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Here, we characterize an uncommon set of telomeres from Streptomyces rimosus ATCC 10970, the parental strain of a lineage of one of the earliest-discovered antibiotic producers. Following the closure of its genome sequence, we compared unusual telomeres from this organism with the other five classes of replicon ends found amongst streptomycetes. Closed replicons of streptomycete chromosomes were organized with respect to their phylogeny and physical orientation, which demonstrated that different telomeres were not associated with particular clades and are likely shared amongst different strains by plasmid-driven horizontal gene transfer. Furthermore, we identified a ~50 kb origin island with conserved synteny that is located at the core of all streptomycete chromosomes and forms an axis around which symmetrical chromosome inversions can take place. Despite this chromosomal bilateral symmetry, a bias in parS sites to the right of oriC is maintained across the family Streptomycetaceae and suggests that the formation of ParB/parS nucleoprotein complexes on the right replichore is a conserved feature in streptomycetes. Consequently, our studies reveal novel features of linear bacterial replicons that, through their manipulation, may lead to improvements in growth and productivity of this important industrial group of bacteria.

Streptomyces clavuligerus is an industrially important actinomycete whose genetic manipulation is limited by low transformation and conjugation efficiencies, low levels of recombination of introduced DNA, and difficulty in obtaining consistent sporulation. We describe the construction and application of versatile vectors for Cas9-mediated genome editing of this strain. To design spacer sequences with confidence, we derived a highly accurate genome assembly for an isolate of the type strain (ATCC 27064). This yielded a chromosome assembly (6.75 Mb) plus assemblies for pSCL4 (1795 kb) and pSCL2 (149 kb). The strain also carries pSCL1 (12 kb), but its small size resulted in only partial sequence coverage. The previously described pSCL3 (444 kb) is not present in this isolate. Using our Cas9 vectors, we cured pSCL4 with high efficiency by targeting the plasmid’s parB gene. Five of the resulting pSCL4-cured isolates were characterized and all showed impaired sporulation. Shotgun genome sequencing of each of these derivatives revealed large deletions at the ends of the chromosomes in all of them, and for two clones sufficient sequence data was obtained to show that the chromosome had circularized. Taken together, these data indicate that pSCL4 is essential for the structural stability of the linear chromosome.

Endophytic actinobacteria offer great potential as a source of novel bioactive compounds. In order to investigate the potential for the production of secondary metabolites by endophytes, we recovered a filamentous microorgansism from the tree Antidesma neurocarpum Miq. After phenotypic analysis and whole genome sequencing we demonstrated that this organism, SUK42 was a member of the actinobacterial genus Kitasatospora. This strain has a small genome in comparison with other type strains of this genus and has lost metabolic pathways associated with Stress Response, Nitrogen Metabolism and Secondary Metabolism. Despite this SUK42 can grow well in a laboratory environment and encodes a core genome that is consistent with other members of the genus. Finally, in contrast to other members of Kitasatospora, SUK42 encodes saccharide secondary metabolite biosynthetic gene clusters, one of which with similarity to the acarviostatin cluster, the product of which displays α-amylase inhibitory activity. As extracts of the host plant demonstrate this inhibitory activity, it suggests that the potential medicinal properties of A. neurocarpum Miq might be provided by the endophytic partner and illustrate the potential for exploitation of endophytes for clinical or industrial uses.

A polyphasic approach was used for evaluating the taxonomic status of strain HST21T isolated from Salar de Huasco in the Atacama Desert. The results of 16S rRNA gene and multilocus sequence phylogenetic analyses assigned strain HST21T to the genus Streptomyceswith Streptomyces albidochromogenes DSM 41800Tand Streptomyces flavidovirens DSM 40150T as its nearest neighbours. Digital DNA–DNA hydridization (dDDH) and average nucleotide identity (ANI) values between the genome sequences of strain HST21T and S. albidochromogenes DSM 41800T (35.6 and 88.2 %) and S. flavidovirens DSM 40105T (47.2 and 88.8 %) were below the thresholds of 70 and 95–96 % for prokaryotic conspecific assignation. Phenotypic, chemotaxonomic and genetic results distinguished strain HST21T from its closest neighbours. Strain HST21T is characterized by the presence of ll-diaminopimelic acid in its peptidoglycan layer; glucose and ribose as whole cell sugars; diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylethanolamine, phosphatidylinositol, glycophospholipids, unknown lipids and phospholipids as polar lipids; and anteiso-C15 : 0 (21.6 %) and anteiso-C17 : 0 (20.5 %) as major fatty acids (>15 %). Based on these results, strain HST21T merits recognition as a novel species, for which the name Streptomyces altiplanensis sp. nov. is proposed. The type strain is HST21T =DSM 107267T=CECT 9647T. While analysing the phylogenies of strain HST21T, Streptomyces chryseus DSM 40420T and Streptomyces helvaticus DSM 40431T were found to have 100 % 16S rRNA gene sequence similarity with digital DNA–DNA hydridization (dDDH) and average nucleotide identity (ANI) values of 95.3 and 99.4 %, respectively. Therefore, S. helvaticus is considered as a later heterotypic synonym of S. chryseus and, consequently, we emend the description of S. chryseus .

To combat the problem of antimicrobial resistance, we are testing the hypothesis that thermophilic Actinobacteria produce novel antimicrobials at higher temperatures, with potential activity against life-threatening infections like invasive aspergillosis caused by the fungus Aspergillus fumigatus. Samples from “windrows” at a green waste processing facility yielded 36 potential thermophilic Actinobacterial strains isolated at 50oC, as well as strains of A. fumigatus. The phylogeny and identities of the bacterial strains were determined by 16S rDNA sequencing. Three strains - DJT 15 Streptomyces thermoviolaceus subsp. apingens, DJT 32 Saccharomonospora viridis and DJT 36 Saccharomonospora glauca - have shown inhibitory activity in bioassays against the ESKAPE pathogens, two of which (DJT 32 and 36) also inhibited the growth of the fungal pathogen Aspergillus fumigatus isolated from the same compost. Strain DJT 32 has also been shown to have an inhibitory effect against azole resistant human pathogenic strains of A. fumigatus. Whole genome Sequencing data of DJT 15 and 32 have been used to identify possible biosynthetic gene clusters for antimicrobial compounds (novel or otherwise) through AntiSmash analysis. Alongside this, bioactive compounds have been extracted from broth cultures of each strain using HP-20 Resin method, and the metabolites will be identified using LC:MS combined with metabolic profiling. Extraction and identification of novel metabolites will provide a path for the development of new antimicrobials for clinical use. This study has shown that thermophilic Actinobacteria produce antimicrobial compounds at higher temperatures, against Staphylococcus aureus and against the highly pathogenic fungus, A. fumigatus.

The genus Streptomyces has proven to be a rich reservoir of specialized metabolites, accounting for 80% of all microbially produced antibiotics including chloramphenicol and nystatin from S. venezuelae and S. noursei respectively. However, the discovery of novel microbial chemistry is still greatly needed to combat antimicrobial resistance. Comparative metabolomics, using platforms such as Global Natural Products Social Molecular Networking (GNPS), as well as tools such as antiSMASH and BiGSCAPE have aided the mining of biosynthetic gene clusters (BGC’s) across datasets but comparing the chemistry to the encoding biosynthetic gene clusters is a significant bottleneck. In this study, ten Streptomyces strains were selected, based on phylogeny and availability of genome sequence. The strains were cultured on 6 types of Actinomycete-specific media to maximise metabolite diversity. Liquid Chromatography tandem Mass Spectrometry (LC-MS/MS) was used to obtain spectral data from crude metabolite extracts enabling comparative metabolomics analysis via the GNPS platform. As the genome sequences were publicly available, genome mining of BGC’s was achieved using antiSMASH resulting in 260 BGC’s across the ten strains. This revealed 53 gene cluster families when analysed using BiGSCAPE, the largest encoding for 8 metabolites. In future, both biosynthetic (BGC’s) and chemistry (parent ions) datasets will be computationally linked based on strain presence/absence. The development of standardised datasets that enable cross-‘omics comparison will aid prioritisation of novel antibiotics, especially when combined with bioactivity data.

The opportunistic pathogen Pseudomonas aeruginosa is a major cause of nosocomial infections, and has been categorised by the World Health Organisation as a “Priority 1: Critical” target for research and development of novel antibiotics owing to its intrinsic multi-resistance and ability to acquire novel resistance mechanisms. One strategy for discovering novel antibiotics is the identification and characterisation of metabolites with antimicrobial activity. Members of the bacterial phylum Actinobacteria are historic source of these metabolites, in particular the genus Streptomyces. However, other genera have not received this same level of interest despite sharing the capacity to biosynthesise a diverse array of metabolites. One such genus is Micromonospora, responsible for production of the broad-spectrum aminoglycoside antibiotic gentamicin (M. purpurea). Here we present three Micromonospora species isolated from the Atacama Desert, Chile; that possess anti-pseudomonad bioactivity inducible by culture on International Streptomyces Project (ISP) Media. In addition, preliminary data indicates that this activity can be affected by the addition of P. aeruginosa conditioned media. In parallel, short-read Illumina sequencing was used to assemble draft genomes for these strains, enabling antiSMASH analysis of putative biosynthetic gene clusters. In addition to this, estimated Average Nucleotide Identity (ANI) as calculated by the autoMLST server indicates that these strains may all be novel Micromonospora species. The results of this work serve to highlight the biosynthetic capacity of an understudied genus of bacteria, as well as the value of examining underexplored environments and habitats in the search for novel bioactive molecules.

Background. Streptomycete bacteria are prolific producers of specialized metabolites, many of which have clinically relevant bioactivity. A striking feature of their genomes is the expansion of gene families that encode the same enzymatic function. Genes that undergo expansion events, either by horizontal gene transfer or duplication, can have a range of fates: genes can be lost, or they can undergo neo-functionalization or sub-functionalization. To test whether expanded gene families in Streptomyces exhibit differential expression, an RNA-Seq approach was used to examine cultures of wild-type Streptomyces coelicolor grown with either glucose or tween as the sole carbon source. Results. RNA-Seq analysis showed that two-thirds of genes within expanded gene families show transcriptional differences when strains were grown on tween compared to glucose. In addition, expression of specialized metabolite gene clusters (actinorhodin, isorenieratane, coelichelin and a cryptic NRPS) was also influenced by carbon source. Conclusions. Expression of genes encoding the same enzymatic function had transcriptional differences when grown on different carbon sources. This transcriptional divergence enables partitioning to function under different physiological conditions. These approaches can inform metabolic engineering of industrial Streptomyces strains and may help develop cultivation conditions to activate the so-called silent biosynthetic gene clusters.

Actinobacteria have provided a rich source of novel antimicrobial compounds throughout the antibiotic era. Thermophilic Actinobacteria growing at higher temperatures (>50 °C), have not been extensively studied, despite producing important antibiotics such as thermomycin and anthramycin. We are testing the hypothesis that thermophilic Actinobacteria produce new and unusual antimicrobials at higher temperatures, potentially leading to the discovery of novel heat stable compounds; especially those active against life-threatening fungal infections in humans such as invasive aspergillosis caused by Aspergillus fumigatus, which has developed resistance to current treatments. Compost is a rich source of thermophilic Actinobacteria responsible for generating the heat required for decomposition and yet this niche has been overlooked in terms of natural product discovery. A. fumigatusis a fungus that also lives in compost and also contributes to the composting process and we reasoned that Actinobacteria living in this environment might display activity against pathogenic strains of the fungus. Samples from a series of ‘windrows’ at a commercial green waste processing facility yielded 13 thermophilic Actinobacteria, and strains of Aspergillus fumigatus. The phylogeny and species identity of the bacterial strains were determined by 16S rRNA sequencing. Candidate strains were screened for the ability to inhibit ESKAPE pathogens as well as A. fumigatus, using agar overlays and MIC assays. Selected strains were analysed by whole genome DNA sequencing and likely antimicrobials predicted. Compound identification using mass spectrometry and metabolic profiling has been undertaken on strains that display antibiotic activity, providing a path for the development of new antimicrobials for clinical use.

With antimicrobial resistance becoming an increasingly severe issue in both the developed and developing regions of the world, new strategies need to be employed to identify and characterise novel antimicrobial activity. Pseudomonas aeruginosa is a Gram negative pathogen and a major cause of opportunistic infections in burn victims and cystic fibrosis patients. Due to a wide repertoire of antibiotic resistance mechanisms, these infections are difficult to cure and thus there is a need to develop novel antimicrobial treatments. Members of the Actinobacterial genus Micromonospora produce a broad range of bioactive secondary metabolites, a number of which possess antimicrobial properties. The goal of this research is to examine the antimicrobial properties of an actinomycete strain – identified by 16S sequencing as Micromonospora –originally isolated from the Atacama desert, Chile, with a focus on identifying and characterising anti-pseudomonad activity. Bioactivity was screened for against Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumanii and Enterococcus faecium. AntiSMASH analysis of assembled Illumina sequencing data was used to identify putative biosynthetic gene clusters. Antimicrobial bioactivity was observed, and potential antimicrobial biosynthetic gene clusters were identified through genome mining. This work has identified antimicrobial activity from a region of underexplored ecology, and highlights the importance of sampling and examining areas which have previously been considered too hostile to support life.

Streptomyces clavuligerus is the producer of clavulanic acid; aβ-lactamaseinhibitor that is used in combination with the antibiotic amoxicillin. Genome sequencing has established that S. clavuligerus contains a linear chromosome and four linear plasmids: pSCL1, pSCL2, pSCL3 and pSCL4. Although pSCL4 carries 20 % of the S. clavuligerus coding sequences, none are thought to be essential with the exception of tap and tpgthat encode terminal proteins necessary for priming of the lagging strand at the telomeres. In order to confirm plasmid essentiality and the genomic architecture of S. clavuligerus, we first corroborated the available genome sequences by physically mapping the genome using Pulsed-field Gel Electrophoresis, this confirmed the presence of the replicons pSCL2, pSCL3 and pSCL4 with sizes of 150, 450 and 1800 kilobases respectively. In addition, bioinformatics and physical analyses of the S. clavuligerus genome allowed the identification of similar non-archetypal telomeres in the chromosome and pSCL4 at one end of each replicon. Despite this, the other telomere remains unidentified, which suggests there is a dynamic chromosome-plasmid relationship in S. clavuligerus. Furthermore, in order to study the essentiality of pSCL4 we first introduced a copy of tap-tpg onto the chromosome prior to plasmid curing and/or tap-tpg deletion. Consequently, targeted genome sequence and further physical analyses, especially of the telomeres, will permit us understand the complex dynamic relationship between the megaplasmid and the chromosome in this important industrial microorganism.

Cotton swab is the conventional swabbing tool that is usually applied for collecting pathogens from contaminated surfaces, followed by cells lysis and DNA extraction before subjecting to genetic analysis. However, such an approach is time consuming as it involves several steps and requires highly trained personnel to perform the experiment. In this study, we developed a new cotton swab-based detection system that involved integrating bacterial collection, preconcentration and detection on Q-tips. The platform is based on a sandwich assay that can detect different pathogens visually by color changes. Lactoferrin-immobilized cotton is used as a general capturing tool to collect various pathogens from surfaces. The presence of particular bacteria is then detected by immersing the cotton in antibodies attached to different colored nanobeads. The target cell is captured between the lactoferrin and specific antibody-conjugated beads which results in certain color development. The effectiveness of this simply fabricated sensor was demonstrated using Salmonella typhimurium, Salmonella enteritidis, Staphylococcus aureus and Campylobacter jejuni. The intensity of the color on the cotton surfaces increased with increasing the concentration of the pathogenic bacteria. The detection limit was as low as 10 cfu/ml for Salmonella typhimurium and Campylobacter jejuni, 100 cfu/ml for Salmonella enteritidis and 100 cfu/ml for Staphylococcus aureus on chicken meat surface. Moreover, this method showed high selectivity and was further confirmed by loop-mediated isothermal amplification (LAMP). The simplicity and the low cost of this colorimetric sensor renders it applicable to a wide range of other pathogens on different surfaces.

Antimicrobial resistance and the shortage of novel antibiotics have led to an urgent need for new antibacterial drug leads. Several existing natural product scaffolds (including chelocardins) have not been developed because their suboptimal pharmacological properties could not be addressed at the time. It is demonstrated here that reviving such compounds through the application of biosynthetic engineering can deliver novel drug candidates. Through a rational approach, the carboxamido moiety of tetracyclines (an important structural feature for their bioactivity) was introduced into the chelocardins, which are atypical tetracyclines with an unknown mode of action. A broad-spectrum antibiotic lead was generated with significantly improved activity, including against all Gram-negative pathogens of the ESKAPE panel. Since the lead structure is also amenable to further chemical modification, it is a platform for further development through medicinal chemistry and genetic engineering.

The draft genome for the type strain Streptomyces phaeoluteigriseus DSM41896 (ISP 5182) is reported. It was classified as a member of the Streptomyces violaceusniger clade; however, a polyphasic study showed it was a separate species based on its distinct spore morphology and 16S rRNA sequence. The genome sequence confirms it as a separate species.

The genome sequences of eight Streptomyces phages are presented, four of which were isolated for this study. Phages R4, TG1, ϕHau3, and SV1 were isolated previously and have been exploited as tools for understanding and genetically manipulating Streptomyces spp. We also extracted five apparently intact prophages from recent Streptomyces spp. genome projects and, together with six phage genomes in the database, we analyzed all 19 Streptomyces phage genomes with a view to understanding their relationships to each other and to other actinophages, particularly the mycobacteriophages. Fifteen of the Streptomyces phages group into four clusters of related genomes. Although the R4-like phages do not share nucleotide sequence similarity with other phages, they clearly have common ancestry with cluster A mycobacteriophages, sharing many protein homologues, common gene syntenies, and similar repressor-stoperator regulatory systems. The R4-like phage ϕHau3 and the prophage StrepC.1 (from Streptomyces sp. strain C) appear to have hijacked a unique adaptation of the streptomycetes, i.e., use of the rare UUA codon, to control translation of the essential phage protein, the terminase. The Streptomyces venezuelae generalized transducing phage SV1 was used to predict the presence of other generalized transducing phages for different Streptomyces species.

The linear chromosome of Streptomyces coelicolor contains two paralogous ssb genes, ssbA and ssbB. Following mutational analysis, we concluded that ssbA is essential, whereas ssbB plays a key role in chromosome segregation during sporulation. In the ssbB mutant, ∼30% of spores lacked DNA. The two ssb genes were expressed differently; in minimal medium, gene expression was prolonged for both genes and significantly upregulated for ssbB. The ssbA gene is transcribed as part of a polycistronic mRNA from two initiation sites, 163 bp and 75 bp upstream of the rpsF translational start codon. The ssbB gene is transcribed as a monocistronic mRNA, from an unusual promoter region, 73 bp upstream of the AUG codon. Distinctive DNA-binding affinities of single-stranded DNA-binding proteins monitored by tryptophan fluorescent quenching and electrophoretic mobility shift were observed. The crystal structure of SsbB at 1.7 Å resolution revealed a common OB-fold, lack of the clamp-like structure conserved in SsbA and previously unpublished S-S bridges between the A/B and C/D subunits. This is the first report of the determination of paralogous single-stranded DNA-binding protein structures from the same organism. Phylogenetic analysis revealed frequent duplication of ssb genes in Actinobacteria, whereas their strong retention suggests that they are involved in important cellular functions.

The fluid mosaic model has recently been amended to account for the existence of membrane domains enriched in certain phospholipids. In rod-shaped bacteria, the anionic phospholipid cardiolipin is enriched at the cell poles but its role in the morphogenesis of the filamentous bacterium Streptomyces coelicolor is unknown. It was impossible to delete clsA (cardiolipin synthase; SCO1389) unless complemented by a second copy of clsA elsewhere in the chromosome. When placed under the control of an inducible promoter, clsA expression, phospholipid profile and morphogenesis became inducer dependent. TLC analysis of phospholipid showed altered profiles upon depletion of clsA expression. Analysis of cardiolipin by mass spectrometry showed two distinct cardiolipin envelopes that reflected differences in acyl chain length; the level of the larger cardiolipin envelope was reduced in concert with clsA expression. ClsA-EGFP did not localize to specific locations, but cardiolipin itself showed enrichment at hyphal tips, branch points and anucleate regions. Quantitative analysis of hyphal dimensions showed that the mycelial architecture and the erection of aerial hyphae were affected by the expression of clsA. Overexpression of clsA resulted in weakened hyphal tips, misshaped aerial hyphae and anucleate spores and demonstrates that cardiolipin synthesis is a requirement for morphogenesis in Streptomyces.

Endophytic bacteria, such as Streptomyces, have the potential to act as a source for novel bioactive molecules with medicinal properties. The present study was aimed at assessing the antimalarial activity of crude extract isolated from various strains of actinobacteria living endophytically in some Malaysian medicinal plants. Using the four day suppression test method on male ICR strain mice, compounds produced from three strains of Streptomyces (SUK8, SUK10, and SUK27) were tested in vivo against Plasmodium berghei PZZ1/100 in an antimalarial screen using crude extracts at four different concentrations. One of these extracts, isolated from Streptomyces SUK10 obtained from the bark of Shorea ovalis tree, showed inhibition of the test organism and was further tested against P. berghei-infected mice for antimalarial activity at different concentrations. There was a positive relationship between the survival of the infected mouse group treated with 50 µg/kg body weight (bw) of ethyl acetate-SUK10 crude extract and the ability to inhibit the parasites growth. The parasite inhibition percentage for this group showed that 50% of the mice survived for more than 90 days after infection with the parasite. The nucleotide sequence and phylogenetic tree suggested that Streptomyces SUK10 may constitute a new species within the Streptomyces genus. As part of the drug discovery process, these promising finding may contribute to the medicinal and pharmaceutical field for malarial treatment.

The model organism Streptomyces coelicolor represents a genus that produces a vast range of bioactive secondary metabolites. We describe a versatile procedure for systematic and comprehensive mutagenesis of the S. coelicolor genome. The high-throughput process relies on in vitro transposon mutagenesis of an ordered cosmid library; mutagenized cosmids with fully characterized insertions are then transferred by intergeneric conjugation into Streptomyces, where gene replacement is selected. The procedure can yield insertions in upward of 90% of genes, and its application to the entire genome is underway. The methodology could be applied to many other organisms that can receive DNA via RK2/RP4-mediated intergeneric conjugation. The system permits introduction of mutations into different genetic backgrounds and qualitative measurement of the expression of disrupted genes as demonstrated in the analysis of a hybrid histidine kinase and response regulator gene pair, osaAB, involved in osmoadaptation in Streptomyces. The independently transcribed response regulator gene, osaB, is essential for osmoadaptation; when grown with supplementary osmolyte, an osaB mutant cannot erect aerial hyphae and produces up to fivefold greater antibiotic yields than the wild-type strain.

The insertion sequence IS6100, belonging to the IS6 family, normally forms a cointegrate as an end product of transposition. The IS6100-based minitransposon, Tn1792, has been developed as a genetic tool to mutagenise antibiotic-producing Streptomyces. Here, we describe resolution of Tn1792 cointegrates in Streptomyces avermitilis that can facilitate both the initial isolation of Tn1792 insertion mutants and also the subsequent rescue of Tn1792-tagged sequences. This is the first reported example of cointegrate resolution for an IS6-type transposable element. As a result of mutagenesis, several putative genes involved in morphological development and antibiotic production have been isolated.

Degeneration of cholinergic neurons in the basal forebrain is a neural marker of Alzheimer's disease and is associated with perceptual and cognitive deficits. An idea that has attracted scientific scutiny is that aging makes the brain more susceptible to neurodegenerative diseases such as Alzheimer's. The purpose of this study was to compare the effects of the loss of cholinergic input from nucleus basalis of Meynert on evoked activity in the posteromedial barrel subfield of the somatosensory cortex in young (2-2.5 months) and aged (28-30 months) male Fisher hybrid rats. The mean firing rate and receptive fields of single neurons in the posteromedial barrel subfield of the somatosensory cortex were examined after selective lesions of cholinergic neurons in the nucleus basalis of Meynert with an immunotoxin. IgG 192-saporin. Functional properties of single neurons in young animals were affected much more significantly by cholinergic depletion than those in aged animals. In cholinergic-depleted young animals, the mean firing rate of evoked activity and receptive field of posteromedial barrel subfield neurons were significantly decreased. Cholinergic depletion caused a 14% decrease in evoked activity and a 33% increase in receptive field size in young animals. The mean firing rate and receptive field of single neurons were not affected by cholinergic depletion in aged animals. It is concluded that functional properties of cortical sensory neurons in young animals are more vulnerable to cholinergic depletion than are those of aged animals and that cholinergic depletion does not further impact the properties of neurons exposed to the processes of aging.

The aim of this study was to assess the distribution of neurons immunoreactive for parvalbumin (PV), calbindin (CaBP), glutamic acid decarboxylase (GAD), and γ-aminobutyric acid (GABA) in the somatosensory thalamus of the raccoon and to compare these features to those of other species, especially primates. Immunohistochemistry was used to study the location of these neurons in the ventroposterior nucleus (VP), ventroposterior inferior nucleus (VPI), posterior group of nuclei (Po), and reticular nucleus (Rt). A consistent differential pattern of PV-positive (PV+) and CaBP-positive (CaBP+) cells was found in the somatosensory thalamus. Many PV+ neurons were observed in VP and Rt, but very few were found in VPI or Po. In contrast, CaBP+ neurons were distributed throughout VP, VPI, and Po but were very sparse or absent in Rt. In the VP nucleus, PV+ cells were distributed in clusters separated by interclusteral regions with a sparse distribution of PV+ cell bodies. The distributions of PV+ and CaBP+ cells tended to be complementary to each other in VP; regions with a high density of PV+ neurons had a low density of CaBP+ cell bodies. Double-labeling experiments revealed very few neurons in which PV and CaBP immunoreactivities were colocalized. Cells immunoreactive for GAD or GABA were found in PV-dense clusters of VP; fewer GABAergic neurons were present in the CaBP-dense interclusteral regions of VP and in VPI and Po. GAD+ and GABA+ neurons were most prominently distributed in Rt. We conclude that the distributions of PV+ and CaBP+ cell bodies in the raccoon somatosensory thalamus are very similar to those in primates. The density of GABAergic neurons in the somatosensory thalamus of the raccoon is less than that in the cat and monkey, but the relative distribution of GABAergic neurons in the different somatosensory nuclei is very similar to that in the cat and monkey. These results are discussed in relation to findings in other species and are related to the functions of lemniscal and nonlemniscal somatosensory pathways. J. Comp. Neurol. 388:120–129, 1997. © 1997 Wiley-Liss, Inc.

Clinical and functional studies have strongly suggested that acetylcholine input from the nucleus basalis of Meynert is important for the cortex's adaptive response to experience. The purpose of this study was to investigate the effects of depletion of acetylcholine inputs from nucleus basalis of Meynert on experience-dependent plasticity in the cortex of young adult male rats. The posteromedial barrel subfield in the primary somatosensory cortex was studied. Experience-dependent plasticity was elicited using a whisker-pairing paradigm in which all whiskers except D2 and D3 were trimmed daily. Plasticity within barrel D2 of the posteromedial barrel subfield was measured using the electrophysiological extracellular recording technique. An index of plasticity was determined in two ways: as an increase in the magnitude of evoked activity to stimulation of whisker D2 and as a bias in the ratio of evoked activity for stimulation of paired whisker D3 and cut whisker D1 (D3/D1). Whiskers D2, D3 and D1 were stimulated (deflected) by a Chubbuck electromechanical stimulator. Cholinergic neurons in the nucleus basalis of Meynert were selectively lesioned with an immunotoxin, 192 IgG-saporin, injected into the left lateral ventricle. Lesions of cholinergic neurons in the nucleus basalis of Meynert were verified using choline acetyltransferase immunocytochemistry and radioenzymatic assay. Experience-dependent plasticity was significantly reduced in cholinergic-depleted animals. The magnitude of evoked activity to stimulation of whisker D2 increased by 16-100% in control animals compared with 0-20% in cholinergic-depleted animals. Similarly, compared to a 60-100% increase in the D3/D1 ratio of evoked activity for phosphate-buffered saline-injected control animals, cholinergic-depleted rats showed no significant increase in the D3/D1 ratio (0-15%) after undergoing the whisker-pairing paradigm. After whisker trimming, the D3/D1 response ratio in immunotoxin-treated animals was essentially the same as in control animals that had not been subjected to the whisker-pairing paradigm. This study showed that no significant plasticity response was observed in the absence of cholinergic input from the nucleus basalis of Meynert. The mechanisms of the action of acetylcholine in cortical plasticity are still not known, but we hypothesize that this type of plasticity is activity dependent and is significantly enhanced in the presence of acetylcholine.

The purpose of these experiments was to determine the effects of cholinergic depletion on the morphology and staining density of barrels formed by glutamic acid decarboxylase-positive neuropil in the posteromedial barrel subfield of the somatosensory cortex. The density and distribution of glutamic acid decarboxylase immunoreactive neuropil were examined after highly selective lesions of cholinergic neurons in the nucleus basalis of Meynert with an immunotoxin, IgG 192-saporin. Glutamic acid decarboxylase immunoreactivity was also examined in animals subjected to a whisker-pairing experience and lesion of acetylcholine inputs from the nucleus basalis of Meynert. Seven to 9 weeks after intraventricular injection of the immunotoxin, animals were perfused with a zinc aldehyde fixative and glutamic acid decarboxylase immunoreactivity was examined in 30-micron tangential sections. Cholinergic depletion caused reduced glutamic acid decarboxylase immunoreactivity in selective regions of the posteromedial barrel subfield. The density of neuropil and cell bodies immunoreactive for glutamic acid decarboxylase was significantly reduced in septa and perimeters of barrel walls. The length, width, and area of barrels were reduced 10-20% in cholinergic-depleted animals compared with controls. The density of glutamic acid decarboxylase immunoreactivity in the hollow of barrels was not affected by this treatment. Whisker pairing did not significantly change the density of glutamic acid decarboxylase immunoreactivity in barrels. These observations are discussed in regard to how long-term cholinergic depletion affects the function of different fiber systems in the posteromedial barrel subfield cortex and how some sensory functions may be comprised.

Tetracyclines (TCs) are medically important antibiotics from the polyketide family of natural products. Chelocardin (CHD), produced by Amycolatopsis sulphurea, is a broad-spectrum tetracyclic antibiotic with potent bacteriolytic activity against a number of Gram-positive and Gram-negative multi-resistant pathogens. CHD has an unknown mode of action that is different from TCs. It has some structural features that define it as 'atypical' and, notably, is active against tetracycline-resistant pathogens. Identification and characterization of the chelocardin biosynthetic gene cluster from A. sulphurea revealed 18 putative open reading frames including a type II polyketide synthase. Compared to typical TCs, the chd cluster contains a number of features that relate to its classification as 'atypical': an additional gene for a putative two-component cyclase/aromatase that may be responsible for the different aromatization pattern, a gene for a putative aminotransferase for C-4 with the opposite stereochemistry to TCs and a gene for a putative C-9 methylase that is a unique feature of this biosynthetic cluster within the TCs. Collectively, these enzymes deliver a molecule with different aromatization of ring C that results in an unusual planar structure of the TC backbone. This is a likely contributor to its different mode of action. In addition CHD biosynthesis is primed with acetate, unlike the TCs, which are primed with malonamate, and offers a biosynthetic engineering platform that represents a unique opportunity for efficient generation of novel tetracyclic backbones using combinatorial biosynthesis., This research was supported in part by grants from the Slovenian Research Agency (7-576-1/2004), the Slovene Human Resources and Scholarship Fund (Ad futura Programs 2006/523-17), the Socrates/Erasmus Program (2006/07420), the British Council (Partnership in Science Award RNP 7/2008), Slovenian Technology Agency TIA (VALOR08-02) and SPIRIT Slovenija (P-MR-09/104).

Next generation sequencing (NGS) has been widely used to study genomic variation in a variety of prokaryotes. Single nucleotide polymorphisms (SNPs) resulting from genomic comparisons need to be annotated for their functional impact on the coding sequences. We have developed a program, TRAMS, for functional annotation of genomic SNPs which is available to download as a single file executable for WINDOWS users with limited computational experience and as a Python script for Mac OS and Linux users. TRAMS needs a tab delimited text file containing SNP locations, reference nucleotide and SNPs in variant strains along with a reference genome sequence in GenBank or EMBL format. SNPs are annotated as synonymous, nonsynonymous or nonsense. Nonsynonymous SNPs in start and stop codons are separated as non-start and non-stop SNPs, respectively. SNPs in multiple overlapping features are annotated separately for each feature and multiple nucleotide polymorphisms within a codon are combined before annotation. We have also developed a workflow for Galaxy, a highly used tool for analysing NGS data, to map short reads to a reference genome and extract and annotate the SNPs. TRAMS is a simple program for rapid and accurate annotation of SNPs that will be very useful for microbiologists in analysing genomic diversity in microbial populations.

This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license.-- et al., We report the draft genome of Streptomyces rimosus (ATCC 10970), a soil isolate that produces oxytetracycline, a commercially important and clinically useful antibiotic., We acknowledge financial support from the National Yang-Ming University, Taiwan, for the sequencing project (R.K.), a British Council for Research Projects Network grant (P.A.H. and H.P.), and the British Council/CUC for a Scotland-China Higher Education Research Partnership for PhD Studies (Z.T.).

The ventral forebrain, including the ventral striatum, the ventral pallidum and the substantia innominata, is an important region involved in the functions of the basal ganglia and the limbic system, as well as the magnocellular corticopetal neurons of the nucleus basalis of Meynert. Although previous studies have shown that this region is richly innervated by GABAergic fibers, little is known with respect to the relative densities of GABAergic to non-GABAergic axon terminals in this region. To address this issue, we have developed a specific rabbit antiserum to GABA and used a postembedding immunocytochemical reaction to analyse the distribution of GABA-like immunoreactive axon terminals in the rat ventral striatum, ventral pallidum and substantia innominata. Of all axon terminals that form identifiable synapses within single ultrathin sections taken from these regions, 11.6% in the ventral striatum, 85.5% in the ventral pallidum and 64.8% in the substantia innominata were GABAergic. Differences were also found in the distribution patterns of these terminals with respect to the size of their synaptic target dendrites. These findings are consistent with previous findings that a majority of inputs to the ventral striatum are excitatory, and that a majority of inputs to the ventral pallidum are inhibitory. Our results provide a first approximation of the anatomical substrate for the physiology and pharmacology of GABA actions in the ventral forebrain region. These results also show that GABA may play an important role in the substantia innominata, where both the cholinergic and the non-cholinergic magnocellular corticopetal neurons reside within a neuropil innervated by many different non-cholinergic fibers.

We report the draft genome sequence of the human pathogen Streptomyces somaliensis (DSM 40738), a pathogen within a genus of largely saprophytic organisms. S. somaliensis causes severe and debilitating deep tissue and bone infections. The genome sequence is deposited in DDBJ/EMBL/GenBank with the accession number AJJM01000000 .

In order to enable microorganisms to manifest their intracellular oxygen levels we constructed a genetic sensor circuitry which converts signals impinging on the cellular redox balance into a reporter gene expression readout. Based on the newly found Streptomyces rimosus redox control system, consisting of Rex modulating Retinopathy of prematurity (ROP)-containing promoters in a NADH-dependent manner, we designed an Escherichia coli sensor transcription control system, which constitutes a Rex transactivator (REDOX) with the ability to bind and activate promoters. When oxygen levels were high and resulted in depleted NADH pools, Rex-specific target promoter (cydP1) driven from the expression of secreted (Green fluorscent protein, GFP) reporter gene was low as a consequence of increased Rex-ROP affinity. Conversely, at hypoxic conditions, it led to high intracellular NADH levels, strongly reduced Rex-ROP interaction and increased GFP expression in E. coli cells. The sensor capacity (oxygen levels) of redox system enabled monitoring of the population's metabolic state in vivo. Our research will not only help to understand the molecular mechanism of the Rex family but also foster progresses in biosensor development.

The aromatic polyketide antibiotic, oxytetracycline (OTC), is produced by Streptomyces rimosus as an important secondary metabolite. High level production of antibiotics in Streptomycetes requires precursors and cofactors which are derived from primary metabolism; therefore it is exigent to engineer the primary metabolism. This has been demonstrated by targeting a key enzyme in the oxidative pentose phosphate pathway (PPP) and nicotinamide adenine dinucleotide phosphate (NADPH) generation, glucose-6-phosphate dehydrogenase (G6PDH), which is encoded by zwf1 and zwf2. Disruption of zwf1 or zwf2 resulted in a higher production of OTC. The disrupted strain had an increased carbon flux through glycolysis and a decreased carbon flux through PPP, as measured by the enzyme activities of G6PDH and phosphoglucose isomerase (PGI), and by the levels of ATP, which establishes G6PDH as a key player in determining carbon flux distribution. The increased production of OTC appeared to be largely due to the generation of more malonyl-CoA, one of the OTC precursors, as observed in the disrupted mutants. We have studied the effect of zwf modification on metabolite levels, gene expression, and secondary metabolite production to gain greater insight into flux distribution and the link between the fluxes in the primary and secondary metabolisms.

We observed movies of replisome trafficking during Streptomyces coelicolor growth. A replisome(s) in the spore served as a replication center(s) until hyphae reached a certain length, when a tip-proximal replisome formed and moved at a fixed distance behind the tip at a speed equivalent to the extension rate of the tip.

A simple and high-throughput transposon mediated mutagenesis system employing in vitro shuttle transposon mutagenesis has been used to systematically mutagenise the Streptomyces coelicolor genome. To achieve the highest coverage, a new ordered cosmid library was also constructed. Individual cosmids from both the existing and new libraries were disrupted using the Tn5-based mini-transposon Tn5062. A total of 35,358 insertions were sequenced resulting in the disruption of 6,482 genes (83% of the predicted open reading frames). Complete information for both the newly generated cosmids as well as all the insertions has been uploaded onto a central database, StrepDB ( http://strepdb.streptomyces.org.uk/ ). All insertions, new cosmids and a range of transposon exchange cassettes are available for study of individual gene function.

Based on available genome sequences, Actinomycetales show significant gene synteny across a wide range of species and genera. In addition, many genera show varying degrees of complex morphological development. Using the presence of gene synteny as a basis, it is clear that an analysis of gene conservation across the Streptomyces and various other Actinomycetales will provide information on both the importance of genes and gene clusters and the evolution of morphogenesis in these bacteria. Genome sequencing, although becoming cheaper, is still relatively expensive for comparing large numbers of strains. Thus, a heterologous DNA/DNA microarray hybridization dataset based on a Streptomyces coelicolor microarray allows a cheaper and greater depth of analysis of gene conservation. This study, using both bioinformatical and microarray approaches, was able to classify genes previously identified as involved in morphogenesis in Streptomyces into various subgroups in terms of conservation across species and genera. This will allow the targeting of genes for further study based on their importance at the species level and at higher evolutionary levels.

Bacteria from the genus Streptomyces are among the most complex of all prokaryotes; not only do they grow as a complex mycelium, they also differentiate to form aerial hyphae before developing further to form spore chains. This developmental heterogeneity of streptomycete microcolonies makes studying the dynamic processes that contribute to growth and development a challenging procedure. As a result, in order to study the mechanisms that underpin streptomycete growth, we have developed a system for studying hyphal extension, protein trafficking, and sporulation by time-lapse microscopy. Through the use of time-lapse microscopy we have demonstrated that Streptomyces coelicolor germ tubes undergo a temporary arrest in their growth when in close proximity to sibling extension sites. Following germination, in this system, hyphae extended at a rate of ∼20 μm h −1 , which was not significantly different from the rate at which the apical ring of the cytokinetic protein FtsZ progressed along extending hyphae through a spiraling movement. Although we were able to generate movies for streptomycete sporulation, we were unable to do so for either the erection of aerial hyphae or the early stages of sporulation. Despite this, it was possible to demonstrate an arrest of aerial hyphal development that we suggest is through the depolymerization of FtsZ-enhanced green fluorescent protein (GFP). Consequently, the imaging system reported here provides a system that allows the dynamic movement of GFP-tagged proteins involved in growth and development of S. coelicolor to be tracked and their role in cytokinesis to be characterized during the streptomycete life cycle.

DNA/DNA genome microarray analysis together with genome sequencing suggests that the genome of members of the genus Streptomyces would seem to have a common structure including a linear genomic structure, a core of common syntenous Actinomycete genes, the presence of species specific terminal regions and two intermediate group of syntenous genes that seem to be genus specific. We analyzed Streptomyces species using DNA/DNA microarray comparative genome analysis. Only Streptomyces rimosus failed to give a congruent genome pattern for the genes found in Streptomyces coelicolor. We expanded the analysis to include a number of strains related to the type strain of S. rimosus and obtained a similar divergence from the main body of Streptomyces species. These strains showed very close identity to the original strain with no gene deletion or duplication detected. The 16S rRNA sequences of these S. rimosus strains were confirmed as very similar to the S. rimosus sequences available from the Ribosomal Database Project. When the SSU ribosomal RNA phylogeny of S. rimosus is analyzed, the species is positioned at the edge of the Streptomyces clade. We conclude that S. rimosus represents a distinct evolutionary lineage making the species a worthy possibility for genome sequencing.

Streptomyces are mycelial, soil-dwelling, differentiating bacteria that are renowned for the production of many clinically-useful antibiotics (e.g. streptomycin, erythromycin, tetracyclines, and a number of the third-generation penicillin-like antibiotics). They also make other useful metabolites (e.g. anticancer agents, immunosuppressants, antihelmintics, growth promoters, herbicides, and insecticides). Their status as simple microbial models of differentiation is well established and, because they are taxonomically related to pathogens such as mycobacteria that cause tuberculosis and leprosy (but are themselves “safe” organisms), they are being cited increasingly as informative models for these life-threatening pathogens. Much effort has gone into investigating the fundamental biology of Streptomyces . In recent years, the dissection of the architecture and regulation of antibiotic production gene clusters has been undertaken, together with studies on the regulatory circuits that control production and integrate with the signaling pathways of the differentiation processes. As clinical infections invariably develop resistance to antibiotics, there is an ongoing need to discover new drugs to combat this problem. The Streptomyces are viewed as a rich source of such drugs and contemporary effort, using recombinant technologies, is aimed at altering the genes for antibiotic production pathways so that they produce new, potentially useful metabolites that may keep them one step ahead of the resistance problem. Recent advances in the molecular biological techniques applicable to streptomycetes have assisted greatly in this task. The genome sequence of the model streptomycete, Streptomyces coelicolor , was published in 2002, followed recently by that of Streptomyces avermitilis . In 2004, a number of other sequencing projects were in progress, so that these resources will grow significantly in the near future. The Streptomyces are remarkable in that their genomes are among the largest (8–9 Mb, compared with E. coli at 4.6 Mb) reported so far for any bacteria. Bioinformatics, combined with postgenomic techniques such as microarrays and proteome gels, are now being used to define and characterize global gene expression under a variety of conditions. Rapid methods have been developed to create mutants for any gene, using its genome-derived DNA sequence to guide the strategy. Mutant libraries generated by random transposon insertion, are also becoming available. These aim to provide a collection of mutants in which each of the almost 8000 genes in Streptomyces is mutated individually. With these recent postgenomic resources building on the robust genetic systems that have been set up for Streptomyces , the next few years promise to be an exciting phase in our understanding of these unique bacteria. Keywords: Auxotroph; Complementation; Cosmid; Generalized Transduction; Lysogeny; Open Reading Frame (ORF); Proteome; Protoplast; Secondary Metabolite; Transcriptome; Sigma (σ) Factor; Transformation/Transformant; Transposition; Transposon

On solid media, the reproductive growth of Streptomyces involves antibiotic biosynthesis coincident with the erection of filamentous aerial hyphae. Following cessation of growth of an aerial hypha, multiple septation occurs at the tip to form a chain of unigenomic spores. A gene, crgA , that coordinates several aspects of this reproductive growth is described. The gene product is representative of a well-conserved family of small actinomycete proteins with two C-terminal hydrophobic-potential membrane-spanning segments. In Streptomyces avermitilis , crgA is required for sporulation, and inactivation of the gene abolished most sporulation septation in aerial hyphae. Disruption of the orthologous gene in Streptomyces coelicolor indicates that whereas CrgA is not essential for sporulation in this species, during growth on glucose-containing media, it influences the timing of the onset of reproductive growth, with precocious erection of aerial hyphae and antibiotic production by the mutant. Moreover, CrgA subsequently acts to inhibit sporulation septation prior to growth arrest of aerial hyphae. Overexpression of CrgA in S. coelicolor , uncoupling any nutritional and growth phase-dependent regulation, results in growth of nonseptated aerial hyphae on all media tested, consistent with a role for the protein in inhibiting sporulation septation.

Transcriptional activation of the thiostrepton-inducible promoter,ptipA, inStreptomyces lividansis mediated by TipAL. This transcriptional activator belongs to the MerR/SoxR family that characteristically binds an operator sequence located between the −10 and −35 hexamers normally occupied by RNA polymerase. As for theEscherichia colimerTpromoter, theptipAhexamers are separated by a long 19 bp spacer and hence a topological transition of the DNA is likely to be a requisite for alignment with RNA polymerase. Growth conditions that could facilitate this conformational change were investigated using transcriptional fusions ofptipAwith reporter genes. Adjustment of growth medium osmolarity led to increased and prolonged TipAL-dependent expression, both with and without the inducer, thiostrepton. These effects correlated with increases in negative DNA supercoiling. Moreover, an inability to induce the promoter with thiostrepton in strain TK64 was corrected by increasing the concentration of osmolyte, compensating for an apparent reduced level of negative DNA supercoiling in the strain. Prolonging the time of activation oftipAin the wild-type by manipulating growth conditions revealed that mycelial autolysis could be induced by thiostrepton in 4-d-old cultures.

The huge genome (8.7 Mb) and developmental complexity of Streptomyces coelicolor A3(2) present significant challenges for functional genomics. Nevertheless, effective microarrays of PCR-generated gene fragments have been generated in two laboratories, and significant information is accruing from proteome analysis. Genomewide transposon mutagenesis and highly efficient PCR-targeted gene disruption are making genetic intervention straightforward.

Exposure to narrowband violet-blue light around 405 nm wavelength can induce lethal oxidative damage to bacteria and fungi, however effects on viruses are unknown. As photosensitive porphyrin molecules are involved in the microbicidal inactivation mechanism, and since porphyrins are absent in viruses, then any damaging effects of 405 nm light on viruses might appear unlikely. This study used the bacteriophage ɸC31, as a surrogate for non-enveloped double-stranded DNA viruses, to establish whether 405 nm light can induce virucidal effects. Exposure of ɸC31 suspended in minimal media, nutrient-rich media, and porphyrin solution, demonstrated differing sensitivity of the phage. Significant reductions in phage titer occurred when exposed in nutrient-rich media, with ~3-, 5- and 7-log10 reductions achieved after exposure to doses of 0.3, 0.5 and 1.4 kJ/cm2, respectively. When suspended in minimal media a 0.3-log10 reduction (P = 0.012) occurred after exposure to 306 J/cm2: much lower than the 2.7- and > 2.5-log10 reductions achieved with the same dose in nutrient-rich, and porphyrin-supplemented media, suggesting inactivation is accelerated by the photo-activation of light-sensitive components in the media. This study provides the first evidence of the interaction of narrowband 405 nm light with viruses, and demonstrates that viral susceptibility to 405 nm light can be significantly enhanced by involvement of exogenous photosensitive components. The reduced susceptibility of viruses in minimal media, compared with that of other microorganisms, provides further evidence that the antimicrobial action of 405 nm light is predominantly due to the photo-excitation of endogenous photosensitive molecules such as porphyrins within susceptible microorganisms.

The purpose of these experiments was to determine the effects of cholinergic depletion on spontaneous and evoked activity of neurons in the different layers of the posteromedial barrel subfield (PMBSF) of the rat somatosensory cortex. Acetylcholine neurons in nucleus basalis of Meynert (NBM) were selectively lesioned with an immunotoxin (IT), 192 IgG-saporin. Spontaneous activity was significantly lower in layers II–III, Va, and VI in IT-injected animals compared to control animals. Evoked activity was significantly lower in layers II–III, IV, Vb, and VI of IT-injected animals compared to control animals. The largest difference was observed in layer Vb. Thus, cholinergic depletion causes significant changes in the magnitude of spontaneous and evoked activity but these differences are not completely in register with one another.

Changes in market-driven health care economics are rapid and of great magnitude. This report describes a study of some of these changes in regard to children's health issues. We used a survey tool to assess long-range plans (next 10 years) and marketing strategies for major freestanding children's hospitals in different regions of the United States. We then used these assessments to draw conclusions about the impact of the plans and strategies on the practice of pediatric physicians and their workforce requirements. This may allow pediatric specialists and their programs to develop strategic plans and to take actions to contend with these market-driven economic changes. The tool was a questionnaire mailed to chief executive officers of 30 randomly chosen but geographically well-distributed children's hospitals. Seventeen children's hospitals responded (57%), providing information concerning each hospital and its current medical economic environment. The data were analyzed and trends were then identified from their responses. All institutions in this study expected to have fewer physicians on staff in the future. These institutions plan: (1) to improve quality and (2) to reduce costs. Quality will be improved by utilizing Continuous Quality Improvement (CQJ) and/or Benchmarking to Best Practices, both of which encourage physicians to follow standardized treatment protocols. Costs will be reduced by decreasing hospital staff size. Some children's hospitals have merged or will merge with larger, fullservice adult hospitals, but most plan to remain autonomous. Many expect a continued decrease in revenues, and almost all expect to downsize both bed number and staff. Restructuring will reduce the number of specialists, particularly in the fields of hematology-oncology, psychiatry, endocrinology, nephrology, and cardiology, and will also reduce the number of surgical specialists. The administrators predicted that more nurse practitioners will be employed at these hospitals in the 21st century, serving the role of physician extenders, if not physician substitutes. To ensure their own survival, physicians must develop their own strategic plans as have the hospitals in which they practice. Optimally they should embrace those activities that parallel their hospital's efforts such as Quality Improvement (CQI, Benchmarking), cost containment, and practice mergers.