Recovery and diversity of heterotrophic bacteria from chlorinated drinking waters.

Heterotrophic bacteria were enumerated from the Seattle drinking water catchment basins and distribution system. The highest bacterial recoveries were obtained by using a very dilute medium containing 0.01% peptone as the primary carbon source. Other factors favoring high recovery were the use of incubation temperatures close to that of the habitat and an extended incubation (28 days or longer provided the highest counts). Total bacterial counts were determined by using acridine orange staining. With one exception, all acridine orange counts in chlorinated samples were lower than those in prechlorinated reservoir water, indicating that chlorination often reduces the number of acridine orange-detectable bacteria. Source waters had higher diversity index values than did samples examined following chlorination and storage in reservoirs. Shannon index values based upon colony morphology were in excess of 4.0 for prechlorinated source waters, whereas the values for final chlorinated tap waters were lower than 2.9. It is not known whether the reduction in diversity was due solely to chlorination or in part to other factors in the water treatment and distribution system. Based upon the results of this investigation, we provide a list of recommendations for changes in the procedures used for the enumeration of heterotrophic bacteria from drinking waters.     

Formation of a native-like subdomain in a partially folded intermediate of bovine pancreatic trypsin inhibitor.

In the folding of bovine pancreatic trypsin inhibitor (BPTI), the single-disulfide intermediate [30-51] plays a key role. We have investigated a recombinant analog of [30-51] using a 2-dimensional nuclear magnetic resonance (2D-NMR). This recombinant analog, named [30-51]Ala, contains a disulfide bond between Cys-30 and Cys-51, but contains alanine in place of the other cysteines in BPTI to prevent the formation of other intermediates. By 2D-NMR, [30-51]Ala consists of 2 regions-one folded and one predominantly unfolded. The folded region resembles a previously characterized peptide model of [30-51], named P alpha P beta, that contains a native-like subdomain with tertiary packing. The unfolded region includes the first 14 N-terminal residues of [30-51] and is as unfolded as an isolated peptide containing these residues. Using protein dissection, we demonstrate that the folded and unfolded regions of [30-51]Ala are structurally independent. The partially folded structure of [30-51]Ala explains many of the properties of authentic [30-51] in the folding pathway of BPTI. Moreover, direct structural characterization of [30-51]Ala has revealed that a crucial step in the folding pathway of BPTI coincides with the formation of a native-like subdomain, supporting models for protein folding that emphasize the formation of cooperatively folded subdomains.     

Recovery of intestinal membrane binding sites for K88 E. coli from pig mucosal organ cultures

Summary Putative receptors for K88+ E. coli from piglet intestinal epithelium were released into the organ culture medium and were demonstrated by direct binding with K88+ E. coli through the utilization of an in vitro binding procedure or by immunoprecipitation with K88 antigen.Incorporation of ¹⁴C-glucosamine by newborn to day old and 3-week to 6-week old piglet jejunal and ileal mucosa, in organ culture, occurred throughout the 24 hr culture period. Uptake in both age groups and both areas of the intestine was similar with a somewhat greater incorporation by the older age group.Secretion of ¹⁴C-glucosamine-labeled components into the culture medium was demonstrated by gel filtration of the concentrated medium. Some large molecular weight components eluted in the void volume in excess of 2 x 10⁶ daltons. A second peak of activity was spread from approximately 690K to 25K daltons. All eluted fractions demonstrated binding to K88+ E. coli.Antibodies to purified brush borders from susceptible pigs produced prominent precipitation bands following double diffusion with concentrated organ culture media which confirmed that the organ culture media contained labeled proteins of brush border origin.Immunoprecipitation of the intestinal mucosal organ culture media with K88+ pili and pilus antisera, followed by electrophoresis with SDS and reduced conditions, demonstrated a subunit of approximately 35K daltons.     

High Numbers of Prosthecate Bacteria in Pulp Mill Waste Aeration Lagoons

Prosthecate bacteria comprised 0.6 to 10.5% of the bacterial community in samples from 11 pulp mill waste aeration lagoons. Because of their distinct morphology, the genera Ancalomicrobium, Caulobacter, Prosthecobacter, Prosthecomicrobium, Stella, and Hyphomicrobium or Hyphomonas could be identified and enumerated by direct microscopic examination. Monthly samples from one lagoon showed that several genera varied from undetectable to predominant among the appendaged organisms. Temperature (season), type of wood pulped, and pulping process did not significantly affect the density of prosthecate bacteria.     

Prosthecobacter fusiformis nov. gen. et sp., the fusiform caulobacter

Four strains of heterotrophic, fusiform caulobacters have been isolated from freshwater sources. A single prostheca extends from one pole of mature cells, and cells attach to various substrata by means of a holdfast located at the distal tip of the appendage. Thus, superficially these bacteria bear a strong resemblance to bacteria in the genus Caulobacter. However, unlike Caulobacter these bacteria do not exhibit a dimorphic life cycle of motile, non-stalked daughter cells and immotile, stalked mother cells. Instead both mother and daughter cells are immotile, and at the time of cell separation the daughter cells are essentially identical mirror-image replicas of the mother cell. In addition, the prosthecae of these fusiform caulobacters do not have crossbands, they are somewhat wider than the stalks of Caulobacter and the pseudostalks of Asticcacaulis, and they terminate in a bulbous tip. The deoxyribonucleic acid (DNA) base composition ranges from 54.6–60.1, well below the 62–67 range for the genus Caulobacter. Based upon these and other differences, a new genus and species, Prosthecobacter fusiformis, is proposed for the fusiform caulobacters.     

Characterization of prostaglandin production in tissue culture of rat renal medullary cells

Renal medullary cells from the rat were used to establish a cell culture line. The morphologic characteristics of these cells were similar to renal medullary interstitial cells. They produced substantial amounts of PGE when provided with arachidonic acid or fetal calf serum. PGE production was inhibited 80–90% by indomethacin or meclofenamate. PGE release by the cells was sensitive to and stimulated by changing the culture media. Stable levels of PGE in the media could be achieved if media changes were avoided during the experimental period.     

The DExD/H-box ATPase Prp2p destabilizes and proofreads the catalytic RNA core of the spliceosome

After undergoing massive RNA and protein rearrangements during assembly, the spliceosome undergoes a final, more subtle, ATP-dependent rearrangement that is essential for catalysis. This rearrangement requires the DEAH-box protein Prp2p, an RNA-dependent ATPase. Prp2p has been implicated in destabilizing interactions between the spliceosome and the protein complexes SF3 and RES, but a role for Prp2p in destabilizing RNA–RNA interactions has not been explored. Using directed molecular genetics in budding yeast, we have found that a cold-sensitive prp2 mutation is suppressed not only by mutations in SF3 and RES components but also by a range of mutations that disrupt the spliceosomal catalytic core element U2/U6 helix I, which is implicated in juxtaposing the 5′ splice site and branch site and in positioning metal ions for catalysis within the context of a putative catalytic triplex; indeed, mutations in this putative catalytic triplex also suppressed a prp2 mutation. Remarkably, we also found that prp2 mutations rescue lethal mutations in U2/U6 helix I. These data provide evidence that RNA elements that comprise the catalytic core are already formed at the Prp2p stage and that Prp2p destabilizes these elements, directly or indirectly, both to proofread spliceosome activation and to promote reconfiguration of the spliceosome to a fully competent, catalytic conformation.     

Phylogenetic Diversity of Numerically Important Arctic Sea-Ice Bacteria Cultured at Subzero Temperature

Heterotrophic bacteria in sea ice play a key role in carbon cycling, but little is known about the predominant players at the phylogenetic level. In a study of both algal bands and clear ice habitats within summertime Arctic pack ice from the Chukchi Sea, we determined the abundance of total bacteria and actively respiring cells in melted ice samples using epifluorescence microscopy and the stains 4', 6'-diamidino-2-phenylindole 2HCl (DAPI) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), respectively. Organic-rich and -poor culturing media were used to determine culturable members by plating (at 0 degrees C and 5 degrees C) and most-probable-number (MPN) analyses (at -1 degrees C). Total bacterial counts ranged from 5.44 x 10(4) ml(-1) in clear ice to 2.41 x 10(6) ml(-1) in algal-band ice samples, with 2-27% metabolically active by CTC stain. Plating and MPN results revealed a high degree of culturability in both types of media, but greater success in oligotrophic media (to 62% of total abundance) and from clear ice samples. The bacterial enumeration anomaly, commonly held to mean 相似文献