Wildfire and charcoal enhance nitrification and ammonium-oxidizing bacterial abundance in dry montane forest soils.

All forest fire events generate some quantity of charcoal, which may persist in soils for hundreds to thousands of years. However, few studies have effectively evaluated the potential for charcoal to influence specific microbial communities or processes. To our knowledge, no studies have specifically addressed the effect of charcoal on ammonia-oxidizing bacteria (AOB) in forest soils. Controlled experiments have shown that charcoal amendment of fire-excluded temperate and boreal coniferous forest soil increases net nitrification, suggesting that charcoal plays a major role in maintaining nitrification for extended periods postfire. In this study, we examined the influence of fire history on gross nitrification, nitrification potential, and the nature and abundance of AOB. Soil cores were collected from sites in the Selway-Bitterroot wilderness area in northern Idaho that had been exposed twice (in 1910, 1934) or three times (1910, 1934, and 1992) in the last 94 yr, allowing us to contrast soils recently exposed to fire to those that experienced no recent fire (control). Charcoal content was determined in the O horizon by hand-separation and in the mineral soil by a chemical digestion procedure. Gross and net nitrification, and potential rates of nitrification were measured in mineral soil. Analysis of the AOB community was conducted using primer sets specific for the ammonia mono-oxygenase gene (amoA) or the 16S rRNA gene of AOB. Denaturing gradient gel electrophoresis was used to analyze the AOB community structure, while AOB abundance was determined by quantitative polymerase chain reaction. Recent (12-yr-old) wildfire resulted in greater charcoal contents and nitrification rates compared with sites without fire for 75 yr, and the more recent fire appeared to have directly influenced AOB abundance and community structure. We predicted and observed greater abundance of AOB in soils recently exposed to fire compared with control soils. Interestingly, sequence data revealed that Clusters 3 and 4, and not Cluster 2, of genus Nitrosospira dominated these forest soils, with a shift toward Cluster 3 in recently burned sites.

Structure and seasonal dynamics of hyporheic zone microbial communities in free-stone rivers of the western United States.

The hyporheic zone of a river is characterized by being nonphotic, exhibiting chemical/redox gradients, and having a heterotrophic food web based on the consumption of organic carbon entrained from surface waters. Hyporheic microbial communities constitute the base of food webs in these environments and are important for maintaining a functioning lotic ecosystem. While microbial communities of rivers dominated by fine-grained sediments are relatively well studied, little is known about the structure and seasonal dynamics of microbial communities inhabiting the predominantly gravel and cobble hyporheic zones of rivers of the western United States. Here, we present the first molecular analysis of hyporheic microbial communities of three different stream types (based on mean base discharge, substratum type, and drainage area), in Montana. Utilizing 16S rDNA phylogeny, DGGE pattern analysis, and qPCR, we have analyzed the prokaryotic communities living on the 1.7 to 2.36 mm grain-size fraction of hyporheic sediments from three separate riffles in each stream. DGGE analysis showed clear seasonal community patterns, indicated similar community composition between different riffles within a stream (95.6-96.6% similarity), and allowed differentiation between communities in different streams. Each river supported a unique complement of species; however, several phylogenetic groups were conserved between all three streams including Pseudomonads and members of the genera Aquabacterium, Rhodoferax, Hyphomicrobium, and Pirellula. Each group showed pronounced seasonal trends in abundance, with peaks during the Fall. The Hyphomicrobium group was numerically dominant throughout the year in all three streams. This work provides a framework for investigating the effects of various environmental factors and anthropogenic effects on microbial communities inhabiting the hyporheic zone.

Percent G+C profiling accurately reveals diet-related differences in the gastrointestinal microbial community of broiler chickens.

Broiler chickens from eight commercial farms in Southern Finland were analyzed for the structure of their gastrointestinal microbial community by a nonselective DNA-based method, percent G+C-based profiling. The bacteriological impact of the feed source and in-farm whole-wheat amendment of the diet was assessed by percent G+C profiling. Also, a phylogenetic 16S rRNA gene (rDNA)-based study was carried out to aid in interpretation of the percent G+C profiles. This survey showed that most of the 16S rDNA sequences found could not be assigned to any previously known bacterial genus or they represented an unknown species of one of the taxonomically heterogeneous genera, such as Ruminococcus or Clostridium. The data from bacterial community profiling were analyzed by t-test, multiple linear regression, and principal-component statistical approaches. The percent G+C profiling method with appropriate statistical analyses detected microbial community differences smaller than 10% within each 5% increment of the percent G+C profiles. Diet turned out to be the strongest determinant of the cecal bacterial community structure. Both the source of feed and local feed amendment changed the bacteriological profile significantly, whereas profiles of individual farms with identical feed regimens hardly differed from each other. This suggests that the management of typical Finnish farms is relatively uniform or that hygiene on the farm, in fact, has little impact on the structure of the cecal bacterial community. Therefore, feed compounders should have a significant role in the modulation of gut microflora and consequently in prevention of gastrointestinal disorders in farm animals.

Comparison of methods for monitoring bacterial transport in the subsurface.

The purpose of this study was to compare in a laboratory experiment, a suite of methods developed to track viable bacteria during field transport experiments. The criteria for development and selection of these methods included: (1) the ability to track bacteria within the environment from which they were isolated; (2) the lack of any effect upon the viability or the transport characteristics of the strain; (3) low detection limits; (4) a quantification range that covered several orders of magnitude; and (5) an analytical cost and turnover time commensurate with the analysis of several thousands of samples in a few months. The approaches developed included: enumeration of bacteria labeled with a vital fluorescent stain (CFDA/SE) using microplate spectrofluorometry, flow cytometry, and ferrographic (immunomagnetic) capture; enumeration of highly (13)C-enriched bacteria using combustion-IRMS; and quantitative PCR. These methods were compared to direct microscopic enumeration and plate counts during a bacterial transport experiment performed in an intact sediment core and designed to simulate the field experiment. Four of the seven methods had equivalent recoveries for the breakthrough of a pulse of bacteria eluting from a 50-cm long sediment core, and all of the methods detected the arrival of cells in the effluent prior to the conservative tracer. Combustion IRMS and ferrographic enumeration had the lowest quantification limits (approximately 2 to 20 cells/ml), whereas microplate spectrofluorometry had the highest quantification limit (approximately 10(5) cells/ml). These methods have the potential for numerous applications beyond tracking bacteria injected into the subsurface.

Monitoring bacterial transport by stable isotope enrichment of cells.

Understanding the transport and behavior of bacteria in the environment has broad implications in diverse areas, ranging from agriculture to groundwater quality, risk assessment, and bioremediation. The ability to reliably track and enumerate specific bacterial populations in the context of native communities and environments is key to developing this understanding. We report a novel bacterial tracking approach, based on altering the stable carbon isotope value (delta(13)C) of bacterial cells, which provides specific and sensitive detection and quantification of those cells in environmental samples. This approach was applied to the study of bacterial transport in saturated porous media. The transport of introduced organisms was indicated by mass spectrometric analysis of groundwater samples, where the presence of (13)C-enriched bacteria resulted in increased delta(13)C values of the samples, allowing specific and sensitive detection and enumeration of the bacteria of interest. We demonstrate the ability to produce highly (13)C-enriched bacteria, present data indicating that results obtained with this approach accurately represent intact introduced bacteria, and include field data on the use of this stable isotope approach to monitor in situ bacterial transport. This detection strategy allows sensitive detection of an introduced, unmodified bacterial strain in the presence of the indigenous bacterial community, including itself in its unenriched form.

Genetic variation among endosymbionts of widely distributed vestimentiferan tubeworms.

Vestimentiferan tubeworms thriving in sulfidic deep-sea hydrothermal vents and cold seeps are constrained by their nutritional reliance on chemoautotrophic endosymbionts. In a recent phylogenetic study using 16S ribosomal DNA, we found that endosymbionts from vent and seep habitats form two distinct clades with little variation within each clade. In the present study, we used two different approaches to assess the genetic variation among biogeographically distinct vestimentiferan symbionts. DNA sequences were obtained for the noncoding, internal transcribed spacer (ITS) regions of the rRNA operons of symbionts associated with six different genera of vestimentiferan tubeworms. ITS sequences from endosymbionts of host genera collected from different habitats and widely distributed vent sites were surprisingly conserved. Because the ITS region was not sufficient for distinguishing endosymbionts from different habitats or locations, we used a DNA fingerprinting technique, repetitive-extragenic-palindrome PCR (REP-PCR), to reveal differences in the distribution of repetitive sequences in the genomes of the bacterial endosymbionts. Most of the endosymbionts displayed unique REP-PCR patterns. A cladogram generated from these fingerprints reflected relationships that may be influenced by a variety of factors, including host genera, geographic location, and bottom type.

Effective recovery of bacterial DNA and percent-guanine-plus-cytosine-based analysis of community structure in the gastrointestinal tract of broiler chickens.

A DNA-based, direct method for initial characterization of the total bacterial community in ileum and cecum of the chicken gastrointestinal (GI) tract was developed. The efficiencies of bacterial extraction and lysis were >95 and >99%, respectively, and therefore the DNA recovered should accurately reflect the bacterial communities of the ileal and cecal digesta. Total bacterial DNA samples were fractionated according to their percent G+C content. The profiles reflecting the composition of the bacterial community were reproducible within each compartment, but different between the compartments of the GI tract. This approach is independent of the culturability of the bacteria in the consortium and can be used to improve our understanding of how diet and other variables modulate the microbial communities of the GI tracts of animals.

Characterization of the sediment bacterial community in groundwater discharge zones of an alkaline fen: a seasonal study.

The cell density, activity, and community structure of the bacterial community in wetland sediments were monitored over a 13-month period. The study was performed at Cedar Bog, an alkaline fen. The objective was to characterize the relationship between the sediment bacterial community in groundwater upwelling zones and the physical and chemical factors which might influence the community structure and activity. DNA, protein, and lipid synthesis were measured at three different upwelling zones by using [3H]thymidine, [14C]leucine, and [14C]glucose incorporation, respectively. The physiological status (apparent stress) of the consortium was assessed by comparing [14C]glucose incorporation into membrane and that into storage lipids. Bacterial cell density was determined by acridine orange direct counts, and gross bacterial community structure was determined by bisbenzimidazole-cesium chloride gradient analysis of total bacterial community DNA. Both seasonal and site-related covariation were observed in all estimates of bacterial biomass and activity. Growth rate estimates and cell density peaked in late July at 2.5 x 10(8) cells/g/day and 2.7 x 10(9) cells/g, respectively, and decreased in December to 2.0 x 10(7) cells/g/day and 1.5 x 10(9) cells/g, respectively. Across sites, membrane-to-storage-lipid ratios were generally highest in late spring and peaked in September for one site. Overall, the data indicate dynamic seasonal differences in sediment bacterial community activity and physiology, possibly in response to changing physical and chemical environmental factors which included the C/N/P ratios of the perfusing groundwater. By contrast, total cell numbers were rather constant, and community structure analysis indicated that the overall community structure was similar throughout the study.

Evidence for Acquisition in Nature of a Chromosomal 2,4-Dichlorophenoxyacetic Acid/(alpha)-Ketoglutarate Dioxygenase Gene by Different Burkholderia spp.

We characterized the gene required to initiate the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) by the soil bacterium Burkholderia sp. strain TFD6, which hybridized to the tfdA gene of the canonical 2,4-D catabolic plasmid pJP4 under low-stringency conditions. Cleavage of the ether bond of 2,4-D by cell extracts of TFD6 proceeded by an (alpha)-ketoglutarate-dependent reaction, characteristic of TfdA (F. Fukumori and R. P. Hausinger, J. Bacteriol. 175:2083-2086, 1993). The TFD6 tfdA gene was identified in a recombinant plasmid which complemented a tfdA transposon mutant of TFD6 created by chromosomal insertion of Tn5. The plasmid also expressed TfdA activity in Escherichia coli DH5(alpha), as evidenced by enzyme assays with cell extracts. Sequence analysis of the tfdA gene and flanking regions from strain TFD6 showed 99.5% similarity to a tfdA gene cloned from the chromosome of a different Burkholderia species (strain RASC) isolated from a widely separated geographical area. This chromosomal gene has 77.2% sequence identity to tfdA from plasmid pJP4 (Y. Suwa, W. E. Holben, and L. J. Forney, abstr. Q-403, in Abstracts of the 94th General Meeting of the American Society for Microbiology 1994.). The tfdA homologs cloned from strains TFD6 and RASC are the first chromosomally encoded 2,4-D catabolic genes to be reported. The occurrence of highly similar tfdA genes in different bacterial species suggests that this chromosomal gene can be horizontally transferred.

Characterization of a chromosomally encoded 2,4-dichlorophenoxyacetic acid/alpha-ketoglutarate dioxygenase from Burkholderia sp. strain RASC.

The findings of previous studies indicate that the genes required for metabolism of the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D) are typically encoded on broad-host-range plasmids. However, characterization of plasmid-cured strains of Burkholderia sp. strain RASC, as well as mutants obtained by transposon mutagenesis, suggested that the 2,4-D catabolic genes were located on the chromosome of this strain. Mutants of Burkholderia strain RASC unable to degrade 2,4-D (2,4-D- strains) were obtained by insertional inactivation with Tn5. One such mutant (d1) was shown to have Tn5 inserted in tfdARASC, which encodes 2,4-D/alpha-ketoglutarate dioxygenase. This is the first reported example of a chromosomally encoded tfdA. The tfdARASC gene was cloned from a library of wild-type Burkholderia strain RASC DNA and shown to express 2,4-D/alpha-ketoglutarate dioxygenase activity in Escherichia coli. The DNA sequence of the gene was determined and shown to be similar, although not identical, to those of isofunctional genes from other bacteria. Moreover, the gene product (TfdARASC) was purified and shown to be similar in molecular weight, amino-terminal sequence, and reaction mechanism to the canonical TfdA of Alcaligenes eutrophus JMP134. The data presented here indicate that tfdA genes can be found on the chromosome of some bacterial species and suggest that these catabolic genes are rather mobile and may be transferred by means other than conjugation.

DNA-based monitoring of total bacterial community structure in environmental samples.

Determining the structure of bacterial communities and their response to stimuli is key to understanding community function and the interactions that occur between micro-organisms and the environment. However, bacterial communities often comprise complex assemblages of large numbers of different bacterial populations. An approach is presented which allows bacterial community structure to be determined by fractionation of the complex mixture of total bacterial community DNA using the DNA-binding dye bisbenzimidazole which imposes G+C-dependent changes in the buoyant density of DNA. Bacterial community structure presented as percentage of total DNA vs. percentage G+C content of DNA is an indication of the relative abundance of phylogenetic groups of bacteria. Changes in the composition of a soil bacterial community in response to perturbations in the form of carbon amendment and altered water status were monitored.

2,4-Dichlorophenoxyacetic acid-degrading bacteria contain mosaics of catabolic genes.

DNA from 32 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria from diverse locations was probed with the first three genes of the well-known 2,4-D degradation pathway found in Alcaligenes eutrophus JMP134(pJP4). The majority of strains did not show high levels of homology to the first three genes of the 2,4-D degradation pathway, tfdA, -B, and -C. Most strains showed combinations of tfdA-, B-, and C-like elements that exhibited various degrees of homology to the gene probes. Strains having the same genomic fingerprints (as determined by repetitive extragenic palindromic PCR) exhibited the same hybridization pattern regardless of the geographic origin of the strain, with the exception of a strain isolated from Puerto Rico. This strain had the same genomic fingerprint as that of numerous other strains in the collection but differed in its hybridization against the tfdA gene probe. Members of the beta subdivision of the Proteobacteria class, specifically Alcaligenes, Burkholderia, and Rhodoferax species, carried DNA fragments with 60% or more sequence similarity to tfdA of pJP4, and most carried fragments showing at least 60% homology to tfdB. However, many strains did not hybridize with tfdC, although they exhibited chlorocatechol dioxygenase activity. Members of the alpha subdivision of the Proteobacteria class, mostly of the genus Sphingomonas, did not hybridize to either tfdA or tfdC, but some hybridized at low stringency to tfdB. The data suggest that extensive interspecies transfer of a variety of homologous degradative genes has been involved in the evolution of 2,4-D-degrading bacteria.

Characterization of diverse 2,4-dichlorophenoxyacetic acid-degradative plasmids isolated from soil by complementation.

The diversity of 2,4-dichlorophenoxyacetic acid (2,4-D)-degradative plasmids in the microbial community of an agricultural soil was examined by complementation. This technique involved mixing a suitable Alcaligenes eutrophus (Rifr) recipient strain with the indigenous microbial populations extracted from soil. After incubation of this mixture, Rifr recipient strains which grow with 2,4-D as the only C source were selected. Two A. eutrophus strains were used as recipients: JMP228 (2,4-D-), which was previously derived from A. eutrophus JMP134 by curing of the 2,4-D-degradative plasmid pJP4, and JMP228 carrying pBH501aE (a plasmid derived from pJP4 by deletion of a large part of the tfdA gene which encodes the first step in the mineralization of 2,4-D). By using agricultural soil that had been treated with 2,4-D for several years, transconjugants were obtained with both recipients. However, when untreated control soil was used, no transconjugants were isolated. The various transconjugants had plasmids with seven different EcoRI restriction patterns. The corresponding plasmids are designated pEMT1 to pEMT7. Unlike pJP4, pEMT1 appeared not to be an IncP1 plasmid, but all the others (pEMT2 to pEMT7) belong to the IncP1 group. Hybridization with individual probes for the tfdA to tfdF genes of pJP4 demonstrated that all plasmids showed high degrees of homology to the tfdA gene. Only pEMT1 showed a high degree of homology to tfdB, tfdC, tfdD, tfdE, and tfdF, while the others showed only moderate degrees of homology to tfdB and low degrees of homology to tfdC.(ABSTRACT TRUNCATED AT 250 WORDS)

Polyphasic characterization of a suite of bacterial isolates capable of degrading 2,4-D.

To develop a better understanding of the ecological aspects of microbial biodegradation, it is important to assess the phenotypic and biochemical diversity of xenobiotic degrading organisms. Forty-six bacterial isolates capable of degrading 2,4-dichlorophenoxyacetic acid (2,4-D) and representing several geographically distinct locations were characterized and placed into taxonomic groups based on the results of several independent analyses. The isolates were characterized based on Gram's reaction, colony morphology, cell morphology, fatty acid methyl ester (FAME) fingerprints, carbon substrate oxidation patterns (BIOLOG), DNA homology to whole-plasmid probes and repetitive extragenic palindromic (REP) fingerprints. Attempts to group organisms taxonomically based on colony morphology and cell morphology were largely unsuccessful. Both FAME and BIOLOG analyses were generally unable to provide reliable genus or species identifications of these environmental isolates by comparison of fingerprints or substrate use patterns to existing data bases. Modification of the standard protocols for these analyses, however, allowed taxonomic grouping of the isolates and the construction of new data bases, comprised solely of 2,4-D-degrading organisms, against which future novel isolates can be compared. Independent cluster analysis of the FAME and BIOLOG data shows that the isolates can be segregated into five taxonomic classes. The collection of 2,4-D-degrading isolates was also separated into five classes based on DNA homology to whole-plasmid probes obtained from individual isolates. REP analysis allowed isolates that likely represent the same (or very similar) organism(s) to be identified and grouped. Each of the analyses used represents a mechanistically different means of classifying organisms, yet the taxonomic groupings obtained by several of the methods (FAME, BIOLOG, DNA homology, and to some degree, REP analysis) were in good agreement. This indicates that the features discriminated by these different methods represent fundamental characteristics that determine phylogenetic groups of bacteria.

Genetic and phenotypic diversity of 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria isolated from 2,4-D-treated field soils.

Forty-seven numerically dominant 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria were isolated at different times from 1989 through 1992 from eight agricultural plots (3.6 by 9.1 m) which were either not treated with 2,4-D or treated with 2,4-D at three different concentrations. Isolates were obtained from the most dilute positive most-probable-number tubes inoculated with soil samples from the different plots on seven sampling dates over the 3-year period. The isolates were compared by using fatty acid methyl ester (FAME) profiles, chromosomal patterns obtained by PCR amplification of repetitive extragenic palindromic (REP) sequences, and hybridization patterns obtained with probes for the tfd genes of plasmid pJP4 and a probe (Spa probe) that detects a distinctly different 2,4-D-degrading isolate, Sphingomonas paucimobilis (formerly Pseudomonas paucimobilis). A total of 57% of the isolates were identified to the species level by the FAME analysis, and these isolates were strains of Sphingomonas, Pseudomonas, or Alcaligenes species. Hybridization analysis revealed four groups. Group I strains, which exhibited sequence homology with tfdA, -B, -C, and -D genes, were rather diverse, as determined by both the FAME analysis and the REP-PCR analysis. Group II, which exhibited homology only with the tfdA gene, was a small group and was probably a subset of group I. All group I and II strains had plasmids. Hybridization analysis revealed that the tfd genes were located on plasmids in 75% of these strains and on the chromosome or a large plasmid in the other 25% of the strains. One strain exhibited tfdA and -B hybridization associated with a plasmid band, while tfdC and -D hybridized with the chromosomal band area. The group III strains exhibited no detectable homology to tfd genes but hybridized to the Spa probe. The members of this group were tightly clustered as determined by both the FAME analysis and the REP-PCR analysis, were distinctly different from group I strains as determined by the FAME analysis, and had very few plasmids; this group contained more of the 47 isolates than any other group. The group III strains were identified as S. paucimobilis. The group IV strains, which hybridized to neither the tft prove nor the Spa probe, were as diverse as the group I strains as determined by the FAME and REP-PCR analyses. Most of group IV strains could not be identified by the FAME analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of gene probes to aid in recovery and identification of functionally dominant 2,4-dichlorophenoxyacetic acid-degrading populations in soil.

The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was applied to soils in microcosms, and degradation was monitored after each of five repeated additions. Total DNAs were isolated from soil bacterial communities after each 2,4-D treatment. The DNA samples were analyzed on slot blots and Southern blots by using a tfdA gene probe subcloned from plasmid pJP4 and a Spa probe derived from a different 2,4-D-degrading isolate, a Sphingomonas paucimobilis strain. 2,4-D applied to soil was quickly degraded by indigenous microbial populations. As determined by slot blot analyses of DNA from a Michigan soil, the increase in hybridization signal in response to 2,4-D treatments was greater with the Spa probe than with the tfdA probe. In contrast, the DNA from a Saskatchewan soil exhibited an increase in hybridization signal with the tfdA probe. This indicated that a population with 2,4-D-degradative gene sequences different from the tfdA gene sequence was dominant in the Michigan site, but not in the Saskatchewan site. A Southern blot analysis of DNA from Michigan soil showed that the dominant 2,4-D-degrading population was S. paucimobilis 1443. A less dominant 2,4-D-degrading population was detected with the tfdA probe; further analysis revealed that this population was a Pseudomonas pickettii 712. These gene probe analyses revealed that an important population carrying out 2,4-D degradation was not detected when the canonical tfdA gene probe was used. After a series of new strains were isolated, we identified a probe to detect and identify the dominant members of this new group.

Analysis of competition in soil among 2,4-dichlorophenoxyacetic acid-degrading bacteria.

Competition among indigenous and inoculated 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria was studied in a native Kansas prairie soil following 2,4-D additions. The soil was inoculated with four different 2,4-D-degrading strains at densities of 10(3) cells per g of soil; the organisms used were Pseudomonas cepacia DBO1(pJP4) and three Michigan soil isolates, strain 745, Sphingomonas paucimobilis 1443, and Pseudomonas pickettii 712. Following 2,4-D additions, total soil DNA was extracted and analyzed on Southern blots by using a tfdA gene probe which detected three of the strains and another probe that detected the fourth strain, S. paucimobilis 1443, which belongs to a different class of 2,4-D degraders. P. cepacia DBO1(pJP4), a constructed strain, outcompeted the other added strains and the indigenous 2,4-D-degrading populations. The S. paucimobilis population was the secondary dominant population, and strain 745 and P. pickettii were not detected. Relative fitness coefficients determined in axenic broth cultures predicted the outcome of competition in soil for some but not all strains. Lag time was shown to be a principal determinant of competitiveness among the strains, but the lag times were significantly reduced in mixed broth cultures, which changed the competitive outcome. Plasmids containing the genes for the 2,4-D pathway were important determinants of competitiveness since plasmid pKA4 in P. cepacia DBO1 resulted in the slower growth characteristic of its original host, P. pickettii, rather than the rapid growth observed when this strain harbors pJP4.

Gene probe analysis of soil microbial populations selected by amendment with 2,4-dichlorophenoxyacetic acid.

Soils with a history of 2,4-dichlorophenoxyacetic acid (2,4-D) treatment at field application rates and control soils with no prior exposure to 2,4-D were amended with 2,4-D in the laboratory. Before and during these treatments, the populations of 2,4-D-degrading bacteria were monitored by most-probable-number (MPN) enumeration and hybridization analyses, using probes for the tfd genes of plasmid pJP4, which encode enzymes for 2,4-D degradation. Data obtained by these alternate methods were compared. Several months after the most recent field application of 2,4-D (approximately 1 ppm), soils with a 42-year history of 2,4-D treatment did not have significantly higher numbers of 2,4-D-degrading organisms than did control soils with no prior history of treatment. In response to laboratory amendments with 2,4-D, both the previously treated soils and those with no prior history of exposure exhibited a dramatic increase in the number of 2,4-D-metabolizing organisms. The MPN data indicate a 4- to 5-log population increase after one amendment with 250 ppm of 2,4-D and ultimately a 6- to 7-log increase after four additional amendments, each with 400 ppm of 2,4-D. Similarly, when total bacterial DNA from the soil microbial community of these samples was analyzed by using a probe for the tfdA gene (2,4-D monoxygenase) or the tfdB gene (2,4-dichlorophenol hydroxylase) a dramatic increase in the level of hybridization was observed in both soils.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection in soil of a deletion in an engineered DNA sequence by using DNA probes.

Two Pseudomonas strains were engineered to contain the nptII gene and plasmid vector sequences in their chromosomes. After incubation of these strains in nonsterile soil, total bacterial DNA was isolated and analyzed by Southern blot hybridization with the nptII gene and the plasmid vector as probes. In addition to the expected bands of hybridization, a new band corresponding to the loss of vector sequences from the chromosome while retaining the nptII gene was observed for one of the strains. The more stressful conditions encountered in soil appeared to increase the frequency of loss of the vector sequences from this strain.

Antitermination by both the promoter and the leader regions of an Escherichia coli ribosomal RNA operon.

RNA polymerase initiating at Escherichia coli ribosomal RNA promoter-leader regions can efficiently read through factor rho-dependent termination signals. Dissection of the promoter-leader region reveals that the ability to read through termination signals is conferred independently by both promoter and leader regions. Events in the leader also affect the transcription rate of structural genes downstream of the leader. When cells are grown in rich medium, the rrnC leader reduces transcription by a factor of approximately 4 when downstream of the rrnC promoters and by a factor of 2 when downstream of the lac promoter.