the presence of two genomic clusters. While the DNA of strains L2T and L3 showed 100% DNA relatedness, strains L2T and H2T shared only 51% DNA relatedness. These two clusters differed in some phenotypic properties, e.g. utilization of melibiose, D-mannitol, adenosine 5'-monophosphate and uridine 5'-monophosphate, and in their fatty acid compositions. Based on the composition of isoprenoid quinones, peptidoglycan, polar lipids and fatty acids, these organisms are members of the genus Exiguobacterium. This is supported by 16S rDNA analyses, which revealed 97-98% similarity to Exiguobacterium acetylicum DSM 20416T and 93.2-93.8% similarity to Exiguobacterium aurantiacum DSM 6208T. E. acetylicum DSM 20416T, the closest phylogenetic neighbour, shows only 39% DNA similarity to strain L2T and 40% DNA similarity to strain H2T. Based on genomic distinctiveness and the clear differences in chemotaxonomy and physiology, two novel species are proposed, Exiguobacterium undae sp. nov. and Exiguobacterium antarcticum sp. nov.

Bacillaceae↗
PubMed2002

Shuttleworthia satelles gen. nov., sp. nov., isolated from the human oral cavity.

Nine strains of anaerobic, non-spore-forming, gram-positive bacilli, isolated from the human oral cavity and provisionally identified as belonging to the genus Eubacterium, were subjected to a comprehensive range of phenotypic and genetic tests. Biochemically, they were found to comprise a homogeneous group, and phylogenetic analysis of their 16S rRNA sequences indicated that they constitute a unique branch within the Clostridium-Bacillus subphylum of the phylum Firmicutes. All of the isolates displayed an unusual colonial morphology after extended incubation. This resembled a contaminated culture in that small, secondary colonies were seen to arise around and from within the primary colony form, and a third, independent, colony type was also seen. However, inspection of the colonies by Gram-staining and scanning electron microscopy together with protein profile analysis and 16S rRNA gene sequence comparison of the two independent colony types revealed that only a single organism was present. A new genus, Shuttleworthia, and the species Shuttleworthia satelles gen. nov., sp. nov., are proposed. The cells are saccharolytic, and acetate, butyrate and lactate are produced as end products of glucose fermentation. Aesculin is hydrolysed and indole is produced. The G+C content of the DNA of the type strain is 51 mol%. The type strain is strain DSM 14600T (= CCUG 45864T = VPI D143K-13T).

Adolescent↗
PubMed2002

Lentibacillus salicampi gen. nov., sp. nov., a moderately halophilic bacterium isolated from a salt field in Korea.

A Gram-variable, aerobic, endospore-forming, rod-shaped bacterial strain, SF-20(T), which was isolated from a salt field in Korea, was subjected to a polyphasic taxonomic study. Cells of this organism were motile by means of single flagella. Strain SF-20(T) grew optimally in the presence of 4-8% NaCl. The cell wall peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. The predominant menaquinone is MK-7. Strain SF-20(T) has a cellular fatty acid profile containing major amounts of branched fatty acids. The major fatty acids are anteiso-C15:0 and iso-C16:0. The cellular phospholipids are phosphatidylglycerol and diphosphatidylglycerol. The G+C content of the DNA is 44 mol%. Strain SF-20(T) is phylogenetically closely related to the genus Bacillus and some related genera and, particularly, formed a coherent cluster with the genera Salibacillus and Virgibacillus. The clustering fidelity between strain SF-20(T) and the cluster comprising these two genera was supported by bootstrap analysis at a confidence level of 67.2%. Strain SF-20(T) exhibited levels of 16S rDNA similarity of 93.0-94.7% to the genus Salibacillus and 94.0-94.1% to the genus Virgibacillus. On the basis of phenotypic and phylogenetic data, strain SF-20(T) should be classified in a novel genus and species, for which the name Lentibacillus salicampi gen. nov., sp. nov. is proposed. The type strain is strain SF-20(T) (= KCCM 41560(T) = JCM 11462(T)).

Bacillaceae↗
PubMed2002

Amplified fragment length polymorphism (AFLP) analysis of Clostridium novyi, C. perfringens and Bacillus cereus isolated from injecting drug users during 2000.

As part of the follow-up investigations associated with an outbreak of severe illness and death among illegal injecting drug users during 2000, 43 cultures of Clostridium novyi type A, 40 C. perfringens type A and 6 isolates of Bacillus cereus were characterised by amplified fragment length polymorphism (AFLP) analysis. Among the 43 C. novyi isolates, 23 different AFLP profiles were detected. The same AFLP profile was detected in isolates from 18 drug users investigated during 2000 from Scotland, England, the Republic of Ireland and Norway and a wound from a patient in 2000 who was not identified as a drug user. Unique AFLP profiles were obtained from four drug users from England and the Republic of Ireland, 10 historical isolates from culture collections, an isolate from food (1989) and three isolates from wounds (1995, 1991, 1988). The 40 C. perfringens isolates were from 13 drug users, the contents of one syringe and two samples of heroin. Sixteen AFLP types of C. perfringens were distinguished and there was little evidence for commonality among the isolates. The AFLP types of C. perfringens from heroin differed and were unique. Six isolates of B. cereus were from four drug users and two samples of heroin. Four different AFLP patterns were distinguished. Three AFLP types were isolated from four drug users. B. cereus isolates from an aspirate and a heroin sample collected from the same drug user were identical, and were also indistinguishable from an isolate from a groin infection in a second drug user. The AFLP type of the isolate from a second and unrelated heroin sample was unique. The AFLP results showed no or very limited evidence for commonality between the different isolates of B. cereus and C. perfringens. In marked contrast, the C. novyi isolates from the majority of the drug users during 2000 were homogeneous, suggesting a common source or clonal selection of a C. novyi type, or both, which either had an adaptive advantage in spore germination, survival or growth following the drug preparation and the injection procedure, or produced a more severe clinical presentation.

Animals↗
PubMed1979

Lipoquinones of some spore-forming rods, lactic-acid bacteria and actinomycetes.

The respiratory quinones of 73 strains of Gram-positive bacteria including spore-forming rods, lactic-acid bacteria and actinomyctes were examined. Menaquinones with seven isoprenoid units (MK-7) were the main quinone type found in representatives of the genus Bacillus and in Sporolactobacillus inulinus. However, a strain of B. thuringiensis produced MK-8 in addition to MK-7, and strains of B. lentus and B. pantothenticus appeared to produce MK-9 and MK-8, respectively, with no MK-7. In the clostridia and lactic-acid bacteria, no quinones were found, except in Pediococcus cerevisiae NCTC 8066 and Lactobacillus casei subsp. rhamnosus ATCC 7469, which contained menaquinones, and Streptococcus faecalis NCTC 775 and HIM 478-1, which contained demethylmenaquinones, in relatively low concentrations. Menaquinones were also found in the actinomycetes (except Actinomyces odontolyticus and Bifidobacterium bifidum which did not produce any quinones) and in Protaminobacter alboflavus ATCC 8458, the so-called Actinobacillus actinoides ATCC 15900 and Noguchia granulosis NCTC 10559.

Actinomycetales↗
PubMed2000

The plcR regulon is involved in the opportunistic properties of Bacillus thuringiensis and Bacillus cereus in mice and insects.

Bacillus thuringiensis has been widely used for 40 years as a safe biopesticide for controlling agricultural pests and mosquitoes because it produces insecticidal crystal proteins. However, spores have also been shown to contribute to overall entomopathogenicity. Here, the opportunistic properties of acrystalliferous B. thuringiensis Cry(-) and Bacillus cereus strains were investigated in an insect species, Galleria mellonella, and in a mammal, BALB/c mice. In both animal models, the pathogenicity of the two bacterial species was similar. Mutant strains were constructed in which the plcR gene, encoding a pleiotropic regulator of extracellular factors, was disrupted. In larvae, co-ingestion of 10(6) spores of the parental strain with a sublethal concentration of Cry1C toxin caused 70% mortality whereas only 7% mortality was recorded if spores of the DeltaplcR mutant strain were used. In mice, nasal instillation of 10(8) spores of the parental strain caused 100% mortality whereas instillation with the same number of DeltaplcR strain spores caused much lower or no mortality. Similar effects were obtained if vegetative cells were used instead of spores. The cause of death is unknown and is unlikely to be due to actual growth of the bacteria in mice. The lesions caused by B. thuringiensis supernatant in infected mice suggested that haemolytic toxins were involved. The cytolytic properties of strains of B. thuringiensis and B. cereus, using sheep, horse and human erythrocytes and G. mellonella haemocytes, were therefore investigated. The level of cytolytic activity is highly reduced in DeltaplcR strains. Together, the results indicate that the pathogenicity of B. thuringiensis strain 407 and B. cereus strain ATCC 14579 is controlled by PlcR.

Animals↗
PubMed2000

A novel thermostable multidomain 1,4-beta-xylanase from 'Caldibacillus cellulovorans' and effect of its xylan-binding domain on enzyme activity.

The nucleotide sequence of the complete xynA gene, encoding a novel multidomain xylanase XynA of 'Caldibacillus cellulovorans', was determined by genomic-walking PCR. The putative XynA comprises an N-terminal domain (D1), recently identified as a xylan-binding domain (XBD), homologous to non-catalytic thermostabilizing domains from other xylanases. D1 is followed by a xylanase catalytic domain (D2) homologous to family 10 glycosyl hydrolases. Downstream of this domain two cellulose-binding domains (CBD), D3 and D4, were found linked via proline-threonine (PT)-rich peptides. Both CBDs showed sequence similarity to family IIIb CBDs. Upstream of xynA an incomplete open reading frame was identified, encoding a putative C-terminal CBD homologous to family IIIb CBDs. Two expression plasmids encoding the N-terminal XBD plus the catalytic domain (XynAd1/2) and the xylanase catalytic domain alone (XynAd2) were constructed and the biochemical properties of the recombinant enzymes compared. The absence of the XBD resulted in a decrease in thermostability of the catalytic domain from 70 degrees C (XynAd1/2) to 60 degrees C (XynAd2). Substrate-specificity experiments and analysis of the main products released from xylan hydrolysis indicate that both recombinant enzymes act as endo-1, 4-beta-xylanases, but differ in their ability to cleave small xylooligosaccharides.

Bacillaceae↗
PubMed2000

Three multidomain esterases from the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17 that carry divergent dockerin sequences.

Three enzymes carrying esterase domains have been identified in the rumen cellulolytic anaerobe Ruminococcus flavefaciens 17. The newly characterized CesA gene product (768 amino acids) includes an N-terminal acetylesterase domain and an unidentified C-terminal domain, while the previously characterized XynB enzyme (781 amino acids) includes an internal acetylesterase domain in addition to its N-terminal xylanase catalytic domain. A third gene, xynE, is predicted to encode a multidomain enzyme of 792 amino acids including a family 11 xylanase domain and a C-terminal esterase domain. The esterase domains from CesA and XynB share significant sequence identity (44%) and belong to carbohydrate esterase family 3; both domains are shown here to be capable of deacetylating acetylated xylans, but no evidence was found for ferulic acid esterase activity. The esterase domain of XynE, however, shares 42% amino acid identity with a family 1 phenolic acid esterase domain identified from Clostridum thermocellum XynZ. XynB, XynE and CesA all contain dockerin-like regions in addition to their catalytic domains, suggesting that these enzymes form part of a cellulosome-like multienzyme complex. The dockerin sequences of CesA and XynE differ significantly from those previously described in R. flavefaciens polysaccharidases, including XynB, suggesting that they might represent distinct dockerin specificities.

Acetylesterase↗