Genetic diversity among Botulinum Neurotoxin-producing clostridial strains.

Clostridium botulinum is a taxonomic designation for many diverse anaerobic spore-forming rod-shaped bacteria that have the common property of producing botulinum neurotoxins (BoNTs). The BoNTs are exoneurotoxins that can cause severe paralysis and death in humans and other animal species. A collection of 174 C. botulinum strains was examined by amplified fragment length polymorphism (AFLP) analysis and by sequencing of the 16S rRNA gene and BoNT genes to examine the genetic diversity within this species. This collection contained representatives of each of the seven different serotypes of botulinum neurotoxins (BoNT/A to BoNT/G). Analysis of the16S rRNA gene sequences confirmed previous identifications of at least four distinct genomic backgrounds (groups I to IV), each of which has independently acquired one or more BoNT genes through horizontal gene transfer. AFLP analysis provided higher resolution and could be used to further subdivide the four groups into subgroups. Sequencing of the BoNT genes from multiple strains of serotypes A, B, and E confirmed significant sequence variation within each serotype. Four distinct lineages within each of the BoNT A and B serotypes and five distinct lineages of serotype E strains were identified. The nucleotide sequences of the seven toxin genes of the serotypes were compared and showed various degrees of interrelatedness and recombination, as was previously noted for the nontoxic nonhemagglutinin gene, which is linked to the BoNT gene. These analyses contribute to the understanding of the evolution and phylogeny within this species and assist in the development of improved diagnostics and therapeutics for the treatment of botulism.

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.

Growth of Pseudomonas mendocina on Fe(III) (hydr)oxides.

Although iron (Fe) is an essential element for almost all living organisms, little is known regarding its acquisition from the insoluble Fe(III) (hydr)oxides in aerobic environments. In this study a strict aerobe, Pseudomonas mendocina, was grown in batch culture with hematite, goethite, or ferrihydrite as a source of Fe. P. mendocina obtained Fe from these minerals in the following order: goethite > hematite > ferrihydrite. Furthermore, Fe release from each of the minerals appears to have occurred in excess, as evidenced by the growth of P. mendocina in the medium above that of the insoluble Fe(III) (hydr)oxide aggregates, and this release was independent of the mineral's surface area. These results demonstrate that an aerobic microorganism was able to obtain Fe for growth from several insoluble Fe minerals and did so with various growth rates.

Fluorescent Amplified Fragment Length Polymorphism Analysis of Norwegian Bacillus cereus and Bacillus thuringiensis Soil Isolates.

We examined 154 Norwegian B. cereus and B. thuringiensis soil isolates (collected from five different locations), 8 B. cereus and 2 B. thuringiensis reference strains, and 2 Bacillus anthracis strains by using fluorescent amplified fragment length polymorphism (AFLP). We employed a novel fragment identification approach based on a hierarchical agglomerative clustering routine that identifies fragments in an automated fashion. No method is free of error, and we identified the major sources so that experiments can be designed to minimize its effect. Phylogenetic analysis of the fluorescent AFLP results reveals five genetic groups in these group 1 bacilli. The ATCC reference strains were restricted to two of the genetic groups, clearly not representative of the diversity in these bacteria. Both B. anthracis strains analyzed were closely related and affiliated with a B. cereus milk isolate (ATCC 4342) and a B. cereus human pathogenic strain (periodontitis). Across the entire study, pathogenic strains, including B. anthracis, were more closely related to one another than to the environmental isolates. Eight strains representing the five distinct phylogenetic clusters were further analyzed by comparison of their 16S rRNA gene sequences to confirm the phylogenetic status of these groups. This analysis was consistent with the AFLP analysis, although of much lower resolution. The innovation of automated genotype analysis by using a replicated and statistical approach to fragment identification will allow very large sample analyses in the future.

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.

Genetic comparison of Bacillus anthracis and its close relatives using amplified fragment length polymorphism and polymerase chain reaction analysis.

Amplified fragment length polymorphism (AFLP) analysis allows a rapid, relatively simple analysis of a large portion of a microbial genome, providing information about the species and its phylogenetic relationship to other microbes (Vos et al. 1995). The method simply surveys the genome for length and sequence polymorphisms. The AFLP pattern identified can be used for comparison to the genomes of other species. Unlike other methods, it does not rely on analysis of a single genetic locus that may bias the interpretation of results and does not require any prior knowledge of the targeted organism. Moreover, a standard set of reagents can be applied to any species without using species-specific information or molecular probes. We are using AFLP analysis to rapidly identify different bacterial species. A comparison of AFLP profiles generated from a large battery of Bacillus anthracis strains shows very little variability among different isolates (Keim et al. 1997). By contrast, there is a significant difference between AFLP profiles generated for any B. anthracis strain and even the most closely related Bacillus species. Sufficient variability is apparent among all known microbial species to allow phylogenetic analysis based on large numbers of genetically unlinked loci. These striking differences among AFLP profiles allow unambiguous identification of previously identified species and phylogenetic placement of newly characterized isolates relative to known species based on a large number of independent genetic loci. Data generated thus far show that the method provides phylogenetic analyses that are consistent with other widely accepted phylogenetic methods. However, AFLP analysis provides a more detailed analysis of the targets and samples a much larger portion of the genome. Consequently, it provides an inexpensive, rapid means of characterizing microbial isolates to further differentiate among strains and closely related microbial species. Such information cannot be rapidly generated by other means. AFLP sample analysis quickly generates a very large amount of molecular information about microbial genomes. However, this information cannot be analysed rapidly using manual methods. We are developing a large archive of electronic AFLP signatures that is being used to identify isolates collected from medical, veterinary, forensic and environmental samples. We are also developing the computational packages necessary to rapidly and unambiguously analyse the AFLP profiles and conduct a phylogenetic comparison of these data relative to information already in our database. We will use this archive and the associated algorithms to determine the species identity of previously uncharacterized isolates and place them phylogenetically relative to other microbes based on their AFLP signatures. This study provides significant new information about microbes with environmental, veterinary and medical significance. This information can be used in further studies to understand the relationships among these species and the factors that distinguish them from one another. It should also allow the identification of unique factors that contribute to important microbial traits, including pathogenicity and virulence. We are also using AFLP data to identify, isolate and sequence DNA fragments that are unique to particular microbial species and strains. The fragment patterns and sequence information provide insights into the complexity and organization of bacterial genomes relative to one another. They also provide the information necessary for the development of species-specific polymerase chain reaction primers that can be used to interrogate complex samples for the presence of B. anthracis, other microbial pathogens or their remnants.

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.