A connection between stress and development in the multicelular prokaryote Streptomyces coelicolor

Morphological changes leading to aerial mycelium formation and sporulation in the mycelial bacterium Streptomyces coelicolor rely on establishing distinct patterns of gene expression in separate regions of the colony. sH was identified previously as one of three paralogous sigma factors associated with stress responses in S. coelicolor. Here, we show that sigH and the upstream gene prsH (encoding a putative antisigma factor of sH) form an operon transcribed from two developmentally regulated promoters, sigHp1 and sigHp2. While sigHp1 activity is confined to the early phase of growth, transcription of sigHp2 is dramatically induced at the time of aerial hyphae formation. Localization of sigHp2 activity using a transcriptional fusion to the green fluorescent protein reporter gene (sigHp2–egfp) showed that sigHp2 transcription is spatially restricted to sporulating aerial hyphae in wild-type S. coelicolor. However, analysis of mutants unable to form aerial hyphae (bld mutants) showed that sigHp2 transcription and sH protein levels are dramatically upregulated in a bldD mutant, and that the sigHp2–egfp fusion was expressed ectopically in the substrate mycelium in the bldD background. Finally, a protein possessing sigHp2 promoter-binding activity was purified to homogeneity from crude mycelial extracts of S. coelicolor and shown to be BldD. The BldD binding site in the sigHp2 promoter was defined by DNase I footprinting. These data show that expression of sH is subject to temporal and spatial regulation during colony development, that this tissue-specific regulation is mediated directly by the developmental transcription factor BldD and suggest that stress and developmental programmes may be intimately connected in Streptomyces morphogenesis

Cell-biological studies of osmotic shock response in Streptomyces spp.

Most bacteria are likely to face osmotic challenges, but there is yet much to learn about how such environmental changes affect the architecture of bacterial cells. Here, we report a cell-biological study in model organisms of the genus Streptomyces, which are actinobacteria that grow in a highly polarized fashion to form branching hyphae. The characteristic apical growth of Streptomyces hyphae is orchestrated by protein assemblies, called polarisomes, which contain coiled-coil proteins DivIVA and Scy, and recruit cell wall synthesis complexes and the stressbearing cytoskeleton of FilP to the tip regions of the hyphae. We monitored cell growth and cell-architectural changes by time-lapse microscopy in osmotic upshift experiments. Hyperosmotic shock caused arrest of growth, loss of turgor, and hypercondensation of chromosomes. The recovery period was protracted, presumably due to the dehydrated state of the cytoplasm, before hyphae could restore their turgor and start to grow again. In most hyphae, this regrowth did not take place at the original hyphal tips. Instead, cell polarity was reprogrammed, and polarisomes were redistributed to new sites, leading to the emergence of multiple lateral branches from which growth occurred. Factors known to regulate the branching pattern of Streptomyces hyphae, such as the serine/threonine kinase AfsK and Scy, were not involved in reprogramming of cell polarity, indicating that different mechanisms may act under different environmental conditions to control hyphal branching. Our observations of hyphal morphology during the stress response indicate that turgor and sufficient hydration of cytoplasm are required for Streptomyces tip growth

Imaging of Streptomyces coelicolor A3(2) with Reduced Autofluorescence Reveals a Novel Stage of FtsZ Localization

Imaging of low abundance proteins in time and space by fluorescence microscopy is typically hampered by host-cell autofluorescence. Streptomycetes are an important model system for the study of bacterial development, and undergo multiple synchronous cell division during the sporulation stage. To analyse this phenomenon in detail, fluorescence microscopy, and in particular also the recently published novel live imaging techniques, require optimal signal to noise ratios. Here we describe the development of a novel derivative of Streptomyces coelicolor A3(2) with strongly reduced autofluorescence, allowing the imaging of fluorescently labelled proteins at significantly higher resolution. The enhanced image detail provided novel localization information for the cell division protein FtsZ, demonstrating a new developmental stage where multiple FtsZ foci accumulate at the septal plane. This suggests that multiple foci are sequentially produced, ultimately connecting to form the complete Z ring. The enhanced imaging properties are an important step forward for the confocal and live imaging of less abundant proteins and for the use of lower intensity fluorophores in streptomycetes

Metabolic reconstruction of sulfur assimilation in the extremophile Acidithiobacillus ferrooxidans based on genome analysis

BACKGROUND: Acidithiobacillus ferrooxidans is a gamma-proteobacterium that lives at pH2 and obtains energy by the oxidation of sulfur and iron. It is used in the biomining industry for the recovery of metals and is one of the causative agents of acid mine drainage. Effective tools for the study of its genetics and physiology are not in widespread use and, despite considerable effort, an understanding of its unusual physiology remains at a rudimentary level. Nearly complete genome sequences of A. ferrooxidans are available from two public sources and we have exploited this information to reconstruct aspects of its sulfur metabolism. RESULTS: Two candidate mechanisms for sulfate uptake from the environment were detected but both belong to large paralogous families of membrane transporters and their identification remains tentative. Prospective genes, pathways and regulatory mechanisms were identified that are likely to be involved in the assimilation of sulfate into cysteine and in the formation of Fe-S centers. Genes and regulatory networks were also uncovered that may link sulfur assimilation with nitrogen fixation, hydrogen utilization and sulfur reduction. Potential pathways were identified for sulfation of extracellular metabolites that may possibly be involved in cellular attachment to pyrite, sulfur and other solid substrates. CONCLUSIONS: A bioinformatic analysis of the genome sequence of A. ferrooxidans has revealed candidate genes, metabolic process and control mechanisms potentially involved in aspects of sulfur metabolism. Metabolic modeling provides an important preliminary step in understanding the unusual physiology of this extremophile especially given the severe difficulties involved in its genetic manipulation and biochemical analysis

DNA recognition and transcriptional regulation by the WhiA sporulation factor

Sporulation in the filamentous bacteria Streptomyces coelicolor is a tightly regulated process involving aerial hyphae growth, chromosome segregation, septation and spore maturation. Genetic studies have identified numerous genes that regulate sporulation, including WhiA and the sigma factor WhiG. WhiA, which has been postulated to be a transcriptional regulator, contains two regions typically associated with DNA binding: an N-terminal domain similar to LAGLIDADG homing endonucleases, and a C-terminal helix-turn-helix domain. We characterized several in vitro activities displayed by WhiA. It binds at least two sporulation-specific promoters: its own and that of parABp2. DNA binding is primarily driven by its HTH domain, but requires full-length protein for maximum affinity. WhiA transcription is stimulated by WhiG, while the WhiA protein binds directly to WhiG (leading to inhibition of WhiG-dependent transcription). These separate activities, which resemble a possible feedback loop, may help coordinate the closely timed cessation of aerial growth and subsequent spore formation

The SsgA-like proteins in actinomycetes: small proteins up to a big task

Several unique protein families have been identified that play a role in the control of developmental cell division in streptomycetes. The SsgA-like proteins or SALPs, of which streptomycetes typically have at least five paralogues, control specific steps of sporulation-specific cell division in streptomycetes, affecting cell wall-related events such as septum localization and synthesis, thickening of the spore wall and autolytic spore separation. The expression level of SsgA, the best studied SALP, has a rather dramatic effect on septation and on hyphal morphology, which is not only of relevance for our understanding of (developmental) cell division but has also been succesfully applied in industrial fermentation, to improve growth and production of filamentous actinomycetes. Recent observations suggest that SsgB most likely is the archetypal SALP, with only SsgB orthologues occurring in all morphologically complex actinomycetes. Here we review 10 years of research on the SsgA-like proteins in actinomycetes and discuss the most interesting regulatory, functional, phylogenetic and applied aspects of this relatively unknown protein family

Constitutive expression of ftsZ overrides the whi developmental genes to initiate sporulation of Streptomyces coelicolor

The filamentous soil bacteria Streptomyces undergo a highly complex developmental programme. Before streptomycetes commit themselves to sporulation, distinct morphological checkpoints are passed in the aerial hyphae that are subject to multi-level control by the whi sporulation genes. Here we show that whi-independent expression of FtsZ restores sporulation to the early sporulation mutants whiA, whiB, whiG, whiH, whiI and whiJ. Viability, stress resistance and high-resolution electron microscopy underlined that viable spores were formed. However, spores from sporulation-restored whiA and whiG mutants showed defects in DNA segregation/condensation, while spores from the complemented whiB mutant had increased stress sensitivity, perhaps as a result of changes in the spore sheath. In contrast to the whi mutants, normal sporulation of ssgB null mutants—which fail to properly localise FtsZ—could not be restored by enhancing FtsZ protein levels, forming spore-like bodies that lack spore walls. Our data strongly suggest that the whi genes control a decisive event towards sporulation of streptomycetes, namely the correct timing of developmental ftsZ transcription. The biological significance may be to ensure that sporulation-specific cell division will only start once sufficient aerial mycelium biomass has been generated. Our data shed new light on the longstanding question as to how whi genes control sporulation, which has intrigued scientists for four decades

Exploring the symbiotic pangenome of the nitrogen-fixing bacterium Sinorhizobium meliloti

<p>Abstract</p> <p>Background</p> <p><it>Sinorhizobium meliloti </it>is a model system for the studies of symbiotic nitrogen fixation. An extensive polymorphism at the genetic and phenotypic level is present in natural populations of this species, especially in relation with symbiotic promotion of plant growth. AK83 and BL225C are two nodule-isolated strains with diverse symbiotic phenotypes; BL225C is more efficient in promoting growth of the <it>Medicago sativa </it>plants than strain AK83. In order to investigate the genetic determinants of the phenotypic diversification of <it>S. meliloti </it>strains AK83 and BL225C, we sequenced the complete genomes for these two strains.</p> <p>Results</p> <p>With sizes of 7.14 Mbp and 6.97 Mbp, respectively, the genomes of AK83 and BL225C are larger than the laboratory strain Rm1021. The core genome of Rm1021, AK83, BL225C strains included 5124 orthologous groups, while the accessory genome was composed by 2700 orthologous groups. While Rm1021 and BL225C have only three replicons (Chromosome, pSymA and pSymB), AK83 has also two plasmids, 260 and 70 Kbp long. We found 65 interesting orthologous groups of genes that were present only in the accessory genome, consequently responsible for phenotypic diversity and putatively involved in plant-bacterium interaction. Notably, the symbiosis inefficient AK83 lacked several genes required for microaerophilic growth inside nodules, while several genes for accessory functions related to competition, plant invasion and bacteroid tropism were identified only in AK83 and BL225C strains. Presence and extent of polymorphism in regulons of transcription factors involved in symbiotic interaction were also analyzed. Our results indicate that regulons are flexible, with a large number of accessory genes, suggesting that regulons polymorphism could also be a key determinant in the variability of symbiotic performances among the analyzed strains.</p> <p>Conclusions</p> <p>In conclusions, the extended comparative genomics approach revealed a variable subset of genes and regulons that may contribute to the symbiotic diversity.</p