Minimal Amino Acid Requirements of the Hyperthermophilic Archaeon Pyrococcus abyssi, Isolated from Deep-Sea Hydrothermal Vents

Vol. 61, no. 3, p. 1138, column 1, line 4 from bottom: "(A(inf600) = 0.1 = 8 x 10(sup8) cells ml(sup-1))" should read "(A(inf600) = 0.1 = 1.8 x 10(sup8) cells ml(sup-1))." [This corrects the article on p. 1138 in vol. 61.].     

Early Interactions of Rhizobium leguminosarum bv. phaseoli and Bean Roots: Specificity in the Process of Adsorption and Its Requirement of Ca(sup2+) and Mg(sup2+) Ions

Roots of Phaseolus vulgaris L. were incubated with dilute suspensions (1 x 10(sup3) to 3 x 10(sup3) bacteria ml(sup-1)) of an antibiotic-resistant indicator strain of Rhizobium leguminosarum bv. phaseoli in mineral medium and washed four times by a standardized procedure prior to quantitation of adsorption (G. Caetano-Anolles and G. Favelukes, Appl. Environ. Microbiol. 52:371-376, 1986). The population of rhizobia remaining adsorbed on roots after washing was homogeneous, as indicated by the first-order course of its desorption by hydrodynamic shear. Rhizobia were maximally active for adsorption in the early stationary phase of growth. The process leading to adsorption was rapid, without an initial lag, and slowed down after 1 h. Adsorption of the indicator strain at 10(sup3) bacteria ml(sup-1) was inhibited to different extents in the presence of 10(sup3) to 10(sup8) antibiotic-sensitive competitor rhizobia ml(sup-1). After a steep rise above 10(sup4) bacteria ml(sup-1), inhibition by heterologous competitors in the concentration range of 10(sup5) to 10(sup7) bacteria ml(sup-1) was markedly less than by homologous strains, while at 10(sup8) bacteria ml(sup-1) it approached the high level of inhibition by the latter. At 10(sup7) bacteria ml(sup-1), all of the heterologous strains tested were consistently less inhibitory than homologous competitors (P < 0.001). These differences in competitive behavior indicate that in the process of adsorption of R. leguminosarum bv. phaseoli to its host bean roots, different modes of adsorption occur and that some of these modes are specific for the microsymbiont (as previously reported for the alfalfa system [G. Caetano-Anolles and G. Favelukes, Appl. Environ. Microbiol. 52:377-381, 1986]). Moreover, whereas the nonspecific process occurred either in the absence or in the presence of Ca(sup2+) and Mg(sup2+) ions, expression of specificity was totally dependent on the presence of those cations. R. leguminosarum bv. phaseoli bacteria adsorbed in the presence of Ca(sup2+) and Mg(sup2+) were more resistant to desorption by shear forces than were rhizobia adsorbed in their absence. These results indicate that (i) symbiotic specificity in the P. vulgaris-R. leguminosarum bv. phaseoli system is expressed already during the early process of rhizobial adsorption to roots, (ii) Ca(sup2+) and Mg(sup2+) ions are required by R. leguminosarum bv. phaseoli for that specificity, and (iii) those cations cause tighter binding of rhizobia to roots.     

Fluorescently Labeled Virus Probes Show that Natural Virus Populations Can Control the Structure of Marine Microbial Communities

Fluorescently stained viruses were used as probes to label, identify, and enumerate specific strains of bacteria and cyanobacteria in mixed microbial assemblages. Several marine virus isolates were fluorescently stained with YOYO-1 or POPO-1 (Molecular Probes, Inc.) and added to seawater samples that contained natural microbial communities. Cells to which the stained viruses adsorbed were easily distinguished from nonhost cells; typically, there was undetectable binding of stained viruses to natural microbial assemblages containing >10(sup6) bacteria ml(sup-1) but to which host cells were not added. Host cells that were added to natural seawater were quantified with 99% (plusmn) 2% (mean (plusmn) range) efficiency with fluorescently labeled virus probes (FLVPs). A marine bacterial isolate (strain PWH3a), tentatively identified as Vibrio natriegens, was introduced into natural microbial communities that were either supplemented with nutrients or untreated, and changes in the abundance of the isolate were monitored with FLVPs. Simultaneously, the concentrations of viruses that infected strain PWH3a were monitored by plaque assay. Following the addition of PWH3a, the concentration of viruses infecting this strain increased from undetectable levels (<1 ml(sup-1)) to 2.9 x 10(sup7) and 8.3 x 10(sup8) ml(sup-1) for the untreated and nutrient-enriched samples, respectively. The increase in viruses was associated with a collapse in populations of strain PWH3a from ca. 30 to 2% and 43 to 0.01% of the microbial communities in untreated and nutrient-enriched samples, respectively. These results clearly demonstrate that FLVPs can be used to identify and quantify specific groups of bacteria in mixed microbial communities. The data show as well that viruses which are present at low abundances in natural aquatic viral communities can control microbial community structure.     

Highly Active Microbial Communities in the Ice and Snow Cover of High Mountain Lakes

An exploratory study carried out in Pyrenean and Alpine lakes shows that a rich, active microbial community lives in the slush layers of the winter cover of such lakes in spite of the low temperature and the seasonal occurrence of the habitat. Bacteria were very diverse in morphology, with filaments reaching up to 100 (mu)m long; flagellates, both autotrophic (chrysophytes, cryptophytes, dinoflagellates, and volvocales) and heterotrophic, and ciliates were abundant, reaching biovolume values up to 2.7 x 10(sup6) (mu)m(sup3) ml(sup-1). Species composition was very variable, with dominance depending on date and depth. Although many species were typical of lake plankton communities, some were restricted to the slush, for instance the predatory ciliates Dileptus sp. and Lacrymaria sp., and others were restricted to the surface pools, such as the snow algae Chlamydomonas nivalis. Microbial biomasses and usually bacterial and algal activities were greater in the slush layers than in the lake water. Photosynthesis rate in the upper cover layers reached values up to 0.5 (mu)g of C liter(sup-1) h(sup-1), and high bacterial activities up to 226 pmol of leucine incorporated liter(sup-1) h(sup-1) and 25 pmol of thymidine incorporated liter(sup-1) h(sup-1) were measured. For most species, lake water flooding the ice and snow cover could provide an inoculum. Differential growth depending on the environmental conditions (nutrients, organic matter, light) of a particular slush layer could provide dominance of different groups or species. However, there was no obvious colonizing mechanism for those species not appearing either in plankton or in communities on top of the snowpack.     

Significance of Bacteriophages for Controlling Bacterioplankton Growth in a Mesotrophic Lake

Bacterium-specific viruses have attracted much interest in aquatic microbial ecology because they have been shown to be about 10 times more abundant than planktonic bacteria. So far most of the studies of interactions of planktonic bacteria and viruses have been done in marine environments, and very little is known about these interactions in lakes. Therefore, we studied phage proliferation in Lake Constance, a large mesotrophic lake in Germany. We enumerated bacteria and quantified the fraction of bacteria with mature intracellular phage particles and the number of free viruses by transmission electron microscopy. Between the end of March and early August 1992, peaks of bacterial abundance were followed in 1 to 2 weeks by peaks in the fraction of bacteria containing visible phage particles (0 to 1.7%) and in the number of free viruses (1 x 10(sup7) to 4 x 10(sup7) ml(sup-1)). We estimated that 1 to 17% +/- 12% of all bacteria were phage infected, implying that phage-induced mortality was <34% +/- 24% of total mortality. A direct comparison between phage-induced mortality, the net decrease of bacterial numbers, and bacterial growth rates indicated that phage-induced mortality accounted for <11% of total bacterial mortality during the phytoplankton spring bloom and 18 to 21% following the bloom. Estimated burst sizes ranged from 21 to 121 phages. Phage production rates of 0.5 x 10(sup6) to 2.5 x 10(sup6) ml(sup-1) day(sup-1) accounted for 70 to 380% of the observed net increase rates of free phages, implying high rates of simultaneous phage decay. The cyclic dynamics between bacteria and phages and the varying size structure of the intracellular mature phage particles suggested that phage infection was important in structuring the bacterial host assemblage during the study period.     

Population Dynamics of Polychlorinated Biphenyl-Dechlorinating Microorganisms in Contaminated Sediments

The growth dynamics of polychlorinated biphenyl (PCB)-dechlorinating microorganisms were determined for the first time, along with those of sulfate reducers and methanogens, by using the most-probable-number technique. The time course of Aroclor 1248 dechlorination mirrored the growth of dechlorinators; dechlorination ensued when the dechlorinating population increased by 2 orders of magnitude from 2.5 x 10(sup5) to 4.6 x 10(sup7) cells g of sediment(sup-1), at a specific growth rate of 6.7 day(sup-1) between 2 and 6 weeks. During this period, PCB-dechlorinating microorganisms dechlorinated Aroclor 1248 at a rate of 3.9 x 10(sup-8) mol of Cl g of sediment(sup-1) day(sup-1), reducing the average number of Cl molecules per biphenyl from 3.9 to 2.8. The growth yield was 4.2 x 10(sup13) cells mol of Cl dechlorinated(sup-1). Once dechlorination reached a plateau, after 6 weeks, the number of dechlorinators began to decrease. On the other hand, dechlorinators inoculated into PCB-free sediments decreased over time from their initial level, suggesting that PCBs are required for their selective enrichment. The numbers of sulfate reducers and methanogens increased in both PCB-free and contaminated sediments, showing little difference between them. The maximum population size of sulfate reducers was about an order of magnitude higher than that of dechlorinators, whereas that of methanogens was slightly less. Unlike those of dechlorinators, however, numbers of both sulfate reducers and methanogens remained high even when dechlorination ceased. The results of this study imply that PCB concentrations may have to exceed a certain threshold to maintain the growth of PCB dechlorinators.     

Development of a Chemically Defined Medium for the Growth of Leuconostoc mesenteroides

A chemically defined medium for the growth of Leuconostoc mesenteroides was developed. This medium contained lactose, Mn(sup2+), Mg(sup2+), 12 amino acids, eight vitamins, adenine, uracil, and Tween 80. We showed the beneficial effect of aerobic conditions on growth and that potassium phosphate (135 mM) is a suitable buffer. The growth rate in this medium was 0.85 (plusmn) 0.10 h(sup-1) for the six strains examined, and cell densities up to 3.5 x 10(sup9) CFU/ml were attained.     

Effect of Parental Growth on Dynamics of Conjugative Plasmid Transfer in the Pea Spermosphere

Plasmid transfer rates for the conjugative plasmid R388::Tn1721 from Pseudomonas cepacia (donor) to Pseudomonas fluorescens (recipient) on agar media, in broth, and in microcosms containing sterile or nonsterile soil, in the presence or absence of germinating pea seeds, were determined. Donors, recipients, and transconjugants were enumerated on selective media after 1 day on agar or in broth culture and over a 7-day period in soil or pea spermosphere microcosms. Donor and recipient growth rates and plasmid transfer rate constants [(gamma), where (gamma) = transconjugants (middot) (donors (middot) recipients)(sup-1) (middot) h(sup-1)] were calculated for three initial parental densities (10(sup4), 10(sup6), or 10(sup8) CFU/g or ml) in each system. For all initial density levels, values of (gamma) in agar and broth matings were higher than those in soil or in the pea spermosphere-rhizosphere microcosms. Values of (gamma) were not influenced by the pea spermosphere or by sterile or nonsterile conditions of the soil. However, (gamma) values in microcosm experiments were inversely related to initial parental density and were directly related to donor growth rates. Values of (gamma) averaged 4 x 10(sup-10), 4 x 10(sup-12), and 3 x 10(sup-14) when initial donor and recipient cell densities were 10(sup4), 10(sup6), and 10(sup8) CFU/g, respectively. These results suggest that the plasmid transfer rate constant is independent of parental cell density only when parental growth is not limited. In a resource-limited environment, intra- or interspecific competition may reduce the transfer rate by limiting parental growth.     

Activity of microorganisms in activated sludge in relation to the concentration of mercury in sewage

Activated sludge from the refinery of a plant producing synthetic rubber was shown to adsorb mercury ions contained in the sewage. As a result, the content of microorganisms in the sludge as well as the activity of dehydrogenase decreased. The quantity of Pseudomonas aeruginosa cells was 40 x 10(6) per 1 ml in a chemically defined medium without mercury ions, 20 x 10(5) per 1 ml in the water after the first sedimentation tank (0.07 to 0.08 mg Hg per litre), and 10 x 10(6) per 1 ml in the water of sewage common to the whole plant (0.13 to 0.14 mg Hg per litre). The use of the phage-resistant P-aeruginosa T-76 culture adapted to synthetic organic pollutants contained in the sewage of the plant together with the activated sludge of the refinery increased the biological activity of the sludge and improved the quality of purification in terms of dehydrogenase activity and chemical uptake of oxygen.     

Factors Limiting Microbial Growth and Activity at a Proposed High-Level Nuclear Repository, Yucca Mountain, Nevada

As part of the characterization of Yucca Mountain, Nev., as a potential repository for high-level nuclear waste, volcanic tuff was analyzed for microbial abundance and activity. Tuff was collected aseptically from nine sites along a tunnel in Yucca Mountain. Microbial abundance was generally low: direct microscopic cell counts were near detection limits at all sites (3.2 x 10(sup4) to 2.0 x 10(sup5) cells g(sup-1) [dry weight]); plate counts of aerobic heterotrophs ranged from 1.0 x 10(sup1) to 3.2 x 10(sup3) CFU g(sup-1) (dry weight). Phospholipid fatty acid concentrations (0.1 to 3.7 pmol g(sup-1)) also indicated low microbial biomasses; diglyceride fatty acid concentrations, indicative of dead cells, were in a similar range (0.2 to 2.3 pmol g(sup-1)). Potential microbial activity was quantified as (sup14)CO(inf2) production in microcosms containing radiolabeled substrates (glucose, acetate, and glutamic acid); amendments with water and nutrient solutions (N and P) were used to test factors potentially limiting this activity. Similarly, the potential for microbial growth and the factors limiting growth were determined by performing plate counts before and after incubating volcanic tuff samples for 24 h under various conditions: ambient moisture, water-amended, and amended with various nutrient solutions (N, P, and organic C). A high potential for microbial activity was demonstrated by high rates of substrate mineralization (as much as 70% of added organic C in 3 weeks). Water was the major limiting factor to growth and microbial activity, while amendments with N and P resulted in little further stimulation. Organic C amendments stimulated growth more than water alone.     

Optimal Renewal Rate and Nutrient Concentration for the Production of the Marine Microalga Phaeodactylum tricornutum in Semicontinuous Cultures

A factorial experiment comprising six nutrient concentrations and six renewal rates was set to optimize semicontinuous cultures of Phaeodactylum tricornutum. Maximal cell density, 85 x 10(sup6) cells ml(sup-1), was obtained with 8 and 16 mmol of N per liter and a renewal rate of 10%. Protein stabilized at about 60% of the organic fraction for nitrogen-sufficient cultures. Both lipids and carbohydrates were stored as energetic reserves.     

Growth Kinetics of Thiobacillus thiooxidans on the Surface of Elemental Sulfur

The growth kinetics of Thiobacillus thiooxidans on elemental sulfur in batch cultures at 30(deg)C and pH 1.5 was studied by measuring the time courses of the concentration of adsorbed cells on sulfur, the concentration of free cells suspended in liquid medium, and the amount of sulfur oxidized. As the elemental sulfur was oxidized to sulfate ions, the surface concentration of adsorbed cells per unit mass of sulfur approached a maximum value (maximum adsorption capacity of sulfur particles) whereas the concentration of free cells continued to increase with time. There was a close relationship between the concentrations of free and adsorbed cells during the microbial sulfur oxidation, and the two cell concentrations were well correlated by the Langmuir isotherm with adsorption equilibrium constant K(infA) and maximum adsorption capacity X(infAm) of 2.10 x 10(sup-9) ml per cell and 4.57 x 10(sup10) cells per g, respectively. The total concentration of free and adsorbed cells increased in parallel with the amount of sulfate formed. The total growth on elemental sulfur gave a characteristic growth curve in which a linear-growth phase followed the period of an initial exponential phase. The batch rate data collected under a wide variety of inoculum levels (about 10(sup5) to 10(sup8) cells per ml) were consistent with a kinetic model assuming that the growth rate of adsorbed bacteria is proportional to the product of the concentration, X(infA), of adsorbed cells and the fraction, (theta)(infV), of adsorption sites unoccupied by cells. The kinetic and stoichiometric parameters appearing in the model were estimated from the experimental data, and the specific growth rate, (mu)(infA), and growth yield, Y(infA), were 2.58 day(sup-1) and 2.05 x 10(sup11) cells per g, respectively. The proposed model and the parameter values allowed us to predict quantitatively the surface attachment of T. thiooxidans cells on elemental sulfur and the bacterial growth in both initial exponential and subsequent linear phases. The transition from exponential to linear growth was a result of two competing factors: an increase in the adsorbed-cell concentration, X(infA), permitted a decrease in the unoccupied-site fraction, (theta)(infV).     

Minimal Amino Acid Requirements of the Hyperthermophilic Archaeon Pyrococcus abyssi, Isolated from Deep-Sea Hydrothermal Vents

A minimal growth medium containing only nine amino acids and vitamins as the sole carbon and energy sources allowed the growth of Pyrococcus abyssi GE 5, a novel hyperthermophilic sulfur-metabolizing archaeon isolated from deep-sea hydrothermal vents. The generation time in this medium was about 40 min, and cell densities up to 5 x 10(sup8) cells ml(sup-1) were attained. These results are similar to those obtained previously with complex proteinaceous media.     

Distribution of Polyunsaturated Fatty Acids in Bacteria Present in Intestines of Deep-Sea Fish and Shallow-Sea Poikilothermic Animals

The lipid and fatty acid compositions in nine obligate and facultative barophilic bacteria isolated from the intestinal contents of seven deep-sea fish were determined. Phospholipid compositions were simple, with phosphatidylethanolamine and phosphatidylglycerol predominating in all strains. Docosahexaenoic acid (DHA; 22:6n-3), which has not been reported in procaryotes except for deep-sea bacteria, was found to be present in eight strains at a level of 8.1 to 21.5% of total fatty acids. In the other strain, eicosapentaenoic acid (EPA; 20:5n-3) was present at a level of 31.5% of total fatty acids. Other fatty acids observed in all strains were typical of marine gram-negative bacteria. Subcultures from pouches prepared from intestinal contents of five deep-sea fish by the most-probable-number (MPN) method were analyzed for fatty acids, and all subcultures contained DHA and/or EPA. Accordingly, viable cell counts of bacteria containing DHA and EPA were estimated at a maximum of 1.3 x 10(sup8) and 2.4 x 10(sup8) cells per ml, respectively, and accounted for 14 and 30%, respectively, of the total cell counts in the intestinal contents of the deep-sea fish. In the case of 10 shallow-sea poikilothermic animals having bacterial populations of 1.1 x 10(sup6) to 1.9 x 10(sup9) CFU per ml in intestinal contents, no DHA was found in the 112 isolates examined, while production of EPA was found in 40 isolates from cold- and temperate-sea samples. These results suggest that DHA and EPA are involved in some adaptations of bacteria to low temperature and high pressure.     

Purification of Pyoverdines of Pseudomonas fluorescens 2-79 by Copper-Chelate Chromatography

Three pyoverdines, Pf-A, Pf-B, and Pf-C, were purified with copper-chelate Sepharose and Sephadex G-15 columns from Pseudomonas fluorescens 2-79, and the yields (per 100 ml of culture supernatant) were 2.8, 21.6, and 3.2 mg, respectively. The absorption and fluorescence spectra of these pyoverdines were strongly pH dependent. Characteristic changes in the maximal absorbance wavelengths were observed when Fe(sup3+) or Cu(sup2+) was added. The addition of Cu(sup2+) shifted the pyoverdine Pf-B absorbance spectrum so that it exhibited a single peak at 410 nm but did not give rise to a new absorbance maximum at approximately 460 nm, which appeared when Fe(sup3+) was added. Fluorescence quenching experiments revealed that the forward reaction rate constant with pyoverdines was much higher with Cu(sup2+) (10(sup4) to 10(sup5) M(sup-1) s(sup-1)) than with Fe(sup3+) (10(sup2) M(sup-1) s(sup-1)). However, Cu(sup2+)-pyoverdine complexes were completely dissociated by EDTA at a low concentration (0.1 mM), while the level of Fe(sup3+)-pyoverdine complex dissociation at the same EDTA concentration was relatively low. The dissociation of Fe(sup3+)-pyoverdine complexes was EDTA concentration dependent. Formation of free pyoverdine was observed when the three types of Fe(sup3+)-pyoverdine complexes were incubated separately with P. fluorescens 2-79 cells, thus demonstrating that pyoverdines Pf-A, Pf-B, and Pf-C mediate iron transport.     

Partitioning of Symbiotic Bacteria between Generations of an Insect: a Quantitative Study of a Buchnera sp. in the Pea Aphid (Acyrthosiphon pisum) Reared at Different Temperatures

The population of symbiotic Buchnera bacteria in parthenogenetic females of the pea aphid Acyrthosiphon pisum was determined by quantitative hybridization of a DNA probe (groESL) to aphid homogenates. The aphids bore 1 x 10(sup7) to 2 x 10(sup7) bacterial cells per mg (fresh weight). In teneral aphids (i.e., aphids that had moulted to adulthood but that had not initiated reproduction), >75% of the bacteria were in the embryos, and the density of bacteria in the embryos was consistently greater than that in the maternal tissues. The bacterial density in teneral aphids increased from 1.3 x 10(sup7) to 2.0 x 10(sup7) cells mg (fresh weight) of aphids(sup-1) with temperature between 15 and 25(deg)C. This variation could be attributed to a temperature-dependent increase in both the density of bacteria in the embryos and embryo content of the aphids.     

Contrasting Bacterial Strategies To Coexist with a Flagellate Predator in an Experimental Microbial Assemblage

We studied predator-induced changes within a slowly growing mixed microbial assemblage that was sustained by algal exudates in a continuous cultivation system. In situ hybridization with fluorescent monolabeled oligonucleotide probes was used for a tentative community analysis. This method also allowed us to quantify the proportions of predators with ingested bacteria of different taxonomic groups. In addition, we determined grazing rates on bacteria with fluorescently labelled prey. Bacteria belonging to the alpha and beta subdivisions of the phylum Proteobacteria ((alpha)- and (beta)-Proteobacteria, respectively) showed very different responses to the addition of a bacterivorous flagellate, Bodo saltans. Within one day, filamentous protist-inedible bacteria developed; these belonged to the (beta)-Proteobacteria and constituted between 8.7 and 34% of bacteria from this subgroup. Total abundance of (beta)-Proteobacteria decreased from 3.05 x 10(sup6) to 0.23 x 10(sup6) cells ml(sup-1), and estimated cell division rates were low. Other morphologically inconspicuous protist-edible bacteria belonging to the (alpha)-Proteobacteria were found to respond to predation by an increase in growth rate. Although these bacteria were heavily grazed upon, as on average >85% of flagellate cells had ingested (alpha)-Proteobacteria, they numerically dominated after the addition of B. saltans (mean, 1.35 x 10(sup6) cells ml(sup-1)). It was thus mainly those fast-dividing strains of (alpha)-Proteobacteria that supported the growth of the flagellate population. We conclude that bacteria in mixed assemblages can adopt at least two distinct strategies as a reaction to intense flagellate predation: to outgrow predation pressure or to develop inedible, inactive filaments. Since these strategies occurred within 24 h after the addition of the flagellate, we hypothesize that chemical stimuli released by the predator may have triggered bacterial responses.     

A rapid luminescent-phage based MPN method for the enumeration of Salmonella typhimurium in environmental samples

Materials such as soils, waters, sewage sludges and foods can contain low numbers of salmonellas. A most-probable-number (MPN) method that utilized a bioluminescent-bacteriophage is described that allowed the specific determination of as few as one Salmonella typhimurium cell/100 ml of material within 24 h. The method was developed with soil, lake water and sewage sludge inoculated with Salm. typhimurium and had an efficiency of 100% when tested against a traditional MPN method. The protocol is rapid, sensitive, inexpensive, has a low operator time compared to the traditional MPN method, allows for the repair of injured cells and is amenable to automation.     

Quantification of Hyphomicrobium populations in activated sludge from an industrial wastewater treatment system as determined by 16S rRNA analysis

The bacterial community structure of the activated sludge from a 25 million-gal-per-day industrial wastewater treatment plant was investigated using rRNA analysis. 16S ribosomal DNA (rDNA) libraries were created from three sludge samples taken on different dates. Partial rRNA gene sequences were obtained for 46 rDNA clones, and nearly complete 16S rRNA sequences were obtained for 18 clones. Seventeen of these clones were members of the beta subdivision, and their sequences showed high homology to sequences of known bacterial species as well as published 16S rDNA sequences from other activated sludge sources. Sixteen clones belonged to the alpha subdivision, 7 of which showed similarity to Hyphomicrobium species. This cluster was chosen for further studies due to earlier work on Hyphomicrobium sp. strain M3 isolated from this treatment plant. A nearly full-length 16S rDNA sequence was obtained from Hyphomicrobium sp. strain M3. Phylogenetic analysis revealed that Hyphomicrobium sp. strain M3 was 99% similar to Hyphomicrobium denitrificans DSM 1869(T) in Hyphomicrobium cluster II. Three of the cloned sequences from the activated sludge samples also grouped with those of Hyphomicrobium cluster II, with a 96% sequence similarity to that of Hyphomicrobium sp. strain M3. The other four cloned sequences from the activated sludge sample were more closely related to those of the Hyphomicrobium cluster I organisms (95 to 97% similarity). Whole-cell fluorescence hybridization of microorganisms in the activated sludge with genus-specific Hyphomicrobium probe S-G-Hypho-1241-a-A-19 enhanced the visualization of Hyphomicrobium and revealed that Hyphomicrobium appears to be abundant both on the outside of flocs and within the floc structure. Dot blot hybridization of activated sludge samples from 1995 with probes designed for Hyphomicrobium cluster I and Hyphomicrobium cluster II indicated that Hyphomicrobium cluster II-positive 16S rRNA dominated over Hyphomicrobium cluster I-positive 16S rRNA by 3- to 12-fold. Hyphomicrobium 16S rRNA comprised approximately 5% of the 16S rRNA in the activated sludge.     

Activity and Distribution of Methane-Oxidizing Bacteria in Flooded Rice Soil Microcosms and in Rice Plants (Oryza sativa)

The activity and distribution of CH(inf4)-oxidizing bacteria (MOB) in flooded rice (Oryza sativa) soil microcosms was investigated. CH(inf4) oxidation was shown to occur in undisturbed microcosms by using (sup14)CH(inf4), and model calculations indicated that almost 90% of the oxidation measured had taken place at a depth where only roots could provide the O(inf2) necessary. Slurry from soil planted with rice had an apparent K(infm) for CH(inf4) of 4 (mu)M and a V(infmax) of 0.1 (mu)mol g (dry weight)(sup-1) h(sup-1). At a depth of 1 to 2 cm, there was no significant difference (P > 0.05) in numbers of MOB between soil from planted and nonplanted microcosms (mean, 7.7 x 10(sup5) g [fresh weight](sup-1)). Thus, the densely rooted soil at 1 to 2 cm deep did not represent rhizospheric soil with respect to the number of MOB. A significantly increased number of MOB was found only in soil immediately around the roots (1.2 x 10(sup6) g [fresh weight](sup-1)), corresponding to a layer of 0.1 to 0.2 mm. Plant-associated CH(inf4) oxidation was shown in a double chamber with carefully washed intact rice plants. Up to 90% of the CH(inf4) supplied to the root compartment was oxidized in the plants. CH(inf4) oxidation on isolated roots was higher and had a larger variability than that in soil slurries. Roots had an apparent K(infm) for CH(inf4) of 6 (mu)M and a V(infmax) of 5 (mu)mol g (dry weight)(sup-1) h(sup-1). The average number of MOB in homogenized roots was larger than on the rhizoplane and increased with plant age. MOB also were found in surface-sterilized roots and basal culms, indicating the ability of these bacteria to colonize the interior of roots and culms.