Workshop on treatment of and postexposure prophylaxis for Burkholderia pseudomallei and B. mallei Infection, 2010.

The US Public Health Emergency Medical Countermeasures Enterprise convened subject matter experts at the 2010 HHS Burkholderia Workshop to develop consensus recommendations for postexposure prophylaxis against and treatment for Burkholderia pseudomallei and B. mallei infections, which cause melioidosis and glanders, respectively. Drugs recommended by consensus of the participants are ceftazidime or meropenem for initial intensive therapy, and trimethoprim/sulfamethoxazole or amoxicillin/clavulanic acid for eradication therapy. For postexposure prophylaxis, recommended drugs are trimethoprim/sulfamethoxazole or co-amoxiclav. To improve the timely diagnosis of melioidosis and glanders, further development and wide distribution of rapid diagnostic assays were also recommended. Standardized animal models and B. pseudomallei strains are needed for further development of therapeutic options. Training for laboratory technicians and physicians would facilitate better diagnosis and treatment options.

The NIH Human Microbiome Project.

The Human Microbiome Project (HMP), funded as an initiative of the NIH Roadmap for Biomedical Research (http://nihroadmap.nih.gov), is a multi-component community resource. The goals of the HMP are: (1) to take advantage of new, high-throughput technologies to characterize the human microbiome more fully by studying samples from multiple body sites from each of at least 250 "normal" volunteers; (2) to determine whether there are associations between changes in the microbiome and health/disease by studying several different medical conditions; and (3) to provide both a standardized data resource and new technological approaches to enable such studies to be undertaken broadly in the scientific community. The ethical, legal, and social implications of such research are being systematically studied as well. The ultimate objective of the HMP is to demonstrate that there are opportunities to improve human health through monitoring or manipulation of the human microbiome. The history and implementation of this new program are described here.

Ribosomal protein S1 promotes transcriptional cycling.

Prokaryotic RNA polymerases are capable of efficient, continuous synthesis of RNA in vivo, yet purified polymerase-DNA model systems for RNA synthesis typically produce only a limited number of catalytic turnovers. Here, we report that the ribosomal protein S1--which plays critical roles in translation initiation and elongation in Escherichia coli and is believed to stabilize mRNA on the ribosome--is a potent activator of transcriptional cycling in vitro. Deletion of the two C-terminal RNA-binding modules--out of a total of six loosely homologous RNA-binding modules present in S1--resulted in a near-loss of the ability of S1 to enhance transcription, whereas disruption of the very last C-terminal RNA-binding module had only a mild effect. We propose that, in vivo, cooperative interaction of multiple RNA-binding modules in S1 may enhance the transcript release from RNA polymerase, alleviating its inhibitory effect and enabling the core enzyme for continuous reinitiation of transcription.

lacP1 promoter with an extended -10 motif. Pleiotropic effects of cyclic AMP protein at different steps of transcription initiation.

The cyclic AMP receptor protein (CRP), which activates transcription from the wild-type lacP1 promoter and most of its mutants, represses productive RNA synthesis from a lacP1 promoter variant that contains an extended -10 element, although CRP enhances RNA polymerase binding as well as open complex formation in both promoters. Moreover, abortive RNA synthesis, which is already higher in the extended -10 variant compared with the parent promoter, was further enhanced by CRP. These results, together with the observed decrease in productive RNA synthesis, indicate that CRP, while facilitating the earlier steps of initiation, inhibits transcription from the extended -10 lacP1 by hindering promoter clearance. We propose that CRP decreases energetic barriers to RNA polymerase binding, isomerization, and abortive RNA synthesis but stabilizes the abortive RNA initiating complex, which results in increasing the activation energy of the transition state before the elongation complex. The results demonstrate for the first time that a DNA-binding regulatory protein acts as an activator or a repressor in different steps of the transcription initiation pathway because of the energetic differences of the intermediate complex in the same promoter.

Interaction of Escherichia coli RNA polymerase with the ribosomal protein S1 and the Sm-like ATPase Hfq.

We report evidence that ribosomal protein S1 and nucleic acid-binding protein Hfq copurify in molar ratios with RNA polymerase (RNAP). Purified S1 associates independently with RNAP, and Hfq binding to polymerase occurs in the presence of S1. Looking for a functional role of the RNAP-S1-Hfq association, we studied the effects of S1 and Hfq on transcription and coupled transcription-translation. S1 was capable of significant stimulation of the RNAP transcriptional activity from a number of promoters; the stimulatory effect was observed on linear as well as supercoiled DNA templates. In addition, we present biochemical and genetic evidence of ATPase activity associated with the Sm-like hexameric nucleic acid-binding protein Hfq. The limited sequence homology between Hfq and known ATP-utilizing enzymes suggests a new class of ATPases.

A mutant spacer sequence between -35 and -10 elements makes the Plac promoter hyperactive and cAMP receptor protein-independent.

To determine whether the spacer region between the -35 and -10 elements plays any sequence-specific role, we randomized the GC-rich sequence ((-20)CCGGCTCG(-13)) within the spacer region of the cAMP-dependent lac promoter and selected an activator-independent mutant, which showed extraordinarily high intrinsic activity. The hyperactive promoter is obtained by incorporation of a specific 10-bp-long AT-rich DNA sequence within the spacer, referred to as the -15 sequence, which must be juxtaposed to the upstream end of the -10 sequence for the hyperactivity. The transcription enhancement functions only in the presence of a -35 element. The spacer sequence enhanced both RNA polymerase binding and open complex formation. Isolated in the lac promoter, it also enhanced transcription when placed at two other unrelated promoters. Sequence analysis shows a low GC content and an abundance of stereochemically flexible TG:CA and TA:TA dimeric steps in the -18/-9 region and a strong correlation between the presence of flexible dimeric steps in this region and the intrinsic strength of the promoter.

Kinetics of transcription initiation at lacP1. Multiple roles of cyclic AMP receptor protein.

The cyclic AMP receptor protein (CRP) acts as a transcription activator at many promoters of Escherichia coli. We have examined the kinetics of open complex formation at the lacP1 promoter using tryptophan fluorescence of RNA polymerase and DNA fragments with 2-aminopurine substituted at specific positions. Apart from the closed complex formation and promoter clearance, we were able to detect three steps. The first step after the closed complex formation leads to a rapid increase of 2-aminopurine fluorescence. This was followed by another rapid step in which quenching of tryptophan fluorescence of RNA polymerase was observed. The slowest step detected by 2-aminopurine fluorescence increase is assigned to the final open complex formation. We have found that CRP not only enhances RNA polymerase binding at the promoter, but also enhances the slowest isomerization step by about 2-fold. Furthermore, potassium permanganate probing shows that the conformation of the open complex in the presence of CRP appears qualitatively and quantitatively different from that in the absence of CRP, suggesting that contact with RNA polymerase is maintained throughout the transcription initiation.

Genetic instability favoring transversions associated with ErbB2-induced mammary tumorigenesis.

It has been argued that genetic instability is required to generate the myriad mutations that fuel tumor initiation and progression and, in fact, patients with heritable cancer susceptibility syndromes harbor defects in specific genes that normally maintain DNA integrity. However, the vast majority of human cancers arise sporadically, in the absence of deficiencies in known "mutator" genes. We used a cII-based mutation detection assay to show that the mean frequency of forward mutations in primary mammary adenocarcinomas arising in mouse mammary tumor virus-c-erbB2 transgenic mice harboring multiple copies of the lambda bacteriophage genome was significantly higher than in aged-matched, wild-type mammary tissue. Analysis of the cII mutational spectrum within the mammary tumor genomic DNA demonstrated a >6-fold elevation in transversion mutation frequency, resulting in a highly unusual inversion of the transition/transversion ratio characteristic of normal epithelium; frameshift mutation frequencies were unaltered. Arising oncogenic point mutations within the c-erbB2 transgene of such tumors were predominantly transversions as well. Data from this model system support the notion that elaboration of a mutator phenotype is a consequential event in breast cancer and suggest that a novel DNA replication/repair gene is a relatively early mutational target in c-erbB2-induced mammary tumorigenesis.