Cytoplasmic contractile injection systems mediate cell death in Streptomyces.

Contractile injection systems (CIS) are bacteriophage tail-like structures that mediate bacterial cell-cell interactions. While CIS are highly abundant across diverse bacterial phyla, representative gene clusters in Gram-positive organisms remain poorly studied. Here we characterize a CIS in the Gram-positive multicellular model organism Streptomyces coelicolor and show that, in contrast to most other CIS, S. coelicolor CIS (CISSc) mediate cell death in response to stress and impact cellular development. CISSc are expressed in the cytoplasm of vegetative hyphae and are not released into the medium. Our cryo-electron microscopy structure enabled the engineering of non-contractile and fluorescently tagged CISSc assemblies. Cryo-electron tomography showed that CISSc contraction is linked to reduced cellular integrity. Fluorescence light microscopy furthermore revealed that functional CISSc mediate cell death upon encountering different types of stress. The absence of functional CISSc had an impact on hyphal differentiation and secondary metabolite production. Finally, we identified three putative effector proteins, which when absent, phenocopied other CISSc mutants. Our results provide new functional insights into CIS in Gram-positive organisms and a framework for studying novel intracellular roles, including regulated cell death and life-cycle progression in multicellular bacteria.

Cap-tivating findings provide insight into bacterial cell division.

In most bacteria, cell division is orchestrated by the tubulin homolog FtsZ. To ensure the correct placement of the division machinery, FtsZ activity needs to be tightly regulated. Corrales-Guerrero et al. now describe the molecular details of how MipZ, an alphaproteobacterial regulator, interacts with FtsZ to promote proper cell division.

A conserved cell division protein directly regulates FtsZ dynamics in filamentous and unicellular actinobacteria.

Bacterial cell division is driven by the polymerization of the GTPase FtsZ into a contractile structure, the so-called Z-ring. This essential process involves proteins that modulate FtsZ dynamics and hence the overall Z-ring architecture. Actinobacteria like Streptomyces and Mycobacterium lack known key FtsZ-regulators. Here we report the identification of SepH, a conserved actinobacterial protein that directly regulates FtsZ dynamics. We show that SepH is crucially involved in cell division in Streptomyces venezuelae and that it binds FtsZ via a conserved helix-turn-helix motif, stimulating the assembly of FtsZ protofilaments. Comparative in vitro studies using the SepH homolog from Mycobacterium smegmatis further reveal that SepH can also bundle FtsZ protofilaments, indicating an additional Z-ring stabilizing function in vivo. We propose that SepH plays a crucial role at the onset of cytokinesis in actinobacteria by promoting the assembly of FtsZ filaments into division-competent Z-rings that can go on to mediate septum synthesis.

Molecular cloning, expression and biochemical characterization of periplasmic nitrate reductase from Campylobacter jejuni.

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.