Paul Dyson

Tris-dependent site-specific cleavage of Streptomyces lividans DNA

During conventional gel electrophoresis, Streptomyces lividans DNA undergoes site-specific double-strand cleavage at the positions of closely opposed unstable modifications introduced into the DNA in vivo. We investigated this electrophoretic instability and demonstrated that it was dependent on Tris. Tris buffer was activated at the anode to generate a nucleolytic species; prior to activation, Tris was not able to cleave the DNA. The nucleolytic species was shown to react with thiourea, which could thus protect the DNA from strand cleavage. Non-degradative electrophoresis of the DNA could also be achieved in an alternative buffer such as Hepes.

Influence of CrgA on Assembly of the Cell Division Protein FtsZ during Development of Streptomyces coelicolor

The product of the crgA gene of Streptomyces coelicolor represents a novel family of small proteins. A single orthologous gene is located close to the origin of replication of all fully sequenced actinomycete genomes and borders a conserved gene cluster implicated in cell growth and division. In S. coelicolor, CrgA is important for coordinating growth and cell division in sporogenic hyphae. In this study, we demonstrate that CrgA is an integral membrane protein whose peak expression is coordinated with the onset of development of aerial hyphae. The protein localizes to discrete foci away from growing hyphal tips. Upon overexpression, CrgA localizes to apical syncytial cells of aerial hyphae and inhibits the formation of productive cytokinetic rings of the bacterial tubulin homolog FtsZ, leading to proteolytic turnover of this major cell division determinant. In the absence of known prokaryotic cell division inhibitors in actinomycetes, CrgA may have an important conserved function influencing Z-ring formation in these bacteria.

Streptomyces coelicolor Dps‐like proteins: differential dual roles in response to stress during vegetative growth and in nucleoid condensation during reproductive cell division

The Dps protein, a member of the ferritin family, contributes to DNA protection during oxidative stress and plays a central role in nucleoid condensation during stationary phase in unicellular eubacteria. Genome searches revealed the presence of three Dps‐like orthologues within the genome of the Gram‐positive bacterium Streptomyces coelicolor. Disruption of the S. coelicolor dpsA, dpsB and dpsC genes resulted in irregular condensation of spore nucleoids in a gene‐specific manner. These irregularities are correlated with changes to the spacing between sporulation septa. This is the first example of these proteins playing a role in bacterial cell division. Translational fusions provided evidence for both developmental control of DpsA and DpsC expression and their localization to sporogenic compartments of aerial hyphae. In addition, various stress conditions induced expression of the Dps proteins in a stimulus‐dependent manner in vegetative hyphae, suggesting stress‐induced, protein‐specific protective functions in addition to their role during reproductive cell division. Unlike in other bacteria, the S. coelicolor Dps proteins are not induced in response to oxidative stress.

Characterization of Changes to the Cell Surface during the Life Cycle of Streptomyces coelicolor: Atomic Force Microscopy of Living Cells

Cell surface changes that accompany the complex life cycle of Streptomyces coelicolor were monitored by atomic force microscopy (AFM) of living cells. Images were obtained using tapping mode to reveal that young, branching vegetative hyphae have a relatively smooth surface and are attached to an inert silica surface by means of a secreted extracellular matrix. Older hyphae, representing a transition between substrate and aerial growth, are sparsely decorated with fibers. Previously, a well-organized stable mosaic of fibers, called the rodlet layer, coating the surface of spores has been observed using electron microscopy. AFM revealed that aerial hyphae, prior to sporulation, possess a relatively unstable dense heterogeneous fibrous layer. Material from this layer is shed as the hyphae mature, revealing a more tightly organized fibrous mosaic layer typical of spores. The aerial hyphae are also characterized by the absence of the secreted extracellular matrix. The formation of sporulation septa is accompanied by modification to the surface layer, which undergoes localized temporary disruption at the sites of cell division. The characteristics of the hyphal surfaces of mutants show how various chaplin and rodlin proteins contribute to the formation of fibrous layers of differing stabilities. Finally, older spores with a compact rodlet layer develop surface concavities that are attributed to a reduction of intracellular turgor pressure as metabolic activity slows.

Transposon-encoded site-specific recombination: nature of the Tn3 DNA sequences which constitute the recombination site res.

The tnpR gene of transposon Tn3 encodes a site‐specific recombination enzyme that acts at res, a DNA region adjacent to tnpR, to convert co‐integrate intermediates of interreplicon transposition to the normal transposition end‐products. We have used two complementary approaches to study the nature of the Tn3 recombination region, res. Firstly, the DNA‐binding sites for tnpR protein were determined in DNase I protection experiments. These identified a 120‐bp region between the tnpA and tnpR genes that can be subdivided into three separate protein‐binding sites. Genetic dissection experiments indicate that few, if any, other sequences in addition to this 120‐bp region are required for res function. Moreover, we have shown that the two directly repeated res regions within a molecule are unequal partners in the recombination reaction: a truncated res region, which is unable to recombine with a second identical res region, can recombine efficiently with an intact res region. This demonstration, along with the observation that tnpR/res recombination acts efficiently on directly repeated res regions within a molecule but inefficiently both on inverted res regions in the same molecule and in the fusion reaction between res regions in different molecules, leads us to propose that one‐dimensional diffusion (tracking) of tnpR protein along DNA is used to locate an initial res region, and then to bring a second directly repeated res region into a position that allows recombination between the res regions.

A heterodimer of EsxA and EsxB is involved in sporulation and is secreted by a type VII secretion system in Streptomyces coelicolor

An esx locus, related to the multiple esx loci of Mycobacterium tuberculosis, is conserved in all sequenced Streptomyces genomes, where it is associated with the developmental regulatory gene bldB. Here we demonstrate that the esxBA operon, comprising part of the locus, has a novel morphogenetic function in the model species Streptomyces coelicolor. This operon encodes two proteins belonging to the WXG-100 superfamily that can form a heterodimer and are secreted in the absence of signal sequences. A mutation in esxBA results in a delay in sporulation, with eventual development of aerial hyphae with chains of abnormally sized spore compartments possessing irregular DNA contents. During early sporulation, expression of the operon is elevated in a bldB mutant. Other genes in the locus, notably SCO5734 and SCO5721, encode components of a type VII secretion system. Disruption of either of these genes prevents secretion of EsxAB but has no effect on sporulation. To explain the morphogenetic function of EsxAB, we propose that the heterodimer sequesters a regulator of expression of genes involved in nucleoid organization during sporulation.

Osmoregulation in Streptomyces coelicolor: modulation of SigB activity by OsaC

As free‐living non‐motile saprophytes, Streptomyces need to adapt to a wide range of environmental conditions and this is reflected by an enormous diversity of regulatory proteins encoded by, for example, the genome of the model streptomycete Streptomyces coelicolor. In this organism, we have identified a new osmoregulation gene, osaC, encoding a member of a novel family of regulatory proteins. Members of the family have a predicted domain composition consisting of an N‐terminal kinase domain related to anti‐sigma factors, sensory Pas and Gaf domains, and a C‐terminal phosphatase domain. osaC is linked to the response regulator gene osaB; expression analysis of the latter revealed that it is induced after osmotic stress in a σB‐dependent manner. OsaC is required to return osaB and sigB expression back to constitutive levels after osmotic stress. From analysis of the activities of OsaCΔPho, lacking the C‐terminal phosphatase domain, and OsaCN92A, with a substitution of a critical asparagine residue in the kinase domain, we infer that this N‐terminal domain functions as a σB anti‐sigma factor. Indeed, co‐purification experiments indicate association of OsaC and σB. These results support a model for post‐osmotic stress modulation of σB activity by OsaC.

FtsW Is a Dispensable Cell Division Protein Required for Z-Ring Stabilization during Sporulation Septation in Streptomyces coelicolor

The conserved rodA and ftsW genes encode polytopic membrane proteins that are essential for bacterial cell elongation and division, respectively, and each gene is invariably linked with a cognate class B high-molecular-weight penicillin-binding protein (HMW PBP) gene. Filamentous differentiating Streptomyces coelicolor possesses four such gene pairs. Whereas rodA, although not its cognate HMW PBP gene, is essential in these bacteria, mutation of SCO5302 or SCO2607 (sfr) caused no gross changes to growth and septation. In contrast, disruption of either ftsW or the cognate ftsI gene blocked the formation of sporulation septa in aerial hyphae. The inability of spiral polymers of FtsZ to reorganize into rings in aerial hyphae of these mutants indicates an early pivotal role of an FtsW-FtsI complex in cell division. Concerted assembly of the complete divisome was unnecessary for Z-ring stabilization in aerial hyphae as ftsQ mutants were found to be blocked at a later stage in cell division, during septum closure. Complete cross wall formation occurred in vegetative hyphae in all three fts mutants, indicating that the typical bacterial divisome functions specifically during nonessential sporulation septation, providing a unique opportunity to interrogate the function and dependencies of individual components of the divisome in vivo.

Osmotic regulation of the Streptomyces lividans thiostrepton-inducible promoter, ptipA.

Transcriptional activation of the thiostrepton-inducible promoter, ptipA, in Streptomyces lividans is 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 the Escherichia coli merT promoter, the ptipA hexamers 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 of ptipA with 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 of tipA in the wild-type by manipulating growth conditions revealed that mycelial autolysis could be induced by thiostrepton in 4-d-old cultures.

A transposon insertion single-gene knockout library and new ordered cosmid library for the model organism Streptomyces coelicolor A3(2)

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.