Changes in Synechococcus population size and cellular ribosomal RNA content in response to predation and nutrient limitation.

A mathematical model of predator-prey interactions was used to predict the relationship between population size and cellular growth rate in a two-tiered trophic system consisting of Synechococcus PCC 6301 and Tetrahymena pyriformis. As predicted, axenic chemostat cultures of Synechococcus responded to increased nutrient availability by expanding the equilibrium population size without a concurrent change in growth rate. Likewise, the addition of the predator Tetrahymena pyriformis decreased the Synechococcus population size by 85% and increased the Synechococcus growth rate. Synechococcus populations in the surface waters of the Gulf of Mexico were sampled to ascertain whether the relationship between population size and cellular 16S rRNA concentration conformed to that predicted by the model. Direct counts of autofluorescent cells in size-fractionated seawater samples provided an estimate of Synechococcus population size. The growth rate of in situ populations was estimated by measuring the extent of hybridization of an oligonucleotide probes complementary to Synechococcus 16S rRNA, based on evidence that ribosomal RNA content increases concurrently with growth rate. The comparison of in situ population sizes and specific growth rates revealed that relatively large Synechococcus populations were growing slowly, indicative of nutrient limitation, and that quickly growing populations were relatively small, as predicted for predator-limited populations.

Bordetella species are distinguished by patterns of substantial gene loss and host adaptation.

Pathogens of the bacterial genus Bordetella cause respiratory disease in humans and animals. Although virulence and host specificity vary across the genus, the genetic determinants of this diversity remain unidentified. To identify genes that may underlie key phenotypic differences between these species and clarify their evolutionary relationships, we performed a comparative analysis of genome content in 42 Bordetella strains by hybridization of genomic DNA to a microarray representing the genomes of three Bordetella species and by subtractive hybridization. Here we show that B. pertussis and B. parapertussis are predominantly differentiated from B. bronchiseptica by large, species-specific regions of difference, many of which encode or direct synthesis of surface structures, including lipopolysaccharide O antigen, which may be important determinants of host specificity. The species also exhibit sequence diversity at a number of surface protein-encoding loci, including the fimbrial major subunit gene, fim2. Gene loss, rather than gene acquisition, accompanied by the proliferation of transposons, has played a fundamental role in the evolution of the pathogenic bordetellae and may represent a conserved evolutionary mechanism among other groups of microbial pathogens.

Sequence variability in the first internal transcribed spacer region within and among Cyclospora species is consistent with polyparasitism.

Cyclospora cayetanensis is a coccidian parasite which causes severe gastroenteritis in humans. Molecular information on this newly emerging pathogen is scarce. Our objectives were to assess genetic variation within and between human-associated C. cayetanensis and baboon-associated Cyclospora papionis by examining the internal transcribed spacer (ITS) region of the ribosomal RNA operon, and to develop an efficient polymerase chain reaction- (PCR)-based method to distinguish C. cayetanensis from other closely related organisms. For these purposes, we studied C. cayetanensis ITS-1 nucleotide variability in 24 human faecal samples from five geographic locations and C. papionis ITS-1 variability in four baboon faecal samples from Tanzania. In addition, a continuous sequence encompassing ITS-1, 5.8S rDNA and ITS-2 was determined from two C. cayetanensis samples. The results indicate that C. cayetanensis and C. papionis have distinct ITS-1 sequences, but identical 5.8S rDNA sequences. ITS-1 is highly variable within and between samples, but variability does not correlate with geographic origin of the samples. Despite this variability, conserved species-specific ITS-1 sequences were identified and a single-round, C. cayetanensis-specific PCR-based assay with a sensitivity of one to ten oocysts was developed. This consistent and remarkable diversity among Cyclospora spp. ITS-1 sequences argues for polyparasitism and simultaneous transmission of multiple strains.

Rhinosporidium seeberi: a human pathogen from a novel group of aquatic protistan parasites.

Rhinosporidium seeberi, a microorganism that can infect the mucosal surfaces of humans and animals, has been classified as a fungus on the basis of morphologic and histochemical characteristics. Using consensus polymerase chain reaction (PCR), we amplified a portion of the R. seeberi 18S rRNA gene directly from infected tissue. Analysis of the aligned sequence and inference of phylogenetic relationships showed that R. seeberi is a protist from a novel clade of parasites that infect fish and amphibians. Fluorescence in situ hybridization and R. seeberi- specific PCR showed that this unique 18S rRNA sequence is also present in other tissues infected with R. seeberi. Our data support the R. seeberi phylogeny recently suggested by another group. R. seeberi is not a classic fungus, but rather the first known human pathogen from the DRIPs clade, a novel clade of aquatic protistan parasites (Ichthyosporea).

Organization, structure, and variability of the rRNA operon of the Whipple's disease bacterium (Tropheryma whippelii).

Whipple's disease is a systemic disorder associated with a cultivation-resistant, poorly characterized actinomycete, Tropheryma whippelii. We determined a nearly complete rRNA operon sequence of T. whippelii from specimens from 3 patients with Whipple's disease, as well as partial operon sequences from 43 patients. Variability was observed in the 16S-23S rRNA spacer sequences, leading to the description of five distinct sequence types. One specimen contained two spacer sequence types, raising the possibility of a double infection. Secondary structure models for the primary rRNA transcript and mature rRNAs revealed rare or unique features.

Bacterial diversity within the human subgingival crevice.

Molecular, sequence-based environmental surveys of microorganisms have revealed a large degree of previously uncharacterized diversity. However, nearly all studies of the human endogenous bacterial flora have relied on cultivation and biochemical characterization of the resident organisms. We used molecular methods to characterize the breadth of bacterial diversity within the human subgingival crevice by comparing 264 small subunit rDNA sequences from 21 clone libraries created with products amplified directly from subgingival plaque, with sequences obtained from bacteria that were cultivated from the same specimen, as well as with sequences available in public databases. The majority (52.5%) of the directly amplified 16S rRNA sequences were <99% identical to sequences within public databases. In contrast, only 21.4% of the sequences recovered from cultivated bacteria showed this degree of variability. The 16S rDNA sequences recovered by direct amplification were also more deeply divergent; 13.5% of the amplified sequences were more than 5% nonidentical to any known sequence, a level of dissimilarity that is often found between members of different genera. None of the cultivated sequences exhibited this degree of sequence dissimilarity. Finally, direct amplification of 16S rDNA yielded a more diverse view of the subgingival bacterial flora than did cultivation. Our data suggest that a significant proportion of the resident human bacterial flora remain poorly characterized, even within this well studied and familiar microbial environment.

A small, dilute-cytoplasm, high-affinity, novel bacterium isolated by extinction culture and having kinetic constants compatible with growth at ambient concentrations of dissolved nutrients in seawater.

Dilutions of raw seawater produced a bacterial isolate capable of extended growth in unamended seawater. Its 2.9-Mb genome size and 40-fg dry mass were similar to values for many naturally occurring aquatic organotrophs, but water and DNA comprised a large portion of this small chemoheterotroph, as compared to Escherichia coli. The isolate used only a few aromatic hydrocarbons and acetate, and glucose and amino acid incorporation were entirely absent, although many membrane and cytoplasmic proteins were inducible; it was named Cycloclasticus oligotrophus. A general rate equation that incorporates saturation phenomena into specific affinity theory is derived. It is used to relate the kinetic constants for substrate uptake by the isolate to its cellular proteins. The affinity constant KA for toluene was low at 1.3 microg/liter under optimal conditions, similar to those measured in seawater, and the low value was ascribed to an unknown slow step such as limitation by a cytoplasmic enzyme; KA increased with increasing specific affinities. Specific affinities, a degreess, were protocol sensitive, but under optimal conditions were 47.4 liters/mg of cells/h, the highest reported in the literature and a value sufficient for growth in seawater at concentrations sometimes found. Few rRNA operons, few cytoplasmic proteins, a small genome size, and a small cell size, coupled with a high a degreess and a low solids content and the ability to grow without intentionally added substrate, are consistent with the isolation of a marine bacterium with properties typical of the bulk of those present.

Phylogenetic relationships among the agent of bacillary angiomatosis, Bartonella bacilliformis, and other alpha-proteobacteria.

Bacillary angiomatosis (BA) and chronic bartonellosis are bacterial infections of humans which result in an unusual vascular proliferative tissue response. In order to determine their phylogenetic relationships, we have determined greater than 95% of the 16S rRNA sequences for these two organisms by amplification directly from infected BA tissue and from a Bartonella bacilliformis lyophilized culture. The BA agent and B. bacilliformis are closely related alpha-proteobacteria (98.5%), although the BA agent is more closely related to Rochalimaea quintana (99.1%). Contrary to previous belief, the BA agent is distinct from, and less closely related to, the cat scratch bacillus (Afipia felis) (90.7%). We propose a novel secondary structure in a hypervariable region of the 16S rRNA which is useful for alignment of primary sequences and which may be useful for design of nucleic acid probes.