Matthew I. Hutchings

Cell envelope stress response in Gram‐positive bacteria

The bacterial cell envelope is the first and major line of defence against threats from the environment. It is an essential and yet vulnerable structure that gives the cell its shape and counteracts the high internal osmotic pressure. It also provides an important sensory interface and molecular sieve, mediating both information flow and the controlled transport of solutes. The cell envelope is also the target for numerous antibiotics. Therefore, the monitoring and maintenance of cell envelope integrity in the presence of envelope perturbating agents and conditions is crucial for survival. The underlying signal transduction is mediated by two regulatory principles, two-component systems and extracytoplasmic function sigma factors, in both the Firmicutes (low-GC) and Actinobacteria (high-GC) branches of Gram-positive bacteria. This study presents a comprehensive overview of cell envelope stress-sensing regulatory systems. This knowledge will then be applied for in-depth comparative genomics analyses to emphasize the distribution and conservation of cell envelope stress-sensing systems. Finally, the cell envelope stress response will be placed in the context of the overall cellular physiology, demonstrating that its regulatory systems are linked not only to other stress responses but also to the overall homeostasis and lifestyle of Gram-positive bacteria.

Streptomyces as symbionts; an emerging and widespread theme?

Streptomyces bacteria are ubiquitous in soil, conferring the characteristic earthy smell, and they have an important ecological role in the turnover of organic material. More recently, a new picture has begun to emerge in which streptomycetes are not in all cases simply free-living soil bacteria but have also evolved to live in symbiosis with plants, fungi and animals. Furthermore, much of the chemical diversity of secondary metabolites produced by Streptomyces species has most likely evolved as a direct result of their interactions with other organisms. Here we review what is currently known about the role of streptomycetes as symbionts with fungi, plants and animals. These interactions can be parasitic, as is the case for scab-causing streptomycetes, which infect plants, and the Streptomyces species Streptomyces somaliensis and Streptomyces sudanensis that infect humans. However, in most cases they are beneficial and growth promoting, as is the case with many insects, plants and marine animals that use streptomycete-produced antibiotics to protect themselves against infection. This is an exciting and newly emerging field of research that will become increasingly important as the search for new antibiotics switches to unusual and under-explored environments.

Lipoprotein biogenesis in Gram-positive bacteria: knowing when to hold ‘em, knowing when to fold ‘em

Gram-positive bacterial lipoproteins are a functionally diverse and important class of peripheral membrane proteins. Recent advances in molecular biology and the availability of whole genome sequence data have overturned many long-held assumptions about the export and processing of these proteins, most notably the recent discovery that not all lipoproteins are exported as unfolded substrates through the general secretion pathway. Here, we review recent discoveries concerning the export and processing of these proteins, their role in virulence in Gram-positive bacteria and their potential as vaccine candidates or targets for new antimicrobials.

A mixed community of actinomycetes produce multiple antibiotics for the fungus farming ant Acromyrmex octospinosus

BackgroundAttine ants live in an intensely studied tripartite mutualism with the fungus Leucoagaricus gongylophorus, which provides food to the ants, and with antibiotic-producing actinomycete bacteria. One hypothesis suggests that bacteria from the genus Pseudonocardia are the sole, co-evolved mutualists of attine ants and are transmitted vertically by the queens. A recent study identified a Pseudonocardia-produced antifungal, named dentigerumycin, associated with the lower attine Apterostigma dentigerum consistent with the idea that co-evolved Pseudonocardia make novel antibiotics. An alternative possibility is that attine ants sample actinomycete bacteria from the soil, selecting and maintaining those species that make useful antibiotics. Consistent with this idea, a Streptomyces species associated with the higher attine Acromyrmex octospinosus was recently shown to produce the well-known antifungal candicidin. Candicidin production is widespread in environmental isolates of Streptomyces, so this could either be an environmental contaminant or evidence of recruitment of useful actinomycetes from the environment. It should be noted that the two possibilities for actinomycete acquisition are not necessarily mutually exclusive.ResultsIn order to test these possibilities we isolated bacteria from a geographically distinct population of A. octospinosus and identified a candicidin-producing Streptomyces species, which suggests that they are common mutualists of attine ants, most probably recruited from the environment. We also identified a Pseudonocardia species in the same ant colony that produces an unusual polyene antifungal, providing evidence for co-evolution of Pseudonocardia with A. octospinosus.ConclusionsOur results show that a combination of co-evolution and environmental sampling results in the diversity of actinomycete symbionts and antibiotics associated with attine ants.

The transcriptional repressor protein NsrR senses nitric oxide directly via a [2Fe-2S] cluster

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.

The NorR protein of Escherichia coli activates expression of the flavorubredoxin gene norV in response to reactive nitrogen species.

The Escherichia coli norVW genes encode a flavorubredoxin and NADH:(flavo)rubredoxin reductase, respectively, which are involved in nitric oxide detoxification under anaerobic growth conditions. Here it is shown that the norVW genes also have a role in protection against reactive nitrogen intermediates generated from nitroprusside. Transcription from the norV promoter is activated by the presence of nitroprusside in the growth medium; activation requires the product of a divergently transcribed regulatory gene, norR.

Characterization of an inducible vancomycin resistance system in Streptomyces coelicolor reveals a novel gene (vanK) required for drug resistance

Vancomycin is the front‐line therapy for treating problematic infections caused by methicillin‐resistant Staphylococcus aureus (MRSA), and the spread of vancomycin resistance is an acute problem. Vancomycin blocks cross‐linking between peptidoglycan intermediates by binding to the d‐Ala‐d‐Ala termini of bacterial cell wall precursors, which are the substrate of transglycosylase/transpeptidase. We have characterized a cluster of seven genes (vanSRJKHAX) in Streptomyces coelicolor that confers inducible, high‐level vancomycin resistance. vanHAX are orthologous to genes found in vancomycin‐resistant enterococci that encode enzymes predicted to reprogramme peptidoglycan biosynthesis such that cell wall precursors terminate in d‐Ala‐d‐Lac rather than d‐Ala‐d‐Ala. vanR and vanS encode a two‐component signal transduction system that mediates transcriptional induction of the seven van genes. vanJ and vanK are novel genes that have no counterpart in previously characterized vancomycin resistance clusters from pathogens. VanK is a member of the Fem family of enzymes that add the cross‐bridge amino acids to the stem pentapeptide of cell wall precursors, and vanK is essential for vancomycin resistance. The van genes are organized into four transcription units, vanRS, vanJ, vanK and vanHAX, and these transcripts are induced by vancomycin in a vanR‐dependent manner. To develop a sensitive bioassay for inducers of the vancomycin resistance system, the promoter of vanJ was fused to a reporter gene conferring resistance to kanamycin. All the inducers identified were glycopeptide antibiotics, but teicoplanin, a membrane‐anchored glycopeptide, failed to act as an inducer. Analysis of mutants defective in the vanRS and cseBC cell envelope signal transduction systems revealed significant cross‐talk between the two pathways.

The Role of the Novel Fem Protein VanK in Vancomycin Resistance in Streptomyces coelicolor

The non-pathogenic, non-glycopeptide-producing actinomycete Streptomyces coelicolor carries a cluster of seven genes (vanSRJKHAX) that confers inducible, high level resistance to vancomycin. The vanK gene has no counterpart in previously characterized vancomycin resistance clusters, yet vanK is required for vancomycin resistance in S. coelicolor. VanK belongs to the Fem family of enzymes, which add the branch amino acid(s) to the stem pentapeptide of peptidoglycan precursors. Upon exposure to vancomycin, the VanRS two-component system switches on expression of all seven van genes, and the VanHAX enzymes reprogram the cell wall such that precursors terminate d-Ala-d-lactate (Lac) rather than d-Ala-d-Ala, thus conferring resistance to vancomycin, which only binds d-Ala-d-Ala-containing precursors. Here we provide biochemical and genetic evidence that VanK is required for vancomycin resistance because the constitutively expressed FemX enzyme, encoded elsewhere on the chromosome, cannot recognize d-Lac-containing precursors as a substrate, whereas VanK can. Consistent with this view, d-Lac-containing precursors carrying the Gly branch are present in the wild type transiently exposed to vancomycin but are undetectable in a vanK mutant treated in the same way. Further, femX null mutants are viable in the presence of vancomycin but die in its absence. Because only VanK can recognize d-Lac-containing precursors, vancomycin-induced expression of VanHAX in a vanK mutant is lethal, and so vanK is required for vancomycin resistance.

The vancomycin resistance VanRS two-component signal transduction system of Streptomyces coelicolor.

We took advantage of the vancomycin‐dependent phenotype of Streptomyces coelicolor femX null mutants to isolate a collection of spontaneous, drug‐independent femX suppressor mutants that expressed the vancomycin‐resistance (van) genes constitutively. All of the suppressor mutations were in vanS but, unexpectedly, many were predicted to be loss‐of‐function mutations. Confirming this interpretation, a constructed vanS deletion mutation also resulted in constitutive expression of the van genes, suggesting that VanS negatively regulated VanR function in the absence of drug. In contrast, a vanS pta ackA triple mutant, which should not be able synthesize acetyl phosphate, failed to express the van genes, whereas a pta ackA double mutant showed wild‐type, regulated induction of the van genes. These results suggest that in the absence of vancomycin, acetyl phosphate phosphorylates VanR, and VanS acts as a phosphatase to suppress the levels of VanR∼P. On exposure to vancomycin, VanS activity switches from a phosphatase to a kinase and vancomycin resistance is induced. In S. coelicolor, the van genes are induced by both vancomycin and the glycopeptide A47934, whereas in Streptomyces toyocaensis (the A47934 producer) resistance is induced by A47934 but not by vancomycin. We exploited this distinction to replace the S. coelicolor vanRS genes with the vanRS genes from S. toyocaensis. The resulting strain acquired the inducer profile of S. toyocaensis, providing circumstantial evidence that the VanS effector ligand is the drug itself, and not an intermediate in cell wall biosynthesis that accumulates as result of drug action. Consistent with this suggestion, we found that non‐glycopeptide inhibitors of the late steps in cell wall biosynthesis such as moenomycin A, bacitracin and ramoplanin were not inducers of the S. coelicolor VanRS system, in contrast to results obtained in enterococcal VanRS systems.

A Single Streptomyces Symbiont Makes Multiple Antifungals to Support the Fungus Farming Ant Acromyrmex octospinosus

Attine ants are dependent on a cultivated fungus for food and use antibiotics produced by symbiotic Actinobacteria as weedkillers in their fungus gardens. Actinobacterial species belonging to the genera Pseudonocardia, Streptomyces and Amycolatopsis have been isolated from attine ant nests and shown to confer protection against a range of microfungal weeds. In previous work on the higher attine Acromyrmex octospinosus we isolated a Streptomyces strain that produces candicidin, consistent with another report that attine ants use Streptomyces-produced candicidin in their fungiculture. Here we report the genome analysis of this Streptomyces strain and identify multiple antibiotic biosynthetic pathways. We demonstrate, using gene disruptions and mass spectrometry, that this single strain has the capacity to make candicidin and multiple antimycin compounds. Although antimycins have been known for >60 years we report the sequence of the biosynthetic gene cluster for the first time. Crucially, disrupting the candicidin and antimycin gene clusters in the same strain had no effect on bioactivity against a co-evolved nest pathogen called Escovopsis that has been identified in ∼30% of attine ant nests. Since the Streptomyces strain has strong bioactivity against Escovopsis we conclude that it must make additional antifungal(s) to inhibit Escovopsis. However, candicidin and antimycins likely offer protection against other microfungal weeds that infect the attine fungal gardens. Thus, we propose that the selection of this biosynthetically prolific strain from the natural environment provides A. octospinosus with broad spectrum activity against Escovopsis and other microfungal weeds.