The McrB restriction system of Escherichia coli K-12 is responsible for the biological inactivation of foreign DNA that contains 5-methylcytosine residues (E. A. Raleigh and G. Wilson, Proc. Natl. Acad. Sci. USA 83:9070-9074, 1986). Within the McrB region of the chromosome is the mcrB gene, which encodes a protein of 51 kilodaltons (kDa) (T. K. Ross, E. C. Achberger, and H. D. Braymer, Gene 61:277-289, 1987), and the mcrC gene, the product of which is 39 kDa (T. K. Ross, E. C. Achberger, and H. D. Braymer, Mol. Gen. Genet., in press). The nucleotide sequence of a 2,695-base-pair segment encompassing the McrB region was determined. The deduced amino acid sequence was used to identify two open reading frames specifying peptides of 455 and 348 amino acids, corresponding to the products of the mcrB and mcrC genes, respectively. A single-nucleotide overlap was found to exist between the termination codon of the mcrB gene and the proposed initiation codon of the mcrC gene. The presence of an additional peptide of 33 kDa in strains containing various recombinant plasmids with portions of the McrB region has been reported by Ross et al. (Gene 61:277-289, 1987). The analysis of frameshift and deletion mutants of one such hybrid plasmid, pRAB-13, provided evidence for a second translational initiation site within the McrB open reading frame. The proposed start codon for translation of the 33-kDa peptide lies 481 nucleotides downstream from the initiation codon for the 51-kDa mcrB gene product. The 33-kDa peptide may play a regulatory role in the McrB restriction of DNA containing 5-methylcytosine.
SYNOPSIS. The filtration mechanics of the gill of the zebra mussel, Dreissena polymorpha, allow this organism to capture particles less than 1 μm. The organization of gill cirri and the architecture of the cirri appear to be important in providing the organism with the ability to filter small particles. Bacteria may provide a useful nutrient source for these animals as bacterial proteins can be digested and assimilated into mussel proteins. Laboratory experiments indicate that D. polymorpha is capable of filtering and assimilating a wide range of bacteria ranging in size from 1–4 μm. Unionid species appear to be at least an order of magnitude less efficient at filtering bacteria than D. polymorpha. Because of its relatively smaller gill size, C. fluminea also filters bacteria less efficiently than D. polymorpha. We suggest that bacterial utilization by freshwater mussel species has important population and evolutionary implications.
Two experiments were conducted to investigate and quantify the weak and strong adsorption of an indicatorbacterium (Escherichia coli) in soilwater systems composed of Tangi silt loam (14% clay) or Commerce clay loam (35%clay). Percent adsorption of E. coli was significantly higher in Commerce clay loam than in Tangi silt loam for both weakand strong adsorption. The distribution coefficient of weak adsorption of E. coli in the Commerce soilwater system was foundto be significantly higher than that of strong adsorption. However, the distribution coefficient of weak adsorption of E. coliin the Tangi soilwater system was found to be significantly lower than that of strong adsorption. For strong adsorption,together with literature data, a high correlation (R 2 = 0.89) was found between percent adsorption and clay content.Significant correlation (R 2 = 0.67) was found between distribution coefficient and clay content. The relationships developedmay be used for modeling purposes.
The ability of curved DNA upstream of the -35 region to affect the interaction of Escherichia coli RNA polymerase and promoter DNA was examined through the use of hybrid promoters. These promoters were constructed by substituting the curved DNA from two Bacillus subtilis bacteriophage SP82 promoters for the comparable DNA of the bacteriophage lambda promoters lambda pR and lambda pL. The SP82 promoters possessed intrinsic DNA curvature upstream of their -35 regions, as characterized by runs of adenines in phase with the helical repeat. In vitro, the relative affinities of purified sigma 70-RNA polymerase for the promoters were determined in a competition binding assay. Hybrid promoters derived from lambda pR that contained curved DNA were bound by E. coli RNA polymerase more efficiently than was the original lambda pR. Binding of E. coli RNA polymerase to these hybrid promoters was favored on superhelical DNA templates according to gel retardation analysis. Both the supercoiled and relaxed forms of the hybrid lambda pL series were better competitors for E. coli RNA polymerase binding than was the original lambda pL. The results of DNase I footprinting analysis provided evidence for the wrapping of the upstream curved DNA of the hybrid lambda pR promoters around the E. coli RNA polymerase in a tight, nucleosomal-like fashion. The tight wrapping of the upstream DNA around the polymerase may facilitate the subsequent steps of DNA untwisting and strand separation.
RNA polymerase was purified from five species of Bacillus, including Bacillus subtilis. Each polymerase had a subunit composition analogous to that reported for B. subtilis, i.e., beta beta '2 alpha sigma delta omega 1 omega 2. The delta subunits from the B. subtilis and Bacillus thuringiensis polymerases were interchangeable, as judged from their effects on promoter selection in the polymerase binding assay.
Earthen basin lagoons are typically used to store and treat waste in dairy operations in the United States. In pasture-based dairy operations, it is often uneconomical to land-apply that waste mainly because of high dilution rates. Anaerobic lagoons are designed to reduce oxygen demand, but those facilities emit methane, ammonia, and odors. Pathogen populations can largely escape anaerobic treatment. Expanding treatment capacity may be more efficient and economical longterm solution. For the past 6 years, the LSU AgCenter Dairy Waste and Nutrient Management Team has been evaluating technologies and practices to improve wastewater treatment using the Southeast Research Station multi-stage treatment system. The system is comprised of a replicated set of anaerobic lagoon, aerobic lagoon, and constructed wetlands set in this sequence. Abatement rates range from 50% to 80% for phosphorus and nitrogen, respectively, and 3 to 4 logarithms of E. coli counts. The objective of the current study was to evaluate flow rates into constructed wetlands on abatement efficiency of nutrient and coliforms. Wastewater flow was controlled in each of the six constructed wetlands to produce three flow rates (5.2 – high; 1.8 – medium; and 0.4 – low, in gallons per minute). The design was a 3x3 Latin-square. Wastewater was sampled bi-weekly in 3 month-periods. Samples were analyzed for pH, chlorophyll-A, chemical oxygen demand (COD), total solids (TS), total dissolved solids (TDS), total suspended solids (TSS), ammonia-N, nitrate-N, nitrite-N, total Kjeldahl nitrogen (TKN), total phosphorus (TP), anions, and DO concentrations. Two thirds or more of TS, COD, TKN, and ammonia-N influent wastewater concentrations decreased throughout the system. Effluent P ranged between 40% and 50% of the influent and E. coli counts were reduced by 2.5 to 4 logs comparing high to low flow rates. Treatment rates decreased sharply from slow to medium flow rates.
the ability of the core isolated from Escherichia coli RNA polymerase to interact with specificity-determining subunits isolated from Bacillus subtilis RNA polymerase has been determined by measuring the transcription of and genes of phage SP82. Two specificity-determining subunits were tested: the sigma subunit and a 28,000 dalton (28 K) peptide isolated from a modified polymerase produced at approximately 8 min after infection of B. subtilis with SP82. Earlier experiments (Spiegelman, G. B. and Whiteley, H. R. (1978) Biochem. Biophys. Res. Commun. 81, 1058-1065) demonstrated that sigma and the 28K peptide are required for the recognition of early and middle gene promoters, respectively, by the B. subtilis core assembly. The present investigation showed that E. coli core interacted more efficiently with the B. subtilis sigma than with the 28K peptide, as judged by the rate of RNA synthesis. Early RNA was produced by the E. coli and B. subtilis holoenzymes and by E. coli core supplenented with B. subtilis sigma and only minor differences were found in comparisons of transcripts by hybridization and by electrophoretic analysis. Measurements of template specificity, the formation of stable enzyme . DNA complexes, and the hybridization of transcripts to fragments of SP82 DNA produced by digestion with restriction endonuclease Hha indicated that E. coli core supplemented with the 28K-supplemented E. coli core with those synthesized by the modified polymerase extracted from B. subtilis 8 min after infection with SP82 suggest that both preparations recognized the same initiation and termination sequences.
Abstract We have examined the effect of the delta subunit of the Bacillus subtilis RNA polymerase on the formation of closed, open, and stably initiated complexes with Hha I restriction fragments of phage SP82 DNA; the effect of delta on the transcription of these DNA fragments has also been investigated. In vitro, the holoenzyme (core-sigma-delta) bound to and transcribed the same regions of the phage genome that are transcribed in vivo early in infection. In the absence of the delta subunit, the polymerase (core-sigma) bound nonspecifically and transcribed regions of the genome other than those containing early phage genes. Addition of delta to preparations of core-sigma restored the pattern of binding and transcription observed with the holoenzyme. Similarly, delta-less preparations of two SP82-modified forms of polymerase (the enzyme isolated at 8 min after infection and the enzyme isolated 20 min after infection) bound nonspecifically and transcribed regions of the genome other than those containing and genes. Addition of delta to these preparations resulted in patterns of binding and transcription expected for enzymes functioning a middle and late times of infection, respectively. Quantitation of polymerase-DNA complexes at various temperatures, NaCl concentrations, and polymerase-DNA ratios supported the conclusion that delta enhanced promoter selection.
