Mobile CRISPRi moves through the complexity of bacterial genetics.

In this issue of Cell Reports Methods, Rachwalski et al. describe a high-throughput method to screen genetic interactions in bacteria using a conjugative CRISPR interference (CRISPRi) plasmid. The method enables systematic studies of gene essentiality in diverse genomic and environmental contexts and is applicable to Escherichia coli as well as other bacteria.

High incidence of carbapenemase-producing clinical isolates from Lagos, Nigeria.

Background: Carbapenem-resistant strains are on the rise worldwide. This study characterized clinical isolates of from three Nigerian hospitals for carbapenem resistance.

Methods: Strains isolated from wounds ( = 88), urine/catheter tips ( = 25), sputum/tracheotomy aspirates ( = 5), ear swabs ( = 4) and vaginal swabs ( = 1) were identified by MALDI-TOF and antibiotic susceptibility testing was performed using the VITEK 2 system.

DYRK kinase Pom1 drives F-BAR protein Cdc15 from the membrane to promote medial division.

In many organisms, positive and negative signals cooperate to position the division site for cytokinesis. In the rod-shaped fission yeast , symmetric division is achieved through anillin/Mid1-dependent positive cues released from the central nucleus and negative signals from the DYRK-family polarity kinase Pom1 at cell tips. Here we establish that Pom1's kinase activity prevents septation at cell tips even if Mid1 is absent or mislocalized.

Arbuscular cell invasion coincides with extracellular vesicles and membrane tubules.

During establishment of arbuscular mycorrhizal symbioses, fungal hyphae invade root cells producing transient tree-like structures, the arbuscules, where exchange of photosynthates for soil minerals occurs. Arbuscule formation and collapse lead to rapid production and degradation of plant and fungal membranes, their spatiotemporal dynamics directly influencing nutrient exchange. We determined the ultra-structural details of both membrane surfaces and the interstitial apoplastic matrix by transmission electron microscopy tomography during growth and senescence of Rhizophagus irregularis arbuscules in rice.

Ustilago maydis effectors and their impact on virulence.

Biotrophic fungal plant pathogens establish an intimate relationship with their host to support the infection process. Central to this strategy is the secretion of a range of protein effectors that enable the pathogen to evade plant immune defences and modulate host metabolism to meet its needs. In this Review, using the smut fungus Ustilago maydis as an example, we discuss new insights into the effector repertoire of smut fungi that have been gained from comparative genomics and discuss the molecular mechanisms by which U.

How filamentous plant pathogen effectors are translocated to host cells.

The interaction of microbes with "signature" plants is largely governed by secreted effector proteins, which serve to dampen plant defense responses and modulate host cell processes. Secreted effectors can function either in the apoplast or within plant cell compartments. How oomycetes and fungi translocate their effectors to plant cells is still poorly understood and controversial.

An assay for entry of secreted fungal effectors into plant cells.

Successful colonization of plants by prokaryotic and eukaryotic pathogens requires active effector-mediated suppression of defense responses and host tissue reprogramming. Secreted effector proteins can either display their activity in the apoplast or translocate into host cells and function therein. Although characterized in bacteria, the molecular mechanisms of effector delivery by fungal phytopathogens remain elusive.

The DYRK-family kinase Pom1 phosphorylates the F-BAR protein Cdc15 to prevent division at cell poles.

Division site positioning is critical for both symmetric and asymmetric cell divisions. In many organisms, positive and negative signals cooperate to position the contractile actin ring for cytokinesis. In rod-shaped fission yeast Schizosaccharomyces pombe cells, division at midcell is achieved through positive Mid1/anillin-dependent signaling emanating from the central nucleus and negative signals from the dual-specificity tyrosine phosphorylation-regulated kinase family kinase Pom1 at the cell poles.

A conserved co-chaperone is required for virulence in fungal plant pathogens.

The maize pathogenic fungus Ustilago maydis experiences endoplasmic reticulum (ER) stress during plant colonization and relies on the unfolded protein response (UPR) to cope with this stress. We identified the U. maydis co-chaperone, designated Dnj1, as part of this conserved cellular response to ER stress.

Experimental approaches to investigate effector translocation into host cells in the Ustilago maydis/maize pathosystem.

The fungus Ustilago maydis is a pathogen that establishes a biotrophic interaction with Zea mays. The interaction with the plant host is largely governed by more than 300 novel, secreted protein effectors, of which only four have been functionally characterized. Prerequisite to examine effector function is to know where effectors reside after secretion.

Fungal effectors and plant susceptibility.

Plants can be colonized by fungi that have adopted highly diverse lifestyles, ranging from symbiotic to necrotrophic. Colonization is governed in all systems by hundreds of secreted fungal effector molecules. These effectors suppress plant defense responses and modulate plant physiology to accommodate fungal invaders and provide them with nutrients.

The novel proteins Rng8 and Rng9 regulate the myosin-V Myo51 during fission yeast cytokinesis.

The myosin-V family of molecular motors is known to be under sophisticated regulation, but our knowledge of the roles and regulation of myosin-Vs in cytokinesis is limited. Here, we report that the myosin-V Myo51 affects contractile ring assembly and stability during fission yeast cytokinesis, and is regulated by two novel coiled-coil proteins, Rng8 and Rng9. Both rng8Δ and rng9Δ cells display similar defects as myo51Δ in cytokinesis.

Myosin Vs organize actin cables in fission yeast.

Myosin V motors are believed to contribute to cell polarization by carrying cargoes along actin tracks. In Schizosaccharomyces pombe, Myosin Vs transport secretory vesicles along actin cables, which are dynamic actin bundles assembled by the formin For3 at cell poles. How these flexible structures are able to extend longitudinally in the cell through the dense cytoplasm is unknown.

Shaping fission yeast cells by rerouting actin-based transport on microtubules.

Kinesins and myosins transport cargos to specific locations along microtubules and actin filaments, respectively. The relative contribution of the two transport systems for cell polarization varies extensively in different cell types, with some cells relying exclusively on actin-based transport while others mainly use microtubules. Using fission yeast, we asked whether one transport system can substitute for the other.

Choice of an adequate promoter for efficient complementation in Saccharomyces cerevisiae: a case study.

Conservation of the function of open reading frames recently identified in fungal genome projects can be assessed by complementation of deletion mutants of putative Saccharomyces cerevisiae orthologs. A parallel complementation assay expressing the homologous wild type S. cerevisiae gene is generally performed as a positive control.

Functional differentiation of tbf1 orthologues in fission and budding yeasts.

In Saccharomyces cerevisiae, TBF1, an essential gene, influences telomere function but also has other roles in the global regulation of transcription. We have identified a new member of the tbf1 gene family in the mammalian pathogen Pneumocystis carinii. We demonstrate by transspecies complementation that its ectopic expression can provide the essential functions of Schizosaccharomyces pombe tbf1 but that there is no rescue between fission and budding yeast orthologues.

Functional characterization of Pneumocystis carinii brl1 by transspecies complementation analysis.

Pneumocystis jirovecii is a fungus which causes severe opportunistic infections in immunocompromised humans. The brl1 gene of P. carinii infecting rats was identified and characterized by using bioinformatics in conjunction with functional complementation in Saccharomyces cerevisiae and Schizosaccharomyces pombe.

Molecular cloning of the human beta1,4 N-acetylgalactosaminyltransferase responsible for the biosynthesis of the Sd(a) histo-blood group antigen: the sequence predicts a very long cytoplasmic domain.

The Sd(a) antigen is a carbohydrate determinant expressed on erythrocytes, the colonic mucosa and other tissues. This epitope, whose structure is Siaalpha2,3[GalNAcbeta1,4]Gal beta1,4GlcNAc, is synthesized by a beta1,4 N-acetylgalactosaminyltransferase (beta4GalNAc-T) that transfers a beta1,4-linked GalNAc to the galactose residue of an alpha2,3-sialylated chain. We have cloned from human colon carcinoma Caco2 cells a cDNA whose transfection in COS cells induces a GalNAc-T active on sialylated but not on asialylated fetuin and putatively represents the human Sd(a) beta4GalNAc-T.