Comparative 16S rRNA sequencing was used to evaluate phylogenetic relationships among selected strains of ammonia-and nitrite-oxidizing bacteria. All characterized strains were shown to be affiliated with the proteobacteria. The study extended recent 16S rRNA-based studies of phylogenetic diversity among nitrifiers by the comparison of eight strains of the genus Nitrobacter and representatives of the genera Nitrospira and Nitrospina. The later genera were shown to be affiliated with the delta subdivision of the proteobacteria but did not share a specific relationship to each other or to other members of the delta subdivision. All characterized Nitrobacter strains constituted a closely related assemblage within the alpha subdivision of the proteobacteria. As previously observed, all ammonia-oxidizing genera except Nitrosococcus oceanus constitute a monophyletic assemblage within the beta subdivision of the proteobacteria. Errors in the 16S rRNA sequences for two strains previously deposited in the databases by other investigators (Nitrosolobus multiformis C-71 and Nitrospira briensis C-128) were corrected. Consideration of physiology and phylogenetic distribution suggested that nitrite-oxidizing bacteria of the alpha and gamma subdivisions are derived from immediate photosynthetic ancestry. Each nitrifier retains the general structural features of the specific ancestor's photosynthetic membrane complex. Thus, the nitrifiers, as a group, apparently are not derived from an ancestral nitrifying phenotype.Biologists have been asking questions concerning the evolutionary origins of, and phylogenetic relationships among, chemolithotrophic microorganisms for well over a century. However, it has been only in the last decade that comparative molecular studies have provided the basis to shape an understanding of their phylogeny. Most notably, the use of comparative rRNA sequencing has provided an all-encompassing phylogenetic framework within which all the chemolithotrophs can be placed. The emerging phylogeny has, in turn, provided insights into their antiquity and the origins of lithotrophic metabolism. For example, sulfur oxidation and iron oxidation appear to be evolutionarily early and widespread metabolic modes that are not confined to a single phylogenetic assemblage of bacteria (27). An University, Evanston, with secondary consideration of cell shape and the highly characteristic cytoplasmic membrane structures (46). All known ammonia-oxidizing bacteria are obligate chemolithoautotrophs. In contrast, some nitrite-oxidizing bacteria are mixotrophs and also can grow heterotrophically (4, 26). Although the existing taxonomy assigns these bacteria to a single family, accumulating biochemical and molecular data do not support their phylogenetic coherence.Physiological and enzymatic data argue against a close relatedness between ammonia and nitrite oxidizers. They employ two very different key enzyme systems for the energygaining oxidation of ammonia and nitrite (5). Comparative sequencing studies based on 16S rRNA oli...

Extended multilocus sequence typing (MLST) analysis of atypical Escherichia isolates was used to identify five novel phylogenetic clades (CI to CV) among isolates from environmental, human, and animal sources. Analysis of individual housekeeping loci showed that E. coli and its sister clade, CI, remain largely indistinguishable and represent nascent evolutionary lineages. Conversely, clades of similar age (CIII and CIV) were found to be phylogenetically distinct. When all Escherichia lineages (named and unnamed) were evaluated, we found evidence that Escherichia fergusonii has evolved at an accelerated rate compared to E. coli, CI, CIII, CIV, and CV, suggesting that this species is younger than estimated by the molecular clock method. Although the five novel clades were phylogenetically distinct, we were unable to identify a discriminating biochemical marker for all but one of them (CIII) with traditional phenotypic profiling. CIII had a statistically different phenotype from E. coli that resulted from the loss of sucrose and sorbitol fermentation and lysine utilization. The lack of phenotypic distinction has likely hindered the ability to differentiate these clades from typical E. coli, and so their ecological significance and importance for applied and clinical microbiology are yet to be determined. However, our sampling suggests that CIII, CIV, and CV represent environmentally adapted Escherichia lineages that may be more abundant outside the host gastrointestinal tract.

Escherichia coli is an important member of the gastrointestinal tract of humans and warm-blooded animals (primary habitat). In the external environment outside the host (secondary habitat), it is often considered to be only a transient member of the microbiota found in water and soil, although recent evidence suggests that some strains can persist in temperate soils and freshwater beaches. Here we quantified the population genetic structure of E. coli from a longitudinal collection of environmental strains isolated from six freshwater beaches along Lake Huron and the St. Clair River in Michigan. Multilocus enzyme electrophoresis (MLEE) and multilocus sequence typing (MLST) revealed extensive genetic diversity among 185 E. coli isolates with an average of 40 alleles per locus. Despite evidence for extensive recombination generating new alleles and genotypic diversity, several genotypes marked by distinct MLEE and MLST profiles were repeatedly recovered from separate sites at different times. A PCR-based phylogrouping technique showed that the persistent, naturalized E. coli belonged to the B1 group. These results support the hypothesis that persistent genotypes have an adaptive advantage in the secondary habitat outside the host.

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Universal oligonucleotide hybridization probes targeting the small-subunit rRNA are commonly used to quantify total microbial representation in environmental samples. Universal probes also serve to normalize results obtained with probes targeting specific phylogenetic groups of microorganisms. In this study, six universal probes were evaluated for stability of probe-target duplexes by using rRNA from nine organisms representing the three domains of Bacteria, Archaea, and Eucarya. Domain-specific variations in dissociation temperatures were observed for all probes. This could lead to a significant bias when these probes are used to quantify microbial populations in environmental samples. We suggest lowering the posthybridization wash stringency for two of the universal probes (S-*-Univ-1390-a-A-18 and S-*-Univ-1392-a-A-15) examined. These two probes were evaluated with traditional and modified hybridization conditions to characterize defined mixtures of rRNAs extracted from pure cultures and rRNA samples obtained from anaerobic digester samples. Probe S-*-Univ-1390-a-A-18 provided excellent estimations of domain-level community composition of these samples and is recommended for future use in microbial ecology studies.