visual-microscopic-evaluation-streptomyces-developmental

Visual and Microscopic Evaluation of <em>Streptomyces</em> Developmental Mutants

Streptomycetes are filamentous soil bacteria belonging to the phylum Actinobacteria that are found throughout the world and produce a wide array of ...

Duquesne University

We have characterized homologues of the bacterial cell division genes ftsL and divIC in the gram-positive mycelial bacterium Streptomyces coelicolor A3(2). We show by deletion-insertion mutations that ftsL and divIC are dispensable for growth and viability in S. coelicolor. When mutant strains were grown on a conventional rich medium (R2YE, containing high sucrose), inactivation of either ftsL or divIC resulted in the formation of aerial hyphae with partially constricted division sites but no clear separation of prespore compartments. Surprisingly, this phenotype was largely suppressed when strains were grown on minimal medium or sucrose-free R2YE, where division sites in many aerial hyphae had finished constricting and chains of spores were evident. Thus, osmolarity appears to affect the severity of the division defect. Furthermore, double mutant strains deleted for both ftsL and divIC are viable and have medium-dependent phenotypes similar to that of the single mutant strains, suggesting that functions performed by FtsL and DivIC are not absolutely required for septation during growth and sporulation. Alternatively, another division protein may partially compensate for the loss of both FtsL and DivIC on minimal medium or sucrose-free R2YE. Finally, based on transmission electron microscopy observations, we propose that FtsL and DivIC are involved in coordinating symmetrical annular ingrowth of the invaginating septum.

Streptomyces coelicolor genes ftsL and divIC play a role in cell division but are dispensable for colony formation.

Streptomyces coelicolor A3(2) ftsI- and ftsW-null mutants produced aerial hyphae with no evidence of septation when grown on a traditional osmotically enhanced medium. This phenotype was partially suppressed when cultures were grown on media prepared without sucrose. We infer that functional FtsZ rings can form in ftsI- and ftsW-null mutants under certain growth conditions.

Medium-dependent phenotypes of Streptomyces coelicolor with mutations in ftsI or ftsW.

The mechanisms by which chromosomes condense and segregate during developmentally regulated cell division are of interest for Streptomyces coelicolor, a sporulating, filamentous bacterium with a large, linear genome. These processes coordinately occur as many septa synchronously form in syncytial aerial hyphae such that prespore compartments accurately receive chromosome copies. Our genetic approach analyzed mutants for ftsK, smc, and parB. DNA motor protein FtsK/SpoIIIE coordinates chromosome segregation with septum closure in rod-shaped bacteria. SMC (structural maintenance of chromosomes) participates in condensation and organization of the nucleoid. ParB/Spo0J partitions the origin of replication using a nucleoprotein complex, assembled at a centromere-like sequence. Consistent with previous work, we show that an ftsK-null mutant produces anucleate spores at the same frequency as the wild-type strain (0.8%). We report that the smc and ftsK deletion-insertion mutants (ftsK' truncation allele) have developmental segregation defects (7% and 15% anucleate spores, respectively). By use of these latter mutants, viable double and triple mutants were isolated in all combinations with a previously described parB-null mutant (12% anucleate spores). parB and smc were in separate segregation pathways; the loss of both exacerbates the segregation defect (24% anucleate spores). For a triple mutant, deletion of the region encoding the FtsK motor domain and one transmembrane segment partially alleviates the segregation defect of the smc parB mutant (10% anucleate spores). Considerable redundancy must exist in this filamentous organism because segregation of some genomic material occurs 90% of the time during development in the absence of three functions with only a fourfold loss of spore viability. Furthermore, we report that scpA and scpAB mutants (encoding SMC-associated proteins) have spore nucleoid organization defects. Finally, FtsK-enhanced green fluorescent protein (EGFP) localized as bands or foci between incipient nucleoids, while SMC-EGFP foci were not uniformly positioned along aerial hyphae, nor were they associated with every condensing nucleoid.

Genetic interactions of smc, ftsK, and parB genes in Streptomyces coelicolor and their developmental genome segregation phenotypes.

In part, members of the genus Streptomyces have been studied because they produce many important secondary metabolites with antibiotic activity and for the interest in their relatively elaborate life cycle. These sporulating filamentous bacteria are remarkably synchronous for division and genome segregation in specialized aerial hyphae. Streptomycetes share some, but not all, of the division genes identified in the historic model rod-shaped organisms. Curiously, normally essential cell division genes are dispensable for growth and viability of Streptomyces coelicolor. Mainly, cell division plays a more important role in the developmental phase of life than during vegetative growth. Dispensability provides an advantageous genetic system to probe the mechanisms of division proteins, especially those with functions that are poorly understood.

Cell division is dispensable but not irrelevant in Streptomyces.

Streptomyces coelicolor is the genetically best characterized species of a populous genus belonging to the gram-positive Actinobacteria. Streptomycetes are filamentous soil organisms, well known for the production of a plethora of biologically active secondary metabolic compounds. The Streptomyces developmental life cycle is uniquely complex and involves coordinated multicellular development with both physiological and morphological differentiation of several cell types, culminating in the production of secondary metabolites and dispersal of mature spores. This review presents a current appreciation of the signaling mechanisms used to orchestrate the decision to undergo morphological differentiation, and the regulators and regulatory networks that direct the intriguing development of multigenomic hyphae first to form specialized aerial hyphae and then to convert them into chains of dormant spores. This current view of S. coelicolor development is destined for rapid evolution as data from '-omics' studies shed light on gene regulatory networks, new genetic screens identify hitherto unknown players, and the resolution of our insights into the underlying cell biological processes steadily improve.

Signals and regulators that govern Streptomyces development.

The study of Streptomyces development has made significant advances in the past few years and ongoing work is poised to add even more. One key to advancing the field has been the application of genome-wide approaches using Streptomyces venezuelae, which is capable of fairly synchronous sporulation in submerged growth conditions. WhiA and WhiB are well-known transcriptional regulators governing the pathway for spore formation in aerial hyphae. Recent ChIP-seq and RNA expression analyses indicated that WhiA and WhiB regulate the same set of genes, each being dependent on the presence of the other to exert control. Functional WhiAB is believed to form when developmental accumulation of WhiB joins constitutive accumulation of WhiA, suggesting that an important developmental decision is the control of WhiB accumulation. Now, a new WhiAB-controlled gene called bldO has been described and characterized. Strikingly, BldO has one target for repression in the entire genome, whiB. BldO now joins pleiotropic repressor BldD to exert a multi-layer control of the temporal and spatial activity of WhiB. BldD activity is controlled by c-di-GMP concentration and BldO potentially responds to an unknown signal. Together BldO and BldD repress developmental genes from being expressed until the appropriate time.

The Streptomyces O-B one connection: a force within layered repression of a key developmental decision.

The central player in bacterial cell division, FtsZ, is essential in almost all organisms in which it has been tested, with the most notable exception being Streptomyces. Streptomycetes differ from many bacteria in growing from the cell tip and undergoing branching, similar to filamentous fungi. Here we show that limited cell damage, either mechanical or enzymatic, leads to near complete destruction of mycelial microcolonies of a Streptomyces venezuelae ftsZ mutant. This result is consistent with a lack of ftsZ-dependent cross-walls and may be inconsistent with a recently proposed role for membrane structures in the proliferation of ftsZ mutants in other Streptomyces species. Rare surviving fragments of mycelium, usually around branches, appear to be the preferred sites of resealing. Restoration of growth in hyphal fragments of both wild-type and ftsZ mutant hyphae can occur at multiple sites, via branch-like outgrowths containing DivIVA protein at their tips. Thus, our results highlight branching as a means of FtsZ-independent cell proliferation.

A mechanism for FtsZ-independent proliferation in Streptomyces.

Campylobacter jejuni, a human gastrointestinal pathogen, uses nitrate for growth under microaerophilic conditions using periplasmic nitrate reductase (Nap). The catalytic subunit, NapA, contains two prosthetic groups, an iron sulfur cluster and a molybdenum cofactor. Here we describe the cloning, expression, purification, and Michaelis-Menten kinetics (kcat of 5.91 ± 0.18 s-1 and a KM (nitrate) of 3.40 ± 0.44 μM) in solution using methyl viologen as an electron donor. The data suggest that the high affinity of NapA for nitrate could support growth of C. jejuni on nitrate in the gastrointestinal tract. Site-directed mutagenesis was used and the codon for the molybdenum coordinating cysteine residue has been exchanged for serine. The resulting variant NapA is 4-fold less active than the native enzyme confirming the importance of this residue. The properties of the C. jejuni enzyme reported here represent the first isolation and characterization of an epsilonproteobacterial NapA. Therefore, the fundamental knowledge of Nap has been expanded.

Joseph R. McCormick

Department of Biological Sciences

Duquesne University

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Joseph R. McCormick

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biological Sciences

Duquesne University

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Department of Biological Sciences