The reactivity of three cytotoxic trans-PtII complexes bearing aliphatic amine ligands, with transferrin and single-stranded oligonucleotides as DNA models, was investigated by ESI-MS and the results obtained are discussed in comparison with cisplatin. Tandem MS studies provided additional information on the preferential Pt binding sites. To determine whether trans-PtII complexes can migrate from a peptide to an oligonucleotide, transfer experiments were also performed using ESI-MS, and competitive binding of the trans-PtII complexes toward a model peptide and different oligonucleotides was also investigated. Significant differences in the reactivity of the trans complexes with respect to cisplatin were observed. In general, adduct formation with the selected peptide is favored for the trans compounds, whereas cisplatin shows a preference for oligonucleotides, especially if adjacent G–G residues are present. The results are discussed in relation to the possible mechanism of action of the trans-PtII complexes.
cancer; mass spectrometry; oligonucleotides; peptides; platinum
Streptomyces violaceusniger strain SPC6 is a halotolerant streptomycete isolated from the Linze desert in China. The strain has a very high growth rate and a short life cycle for a streptomycete. For surface-grown cultures, the period from spore germination to formation of colonies with mature spore chains is only 2 days at 37°C. Additionally, the strain is remarkably resistant to osmotic, heat, and UV stress compared with other streptomycetes. Analysis of the draft genome sequence indicates that the strain has the smallest reported genome (6.4 Mb) of any streptomycete. The availability of this genome sequence allows us to investigate the genetic basis of adaptation for growth in an extremely arid environment.
We report the 4,385,577-bp high-quality draft assembly of the bacterial symbiont Rhodococcus rhodnii strain LMG5362, isolated from the gut of Rhodnius prolixus (Hemiptera, Reduviidae, Triatominae), the principle vector of the protozoan Trypanosoma cruzi, the etiological agent of Chagas disease. This sequence might provide useful information for subsequent studies of the symbiotic relationship between Rd. prolixus and Rc. rhodnii, while also providing a starting point for the development of biotechnological applications for the control of Rd. prolixus.
BTZ043, a tuberculosis drug candidate with nanomolar whole-cell activity, targets the DprE1 enzyme of the essential decaprenylphosphoryl-β-D-ribofuranose-2′-epimerase thus blocking biosynthesis of arabinans, vital cell-wall components of mycobacteria. Crystal structures of DprE1, in its native form and in complex with BTZ043, unambiguously reveal formation of a semimercaptal adduct between the drug and an active-site cysteine, as well as contacts to a neighbouring catalytic lysine residue. Kinetic studies confirm BTZ043 as a mechanism-based, covalent inhibitor. This explains the exquisite potency of BTZ043, which, when fluorescently labelled, localizes DprE1 at the poles of growing bacteria. Menaquinone can reoxidize the FAD cofactor in DprE1 and may be the natural electron acceptor for this reaction in the cell. Our structural and kinetic analysis provides both insight into a critical epimerization reaction and a platform for structure-based design of improved inhibitors. Surprisingly, given the colossal tuberculosis burden globally, BTZ043 is the only new drug candidate to have been co-crystallized with its target.
Dps proteins are found almost ubiquitously in bacterial genomes and there is now an appreciation of their multifaceted roles in various stress responses. Previous studies have shown that this family of proteins assemble into dodecamers and their quaternary structure is entirely critical to their function. Moreover, the numbers of dps genes per bacterial genome is variable; even amongst closely related species - however, for many genera this enigma is yet to be satisfactorily explained. We reconstruct the most probable evolutionary history of Dps in Streptomyces genomes. Typically, these bacteria encode for more than one Dps protein. We offer the explanation that variation in the number of dps per genome among closely related Streptomyces can be explained by gene duplication or lateral acquisition, and the former preceded a subsequent shift in expression patterns for one of the resultant paralogs. We show that the genome of S. coelicolor encodes for three Dps proteins including a tailless Dps. Our in vivo observations show that the tailless protein, unlike the other two Dps in S. coelicolor, does not readily oligomerise. Phylogenetic and bioinformatic analyses combined with expression studies indicate that in several Streptomyces species at least one Dps is significantly over-expressed during osmotic shock, but the identity of the ortholog varies. In silico analysis of dps promoter regions coupled with gene expression studies of duplicated dps genes shows that paralogous gene pairs are expressed differentially and this correlates with the presence of a sigB promoter. Lastly, we identify a rare novel clade of Dps and show that a representative of these proteins in S. coelicolor possesses a dodecameric quaternary structure of high stability.
Phylogenetic reconstruction revealed that most Actinobacterial orthologs of S. coelicolor SCO2837, encoding a metal-dependent galactose oxidase-like protein, are found within Streptomyces and were probably acquired by horizontal gene transfer from fungi. Disruption of SCO2837 (glxA) caused a conditional bld phenotype that could not be reversed by extracellular complementation. Studies aimed at characterising the regulation of expression of glxA showed that it is not a target for other bld genes. We provide evidence that glxA is required for osmotic adaptation, although independently from the known osmotic stress response element SigB. glxA has been predicted to be part of an operon with the transcription unit comprising the upstream cslA gene and glxA. However, both phenotypic and expression studies indicate that it is also expressed from an independent promoter region internal to cslA. GlxA displays an in situ localisation pattern similar to that one observed for CslA at hyphal tips, but localisation of the former is independent of the latter. The functional role of GlxA in relation to CslA is discussed.
Following our strategy of coupling cyclin-dependent kinase
inhibitors with organometallic moieties to improve their physicochemical
properties and bioavailability, five organoruthenium complexes (1c–5c) of the general formula [RuCl(η6-arene)(L)]Cl have been synthesized in which the arene is
4-formylphenoxyacetyl-η6-benzylamide and L is a Cdk
inhibitor [3-(1H-benzimidazol-2-yl)-1H-pyrazolo[3,4-b]pyridines (L1–L3) and indolo[3,2-d]benzazepines (L4 and L5)]. All of the compounds were characterized
by spectroscopic and analytical methods. Upon prolonged standing (2–3
months) at room temperature, the dimethyl sulfoxide (DMSO) solutions
of 1c and 2c–HCl afforded residues, which after recrystallization from EtOH
and EtOH/H2O, respectively, were shown by X-ray diffraction
to be cis,cis-[RuIICl2(DMSO)2(L1)]·H2O and mer-[RuIICl(DMSO)3(L2–H)]·H2O. Compound 5c, with a
coordinated amidine unit, undergoes E/Z isomerization in solution. The antiproliferative activities and
effects on the cell cycle of the new compounds were evaluated. Complexes 1c–5c are moderately cytotoxic to cancer
cells (CH1, SW480, A549, A2780, and A2780cisR cell lines). Therefore,
in order to improve their antiproliferative effects, as well as their
drug targeting and delivery to cancer cells, 1c–5c were conjugated to recombinant human serum albumin, potentially
exploiting the so-called “enhanced permeability and retention”
effect that results in the accumulation of macromolecules in tumors.
Notably, a marked increase in cytotoxicity of the albumin conjugates
was observed in all cases.
Five organoruthenium complexes
[RuCl(η6-arene)(L)]Cl with a modified arene ligand,
namely, 4-formylphenoxyacetyl-η6-benzylamide, and
L = 3-(1H-benzimidazol-2-yl)-1H-pyrazolo[3,4-b]pyridines or indolo[3,2-d]benzazepines
were synthesized and conjugated to recombinant
human serum albumin in order to improve their drug targeting and delivery
to cancer cells, and a marked increase in cytotoxicity was observed.
The ruthenium-based complex [Ru(η6-p-phenylethacrynate)Cl2(pta)] (pta = 1,3,5-triaza-7-phosphatricyclo-[220.127.116.11]decane), termed ethaRAPTA, is an interesting antitumor compound. The elucidation of the molecular mechanism of drug activity is central to the drug development program. To this end, we have characterized the ethaRAPTA interaction with DNA, including probing the sequence specific modified DNA structural stability and DNA amplification using the breast cancer suppressor gene 1 (BRCA1) of human breast and colon adenocarcinoma cell lines as models. The preference of ethaRAPTA base binding is in the order A > G > T > C. Once modified, the ethaRAPTA-induced BRCA1 structure has higher thermal stability than the modified equivalents of its related compound, RAPTA-C. EthaRAPTA exhibits a higher efficiency than RAPTA-C in inhibiting BRCA1 amplification. With respect to both compounds, the inhibition of BRCA1 amplification is more effective in an isolated system than in cell lines. These data provide evidence that will help to understand the process of elucidating the pathways involved in the response induced by ethaRAPTA.
BRCA1; DNA adducts; DNA amplification; ethaRAPTA; tumor suppressor gene
The DpsA protein plays a dual role in Streptomyces coelicolor, both as part of the stress response and contributing to nucleoid condensation during sporulation. Promoter mapping experiments indicated that dpsA is transcribed from a single, sigB-like dependent promoter. Expression studies implicate SigH and SigB as the sigma factors responsible for dpsA expression while the contribution of other SigB-like factors is indirect by means of controlling sigH expression. The promoter is massively induced in response to osmotic stress, in part due to its sensitivity to changes in DNA supercoiling. In addition, we determined that WhiB is required for dpsA expression, particularly during development. Gel retardation experiments revealed direct interaction between apoWhiB and the dpsA promoter region, providing the first evidence for a direct WhiB target in S. coelicolor.
Heavy metal compounds have toxic and medicinal potential through capacity to form strong specific bonds with macromolecules, and the interaction of platinum drugs at the major groove nitrogen atom of guanine bases primarily underlies their therapeutic activity. By crystallographic analysis of transition metal–and in particular platinum compound–DNA site selectivity in the nucleosome core, we establish that steric accessibility, which is controlled by specific structural parameters of the double helix, modulates initial guanine–metal bond formation. Moreover, DNA conformational features can be linked to both similarities and distinctions in platinum drug adduct formation between the naked and nucleosomal DNA states. Notably, structures that facilitate initial platinum–guanine bond formation can oppose cross-link generation, rationalizing the occurrence of long-lived therapeutically ineffective monofunctional adducts. These findings illuminate DNA structure-dependent reactivity and provide a novel framework for understanding metal–double helix interactions, which should facilitate the development of improved chromatin-targeting medicinal agents.
To date, the function of only two of the 34 predicted serine/threonine protein kinases (STPKs) of Streptomyces coelicolor has been described. Here we report functional analysis of pknB and two linked genes, fhaAB, encoding forkhead‐associated (FHA) domain proteins that are part of a highly conserved gene locus in actinobacteria. In contrast to the homologous gene of Mycobacterium tuberculosis, pknB in S. coelicolor is not essential and has no apparent role in defining cell shape. Phosphorylation of recombinant forms of both the full‐length protein and N‐terminal kinase domain suggest that PknB‐mediated signalling in S. coelicolor may be modulated by another factor(s). FhaAB are candidate interacting partners of PknB and loss of their function resulted in deregulation of central carbon metabolism, with carbon flux diverted to synthesis of the antibiotic actinorhodin. The substrate hyphae of the fhaAB mutant also exhibited an unusual cording morphology. The results indicate that inactivation of FHA ‘brake’ proteins can potentially amplify the function of STPKs and, in this case, provide a means to overproduce antibiotics.
A method based on the coupling of high resolution size-exclusion liquid chromatography using a polymer stationary phase with inductively coupled plasma mass spectrometry was developed to study the interactions of two metallodrugs – cisplatin and RAPTA-T – with the serum proteins albumin and transferrin. In contrast to previous approaches, the technique allowed the total recovery of the metals from the column and was able to discriminate between the different species of the metallodrugs and their complexes with the proteins at femtomolar detection levels. Metal binding was found to be dependent on the protein concentration and on the incubation time of the sample. Cisplatin was found to bind the serum proteins to the same extent, whereas RAPTA-T showed marked preference for transferrin. The affinity of the ruthenium complex for holo-transferrin was higher than for the apo-form suggesting a cooperative iron-mediated metal binding mechanism. RAPTA-T binding to holo-transferrin was further investigated by electrospray mass spectrometry using both the intact protein and a model peptide mimicking the iron-binding pocket.
The gene from Streptomyces coelicolor A3(2) encoding CYP102B1, a recently discovered CYP102 subfamily which exists solely as a single P450 heme domain, has been cloned, expressed in Escherichia coli, purified, characterized, and compared to its fusion protein family members. Purified reconstitution metabolism experiments with spinach ferredoxin, ferredoxin reductase, and NADPH revealed differences in the regio- and stereoselective metabolism of arachidonic acid compared to that of CYP102A1, exclusively producing 11,12-epoxyeicosa-5,8,14-trienoic acid in addition to the shared metabolites 18-hydroxy arachidonic acid and 14,15-epoxyeicosa-5,8,11-trienoic acid. Consequently, in order to elucidate the physiological function of CYP102B1, transposon mutagenesis was used to generate an S. coelicolor A3(2) strain lacking CYP102B1 activity and the phenotype was assessed.
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.
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.
A series of sulfonium halides bearing allyl groups have been prepared and characterized. Anion metathesis with Li[Tf2N] and Ag[N(CN)2] resulted in sulfonium-based ionic liquids which exhibit low viscosities at room temperature. The solid state structure of one of the halide salts was determined by single crystal X-ray diffraction.
Sulfonium salts; ionic liquids; allyl group; viscosity; X-ray structure
Members of a family of serine/threonine protein kinases (STPKs), unique to gram-positive bacteria, comprise an intracellular kinase domain and reiterated extracellular PASTA (for “penicillin-binding protein and serine/threonine kinase associated”) domains. PASTA domains exhibit low affinity for β-lactam antibiotics that are structurally similar to their likely normal ligands: stem peptides of unlinked peptidoglycan. The PASTA-domain STPKs are found in the actinobacteria and firmicutes and, as exemplified by PknB of Mycobacterium tuberculosis, they are functionally implicated in aspects of growth, cell division, and development. Whereas the kinase domains are well conserved, there is a wide divergence in the sequences of the multiple PASTA domains. Closer inspection reveals position-dependent evolution of individual PASTA domains: a domain at one position within a gene has a close phylogenetic relationship with a domain at a similar position in an orthologous gene, whereas neighboring domains have clearly diverged one from one another. A similar position-dependent relationship is demonstrated in the second family of proteins with multiple PASTA domains: the high-molecular-weight type II penicillin-binding protein (PBP2x) family. These transpeptidases are recruited to the division site by a localized pool of unlinked peptidoglycan. We infer that protein localization is guided by low-affinity interactions between structurally different unlinked peptidoglycan stem peptides and individual PASTA domains. The STPKs possess a greater multiplicity and diversity of PASTA domains, allowing interactions with a wider range of stem-peptide ligands. These interactions are believed to activate the intracellular kinase domain, allowing an STPK to coordinate peptidoglycan remodeling and reproduction of a complex cell wall structure.
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.
Comprehensive mutant libraries can be readily constructed by transposon mutagenesis. To systematically mutagenise the genome of the Gram-positive bacterium Streptomyces coelicolor A3(2), we have employed high-throughput shuttle transposon mutagenesis of a cosmid library prepared in Escherichia coli. The location of transposon insertions is determined using automated procedures for cosmid isolation and DNA sequencing. However, a major bottleneck was the subsequent analysis of DNA sequence files. To overcome this limitation, a software application, Transposon Express, was written to allow the rapid location of transposon insertions in a sequenced genome (available at http://www.swan.ac.uk/genetics/dyson/InstallTE). Transposon Express determines the identity both of a disrupted open reading frame (ORF), and the short target site duplication created by transposition. Transposon Express also reports the orientation of the transposon and can therefore predict transcriptional coupling between an upstream promoter and a promoter-less reporter gene carried by the transposon. Analysis of a large dataset of independent insertions created using a Tn5-based transposon revealed an insertional preference for GC-rich streptomycete DNA compared to E.coli vector DNA. In addition to demonstrating the value of Transposon Express as a generic tool supporting genome-wide transposon mutagenesis programs, these data provide insight into target site selection by Tn5.
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.