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.

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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.

Genetic instability is thought to be responsible for the numerous genotypic changes that occur during neoplastic transformation and metastatic progression. To explore the role of genetic instability at the level of point mutations during mammary tumor development and malignant progression, we combined transgenic mouse models of mutagenesis detection and oncogenesis. Bitransgenic mice were generated that carried both a bacteriophage A transgene to assay mutagenesis and a polyomavirus middle T oncogene, mammary gland-targeted expression of which led to metastatic mammary adenocarcinomas. We developed a novel assay for the detection of mutations in the A transgene that selects for phage containing forward mutations only in the A cII gene, using an hfl-bacterial host. In addition to the relative ease of direct selection, the sensitivity of this assay for both spontaneous and chemically induced mutations was comparable to the widely used mutational target gene, A lacI, making the cII assay an attractive alternative for mutant phage recovery for any A-based mouse mutagenesis assay system. The frequencies of A cII mutants were not significantly different in normal mammary epithelium, primary mammary adenocarcinomas, and pulmonary metastases. The cII mutational spectra in these tissues consisted mostly of G/C -> A/T transitions, a large fraction ofwhich occurred at CpG dinucleotides. These data suggest that, in this middle T oncogene model of mammary tumor progression, a significant increase in mutagenesis is not required for tumor development or for metastatic progression.

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.

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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 activatorindependent 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 ؊18͞؊9 region and a strong correlation between the presence of flexible dimeric steps in this region and the intrinsic strength of the promoter.A promoter is a stretch of DNA sequence that encodes information for RNA polymerase binding and initiation of transcription. Genetic and statistical analysis of promoters in Escherichia coli defined two kinds of E 70 RNA polymerase holoenzyme-dependent promoters (1). Both kinds require a 6-bp Ϫ10 sequence (consensus 5Ј-TATAAT-3Ј) located Ϸ7 bp 5Ј to the transcription start site. Functionally, the Ϫ10 element participates in RNA polymerase binding by interacting with the region 2.3-2.4 of 70 (2-10) and is part of an Ϸ15-nt putative single-stranded region in the open complex (4,8,(11)(12)(13)(14)(15). The first kind of promoters (Ϫ35 promoters) is more common and characterized by the presence of the Ϫ10 element as well as a 6-bp (consensus sequence 5Ј-TTGACA-3Ј) in the Ϫ35 position (16). The Ϫ35 element also helps RNA polymerase binding through interaction with region 4.2 of 70 (2,5,17). It is believed that the spacer region between Ϫ35 and Ϫ10 (optimal length 17 bp) does not have any specific sequence requirement and simply facilitates the spatial alignment of the Ϫ10 and Ϫ35 elements in binding to the 2.4 and 4.2 regions of the factor (7, 17, 18). The second kind, called ''extended Ϫ10'' promoters, contains an extra 2-bp 5Ј-TG-3Ј sequence located 1 bp 5Ј to the Ϫ10 element, (consensus sequence Ϫ15 TGNTATAAT Ϫ7 ) (19). The DNA sequence immediately upstream of an extended Ϫ10 element may enhance the activity of the extended Ϫ10 promoter (20, 21). The TG element also binds to RNA polymerase by contacting the 3.0 (formerly 2.5) segment of 70 (22). The Ϫ35 promoters may be improved by the presence of the extended Ϫ10 sequence and vice versa.Many naturally occurring promoters are more or less inefficient because of the presence of non-consensus sequence elements or suboptimal spacer lengths and are resurrected by extra regulatory factors (23). The extra factor may be a DNA sequence, e.g., an UP element (24-26). The UP element, located imme...

The cAMP receptor protein (CRP) of Escherichia coli is a dimer of a two-domain subunit. It requires binding of cAMP for a conformational change in order to function as a site-specific DNA-binding protein that regulates gene activity. The hinge region connecting the cAMP-binding domain to the DNA-binding domain is involved in the cAMPinduced allosteric change. We studied the structural changes in CRP that are required for gene regulation by making a large number of single and double amino acid substitutions at four different positions in or near the hinge. To achieve cAMPindependent transcription by CRP, amino acid residues 138 (located within the hinge region) and 141 (located in the D a-helix adjacent to the hinge) must be polar. This need for polar residues at positions 138 and 141 suggests an interaction that causes the C and D a-helices to come together. As a consequence, the F a-helix is released from the D a-helix and can interact with DNA. At position 144 in the D a-helix and within interacting distances of the F a-helix, replacement of alanine by an amino acid with a larger side chain, regardless of its nature, allows cAMP independence. This result indicates that pushing against the F a-helix may be a way of making the helix available for DNA binding. We believe that the cAMPinduced allosteric change involves similar hinge reorientation to adjust the C and D a-helices, allowing outward movement of the F a-helix.The cAMP receptor protein (CRP), when bound to cAMP, regulates expression of many genes in Escherichia coli (1-3). Free CRP binds only nonspecifically to DNA at much lower efficiency. From a variety of biochemical and biophysical studies such as limited proteolysis (4-6), chemical crosslinking (7), small-angle x-ray scattering (8), and fluorescence studies (9, 10), it appears that cAMP binding to CRP alters CRP conformation allosterically. The altered conformation binds to DNA with higher affinity and sequence specificity. Steitz and coworkers (11,12) have determined the structure of the dimeric CRP-cAMP complex (Fig. 1). Each subunit is composed of two domains: the large amino-terminal domain binds a molecule of cAMP and participates in the dimerization. The small carboxyl-terminal domain contains the DNAbinding segment, which has the helix-turn-helix DNArecognizing structure, similar to other DNA-binding proteins (12,13). The two domains are connected by a hinge polypeptide (residues 135-138). Recently, Schultz et al. (14) have also solved the structure ofa CRP-cAMP DNA cocrystal, and so it is known precisely how CRP binds to DNA. No one has solved the structure of CRP without a cAMP molecule bound, making it difficult to predict what structural effect cAMP binding to one domain would have on the other DNA-binding portion of the protein.Taking a genetic approach to understand the structural changes that ensue following cAMP binding, we and others (15-17) have isolated and mapped mutations in the crp gene that allow cAMP independence. One of these mutations caused substitution of a serine...