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gE, an RNA polymerase ofactor ofapparent Mr 28,000, was previously identified by its ability to direct transcription from the P2 promoter of the agarase gene (dagA) was grown in YEME medium containing 34% sucrose and 5 mM MgCl2 to OD6oo = 0.6-0.8, and o-E-containing RNA polymerase holoenzyme was purified from approximately 50 g of mycelium as described (2,4). In vitro transcription reactions on the dagA promoter region carried on a 589-bp Sma I-Ava II DNA fragment (4) were performed as described previously (2, 4) and oE was renatured for holoenzyme reconstitution experiments by using the GroEL-assisted protocol ofBrown et al. (2). Fractions ofRNA polymerase eluted from the final Superose 6 (Pharmacia) gel filtration column with peak levels of dagA P2 transcribing activity were pooled, electrophoresed on a 7.5% polyacrylamide/SDS gel, and electroblotted for 30 min at 0.5 A onto Trans-Blot [Trans-Blot Transfer Medium polyvinylidene difluoride (PVDF) protein sequencing membrane; Bio-Rad] in 10 mM cyclohexylaminopropanesulfonic acid (Caps), pH 11.0/10%o (vol/vol) aqueous methanol. The membrane was subsequently stained and treated as described (2) to visualize the yE polypeptide. The sequence of the first 27 residues of aE was determined by sequential N-terminal Edman degradation using a gas-phase sequenator.Cloning and Nucleotide Sequencing of the sigE Gene. The 256-fold-degenerate 49-mer G332, designed from the sequence of the first 17 N-terminal residues of o-E, was

Inhibition of the mammalian target of rapamycin (mTOR) pathway extends life span in all species studied to date, and in mice delays the onset of age-related diseases and comorbidities. However, it is unknown if mTOR inhibition affects aging or its consequences in humans. To begin to assess the effects of mTOR inhibition on human aging-related conditions, we evaluated whether the mTOR inhibitor RAD001 ameliorated immunosenescence (the decline in immune function during aging) in elderly volunteers, as assessed by their response to influenza vaccination. RAD001 enhanced the response to the influenza vaccine by about 20% at doses that were relatively well tolerated. RAD001 also reduced the percentage of CD4 and CD8 T lymphocytes expressing the programmed death-1 (PD-1) receptor, which inhibits T cell signaling and is more highly expressed with age. These results raise the possibility that mTOR inhibition may have beneficial effects on immunosenescence in the elderly.

SummaryStreptococcus pneumoniae remains a serious cause of morbidity and mortality in humans, but relatively little is known about the molecular basis of its pathogenesis. We used signature-tagged mutagenesis together with an analysis of S. pneumoniae genome sequence to identify and characterize genes required for pathogenesis. A library of signaturetagged mutants was created by insertion±duplication mutagenesis, and 1786 strains were analysed for their inability to survive and replicate in murine models of pneumonia and bacteraemia. One hundred and eightysix mutant strains were identified as attenuated, and 56 were selected for further genetic characterization based on their ability to excise the integrated plasmid spontaneously. The genomic DNA inserts of the plasmids were cloned in Escherichia coli and sequenced. These sequences were subjected to database searches, including the S. pneumoniae genome sequence, which allowed us to examine the chromosomal regions flanking these genes. Most of the insertions were in probable operons, but no pathogenicity islands were found. Forty-two novel virulence loci were identified. Five strains mutated in genes involved in gene regulation, cation transport or stress tolerance were shown to be highly attenuated when tested individually in a murine respiratory tract infection model. Additional experiments also suggest that induction of competence for genetic transformation has a role in virulence.

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A comparative genomic approach was used to identify Helicobacter pylori 26695 open reading frames (ORFs) which are conserved in H. pylori J99 but highly diverged in other eubacteria. A survey of selected pathways of central intermediary metabolism was also carried out, and genes with a potentially selective role in H. pylori were identified. Forty-five ORFs identified in these two analyses were screened using a rapid vector-free allelic replacement mutagenesis technique, and 33 were shown to be essential in vitro. Notably, 13 ORFs gave essentiality results which are unexpected in view of their known or proposed functions, and phylogenetic analysis was used to investigate the annotation of 7 such ORFs which are highly diverged. We propose that the products of a number of these H. pylori-specific essential genes may be suitable targets for novel anti-H. pylori therapies.

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The promoter of the Streptococcus pneumoniae putative fuculose kinase gene (fcsK), the first gene of a novel fucose utilization operon, is induced by fucose and repressed by glucose or sucrose. When the streptococcal polypeptide deformylase (PDF) gene (def1, encoding PDF) was placed under the control of P fcsK , fucosedependent growth of the S. pneumoniae (P fcsK ::def1) strain was observed, confirming the essential nature of PDF in this organism. The mode of antibacterial action of actinonin, a known PDF inhibitor, was also confirmed with this strain. The endogenous fuculose kinase promoter is a tightly regulated, titratable promoter which will be useful for target validation and for confirmation of the mode of action of novel antibacterial drugs in S. pneumoniae.Streptococcus pneumoniae is a widespread human pathogen and a major cause of community-acquired diseases such as pneumonia, otitis media, sinusitis, and meningitis (12). Established antibiotic treatments of pneumococcal infections have become less effective due to the emergence of drug-resistant isolates (34). A genomics-based strategy has been applied in the search for new drug targets to identify inhibitors active against this pathogen (25,33). Once a lead antimicrobial compound has been discovered, it is fundamentally important to demonstrate that during chemical optimization the antibacterial activity continues to be related to inhibition of the specific target (25). Tightly regulated, titratable promoter systems that are able to modulate the levels of the protein target have proven to be invaluable tools for tracking the mechanism of antibacterial activity of novel inhibitors (2, 37). In addition, inducible promoters have been used in antimicrobial drug discovery for establishing gene essentiality and characterizing the function of essential drug targets (1, 9, 31). For S. pneumoniae only a limited number of regulated promoters have been studied. Heterologous promoter systems derived from nisin and tetracycline genes have been analyzed as tools for regulating gene expression, but their narrow titratable range and high basal levels of expression have compromised their use (1, 2, 7). The streptococcal promoter of the maltose operon has been characterized at the molecular level and shown to be inducible by maltose and repressible by sucrose in S. pneumoniae (23), but its use has been limited by its high basal expression levels.More recently, the raffinose operon has been identified and its promoter has been shown to be regulated, though its application for target validation or mode-of-action analysis has yet to be demonstrated (27). The expression of genes involved in sugar metabolism is known to be a regulated process in many bacterial species. With the availability of genomic sequence data for S. pneumoniae, it is now possible to identify and study many, and perhaps all, putative sugar metabolic genes and their associated promoter sequences.Identification and bioinformatic analysis of the fucose gene cluster of S. pneumoniae. In an attempt to ident...