Measurement of hydrocarbon-degrading microbial populations by a 96-well plate most-probable-number procedure

A 96-well microtiter plate most-probable-number (MPN) procedure was developed to enumerate hydrocarbondegrading microorganisms. The performance of this method, which uses number 2 fuel oil (F2) as the selective growth substrate and reduction of iodonitrotetrazolium violet (INT) to detect positive wells, was evaluated by comparison with an established 24-well microtiter plate MPN procedure (the Sheen Screen), which uses weathered North Slope crude oil as the selective substrate and detects positive wells by emulsification or dispersion of the oil. Both procedures gave similar estimates of the hydrocarbon-degrader population densities in several oil-degrading enrichment cultures and sand samples from a variety of coastal sites. Although several oils were effective substrates for the 96-well procedure, the combination of F2 with INT was best, because the color change associated with INT reduction was more easily detected in the small wells than was disruption of the crude oil slick. The method's accuracy was evaluated by comparing hydrocarbon-degrader MPNs with heterotrophic plate counts for several pure and mixed cultures. For some organisms, it seems likely that a single cell cannot initiate sufficient growth to produce a positive result. Thus, this and other hydrocarbon-degrader MPN procedures might underestimate the hydrocarbon-degrading population, even for culturable organisms.     

Seasonal Dynamics of Dimethylarsenic Acid Degrading Bacteria Dominated in Lake Kibagata

Degradation processes of organoarsenic compounds significantly influence arsenic cycles in aquatic environments and would depend on the bacterial activities. The bacterial population involving dimethylarsinic acid (DMAA) degradation was investigated in Lake Kibagata from April to December in 2003. During the experimental period, the methylated arsenic was not detected, although the inorganic arsenic concentration ranged from 3.4 nM to 9.2 nM. Moreover, in the sample water of Lake Kibagata to which DMAA added, DMAA decreased while inorganic arsenic increased for 25 days. These facts suggested that the bacteria remineralized methylate arsenic species to inorganic arsenic. In fact, monitoring the use of Most Probable Number (MPN) procedure demonstrated that the DMAA-degrading bacteria exist at cell densities ranged from 41 cells/ml to 510 cells/ml. To determine the composition of DMAA-degrading bacteria, the total 110 isolates obtained as dominated bacterial species were analyzed by the restriction-fragment-length polymorphism (RFLP) analysis of 16S rDNA. As a result, total 110 isolates were classified into 12 types, of which 4 types dominated during the spring and/or fall seasons, and the rest 8 types dominated during summer season. DMAA degrading activities of the 110 isolates ranged at various degrees. Especially, the some isolates of fall season tend to show high degradation activities. The phylogenetic analysis using 16S rDNA revealed that the representative isolates formed several clusters in the gram-positive bacterial group and the proteobacteria subdivision. The diverse compositions of DMAA-degrading bacteria would seasonally change to control the rates of organoarsenic degradation in Kibagata.     

Quantitative analysis of bacterial communities from Mediterranean sapropels based on cultivation-dependent methods

Microbial communities of ancient Mediterranean sapropels, buried sediment layers of high organic matter, were analyzed by most probable number (MPN) approaches. Mineral media containing different carbon sources in sub-millimolar concentrations were used. MPN numbers were elevated in sapropels and at the sediment surface, which mirrored total cell count distributions. Highest MPN counts were obtained with a mixture of different monomeric and polymeric substrates, with amino acids or with long-chain fatty acids as sole carbon sources. These values reached up to 2 x 10(7) cm(-3), representing 3.3% of the total cell count. A total of 98 pure cultures were isolated from the highest positive dilutions of the MPN series, representing the most abundant microorganisms culturable by the methods used. The strains were identified by molecular biological methods and could be grouped into 19 different phylotypes. They belonged to the alpha-, beta-, gamma-, and delta-Proteobacteria, to the Actinobacteria and the Firmicutes. However, about half of the number of isolates was closely related to the genera Photobacterium and Agrobacterium. Regarding the high cultivation success, these organisms can be assumed to be typical sapropel bacteria, representing a substantial part of the culturable indigenous microbial community.     

PELAGIC AND BENTHIC ENVIRONMENTAL CONTROLS ON THE SPATIAL DISTRIBUTION OF A VIABLE DIATOM PROPAGULE BANK ON THE SWEDISH WEST COAST1

The distribution of viable diatom resting stages in sediments on the Swedish west coast was assessed by the most probable number (MPN) culture technique. Multivariate analyses correlated benthic and pelagic environmental factors to the observed spatial variations in the size and taxonomic composition of the propagule bank. Viable diatom resting stages were plentiful (0.2–4.8 million cells·g ^{? 1} 1 Received 11 September 2001. Accepted 12 June 2002.
dry weight) and were dominated by the genera Skeletonema, Detonula, Chaetoceros, and Thalassiosira. Size of the propagule bank was primarily related to planktonic biomass (measured as chl a) and was highest in the Orust‐Tjörn fjord system. Species composition in this fjord system was dominated by D. confervacea (Cleve) Gran and T. nordenskioeldii Cleve in contrast to stations on the outer coast, which contained more cells of T. minima Gaarder, Asterionellopsis glacialis (Castracane) Round, and Leptocylindrus danicus Cleve. These taxonomic variations were principally influenced by deep water oxygen concentrations and water column stability. Benthic resting cells of S. costatum (Greville) Cleve were abundant all along the coast but showed reduced viability in low oxygen environments. Calculations based on MPN values estimated that resuspension of sediment could provide a sizable inoculum to the plankton, although the development of planktonic blooms will also depend on forces of hydrography and weather. Although benthic resting stages may not be absolutely necessary for survival of all diatoms, these cells may be important in determining species cycles, succession, and the spatial distribution of diatoms.     

Identification of the synaptic vesicle glycoprotein 2 receptor binding site in botulinum neurotoxin A

Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release by hydrolysing SNARE proteins. The most important serotype BoNT/A employs the synaptic vesicle glycoprotein 2 (SV2) isoforms A-C as neuronal receptors. Here, we identified their binding site by blocking SV2 interaction using monoclonal antibodies with characterised epitopes within the cell binding domain (H_C). The site is located on the backside of the conserved ganglioside binding pocket at the interface of the H_CC and H_CN subdomains. The dimension of the binding pocket was characterised in detail by site directed mutagenesis allowing the development of potent inhibitors as well as modifying receptor binding properties.     

Evaluation of Petrifilm™ EC method for enumeration of E. coli from soil

Aims: To evaluate the suitability of commercially available Petrifilm? EC plates for enumeration of Escherichia coli from soil. Methods and Results: A confirmed E. coli strain isolated from liquid swine manure was inoculated into sterilized sandy clay loam and loam soils at the concentrations of 10², 10³, 10⁵ CFU g^?1 of soil. The efficiency of recovery on Petrifilm? EC plates for soils spiked with E. coli was compared with standard membrane filtration techniques on m‐FC basal medium supplemented with 3‐bromo‐4‐chloro‐5‐indoyl‐β‐d ‐glucopyranoside (BCIG) and most probable numbers (MPN) techniques in E. coli medium with 4‐methylumbelliferyl‐β‐d ‐glucuronide (EC‐MUG) broth. Petrifilm? EC and m‐FC (BCIG) methods were then assessed for the ability to recover E. coli from field soils applied with swine manure. No significant differences (P > 0·05) were observed between Petrifilm? EC, m‐FC (BCIG) and MPN methods for the recovery of E. coli from spiked samples, irrespective of soil type. However, recovery of E. coli from manure‐applied field soil samples showed a significant difference (P < 0·05) between the Petrifilm? EC method and the m‐FC method in enumerating E. coli possibly as a result of false positives on m‐FC. Conclusion: The Petrifilm? EC method is suitable for the enumeration of E. coli from soil with a detection limit of 10 CFU g^?1 soil. Significance and Impact of the Study: The commercially available Petrifilm? EC method is comparatively low cost, easy to use method for the enumeration of E. coli from soil without the need for further confirmation tests.     

Base-CP proteasome can serve as a platform for stepwise lid formation

26S proteasome, a major regulatory protease in eukaryotes, consists of a 20S proteolytic core particle (CP) capped by a 19S regulatory particle (RP). The 19S RP is divisible into base and lid sub-complexes. Even within the lid, subunits have been demarcated into two modules: module 1 (Rpn5, Rpn6, Rpn8, Rpn9 and Rpn11), which interacts with both CP and base sub-complexes and module 2 (Rpn3, Rpn7, Rpn12 and Rpn15) that is attached mainly to module 1. We now show that suppression of RPN11 expression halted lid assembly yet enabled the base and 20S CP to pre-assemble and form a base-CP. A key role for Regulatory particle non-ATPase 11 (Rpn11) in bridging lid module 1 and module 2 subunits together is inferred from observing defective proteasomes in rpn11–m1, a mutant expressing a truncated form of Rpn11 and displaying mitochondrial phenotypes. An incomplete lid made up of five module 1 subunits attached to base-CP was identified in proteasomes isolated from this mutant. Re-introducing the C-terminal portion of Rpn11 enabled recruitment of missing module 2 subunits. In vitro, module 1 was reconstituted stepwise, initiated by Rpn11–Rpn8 heterodimerization. Upon recruitment of Rpn6, the module 1 intermediate was competent to lock into base-CP and reconstitute an incomplete 26S proteasome. Thus, base-CP can serve as a platform for gradual incorporation of lid, along a proteasome assembly pathway. Identification of proteasome intermediates and reconstitution of minimal functional units should clarify aspects of the inner workings of this machine and how multiple catalytic processes are synchronized within the 26S proteasome holoenzymes.     

Three manganese oxide-rich marine sediments harbor similar communities of acetate-oxidizing manganese-reducing bacteria

Dissimilatory manganese reduction dominates anaerobic carbon oxidation in marine sediments with high manganese oxide concentrations, but the microorganisms responsible for this process are largely unknown. In this study, the acetate-utilizing manganese-reducing microbiota in geographically well-separated, manganese oxide-rich sediments from Gullmar Fjord (Sweden), Skagerrak (Norway) and Ulleung Basin (Korea) were analyzed by 16S rRNA-stable isotope probing (SIP). Manganese reduction was the prevailing terminal electron-accepting process in anoxic incubations of surface sediments, and even the addition of acetate stimulated neither iron nor sulfate reduction. The three geographically distinct sediments harbored surprisingly similar communities of acetate-utilizing manganese-reducing bacteria: 16S rRNA of members of the genera Colwellia and Arcobacter and of novel genera within the Oceanospirillaceae and Alteromonadales were detected in heavy RNA-SIP fractions from these three sediments. Most probable number (MPN) analysis yielded up to 10⁶ acetate-utilizing manganese-reducing cells cm⁻³ in Gullmar Fjord sediment. A 16S rRNA gene clone library that was established from the highest MPN dilutions was dominated by sequences of Colwellia and Arcobacter species and members of the Oceanospirillaceae, supporting the obtained RNA-SIP results. In conclusion, these findings strongly suggest that (i) acetate-dependent manganese reduction in manganese oxide-rich sediments is catalyzed by members of taxa (Arcobacter, Colwellia and Oceanospirillaceae) previously not known to possess this physiological function, (ii) similar acetate-utilizing manganese reducers thrive in geographically distinct regions and (iii) the identified manganese reducers differ greatly from the extensively explored iron reducers in marine sediments.     

Microbial Population Analysis as a Measure of Ecosystem Restoration

During a controlled oil spill study in a freshwater wetland, four methods were used to track changes in microbial populations in response to in situ remediation treatments, including nutrient amendments and the removal of surface vegetation. Most probable number (MPN) estimates of alkane and aromatic hydrocarbon degraders showed divergence of the alkane and aromatic degrading populations during the first summer of the experiment. Alkane degraders increased in all plots by 1.5 orders of magnitude and aromatic degraders increased in oiled plots by 3.5 orders of magnitude. Phospholipid fatty acid (PLFA) analysis of biomass and community composition showed no essential differences among treatments. Denaturing gradient gel electrophoresis (DGGE) analysis of the sediment microbial community showed some differences in specific populations of organisms with respect to oiled and unoiled plots. Some organisms were only found in the oiled plots. Sediment toxicity measured against single celled algae showed that the oiled sediments were toxic into the second year of the study, but that nutrient addition relieved the toxicity more rapidly than natural attenuation of the oil.     

Inhibition of Acetoclastic Methanogenesis in Crude Oil- and Creosote-Contaminated Groundwater

Results from a series of studies of methanogenic processes in crude oil- and creosote-contaminated aquifers indicate that acetoclastic methanogenesis is inhibited near non-aqueous sources. At a crude oil-contaminated site, numbers of acetoclastic methanogens found close to crude oil were one hundred times fewer than those of hydrogen- and formate-utilizing methanogens. In laboratory toxicity assays, crude oil collected from the site inhibited methane production from acetate but not from formate or hydrogen. Toxicity assays with aqueous creosote extract completely inhibited acetate utilization over the range of tested dilutions but only mildly affected formate and hydrogen utilization. The combined results from the laboratory and field studies suggest that in methanogenic contaminated aquifers, inhibition of acetoclastic methanogenesis may lead to a buildup of acetate relative to dissolved organic carbon.