Showing papers in "Applied and Environmental Microbiology in 1986"

Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics

Abstract: Methodes d'extraction d'enzymes, d'electrophorese en gel et de coloration specifique des enzymes utilisees pour etudier la variation genetique chez Escherichia coli et d'autres bacteries. Les procedures decrites peuvent s'appliquer, avec quelques modifications mineures, a toute espece de bacterie

Organic matter mineralization with reduction of ferric iron in anaerobic sediments.

Abstract: The potential for ferric iron reduction with fermentable substrates, fermentation products, and complex organic matter as electron donors was investigated with sediments from freshwater and brackish water sites in the Potomac River Estuary. In enrichments with glucose and hematite, iron reduction was a minor pathway for electron flow, and fermentation products accumulated. The substitution of amorphous ferric oxyhydroxide for hematite in glucose enrichments increased iron reduction 50-fold because the fermentation products could also be metabolized with concomitant iron reduction. Acetate, hydrogen, propionate, butyrate, ethanol, methanol, and trimethylamine stimulated the reduction of amorphous ferric oxyhydroxide in enrichments inoculated with sediments but not in uninoculated or heat-killed controls. The addition of ferric iron inhibited methane production in sediments. The degree of inhibition of methane production by various forms of ferric iron was related to the effectiveness of these ferric compounds as electron acceptors for the metabolism of acetate. The addition of acetate or hydrogen relieved the inhibition of methane production by ferric iron. The decrease of electron equivalents proceeding to methane in sediments supplemented with amorphous ferric oxyhydroxides was compensated for by a corresponding increase of electron equivalents in ferrous iron. These results indicate that iron reduction can outcompete methanogenic food chains for sediment organic matter. Thus, when amorphous ferric oxyhydroxides are available in anaerobic sediments, the transfer of electrons from organic matter to ferric iron can be a major pathway for organic matter decomposition.

Viable but nonculturable stage of Campylobacter jejuni and its role in survival in the natural aquatic environment.

Abstract: Conditions influencing the survival of Campylobacter jejuni in the natural aquatic environment have been determined Release of Campylobacter spp into natural waters by animal hosts is postulated to play a key role in the maintenance of viability and transmission of the organism in the environment Laboratory flask microcosms containing filter-sterilized stream water were used to test C jejuni for the ability to remain viable in simulated natural systems The microcosms were compared with the biphasic and shaking broth procedures used routinely for growth of Campylobacter spp in the research laboratory The stream-water microcosms were analyzed to determine effects of temperature and aeration on the survival of a well-characterized C jejuni strain isolated originally from a human campylobacteriosis patient Morphological characteristics were evaluated by phase-contrast microscopy and scanning or transmission electron microscopy Survival curves were quantified on the basis of plate counts, epifluorescent microscopy, optical density measurements, and direct viable counts associated with protein synthesis in the absence of DNA replication A significant difference was observed between results of direct enumeration, ie, direct viable counts or acridine orange direct counts, and those from spread plate cultures In all cases, increasing temperature of cultivation resulted in decreased recoverability on laboratory media, due possibly to an increased metabolic rate, as analyzed by CO2 evolution in the presence of radiolabeled glutamate Stream water held at low temperature (4 degrees C) sustained significant numbers of campylobacters for greater than 4 months Microcosms, aerated with shaking, exhibited logarithmic decline in recoverable C jejuni, while stationary systems underwent a more moderate rate of decrease to the nonculturable state(ABSTRACT TRUNCATED AT 250 WORDS)

Availability of ferric iron for microbial reduction in bottom sediments of the freshwater tidal potomac river

Abstract: The distribution of Fe(III), its availability for microbial reduction, and factors controlling Fe(III) availability were investigated in sediments from a freshwater site in the Potomac River Estuary. Fe(III) reduction in sediments incubated under anaerobic conditions and depth profiles of oxalate-extractable Fe(III) indicated that Fe(III) reduction was limited to depths of 4 cm or less, with the most intense Fe(III) reduction in the top 1 cm. In incubations of the upper 4 cm of the sediments, Fe(III) reduction was as important as methane production as a pathway for anaerobic electron flow because of the high rates of Fe(III) reduction in the 0- to 0.5-cm interval. Most of the oxalate-extractable Fe(III) in the sediments was not reduced and persisted to a depth of at least 20 cm. The incomplete reduction was not the result of a lack of suitable electron donors. The oxalate-extractable Fe(III) that was preserved in the sediments was considered to be in a form other than amorphous Fe(III) oxyhydroxide, since synthetic amorphous Fe(III) oxyhydroxide, amorphous Fe(III) oxyhydroxide adsorbed onto clay, and amorphous Fe(III) oxyhydroxide saturated with adsorbed phosphate or fulvic acids were all readily reduced. Fe3O4 and the mixed Fe(III)-Fe(II) compound(s) that were produced during the reduction of amorphous Fe(III) oxyhydroxide in an enrichment culture were oxalate extractable but were not reduced, suggesting that mixed Fe(III)-Fe(II) compounds might account for the persistence of oxalate-extractable Fe(III) in the sediments. The availability of microbially reducible Fe(III) in surficial sediments demonstrates that microbial Fe(III) reduction can be important to organic matter decomposition and iron geochemistry. However, the overall extent of microbial Fe(III) reduction is governed by the inability of microorganisms to reduce most of the Fe(III) in the sediment.

Phospholipid ester-linked fatty acid profile changes during nutrient deprivation of Vibrio cholerae: increases in the trans/cis ratio and proportions of cyclopropyl fatty acids

Abstract: The phospholipid ester-linked fatty acids of 0-day-, 7-day-, and 30-day-starved cultures of Vibrio cholerae were compared. Statistically significant trends were noted in the fatty acid profiles as the cells starved. The amount of the cis-monoenoic fatty acids declined (e.g., 16:1 omega 7c: 0 day, 39%; 7 day, 18%; 30 day, 11%). In contrast, the saturated fatty acids, the cyclopropyl derivatives of the cis-monoenoic fatty acids, and trans-monoenoic fatty acids increased during starvation. For instance, the amounts of 16:1 omega 7t were: 0 day, 1%; 7 day, 13%; 30 day, 17%; which increased the trans/cis ratio for 16:1 omega 7 from 0.02 (0 day) to 0.70 (7 day) to 1.56 (30 day). This may be due to the reported high turnover rates of cis-monoenoic fatty acids of membrane phospholipids and the availability of enzymes for the metabolism of these isomers. During starvation-induced phospholipid loss, the cis-monoenoic fatty acids would, therefore, be preferentially utilized. The ability to either synthesize trans-monoenoic acids (which are not easily metabolized by bacteria) or modify the more volatile cis-monoenoic acids to their cyclopropyl derivatives may be a survival mechanism which helps maintain a functional (although structurally altered) membrane during starvation-induced lipid utilization. In addition, a trans/cis fatty acid ratio significantly greater than that reported for most bacterial cultures and environmental samples (less than 0.1) may be used as a starvation or stress lipid index. Such a ratio could help determine the nutritional status of ultramicrobacteria and other reported dormant cells in natural aquatic environments.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid assay for screening and characterizing microorganisms for the ability to degrade polychlorinated biphenyls.

Abstract: We designed a rapid assay that assesses the polychlorinated biphenyl (PCB)-degradative competence and congener specificity of aerobic microorganisms, identifies strains capable of degrading highly chlorinated biphenyls, and distinguishes among those that degrade PCBs by alternative pathways. Prior attempts to assay PCB-degradative competence by measuring disappearance of Aroclors (commercial PCB mixtures) have frequently produced false-positive findings because of volatilization, adsorption, or absorption losses. Furthermore, these assays have generally left the chemical nature of the competence obscure because of incomplete gas chromatographic resolution and uncertain identification of Aroclor peaks. We avoided these problems by using defined mixtures of PCB congeners and by adopting incubation and extraction methods that prevent physical loss of PCBs. Our assay mixtures include PCB congeners ranging from dichloro- to hexachlorobiphenyls and representing various structural classes, e.g., congeners chlorinated on a single ring (2,3-dichlorobiphenyl), blocked at 2,3 sites (2,5,2'5'-tetrachlorobiphenyl), blocked at 3,4 sites (4,4'-dichlorobiphenyl), and lacking adjacent unchlorinated sites (2,4,5,2',4',5'-hexachlorobiphenyl). The PCB-degrative ability of microorganisms is assessed by packed-column gas chromatographic analysis of these defined congener mixtures following 24-h incubation with resting cells. When tested with 25 environmental isolates, this assay revealed a broad range of PCB-degradative competence, highlighted differences in congener specificity and in the extent of degradation of individual congeners, predicted degradative competence on commercial PCBs, and (iv) identified strains with superior PCB-degradative ability.

Relative Rates of Nitric Oxide and Nitrous Oxide Production by Nitrifiers, Denitrifiers, and Nitrate Respirers

Abstract: Biogenic emissions of nitric and nitrous oxides have important impacts on the photochemistry and chemistry of the atmosphere. Although biogenic production appears to be the overwhelming source of N2O, the magnitude of the biogenic emission of NO is very uncertain. In soils, possible sources of NO and N2O include nitrification by autotrophic and heterotrophic nitrifiers, denitrification by nitrifiers and denitrifiers, nitrate respiration by fermenters, and chemodenitrification. The availability of oxygen determines to a large extent the relative activities of these various groups of organisms. To better understand this influence, we investigated the effect of the partial pressure of oxygen (pO2) on the production of NO and N2O by a wide variety of common soil nitrifying, denitrifying, and nitrate-respiring bacteria under laboratory conditions. The production of NO per cell was highest by autotrophic nitrifiers and was independent of pO2 in the range tested (0.5 to 10%), whereas N2O production was inversely proportional to pO2. Nitrous oxide production was highest in the denitrifier Pseudomonas fluorescens, but only under anaerobic conditions. The molar ratio of NO/N2O produced was usually greater than unity for nitrifiers and much less than unity for denitrifiers. Chemodenitrification was the major source of both the NO and N2O produced by the nitrate respirer Serratia marcescens. Chemodenitrification was also a possible source of NO and N2O in nitrifier cultures but only when high concentrations of nitrite had accumulated or were added to the medium. Although most of the denitrifiers produced NO and N2O only under anaerobic conditions, chemostat cultures of Alcaligenes faecalis continued to emit these gases even when the cultures were sparged with air. Based upon these results, we predict that aerobic soils are primary sources of NO and that N2O is produced only when there is sufficient soil moisture to provide the anaerobic microsites necessary for denitrification by either denitrifiers or nitrifiers.

Biochemical function and ecological significance of novel bacterial lipids in deep-sea procaryotes.

Abstract: The fatty acid composition of the membrane lipids in 11 deep-sea bacterial isolates was determined. The fatty acids observed were typical of marine vibrios except for the presence of large amounts of long-chain polyunsaturated fatty acids (PUFAs). These long-chain PUFAs were previously thought to be absent in procaryotes, with the notable exception of a single marine Flexibacter sp. In three barophilic strains tested at 2°C, there was a general increase in the relative amount of PUFAs as pressure was increased from a low growth pressure towards the optimal growth pressure. In Vibrio marinus MP-1, a psychrophilic strain, PUFAs were found to increase as a function of decreasing temperature at constant atmospheric pressure. These results suggest the involvement of PUFAs in the maintenance of optimal membrane fluidity and function over environmentally relevant temperatures and pressures. Furthermore, since these lipids are essential nutrients for higher taxa and are found in large amounts in the lipids of deep-sea vertebrates and invertebrates, an important, specific role for deep-sea bacteria in abyssal food webs is implicated.

Automatic Determination of Bacterioplankton Biomass by Image Analysis

Abstract: Image analysis was applied to epifluorescense microscopy of acridine orange-stained plankton samples. A program was developed for discrimination and binary segmentation of digitized video images, taken by an ultrasensitive video camera mounted on the microscope. Cell volumes were estimated from area and perimeter of the objects in the binary image. The program was tested on fluorescent latex beads of known diameters. Biovolumes measured by image analysis were compared with directly determined carbon biomasses in batch cultures of estuarine and freshwater bacterioplankton. This calibration revealed an empirical conversion factor from biovolume to biomass of 0.35 pg of C mum (+/- 0.03 95% confidence limit). The deviation of this value from the normally used conversion factors of 0.086 to 0.121 pg of C mum is discussed. The described system was capable of measuring 250 cells within 10 min, providing estimates of cell number, mean cell volume, and biovolume with a precision of 5%.

Biodegradation of chlorinated ethenes by a methane-utilizing mixed culture.

Abstract: Chlorinated ethenes are toxic substances which are widely distributed groundwater contaminants and are persistent in the subsurface environment. Reports on the biodegradation of these compounds under anaerobic conditions which might occur naturally in groundwater show that these substances degrade very slowly, if at all. Previous attempts to degrade chlorinated ethenes aerobically have produced conflicting results. A mixed culture containing methane-utilizing bacteria was obtained by methane enrichment of a sediment sample. Biodegradation experiments carried out in sealed culture bottles with radioactively labeled trichloroethylene (TCE) showed that approximately half of the radioactive carbon had been converted to 14CO2 and bacterial biomass. In addition to TCE, vinyl chloride and vinylidene chloride could be degraded to products which are not volatile chlorinated substances and are therefore likely to be further degraded to CO2. Two other chlorinated ethenes, cis and trans-1,2-dichloroethylene, were shown to degrade to chlorinated products, which appeared to degrade further. A sixth chlorinated ethene, tetrachloroethylene, was not degraded by the methane-utilizing culture under these conditions. The biodegradation of TCE was inhibited by acetylene, a specific inhibitor of methane oxidation by methanotrophs. This observation supported the hypothesis that a methanotroph is responsible for the observed biodegradations.

Alginate Beads as Synthetic Inoculant Carriers for Slow Release of Bacteria That Affect Plant Growth

Abstract: Uniform synthetic beads were developed as carriers for the bacterial inoculation of plants The beads are made of sodium alginate and skim milk and contain a large reservoir of bacterial culture which releases the bacteria at a slow and constant rate The beads are biodegradable and produce no environmental pollution The strength of the beads, the rate of bacterial release, and the time of their survival in the soil can be controlled by several hardening treatments The final product, lyophilized beads, is simple to use and is applied to the seeds concomitantly with sowing The released bacteria are available for root colonization immediately at seed germination Dry beads containing bacteria can be stored at ambient temperature over a long period without loss of bacterial content; storage requires a limited space, and the quality control of a number of bacteria in the bead is simple The level of plant inoculation with beads was similar to that with previously used peat inoculants, but the former method yielded more consistent results, as the frequency of inoculated plants was much higher The former method provides a different approach for inoculation of plants with beneficial rhizosphere bacteria

Generation and Characterization of Tn5 Insertion Mutations in Pseudomonas syringae pv. tomato.

Abstract: Tn5-induced insertion mutations were generated in the Pseudomonas syringae pv. tomato genome by mating this plant pathogen with an Escherichia coli strain carrying the suicide plasmid vector for Tn5, pGS9. Km transconjugants occurred at frequencies ranging from 2 x 10 to 9 x 10; approximately 5.5% of these transconjugants were also Cm, indicating the presence of additional pGS9 DNA sequences. Approximately 1% of the Km Cm mutants were auxotrophic. Southern blot analysis revealed that the Tn5 element had inserted into one unique site on the chromosome for each Km Cm transconjugant examined. Physical and genetic tests of Tn5-induced auxotrophs showed that Tn5 mutations in P. syringae pv. tomato were very stable and that secondary transposition of Tn5 or its insertion sequence IS50 was a rare event. Nine of 920 Km Cm transconjugants screened on tomato seedlings either were avirulent or produced very mild symptoms. Each of the virulence mutants was the result of a unique single-site Tn5 insertion. Five mutants also failed to induce a hypersensitivity reaction on tobacco.

Complete reductive dechlorination and mineralization of pentachlorophenol by anaerobic microorganisms.

Abstract: Anaerobically digested municipal sewage sludge which had been acclimated to monochlorophenol degradation for more than 2 years was shown to degrade pentachlorophenol (PCP). Di-, tri-, and tetrachlorophenols accumulated when PCP was added to the individual acclimated sludges. When the 2-chlorophenol- (2-CP), 3-CP-, and 4-CP-acclimated sludges were mixed in equal volumes, PCP was completely dechlorinated. The same results were obtained in sludge acclimated to the three monochlorophenol isomers simultaneously. With repeated PCP additions, 3,4,5,-trichlorophenol, 3,5-dichlorophenol, and 3-CP accumulated in less than stoichiometric amounts. All chlorinated compounds disappeared after PCP additions were stopped. All chlorinated compounds disappeared after PCP additions were stopped. Incubations with [14C]PCP resulted in 66% of the added 14C being mineralized to 14CO2 and 14CH4. Technical-grade PCP was found to be degraded initially at a rate very similar to that of reagent-grade PCP, but after repeated additions, the technical PCP was degraded more slowly. Pentabromophenol was also rapidly degraded by the mixture of acclimated sludges. These results clearly show the complete reductive dechlorination of PCP by the combined activities of three chlorophenol-degrading populations.

Sulfate reduction relative to methane production in high-rate anaerobic digestion: microbiological aspects.

Abstract: In the high-rate anaerobic reactors studied (ca. 10 g of chemical oxygen demand [COD] removed per liter of reactor per day), the sulfate-reducing bacteria (SRB) were poor competitors of methane-producing bacteria (MPB), scavenging only on the order of 10 to 20% of the total electron flow. The relatively noncompetitive nature of the SRB in this type of reactor is in sharp contrast to the tendency of the SRB to dominate in natural environments and in other types of anaerobic digesters. Various factors such as the feedback inhibition of H(2)S on the SRB, iron limitation, the origin of the SRB inocula, biokinetics, and thermodynamics were investigated. The outcome of the SRB-MPB competition under the reactor conditions studied appeared to be particularly determined by two factors. The SRB, as predicted by the V(max)-K(m) kinetics, competed most effectively at low substrate levels (<0.5 g of COD per liter). The MPB, however, appeared to colonize and adhere much more effectively to the polyurethane carrier matrix present in the reactor, thus compensating for the apparent lower growth rates. Even if the reactor was initially allowed to be predominantly colonized by SRB, the MPB could regain dominance.

Incorporation of Oxygen from Water into Toluene and Benzene during Anaerobic Fermentative Transformation

Abstract: Toluene and benzene were anaerobically transformed and eventually mineralized in mixed methanogenic cultures. However, the source of oxygen for the initial oxidation step had been unknown, owing to the presence of both methanol and water. No exogenous electron acceptors other than carbon dioxide, toluene, and benzene were present in the defined mineral medium. Through the use of 18O-labeled water, the oxygen incorporated into the monoaromatic compounds was shown to come from water. The cresol from the toluene and the phenol from the benzene contained up to 8% 18O label after incubation in 9% 18O-labeled medium. Gas chromatography-mass spectrometry was used to detect the 18O-labeled aromatic metabolites.

Sulfate reduction relative to methane production in high-rate anaerobic digestion: technical aspects.

Abstract: The effect of different substrates and different levels of sulfate and sulfide on methane production relative to sulfate reduction in high-rate anaerobic digestion was evaluated. Reactors could be acclimated so that sulfate up to a concentration of 5 g of sulfate S per liter did not significantly affect methanogenesis. Higher levels gave inhibition because of salt toxicity. Sulfate reduction was optimal at a relatively low level of sulfate, i.e., 0.5 g of sulfate S per liter, but was also not significantly affected by higher levels. Both acetoclastic and hydrogenotrophic methane-producing bacteria adapted to much higher levels of free H2S than the values reported in the literature (50% inhibition occurred only at free H2S levels of more than 1,000 mg/liter). High levels of free H2S affected the sulfate-reducing bacteria only slightly. Formate and acetate supported the sulfate-reducing bacteria very poorly. In the high-rate reactors studied, intensive H2S formation occurred only when H2 gas or an H2 precursor such as ethanol was supplied.

Adaptation to Hydrogen Sulfide of Oxygenic and Anoxygenic Photosynthesis among Cyanobacteria.

Abstract: Four different types of adaptation to sulfide among cyanobacteria are described based on the differential toxicity to sulfide of photosystems I and II and the capacity for the induction of anoxygenic photosynthesis. Most cyanobacteria are highly sensitive to sulfide toxicity, and brief exposures to low concentrations cause complete and irreversible cessation of CO2 photoassimilation. Resistance of photosystem II to sulfide toxicity, allowing for oxygenic photosynthesis under sulfide, is found in cyanobacteria exposed to low H2S concentrations in various hot springs. When H2S levels exceed 200 μM another type of adaptation involving partial induction of anoxygenic photosynthesis, operating in concert with partially inhibited oxygenic photosynthesis, is found in cyanobacterial strains isolated from both hot springs and hypersaline cyanobacterial mats. The fourth type of adaptation to sulfide is found at H2S concentrations higher than 1 mM and involves a complete replacement of oxygenic photosynthesis by an effective sulfide-dependent, photosystem II-independent anoxygenic photosynthesis. The ecophysiology of the various sulfide-adapted cyanobacteria may point to their uniqueness within the division of cyanobacteria.

Metabolism of reduced methylated sulfur compounds in anaerobic sediments and by a pure culture of an estuarine methanogen.

Abstract: Addition of dimethylsulfide (DMS), dimethyldisulfide (DMDS), or methane thiol (MSH) to a diversity of anoxic aquatic sediments (eg, fresh water, estuarine, alkaline/hypersaline) stimulated methane production The yield of methane recovered from DMS was often 52 to 63%, although high concentrations of DMS (as well as MSH and DMDS) inhibited methanogenesis in some types of sediments Production of methane from these reduced methylated sulfur compounds was blocked by 2-bromoethanesulfonic acid Sulfate did not influence the metabolism of millimolar levels of DMS, DMDS, or MSH added to sediments However, when DMS was added at ∼2-μM levels as [14C]DMS, metabolism by sediments resulted in a 14CH4/14CO2 ratio of only 006 Addition of molybdate increased the ratio to 18, while 2-bromoethanesulfonic acid decreased it to 0, but did not block 14CO2 production These results indicate the methanogens and sulfate reducers compete for DMS when it is present at low concentrations; however, at high concentrations, DMS is a “noncompetitive” substrate for methanogens Metabolism of DMS by sediments resulted in the appearance of MSH as a transient intermediate A pure culture of an obligately methylotrophic estuarine methanogen was isolated which was capable of growth on DMS Metabolism of DMS by the culture also resulted in the transient appearance of MSH, but the organism could grow on neither MSH nor DMDS The culture metabolized [14C]-DMS to yield a 14CH4/14CO2 ratio of ∼28 Reduced methylated sulfur compounds represent a new class of substrates for methanogens and may be potential precursors of methane in a variety of aquatic habitats

Method for flow cytometric detection of Listeria monocytogenes in milk

Abstract: This report describes a method for the detection of Listeria monocytogenes in raw milk by flow cytometric analysis of fluorescently labeled bacterial populations. The use of immunofluorescence in combination with measures of DNA content by propidium iodide labeling and size by light scattering enabled specific identification of L. monocytogenes from Streptococcus faecalis, Streptococcus agalactiae, Streptococcus uberis, Staphylococcus epidermidis, and Staphylococcus hyicus. Additional specific resolution of L. monocytogenes populations was achieved through selective enrichment of raw milk in Listeria enrichment broth. These procedures should permit the rapid screening of milk and other food samples for L. monocytogenes and eliminate many of the short-comings associated with conventional fluorescent-antibody procedures.

Isolation of protozoa from water associated with a legionellosis outbreak and demonstration of intracellular multiplication of Legionella pneumophila.

Abstract: At the site of a legionellosis outbreak, amoebae and two ciliates, Tetrahymena sp. and Cyclidium sp., were isolated from cooling-tower water containing Legionella pneumophila. The Tetrahymena sp. and the amoebae repeatedly showed the ability to support intracellular multiplication of L. pneumophila. Both were isolated from cooling towers specifically implicated as the source for the spread of legionellosis. These protozoa may be reservoirs supporting the survival and multiplication of virulent legionellae in cooling-tower water.

Identification of a plasmid-borne parathion hydrolase gene from Flavobacterium sp. by southern hybridization with opd from Pseudomonas diminuta.

Abstract: Parathion hydrolases have been previously described for an American isolate of Pseudomonas diminuta and a Philippine isolate of Flavobacterium sp. (ATCC 27551). The gene which encodes the broad-spectrum organophosphate phosphotriesterase in P. diminuta has been shown by other investigators to be located on a 66-kilobase (kb) plasmid. The intact gene (opd, organophosphate-degrading gene) from this degradative plasmid was cloned into M13mp10 and found to express parathion hydrolase under control of the lac promoter in Escherichia coli. In Flavobacterium sp. strain ATCC 27551, a 43-kb plasmid was associated with the production of parathion hydrolase by curing experiments. The M13mp10-cloned fragment of the opd gene from P. diminuta was used to identify a homologous genetic region from Flavobacterium sp. strain ATCC 27551. Southern hybridization experiments demonstrated that a genetic region from the 43-kb Flavobacterium sp. plasmid possessed significant homology to the opd sequence. Similar hybridization did not occur with three other native Flavobacterium sp. plasmids (approximately 23, 27, and 51 kb) present within this strain or with genomic DNA from cured strains. Restriction mapping of various recombinant DNA molecules containing subcloned fragments of both opd plasmids revealed that the restriction maps of the two opd regions were similar, if not identical, for all restriction endonucleases tested thus far. In contrast, the restriction maps of the cloned plasmid sequences outside the opd regions were not similar. Thus, it appears that the two discrete bacterial plasmids from parathion-hydrolyzing soil bacteria possess a common but limited region of sequence homology within potentially nonhomologous plasmid structures.

Acetate Synthesis from H(2) plus CO(2) by Termite Gut Microbes.

Abstract: Gut microbiota from Reticulitermes flavipes termites catalyzed an H2-dependent total synthesis of acetate from CO2. Rates of H2-CO2 acetogenesis in vitro were 1.11 ± 0.37 μmol of acetate g (fresh weight)−1 h−1 (equivalent to 4.44 ± 1.47 nmol termite−1 h−1) and could account for approximately 1/3 of all the acetate produced during the hindgut fermentation. Formate was also produced from H2 + CO2, as were small amounts of propionate, butyrate, and lactate-succinate. However, H2-CO2 formicogenesis seemed largely unrelated to acetogenesis and was believed not to be a significant reaction in situ. Little or no CH4 was formed from H2 + CO2 or from acetate. H2-CO2 acetogenesis was inhibited by O2, KCN, CHCl3, and iodopropane and could be abolished by prefeeding R. flavipes with antibacterial drugs. By contrast, prefeeding R. flavipes with starch resulted in almost complete defaunation but had little effect on H2-CO2 acetogenesis, suggesting that bacteria were the acetogenic agents in the gut. H2-CO2 acetogenesis was also observed with gut microbiota from Prorhinotermes simplex, Zootermopsis angusticollis, Nasutitermes costalis, and N. nigriceps; from the wood-eating cockroach Cryptocercus punctulatus; and from the American cockroach Periplaneta americana. Pure cultures of H2-CO2-acetogenic bacteria were isolated from N. nigriceps, and a preliminary account of their morphological and physiological properties is presented. Results indicate that in termites, CO2 reduction to acetate, rather than to CH4, represents the main electron sink reaction of the hindgut fermentation and can provide the insects with a significant fraction (ca. 1/3) of their principal oxidizable energy source, acetate.

Growth Pattern and Yield of a Chemoautotrophic Beggiatoa sp. in Oxygen-Sulfide Microgradients.

Abstract: Recently developed techniques involving opposed, gel-stabilized gradients of O2 and H2S permit cultivation of a marine Beggiatoa strain as a chemolithoautotroph which uses gliding motility to precisely track the interface between H2S and O2. In the current study with microelectrodes, vertical profiles of H2, O2, and pH were measured in replicate cultures grown for various intervals. After an initial period of exponential biomass increase (doubling time, 11 h), linear growth prevailed throughout much of the time course. This H2S-limited growth was followed by a transition to stationary phase when the declining H2S flux was sufficient only to supply maintenance energy. During late-exponential and linear growth phases, the Beggiatoa sp. consumed a constant 0.6 mol of H2S for each 1.0 mol of O2, the ratio anticipated for balanced lithoautotrophic growth at the expense of complete oxidation of H2S to SO42−. Over the entire range of conditions studied, this consumption ratio varied by approximately twofold. By measuring the extent to which the presence of the bacterial plate diminished the overlap of O2 and H2S, we demonstrated that oxidation of H2S by Beggiatoa sp. is approximately 3 orders of magnitude faster than spontaneous chemical oxidation. By integrating sulfide profiles and comparing sulfide consumed with biomass produced, a growth yield of 8.4 g (dry weight) mol−1 of H2S was computed. This is higher than that found for sulfide-grown thiobacilli, indicating very efficient growth of Beggiatoa sp. as a chemoautotroph. The methods used here offer a unique opportunity to determine the yield of H2S-oxidizing chemolithoautotrophs while avoiding several problems inherent in the use of homogeneous liquid culture. Finally, by monitoring time-dependent formation of H2S profiles under anoxic conditions, we demonstrate a method for calculating the molecular diffusion coefficient of soluble substrates in gel-stabilized media.

Aerobic metabolism of trichloroethylene by a bacterial isolate.

Abstract: A number of soil and water samples were screened for the biological capacity to metabolize trichloroethylene. One water sample was found to contain this capacity, and a gram-negative, rod-shaped bacterium which appeared to be responsible for the metabolic activity was isolated from this sample. The isolate degraded trichloroethylene to CO2 and unidentified, nonvolatile products. Oxygen and water from the original site of isolation were required for degradation.

Distinguishing between Nitrification and Denitrification as Sources of Gaseous Nitrogen Production in Soil.

Abstract: The source of N2O produced in soil is often uncertain because denitrification and nitrification can occur simultaneously in the same soil aggregate. A technique which exploits the differential sensitivity of these processes to C2H2 inhibition is proposed for distinguishing among gaseous N losses from soils. Denitrification N2O was estimated from 24-h laboratory incubations in which nitrification was inhibited by 10-Pa C2H2. Nitrification N2O was estimated from the difference between N2O production under no C2H2 and that determined for denitrification. Denitrification N2 was estimated from the difference between N2O production under 10-kPa C2H2 and that under 10 Pa. Laboratory estimates of N2O production were significantly correlated with in situ N2O diffusion measurements made during a 10-month period in two forested watersheds. Nitrous oxide production from nitrification was most important on well-drained sites of a disturbed watershed where ambient NO3− was high. In contrast, denitrification N2O was most important on poorly drained sites near the stream of the same watershed. Distinction between N2O production from nitrification and denitrification was corroborated by correlations between denitrification N2O and water-filled pore space and between nitrification N2O and ambient NO3−. This technique permits qualitative study of environmental parameters that regulate gaseous N losses via denitrification and nitrification.

Effect of temperature, sodium chloride, and pH on growth of Listeria monocytogenes in cabbage juice.

Abstract: Human illness and death have resulted from the consumption of milk, cheese, and cole slaw contaminated with Listeria monocytogenes. Since the effects of temperature, NaCl, and pH on the growth of the organism in cabbage were unknown, a series of experiments was designed to investigate these factors. Two strains (LCDC 81-861 and Scott A, both serotype 4b) were examined. At 30 degrees C, the viable population of the LCDC 81-861 strain increased in sterile unclarified cabbage juice (CJ) containing 0 to 1.5% NaCl; a decrease in the population of both strains occurred in juice containing greater than or equal to 2% NaCl. At 5 degrees C, the population of the Scott A strain in CJ containing up to 5% NaCl was reduced by about 90% over a 70-day period; the LCDC 81-861 strain was more sensitive to refrigeration but remained viable in CJ containing less than or equal to 3.5% NaCl for 70 days. Growth in CJ at 30 degrees C resulted in a decrease in pH from 5.6 to 4.1 within 8 days. Death of L. monocytogenes occurred at 30 degrees C when the organism was inoculated into sterile CJ adjusted to pH less than or equal to 4.6 with lactic acid. No viable cells were detected after 3 days at pH less than or equal to 4.2. At 5 degrees C, the rate of death at pH less than or equal to 4.8 was slower than at 30 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon and nitrogen content of natural planktonic bacteria.

Abstract: A method of estimating carbon and nitrogen content per unit of natural bacterial cell volume was developed. This method is based on the difference in the retentiveness of bacteria between two kinds of glass fiber filter, GF/C and GF/F (Whatman, Inc., Clifton, N.J.). Biovolume and biomass (carbon and nitrogen content) of bacteria which passed through the GF/C but not the GF/F filter were estimated with an epifluorescence microscopy and a CHN analyzer, respectively. From seasonal determinations of natural planktonic bacteria in epilimnetic waters of a mesotrophic lake, the conversion factors of 106 fg of C/mum and 25 fg of N/mum were derived as average values. By using these values, the contribution of bacteria to the biomass of lake plankton is discussed.

Serotyping of Vibrio anguillarum.

Abstract: A serotyping scheme based on the detection of O antigens by slide agglutination in fish-pathogenic strains of Vibrio anguillarum is presented. Over a period of 5 years 270 Vibrio strains from feral and cultured fish, 189 strains from the environment, and 36 strains from invertebrates were collected. The strains were divided into 10 distinct serotypes (O1 through O10). More than 90% of the fish-pathogenic strains, but only 40% of the environmental strains, were typable; 71% of the strains isolated from cultured rainbow trout were serotype O1, whereas 78% of the strains isolated from feral fish were serotype O2. No dominating environmental serotype was found. A serotyping system for V. anguillarum is proposed. A total of 90 strains received from culture collections and laboratories in different countries were typed according to the present system.

Extrapolation of biodegradation results to groundwater aquifers: reductive dehalogenation of aromatic compounds.

Abstract: The reductive biodegradation of a variety of haloaromatic substrates was monitored in samples from two sites within a shallow anoxic aquifer and was compared with freshwater sediment and sewage sludge. The metabolic capacity existing in methane-producing aquifer material was very similar to that in sediment in that three of four chlorobenzoates, five of seven chlorophenols, and one of two chlorophenoxyacetate herbicides were reductively dehalogenated in both types of incubations. The 2,4-dichlorophenoxyacetate was first converted to a dichlorophenol before dehalogenation occurred. Sewage sludge microorganisms dehalogenated four of seven chlorophenols tested and degraded both phenoxyacetate herbicides by first converting them to the corresponding chlorophenols, but the microorganisms did not transform the chlorobenzoates. In general, the same suite of initial metabolites were produced from a test substrate in all types of samples, as confirmed by cochromatography of the intermediates with authentic material. Aquifer microbiota from a sulfate-reducing site was unable to significantly degrade any of the haloaromatic substrates tested. Biological removal of the sulfate in samples from this site permitted dehalogenation of a model substrate, while stimulation of methanogenesis without removal of sulfate did not. These results demonstrate that dehalogenating microorganisms were present at this site but that their activity was at least partially inhibited by the high sulfate levels.

Close association of azospirillum and diazotrophic rods with different root zones of kallar grass.

Abstract: The populations of diazotrophic and nondiazotrophic bacteria were estimated in the endorhizosphere and on the rhizoplane of Kallar grass (Leptochloa fusca) and in nonrhizosphere soil. Microaerophilic diazotrophs were counted by the most-probable-number method, using two semisolid malate media, one of them adapted to the saline-sodic Kallar grass soil. Plate counts of aerobic heterotrophic bacteria were done on nutrient agar. The dominating N2-fixing bacteria were differentiated by morphological, serological, and physiological criteria. Isolates, which could not be assigned to a known species, were shown to fix nitrogen unequivocally by 15N2 incorporation. On the rhizoplane we found 2.0 × 107 diazotrophs per g (dry weight) of root, which consisted in equal numbers of Azospirillum lipoferum and Azospirillum-like bacteria showing characteristics different from those of known Azospirillum species. Surface sterilization by NaOCI treatment effectively reduced the rhizoplane population, so that bacteria released by homogenization of roots could be regarded as endorhizosphere bacteria. Azospirillum spp. were not detected in the endorhizosphere, but diazotrophic, motile, straight rods producing a yellow pigment occurred with 7.3 × 107 cells per g (dry weight) of root in the root interior. In nonrhizosphere soil we found 3.1 × 104 nitrogen-fixing bacteria per g. Diazotrophs were preferentially enriched in the Kallar grass rhizosphere. In nonrhizosphere soil they made up 0.2% of the total aerobic heterotrophic microflora, on the rhizoplane they made up 7.1%, and in the endorhizosphere they made up 85%. Owing to high numbers in and on roots and their preferential enrichment, we concluded that diazotrophs are in close association with Kallar grass. They formed entirely different populations on the rhizoplane and in the endorhizosphere.