Heterogeneity of vaginal microbial communities within individuals.

Recent culture-independent studies have revealed that a healthy vaginal ecosystem harbors a surprisingly complex assemblage of microorganisms. However, the spatial distribution and composition of vaginal microbial populations have not been investigated using molecular methods. Here, we evaluated site-specific microbial composition within the vaginal ecosystem and examined the influence of sampling technique in detection of the vaginal microbiota. 16S rRNA gene clone libraries were prepared from samples obtained from different locations (cervix, fornix, outer vaginal canal) and by different methods (swabbing, scraping, lavaging) from the vaginal tracts of eight clinically healthy, asymptomatic women. The data reveal that the vaginal microbiota is not homogenous throughout the vaginal tract but differs significantly within an individual with regard to anatomical site and sampling method used. Thus, this study illuminates the complex structure of the vaginal ecosystem and calls for the consideration of microenvironments when sampling vaginal microbiota as a clinical predictor of vaginal health.

A whole-genome approach to identifying protein binding sites: promoters in Methanocaldococcus (Methanococcus) jannaschii.

We have adapted an electrophoretic mobility shift assay (EMSA) to isolate genomic DNA fragments that bind the archaeal transcription initiation factors TATA-binding protein (TBP) and transcription factor B (TFB) to perform a genome-wide search for promoters. Mobility-shifted fragments were cloned, tested for their ability to compete with known promoter-containing fragments for a limited concentration of transcription factors, and sequenced. We applied the method to search for promoters in the genome of Methanocaldococcus jannaschii. Selection was most efficient for promoters of tRNA genes and genes for several presumed small non-coding RNAs (ncRNA). Protein-coding gene promoters were dramatically underrepresented relative to their frequency in the genome. The repeated isolation of these genomic regions was partially rectified by including a hybridization-based screening. Sequence alignment of the affinity-selected promoters revealed previously identified TATA box, BRE, and the putative initiator element. In addition, the conserved bases immediately upstream and downstream of the BRE and TATA box suggest that the composition and structure of archaeal natural promoters are more complicated.

Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes.

rRNA-based studies, which have become the most common method for assessing microbial communities, rely upon faithful amplification of the corresponding genes from the original DNA sample. We report here an analysis and reevaluation of commonly used primers for amplifying the DNA between positions 27 and 1492 of bacterial 16S rRNA genes (numbered according to the Escherichia coli rRNA). We propose a formulation for a forward primer (27f) that includes three sequences not usually present. We compare our proposed formulation to two common alternatives by using linear amplification-providing an assessment that is independent of a reverse primer-and in combination with the 1492 reverse primer (1492r) under the PCR conditions appropriate for making community rRNA gene clone libraries. For analyses of DNA from human vaginal samples, our formulation was better at maintaining the original rRNA gene ratio of Lactobacillus spp. to Gardnerella spp., particularly under stringent amplification conditions. Because our 27f formulation remains relatively simple, having seven distinct primer sequences, there is minimal loss of overall amplification efficiency and specificity.

Genomic minimalism in the early diverging intestinal parasite Giardia lamblia.

The genome of the eukaryotic protist Giardia lamblia, an important human intestinal parasite, is compact in structure and content, contains few introns or mitochondrial relics, and has simplified machinery for DNA replication, transcription, RNA processing, and most metabolic pathways. Protein kinases comprise the single largest protein class and reflect Giardia's requirement for a complex signal transduction network for coordinating differentiation. Lateral gene transfer from bacterial and archaeal donors has shaped Giardia's genome, and previously unknown gene families, for example, cysteine-rich structural proteins, have been discovered. Unexpectedly, the genome shows little evidence of heterozygosity, supporting recent speculations that this organism is sexual. This genome sequence will not only be valuable for investigating the evolution of eukaryotes, but will also be applied to the search for new therapeutics for this parasite.

Shotgun proteomics of Methanococcus jannaschii and insights into methanogenesis.

Methanococcus jannaschii is an autotrophic hyperthermophilic archaeon isolated from an oceanic hydrothermal vent. Its primary pathway for energy production is methanogenesis from H2 and CO2. High-throughput Multidimensional Protein Identification Technology based on microcapillary LC/LC/ MS/MS was used to investigate the proteome of M. jannaschii and the methanogenesis pathway in cells grown in complex medium with high H2 supply. A total of 963 proteins have been unambiguously identified. The identified proteins represent approximately 54% of the whole genome of M. jannaschii. About 44% of the identified proteins are either conserved hypothetical or hypothetical proteins. We identified 83-95% of the proteins predicted to be involved in amino acid biosynthesis, cellular processes, central intermediary metabolism, energy metabolism, protein synthesis, transcription, and purine, pyridine, nucleoside, and nucleotide synthesis. Over 40% of these proteins have better than 50% sequence coverage. Approximately 90% of the predicted methanogenesis proteins were detected. In contrast, only 27-37% of predicted hypothetical proteins, proteins involved in transport and binding, and proteins with regulatory functions were identified. High peptide number, spectrum count, and sequence coverage have been used as indicators of high expression levels and are in good agreement with codon bias analysis. Predicted intein peptides were detected in MJ1043 (DNA-directed RNA polymerase, subunit A"), MJ0542 (phosphoenolpyruvate synthase), MJ0782 (transcription initiation factor IIB), and MJ1422 (putative replication factor C subunit). New peptides created by protein splicing were detected in MJ0885 (DNA dependent DNA polymerase), MJ0542, and MJ0782. The methanogenesis pathway and the enzymes involved are also discussed.

Identification of 2D-gel proteins: a comparison of MALDI/TOF peptide mass mapping to mu LC-ESI tandem mass spectrometry.

A comparative analysis of protein identification for a total of 162 protein spots separated by two-dimensional gel electrophoresis from two fully sequenced archaea, Methanococcus jannaschii and Pyrococcus furiosus, using MALDI-TOF peptide mass mapping (PMM) and mu LC-MS/MS is presented. 100% of the gel spots analyzed were successfully matched to the predicted proteins in the two corresponding open reading frame databases by mu LC-MS/MS while 97% of them were identified by MALDI-TOF PMM. The high success rate from the PMM resulted from sample desalting/concentrating with ZipTip(C18) and optimization of several PMM search parameters including a 25 ppm average mass tolerance and the application of two different protein molecular weight search windows. By using this strategy, low-molecular weight (<23 kDa) proteins could be identified unambiguously with less than 5 peptide matches. Nine percent of spots were identified as containing multiple proteins. By using mu LC-MS/MS, 50% of the spots analyzed were identified as containing multiple proteins. mu LC-MS/MS demonstrated better protein sequence coverage than MALDI-TOF PMM over the entire mass range of proteins identified. MALDI-TOF and PMM produced unique peptide molecular weight matches that were not identified by mu LC-MS/MS. By incorporating amino acid sequence modifications into database searches, combined sequence coverage obtained from these two complimentary ionization methods exceeded 50% for approximately 70% of the 162 spots analyzed. This improved sequence coverage in combination with enzymatic digestions of different specificity is proposed as a method for analysis of post-translational modification from 2D-gel separated proteins.