Genome of the bacterium Streptococcus pneumoniae strain R6.

Streptococcus pneumoniae is among the most significant causes of bacterial disease in humans. Here we report the 2,038,615-bp genomic sequence of the gram-positive bacterium S. pneumoniae R6. Because the R6 strain is avirulent and, more importantly, because it is readily transformed with DNA from homologous species and many heterologous species, it is the principal platform for investigation of the biology of this important pathogen. It is also used as a primary vehicle for genomics-based development of antibiotics for gram-positive bacteria. In our analysis of the genome, we identified a large number of new uncharacterized genes predicted to encode proteins that either reside on the surface of the cell or are secreted. Among those proteins there may be new targets for vaccine and antibiotic development.

Cloning and analysis of the spinosad biosynthetic gene cluster of Saccharopolyspora spinosa.

BACKGROUND: Spinosad is a mixture of novel macrolide secondary metabolites produced by Saccharopolyspora spinosa. It is used in agriculture as a potent insect control agent with exceptional safety to non-target organisms. The cloning of the spinosyn biosynthetic gene cluster provides the starting materials for the molecular genetic manipulation of spinosad yields, and for the production of novel derivatives containing alterations in the polyketide core or in the attached sugars. RESULTS: We cloned the spinosad biosynthetic genes by molecular probing, complementation of blocked mutants, and cosmid walking, and sequenced an 80 kb region. We carried out gene disruptions of some of the genes and analyzed the mutants for product formation and for the bioconversion of intermediates in the spinosyn pathway. The spinosyn gene cluster contains five large open reading frames that encode a multifunctional, multi-subunit type I polyketide synthase (PKS). The PKS cluster is flanked on one side by genes involved in the biosynthesis of the amino sugar forosamine, in O-methylations of rhamnose, in sugar attachment to the polyketide, and in polyketide cross-bridging. Genes involved in the early common steps in the biosynthesis of forosamine and rhamnose, and genes dedicated to rhamnose biosynthesis, were not located in the 80 kb cluster. CONCLUSIONS: Most of the S. spinosa genes involved in spinosyn biosynthesis are found in one 74 kb cluster, though it does not contain all of the genes required for the essential deoxysugars. Characterization of the clustered genes suggests that the spinosyns are synthesized largely by mechanisms similar to those used to assemble complex macrolides in other actinomycetes. However, there are several unusual genes in the spinosyn cluster that could encode enzymes that generate the most striking structural feature of these compounds, a tetracyclic polyketide aglycone nucleus.

Group III human metabotropic glutamate receptors 4, 7 and 8: molecular cloning, functional expression, and comparison of pharmacological properties in RGT cells.

Cloning and expression in a stable mammalian cell line co-transfected with a glutamate transporter (RGT cells) were used as tools for studying the functions and pharmacological properties of group III metabotropic glutamate receptors (mGluRs). Complementary DNAs (cDNAs) encoding the human mGluR4, human mGluR7, and human mGluR8 were isolated from human cerebellum, fetal brain or retinal cDNA libraries. The human mGluR4, mGluR7 and mGluR8 receptors were 912, 915 and 908 amino acid residues long and share 67-70% amino acid similarity with each other and 42-45% similarity with the members of mGluR subgroups I and II. The human mGluR4 and mGluR7 had amino acid identity of 96% and 99.5% with rat mGluR4 and 7, respectively, whereas the human mGluR8 has 98.8% amino acid identity with the mouse mGluR8. The nucleotide and amino acid sequences in the coding region of human mGluR4 and mGluR7 were found to be identical to the previously published sequences by Flor et al. and Makoff et al. Following stable expression in RGT cells, highly significant inhibitions of forskolin stimulation of cAMP production by group III agonists were found for each receptor. The relative potencies of the group III agonist L-AP4 varied greatly between the group III clones, being mGluR8>mGluR4 >> mGluR7. The reported group II mGluR agonist L-CCG-I was a highly potent mGluR8 agonist (EC50=0.35 microM), with significant agonist activities at both mGluR4 (EC50=3.7 microM) and mGluR7 (EC50=47 microM). The antagonist potency of the purported group III mGluR antagonist MPPG also varied among the receptors being human mGluR8 >> mGluR4 = mGluR7. The expression and second messenger coupling of human group III mGluRs expressed in the RGT cell line are useful to clearly define the subtype selectivities of mGluR ligands.

DNA sequence sampling of the Streptococcus pneumoniae genome to identify novel targets for antibiotic development.

We initiated a survey of the Streptococcus pneumoniae genome by DNA sequence sampling. More than 9,500 random DNA sequences of approximately 500 bases average length were determined. Partial sequences sufficient to identify approximately 95% of the aminoacyl tRNA synthetase genes and ribosomal protein (rps) genes were found by comparing the database of partial sequences to known sequences from other organisms. Many genes involved in DNA replication, repair, and mutagenesis are present in S. pneumoniae. Genes for the major subunits of RNA polymerase are also present, as are genes for two alternative sigma factors, rpoD and rpoN. Many genes necessary for amino acid or cofactor biosynthesis and aerobic energy metabolism in other bacteria appear to be absent from the S. pneumoniae genome. A number of genes involved in cell wall biosynthesis and septation were identified, including six homologs to different penicillin binding proteins. Interestingly, four genes involved in the addition of D-alanine to lipoteicoic acid in other gram positive bacteria were found, even though the lipoteicoic acid in S. pneumoniae has not been shown to contain D-alanine. The S. pneumoniae genome contains a number of chaperonin genes similar to those found in other bacteria, but apparently does not contain genes involved in the type III secretion commonly observed in gram negative pathogens. The G+C content of S. pneumoniae genomic DNA is approximately 43 mole percent and the size of the genome is approximately 2.0 Mb as determined by pulsed-field gel electrophoresis. Many of the genes identified by sequence sampling have been physically mapped to the 19 different SmaI fragments derived from the S. pneumoniae genome. The database of random genome sequence tags (GSTs) provides the starting material for determining the complete genome sequence, gene disruption analysis, and comparative genomics to identify novel targets for antibiotic development.

Analysis of differential gene expression in rat tibia after an osteogenic stimulus in vivo: mechanical loading regulates osteopontin and myeloperoxidase.

The skeleton has the ability to alter its mass, geometry, and strength in response to mechanical stress. In order to elucidate the molecular mechanisms underlying this phenomenon, differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR) was used to analyze gene expression in endocortical bone of mature female rats. Female Sprague-Dawley rats, approximately 8 months old, received either a sham or bending load using a four-point loading apparatus on the right tibia. RNA was collected at 1 h and 24 h after load was applied, reverse-transcribed into cDNA, and used in DDRT-PCR. Parallel display of samples from sham and loaded bones on a sequencing gel showed several regulated bands. Further analysis of seven of these bands allowed us to isolate two genes that are regulated in response to a loading stimulus. Nucleotide analysis showed that one of the differentially expressed bands shares 99% sequence identity with rat osteopontin (OPN), a noncollagenous bone matrix protein. Northern blot analysis confirms that OPN mRNA expression is increased by nearly 4-fold, at 6 h and 24 h after loading. The second band shares 90% homology with mouse myeloperoxidase (MPO), a bactericidal enzyme found primarily in neutrophils and monocytes. Semiquantitative PCR confirms that MPO expression is decreased 4- to 10-fold, at 1 h and 24 h after loading. Tissue distribution analysis confirmed MPO expression in bone but not in other tissues examined. In vitro analysis showed that MPO expression was not detectable in total RNA from UMR 106 osteoblastic cells or in confluent primary cultures of osteoblasts derived from either rat primary spongiosa or diaphyseal marrow. Database analysis suggests that MPO is expressed by osteocytes. These findings reinforce the association of OPN expression to bone turnover and describes for the first time, decreased expression of MPO during load-induced bone formation. These results suggest a role for both OPN and MPO expression in bone cell function.

Molecular cloning and physical mapping of the daptomycin gene cluster from Streptomyces roseosporus.

The daptomycin biosynthetic gene cluster of Streptomyces roseosporus was analyzed by Tn5099 mutagenesis, molecular cloning, partial DNA sequencing, and insertional mutagenesis with cloned segments of DNA. The daptomycin biosynthetic gene cluster spans at least 50 kb and is located about 400 to 500 kb from one end of the approximately 7,100-kb linear chromosome. We identified two peptide synthetase coding regions interrupted by a 10- to 20-kb region that may encode other functions in lipopeptide biosynthesis.

Cloning and expression of a rat brain interleukin-1beta-converting enzyme (ICE)-related protease (IRP) and its possible role in apoptosis of cultured cerebellar granule neurons.

Several members of the IL-1beta-converting enzyme (ICE) family of proteases recently have been implicated in the intracellular cascade mediating the apoptotic death of various cell types. It is unclear, however, whether ICE-related proteases are involved in apoptosis of mammalian neurons and, if so, how they are activated. Here we report the cloning of an ICE-related protease (IRP) from rat brain, which displays strong sequence identity to human CPP32. In situ hybridization histochemistry reveals that this IRP mRNA is expressed in neuron-enriched regions of the developing and adult rat brain but is profoundly downregulated in the adult (compared with developing) brain. To investigate whether this IRP is involved in the death of neurons in the developing brain, we studied IRP expression in cultured cerebellar granule neurons. In cultured cerebellar granule neurons, reduction of extracellular K+ reliably induces apoptosis and stimulates overexpression of IRP mRNA. The latter is especially prominent 4 hr after switching from high K+ to low K+ medium. The expression of IRP mRNA was maintained at this level for at least 8 hr and was followed by apoptotic death of these neurons. Induction of IRP mRNA and cell death are blocked completely by adding depolarizing concentrations of K+

Molecular cloning, expression, and chromosomal localization of a human brain-specific Na(+)-dependent inorganic phosphate cotransporter.

We describe the molecular cloning of a cDNA encoding a human brain Na(+)-dependent inorganic phosphate (P(i)) cotransporter (hBNPI). The nucleotide and deduced amino acid sequences of hBNPI reveal a protein of 560 amino acids with six to eight putative transmembrane segments. hBNPI shares a high degree of homology with other Na(+)-dependent inorganic P(i) cotransporters, including those found in rat brain and human and rabbit kidney. Expression of hBNPI in COS-1 cells results in Na(+)-dependent P(i) uptake. Northern blot analysis demonstrates that hBNPI mRNA is expressed predominantly in brain and most abundantly in neuron-enriched regions such as the amygdala and hippocampus. Moderate levels of expression are also observed in glia-enriched areas such as the corpus callosum, and low levels are observed in the substantia nigra, subthalamic nuclei, and thalamus. In situ hybridization histochemistry reveals relatively high levels of hBNPI mRNA in pyramidal neurons of the cerebral cortex and hippocampus and in granule neurons of dentate gyrus. The level of hBNPI mRNA is quite low in fetal compared with adult human brain, suggesting developmental regulation of hBNPI gene expression. Southern analyses of nine eukaryotic genomic DNAs probed under stringent conditions with hBNPI cDNA revealed that the hBNPI gene is highly conserved during vertebrate evolution and that each gene is most likely present as a single copy. Using fluorescent in situ hybridization, we localized hBNPI to the long arm of chromosome 19 (19q13) in close proximity to the late-onset familial Alzheimer's disease locus.

Sequence, genetic analysis, and expression of Actinobacillus pleuropneumoniae transferrin receptor genes.

The tbpA and tbpB genes encoding the transferrin receptor proteins Tbp1 and Tbp2 from a serotype 7 strain of Actinobacillus pleuropneumoniae were cloned, sequenced, and expressed in Escherichia coli. The tbpB gene was preceded by putative promoter and regulatory sequences and was separated from the downstream tbpA gene by a 13 bp intercistronic sequence suggesting that the two genes may be coordinately transcribed. Determination of the nucleotide sequence of this region facilitated PCR amplification of the tbp region from a serotype 1 strain for comparative purposes. The deduced amino acid sequences of the Tbp1 proteins had regions of homology with Neisseria Lbp and Tbp1s and with TonB-dependent outer membrane (OM) receptors of E. coli. The deduced amino acid sequences of the Tbp2 proteins were nearly identical to those presented in previous studies. Upon high-level expression of the tbpA gene, a large proportion of the recombinant Tbp1 was found in inclusion bodies and could not be affinity-isolated with immobilized porcine transferrin. Most of the remaining expressed Tbp1 was present in the OM fraction, was expressed at the surface of E. coli cells, and retained binding activity that was specific for the C-lobe of porcine transferrin. Although recombinant Tbp2 was found in inclusion bodies during high-level expression, a significant proportion was associated with a novel OM fraction that appeared in sucrose density gradients which was distinct from the OM fraction containing recombinant Tbp1. The recombinant Tbp2 was accessible at the surface yet was unable to bind porcine transferrin. In contrast to previous observations, the binding by recombinant Tbp2 was specific for the C-lobe of porcine transferrin. These results indicate that the A. pleuropneumoniae transferrin receptor proteins have similar properties to the receptor proteins in Neisseria spp. and Haemophilus influenzae, and that functional studies performed with recombinant receptor proteins need to consider differences in processing and export of these proteins when expressed in heterologous hosts.

Software trapping: a strategy for finding genes in large genomic regions.

We present an approach to the gene identification phase of positional cloning that combines sparse sampling of DNA sequences from large genomic regions with computational analysis. We call the method "software trapping." The goal is to find coding exons while avoiding massive DNA sequence determination and contig assembly. Instead, rapid sequence sampling is combined with exon screening software such as a newly developed package called XPOUND to identify coding sequences. We have tested the approach using a set of model genomic sequences with known intron/exon structures as well as with bona fide P1 genomic clones. The results suggest that the strategy is a useful complement to other methods for finding genes in poorly characterized regions of genomes.

A physical map encompassing GP2B, EPB3, D17S183, D17S78, D17S1183, and D17S1184.

The q21 region of chromosome 17 contains the gene BRCA1, which is involved in familial early-onset breast and ovarian cancers. A physical map of a region that extends from a distal boundary of the BRCA1 region, D17S78, to GP2B has been constructed. The map consists of 30 STSs, including 2 new short tandem repeat polymorphic markers. The contig is composed of a mixture of 7 YACs, 5 P1 plasmids, and 14 cosmids and was ordered by STS-content mapping.

The Bacillus subtilis pnbA gene encoding p-nitrobenzyl esterase: cloning, sequence and high-level expression in Escherichia coli.

p-Nitrobenzyl esters serve as protecting groups on intermediates in the manufacture of clinically important oral beta-lactam antibiotics; de-esterification of the intermediates is required for synthesis of the final product. A Bacillus subtilis PNB carboxy-esterase (PNBCE) catalyzes hydrolysis of several beta-lactam antibiotic PNB esters to the corresponding free acid and PNB alcohol. This communication (i) describes cloning the pnbA gene, which encodes PNBCE, (ii) provides the nucleotide sequence of the pnbA open reading frame (ORF) and (iii) describes a method for efficiently expressing the ORF in Escherichia coli. The amino acid (aa) sequence, deduced from the nucleotide sequence of the pnbA ORF, matched an experimentally determined N-terminal aa sequence of B. subtilis PNBCE and also matched an active site sequence previously identified by biochemical analyses. Specific activity of PNBCE in crude extracts was more than 90-fold greater in recombinant E. coli, as compared to B. subtilis. This increase in expression led to more than a 500-fold improvement in the efficiency of purification of PNBCE.

The human genome project: genetic and physical mapping.

The modern tools of molecular biology, recombinant DNA techniques, have given scientists the ability to isolate and study individual genes from even complex eukaryotic genomes. The availability of genes enables the study o f their structure and biologic function, and their role in normal and abnormal physiologic processes. A worldwide effort to study and understand the entire human genome is under way, which will result in information on the location of all genes, their sequences, and their complex regulation and interactions. As this knowledge becomes available, it will be rapidly applied to the practice of medicine through use in the development of diagnostic tests for genetic-based diseases and in the development of therapeutics.

Cloning and expression of a cDNA encoding a brain-specific Na(+)-dependent inorganic phosphate cotransporter.

We have isolated a brain-specific cDNA that encodes a Na(+)-dependent inorganic phosphate (Pi) cotransporter (BNPI). The nucleotide sequence of BNPI predicts a protein of 560 amino acids with 6-8 putative transmembrane-spanning segments that is approximately 32% identical to the rabbit kidney Na(+)-dependent Pi cotransporter. Expression of BNPI mRNA in Xenopus oocytes results in Na(+)-dependent Pi transport similar to that reported for the recombinantly expressed or native kidney Na(+)-dependent cotransporter. RNA blot analysis reveals that BNPI mRNA is expressed predominantly (if not exclusively) in brain, and in situ hybridization histochemistry reveals BNPI transcripts in neurons of the cerebral cortex, hippocampus, and cerebellum. Furthermore, we have confirmed the presence of saturable Na(+)-dependent Pi cotransport in cultured cerebellar granule cells. Together, these data demonstrate the presence of a specific neuronal Na(+)-dependent transport system for Pi in brain.

Improved expression of a hybrid Streptomyces clavuligerus cefE gene in Penicillium chrysogenum.

A hybrid cefE gene, encoding penicillin N expandase, was constructed by fusing the promoter sequences, Pcp, and terminator sequences, Pct from the Penicillium chrysogenum pcbC gene to the open reading frame (orf), cefEorf, from the Streptomyces clavuligerus cefE gene. The resulting hybrid gene, Pcp/cefE'orf/Pct, differed from a previously reported hybrid cefE gene contained on plasmid pPS65. The latter gene, Pcp/cefE'orf/Sct, contained the Pcp sequences fused to the S. clavuligerus cefE orf still attached to the S. clavuligerus terminator sequences, Sct. The new hybrid gene was transformed into P. chrysogenum on plasmid vector pRH6. Transformants were selected by phleomycin resistance conferred by a hybrid ble gene present on plasmid pRH6. The hybrid ble gene was formed by attaching Pcp sequences to the ble orf. Among transformants obtained with pRH6, one exhibited a 70-fold higher level of activity of penicillin N expandase than the best transformant previously obtained from a 10-fold larger population of pPS65 transformants. The penicillin N expandase activity in pRH6 transformant, 9EN-5-1, was fourfold higher than the activity in the S. clavuligerus strain used as the source of the cefE orf and 75% of the activity observed in an industrial strain of Cephalosporium acremonium. Sequencing of the junctions of the heterologous DNA in Pcp/cefEorf/Pct uncovered a modification of the cefE open reading frame introduced during construction of the hybrid gene; the modified open reading frame is designated cefE'orf.

Association of intestinal peptide transport with a protein related to the cadherin superfamily.

The first step in oral absorption of many medically important peptide-based drugs is mediated by an intestinal proton-dependent peptide transporter. This transporter facilitates the oral absorption of beta-lactam antibiotics and angiotensin-converting enzyme inhibitors from the intestine into enterocytes lining the luminal wall. A monoclonal antibody that blocked uptake of cephalexin was used to identify and clone a gene that encodes an approximately 92-kilodalton membrane protein that was associated with the acquisition of peptide transport activity by transport-deficient cells. The amino acid sequence deduced from the complementary DNA sequence of the cloned gene indicated that this transport-associated protein shares several conserved structural elements with the cadherin superfamily of calcium-dependent, cell-cell adhesion proteins.

The human genome project Genetic and physical mapping.

The modern tools of molecular biology, recombinant DNA techniques, have given scientists the ability to isolate and study individual genes from even complex eukaryotic genomes. The availability of genes enables the study of their structure and biologic function, and their role in normal and abnormal physiologic processes. A worldwide effort to study and understand the entire human genome is under way, which will result in information on the location of all genes, their sequences, and their complex regulation and interactions. As this knowledge becomes available, it will be rapidly applied to the practice of medicine through use in the development of diagnostic tests for genetic-based diseases and in the development of therapeutics.

Structure and functional expression of a complementary DNA for porcine growth hormone-releasing hormone receptor.

Growth hormone-releasing hormone (GHRH) belongs to the family of gut-neuropeptide hormones which also includes glucagon, secretin and vasoactive intestinal peptide (VIP). All receptors for this peptide hormone family seem to involve similar signal transduction pathways. Upon hormone binding, these receptors interact with guanine nucleotide binding protein 'Gs' and cause the stimulation of adenylate cyclase. The secretin and VIP receptor cDNAs have recently been cloned and found to be homologous to those of calcitonin and parathyroid hormone receptors. Based on cDNA sequences of these receptors, we designed several oligonucleotide primers which were used to amplify two novel porcine pituitary cDNA fragments by the polymerase chain reaction. One novel receptor cDNA fragment was used to screen a porcine pituitary cDNA library and a full-length cDNA encoding a putative porcine GHRH receptor of 451 amino acids was isolated. This putative receptor mRNA is present specifically in porcine anterior pituitary cells and not in eight other porcine tissues as shown by Northern hybridization analysis. The receptor cDNA was subsequently cloned into a mammalian cell expression vector containing the cytomegalovirus promoter. A human kidney tumor cell line (293) stably transfected with this vector was found to express the receptor efficiently and to bind [125I]-GHRH specifically. Furthermore, challenge of the 293 cells expressing the receptor by GHRH leads to efficient stimulation of cytoplasmic cAMP production.

Sequence similarity between macrolide-resistance determinants and ATP-binding transport proteins.

The three macrolide-resistance-encoding genes, tlrC from Streptomyces fradiae, srmB from Streptomyces ambofaciens, and carA from Streptomyces thermotolerans, encode proteins that possess significant sequence similarity to ATP-dependent transport proteins. The N-terminal and C-terminal halves of these proteins are very similar to each other and contain highly conserved regions that resemble ATP-binding domains typically present within the superfamily of ATP-dependent transport proteins. These observations suggest that the mechanism by which these genes confer resistance to macrolides is due to export of the antibiotics, a process that is driven by energy derived from ATP hydrolysis.

Homology between proteins controlling Streptomyces fradiae tylosin resistance and ATP-binding transport.

A tylosin(Ty)-producing strain of Streptomyces fradiae contains at least three genes, tlrA, tlrB, tlrC, specifying resistance to Ty (TyR). The complete nucleotide sequence of the TyR-encoding gene, tlrC, and the transcription start point of the gene were determined. The sequence contains an open reading frame coding for a protein of 548 amino acids (aa) with an Mr of 59129. The TlrC protein was identified by expression of the cloned gene by in vitro coupled transcription and translation in cell-free extracts derived from Streptomyces lividans. The N- and C-terminal halves of TlrC share extensive homology, suggesting that the protein evolved through tandem gene duplication. Each half of the deduced TlrC aa sequence also shows significant homology to numerous eukaryotic and prokaryotic membrane-associated, active-transport protein subunits. The homologous proteins include examples from the systems responsible for efflux of cytotoxic drugs from multidrug-resistant human cells and for export of hemolysin from Escherichia coli. The greatest similarity to TlrC is in regions containing the ATP-binding sites found in these proteins. These results suggest a role for the tlrC gene product as part of a multiple component, ATP-dependent transport system for the active excretion of Ty from the producing organism.