Search for methanotrophic producers of exopolysaccharides]

Bacteria that produce exopolysaccharides (EPS) and use methane as the only source of carbon were selected by studying a collection of methanotroph strains: Methylococcus capsulatus E 494, 874, and 3009; M. thermophilus 111p, 112p, and 119p; Methylobacter ucrainicus 159 and 161; M. luteus 57v and 12b; Methylobacter sp. 100; Methylomonas rubra 15 sh and SK-32; Methylosinus trichosporium OV3b, OV5b and 4e; M. sporium 5, 12, A20d, and 90v; and Methylocystis parvus OVVP. Mesophilic methanotroph strains with the ribulose monophosphate way of C1-compound assimilation synthesized EPS more actively than bacteria operating the serine cycle. The dynamics of EPS synthesis by methanotrophs during chemostat cultivation was studied.     

Role of CO-binding cytochrome c in enzymatic oxidation of methane by the bacterium Methylococcus capsulatus

The cytochrome c spectrally related to cco cytochromes has been isolated and purified from the methane-oxidizing bacterium Methylococcus capsulatus. The cytochrome binds CO but does not bind other substrates of methane monooxygenase, does not activate the methane monooxygenase reaction and is not a component of methane monooxygenase. In the methanol dehydrogenase enzymatic system cytochrome cco functions as electron acceptor. A possible role of cytochrome cco as electron carrier intermediate in the sequence of the dehydrogenase and oxidase enzymatic systems of M. capsulatus is discussed.     

Soluble methane monooxygenase component B gene probe for identification of methanotrophs that rapidly degrade trichloroethylene.

Restriction fragment length polymorphisms, Western blot (immunoblot) analysis, and fluorescence-labelled signature probes were used for the characterization of methanotrophic bacteria as well as for the identification of methanotrophs which contained the soluble methane monooxygenase (MMO) gene and were able to degrade trichloroethylene (TCE). The gene encoding a soluble MMO component B protein from Methylosinus trichosporium OB3b was cloned. It contained a 2.2-kb EcoRI fragment. With this cloned component B gene as probe, methanotroph types I, II, and X and environmental and bioreactor samples were screened for the presence of the gene encoding soluble MMO. Fragments produced by digestion of DNA with rare cutting restriction endonucleases were separated by pulsed-field gel electrophoresis and transferred to Zeta-Probe membrane (Bio-Rad) for Southern blot analysis. Samples were also analyzed for the presence of soluble MMO by Western blot analysis and the ability to degrade TCE. The physiological groups of methanotrophs in each sample were determined by hybridizing cells with fluorescence-labelled signature probes. Among twelve pure or mixed cultures, DNA fragments of seven methanotrophs hybridized with the soluble MMO B gene probe. When grown in media with limited copper, all of these bacteria degraded TCE. All of them are type II methanotrophs. The soluble MMO component B gene of the type X methanotroph, Methylococcus capsulatus Bath, did not hybridize to the M. trichosporium OB3b soluble MMO component B gene probe, although M. capsulatus Bath also produces a soluble MMO.     

Soluble methane monooxygenase component B gene probe for identification of methanotrophs that rapidly degrade trichloroethylene.

Restriction fragment length polymorphisms, Western blot (immunoblot) analysis, and fluorescence-labelled signature probes were used for the characterization of methanotrophic bacteria as well as for the identification of methanotrophs which contained the soluble methane monooxygenase (MMO) gene and were able to degrade trichloroethylene (TCE). The gene encoding a soluble MMO component B protein from Methylosinus trichosporium OB3b was cloned. It contained a 2.2-kb EcoRI fragment. With this cloned component B gene as probe, methanotroph types I, II, and X and environmental and bioreactor samples were screened for the presence of the gene encoding soluble MMO. Fragments produced by digestion of DNA with rare cutting restriction endonucleases were separated by pulsed-field gel electrophoresis and transferred to Zeta-Probe membrane (Bio-Rad) for Southern blot analysis. Samples were also analyzed for the presence of soluble MMO by Western blot analysis and the ability to degrade TCE. The physiological groups of methanotrophs in each sample were determined by hybridizing cells with fluorescence-labelled signature probes. Among twelve pure or mixed cultures, DNA fragments of seven methanotrophs hybridized with the soluble MMO B gene probe. When grown in media with limited copper, all of these bacteria degraded TCE. All of them are type II methanotrophs. The soluble MMO component B gene of the type X methanotroph, Methylococcus capsulatus Bath, did not hybridize to the M. trichosporium OB3b soluble MMO component B gene probe, although M. capsulatus Bath also produces a soluble MMO.     

Restriction endonuclease DNA analysis of Leptospira interrogans serovars Icterohaemorrhagiae and Copenhageni

Leptospira interrogans serovar icterohaemorrhagiae strains Ictero No. I and RGA and serovar copenhageni strains M20, Shiromizu and Shibaura were examined by restriction endonuclease DNA analysis. Fifteen endonucleases (AluI, BamHI, BglII, EcoRI, HaeIII, HhaI, HindIII, KpnI, PstI, SacI, SalI, SmaI, StyI, XbaI and XhoI) were used as the digesting enzymes. Strain Ictero No. I showed endonuclease cleavage patterns which differed from those of the other four strains only when it was digested with enzymes KpnI and HindIII. When digested with KpnI, an extra band of about 5.4 kb was clearly produced, and when digested with HindIII, an extra band of about 25 kb was produced. When the other 13 enzymes were used, no differences were found between the endonuclease cleavage patterns among the five strains. Moreover, strains RGA, M20, Shiromizu and Shibaura could not be distinguished by the restriction endonuclease DNA analysis using all 15 endonucleases. In addition, six newly isolated leptospires from patients with leptospirosis and from Rattus norvegicus were compared with the Ictero No. I and M20 strains, by restriction endonuclease DNA analysis using enzymes KpnI and HindIII. Three leptospires belonging to serovar icterohaemorrhagiae showed the same endonuclease cleavage patterns as the M20 strain. The other three strains, which belong to serovar copenhageni, showed almost the same endonuclease cleavage patterns as the M20 strain; only the Kai ima 702 strain produced an extra band which was not identical to the Ictero No. I-specific extra band when digested with HindIII. The leptospiral restriction endonuclease DNA analysis has revealed taxonomic structures that are unrecognized by serology alone.     

DNA restriction endonuclease analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG

DNA preparations from two reference (H37Ra and H37Rv) and two wild strains of Mycobacterium tuberculosis and one re-isolated strain of Mycobacterium bovis BCG were analysed using 17 restriction endonucleases. The enzyme BstEII revealed the greatest differences between strains. Electrophoretic DNA patterns from the wild M. tuberculosis strains differed from each other and from the reference strains at relatively few positions. At the highest resolution attained, patterns from the two reference strains remained indistinguishable from each other. The pattern of the M. bovis BCG strain was substantially different from, but had many bands in common with, the M. tuberculosis patterns.     

Taxonomic significance of differences in DNA methylation within the 'Mycoplasma mycoides cluster' detected with restriction endonucleases MboI and DpnI

DNA from 22 strains belonging to the ' Mycoplasma mycoides cluster' was tested for methylation of adenine in GATC sequences by its resistance or susceptibility to digestion by the restriction endonucleases MboI , which is inhibited by the methylation, or DpnI , which requires the methylation. Strains of bovine serogroup 7, M. mycoides subsp. mycoides SC, and some M. mycoides subsp. capri strains plus M. capricolum strain Cal Kid lacked the methylation, whereas other strains of M. mycoides subsp. capri and of M. capricolum , plus the F38-like and M. mycoides subsp. mycoides LC strains, possessed it. We conclude that this simple test could provide a valuable criterion in identifying these mycoplamas.     

High-frequency mobilization of broad-host-range plasmids into Neisseria gonorrhoeae requires methylation in the donor.

Antibiotic resistance in Neisseria gonorrhoeae has been associated with the acquisition of R plasmids from heterologous organisms. The broad-host-range plasmids of incompatibility groups P (IncP) and Q (IncQ) have played a role in this genetic exchange in nature. We have utilized derivatives of RSF1010 (IncQ) and RP1 (IncP) to demonstrate that the plethora of restriction barriers associated with the gonococci markedly reduces mobilization of plasmids from Escherichia coli into strains F62 and PGH 3-2. Partially purified restriction endonucleases from these gonococcal strains can digest RSF1010 in vitro. Protection of RSF1010-km from digestion by gonococcal enzymes purified from strain F62 is observed when the plasmid is isolated from E. coli containing a coresident plasmid, pCAL7. Plasmid pCAL7 produces a 5'-MECG-3' cytosine methylase (M.SssI). The M.SssI methylase only partially protects RSF1010-km from digestion by restriction enzymes from strain PGH 3-2. Total protection of RSF1010-km from PGH 3-2 restriction requires both pCAL7 and a second coresident plasmid, pFnuDI, which produces a 5'-GGMECC-3' cytosine methylase. When both F62 and PGH 3-2 are utilized as recipients in heterospecific matings with E. coli, mobilization of RSF1010 from strains containing the appropriate methylases into the gonococci occurs at frequencies 4 orders of magnitude higher than from strains without the methylases. Thus, protection of RSF1010 from gonococcal restriction enzymes in vitro correlates with an increase in the conjugal frequency. These data indicate that restriction is a major barrier against efficient conjugal transfer between N. gonorrhoeae and heterologous hosts.     

A rapid purification method of restriction endonucleases from Haemophilus strains.

A simple and rapid method of purification of restriction endonucleases from different Haemophilus strains is presented. By this method highly purified and stable enzymes can be obtained. Separation of different restriction activities present in the same strain is possible. This method was so far successfully used with Haemophilus influenzae, Haemophilus parainfluenzae and Haemophilus aegyptius strains. The main advantages over previously published procedures reside in the simplication of certain purification steps (for instance the BioGel A 0.5 M filtration is replaced by a hydroxyapatite batch step), elimination of exonuclease activity by fractionation with (NH4) 2SO4, separation of different restriction activities by phosphocellulose chromatography, application of this method to various strains and high purification degree of enzymes.     

Type II restriction modification systems of Prevotella bryantii TC1-1 and Prevotella ruminicola 23 strains and their effect on the efficiency of DNA introduction via electroporation

The restriction endonucleases PbrTI and Pru2I, isoschizomers of Sau3AI and HaeIII, were partially purified and characterized from anaerobic rumen bacteria Prevotella bryantii TC1-1 and Prevotella ruminicola 23, respectively. These are the first type II restriction endonucleases discovered in strains of the genus Prevotella, and they represent one of the barriers hindering gene transfer in these microorganisms. Heterologous DNA was protected against the action of the PbrTI or Pru2I by incubation in a cell-free extract of the respective strain which contained 20 mM EDTA. This led to the development of a protocol enabling successful electrotransformation of the P. bryantii TC1-1 strain with a pRH3 Bacteroides--Escherichia coli shuttle vector containing up to 7-kb long DNA inserts. Plasmid DNA isolated from the transformed strain facilitated the transfer with further increased efficiency and made possible the introduction of ligation reaction products directly to P. bryantii TC1-1 without passing them first through E. coli.     

The binuclear iron site of the membrane-bound methane hydroxylase from Methylococcus capsulatus (strain M)

The particulate membrane-bound methane hydroxylase (pMMOH) was isolated from methane-oxidizing cells of Methylococcus capsulatus (strain M). At SDS PAGE, pMMOH displays three bands: 47 (alpha), 27 (beta), and 25 kDa (gamma). The ESR spectrum of pMMOH incubated with hydrogen peroxide (final concentration 20 mM) at 4 degrees C exhibited, along with the copper signal of type I with g = 2.05, signals of cytochrome with g = 3.0 and of high-spin ferriheme with g = 6.00. After incubation at -30 degrees C, additional signals with g 8.5 and 13.5 were observed. These signals, which have not been recorded previously in pMMOH preparations, are due to an intermediate of the pMMOH active site, which arises in the reaction of hydrogen peroxide with pMMOH at -30 degrees C. It was established that this intermediate is a high-spin dimer [Fe(IlI)-Fe(IV)] with S = 9/2 and different degree of rhombic distortion of structure (it is responsible for both signals). Presumably, the signal with g = 8.5 also arises from the same dimer [Fe(III)-Fe(IV)], but with S = 7/2. The presence of the intermediate [Fe(lII)-Fe(IV)] in pMMOH preparations suggests that the original state of the pMMOH active site is the dimer [Fe(III)-Fe(III)] which is located in the beta-subunit and cannot be detected by ESR. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http:// www.maik.ru.     

Restriction analysis of the Frankia spp. genome

Abstract Total cellular DNAs of 10 Frankia isolates from Alnus, Elaeagnus and Colletia spp. root nodules were cleaved with ten site-specific restriction endonucleases and analysed by agarose gel electrophoresis. Among the endonucleases tested, Bam HI, Bgl II, Sal I and Sma I proved to be the most suitable for the genome analysis in Frankia spp. DNA restriction banding patterns were reproducible and characteristic of each Frankia strain. The patterns of different strains differed marked indicating considerable genotypic heterogeneity among the isolates. The approach can be used for strain identification in Frankia spp. as well as for differentiation between phenotypically similar strains.     

Occurrence of small Hsd plasmids in Salmonella typhi, Shigella boydii, and Escherichia coli.

The natural occurrence of small Hsd (host specificity for DNA) plasmids was demonstrated in restriction endonuclease-producing strains of Salmonella typhi, Shigella boydii, and Escherichia coli. The five Hsd plasmids isolated were between 5.0 and 12.2 kilobases long. The copy number of all the Hsd plasmids was high (more than 10 copies per cell). Introduction of these small plasmids into E. coli strain 0 drastically lowered the efficiency of plating of the lambda.0 phages (the efficiency of plating was less than 5 X 10(-5) PFU-1). High restriction endonuclease activities were detected in the Hsd plasmid-positive strains because of the elevated copy numbers of the hsdR+ gene. The advantages of using E. coli strains containing the small Hsd plasmids for purification of type II restriction endonucleases are discussed.     

Reinfection of guinea pigs with herpes simplex virus type 2: failure to establish a second latent ganglionic infection

Guinea pigs were infected with herpes simplex virus type 2 (HSV 2) and at various times thereafter reinfected at a different site with the same or a different strain of HSV 2. Viruses were isolated from infected tissues either from the site of primary or secondary infection or from regional ganglia. Viral isolates were characterised by DNA fingerprinting using restriction endonucleases capable of differentiating between the virus strains used. The second infection resulted in a second site of peripheral persistence of the virus, but did not establish a second site of ganglionic infection.     

Enzymatic recognition of DNA modifications induced by singlet oxygen and photosensitizers.

DNA modifications induced either by photosensitization (illumination in the presence of methylene blue) or by chemically generated singlet oxygen (thermal decomposition of an 1,4-etheno-2,3-benzodioxin) are recognized and incised by repair endonucleases present in crude bacterial cell extracts. Only a small fraction of the incised modifications are sites of base loss (AP-sites) sensitive to exonuclease III, endonuclease IV from E. coli or to the UV-endonuclease from M. luteus. Cell extracts from E. coli strains overproducing or defective in endonuclease III recognize the modifications induced by illumination in the presence of methylene blue just as well as do those from wild-type E. coli strains. This indicates that dihydropyrimidine derivatives, which are characteristic of hydroxyl radical-induced DNA modifications, are absent. In contrast, most of the modifications induced are not recognized by a cell extract from a fpg strain defective in formamidopyrimidine-DNA glycosylase FPG protein). Furthermore, incision by a cell extract from an E. coli strain overproducing FPG protein takes place at much lower protein concentration than with the wild-type strain. Experiments with purified FPG protein confirm that this enzyme is responsible for the recognition of singlet oxygen-induced DNA base modifications.     

Involvement of a Spiroplasma citri plasmid in the erythromycin-resistance transfer

An erythromycin-resistant strain (M4 Er-1) was selected from Spiroplasma citri M4+. The transfer by transformation of the erythromycin-resistance character to the erythromycin-sensitive S. citri strain R8A2+ was studied. Transfer became effective and reproducible when cells were treated with alkali cations plus polyethylene glycol. Comparison of the efficiency of transformation of the erythromycin-sensitive strain S. citri R8A2+ by total and extrachromosomal DNA purified from the erythromycin-resistant strain M4 Er-1 showed that the plasmid pM42 was able to transfer the erythromycin-resistance. pM42 was mapped with restriction endonucleases and found to be related to the pMH1 plasmid previously isolated from S. citri MH. Hybridization analysis of DNA from sensitive and resistant strains has shown that a sequence from pM42, analogous to a sequence from pMH1, was integrated at a specific locus in the chromosome of the erythromycin-resistant cells, i.e., of the transformed R8A2 cells and of the spontaneous mutant M4 Er-1 strain.