Isolation and characterization of a new poly(3-hydroxybutyrate)-degrading, denitrifying bacterium from activated sludge

One hundred and forty isolates of thermophilic bacteria from the genus Thermus were screened for the presence of restriction endonuclease activity. Thermostable isoschizomers of restriction endonucleases, such as AceIII, BbvI, BglI, BsePI, FnuDII, HgiAI, MaeII, MboI, MseI, PvuII, StuI, TaqI, Tsp4CI, TspEI, XhoI and XmaIII, were isolated. Two restriction enzymes, TatI and TauI, recognizing novel degenerate sequences 5'-W (downward arrow)GTACW-3' and 5'-GCSG (downward arrow)C-3' respectively were partially purified and the recognition and cleavage sites were determined.     

Occurrence of restriction-modification systems in ruminal butyrate-producing bacteria

Thirty-five strains of ruminal bacteria belonging to the former Butyrivibrio fibrisolvens species were screened for the presence of site-specific restriction endonuclease and modification methyltransferase activities. Seven strains possessed endonuclease activities detectable in crude cell extracts. The recognition sequences and optimal reaction conditions for seven of them were determined. Five enzymes were found to be isoschizomers of type II endonucleases (EcoRV, NsiI, AseI (2x) and SauI), one was type IIS (FokI) and two remained unknown. The optimal reaction buffer was found to be a low ionic strength buffer and all enzymes possessed sufficient activity at 39 degrees C. The presence of DNA modification among all strains was also determined. Most of the methylation activities correlated with restriction activities, yet some strains possessed unaccompanied modification methyltransferases.     

Sequence-specific endonucleases in strains of Anabaena and Nostoc

The complements of restriction endonucleases of 12 strains of cyanobacteria were determined in cell-free extracts, and were compared with the complements of restriction activities assessed by measuring the relative efficiencies of plating of cyanophages on those cyanobacteria. The hosts which were susceptible to all of the phages contained endo R · AvaI and endo R · AvaII, and in several cases probably endo R · AvaIII, or isoschizomers of these enzymes. Three hosts which were lysed by only a subset (1 or 3) of the phages contained different restriction endonuclease. Anabaena sp. PCC 7120 showed apparent phenotypic restriction of phage An-22 grown in hosts with (isoschizomers of) AvaI, II and III, but no corresponding endonuclease has yet been detected in vitro. Nostoc sp. ATCC 29131 (PCC 6705) was found to contain a restriction enzyme, NspBII, with hitherot unknown specificity, C(A/C)GC(T/G)G.     

A method for detecting new strains producing DNA modification and restriction enzymes--isoschizomers and isomethylomers of known restrictases and methylases

The paper describes a technique for the detection of new strains producing enzymes which mediate DNA modification and restriction, and isoschizomers and isomethylomers of the known restriction endonucleases and methylases. Three Bacillus subtilis strains whose DNA carries a BamH1 modification have been found. Two of these strains exert the restrictase activity with an R BamH1 specificity.     

A simple strategy for the purification of large thermophilic proteins overexpressed in mesophilic microorganisms: application to multimeric enzymes from Thermus sp. strain T2 expressed in Escherichia coli

The heating of protein preparations of mesophilic organism (e.g., E. coli) produces the obliteration of all soluble multimeric proteins from this organism. In this way, if a multimeric enzyme from a thermophilic microorganism is expressed in these mesophilic hosts, the only large protein remaining soluble in the preparation after heating is the thermophilic enzyme. These large proteins may be then selectively adsorbed on lowly activated anionic exchangers, enabling their full purification in just these two simple steps. This strategy has been applied to the purification of an alpha-galactosidase and a beta-galactosidase from Thermus sp. strain T2, both expressed in E. coli, achieving the almost full purification of both enzymes in only these two simple steps. This very simple strategy seems to be of general applicability to the purification of any thermophilic multimeric enzyme expressed in a mesophilic host.     

Endophytic bacilli--producers of type II restriction endonucleases

Two of thirteen bacillar strains isolated from the inner tissues of cotton plants were found to produce type II restriction endonucleases. The investigation of the site specificity of these enzymes showed that they are AsuI and Eco31I isoschizomers.     

Genomic restriction map of the extremely thermophilic bacterium Thermus thermophilus HB8.

A physical map of the chromosome of the extremely thermophilic eubacterium Thermus thermophilus HB8 has been constructed by using pulsed-field gel electrophoresis techniques. A total of 26 cleavage sites for the rarely cutting restriction endonucleases HpaI, MunI, and NdeI were located on the genome. On the basis of the sizes of the restriction fragments generated, the genome size was estimated to be 1.74 Mbp, which is significantly smaller than the chromosomes of Escherichia coli and other mesophiles. Partial digestion experiments revealed the order of the six HpaI bands on the chromosome. Hybridization of isolated restriction fragments to pulsed-field gel-separated restriction digestions confirmed the deduced order of the HpaI fragments and allowed ordering and alignment of the NdeI and MunI fragments. In addition, 16 genes or gene clusters cloned from several different Thermus strains were located on the T. thermophilus HB8 chromosomal map by hybridization of gene probes to pulsed-field gel-resolved restriction digestions.     

Eight new restriction endonucleases fröm Herpetosiphon giganteus--divergent evolution in a family of enzymes.

Characterization of eight restriction endonucleases isolated from five strains of Herpetosiphon giganteus is described. HgiCI from strain Hpg9 recognizes and cleaves the degenerate sequence: GGPyPuCC, producing 5'-hexanucleotide protruding ends. Endonucleases HgiBI, HgiCII and HgiEI are isoschizomers of AvaII; HgiCIII and HgiDII are isoschizomers of SalI; and HgiDI and HgiGI are isoschizomers of AcyI. Based upon their closely related and in part overlapping recognition specificities a close evolutionary relationship is proposed for all known Hgi restriction endonucleases.     

Endophytic Bacilli Producing Type II Restriction Endonucleases

Two of thirteen bacillar strains isolated from the inner tissues of cotton plants were found to produce type II restriction endonucleases. The investigation of the site specificity of these enzymes showed that they are AsuI and Eco31I isoschizomers.     

Site-specific restriction endonucleases in cyanobacteria

AIM: Planktic cyanobacteria were screened for endodeoxyribonucleases. Principal component analysis (PCA) was employed to demonstrate a potential relationship between certain enzymes and a group of cyanobacteria. The data were obtained from a data bank and this study. METHODS AND RESULTS: Enzymes were partially purified using column chromatography. Anabaena strains contained Asp83/1I (5'-TTCGAA-3'), Asp83/1II (5'-GGCC-3'), Asp90I (5'-ACRYGT-3') and five isoschizomeric enzymes (5'-ATCGAT-3'). Aphanizomenon and Microcystis strains contained ApcTR183I (5'-TGCGCA-3') and Msp199I (5'-CCGG-3'), respectively. Planktothrix strains possessed Psc2I (5'-GAANNNNTTC-3'), Psc27I and Psc28I (5'-TTCGAA-3'). PCA showed that the most common cyanobacterial endonuclease types were AvaII, AvaI and AsuII. CONCLUSIONS: All planktic cyanobacteria studied contained restriction endonucleases. The defined restriction endonucleases were isoschizomers of known enzymes. The Nostoc and the Spirulina genera had an association, while the majority of the genera had no association with certain endonuclease type(s). SIGNIFICANCE AND IMPACT OF THE STUDY: The defined enzymes in this study and the estimated trend in the endonuclease type distribution allow more efficient avoidance of cyanobacterial restriction barriers.     

Thermophilic fungi: their physiology and enzymes.

Thermophilic fungi are a small assemblage in mycota that have a minimum temperature of growth at or above 20 degrees C and a maximum temperature of growth extending up to 60 to 62 degrees C. As the only representatives of eukaryotic organisms that can grow at temperatures above 45 degrees C, the thermophilic fungi are valuable experimental systems for investigations of mechanisms that allow growth at moderately high temperature yet limit their growth beyond 60 to 62 degrees C. Although widespread in terrestrial habitats, they have remained underexplored compared to thermophilic species of eubacteria and archaea. However, thermophilic fungi are potential sources of enzymes with scientific and commercial interests. This review, for the first time, compiles information on the physiology and enzymes of thermophilic fungi. Thermophilic fungi can be grown in minimal media with metabolic rates and growth yields comparable to those of mesophilic fungi. Studies of their growth kinetics, respiration, mixed-substrate utilization, nutrient uptake, and protein breakdown rate have provided some basic information not only on thermophilic fungi but also on filamentous fungi in general. Some species have the ability to grow at ambient temperatures if cultures are initiated with germinated spores or mycelial inoculum or if a nutritionally rich medium is used. Thermophilic fungi have a powerful ability to degrade polysaccharide constituents of biomass. The properties of their enzymes show differences not only among species but also among strains of the same species. Their extracellular enzymes display temperature optima for activity that are close to or above the optimum temperature for the growth of organism and, in general, are more heat stable than those of the mesophilic fungi. Some extracellular enzymes from thermophilic fungi are being produced commercially, and a few others have commercial prospects. Genes of thermophilic fungi encoding lipase, protease, xylanase, and cellulase have been cloned and overexpressed in heterologous fungi, and pure crystalline proteins have been obtained for elucidation of the mechanisms of their intrinsic thermostability and catalysis. By contrast, the thermal stability of the few intracellular enzymes that have been purified is comparable to or, in some cases, lower than that of enzymes from the mesophilic fungi. Although rigorous data are lacking, it appears that eukaryotic thermophily involves several mechanisms of stabilization of enzymes or optimization of their activity, with different mechanisms operating for different enzymes.     

Quick identification of Type I restriction enzyme isoschizomers using newly developed pTypeI and reference plasmids

Although DNA-recognition sequences are among the most important characteristics of restriction enzymes and their corresponding methylases, determination of the recognition sequence of a Type-I restriction enzyme is a complicated procedure. To facilitate this process we have previously developed plasmid R-M tests and the computer program RM search. To specifically identify Type-I isoschizomers, we engineered a pUC19 derivative plasmid, pTypeI, which contains all of the 27 Type-I recognition sequences in a 248-bp DNA fragment. Furthermore, a series of 27 plasmids (designated 'reference plasmids'), each containing a unique Type-I recognition sequence, were also constructed using pMECA, a derivative of pUC vectors. In this study, we tried those vectors on 108 clinical E. coli strains and found that 48 strains produced isoschizomers of Type I enzymes. A detailed study of 26 strains using these 'reference plasmids' revealed that they produce seven different isoschizomers of the prototypes: EcoAI, EcoBI, EcoKI, Eco377I, Eco646I, Eco777I and Eco826I. One strain EC1344 produces two Type I enzymes (EcoKI and Eco377I).     

Screening for restriction endonucleases in methane-oxidizing bacteria.

51 methane-oxidizing bacteria strains such as Methylomonas methanica, M. rubra, Methylococcus capsulatus, M. thermophilus, M. luteus, M. ucrainicus, M. whittenburyi, Methylosinus trichosporium, M. sporium, Methylocystis parvus isolated from various ecological niches and geographical regions of the Ukraine and also the strains received from R. Whittenbury and Y. Heyer were screened for restriction endonucleases. Type II restriction endonucleases were detected in IMV B-3112 (= 12 b), IMV B-3027 (= 26), IMV B-3019 (= 9 c), IMV B-3017 (= 17 c), IMV B-3226 (= 26 v), IMV B-3033 (= Y), IMV B-3100 (= 100) and IMV B-3494 (= 1E494). The results obtained were indicative of relatively high frequency of restriction enzymes occurrence in methane-oxidizing bacteria. There were Kpn I (Asp 7181) restriction endonuclease isoschizomers in crude extracts of IMV B-3112, B-3017, B-3019, B-3027 isolated from fresh-water silt as well as in IMV B-3226 strain isolated from waste-water silt. Although these isolates had bee previously considered as untypical strains of M. ucrainicus, more detailed study of their properties allowed placing them with Methylovarius luteus (= Methylococcus luteus). IMV B-3494 strain was identified as Methylococcus capsulatus. Strain IMV B-3033 had earlier been allocated to Methylovarius whittenburyi (= Methylococcus whittenburyi). Specificity of restriction endonucleases of this strain was not tested. Therefore, for the first time restriction endonucleases were detected in methane-oxidizing bacteria. 8 strains (3 species) among 51 strains (13 species) were found to produce restriction endonucleases displaying three different types of specificity in the least. Producers of restriction endonucleases having Kpn I (Asp 7181) specificity were isolated from different water and silt samples of the Dnieper flood-land more than 20 years ago.     

Testing and isolation of high-purity restriction endonucleases

A new method of testing restriction nucleases is proposed. This method is based on high-temperature treatment of crude cell extracts. Disrupted cells were heated at 50-60 degrees C, centrifuged, and assayed for restrictases. This method provides the opportunity for screening new enzymes in microbial strains enriched with nonspecific restrictases. High-temperature treatment of cell extracts of certain producers reduces the number of steps of the procedure used for isolating high-purity restrictases; the resulting preparations are capable of maintaining high enzymatic activity during long-term storage. It was shown that high-temperature treatment can be applied not only to thermophilic but also to mesophilic strains of microorganisms of different taxa.     

Restriction of bacteriophage plaque formation in Streptomyces spp.

Several Streptomyces species that produce restriction endonucleases were characterized for their ability to propagate 10 different broad host range bacteriophages. Each species displayed a different pattern of plaque formation. A restrictionless mutant of S. albus G allowed plaque formation by all 10 phages, whereas the wild-type strain showed plaques with only 2 phages. DNA isolated from three of the phages was analyzed for the presence of restriction sites for Streptomyces species-encoded enzymes, and a very strong correlation was established between the failure to form plaques on Streptomyces species that produced particular restriction enzymes and the presence of the corresponding restriction sites in the phage DNA. Also, the phages that lacked restriction sites in their DNA generally formed plaques on the corresponding restriction endonuclease-producing hosts at high efficiency. The DNAs from the three phages analyzed also generally contained either many or no restriction sites for the Streptomyces species-produced enzymes, suggesting a strong evolutionary trend to either eliminate all or tolerate many restriction sites. The data indicate that restriction plays a major role in host range determination for Streptomyces phages. Analysis of bacteriophage host ranges of many other uncharacterized Streptomyces hosts has identified four relatively nonrestricting hosts, at least two of which may be suitable hosts for gene cloning. The data also suggest that several restriction systems remain to be identified in the genus Streptomyces.     

Random amplified ribosomal DNA restriction analysis for rapid identification of thermophilic Actinomycete-like bacteria involved in hypersensitivity pneumonitis

Hypersensitivity pneumonitis (HP) is a pulmonary disease characterised by inflammation that can be caused by, amongst other substances, a subset of 4 thermophilic mycelial bacteria: Saccharopolyspora rectivirgula, Saccharomonospora viridis, Thermoactinomyces sacchari, and Thermoactinomyces vulgaris. Air sampling analyses in highly contaminated environments are often performed to evaluate exposure to these species which are difficult and fastidious to identify by conventional techniques. The aim of this study was to use amplified ribosomal DNA restriction analysis (ARDRA) to develop a method of identification for those thermophilic organisms that would be more rapid and simple. Strains of these 4 species were obtained from the American type culture collection (ATCC) and were characterized using biochemical tests and ARDRA patterns obtained on their partial-lenght amplified 16S rDNAs. To validate this approach, ARDRA with two restriction enzymes, TaqI and HhaI, was applied to 49 thermophilic actinomycete-like strains from environmental samples (sawmills). The results obtained show that combining some cultural characteristics and biochemical tests, such as xanthine or hypoxanthine decomposition, growth in the presence of NaCl, lysozyme or novobiocin, and spore resistance over 100 degrees C provide a rough identification and selection of the genera of interest. Consequently, target species could be confirmed by digestion of partial-lenght 16S rDNA with the use of Taql and HhaI restriction enzymes that gave specific restriction patterns. ARDRA analyses on the 49 environmental actinomycete-like organisms revealed the presence of 8 Saccharopolyspora rectivirgula, 2 Saccharomonospora viridis, and 15 Thermoactinomyces vulgaris strains, the other strains had restriction patterns different than those of the species of interest. Results of the present study will be applicable to other potential HP environments such as dairy barns, peat bogs and compost plants.     

Restriction fragment fingerprint and genome sizes of Staphylococcus species using pulsed-field gel electrophoresis and infrequent cleaving enzymes.

Large restriction fragments of genomic DNA from Staphylococcus species were separated by pulsed-field gel electrophoresis (PFGE). Five different strains of S. aureus (ISP8, SAU3A, PS96, ATCC 6538, ATCC 15564) and three representative strains of S. haemolyticus SM102, S. warneri MCS4, S. cohnii LK478 from human hosts, and one strain of S. aureus (ATCC 8432) from an avian host were used in this study. Since Staphylococcus is A + T rich (approximately 67%), restriction fragments were obtained by digesting chromosomal DNA with endonucleases that recognize GC-rich sequences. Five enzymes Csp I, Sma I, Ecl XI, Ksp I, or Sac II were used for generation of few (7 to 16) distinctly separated fragments, with average sizes in the range of 200-300 kb. The size distribution of restriction fragments for each enzyme for each strain produced a strain-identifying fingerprint, and the genome size of each strain was determined from such restriction fragments separated by PFGE.     

Screening for restriction endonucleases in aerobic,thermophilic eubacteria

Summary A total of 216 Icelandic aerobic, heterotrophic, thermophiles belonging to three different genera were screened for type II restriction endonucleases. The frequency of positive strains was 44% for both Thermus and Bacillus but 63% for Rhodothermus. Approximately half of the enzymes from each group were characterised and a total of 14 different restriction enzymes were found. In all cases they were isoschizomers of known enzymes. Thermus contained 9 different types, Bacillus 6 and Rhodothermus had 3. This is the first time that isoschizomers of BspEI, BglI, EagI and EcoRV are found in Thermus and BstBI and EcoRV are found in Rhodothermus.     

Prevalence of CTGCAG recognizing restriction and modification systems in ruminal selenomonades

Analysis of restriction and modification activities in natural population of Selenomonas ruminantium have revealed the prevalence of CTGCAG (Pst I isoschizomers) recognizing restriction and/or modification systems in these bacteria. Pst I isoschizomeric restriction endonucleases were detected in 4 out of 15 strains tested. In one strain, the Pst I isoschizomeric restriction system was accompanied by another restriction and modification system recognizing GAATTC sequence (Eco RI isoschizomer). Four other strains contained CTGCAG specific methylases which lacked cognate endo-nuclease activities. Presence of identical restriction and modification systems in both of subspecies of S. ruminantium, as well as the occurrence of Pst I isoschizomers in various combinations, indicate the possibility of horizontal transfer of genes coding for these systems.     

The MmeI family: type II restriction–modification enzymes that employ single-strand modification for host protection

The type II restriction endonucleases form one of the largest families of biochemically-characterized proteins. These endonucleases typically share little sequence similarity, except among isoschizomers that recognize the same sequence. MmeI is an unusual type II restriction endonuclease that combines endonuclease and methyltransferase activities in a single polypeptide. MmeI cuts DNA 20 bases from its recognition sequence and modifies just one DNA strand for host protection. Using MmeI as query we have identified numerous putative genes highly similar to MmeI in database sequences. We have cloned and characterized 20 of these MmeI homologs. Each cuts DNA at the same distance as MmeI and each modifies a conserved adenine on only one DNA strand for host protection. However each enzyme recognizes a unique DNA sequence, suggesting these enzymes are undergoing rapid evolution of DNA specificity. The MmeI family thus provides a rich source of novel endonucleases while affording an opportunity to observe the evolution of DNA specificity. Because the MmeI family enzymes employ modification of only one DNA strand for host protection, unlike previously described type II systems, we propose that such single-strand modification systems be classified as a new subgroup, the type IIL enzymes, for Lone strand DNA modification.