Interactive effects of CO2 and O3 on a ponderosa pine plant/litter/soil mesocosm.

To study individual and combined impacts of two important atmospheric trace gases, CO2 and O3, on C and N cycling in forest ecosystems; a multi-year experiment using a small-scale ponderosa pine (Pinus ponderosa Laws.) seedling/soil/litter system was initiated in April 1998. The experiment was conducted in outdoor, sun-lit chambers where aboveground and belowground ecological processes could be studied in detail. This paper describes the approach and methodology used, and presents preliminary data for the first two growing seasons. CO2 treatments were ambient and elevated (ambient + 280 ppm). O3 treatments were elevated (hourly averages to 159 ppb, cumulative exposure > 60 ppb O3, SUM 06 approximately 10.37 ppm h), and a low control level (nearly all hourly averages <40 ppb. SUM 06 approximately 0.07 ppm h). Significant (P < 0.05) individual and interactive effects occurred with elevated CO2 and elevated O3. Elevated CO2 increased needle-level net photosynthetic rates over both seasons. Following the first season, the highest photosynthetic rates were for trees which had previously received elevated O3 in addition to elevated CO2. Elevated CO2 increased seedling stem diameters, with the greatest increase at low O3. Elevated CO2 decreased current year needle % N in the summer. For 1-year-old needles measured in the fall there was a decrease in % N with elevated CO2 at low O3, but an increase in % N with elevated CO2 at elevated O3. Nitrogen fixation (measured by acetylene reduction) was low in ponderosa pine litter and there were no significant CO2 or O3 effects. Neither elevated CO2 nor elevated O3 affected standing root biomass or root length density. Elevated O3 decreased the % N in coarse-fine (1-2 mm diameter) but not in fine (< 1 mm diameter) roots. Both elevated CO2 and elevated O3 tended to increase the number of fungal colony forming units (CFUs) in the AC soil horizon, and elevated O3 tended to decrease bacterial CFUs in the C soil horizon. Thus, after two growing seasons we showed interactive effects of O3 and CO2 in combination, in addition to responses to CO2 or O3 alone for a ponderosa pine plant/litter/soil system.

Fingerprinting of mixed bacterial strains and BIOLOG gram-negative (GN) substrate communities by enterobacterial repetitive intergenic consensus sequence-PCR (ERIC-PCR).

PCR-based genomic fingerprinting by use of enterobacterial repetitive intergenic consensus primers (ERIC-PCR) was evaluated for its use in fingerprinting DNA of mixed Gram-negative bacterial strains and BIOLOG Gram-negative (GN) microplate substrate communities. ERIC-PCR fingerprints of six different pure bacterial strains and a combined mixture of the strains were compared with fingerprints obtained by two more established methods: amplified ribosomal DNA restriction analysis (ARDRA) and random amplified polymorphic DNA analysis (RAPD-PCR). The ERIC-PCR fingerprint of the mixed strains was highly reproducible and was more species-specific and representative of the individual strain fingerprints than the ARDRA and RAPD-PCR fingerprints, respectively. ERIC-PCR fingerprinting of model and rhizosphere BIOLOG GN substrate communities also provided clearly distinguishable fingerprints. Results of this study suggest that ERIC-PCR represents a rapid and highly discriminating method for fingerprinting DNA of mixed Gram-negative bacterial strains and BIOLOG GN substrate communities.

Analysis of nifH gene pool complexity in soil and litter at a Douglas fir forest site in the Oregon cascade mountain range.

Nitrogen-fixing microbial populations in a Douglas fir forest on the western slope of the Oregon Cascade Mountain Range were analyzed. The complexity of the nifH gene pool (nifH is the marker gene which encodes nitrogenase reductase) was assessed by performing nested PCR with bulk DNA extracted from plant litter and soil. The restriction fragment length polymorphisms (RFLPs) of PCR products obtained from litter were reproducibly different than the RFLPs of PCR products obtained from the underlying soil. The characteristic differences were found during the entire sampling period between May and September. RFLP analyses of cloned nifH PCR products also revealed characteristic patterns for each sample type. Among 42 nifH clones obtained from a forest litter library nine different RFLP patterns were found, and among 64 nifH clones obtained from forest soil libraries 13 different patterns were found. Only two of the patterns were found in both the litter and the soil, indicating that there were major differences between the nitrogen-fixing microbial populations. A sequence analysis of clones representing the 20 distinct patterns revealed that 19 of the patterns had a proteobacterial origin. All of the nifH sequences obtained from the Douglas fir forest litter localized in a distinct phylogenetic cluster characterized by the nifH sequences of members of the genera Rhizobium, Sinorhizobium, and Azospirillum. The nifH sequences obtained from soil were found in two additional clusters, one characterized by sequences of members of the genera Bradyrhizobium, Azorhizobium, Herbaspirillum, and Thiobacillus and the other, represented by a single nifH clone, located between the gram-positive bacteria and the cyanobacteria. Our results revealed the distinctness of the nitrogen-fixing microbial populations in litter and soil in a Douglas fir forest; the differences may be related to special requirements for degradation and mineralization processes in the plant litter.

A highly selective PCR protocol for detecting 16S rRNA genes of the genus Pseudomonas (sensu stricto) in environmental samples.

Pseudomonas species are plant, animal, and human pathogens; exhibit plant pathogen-suppressing properties useful in biological control; or express metabolic versatilities valued in biotechnology and bioremediation. Specific detection of Pseudomonas species in the environment may help us gain a more complete understanding of the ecological significance of these microorganisms. The objective of this study was to develop a PCR protocol for selective detection of Pseudomonas (sensu stricto) in environmental samples. Extensive database searches identified a highly selective PCR primer pair for amplification of Pseudomonas 16S rRNA genes. A protocol that included PCR amplification and restriction analysis, a general cloning and sequencing strategy, and phylogenetic analyses was developed. The PCR protocol was validated by testing 50 target and 14 nontarget pure cultures, which confirmed the selectivity to 100%. Further validation used amplification of target sequences from purified bulk soil DNA followed by cloning of PCR products. Restriction analysis with HaeIII revealed eight different fragmentation patterns among 36 clones. Sequencing and phylogenetic analysis of 8 representative clones indicated that 91.7% of the products were derived from target organisms of the PCR protocol. Three patterns, representing only 8.3% of the 36 clones, were derived from non-Pseudomonas or chimeric PCR artifacts. Three patterns, representing 61.1% of the clones, clustered with sequences of confirmed Pseudomonas species, whereas two patterns, representing 30.6% of the clones, formed a novel phylogenetic cluster closely associated with Pseudomonas species. The results indicated that the Pseudomonas-selective PCR primers were highly specific and may represent a powerful tool for Pseudomonas population structure analyses and taxonomic confirmations.

Relative expression and stability of a chromosomally integrated and plasmid-borne marker gene fusion in environmentally competent bacteria.

A xylE-iceC transcriptional fusion was created by ligatinga DNA fragment harboring the cloned xylE structural gene from the TOL plasmid of Pseudomonas putida mt-2 into the cloned iceC gene of Pseudomonas syringae Cit7. This fusion construct was integrated into the chromosome of Pseudomonas syringae Cit7 by homologous recombination. Both cis-merodiploid strain Cit7m17 and marker exchange strain Cit7h69 produced the XylE gene product, catechol2,3-dioxygenase. Strain Cit7m17, in which XylE was influenced by transcription initiated by the amp promoter on pBR322, exhibited XylE activity in stationary phase at levels about 45 times higher than strain Cit7h69, permitting detection of 10(7) Cit7m17 cells in the spectrophotometric assay and 10(3) cells in HPLC measurements. The stability of xylE in both Cit7m17 and Cit7h69 was compared with maintenance of xylE in several plasmid-borne constructs in P.aeruginosa, Erwinia herbicola, and Escherichia coli. Only the xylE-iceC fusion in the chromosome of Cit7h69 and Cit7m17was stable in plate assays over the course of these studies. Even though strain Cit7h69 stably expressed xylE, the low level of expression precludes its use in direct spectrophotometric or HPLC assays as a means for detecting cells in environmental samples. However, expression of xylEin Cit7h69 is sufficient for identification of colonies harboring this marker gene which is useful in laboratory plate assays, and as a marker gene system for the detection of environmentally-competent strains chromosomally taggedwith xylE for use in autecological studies.

An effective method to extract DNA from environmental samples for polymerase chain reaction amplification and DNA fingerprint analysis.

A rapid direct-extraction method was used to obtain DNA from environmental soil samples. Heat, enzymes, and guanidine isothiocyanate were utilized to lyse cells. The DNA was purified by agarose gel electrophoresis, amplified with 16S rRNA-based primers by use of the polymerase chain reaction, and then digested with the restriction endonuclease PalI. The extraction method was used to obtain DNA from a variety of plants, bacteria, and fungi including Gossypium hirsucum (cotton), Pseudomonas, Bacillus, Streptomyces, and Colletotrichum. Up to 100 micrograms DNA/g (wet weight) of soil and 400 micrograms DNA/g of plant material were recovered. Restriction endonuclease analysis patterns of amplified rDNA from pure microbial cultures and plant species contained three to five different DNA fragments. Amplified rDNA of mixed population DNA extracts from soil samples, digested with the restriction endonuclease PalI, contained 12-20 DNA fragments, appearing as sample "fingerprints." Results from eight environmental soil samples that were analyzed suggest that the amplified rDNA fingerprints can be used to help characterize the genetic and biological diversity of the microbial populations in these samples.

Biodegradation of phenoxyacetic acid in soil by Pseudomonas putida PP0301(pR0103), a constitutive degrader of 2,4-dichlorophenoxyacetate.

The efficacy of using genetically engineered microbes (GEMs) to degrade recalcitrant environmental toxicants was demonstrated by the application of Pseudomonas putida PP0301(pR0103) to an Oregon agricultural soil amended with 500 micrograms/g of a model xenobiotic, phenoxyacetic acid (PAA). P. putida PP0301(pR0103) is a constitutive degrader of 2,4-dichlorophenoxyacetate (2,4-D) and is also active on the non-inducing substrate, PAA. PAA is the parental compound of 2,4-dichlorophenoxyacetic acid (2,4-D) and whilst the indigenous soil microbiota degraded 500 micrograms/g 2,4-D to less than 10 micrograms/g, PAA degradation was insignificant during a 40-day period. No significant degradation of PAA occurred in soil inoculated with the parental strain P. putida PP0301 or the inducible 2,4-D degrader P. putida PP0301(pR0101). Moreover, co-amendment of soil with 2,4-D and PAA induced the microbiota to degrade 2,4-D; PAA was not degraded. P. putida PP0301-(pR0103) mineralized 500-micrograms/g PAA to trace levels within 13 days and relieved phytotoxicity of PAA to Raphanus sativus (radish) seeds with 100% germination in the presence of the GEM and 7% germination in its absence. In unamended soil, survival of the plasmid-free parental strain P. putida PP0301 was similar to the survival of the GEM strain P. putida PP0301(pR0103). However, in PAA amended soil, survival of the parent strain was over 10,000-fold lower (< 3 colony forming units per gram of soil) than survival of the GEM strain after 39 days.

Evaluation of methods for detecting ecological effects from genetically engineered microorganisms and microbial pest control agents in terrestrial systems.

This report summarizes and evaluates research from several laboratories that deals with the detection of ecological effects induced through exposure of microbes or plants to genetically engineered microorganisms (GEMs) and microbial pest control agents (MPCAs). Some 27 potential endpoints for measuring effects have been studied. Perturbations induced by GEMs have been detected in about one-half of these endpoints. Detectable effects have been recorded for over half of the 16 species of bacteria and fungi studied. The effects caused by GEMs and MPCAs include inhibition of beneficial mycorrhizal fungi growing on Douglas fir seedling roots, depression in plant root and shoot growth, inhibition of predatory soil protozoa, accumulation of a toxic metabolite during biodegradation that inhibits soil fungi, increased microbial community respiration due to rapid lignin breakdown in soil, and the displacement of a broad group of gram-negative bacteria that inhabit the root surface of cereal crops. These effects were usually, but not always, of short duration. However, some of the changes were irreversible during the observation time of days, weeks, or in one case, months.

Ecologically significant effects of Pseudomonas putida PPO301(pRO103), genetically engineered to degrade 2,4-dichlorophenoxyacetate, on microbial populations and processes in soil.

Pseudomonas putida PPO301 (pRO103), genetically engineered to degrade 2,4-dichlorophenoxyacetate, affected microbial populations and processes in a nonsterile xeric soil. In soil amended with 2,4-dichlorophenoxyacetate (500 micrograms/g soil) and inoculated with PPO301 (pRO103), the rate of evolution of carbon dioxide was retarded for approximately 35 days; there was a transient increase in dehydrogenase activity; and the number of fungal propagules decreased below detection after 18 days. In unamended soil inoculated with PPO301(pRO103), the rate of evolution of carbon dioxide and the dehydrogenase activity were unaffected, and the numbers of fungal propagules were reduced by about two orders of magnitude. The numbers of total, spore-forming, and chitin-utilizing bacteria were reduced transiently in soil either amended or unamended with 2,4-dichlorophenoxyacetate and inoculated with PPO301(pRO103). The activities of arylsulfatases and phosphatases in soil were not affected by the presence of PPO301(pRO103), either in the presence or absence of 2,4-dichlorophenoxyacetate. In soil amended with 2,4-dichlorophenoxyacetate and inoculated with the parental strain (PPO301) or not inoculated, the evolution of carbon dioxide, the numbers of fungal propagules and of total, spore-forming, and chitin-utilizing bacteria, and the dehydrogenase activity were not affected as in soil inoculated with PPO301(pRO103). These results demonstrated that a genetically engineered microorganism, in the presence of the substrate on which its novel genes can function, is capable of inducing measurable ecological effects in soil.

Assay for detection and enumeration of genetically engineered microorganisms which is based on the activity of a deregulated 2,4-dichlorophenoxyacetate monooxygenase.

An assay system was developed for the enumeration of genetically engineered microorganisms expressing a deregulated 2,4-dichlorophenoxyacetate (TFD) monooxygenase, which converts phenoxyacetate (PAA) to phenol. In PAA-amended cultures of Pseudomonas aeruginosa PAO1C(pRO103) and Pseudomonas putida PPO301(pRO103), strains which express a deregulated TFD monooxygenase, phenol production was proportional to cell number. Phenol was reacted, under specific conditions, with a 4-aminoantipyrine dye to form an intensely colored dye-phenol complex (AAPPC), which when measured spectrophotometrically could detect as few as 10(3) cells per ml. This assay was corroborated by monitoring the disappearance of PAA and the accumulation of phenol by high-performance liquid chromatography and gas chromatography. The AAPPC assay was modified for use with plate cultures and clearly distinguished colonies of PPO301(pRO103) and PAO1C(pRO103) from a strain expressing a regulated TFD monooxygenase. Colonies of P. putida PPO301(pRO101) remained cream colored, while colonies of PPO301(pRO103) and PAO1C(pRO103) turned a distinct red.

Effects of 2,4-dichlorophenol, a metabolite of a genetically engineered bacterium, and 2,4-dichlorophenoxyacetate on some microorganism-mediated ecological processes in soil.

A genetically engineered microorganism, Pseudomonas putida PPO301(pRO103), and the plasmidless parent strain, PPO301, were added at approximately 10 CFU/g of soil amended with 500 ppm of 2,4-dichlorophenoxyacetate (2,4-D) (500 mug/g). The degradation of 2,4-D and the accumulation of a single metabolite, identified by gas chromatography-mass spectrophotometry as 2,4-dichlorophenol (2,4-DCP), occurred only in soil inoculated with PPO301(pRO103), wherein 2,4-DCP accumulated to >70 ppm for 5 weeks and the concentration of 2,4-D was reduced to <100 ppm. Coincident with the accumulation of 2,4-DCP was a >400-fold decline in the numbers of fungal propagules and a marked reduction in the rate of CO(2) evolution, whereas 2,4-D did not depress either fungal propagules or respiration of the soil microbiota. 2,4-DCP did not appear to depress the numbers of total heterotrophic, sporeforming, or chitin-utilizing bacteria. In vitro and in situ assays conducted with 2,4-DCP and fungal isolates from the soil demonstrated that 2,4-DCP was toxic to fungal propagules at concentrations below those detected in the soil.

Survival of bacteria during aerosolization.

One form of commercial application of microorganisms, including genetically engineered microorganisms is as an aerosol. To study the effect of aerosol-induced stress on bacterial survival, nonrecombinant spontaneous antibiotic-resistant mutants of four organisms, Enterobacter cloacae, Erwinia herbicola, Klebsiella planticola, and Pseudomonas syringae, were sprayed in separate experiments in a greenhouse. Samples were collected over a distance of 15 m from the spray site for enumeration. Spores of Bacillus subtilis were used as tracers to estimate the effects of dilution on changes in population over distance. Viable counts of P. syringae, Enterobacter cloacae, and K. planticola decreased significantly over a distance of 15 m. Erwinia herbicola showed no significant decline in counts over the same distance. The degree of survival of P. syringae during aerosolization was dependent on ambient environmental conditions (i.e., temperature, relative humidity), droplet size of the aerosol, and prior preparative conditions. Survival was greatest at high relative humidities (70 to 80%) and low temperatures (12 degrees C). Survival was reduced when small droplet sizes were used. The process of washing the cells prior to aerosolization also caused a reduction in their survival. Results from these experiments will be useful in developing sound methodologies to optimize enumeration and for predicting the downwind dispersal of airborne microorganisms, including genetically engineered microorganisms.

Phenoxyacetic acid degradation by the 2,4-dichlorophenoxyacetic acid (TFD) pathway of plasmid pJP4: mapping and characterization of the TFD regulatory gene, tfdR.

Plasmid pJP4 enables Alcaligenes eutrophus JMP134 to degrade 3-chlorobenzoate and 2,4-dichlorophenoxyacetic acid (TFD). Plasmid pRO101 is a derivative of pJP4 obtained by insertion of Tn1721 into a nonessential region of pJP4. Plasmid pRO101 was transferred by conjugation to several Pseudomonas strains and to A. eutrophus AEO106, a cured isolate of JMP134. AEO106(pRO101) and some Pseudomonas transconjugants grew on TFD. Transconjugants with a chromosomally encoded phenol hydroxylase also degraded phenoxyacetic acid (PAA) in the presence of an inducer of the TFD pathway, namely, TFD or 3-chlorobenzoate. A mutant of one such phenol-degrading strain, Pseudomonas putida PPO300(pRO101), grew on PAA as the sole carbon source in the absence of inducer. This isolate carried a mutant plasmid, designated pRO103, derived from pRO101 through the deletion of a 3.9-kilobase DNA fragment. Plasmid pRO103 constitutively expressed the TFD pathway, and this allowed the metabolism of PAA in the absence of the inducer, TFD. Complementation of pRO103 in trans by a DNA fragment corresponding to the fragment deleted in pRO101 indicates that a negative control-regulatory gene (tfdR) is located on the BamHI E fragment of pRO101. Other subcloning experiments resulted in the cloning of the tfdA monooxygenase gene on a 3.5-kilobase fragment derived from pRO101. This subclone, in the absence of other pRO101 DNA, constitutively expressed the tfdA gene and allowed PPO300 to grow on PAA. Preliminary evidence suggests that the monooxygenase activity encoded by this DNA fragment is feedback-inhibited by phenols.

Aerial Dispersal and Epiphytic Survival of Pseudomonas syringae during a Pretest for the Release of Genetically Engineered Strains into the Environment.

Prospective experimental field evaluation of genetically engineered microorganisms, such as microbial pest control agents, raises issues of how to properly ascertain their fate and survival in the environment. Field trials with recombinant organisms must reflect requirements for sampling and monitoring. Field trials were conducted at Tulelake, Calif., to monitor the numbers of viable cells of a nonrecombinant strain of Pseudomonas syringae that entered the atmosphere and landed on plants and soil during and after an aerosol spray application. An exponential decrease in numbers of viable cells deposited at increasing distances from three sprayed plots was observed. The relative rate of survival of cells sprayed directly on plants was more than 10 times higher than that of cells dispersed through the air to similar adjacent plants. Results are being used to gain experience with the characteristics of a release site that influence containment or dispersal and to develop appropriate sampling methodologies for evaluating survival and dispersal characteristics of genetically engineered bacteria released into the environment. The ability to make predictions about microbial dispersal and survival will reduce the uncertainties associated with environmental releases of recombinant organisms.

Predictive model of conjugative plasmid transfer in the rhizosphere and phyllosphere.

A computer simulation model was used to predict the dynamics of survival and conjugation of Pseudomonas cepacia (carrying the transmissible recombinant plasmid R388:Tn1721) with a nonrecombinant recipient strain in simple rhizosphere and phyllosphere microcosms. Plasmid transfer rates were derived for a mass action model, and donor and recipient survival were modeled as exponential growth and decay processes or both. Rate parameters were derived from laboratory studies in which donor and recipient strains were incubated in test tubes with a peat-vermiculite solution or on excised radish or bean leaves in petri dishes. The model predicted donor, recipient, and transconjugant populations in hourly time steps. It was tested in a microcosm planted with radish seeds and inoculated with donor and recipient strains and on leaf surfaces of radish and bean plants also growing in microcosms. Bacteria were periodically enumerated on selective media over 7 to 14 days. When donor and recipient populations were 10(6) to 10(8) CFU/g (wet weight) of plant or soil, transconjugant populations of about 10(1) to 10(4) were observed after 1 day. An initial rapid increase and a subsequent decline in numbers of transconjugants in the rhizosphere and on leaf surfaces were correctly predicted.

Measuring genetic stability in bacteria of potential use in genetic engineering.

Four commonly used conjugation techniques, colony cross streak (CCS), broth mating (BM), combined spread plate (CSP), and membrane filtration (MF), were compared with each other regarding reliability, sensitivity, and complexity in evaluating the transfer of conjugative plasmids. Five plasmids representing several incompatibility groups plus a variety of laboratory and environmental isolates were used as mating pairs. The suitability of each method was evaluated for use in a routine assessment of the genetic stability of genetically engineered microorganisms. By the CSP and MF techniques with laboratory strains such as Escherichia coli and Pseudomonas species as recipients, transconjugants were usually produced in 100% of the mating trials. However, when environmental strains isolated from plants and soil were used as recipients, transconjugants were detected in 100% of some crosses and in as little as 30% in other crosses depending on the plasmid and recipient used. In general, differences in the percentage of successful matings between the CSP and MF techniques compared with the BM and CCS techniques were not statistically significant at the P less than or equal to 0.05 level. Occasionally, certain mating pairs consistently produced transconjugants by CCS or BM but not by CSP or MF. Since any single conjugation mating technique is not completely reliable in detecting transconjugants, we have developed a combined mating technique which integrates the CCS, CSP, BM, and MF methods as a single procedure to assess the mobility of plasmid DNA of genetically engineered microorganisms.

Isolation of Non-O1 Vibrio cholerae Serovars from Oregon Coastal Environments.

Water, sediment, and shellfish from three Oregon estuaries were cultured for pathogenic Vibrio species. Non-O1 serovars of V. cholerae were the most common pathogenic Vibrio species recovered. Non-O1 V. cholerae were isolated from all three estuaries sampled, covering an area of about 170 miles along the Oregon coast. Non-O1 V. cholerae were isolated from water and sediment, but not shellfish, at temperatures ranging from 11 to 19 degrees C and salinities of 2.3 to 26 per thousand. Sixteen isolates representing 12 different non-O1 serovars were identified, while four non-O1 V. cholerae isolates failed to react with any of the 54 antisera tested. These results indicate that non-O1 V. cholerae serovars can be found over a large geographic area and under a variety of environmental conditions. These organisms are apparently an autochthonous component of these estuarine microbial communities.

Nucleic acids of species of Lactobacillus.

Nucleic acid composition of 17 cultures representing the type or neotype strains of 15 named Lactobacillus species was studied. Nucleic acid characterization of these isolates was accompanied by a comparative study of conventional phenotypic reactions. The overall guanine plus cytosine mean deoxyribonucleic acid base composition ranged from 33 to 50% and genome sizes varied between 700 and 1500 X 10(6) daltons. Sporolactobacillus inulinus contained 2500 X 10(6) daltons of chromosomal deoxyribonucleic acid and was therefore similar in size to members of the genus Bacillus. Deoxyribonucleic acid hybridization at a temperature 15 degrees C below the thermal melting temperature confirmed the extreme molecular heterogeneity of many species. The genus can be divided into three major groups, each containing four or more species based on a combination of nucleic acid characteristics and conventional phenotypic reactions. Group I (33 to 39% guanine plus cytosine) contains Lactobacillus jensenii, Lactobacillus helveticus, Lactobacillus helveticus nov. ssp. jugurt, Lactobacillus acidophilus, Lactobacillus salivarius, and Lactobacillus sanfrancisco. Group II (42 to 48% guanine plus cytosine contains Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, and Lactobacillus brevis, and Group III (48 to 50% guanine plus cytosine) contains Lactobacillus delbrueckii, Lactobacillus delbrueckii nov. ssp. bulgaricus, Lactobacillus delbrueckii nov. ssp. lactis, Lactobacillus delbrueckii nov. ssp. leichmanii, Lactobacillus fermentum, and Lactobacillus fermentum nov. ssp. cellobiosis.

Characterization of aquatic bacteria and cloning of genes specifying partial degradation of 2,4-dichlorophenoxyacetic acid.

Water samples from rivers, streams, ponds, and activated sewage were tested for the presence of bacteria which utilize 2,4-dichlorophenoxyacetic acid (2,4-D) as a sole source of carbon. Seventy percent of the attempted enrichments yielded pure cultures of 2,4-D-metabolizing bacteria. All but 1 of the 30 isolates were gram-negative rods, all but 2 were motile, and all were nonfermentative and oxidase and catalase positive. Nine isolates had DNA guanine-plus-cytosine values of 61.1 to 65 mol%. One isolate had a 67 mol% guanine-plus-cytosine value. The results suggest that these 2,4-D-metabolizing bacteria belong to the genus Alcaligenes. Fourteen of 23 isolates contained one or more detectable plasmids of about 20, 60, or 100 megadaltons. HindIII restriction fragment patterns showed these plasmids to be different from each other with one exception. Very similar restriction fragment patterns were revealed with a plasmid isolated from an Alcaligenes eutrophus strain obtained from Australia (pJMP397) and in an Alcaligenes sp. isolated in Oregon (pEML159). These two plasmids were about 56 megadaltons, had the same guanine-plus-cytosine value, were transmissable, and coded for 2,4-D metabolism and resistance to HgCl2. Hybridization of these two plasmids was demonstrated by using nick-translated 32P-labeled pJMP397. The vector pBR325 was used to clone HindIII fragments from pEML159. One cloned fragment of 14.8 megaldaltons expressed in Escherichia coli the ability to release 14CO2 from 2,4-D labeled in the acetate portion.

Rapid microimmunodiffusion method with species-specific antiserum raised to purified antigen for identification of Vibrio vulnificus.

An antigen common to Vibrio vulnificus strains, designated VVA, was purified by ammonium sulfate precipitation, gel filtration, ion-exchange column chromatography, and preparative gel electrophoresis. The molecular weight of VVA was 64,000 when estimated by gel filtration and 40,000 when measured by denaturing polyacrylamide gel electrophoresis. Antiserum prepared against purified VVA (anti-VVA serum) did not agglutinate whole cells of V. vulnificus. Therefore, VVA was considered a possible internal antigen. By using anti-VVA serum, a microimmunodiffusion method was designed to detect the antigen VVA in bacterial cell lysates prepared from a single colony. This simple method allowed the specific identification of V. vulnificus as soon as 10 h after antigen preparation and therefore can be a useful tool in the identification of V. vulnificus from environmental or clinical specimens. VVA was not detected as a line of complete identity in some 20 other Vibrio species or in 7 other bacterial genera. VVA was present in all 63 isolates of V. vulnificus obtained from clinical and nonclinical sources.