Unusual chromatographic behavior of oligonucleotide sequence isomers on two different anion exchange HPLC columns.

The retention behavior of the unmodified phosphodiester oligonucleotide sequence isomers was investigated on two different anion exchange columns: Biospher GMB 1000Q (based on DEAE-modified glycidyl methacrylate) and PolyWAX LP (based on silica with a crosslinked coating of linear polyethyleneimine). There was a notable difference in retention of oligonucleotides of the same composition but differing in the position of a single base. The most pronounced difference was observed between the oligonucleotides with the variable base in the end and in the center of the sequence. The use of either acetonitrile or 2-propanol as a mobile phase organic modifier did not markedly affect the retention time patterns. Prediction of the retention times of oligonucleotides must take into account the base position as well as identity. This is the first report of such a "same composition different sequence" effect, described for the short peptides, for synthetic oligonucleotides.

Metabolism of 2-chlorobenzoic acid in Pseudomonas stutzeri.

3-Chloropyrocatechol is formed as a result of oxidation of 2-chlorobenzoate by Pseudomonas stutzeri. 2-Chloro-cis,cis-muconic acid is the product of oxidation of 3-chloropyrocatechol. A catabolic pathway for the degradation of 2-chlorobenzoate by a newly isolated strain of P. stutzeri is proposed.

Degradation of 2-chlorobenzoic and 2,5-dichlorobenzoic acids in pure culture by Pseudomonas stutzeri.

A strain of Pseudomonas stutzeri KS25 utilizing 2-chlorobenzoic and 2,5-dichlorobenzoic acids as the sole carbon and energy source was isolated from polychlorophenol-contaminated soil and sewage, using the method of enrichment cultures. This strain was also able to grow on 2-fluoro-, 2-iodo-, 2-bromo- and 2,5-dihydroxybenzoate, but did not utilize 3-, 4-chloro-, 2,4- and 2,6-dichlorobenzoates as the sole carbon and energy source, however, it cometabolized 3-chloro-, 2,4- and 2,6-dichlorobenzoates, but not 4-chlorobenzoate. The yield of released chlorine during utilization of 2-chloro- and 2,5-dichlorobenzoates amounted to 100% of the theoretical. The concentration of 2-chloro- and 2,5-dichlorobenzoates, not substantially inhibiting the isolated microorganism, was within the range 0.25-0.5 and 2.5-3.0 g/L, respectively.

Degradation of 2-chlorobenzoic and 2,5-dichlorobenzoic acids in soil columns by Pseudomonas stutzeri.

The heterocontinuous flow cultivation technique was used for the study of 2-chlorobenzoic and 2,5-dichlorobenzoic acid degradation in soil columns inoculated with Pseudomonas stutzeri. 2-Chlorobenzoic and 2,5-dichlorobenzoic acids disappeared from the soil columns within 8 and 12 d, respectively. The presence of the haloaromatics increased the survival of strain KS25 in soil. Viable cell numbers in the soil columns flushed with 2-chlorobenzoic and 2,5-dichlorobenzoic acids were 1.3 and 2 times higher, respectively, than those without the chlorobenzoic acids after 30 d of incubation.

Degradation of the herbicide bromoxynil in Pseudomonas putida.

Biological conversion of the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) was studied in a batch culture of Pseudomonas putida by using HPLC. The process had a cometabolic character and proceeded only in the presence of another, simultaneously metabolizable, carbon and energy source. The intensity of degradation correlated with the growth rate, the degradation stopping when the cosubstrate becomes exhausted or the pH value of the medium falls below 6.5. In a medium with glucose, no lag phase longer than one day was observed concerning growth, sugar and herbicide consumption and formation of metabolic herbicide derivatives (3,5-dibromo-4-hydroxybenzamide and 3,5-dibromo-4-hydroxybenzoic acid). In a medium with ribose, the initial lag of the above processes took 2 d. No formation of other degradation products was detected. Growth inhibition was proportional to the concentration of bromoxynil.

Effect of glucose and ribose on microbial degradation of the herbicide bromoxynil continuously added to soil.

Bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) was continuously added to chernozem (Haplic typic) soil inoculated with a suspension of Pseudomonas putida capable of cometabolic decomposition of the compound in a hetero-continuous-flow cultivation setup. In the steady state, when glucose or ribose were simultaneously added, 90 and 47% of the added herbicide was degraded per day, respectively. If the saccharides were absent, only 10-27% of the herbicide was decomposed. Addition and removal of glucose feeding resulted in an increase and decrease, respectively, of the degradation intensity, irrespective of the amount of the bacterial decomposers present. Two degradation products, 3,5-dibromo-4-hydroxy-benzamide and 3,5-dibromo-4-hydroxybenzoic acid, were formed during cultivation. The total amount of bromine-containing compounds was reduced only in the presence of glucose.

Mineralization of 2,4-dichlorophenoxyacetic acid in soil simultaneously enriched with saccharides.

Detoxication of 2,4-dichlorophenoxyacetic acid (2,4-D) in samples of chernozem soil was determined by a biological test and the time course of production of 14CO2, a product of microbial degradation of 2-14C-2-4-D, was measured during 38-d incubation at 28 degrees C in the dark. Enrichment of the soil with glucose (1000 ppm), two exocellular bacterial glucan and glucomannan polysaccharides (750 ppm), or a mixture of glucose with (NH4)2SO4 (C : N = 5 : 1) brought about acceleration of both detoxication and mineralization of 2,4-D (50 ppm) added simultaneously with the saccharides. Mineralization of the saccharides always preceded the degradation of the herbicide. The lag phase of 2,4-D mineralization did not exceed 3 d. In samples with saccharides the doubling time of the mineralization activity in the exponential phase of the process was substantially shortened and the mineralization of 2,4-D was accelerated even when the soil was inoculated with a suspension of soil in which microbial 2,4-D decomposers had accumulated. The extent of mineralization was not affected by the presence of saccharides (about 1/3 of the introduced radioactive carbon was transformed into 14CO2). All saccharides had a similar effect which reflected an increase in the overall bacterial count and in the relative abundance of bacterial 2,4-D decomposers . The role of other mechanisms such as co-metabolism in the stimulation of the degradation process is discussed.

Mineralization of 2,4-dichlorophenoxyacetic acid in soil previously enriched with organic substrates.

Samples of chernozem soil were enriched with vanillic acid, protocatechuic acid glucose, a mixture of glucose and (NH4)2SO4 (C : N = 5 : 1), ethanol and 2,4-dichlorophenoxyacetic acid (2,4-D). After a 6-d (with 2,4-D 35-d) incubation during which primary oxidation of the introduced substrates occurred, the soil was supplied with a solution of 2-14C-2,4-D (50 ppm; 6.7 kBq) and production of 14CO2 (product of microbial degradation of 2,4-D) was measured. Previously enriched samples exhibited a higher degradation rate; both the lag phase and doubling time of mineralization activity in the exponential phase of the process were markedly higher. This reflected an overall proliferation of bacteria and the increased relative proportion of bacterial strains capable of mineralizing 2,4-D in enriched samples. The stimulation of 2,4-D degradation may involve specific adaptation and selection mechanisms (as in the case with samples previously enriched with 2,4-D or its structural analogues--aromatic monomers, ethanol) as well as non-specific mechanisms. The extent of mineralization of 2,4-D was not affected by soil pretreatment, about 1/3 of introduced radioactive carbon being invariably transformed to 14CO2.

Degradation of 3-chlorobenzoate in soil by pseudomonads carrying biodegradative plasmids.

Degradation of continuously added 3-chlorobenzoate (3-CB) was studied in samples of chernozem soil. Soil columns were inoculated with Pseudomonas putida growing on 3-CB and carrying the biodegradation plasmid and with Pseudomonas aeruginosa incapable of growth on 3-CB and carrying the inserted biodegradation plasmid pBS 2 determining ortho-cleavage of the aromatic ring. While the 3-CB degradation was observed in both inoculated variants, the native microflora of the soil under study was incapable to degrade 3-CB. Among pseudomonads isolated from inoculated soil at different stages of cultivation and growth on 3-CB, some had the taxonomic features of P. putida as well as those differing in 1-5 characteristics. The study of the activities of the enzymes cleaving the aromatic ring revealed the presence of pyrocatechol 1,2-dioxygenase in the isolated strains only, as estimated by means of benzoate and 3-CB as substrates.

Effect of glucose on the amount of bacteria mineralizing 2,4-dichlorophenoxyacetic acid in soil.

Plate numbers of bacteria and relative incidence of strains capable of mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D) in chernozem samples incubated for 14d with the herbicide (50 ppm) in the presence or absence of glucose (1000 ppm) were compared. Whereas the total number of bacteria increased 1.2-fold in the variant with 2,4-D and 2.4-fold in the variant with glucose and the herbicide, the number of 2,4-D-mineralizing bacteria increased 12.1-fold and 34.2-fold, respectively. In a collection of 96 isolates of soil bacteria substantially more strains capable of degradation of 2,4-D in the presence of glucose were detected as compared with the variant without it, indicating that processes of cometabolic type are involved during the degradation of this herbicide in the soil.

Microbial transformations of inorganic sulphur compounds in soil under conditions of heterocontinuous cultivation.

Development and activity of the association of the sulphur cycle bacteria, represented by Thiobacillus thioparus and Desulfovibrio sp., were followed in chernozem soil continuously supplemented with sodium thiosulphate. The technique of heterocontinuous cultivation made it possible (i) to determine changes in the individual components of microflora involved in successive metabolic steps, their time and space sequence, (ii) to follow changes in the transformations of substrate and formation of metabolic products, and (iii) to reach a steady state in the system. A possible use of this approach for the evaluation of the effect of ecological factors, for modelling microbiological processes of the sulphur cycle, for the investigation of trophic relationships among microorganisms in natural and artificial association and for the evaluation of the geochemical activity of sulphur bacteria is discussed.

The priming effect of glucose in soil sterilized by gamma-radiation and reinoculated with Cellulomonas sp.

Mineralization of native organic matter and U-14C-glucose was followed by measuring the formation of CO2 and its radioactivity in chernozem soil presterilized by gamma-radiation and inoculated with a washed suspension of Cellulomonas sp. cells. The introduced bacteria mineralized the soil organic component to a higher extent in variants enriched with glucose. This so-called priming effect of glucose was observed also in the presence of chloramphenicol, inhibiting the growth of the bacteria. The increased mineralization of the native soil organic fraction was also detected in samples that were not enriched with glucose when the bacterial suspension was first disintegrated ultrasonically and the material then used for the inoculation. Possible participation of phenomena of the type of cometabolism and activation of cell membrane transport mechanisms on the occurrence of the priming effect of glucose in the soil is discussed.

Acceleration by montmorillonite of nitrification in soil.

A soil naturally containing montmorillonite (M) was amended with 10% M and sequentially perfused with glyeme, with fresh glyeme being added every 16--17d after nitrification of the previously added glycine-nitrogen had reached a plateau. In some systems, the old perfusates were replaced each time with a fresh glycine solution; in others, the initial perfusate was not replaced but only adjusted each time to the original 200 ml volume and a comparable glycine concentration (140 micrograms NH2-N/ml). The incorporation of M enhanced the rates of heterotrophic degradation of glycine and subsequent autotrophic nitrification, but these stimulatory effects decreased with each successive perfusion. The reasons for these decreases are not known, but they did not appear to be related to inorganic nutrition, as perfusion with a mixed cation solution after five perfusion cycles did not significantly enhance nitrification in either the check or M-amended soils during three subsequent perfusions with glycine. The enhancement of nitrification by M appeared to be a result, in part, of the greater buffering capacity of the M-amended soil, as indicated by lesser reductions in the pH of perfusates from the M-amended soil, by titration curves of the soils, and by the greater and longer stimulation of nitrification in the check soil amended with 1% CaCO3, which had a greater buffering capacity than did M. The stimulation by CaCO3 may also have been partially the result of providing CO2 for the autotrophic nitrifyers. Significant concentrations of nitrite accumulated only in perfusates from soil amended with CaCO3. Air-drying and remoistening the soils enhanced nitrification of subsequently added glycine, especially in the check soil. The importance of pH-mediation, of the production of inhibitors, and/or of feed-back inhibition was indicated by the lower rate and extent of nitrification in systems wherein the perfusates were not replaced between successive additions of glycine. Although the results of these studies confirmed previous observations that M enhances the rate of nitrification in soil, the mechanisms responsible for this stimulation are still not known.

Microbial activity in soil enriched with preparations of AS-lignin and lignofulvonic acid.

In chernozem soil, enriched with preparations of AS-lignin or lignofulvonic acid, an increased production of carbon dioxide was observed during a 4-week incubation, and, as compared with the non-amended control, an increased number of bacteria but not of actinomycetes was detected. Increased numbers of fungi were detected only in the variant with AS-lignin at the end of the incubation. The relative incidence of bacteria utilizing vanillin, syringic acid or protocatechuic acid as the only carbon sources increased in the enriched medium. Oxidation of vanillic acid, syringic acid and to a lesser extent of coumarin increased in suspensions of soils incubated with AS-lignin or fulvic acid. The results obtained indicate that bacteria are involved in the mineralization of the added substrates and confirm the relationship between metabolism of these compounds and simple aromatic derivatives.

Biological decomposition of fulvic acid preparations.

Decomposition of preparations of various fractions of fulvic acids in pure cultures of bacteria and in the soil was investigated. In the soils enriched with fulvic acids the amount of bacteria increased, oxygen consumption and formation of carbon dioxide followed a typical sigmoid curve. The above measurements indicated that mineralization occurred after a very short or negligible lag phase. During the decomposition of fulvic acids the ability of microorganisms to oxidize aromatic compounds, e.g. vanillic and p-hydroxybenzoic acid, increased. The presence of aromatic structures in the used preparations of fulvic acids was demonstrated on the basis of their IR spectra and according to the results of chromatographic analyses of their hydrolysates. The results indicated a relationship between metabolism of fulvic acids and aromatic compounds. In samples of the soil preincubated with glucose with fulvic acids decomposed more rapidly than in untreated samples.