- Volume 165, Issue 10, 2019

Gram-positive Actinobacteria from the genus Streptomyces are best known for their morphological complexity and for their ability to produce numerous bioactive specialized metabolites with useful applications in human and veterinary medicine and in agriculture. In contrast, the ability to infect living plant tissues and to cause diseases of root and tuber crops such as potato common scab (CS) is a rare attribute among members of this genus. Research on the virulence mechanisms of plant-pathogenic Streptomyces spp. has revealed the importance of the thaxtomin phytotoxins as key pathogenicity determinants produced by several species. In addition, other phytotoxic specialized metabolites may contribute to the development or severity of disease caused by Streptomyces spp., along with the production of phytohormones and secreted proteins. A thorough understanding of the molecular mechanisms of plant pathogenicity will enable the development of better management procedures for controlling CS and other plant diseases caused by the Streptomyces .

In radiating populations of Pseudomonas fluorescens SBW25, adaptive wrinkly spreader (WS) mutants are able to gain access to the air–liquid (A–L) interface of static liquid microcosms and achieve a significant competitive fitness advantage over other non-biofilm-forming competitors. Aerotaxis and flagella-based swimming allows SBW25 cells to move into the high-O2 region located at the top of the liquid column and maintain their position by countering the effects of random cell diffusion, convection and disturbance (i.e. physical displacement). However, wild-type cells showed significantly lower levels of enrichment in this region compared to the archetypal WS, indicating that WS cells employ an additional mechanism to transfer to the A–L interface where displacement is no longer an issue and a biofilm can develop at the top of the liquid column. Preliminary experiments suggest that this might be achieved through the expression of an as yet unidentified surface active agent that is weakly associated with WS cells and alters liquid surface tension, as determined by quantitative tensiometry. The effect of physical displacement on the colonization of the high-O2 region and A–L interface was reduced through the addition of agar or polyethylene glycol to increase liquid viscosity, and under these conditions the competitive fitness of the WS was significantly reduced. These observations suggest that the ability to transfer to the A–L interface from the high-O2 region and remain there without further expenditure of energy (through, for example, the deployment of flagella) is a key evolutionary innovation of the WS, as it allows subsequent biofilm development and significant population increase, thereby affording these adaptive mutants a competitive fitness advantage over non-biofilm-forming competitors located within the liquid column.

Fusarium verticillioides is one of the key fungal pathogens responsible for maize stalk rot. While stalk rot pathogens are prevalent worldwide, our understanding of the stalk rot virulence mechanism in pathogenic fungi is still very limited. We previously identified the F. verticillioides FvSYN1 gene, which was demonstrated to play an important role in maize stalk rot virulence. FvSyn1 belongs to a family of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins that play critical roles in a variety of developmental processes. In this study, we further characterized the cellular features of the FvSyn1 protein, namely how different motifs contribute to development and virulence in F. verticillioides by generating motif-specific deletion mutants. Microscopic observation showed that the ∆Fvsyn1 mutant exhibits rough and hyper-branched hyphae when compared to the wild-type progenitor. Moreover, the ∆Fvsyn1 mutant was sensitive to cell wall stress agents, resulting in vegetative growth reduction. We showed that the FvSyn1::GFP protein is associated with the endomembrane, but this did not clarify why the deletion of FvSyn1 led to stress sensitivity and aberrant hyphal development. Characterization of the FvSyn1 domains indicated that both the syntaxin N-terminus (SynN) domain and the SNARE C-terminus domain play distinct roles in fungal development, but also function collectively in the context of virulence. We also determined that two domains in FvSyn1 are not required for fumonisin production. Interestingly, these two domains were involved in carbon nutrient utilization, including pectin, starch and sorbitol. This study further characterized the role of FvSyn1 domains in hyphal growth, cell wall stress response and virulence in F. verticillioides.

Reconciling transcription and DNA replication in the growing hyphae of the filamentous bacterium Streptomyces presents several physical constraints on growth due to their apically extending and branching, multigenomic cells and chromosome replication being independent of cell division. Using a GFP translational fusion to the β′-subunit of RNA polymerase (rpoC-egfp), in its native chromosomal location, we observed growing Streptomyces hyphae using time-lapse microscopy throughout the lifecycle and under different growth conditions. The RpoC-eGFP fusion co-localized with DNA around 1.8 µm behind the extending tip, whereas replisomes localize around 4–5 µm behind the tip, indicating that at the growing tip, transcription and chromosome replication are to some degree spatially separated. Dual-labelled RpoC-egfp/DnaN-mCherry strains also indicate that there is limited co-localization of transcription and chromosome replication at the extending hyphal tip. This likely facilitates the use of the same DNA molecule for active transcription and chromosome replication in growing cells, independent of cell division. This represents a novel, but hitherto unknown mechanism for reconciling two fundamental processes that utilize the same macromolecular template that allows for rapid growth without compromising chromosome replication in filamentous bacteria and may have implications for evolution of filamentous growth in micro-organisms, where uncoupling of DNA replication from cell division is required.

The Agrobacterium tumefaciens ActSR two-component regulatory system is a member of a homologous group of global redox-responsive regulatory systems that adjust the expression of energy-consuming and energy-supplying metabolic pathways in order to maintain cellular redox balance. In this study, the transcriptional organization of the hrpB-actSR locus was determined and the effect of actSR system inactivation on stress resistance was investigated. It was found that hrpB is transcribed as a monocistronic mRNA and actS is transcribed along with actR as a bicistronic mRNA, while actR is also transcribed as a monocistronic message. Each message is initiated from a separate promoter. Inactivation of actR resulted in decreased resistance to membrane stress (sodium dodecyl sulfate), acid stress (pH 5.5), iron starvation (bipyridyl) and iron excess (FeCl3), and antibiotic stress (tetracycline and ciprofloxacin). Resistance to oxidative stress in the form of organic peroxide (cumene hydroperoxide) increased, while resistance to inorganic peroxide (H2O2) decreased. An actR insertion mutant displayed reduced catalase activity, even though transcription of katA and catE remained unchanged. Complementation of the actR inactivation mutant with plasmid-encoded actR or overexpression of hemH, encoding ferrochelatase, restored wild-type catalase activity and H2O2 resistance levels. Gel mobility shift and hemH promoter–lacZ fusion results indicated that ActR is a positive regulator of hemH that binds directly to the hemH promoter region. Thus, inactivation of the A. tumefaciens ActSR system affects resistance to multiple stresses, including reduced resistance to H2O2 resulting from a reduction in catalase activity due to reduced expression of hemH.