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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...

Pantothenate kinase (CoaA) catalyzes the first step of the coenzyme A biosynthetic pathway. Here we report the identification of the Staphylococcus aureus coaA gene and characterization of the enzyme. We have also identified a series of low-molecular-weight compounds which are effective inhibitors of S. aureus CoaA.Increasing reports of antibiotic resistance involving opportunistic gram-positive pathogens, including methicillin-resistant Staphylococcus aureus, have emphasized the critical need for the development of antimicrobial compounds with novel modes of action. Coenzyme A (CoA), an essential cofactor for maintaining life, is used in a multitude of biochemical reactions. In most bacteria, CoA is synthesized from pantothenic acid (vitamin B 5 ) in 5 steps (5), with the first step being the phosphorylation of pantothenate by pantothenate kinase (CoaA). Although this pathway also exists in eukaryotes, in most cases there is no sequence homology between the prokaryotic and eukaryotic CoA biosynthetic enzymes (7,9,12,18,24,27). Thus, there is the potential for developing highly specific inhibitors of bacterial CoA enyzmes.Unlike the case for other biosynthetic pathways of bacteria, the genes involved in CoA biosynthesis are not organized as operons. This has delayed the identification of the enzymes responsible for CoA synthesis, even though the intermediate chemical steps have been known since the 1960s (1). With the recent identification of the Escherichia coli genes encoding the enzymes CoaBC and CoaE, the entire pathway is now known for this organism (9,10,13,19,21). Interestingly, the gene coaA, which encodes the first enzyme in the pathway, has no homolog in the complete genome sequences of the S. aureus strains Mu50 and N315 (11).Cloning and purification of S. aureus CoaA. Initially, the coaA gene sequences in S. aureus strains Mu50 and N15 (GenBank accession numbers BA000017 and BA000018, respectively) were identified through searches of the ERGO comparative genomic database (previously WIT) (http://ergo .integratedgenomics.com/ERGO/) (8). We cloned the S. aureus RN4220 coaA gene and overexpressed it using standard techniques (4, 17). S. aureus RN4220 coaA was amplified by PCR, introducing an NdeI site at the start codon and an XhoI site after the stop codon, and cloned into pSTBlue1 using the Perfectly Blunt Cloning kit. The gene was excised by digestion with NdeI and XhoI and ligated into similarly digested pET28a. The final construct encoded the N-terminal six-His-tagged S. aureus CoaA.Tuner (DE3) cells were transformed with this construct and grown at 37°C in Luria-Bertani medium-50-g/ml kanamycin. Protein expression was induced by 500 M isopropylthio-␤-Dgalactoside, and cells were harvested 3 h postinduction. The cell pellet was resuspended and sonicated, and cell debris was removed by centrifugation. The supernatant was subjected to Ni-chelating column chromatography followed by a HiTrap Q Sepharose ion exchange column. Enzyme identity was confirmed through N-terminal sequencing and matrix-assisted...

The in vitro activity of retapamulin was determined and compared to that of topical and community antibiotics. The MIC 90 s of retapamulin against Staphylococcus aureus and Streptococcus pyogenes were 0.12 g/ml and 0.016 g/ml, respectively. Retapamulin has a low propensity to select resistance and produces an in vitro postantibiotic effect.The emergence and spread of antibiotic resistance in hospital and community pathogens has significantly eroded the utility of established antibiotics, a problem that has been widely publicized and poses a serious threat to public health worldwide (6,8). Novel mechanism antibiotics are needed to address rising resistance to established classes of both systemic and topical agents. Resistance has developed to two of the most commonly used topical antibiotics, fusidic acid and mupirocin, and thus dictates the need for novel mechanism topical agents for managing the treatment of bacterial skin infections.Retapamulin {mutilin 14-(exo-8-methyl-8-azabicyclo[3.2.1]oct-3-yl-sulfanyl)-acetate} is a novel semisynthetic pleuromutilin that was discovered by GlaxoSmithKline as part of a program to identify novel compounds with the appropriate balance of drug developability and microbiological attributes to be formulated and developed as a topical antibiotic. In this work, we describe the initial microbiology evaluation that led to the selection of retapamulin for development.The isolates evaluated were obtained from the GlaxoSmithKline culture collection (Collegeville, PA). The panel of methicillin-resistant Staphylococcus aureus isolates (based on oxacillin MICs) was supplemented with 32 isolates obtained from International Health Management Associates (Schaumburg, IL). Broth microdilution, agar dilution, and cidality experiments were performed using the CLSI recommended procedures (3, 4). In the absence of approved quality control limits for retapamulin and specific comparator antibiotics, correlation with previous internal testing of these compounds was used. Modifications were made to the standard broth microdilution method (pH, inoculum density, serum) to identify potential factors that might influence the in vitro activity of retapamulin. Antibiotics were obtained as follows: retapamulin, amoxicillin, and mupirocin were from GlaxoSmithKline Pharmaceuticals, Harlow, United Kingdom; bacitracin, cefaclor, and fusidic acid were from Sigma Chemical Co., St. Louis, MO; levofloxacin and azithromycin were deformulated by GlaxoSmithKline Pharmaceuticals, Harlow, United Kingdom. The postantibiotic effect (PAE) of retapamulin and mupirocin were determined using a filtration method (staphylococci) or a dilution method (Streptococcus pyogenes) as previously described (7). The spontaneous frequencies of resistance in S. aureus and S. pyogenes were determined by plating the test isolate on agar containing 4ϫ and 10ϫ MIC of retapamulin and mupirocin. Detection of resistant colonies was performed 48 h after inoculation. The development of resistance was determined by daily passage of the test isolates ...

The nucleotide sequence of a 1467 bp fragment of Streptomyces hygroscopicus DNA containing the gene (hyg) encoding a hygromycin B phosphotransferase (HPH) has been determined. The N-terminal amino acid sequence of HPH determined by automated Edman degradation has allowed the coding sequence of the hyg gene to be identified. The translation initiation triplet is GTG and 5 bp preceding it there is a sequence complementary to the 3'-end of 16S rRNA from S. lividans. The transcriptional start and termination sites have been determined; the presumptive promoter region has only partial homology to that of the Streptomyces vinaceus vph gene and is different to the promoter sequences of other Streptomyces genes.

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.

Ribosomes from Streptomyces griseofuscus expressing tlrA, a resistance gene isolated from the tylosin producer Streptomyces fradiae, are resistant to macrolide and lincosamide antibiotics in vitro. The tlrA product was found to be a methylase that introduces two methyl groups into a single base within 23S rRNA, generating N6,N6-dimethyladenine at position 2058. This activity is therefore similar to the ermE resistance mechanism in Saccharopolyspora erythraea (formerly Streptomyces erythraeus).

Nucleotide sequence analysis of a 5,000-bp region of the bialaphos antibiotic production (bap) gene cluster defined five open reading frames (ORFs) which predicted structural genes in the order bah, ORF1, ORF2, and ORF3 followed by the regulatory gene, brpA (H. Anzai, T. Murakami, S. Imai, A. Satoh, K. Nagaoka, and C. J. Thompson, J. Bacteriol. 169:3482-3488, 1987). The four structural genes were translationally coupled and apparently cotranscribed from an undefined promoter(s) under the positive control of the brpA gene product. S1 mapping experiments indicated that brpA was transcribed by two promoters (brpAp1 and brpAp2) which initiate transcription 150 and 157 bp upstream of brpA within an intergenic region and at least one promoter further upstream within the bap gene cluster (brpAp3). All three transcripts were present at low levels during exponential growth and increased just before the stationary phase. The levels of the brpAp3 band continued to increase at the onset of stationary phase, whereas brpApl-and brpAp2-protected fragments showed no further change. BrpA contained a possible helix-turn-helix motif at its C terminus which was similar to the C-terminal regulatory motif found in the receiver component of a family of two-component transcriptional activator proteins. This motif was not associated with the N-terminal domain conserved in other members of the family. The structural gene cluster sequenced began with bah, encoding a bialaphos acetylhydrolase which removes the N-acetyl group from bialaphos as one of the final steps in the biosynthetic pathway. The observation that Bah was similar to a rat and to a bacterial (Acinetobacter calcoaceticus) lipase probably reflects the fact that the ester bonds of triglycerides and the amide bond linking acetate to phosphinothricin are similar and hydrolysis is catalyzed by structurally related enzymes. This was followed by two regions encoding ORF1 and ORF2 which were similar to each other (48% nucleotide identity, 31% amino acid identity), as well as to GrsT, a protein encoded by a gene located adjacent to gramicidin S synthetase in Bacillus brevis, and to vertebrate (mallard duck and rat) thioesterases. The amino acid sequence and hydrophobicity proffle of ORF3 indicated that it was related to a family of membrane transport proteins. It was strikingly similar to the citrate uptake protein encoded by the transposon Tn3411.Bialaphos, a linear tripeptide produced as a secondary metabolite by Streptomyces hygroscopicus, is composed of two L-alanine residues and the glutamate analog phosphinothricin (PT) (4, 29). After cleavage of bialaphos by intracellular nonspecific peptidases, PT acts as an inhibitor of glutamine synthetase (4, 29). For this reason, bialaphos has herbicide and antibiotic activities. The biosynthetic pathway has been well characterized by analyzing intermediates accumulated in cultures of blocked mutants (19). Primary metabolic substrates are converted to the demethylated form of PT, which is then condensed with two alanine residues. Althoug...