Govind Chandra - Academia.edu (original) (raw)
Papers by Govind Chandra
Bacterial lipoproteins are extracellular proteins tethered to cell membranes by covalently attach... more Bacterial lipoproteins are extracellular proteins tethered to cell membranes by covalently attached lipids. Deleting the lipoprotein signal peptidase (lsp) gene in Streptomyces coelicolor results in growth and developmental defects that cannot be restored by reintroducing lsp. This led us to hypothesise that lsp is essential and that the lsp mutant we isolated previously had acquired compensatory secondary mutations. Here we report resequencing of the genomes of wild-type M145 and the cis-complemented ∆lsp mutant (BJT1004) to map and identify these secondary mutations but we show that they do not increase the efficiency of disrupting lsp and are not lsp suppressors. We provide evidence that they are induced by introducing the cosmid St4A10∆lsp, as part of ReDirect PCR mutagenesis protocol, which transiently duplicates a number of important cell division genes. Disruption of lsp using a suicide vector (which does not result in gene duplication) still results in growth and developmental delays and we conclude that loss of Lsp function results in developmental defects due to the loss of all lipoproteins from the cell membrane. Significantly, our results also indicate the use of cosmid libraries for the genetic manipulation of bacteria can lead to phenotypes not necessarily linked to the gene(s) of interest.
Microbiol Mol Biol Rev, 2007
Advan Appl Microbiol, 2004
This chapter explores the importance of λ Red recombination to be used in Streptomyces. This rapi... more This chapter explores the importance of λ Red recombination to be used in Streptomyces. This rapid and highly efficient method has made the generation of gene disruptions more precise and allows the construction of in-frame deletions. So far, more than 100 segments of the S. coelicolor genome ranging in size between 4 bp and over 7 kb have been replaced by PCR-targeting. The technique has also succeeded in other Streptomyces species. The chapter discusses the use of this technology for various other DNA modifications such as introducing point mutations, promoter replacements, and gene fusions. Combining the different approaches helps manipulate Streptomyces DNA more rapidly and precisely than using traditional techniques. The facile integration of whole antibiotic gene clusters into Streptomyces chromosomes makes high-throughput manipulation of the clusters possible. The genes for synthesis of any one antibiotic in streptomycetes are invariably clustered together on the chromosome (or sometimes on a plasmid). The availability of plasmid vectors, which can efficiently carry stable large inserts into different Streptomyces spp. has been exploited in a number of laboratories to allow production in a heterologous host.
![Research paper thumbnail of The transcriptional repressor protein NsrR senses nitric oxide directly via a [2Fe-2S] cluster](https://attachments.academia-assets.com/48213740/thumbnails/1.jpg)
](PLoS ONE, 2008
The regulatory protein NsrR, a member of the Rrf2 family of transcription repressors, is specific... more The regulatory protein NsrR, a member of the Rrf2 family of transcription repressors, is specifically dedicated to sensing nitric oxide (NO) in a variety of pathogenic and non-pathogenic bacteria. It has been proposed that NO directly modulates NsrR activity by interacting with a predicted [Fe-S] cluster in the NsrR protein, but no experimental evidence has been published to support this hypothesis. Here we report the purification of NsrR from the obligate aerobe Streptomyces coelicolor. We demonstrate using UV-visible, near UV CD and EPR spectroscopy that the protein contains an NO-sensitive [2Fe-2S] cluster when purified from E. coli. Upon exposure of NsrR to NO, the cluster is nitrosylated, which results in the loss of DNA binding activity as detected by bandshift assays. Removal of the [2Fe-2S] cluster to generate apo-NsrR also resulted in loss of DNA binding activity. This is the first demonstration that NsrR contains an NO-sensitive [2Fe-2S] cluster that is required for DNA binding activity.
Molecular Microbiology, 2010
Streptomyces scabies is one of a group of organisms that causes the economically important diseas... more Streptomyces scabies is one of a group of organisms that causes the economically important disease potato scab. Analysis of the S. scabies genome sequence indicates that it is likely to secrete many proteins via the twin arginine protein transport (Tat) pathway, including several proteins whose coding sequences may have been acquired through horizontal gene transfer and share a common ancestor with proteins in other plant pathogens. Inactivation of the S. scabies Tat pathway resulted in pleiotropic phenotypes including slower growth rate and increased permeability of the cell envelope. Comparison of the extracellular proteome of the wild type and DtatC strains identified 73 predicted secretory proteins that were present in reduced amounts in the tatC mutant strain, and 47 Tat substrates were verified using a Tat reporter assay. The DtatC strain was almost completely avirulent on Arabidopsis seedlings and was delayed in attaching to the root tip relative to the wild-type strain. Genes encoding 14 candidate Tat substrates were individually inactivated, and seven of these mutants were reduced in virulence compared with the wild-type strain. We conclude that the Tat pathway secretes multiple proteins that are required for full virulence.
Molecular Microbiology, 2011
Following translocation, bacterial lipoproteins are lipidated by lipoprotein diacylglycerol trans... more Following translocation, bacterial lipoproteins are lipidated by lipoprotein diacylglycerol transferase (Lgt) and cleaved of their signal peptides by lipoprotein signal peptidase (Lsp). In Gram-negative bacteria and mycobacteria, lipoproteins are further lipidated by lipoprotein N-acyl transferase (Lnt), to give triacylated lipoproteins. Streptomyces are unusual amongst Gram-positive bacteria because they export large numbers of lipoproteins via the twin arginine protein transport (Tat) pathway. Furthermore, some Streptomyces species encode two Lgt homologues and all Streptomyces species encode two homologues of Lnt. Here we characterize lipoprotein biogenesis in the plant pathogen Streptomyces scabies and report that lgt and lsp mutants are defective in growth and development while only moderately affected in virulence. Lipoproteins are lost from the membrane in an S. scabies lgt mutant but restored by expression of Streptomyces coelicolor lgt1 or lgt2 confirming that both encode functional Lgt enzymes. Furthermore, lipoproteins are N-acylated in Streptomyces with efficient N-acylation dependent on Lnt1 and Lnt2. However, deletion of lnt1 and lnt2 has no effect on growth, development or virulence. We thus present a detailed study of lipoprotein biogenesis in Streptomyces, the first study of Lnt function in a monoderm bacterium and the first study of bacterial lipoproteins as virulence factors in a plant pathogen.
Molecular Microbiology, 2010
Lipoproteins are a distinct class of bacterial membrane proteins that are translocated across the... more Lipoproteins are a distinct class of bacterial membrane proteins that are translocated across the cytoplasmic membrane primarily by the Sec general secretory pathway and then lipidated on a conserved cysteine by the enzyme lipoprotein diacylglycerol transferase (Lgt). The signal peptide is cleaved by lipoprotein signal peptidase (Lsp) to leave the lipidmodified cysteine at the N-terminus of the mature lipoprotein. In all Gram-positive bacteria tested to date this pathway is non-essential and the lipid attaches the protein to the outer leaflet of the cytoplasmic membrane. Here we identify lipoproteins in the model Gram-positive bacterium Streptomyces coelicolor using bioinformatics coupled with proteomic and downstream analysis. We report that Streptomyces species translocate large numbers of lipoproteins out via the Tat (twin arginine translocase) pathway and we present evidence that lipoprotein biogenesis might be an essential pathway in S. coelicolor. This is the first analysis of lipoproteins and lipoprotein biogenesis in Streptomyces and provides the first evidence that lipoprotein biogenesis could be essential in a Gram-positive bacterium. This report also provides the first experimental evidence that Tat plays a major role in the translocation of lipoproteins in a specific bacterium.
Microbiology and Molecular Biology Reviews, 2007
Microbiology, 2004
Streptomyces coelicolor is a Gram-positive soil bacterium that undergoes a complex developmental ... more Streptomyces coelicolor is a Gram-positive soil bacterium that undergoes a complex developmental life cycle. The genome sequence of this organism was recently completed and has revealed the presence of over 60 sigma factors and a multitude of other transcriptional regulators, with a significant number of these being putative two-component signal transduction proteins. The authors have used the criteria established by Hoch and co-workers , J Bacteriol 181, 1975-1983 to identify sensor kinase and response regulator genes encoded within the S. coelicolor genome. This analysis has revealed the presence of 84 sensor kinase genes, 67 of which lie adjacent to genes encoding response regulators. This strongly suggests that these paired genes encode two-component systems. In addition there are 13 orphan response regulators encoded in the genome, several of which have already been characterized and are implicated in development and antibiotic production, and 17 unpaired and as yet uncharacterized sensor kinases. This article attempts to infer useful information from sequence analysis and reviews what is currently known about the two-component systems, unpaired sensor kinases and orphan response regulators of S. coelicolor from both published reports and the authors' own unpublished data.
Journal of Virological Methods, 1995
DNA-A and DNA-B components of the genome of a whitefly transmitted virus causing yellowing and le... more DNA-A and DNA-B components of the genome of a whitefly transmitted virus causing yellowing and leaf curl in tomato (ITLCV) were cloned following a simple procedure for isolation of the double stranded replicative form of viral DNA from infected tomato plants. The method is based on extraction of total DNA from infected plants followed by concentration of the double stranded replicative form of viral DNA by an alkaline denaturation procedure identical to that used for isolation of plasmid DNA from Escherichiu coli. The attempted cloning of DNA showed that 95% of the transformants contained plasmids with an insert of either DNA-A (2.75 kb) or DNA-B (2.55 kb). Cloned DNA-A and DNA-B when used as probes could detect DNA-A/DNA-B in total nucleic acid obtained from fresh diseased tissue. Both DNA-A and DNA-B are needed for infection and they have a common region of 166 bases with about 94% nucleotide sequence homology, a characteristic of all bipartite geminiviruses. Comparison of the amino acid sequence of the putative coat protein product of ITLCV with some other mono-and bipartite geminiviruses revealed a maximum of 86% homology with Indian cassava mosaic virus. * Corresponding author. Fax: +91 522 282849. 0166-0934/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0166-0934(94)00122-7 298 K.M. Sriuastaua et al./Joumal of Virological Methods 51 (1995) 297-304
Journal of Bacteriology, 2013
Streptomyces species are bacteria that resemble filamentous fungi in their hyphal mode of growth ... more Streptomyces species are bacteria that resemble filamentous fungi in their hyphal mode of growth and sporulation. In Streptomyces coelicolor, the conversion of multigenomic aerial hyphae into chains of unigenomic spores requires synchronized septation accompanied by segregation of tens of chromosomes into prespore compartments. The chromosome segregation is dependent on ParB protein, which assembles into an array of nucleoprotein complexes in the aerial hyphae. Here, we report that nucleoprotein ParB complexes are bound in vitro and in vivo by topoisomerase I, TopA, which is the only topoisomerase I homolog found in S. coelicolor. TopA cannot be eliminated, and its depletion inhibits growth and blocks sporulation. Surprisingly, sporulation in the TopA-depleted strain could be partially restored by deletion of parB. Furthermore, the formation of regularly spaced ParB complexes, which is a prerequisite for proper chromosome segregation and septation during the development of aerial hyphae, has been found to depend on TopA. We hypothesize that TopA is recruited to ParB complexes during sporulation, and its activity is required to resolve segregating chromosomes.
FEMS Microbiology Reviews, 2012
The P II proteins are one of the most widely distributed families of signal transduction proteins... more The P II proteins are one of the most widely distributed families of signal transduction proteins in nature. They are pivotal players in the control of nitrogen metabolism in bacteria and archaea, and are also found in the plastids of plants. Quite remarkably, P II proteins control the activities of a diverse range of enzymes, transcription factors and membrane transport proteins, and in recent years the extent of these interactions has been recognized to be much greater than heretofore described. Major advances have been made in structural studies of P II proteins, including the solution of the first structures of P II proteins complexed with their targets. We have also begun to gain insights into how the key effector molecules, 2-oxoglutarate and ATP/ADP, influence the activities of P II proteins. In this review, we have set out to summarize our current understanding of P II biology and to consider where future studies of these extraordinarily adaptable proteins might lead us.
mBio, 2016
WhiB is the founding member of a family of proteins (theWhiB-like [Wbl] family) that carry a [4Fe... more WhiB is the founding member of a family of proteins (theWhiB-like [Wbl] family) that carry a [4Fe-4S] iron-sulfur cluster and play key roles in diverse aspects of the biology of actinomycetes, including pathogenesis, antibiotic resistance, and the control of development. InStreptomyces, WhiB is essential for the process of developmentally controlled cell division that leads to sporulation. The biochemical function of Wbl proteins has been controversial; here, we set out to determine unambiguously if WhiB functions as a transcription factor using chromatin immunoprecipitation sequencing (ChIP-seq) inStreptomyces venezuelae In the first demonstration ofin vivogenome-wide Wbl binding, we showed that WhiB regulates the expression of key genes required for sporulation by binding upstream of ~240 transcription units. Strikingly, the WhiB regulon is identical to the previously characterized WhiA regulon, providing an explanation for the identical phenotypes ofwhiAandwhiBmutants. Using ChIP-seq, we demonstrated thatin vivoDNA binding by WhiA depends on WhiB and vice versa, showing that WhiA and WhiB function cooperatively to control expression of a common set of WhiAB target genes. Finally, we show that mutation of the cysteine residues that coordinate the [4Fe-4S] cluster in WhiB prevents DNA binding by both WhiB and WhiAin vivo Despite the central importance ofWhiB-like (Wbl) proteins in actinomycete biology, a conclusive demonstration of their biochemical function has been elusive, and they have been difficult to study, particularlyin vitro, largely because they carry an oxygen-sensitive [4Fe-4S] cluster. Here we used genome-wide ChIP-seq to investigate the function ofStreptomycesWhiB, the founding member of the Wbl family. The advantage of this approach is that the oxygen sensitivity of the [4Fe-4S] cluster becomes irrelevant once the protein has been cross-linked to DNAin vivo Our data provide the most compellingin vivoevidence to date that WhiB, and, by extension, probably all Wbl proteins, function as transcription factors. Further, we show that WhiB does not act independently but rather coregulates its regulon of sporulation genes with a partner transcription factor, WhiA.
Journal of the Indian Botanical Society, 1986
Environmental Microbiology, 2015
Manipulation of the soil microbiota associated with crop plants has huge promise for the control ... more Manipulation of the soil microbiota associated with crop plants has huge promise for the control of crop pathogens. However, to fully realize this potential we need a better understanding of the relationship between the soil environment and the genes and phenotypes that enable microbes to colonize plants and contribute to biocontrol. A recent 2 years of investigation into the effect of wheat variety on second year crop yield in the context of take-all fungal infection presented the opportunity to examine soil microbiomes under closely defined field conditions. Amplicon sequencing of second year soil samples showed that Pseudomonas spp. were particularly affected by the wheat cultivar grown in year one. Consequently, 318 rhizosphere-associated Pseudomonas fluorescens strains were isolated and characterized across a variety of genetic and phenotypic traits. Again, the wheat variety grown in the first year of the study was shown to exert considerable selec-tive pressure on both the extent and nature of Pseudomonas genomic diversity. Furthermore, multiple significant correlations were identified within the phenotypic/genetic structure of the Pseudomonas population, and between individual genotypes and the external wheat field environment. The approach outlined here has considerable future potential for our understanding of plant-microbe interactions, and for the broader analysis of complex microbial communities.
Advances in Applied Microbiology, Feb 1, 2004
Advances in Applied Microbiology, 2004
BMC genomics, 2015
Next Generation DNA Sequencing (NGS) and genome mining of actinomycetes and other microorganisms ... more Next Generation DNA Sequencing (NGS) and genome mining of actinomycetes and other microorganisms is currently one of the most promising strategies for the discovery of novel bioactive natural products, potentially revealing novel chemistry and enzymology involved in their biosynthesis. This approach also allows rapid insights into the biosynthetic potential of microorganisms isolated from unexploited habitats and ecosystems, which in many cases may prove difficult to culture and manipulate in the laboratory. Streptomyces leeuwenhoekii (formerly Streptomyces sp. strain C34) was isolated from the hyper-arid high-altitude Atacama Desert in Chile and shown to produce novel polyketide antibiotics. Here we present the de novo sequencing of the S. leeuwenhoekii linear chromosome (8 Mb) and two extrachromosomal replicons, the circular pSLE1 (86 kb) and the linear pSLE2 (132 kb), all in single contigs, obtained by combining Pacific Biosciences SMRT (PacBio) and Illumina MiSeq technologies. W...
BMC Genomics
GlnR is an atypical response regulator found in actinomycetes that modulates the transcription of... more GlnR is an atypical response regulator found in actinomycetes that modulates the transcription of genes in response to changes in nitrogen availability. We applied a global in vivo approach to identify the GlnR regulon of Streptomyces venezuelae, which, unlike many actinomycetes, grows in a diffuse manner that is suitable for physiological studies. Conditions were defined that facilitated analysis of GlnR-dependent induction of gene expression in response to rapid nitrogen starvation. Microarray analysis identified global transcriptional differences between glnR+ and glnR mutant strains under varying nitrogen conditions. To differentiate between direct and indirect regulatory effects of GlnR, chromatin immuno-precipitation (ChIP) using antibodies specific to a FLAG-tagged GlnR protein, coupled with microarray analysis (ChIP-chip), was used to identify GlnR binding sites throughout the S. venezuelae genome. GlnR bound to its target sites in both transcriptionally active and apparentl...
The ISME journal, Jan 24, 2015
We examined succession of the rhizosphere microbiota of three model plants (Arabidopsis, Medicago... more We examined succession of the rhizosphere microbiota of three model plants (Arabidopsis, Medicago and Brachypodium) in compost and sand and three crops (Brassica, Pisum and Triticum) in compost alone. We used serial inoculation of 24 independent replicate microcosms over three plant generations for each plant/soil combination. Stochastic variation between replicates was surprisingly weak and by the third generation, replicate microcosms for each plant had communities that were very similar to each other but different to those of other plants or unplanted soil. Microbiota diversity remained high in compost, but declined drastically in sand, with bacterial opportunists and putative autotrophs becoming dominant. These dramatic differences indicate that many microbes cannot thrive on plant exudates alone and presumably also require carbon sources and/or nutrients from soil. Arabidopsis had the weakest influence on its microbiota and in compost replicate microcosms converged on three alt...
Bacterial lipoproteins are extracellular proteins tethered to cell membranes by covalently attach... more Bacterial lipoproteins are extracellular proteins tethered to cell membranes by covalently attached lipids. Deleting the lipoprotein signal peptidase (lsp) gene in Streptomyces coelicolor results in growth and developmental defects that cannot be restored by reintroducing lsp. This led us to hypothesise that lsp is essential and that the lsp mutant we isolated previously had acquired compensatory secondary mutations. Here we report resequencing of the genomes of wild-type M145 and the cis-complemented ∆lsp mutant (BJT1004) to map and identify these secondary mutations but we show that they do not increase the efficiency of disrupting lsp and are not lsp suppressors. We provide evidence that they are induced by introducing the cosmid St4A10∆lsp, as part of ReDirect PCR mutagenesis protocol, which transiently duplicates a number of important cell division genes. Disruption of lsp using a suicide vector (which does not result in gene duplication) still results in growth and developmental delays and we conclude that loss of Lsp function results in developmental defects due to the loss of all lipoproteins from the cell membrane. Significantly, our results also indicate the use of cosmid libraries for the genetic manipulation of bacteria can lead to phenotypes not necessarily linked to the gene(s) of interest.
Microbiol Mol Biol Rev, 2007
Advan Appl Microbiol, 2004
This chapter explores the importance of λ Red recombination to be used in Streptomyces. This rapi... more This chapter explores the importance of λ Red recombination to be used in Streptomyces. This rapid and highly efficient method has made the generation of gene disruptions more precise and allows the construction of in-frame deletions. So far, more than 100 segments of the S. coelicolor genome ranging in size between 4 bp and over 7 kb have been replaced by PCR-targeting. The technique has also succeeded in other Streptomyces species. The chapter discusses the use of this technology for various other DNA modifications such as introducing point mutations, promoter replacements, and gene fusions. Combining the different approaches helps manipulate Streptomyces DNA more rapidly and precisely than using traditional techniques. The facile integration of whole antibiotic gene clusters into Streptomyces chromosomes makes high-throughput manipulation of the clusters possible. The genes for synthesis of any one antibiotic in streptomycetes are invariably clustered together on the chromosome (or sometimes on a plasmid). The availability of plasmid vectors, which can efficiently carry stable large inserts into different Streptomyces spp. has been exploited in a number of laboratories to allow production in a heterologous host.
PLoS ONE, 2008
The regulatory protein NsrR, a member of the Rrf2 family of transcription repressors, is specific... more The regulatory protein NsrR, a member of the Rrf2 family of transcription repressors, is specifically dedicated to sensing nitric oxide (NO) in a variety of pathogenic and non-pathogenic bacteria. It has been proposed that NO directly modulates NsrR activity by interacting with a predicted [Fe-S] cluster in the NsrR protein, but no experimental evidence has been published to support this hypothesis. Here we report the purification of NsrR from the obligate aerobe Streptomyces coelicolor. We demonstrate using UV-visible, near UV CD and EPR spectroscopy that the protein contains an NO-sensitive [2Fe-2S] cluster when purified from E. coli. Upon exposure of NsrR to NO, the cluster is nitrosylated, which results in the loss of DNA binding activity as detected by bandshift assays. Removal of the [2Fe-2S] cluster to generate apo-NsrR also resulted in loss of DNA binding activity. This is the first demonstration that NsrR contains an NO-sensitive [2Fe-2S] cluster that is required for DNA binding activity.
Molecular Microbiology, 2010
Streptomyces scabies is one of a group of organisms that causes the economically important diseas... more Streptomyces scabies is one of a group of organisms that causes the economically important disease potato scab. Analysis of the S. scabies genome sequence indicates that it is likely to secrete many proteins via the twin arginine protein transport (Tat) pathway, including several proteins whose coding sequences may have been acquired through horizontal gene transfer and share a common ancestor with proteins in other plant pathogens. Inactivation of the S. scabies Tat pathway resulted in pleiotropic phenotypes including slower growth rate and increased permeability of the cell envelope. Comparison of the extracellular proteome of the wild type and DtatC strains identified 73 predicted secretory proteins that were present in reduced amounts in the tatC mutant strain, and 47 Tat substrates were verified using a Tat reporter assay. The DtatC strain was almost completely avirulent on Arabidopsis seedlings and was delayed in attaching to the root tip relative to the wild-type strain. Genes encoding 14 candidate Tat substrates were individually inactivated, and seven of these mutants were reduced in virulence compared with the wild-type strain. We conclude that the Tat pathway secretes multiple proteins that are required for full virulence.
Molecular Microbiology, 2011
Following translocation, bacterial lipoproteins are lipidated by lipoprotein diacylglycerol trans... more Following translocation, bacterial lipoproteins are lipidated by lipoprotein diacylglycerol transferase (Lgt) and cleaved of their signal peptides by lipoprotein signal peptidase (Lsp). In Gram-negative bacteria and mycobacteria, lipoproteins are further lipidated by lipoprotein N-acyl transferase (Lnt), to give triacylated lipoproteins. Streptomyces are unusual amongst Gram-positive bacteria because they export large numbers of lipoproteins via the twin arginine protein transport (Tat) pathway. Furthermore, some Streptomyces species encode two Lgt homologues and all Streptomyces species encode two homologues of Lnt. Here we characterize lipoprotein biogenesis in the plant pathogen Streptomyces scabies and report that lgt and lsp mutants are defective in growth and development while only moderately affected in virulence. Lipoproteins are lost from the membrane in an S. scabies lgt mutant but restored by expression of Streptomyces coelicolor lgt1 or lgt2 confirming that both encode functional Lgt enzymes. Furthermore, lipoproteins are N-acylated in Streptomyces with efficient N-acylation dependent on Lnt1 and Lnt2. However, deletion of lnt1 and lnt2 has no effect on growth, development or virulence. We thus present a detailed study of lipoprotein biogenesis in Streptomyces, the first study of Lnt function in a monoderm bacterium and the first study of bacterial lipoproteins as virulence factors in a plant pathogen.
Molecular Microbiology, 2010
Lipoproteins are a distinct class of bacterial membrane proteins that are translocated across the... more Lipoproteins are a distinct class of bacterial membrane proteins that are translocated across the cytoplasmic membrane primarily by the Sec general secretory pathway and then lipidated on a conserved cysteine by the enzyme lipoprotein diacylglycerol transferase (Lgt). The signal peptide is cleaved by lipoprotein signal peptidase (Lsp) to leave the lipidmodified cysteine at the N-terminus of the mature lipoprotein. In all Gram-positive bacteria tested to date this pathway is non-essential and the lipid attaches the protein to the outer leaflet of the cytoplasmic membrane. Here we identify lipoproteins in the model Gram-positive bacterium Streptomyces coelicolor using bioinformatics coupled with proteomic and downstream analysis. We report that Streptomyces species translocate large numbers of lipoproteins out via the Tat (twin arginine translocase) pathway and we present evidence that lipoprotein biogenesis might be an essential pathway in S. coelicolor. This is the first analysis of lipoproteins and lipoprotein biogenesis in Streptomyces and provides the first evidence that lipoprotein biogenesis could be essential in a Gram-positive bacterium. This report also provides the first experimental evidence that Tat plays a major role in the translocation of lipoproteins in a specific bacterium.
Microbiology and Molecular Biology Reviews, 2007
Microbiology, 2004
Streptomyces coelicolor is a Gram-positive soil bacterium that undergoes a complex developmental ... more Streptomyces coelicolor is a Gram-positive soil bacterium that undergoes a complex developmental life cycle. The genome sequence of this organism was recently completed and has revealed the presence of over 60 sigma factors and a multitude of other transcriptional regulators, with a significant number of these being putative two-component signal transduction proteins. The authors have used the criteria established by Hoch and co-workers , J Bacteriol 181, 1975-1983 to identify sensor kinase and response regulator genes encoded within the S. coelicolor genome. This analysis has revealed the presence of 84 sensor kinase genes, 67 of which lie adjacent to genes encoding response regulators. This strongly suggests that these paired genes encode two-component systems. In addition there are 13 orphan response regulators encoded in the genome, several of which have already been characterized and are implicated in development and antibiotic production, and 17 unpaired and as yet uncharacterized sensor kinases. This article attempts to infer useful information from sequence analysis and reviews what is currently known about the two-component systems, unpaired sensor kinases and orphan response regulators of S. coelicolor from both published reports and the authors' own unpublished data.
Journal of Virological Methods, 1995
DNA-A and DNA-B components of the genome of a whitefly transmitted virus causing yellowing and le... more DNA-A and DNA-B components of the genome of a whitefly transmitted virus causing yellowing and leaf curl in tomato (ITLCV) were cloned following a simple procedure for isolation of the double stranded replicative form of viral DNA from infected tomato plants. The method is based on extraction of total DNA from infected plants followed by concentration of the double stranded replicative form of viral DNA by an alkaline denaturation procedure identical to that used for isolation of plasmid DNA from Escherichiu coli. The attempted cloning of DNA showed that 95% of the transformants contained plasmids with an insert of either DNA-A (2.75 kb) or DNA-B (2.55 kb). Cloned DNA-A and DNA-B when used as probes could detect DNA-A/DNA-B in total nucleic acid obtained from fresh diseased tissue. Both DNA-A and DNA-B are needed for infection and they have a common region of 166 bases with about 94% nucleotide sequence homology, a characteristic of all bipartite geminiviruses. Comparison of the amino acid sequence of the putative coat protein product of ITLCV with some other mono-and bipartite geminiviruses revealed a maximum of 86% homology with Indian cassava mosaic virus. * Corresponding author. Fax: +91 522 282849. 0166-0934/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0166-0934(94)00122-7 298 K.M. Sriuastaua et al./Joumal of Virological Methods 51 (1995) 297-304
Journal of Bacteriology, 2013
Streptomyces species are bacteria that resemble filamentous fungi in their hyphal mode of growth ... more Streptomyces species are bacteria that resemble filamentous fungi in their hyphal mode of growth and sporulation. In Streptomyces coelicolor, the conversion of multigenomic aerial hyphae into chains of unigenomic spores requires synchronized septation accompanied by segregation of tens of chromosomes into prespore compartments. The chromosome segregation is dependent on ParB protein, which assembles into an array of nucleoprotein complexes in the aerial hyphae. Here, we report that nucleoprotein ParB complexes are bound in vitro and in vivo by topoisomerase I, TopA, which is the only topoisomerase I homolog found in S. coelicolor. TopA cannot be eliminated, and its depletion inhibits growth and blocks sporulation. Surprisingly, sporulation in the TopA-depleted strain could be partially restored by deletion of parB. Furthermore, the formation of regularly spaced ParB complexes, which is a prerequisite for proper chromosome segregation and septation during the development of aerial hyphae, has been found to depend on TopA. We hypothesize that TopA is recruited to ParB complexes during sporulation, and its activity is required to resolve segregating chromosomes.
FEMS Microbiology Reviews, 2012
The P II proteins are one of the most widely distributed families of signal transduction proteins... more The P II proteins are one of the most widely distributed families of signal transduction proteins in nature. They are pivotal players in the control of nitrogen metabolism in bacteria and archaea, and are also found in the plastids of plants. Quite remarkably, P II proteins control the activities of a diverse range of enzymes, transcription factors and membrane transport proteins, and in recent years the extent of these interactions has been recognized to be much greater than heretofore described. Major advances have been made in structural studies of P II proteins, including the solution of the first structures of P II proteins complexed with their targets. We have also begun to gain insights into how the key effector molecules, 2-oxoglutarate and ATP/ADP, influence the activities of P II proteins. In this review, we have set out to summarize our current understanding of P II biology and to consider where future studies of these extraordinarily adaptable proteins might lead us.
mBio, 2016
WhiB is the founding member of a family of proteins (theWhiB-like [Wbl] family) that carry a [4Fe... more WhiB is the founding member of a family of proteins (theWhiB-like [Wbl] family) that carry a [4Fe-4S] iron-sulfur cluster and play key roles in diverse aspects of the biology of actinomycetes, including pathogenesis, antibiotic resistance, and the control of development. InStreptomyces, WhiB is essential for the process of developmentally controlled cell division that leads to sporulation. The biochemical function of Wbl proteins has been controversial; here, we set out to determine unambiguously if WhiB functions as a transcription factor using chromatin immunoprecipitation sequencing (ChIP-seq) inStreptomyces venezuelae In the first demonstration ofin vivogenome-wide Wbl binding, we showed that WhiB regulates the expression of key genes required for sporulation by binding upstream of ~240 transcription units. Strikingly, the WhiB regulon is identical to the previously characterized WhiA regulon, providing an explanation for the identical phenotypes ofwhiAandwhiBmutants. Using ChIP-seq, we demonstrated thatin vivoDNA binding by WhiA depends on WhiB and vice versa, showing that WhiA and WhiB function cooperatively to control expression of a common set of WhiAB target genes. Finally, we show that mutation of the cysteine residues that coordinate the [4Fe-4S] cluster in WhiB prevents DNA binding by both WhiB and WhiAin vivo Despite the central importance ofWhiB-like (Wbl) proteins in actinomycete biology, a conclusive demonstration of their biochemical function has been elusive, and they have been difficult to study, particularlyin vitro, largely because they carry an oxygen-sensitive [4Fe-4S] cluster. Here we used genome-wide ChIP-seq to investigate the function ofStreptomycesWhiB, the founding member of the Wbl family. The advantage of this approach is that the oxygen sensitivity of the [4Fe-4S] cluster becomes irrelevant once the protein has been cross-linked to DNAin vivo Our data provide the most compellingin vivoevidence to date that WhiB, and, by extension, probably all Wbl proteins, function as transcription factors. Further, we show that WhiB does not act independently but rather coregulates its regulon of sporulation genes with a partner transcription factor, WhiA.
Journal of the Indian Botanical Society, 1986
Environmental Microbiology, 2015
Manipulation of the soil microbiota associated with crop plants has huge promise for the control ... more Manipulation of the soil microbiota associated with crop plants has huge promise for the control of crop pathogens. However, to fully realize this potential we need a better understanding of the relationship between the soil environment and the genes and phenotypes that enable microbes to colonize plants and contribute to biocontrol. A recent 2 years of investigation into the effect of wheat variety on second year crop yield in the context of take-all fungal infection presented the opportunity to examine soil microbiomes under closely defined field conditions. Amplicon sequencing of second year soil samples showed that Pseudomonas spp. were particularly affected by the wheat cultivar grown in year one. Consequently, 318 rhizosphere-associated Pseudomonas fluorescens strains were isolated and characterized across a variety of genetic and phenotypic traits. Again, the wheat variety grown in the first year of the study was shown to exert considerable selec-tive pressure on both the extent and nature of Pseudomonas genomic diversity. Furthermore, multiple significant correlations were identified within the phenotypic/genetic structure of the Pseudomonas population, and between individual genotypes and the external wheat field environment. The approach outlined here has considerable future potential for our understanding of plant-microbe interactions, and for the broader analysis of complex microbial communities.
Advances in Applied Microbiology, Feb 1, 2004
Advances in Applied Microbiology, 2004
BMC genomics, 2015
Next Generation DNA Sequencing (NGS) and genome mining of actinomycetes and other microorganisms ... more Next Generation DNA Sequencing (NGS) and genome mining of actinomycetes and other microorganisms is currently one of the most promising strategies for the discovery of novel bioactive natural products, potentially revealing novel chemistry and enzymology involved in their biosynthesis. This approach also allows rapid insights into the biosynthetic potential of microorganisms isolated from unexploited habitats and ecosystems, which in many cases may prove difficult to culture and manipulate in the laboratory. Streptomyces leeuwenhoekii (formerly Streptomyces sp. strain C34) was isolated from the hyper-arid high-altitude Atacama Desert in Chile and shown to produce novel polyketide antibiotics. Here we present the de novo sequencing of the S. leeuwenhoekii linear chromosome (8 Mb) and two extrachromosomal replicons, the circular pSLE1 (86 kb) and the linear pSLE2 (132 kb), all in single contigs, obtained by combining Pacific Biosciences SMRT (PacBio) and Illumina MiSeq technologies. W...
BMC Genomics
GlnR is an atypical response regulator found in actinomycetes that modulates the transcription of... more GlnR is an atypical response regulator found in actinomycetes that modulates the transcription of genes in response to changes in nitrogen availability. We applied a global in vivo approach to identify the GlnR regulon of Streptomyces venezuelae, which, unlike many actinomycetes, grows in a diffuse manner that is suitable for physiological studies. Conditions were defined that facilitated analysis of GlnR-dependent induction of gene expression in response to rapid nitrogen starvation. Microarray analysis identified global transcriptional differences between glnR+ and glnR mutant strains under varying nitrogen conditions. To differentiate between direct and indirect regulatory effects of GlnR, chromatin immuno-precipitation (ChIP) using antibodies specific to a FLAG-tagged GlnR protein, coupled with microarray analysis (ChIP-chip), was used to identify GlnR binding sites throughout the S. venezuelae genome. GlnR bound to its target sites in both transcriptionally active and apparentl...
The ISME journal, Jan 24, 2015
We examined succession of the rhizosphere microbiota of three model plants (Arabidopsis, Medicago... more We examined succession of the rhizosphere microbiota of three model plants (Arabidopsis, Medicago and Brachypodium) in compost and sand and three crops (Brassica, Pisum and Triticum) in compost alone. We used serial inoculation of 24 independent replicate microcosms over three plant generations for each plant/soil combination. Stochastic variation between replicates was surprisingly weak and by the third generation, replicate microcosms for each plant had communities that were very similar to each other but different to those of other plants or unplanted soil. Microbiota diversity remained high in compost, but declined drastically in sand, with bacterial opportunists and putative autotrophs becoming dominant. These dramatic differences indicate that many microbes cannot thrive on plant exudates alone and presumably also require carbon sources and/or nutrients from soil. Arabidopsis had the weakest influence on its microbiota and in compost replicate microcosms converged on three alt...