Nasopharyngeal carriage is the reservoir from which most disease with Streptococcus pneumoniae arises. Survival as a commensal in this environment is likely to require a set of adaptations distinct from those needed to cause disease, some of which may be mediated by two-component signal transduction systems (TCSTS). We examined the contributions of nine pneumococcal TCSTS to the process of nasopharyngeal colonization by using an infant rat model. Whereas deletions in all but one of these systems have been associated previously with a high degree of attenuation in a murine model of pneumonia, only the CiaRH system was necessary for efficient carriage. Transcriptional analysis by using microarray hybridization identified a locus consisting of two adjacent genes, htrA and spoJ, that was specifically and strongly downregulated in a ⌬ciaRH-null mutant. A S. pneumoniae strain lacking the htrA gene encoding a putative serine protease, but not one lacking spoJ, showed decreased fitness in a competitive model of colonization, a finding consistent with this gene mediating a portion of the carriage deficit observed with the ⌬ciaRH strain.Streptococcus pneumoniae exists in nature both as a commensal on the mucosal surface of the human upper respiratory tract and as a pathogen causing disease in a wide range of sites, including the lung, middle ear, sinuses, blood, and meninges. Survival in these different host environments is likely to require adaptive responses, some of which may be mediated by twocomponent signal transduction systems (TCSTS). These signaling systems (reviewed in reference 27) are found in a wide range of bacteria where they have been shown to regulate diverse processes, including chemotaxis, nutrient utilization, surface adhesion, and the switch between aerobic and anaerobic metabolism. TCSTS consist of a sensor kinase that autophosphorylates in response to an environmental stimulus and a cognate response regulator to which an activated phosphate is transferred and that then mediates a downstream response, often acting as a DNA-binding protein to cause changes in gene expression.Two genomic surveys of S. pneumoniae have identified 13 pairs of genes encoding putative TCSTS as well as a single unpaired putative response regulator (12, 29). The ability of null mutants in these systems to cause disease has been previously examined in two studies of virulence. In a murine model of pneumonia developing after intranasal inoculation in which all of the TCSTS except ComDE were tested, deletions in eight of the systems resulted in an attenuation of at least 3 orders of magnitude (29). In contrast, a second study testing a collection of single-crossover mutants in a different genetic background showed no evidence of decreased virulence for any of the strains in a murine model of intraperitoneal infection in which the requirements of adaptation to the mucosal surface of the airway were circumvented (12). Despite the importance of nasal carriage both as the first step in the pathogenesis of pneumococcal disease and in...

Antibiotic efflux is an important mechanism of resistance in pathogenic bacteria. Here we describe the identification and characterization of a novel chromosomally encoded multidrug resistance efflux protein in Staphylococcus aureus, MdeA (multidrug efflux A). MdeA was identified from screening an S. aureus open reading frame expression library for resistance to antibiotic compounds. When overexpressed, MdeA confers resistance on S. aureus to a range of quaternary ammonium compounds and antibiotics, but not fluoroquinolones. MdeA is a 52-kDa protein with 14 predicted transmembrane segments. It belongs to the major facilitator superfamily and is most closely related, among known efflux proteins, to LmrB of Bacillus subtilis and EmrB of Escherichia coli. Overexpression of mdeA in S. aureus reduced ethidium bromide uptake and enhanced its efflux, which could be inhibited by reserpine and abolished by an uncoupler. The mdeA promoter was identified by primer extension. Spontaneous mutants selected for increased resistance to an MdeA substrate had undergone mutations in the promoter for mdeA, and their mdeA transcription levels were increased by as much as 15-fold. The mdeA gene was present in the genomes of all six strains of S. aureus examined. Uncharacterized homologs of MdeA were present elsewhere in the S. aureus genome, but their overexpression did not mediate resistance to the antibacterials tested. However, MdeA homologs were identified in other bacteria, including Bacillus anthracis, some of which were shown to be functional orthologs of MdeA.

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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...

There is a global emergence of multidrug-resistant (MDR) strains of Klebsiella pneumoniae, a Gram-negative enteric bacterium that causes nosocomial and urinary tract infections. While the epidemiology of K. pneumoniae strains and occurrences of specific antibiotic resistance genes, such as plasmid-borne extendedspectrum ␤-lactamases (ESBLs), have been extensively studied, only four complete genomes of K. pneumoniae are available. To better understand the multidrug resistance factors in K. pneumoniae, we determined by pyrosequencing the nearly complete genome DNA sequences of two strains with disparate antibiotic resistance profiles, broadly drug-susceptible strain JH1 and strain 1162281, which is resistant to multiple clinically used antibiotics, including extended-spectrum ␤-lactams, fluoroquinolones, aminoglycosides, trimethoprim, and sulfamethoxazoles. Comparative genomic analysis of JH1, 1162281, and other published K. pneumoniae genomes revealed a core set of 3,631 conserved orthologous proteins, which were used for reconstruction of whole-genome phylogenetic trees. The close evolutionary relationship between JH1 and 1162281 relative to other K. pneumoniae strains suggests that a large component of the genetic and phenotypic diversity of clinical isolates is due to horizontal gene transfer. Using curated lists of over 400 antibiotic resistance genes, we identified all of the elements that differentiated the antibiotic profile of MDR strain 1162281 from that of susceptible strain JH1, such as the presence of additional efflux pumps, ESBLs, and multiple mechanisms of fluoroquinolone resistance. Our study adds new and significant DNA sequence data on K. pneumoniae strains and demonstrates the value of whole-genome sequencing in characterizing multidrug resistance in clinical isolates.

Attempted allelic replacement of 144 Streptococcus pneumoniae open reading frames of previously uncharacterized function led to the identification of 36 genes essential for growth under laboratory conditions. Of these, 14 genes (obg, spoIIIJ2, trmU, yacA, yacM, ydiC, ydiE, yjbN, yneS, yphC, ysxC, ytaG, yloI and yxeH4) were also essential in Staphylococcus aureus and Haemophilus influenzae or Escherichia coli, 2 genes (yrrK and ydiB) were only essential in H. influenzae as well as S. pneumoniae and 8 genes were necessary for growth of S.pneumoniae and S. aureus and did not have a homolog in H. influenzae(murD2, ykqC, ylqF, yqeH, ytgP, yybQ) or were not essential in that organism (yqeL, yhcT). The proteins encoded by these genes could represent good targets for novel antibiotics covering different therapeutic profiles. The putative functions of some of these essential proteins, inferred by bioinformatic analysis, are presented. Four mutants, with deletions of loci not essential for in vitro growth, were found to be severely attenuated in a murine respiratory tract infection model, suggesting that not all targets for antibacterial therapeutics are revealed by simple in vitro essentiality testing. The results of our experiments together with those collated from previously reported studies including Bacillus subtilis, E. coli and Mycoplasma sp. demonstrate that gene conservation amongst bacteria does not necessarily indicate that essentiality in one organism can be extrapolated to others. Moreover, this study demonstrates that different experimental procedures can produce apparently contradictory results.

BackgroundEvaluate and compare the utility of serum folate receptor alpha (FRA) and megakaryocyte potentiating factor (MPF) determinations relative to serum CA125, mesothelin (MSLN) and HE4 for the diagnosis of epithelial ovarian cancer (EOC).MethodsElectrochemiluminescent assays were developed for FRA, MSLN and MPF and used to assess the levels of these biomarkers in 258 serum samples from ovarian cancer patients. Commercial assays for CA125 and HE4 were run on a subset of 176 of these samples representing the serous histology. Data was analyzed by histotype, stage and grade of disease. A comparison of the levels of the FRA, MSLN and MPF biomarkers in serum, plasma and urine was also performed in a subset of 57 patients.ResultsSerum and plasma levels of FRA, MSLN and MPF were shown to be highly correlated between the two matrices. Correlations between all pairs of markers in 318 serum samples were calculated and demonstrated the highest correlation between HE4 and MPF, and the lowest between FRA and MPF. Serum levels of all markers showed a dependence on both stage and grade of disease. A multi-marker logistic regression model was developed resulting in an AUC=0.91 for diagnosis of serous ovarian cancer, a significant improvement over the AUC for any of the individual markers, including CA125 (AUC=0.84).ConclusionsFRA has significant potential as a biomarker for ovarian cancer, both as a stand-alone marker and in combination with other known markers for EOC. The lack of correlation between the various markers analyzed in the present study suggests that a panel of markers can aid in the detection and/or monitoring of this disease.

We have examined the accumulation of polyphosphorylated nucleotides in Bacillus subtilis in relation to the function of the rel gene. Our results are as follows. (i) During inhibition of isoleucine activation by O-methylthreonine, wildtype B. subtilis cells accumulate unusual nucleotides with the chromatographic and chemical properties of pppApp, ppApp, pppGpp, ppGpp, pGpp, and ppGp. (ii) During the carbon source downshift elicited by inhibiting glucose uptake, we observed accumulation of the polyphosphorylated guanosine but not adenosine nucleotides. (iii) At the end of log phase in sporulation medium, we observed a small transient accumulation of the polyphosphorylated guanosine but not adenosine nucleotides. (iv) We were unable to detect a nucleotide with chromatographic behavior expected for pppAppp under any conditions. (v) The rel mutant of Swanton and Edlin (Biochem. Biophys. Res. Commun. 46:583-588, 1972) did not accumulate any of these polyphosphorylated nucleotides under any of the conditions examined. (vi) The rel mutant is unimpaired in sporulation. We conclude that one or more of the nucleotides we have detected may be involved in controlling the specificity of transcription during the stringent response, but none of them are required for sporogenesis.