The pinyon rhizosphere, plant stress, and herbivory affect the abundance of microbial decomposers in soils.

In terrestrial ecosystems, changes in environmental conditions that affect plant performance cause a cascade of effects through many trophic levels. In a 2-year field study, seasonal abundance measurements were conducted for fast-growing bacterial heterotrophs, humate-degrading actinomycetes, fungal heterotrophs, and fluorescent pseudomonads that represent the decomposers in soil. Links between plant health and soil microbiota abundance in pinyon rhizospheres were documented across two soil types: a dry, nutrient-poor volcanic cinder field and a sandy-loam soil. On the stressful cinder fields, we identified relationships between soil decomposer abundance, pinyon age, and stress due to insect herbivory. Across seasonal variation, consistent differences in microbial decomposer abundance were identified between the cinders and sandy-loam soil. Abundance of bacterial heterotrophs and humate-degrading actinomycetes was affected by both soil nutritional status and the pinyon rhizosphere. In contrast, abundance of the fungal heterotrophs and fluorescent pseudomonads was affected primarily by the pinyon rhizosphere. On the cinder field, the three bacterial groups were more abundant on 150-year-old trees than on 60-year-old trees, whereas fungal heterotrophs were unaffected by tree age. Fungal heterotrophs and actinomycetes were more abundant on insect-resistant trees than on susceptible trees, but the opposite was true for the fluorescent pseudomonads. Although all four groups were present in all the environments, the four microbial groups were affected differently by the pinyon rhizosphere, by tree age, and by tree stress caused by the cinder soil and insect herbivory.

Phylogenetic specificity and reproducibility and new method for analysis of terminal restriction fragment profiles of 16S rRNA genes from bacterial communities.

Terminal restriction fragment (TRF) analysis of 16S rRNA genes is an increasingly popular method for rapid comparison of microbial communities, but analysis of the data is still in a developmental stage. We assessed the phylogenetic resolution and reproducibility of TRF profiles in order to evaluate the limitations of the method, and we developed an essential analysis technique to improve the interpretation of TRF data. The theoretical phylogenetic resolution of TRF profiles was determined based on the specificity of TRFs predicted from 3,908 16S rRNA gene sequences. With sequences from the Proteobacteria or gram-positive division, as much as 73% of the TRFs were phylogenetically specific (representing strains from at most two genera). However, the fraction decreased when sequences from the two divisions were combined. The data show that phylogenetic inference will be most effective if TRF profiles represent only a single bacterial division or smaller group. The analytical precision of the TRF method was assessed by comparing nine replicate profiles of a single soil DNA sample. Despite meticulous care in producing the replicates, numerous small, irreproducible peaks were observed. As many as 85% of the 169 distinct TRFs found among the profiles were irreproducible (i.e., not present in all nine replicates). Substantial variation also occurred in the height of synonymous peaks. To make comparisons of microbial communities more reliable, we developed an analytical procedure that reduces variation and extracts a reproducible subset of data from replicate TRF profiles. The procedure can also be used with other DNA fingerprinting techniques for microbial communities or microbial genomes.

Assessment of microbial diversity in four southwestern United States soils by 16S rRNA gene terminal restriction fragment analysis.

The ability of terminal restriction fragment (T-RFLP or TRF) profiles of 16S rRNA genes to provide useful information about the relative diversity of complex microbial communities was investigated by comparison with other methods. Four soil communities representing two pinyon rhizosphere and two between-tree (interspace) soil environments were compared by analysis of 16S rRNA gene clone libraries and culture collections (Dunbar et al., Appl. Environ. Microbiol. 65:1662-1669, 1998) and by analysis of 16S rDNA TRF profiles of community DNA. The TRF method was able to differentiate the four communities in a manner consistent with previous comparisons of the communities by analysis of 16S rDNA clone libraries. TRF profiles were not useful for calculating and comparing traditional community richness or evenness values among the four soil environments. Statistics calculated from RsaI, HhaI, HaeIII, and MspI profiles of each community were inconsistent, and the combined data were not significantly different between samples. The detection sensitivity of the method was tested. In standard PCRs, a seeded population comprising 0.1 to 1% of the total community could be detected. The combined results demonstrate that TRF analysis is an excellent method for rapidly comparing the relationships between bacterial communities in environmental samples. However, for highly complex communities, the method appears unable to provide classical measures of relative community diversity.

Generation of novel bacterial regulatory proteins that detect priority pollutant phenols.

The genetic systems of bacteria that have the ability to use organic pollutants as carbon and energy sources can be adapted to create bacterial biosensors for the detection of industrial pollution. The creation of bacterial biosensors is hampered by a lack of information about the genetic systems that control production of bacterial enzymes that metabolize pollutants. We have attempted to overcome this problem through modification of DmpR, a regulatory protein for the phenol degradation pathway of Pseudomonas sp. strain CF600. The phenol detection capacity of DmpR was altered by using mutagenic PCR targeted to the DmpR sensor domain. DmpR mutants were identified that both increased sensitivity to the phenolic effectors of wild-type DmpR and increased the range of molecules detected. The phenol detection characteristics of seven DmpR mutants were demonstrated through their ability to activate transcription of a lacZ reporter gene. Effectors of the DmpR derivatives included phenol, 2-chlorophenol, 2,4-dichlorophenol, 4-chloro-3-methylphenol, 2,4-dimethylphenol, 2-nitrophenol, and 4-nitrophenol.

Phylogeny, ribosomal RNA gene typing and relative abundance of new Pseudomonas species (sensu stricto) isolated from two pinyon-juniper woodland soils of the arid southwest U.S.

Rhizosphere-inhabiting Pseudomonas species interact with plant roots and may be important for plant performance under stressful environmental conditions. A comparison was conducted of culturable Pseudomonas isolates associated with pinyon rhizosphere and between-tree interspace areas in a hot, dry, volcanic cinder field and an adjacent sandy loam soil, in order to identify Pseudomonas species which may be involved in pinyon pine survival under stressful conditions. From a collection of 800 isolates, eleven isolates exhibiting different colony morphology were selected for 16S ribosomal RNA gene sequencing. Phylogenetic analysis of rDNA sequences from the eleven field isolates, forty-six described Pseudomonas species, and thirty-four previously characterized environmental isolates indicated that the isolates from the cinders and sandy loam soil clustered into three groups. The field isolates were distinct from any of the named species or other environmental isolates. Oligonucleotide primer pairs that differentiated three field isolate groups were designed from the 16S rDNA sequences, and eight hundred Pseudomonas field isolates cultured from pinyon rhizospheres and interspaces in the cinders and sandy loam soils were typed into the three groups using PCR assays. The composition of Pseudomonas populations in four environments was significantly different. The relative abundance of the three rDNA-based groups appeared to be affected by both the soil type and the pinyon rhizosphere.

Levels of bacterial community diversity in four arid soils compared by cultivation and 16S rRNA gene cloning.

Techniques based on amplification of 16S rRNA genes for comparing bacterial communities are now widely used in microbial ecology, but calibration of these techniques with traditional tools, such as cultivation, has been conspicuously absent. In this study, we compared levels of bacterial community diversity in two pinyon rhizosphere soil samples and two between-tree (interspace) soil samples by analyzing 179 cultivated bacterial isolates and 801 16S rRNA genes amplified from extracted soil DNA. Phylotypes were defined by performing a restriction fragment length polymorphism analysis of 16S rRNA gene sequences with the enzymes RsaI and BstUI. The average level of 16S rRNA gene sequence similarity of members of a phylotype was 86.6% based on an analysis of partial sequences. A total of 498 phylotypes were identified among the 16S ribosomal DNA (rDNA) clones, while 34 phylotypes occurred among the cultivated isolates. Analysis of sequences from a subset of the phylotypes showed that at least seven bacterial divisions were represented in the clone libraries, whereas the isolates represented only three. The phylotype richness, frequency distribution (evenness), and composition of the four culture collections and the four clone libraries were investigated by using a variety of diversity indices. Although cultivation and 16S rRNA cloning analyses gave contradictory descriptions of the relative phylotype richness for one of the four environments, the two methods identified qualitatively consistent relationships when levels of evenness were compared. The levels of phylotype similarity between communities were uniformly low (15 to 31%). Both methods consistently indicated that one environment was distinct from the other three. Our data illustrate that while 16S rDNA cloning and cultivation generally describe similar relationships between soil microbial communities, significant discrepancies can occur.

Wide distribution and diversity of members of the bacterial kingdom Acidobacterium in the environment.

To assess the distribution and diversity of members of the recently identified bacterial kingdom Acidobacterium, members of this kingdom present in 43 environmental samples were surveyed by PCR amplification. A primer designed to amplify rRNA gene sequences (ribosomal DNAs [rDNAs]) from most known members of the kingdom was used to interrogate bulk DNA extracted from the samples. Positive PCR results were obtained with all temperate soil and sediment samples tested, as well as some hot spring samples, indicating that members of this kingdom are very widespread in terrestrial environments. PCR primers specific for four phylogenetic subgroups within the kingdom were used in similar surveys. All four subgroups were detected in most neutral soils and some sediments, while only two of the groups were seen in most low-pH environments. The combined use of these primers allowed identification of a novel lineage within the kingdom in a hot spring environment. Phylogenetic analysis of rDNA sequences from our survey and the literature outlines at least six major subgroups within the kingdom. Taken together, these data suggest that members of the Acidobacterium kingdom are as genetically and metabolically diverse, environmentally widespread and perhaps as ecologically important as the well-known Proteobacteria and gram-positive bacterial kingdoms.

PCR analysis of tissue samples from the 1979 Sverdlovsk anthrax victims: the presence of multiple Bacillus anthracis strains in different victims.

An outbreak of human anthrax occurred in Sverdlovsk, Union of Soviet Socialists Republic (now Ekaterinburg, Russia) in April 1979. Officials attributed this to consumption of contaminated meat, but Western governments believed it resulted from inhalation of spores accidentally released from a nearby military research facility. Tissue samples from 11 victims were obtained and methods of efficiently extracting high-quality total DNA from these samples were developed. Extracted DNA was analyzed by using PCR to determine whether it contained Bacillus anthracis-specific sequences. Double PCR using "nested primers" increased sensitivity of the assay significantly. Tissue samples from 11 persons who died during the epidemic were examined. Results demonstrated that the entire complement of B. anthracis toxin and capsular antigen genes required for pathogenicity were present in tissues from each of these victims. Tissue from a vaccination site contained primarily nucleic acids from a live vaccine, although traces of genes from the infecting organisms were also present. PCR analysis using primers that detect the vrrA gene variable region on the B. anthracis chromosome demonstrated that at least four of the five known strain categories defined by this region were present in the tissue samples. Only one category is found in a single B. anthracis strain.

Diverse uncultivated bacterial groups from soils of the arid southwestern United States that are present in many geographic regions.

We have performed a phylogenetic survey of microbial species present in two soils from northern Arizona. Microbial DNA was purified directly from soil samples and subjected to PCR amplification with primers specific for bacterial 16S rRNA gene sequences (rDNAs). Clone libraries from the two soils were constructed, and 60 clone inserts were partially sequenced. Phylogenetic analysis of these sequences revealed extensive diversity. Most of the analyzed sequences (64%) fell into five novel clusters having no known cultured members. Extensive analysis of 10 nearly full-length rDNAs from clones representative of the novel groups indicated that four of the five groups probably cluster into a large "supergroup" which is as distinct from currently recognized bacterial divisions as the latter are from each other. From this we postulate the existence of a major bacterial lineage, previously known only from a single cultured representative, whose diversity and ecology we are only beginning to explore. Analysis of our data and that from other rDNA sequence-based studies of soils from different geographic regions shows considerable overlap of sequence types. Taken together, these groups encompass most of the novel rDNA sequences recovered in each comparable analysis reported to date, despite large differences in soil types and geographic sources. Our results indicate that members of these new groups comprise a phylogenetically diverse, geographically widespread, and perhaps numerically important component of the soil microbiota.

Characterization of the variable-number tandem repeats in vrrA from different Bacillus anthracis isolates.

PCR analysis of 198 Bacillus anthracis isolates revealed a variable region of DNA sequence differing in length among the isolates. Five polymorphisms differed by the presence of two to six copies of the 12-bp tandem repeat 5'-CAATATCAACAA-3'. This variable-number tandem repeat (VNTR) region is located within a larger sequence containing one complete open reading frame that encodes a putative 30-kDa protein. Length variation did not change the reading frame of the encoded protein and only changed the copy number of a 4-amino-acid sequence (QYQQ) from 2 to 6. The structure of the VNTR region suggests that these multiple repeats are generated by recombination or polymerase slippage. Protein structures predicted from the reverse-translated DNA sequence suggest that any structural changes in the encoded protein are confined to the region encoded by the VNTR sequence. Copy number differences in the VNTR region were used to define five different B. anthracis alleles. Characterization of 198 isolates revealed allele frequencies of 6.1, 17.7, 59.6, 5.6, and 11.1% sequentially from shorter to longer alleles. The high degree of polymorphism in the VNTR region provides a criterion for assigning isolates to five allelic categories. There is a correlation between categories and geographic distribution. Such molecular markers can be used to monitor the epidemiology of anthrax outbreaks in domestic and native herbivore populations.

Molecular evolution and diversity in Bacillus anthracis as detected by amplified fragment length polymorphism markers.

Bacillus anthracis causes anthrax and represents one of the most molecularly monomorphic bacteria known. We have used AFLP (amplified fragment length polymorphism) DNA markers to analyze 78 B. anthracis isolates and six related Bacillus species for molecular variation. AFLP markers are extremely sensitive to even small sequence variation, using PCR and high-resolution electrophoresis to examine restriction fragments. Using this approach, we examined ca. 6.3% of the Bacillus genome for length mutations and ca. 0.36% for point mutations. Extensive variation was observed among taxa, and both cladistic and phenetic analyses were used to construct a phylogeny of B. anthracis and its closest relatives. This genome-wide analysis of 357 AFLP characters (polymorphic fragments) indicates that B. cereus and B. thuringiensis are the closest taxa to B. anthracis, with B. mycoides slightly more distant. B. subtilis, B. polymyxa, and B. stearothermophilus shared few AFLP markers with B. anthracis and were used as outgroups to root the analysis. In contrast to the variation among taxa, only rare AFLP marker variation was observed within B. anthracis, which may be the most genetically uniform bacterial species known. However, AFLP markers did establish the presence or absence of the pXO1 and pXO2 plasmids and detected 31 polymorphic chromosomal regions among the 79 B. anthracis isolates. Cluster analysis identified two very distinct genetic lineages among the B. anthracis isolates. The level of variation and its geographic distribution are consistent with a historically recent African origin for this pathogenic organism. Based on AFLP marker similarity, the ongoing anthrax epidemic in Canada and the northern United States is due to a single strain introduction that has remained stable over at least 30 years and a 1,000-mile distribution.

Subcellular Location of O-Acetylserine Sulfhydrylase Isoenzymes in Cell Cultures and Plant Tissues of Datura innoxia Mill.

O-Acetylserine sulfhydrylase (OASS; EC 4.2.99.8) catalyzes the formation of L-cysteine from O-acetylserine and inorganic sulfide. Three OASS isoenzymes that differ in molecular mass and subunit structure are present in shoot and root tissues and in cadmium-resistant and cadmium-susceptible cell cultures of Datura innoxia Mill. Different OASS forms predominate in leaves, roots, and suspension-cell cultures. To determine the subcellular location of the OASS isoenzymes, purified mitochondria, chloroplasts, and cytosolic fractions from protoplasts were obtained. The isoenzymes are compartmentalized in D. innoxia cells, with a different isoenzyme predominant in the chloroplast, cytosol, and mitochondria, suggesting that they serve different functions in the plant cell. The chloroplast form is most abundant in green leaves and leaf protoplasts. The cytosolic form is most abundant in roots and cell cultures. A mitochondrial form is abundant in cell cultures, but is a minor form in leaves or roots. Cadmium-tolerant cell cultures contain 1.8 times as much constitutive OASS activity as the wild-type cell line, and 2.9 times more than the cadmium-hypersensitive cell line. This may facilitate rapid production of glutathione and metal-binding phytochelatins when these cultures are exposed to cadmium.

Purification and characterization of O-acetylserine sulfhydrylase isoenzymes from Datura innoxia.

Three isoenzyme forms (designated A, B, and C) of O-acetylserine sulfhydrylase were purified from Datura innoxia suspension cultures. Isoenzyme A is the most abundant form, comprising 45-60% of the total activity. Isoenzymes C and B comprise 35-40% and 10-20% of the activity, respectively. The specific activities of the purified isoenzymes are similar (870-893 mumol of cysteine/min/mg of protein). Molecular masses for isoenzymes A, B, and C, estimated by analytical size exclusion high performance liquid chromatography, are 63, 86, and 63 kDa, respectively. Isoenzymes A and B are homodimers; isoenzyme C is a heterodimer. Spectral analysis indicates that these isoenzymes possess a pyridoxal 5'-phosphate cofactor that binds the O-acetylserine substrate. Binding is reversible by addition of the sulfide substrate. The O-acetylserine sulfhydrylase isoenzymes are active over a broad temperature range, with maximum activity between 42 and 58 degrees C. They are active only between pH 7 and 8, with optimal activity at pH 7.6. Kinetic analysis indicates these enzymes are allosterically regulated and exhibit positive cooperativity with respect to both substrates. They are inhibited by sulfide concentrations above 200 microM. The kinetic analysis together with the physical and spectrophotometric characteristics indicate that the O-acetylserine sulfhydrylase enzymes have two active sites.

Phylogenetic relationships between the western aster yellows mycoplasmalike organism and other prokaryotes established by 16S rRNA gene sequence.

Restriction fragments containing the 16S rRNA gene of the western aster yellow mycoplasmalike organism (SAY-MLO) were identified in Southern blots probed with cloned fragments of the western X-disease mycoplasmalike organism 16S rRNA gene. Two fragments which contained the entire SAY-MLO 16S rRNA gene and flanking DNA were cloned in M13 and sequenced. The SAY-MLO 16S rRNA gene is approximately 1,535 bp long, has a G+C content of 47 mol%, and has an overall secondary structure similar to that proposed for Escherichia coli. Putative rRNA promoter sequences and sequences involved in processing of the primary rRNA transcript were similar in the SAY-MLO, two Mycoplasma species, and Bacillus subtilis, suggesting that these prokaryotes and the mycoplasmalike organisms may have similar transcriptional and processing enzymes. We identified two tRNA genes, a tRNA(Tyr) (GTA) gene upstream from the 16S rRNA gene and a tRNA(Ile) (GAT) gene in the spacer region between the 16S and 23S rRNA genes. Comparisons of the SAY-MLO 16S rRNA nucleotide sequence with 16S rRNA sequences of other organisms indicated that the SAY-MLO is phylogenetically related most closely to other plant-pathogenic mycoplasmalike organisms, followed by Anaeroplasma species, Acholeplasma species, and some Mycoplasma species.

Identification and characterization of plasmids from the western aster yellows mycoplasmalike organism.

Supercoiled double-stranded DNA molecules (plasmids) were isolated from plants infected with three laboratory strains of western aster yellows mycoplasma-like organism (AY-MLO) by using cesium chloride-ethidium bromide density gradients. Southern blot analysis, using plasmids from the severe strain of AY-MLO (SAY-MLO) as the probe, identified at least four plasmids in celery, aster, and periwinkle plants and in Macrosteles severini leafhopper vectors infected with either the dwarf AY-MLO, Tulelake AY-MLO, or SAY-MLO strain. Plasmids were also detected in two California field isolates of AY-MLO but not in plants infected with the beet leafhopper-transmitted virescence agent, western X, or elm yellows MLOs. SAY-MLO plasmids were 5.2, 4.9, 3.4, and 1.7 kilobase pairs in size. Plasmids isolated from dwarf AY- and Tulelake AY-MLOs were 7.4, 5.1, 3.5, and 1.7 kilobase pairs in size. No evidence was obtained for integration of SAY-MLO plasmids into the MLO chromosome.