Dreissena polymorpha consumed about 6 x 108 Escherichia coli from 20 ml of artificial pondwater (APW) in 30 min under laboratory conditions. The clearance rate per mussel was 143 +/- 25 ml g-1 dry tissue min-1. The E. coli used in these studies ranged from about 1.7 to 2.9 {mu}m in length. 35S-labeled E. coli were used to demonstrate that bacteria-derived nutrients were incorporated into mussel tissue. Electrophoretic analysis of mussel and bacterial proteins on 12% polyacrylamide gels allowed the visual determination of incorporation of labeled amino acids into bivalve proteins and demonstrated that intact bacteria were not simply trapped in mussel tissues. The conversion of bacterial-labeled amino acids into mussel protein was about 26%. Similarly, we demonstrated that D. polymorpha can use other bacterial species ranging in size from about 1.3 to 4.1 μm, including Citrobacter freundii, Enterobacter aerogenes, Serratia marcescens, Bacillus megaterium, and B. subtilus. The ability of D. polymorpha to take up E. coli was compared with that of two other freshwater mussels, Corbicula fluminea and Carunculina texasensis. On a mussel-dry-weight basis, D. polymorpha cleared bacteria 30 to 100 times faster than Corbicula fluminea and Carunculina texasensis, respectively. The ability to filter E. coli appears to be related to the architecture of the cirri on the latero-frontal cells of the gill. Cirri from Corbicula and Dreissena are similar in size, but Dreissena has a larger gill compared to the tissue dry-weight, and has 102 times more cirri than found in Corbicula. Carunculina, the unionid representative, has smaller and fewer cirri, and has relatively limited ability to capture E. coli.

Nine species of unionids cleared laboratory-raised Escherichia coli from artificial pond water. The six unionid species collected from rivers had higher clearance rates than the three species collected from ponds, when clearance was normalized to millilitres per gram of dry tissue mass per minute. Analysis of variance indicated that all lotic unionids examined form a group with similar clearance rates. When normalized on the basis of gill surface area, rates of clearance by all of the lotic unionids become remarkably similar to one another regardless of mass, but differ significantly from those of the lentic unionids. The cirri found on the laterofrontal cells of the gills of lotic unionids tend to be complex, containing >25 cilia per cirral plate, while the cirri of the unionid species collected from ponds have smaller cirri ( < 16 cilia per cirral plate). There was a strong correlation between cirral surface area (mm2) per milligram of dry tissue and clearance rate among the unionid species studied. As a comparison, Corbicula jluminea and Dreissena polymorpha were also examined and both tended to clear bacteria more rapidly than the lotic unionids.RCsumC : Neuf espkces d'unionidae ont absorb6 tous les Escherichia coli eleves en laboratoire dans I'eau d'un etang artificiel. Les six espkces recoltees dans des rivikres avaient des taux de clearance plus rapides que les trois espkces provenant des ktangs aprks normalisation de la clearance pour l'exprimer en millilitres par gramme de tissu sec par minute. Les resultats d'une analyse de variance indiquent que tous les unionidits lotiques examines forment un groupe dont les taux de clearance sont semblables. En normalisant les donnees en fonction de la surface des branchies, les taux de clearance de tous les unionides lotiques deviennent remarquablement semblables les uns aux autres, quelle que soit la masse, mais diffkrent significativement de ceux des unionides Ienitiques. Les cirres observes sur les cellules latero-frontales des branchies chez les unionides lotiques tendent a etre complexes, contenant >25 cils par plaque, alors que les espkces provenant des etangs ont des cirres plus petits ( > 16 cils par plaque). I1 y a une forte correlation entre la surface du cirre (mm2) par milligramme de tissu sec et le taux de clearance chez les espkces etudiees. Pour fins de comparaison, Corbicula jluminea et Dreissena polymorpha ont egalement ete etudies et les deux espkces ont des taux de clearance de batteries plus rapides que les unionides lotiques. [Traduit par la Redaction]

c Pathogenic enteric viruses are responsible for a wide range of infections in humans, with diverse symptoms. Raw and partially treated wastewaters are major sources of environmental contamination with enteric viruses. We monitored a municipal secondary wastewater treatment plant (New Orleans, LA) on a monthly basis for norovirus (NoV) GI and GII and enterovirus serotypes using multiplex reverse transcription-quantitative PCR (RT-qPCR) and microbial indicators of fecal contamination using standard plating methods. Densities of indicator bacteria (enterococci, fecal coliforms, and Escherichia coli) did not show monthly or seasonal patterns. Norovirus GII was more abundant than GI and, along with enterovirus serotypes, increased in influent during fall and spring. The highest NoV GI density in influent was in the fall, reaching an average of 4.0 log 10 genomic copies/100 ml. Norovirus GI removal (0.95 log 10 ) was lower than that for GII, enterovirus serotypes, and male-specific coliphages (1.48 log 10 ) or for indicator bacteria (4.36 log 10 ), suggesting higher resistance of viruses to treatment. Male-specific coliphages correlated with NoV GII densities in influent and effluent (r ‫؍‬ 0.48 and 0.76, respectively) and monthly removal, indicating that male-specific coliphages can be more reliable than indicator bacteria to monitor norovirus GII load and microbial removal. Dominant norovirus genotypes were classified into three GI genotypes (GI.1, GI.3, and GI.4) and four GII genotypes (GII.3, GII.4, GII.13, and GII.21), dominated by GI.1 and GII.4 strains. Some of the seasonal and temporal patterns we observed in the pathogenic enteric viruses were different from those of epidemiological observations. E nteric viruses are responsible for a wide range of infections in humans with diverse symptoms. Infected individuals shed millions of virus particles in their feces or body fluids, which eventually enter sewage systems. Enteric viruses may naturally occur in aquatic environments as well, but human activities, in particular, sewage discharge, is the primary source of environmental contaminants (1-3). Among the pathogenic enteric viruses, norovirus (NoV), enterovirus (EV), adenovirus, astrovirus, and rotavirus have been found frequently in municipal wastewaters worldwide (1, 4-6). Several gastroenteritis outbreaks have been linked directly or indirectly to human exposure of raw or partially treated sewage-contaminated water or foods (2).Municipal wastewaters usually undergo a secondary treatment before being discharged into the environment. The process involves a mechanical treatment for removing solids followed by biological and chemical treatments, nutrient removal, and discharge (7). Primary-treated (physically processed) wastewater or the effluent water that does not undergo a disinfection process may still harbor infectious enteric viruses, similar to the raw sewage (4, 8). To protect water quality and public safety, fecal coliforms and Escherichia coli have been used to monitor fecal pollution in wastewater dis...

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.

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 promoters p R and p L . 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 70 -RNA polymerase for the promoters were determined in a competition binding assay. Hybrid promoters derived from p R that contained curved DNA were bound by E. coli RNA polymerase more efficiently than was the original p R . 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 p L series were better competitors for E. coli RNA polymerase binding than was the original p L . The results of DNase I footprinting analysis provided evidence for the wrapping of the upstream curved DNA of the hybrid p R 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.Curved DNA upstream of the Ϫ35 region has been implicated in the function of numerous promoters (11,21,27). Studies of promoters associated with upstream curved DNA have demonstrated a strong correlation between promoter function and the presence of altered DNA structure, such as sequence-dependent curved DNA (4, 6, 13, 16-18, 21, 26, 27). The presence of curved DNA, as characterized by in-phase adenine tracts, has been found upstream of several promoters from Escherichia coli, including the ompF promoter (23), the his promoter, and the lpp promoters (29) and a subset of rRNA promoters (8) and tRNA promoters (5,6,16,18,25). Deletion analysis of the upstream curved DNA has suggested that promoter activation is intimately related to sequence-dependent curvature (16,21). In addition, sequence-dependent DNA curvature has been demonstrated to stimulate transcription when curved DNA is used to replace the upstream binding sites for the catabolite activator protein (CAP) (7,14). This suggests that protein-mediated DNA bending is functionally related to sequence-dependent DNA curvature in transcription initiation.Some of the most profound effects of curved DNA have been documented in promoters utilized by the Bacillus subtilis RNA polymerase. Intrinsically curved DNA upstream of the RNA polymerase binding site strongly influences transcription in B. subtilis. Indeed, deletions which eliminate the upstream A tracts have been shown to dramatically reduce expression from several promoters in B. subtilis (4,19,21). Furthermore, the optimal positioning of the curvature relative to the position of the promoter is essenti...

The McrB restriction system in Escherichia coli K12 causes sequence-specific recognition and inactivation of DNA containing 5-methylcytosine residues. We have previously located the mcrB gene near hsdS at 99 min on the E. coli chromosome and demonstrated that it encodes a 51 kDa polypeptide required for restriction of M.AluI methylated (A-G-5mC-T) DNA. We show here, by analysis of maxicell protein synthesis of various cloned fragments from the mcrB region, that a second protein of approximately 39 kDa is also required for McrB-directed restriction. The new gene, designated mcrC, is adjacent to mcrB and located distally to hsdS. The McrB phenotype has been correlated previously with restriction of 5-hydroxy-methyl-cytosine (HMC)-containing T-even phage DNA that lacks the normal glucose modification of HMC, formally designated RglB (for restriction of glucoseless phage). This report reveals a difference between the previously correlated McrB and RglB restriction systems: while both require the mcrB gene product only the McrB system requires the newly identified mcrC-encoded 39-kDa polypeptide.

No abstract

We have examined the effect of the delta subunit on the interaction of the Bacillus subtilis RNA polymerase with an early gene promotor of phase SP82. Methylation by dimethyl sulfate, used to probe close approaches of polymerase to purines, revealed that noninitiated complexes formed by holo-enzyme (core-sigma-delta) had significantly fewer contacts than complexes formed by core-sigma. The presence or absence of delta had little or no effect on close approaches to purines in initiated complexes. DNAase I footprinting indicated that core-sigma was bound to the same region regardless of whether delta, initiating nucleotides, or both, were present. These data support the conclusion that delta acts prior to initiation to enhance promoter selectivity by limiting the number of possible interactions that the polymerase can make with DNA.