Molecular Microbiology

doi: 10.1111/mmi.14303pmid: N/A

doi: 10.1111/mmi.14304pmid: N/A

Balalovski, Phillip; Grainge, Ian

doi: 10.1111/mmi.14563pmid: 32594594

XerCD‐dif site‐specific recombination is a well characterized system, found in most bacteria and archaea. Its role is resolution of chromosomal dimers that arise from homologous recombination. Xer‐mediated recombination is also used by several plasmids for multimer resolution to enhance stability and by some phage for integration into the chromosome. In the past decade, it has been hypothesized that an alternate and novel function exists for this system in the dissemination of genetic elements, notably antibiotic resistance genes, in Acinetobacter species. Currently the mechanism underlying this apparent genetic mobility is unknown. Multidrug resistant Acinetobacter baumannii is an increasingly problematic pathogen that can cause recurring infections. Sequencing of numerous plasmids from clinical isolates of A. baumannii revealed the presence of possible mobile modules: genes were found flanked by pairs of Xer recombination sites, called plasmid‐dif (pdif) sites. These modules have been identified in multiple otherwise unrelated plasmids and in different genetic contexts suggesting they are mobile elements. In most cases, the pairs of sites flanking a gene (or genes) are in inverted repeat, but there can be multiple modules per plasmid providing pairs of recombination sites that can be used for inversion or fusion/deletion reactions; as many as 16 pdif sites have been seen in a single plasmid. Similar modules including genes for surviving environmental toxins have also been found in strains of Acinetobacter Iwoffi isolated from permafrost cores; this suggests that these mobile modules are an ancient adaptation and not a novel response to antibiotic pressure. These modules bear all the hallmarks of mobile genetic elements, yet, their movement has never been directly observed to date. This review gives an overview of the current state of this novel research field.

Garai, Preeti; Blanc‐Potard, Anne

doi: 10.1111/mmi.14564pmid: 32602138

Bacterial small proteins (below 50 amino acids) encoded by small open reading frames (sORFs) are recognized as an emerging class of functional molecules that have been largely overlooked in the past. While some were uncovered serendipitously, global approaches have recently been developed to detect these sORFs. A large portion of small proteins appears to be hydrophobic and located in the bacterial membrane. In the present review, we describe functional small hydrophobic proteins discovered in pathogenic bacteria and report recent advances in the discovery of additional ones. Small membrane proteins contribute to bacterial adaptation to changing environments and often appear to be implicated in negative feedback regulation loops by modulating the function or stability of larger membrane proteins. A subset of these proteins belongs to toxin‐antitoxin modules. We highlight the features of characterized hydrophobic small proteins that may pave the way for identification of the functional small proteins among novel sORFs discovered. Besides providing new insights into bacterial pathogenesis, identification of naturally occurring small hydrophobic proteins of pathogenic bacteria can lead to new therapeutic interventions, as recently shown with the development of synthetic peptides derived from natural small proteins that display antibacterial or antivirulence properties.

Bermas, Arianne; Geddes‐McAlister, Jennifer

doi: 10.1111/mmi.14565pmid: 32697029

Fungal infections are a global concern and the evolution of intrinsic resistance to current antifungals presents an alarming problem. For Cryptococcus neoformans, a human fungal pathogen of primarily immunocompromised individuals, resistance toward treatment strategies demands alternative approaches. Given the prevalence of virulence factor production during cryptococcal infection, an emerging and important field of research encompasses the development of novel antivirulence therapies proposed to improve host immune responses and promote fungal clearance. To accomplish this task, information regarding the presence and role of virulence factors, the mechanisms of action within the host, and the ability to influence fungal susceptibility to antifungals is pertinent. Research into mechanisms of antifungal resistance for C. neoformans is limited but extrapolation from successful studies in other fungal species can improve our understanding of mechanisms employed by C. neoformans and suggest targeted strategies to enhance our ability to combat the pathogen. In this Review, we highlight antifungal therapy options against Cryptococcus, explore current knowledge of underlying mechanisms promoting resistance, and present new opportunities for novel and effective strategies to overcome fungal infections and reduce, or possibly even reverse, the effects of resistance evolution.

Eichler, Jerry

doi: 10.1111/mmi.14569pmid: 32633872

Genome analysis points to N‐glycosylation as being an almost universal posttranslational modification in Archaea. Although such predictions have been confirmed in only a limited number of species, such studies are making it increasingly clear that the N‐linked glycans which decorate archaeal glycoproteins present diversity in terms of both glycan composition and architecture far beyond what is seen in the other two domains of life. In addition to continuing to decipher pathways of N‐glycosylation, recent efforts have revealed how Archaea exploit this variability in novel roles. As well as encouraging glycoprotein synthesis, folding and assembly into properly functioning higher ordered complexes, N‐glycosylation also provides Archaea with a strategy to cope with changing environments. Archaea can, moreover, exploit the apparent species‐specific nature of N‐glycosylation for selectivity in mating, and hence, to maintain species boundaries, and in other events where cell‐selective interactions are required. At the same time, addressing components of N‐glycosylation pathways across archaeal phylogeny offers support for the concept of an archaeal origin for eukaryotes. In this MicroReview, these and other recent discoveries related to N‐glycosylation in Archaea are considered.

Christensen, David G.; Marsden, Anne E.; Hodge‐Hanson, Kelsey; Essock‐Burns, Tara; Visick, Karen L.

doi: 10.1111/mmi.14573pmid: 32654271

Efficient symbiotic colonization of the squid Euprymna scolopes by the bacterium Vibrio fischeri depends on bacterial biofilm formation on the surface of the squid’s light organ. Subsequently, the bacteria disperse from the biofilm via an unknown mechanism and enter through pores to reach the interior colonization sites. Here, we identify a homolog of Pseudomonas fluorescens LapG as a dispersal factor that promotes cleavage of a biofilm‐promoting adhesin, LapV. Overproduction of LapG inhibited biofilm formation and, unlike the wild‐type parent, a ΔlapG mutant formed biofilms in vitro. Although V. fischeri encodes two putative large adhesins, LapI (near lapG on chromosome II) and LapV (on chromosome I), only the latter contributed to biofilm formation. Consistent with the Pseudomonas Lap system model, our data support a role for the predicted c‐di‐GMP‐binding protein LapD in inhibiting LapG‐dependent dispersal. Furthermore, we identified a phosphodiesterase, PdeV, whose loss promotes biofilm formation similar to that of the ΔlapG mutant and dependent on both LapD and LapV. Finally, we found a minor defect for a ΔlapD mutant in initiating squid colonization, indicating a role for the Lap system in a relevant environmental niche. Together, these data reveal new factors and provide important insights into biofilm dispersal by V. fischeri.

Lu, Hua; Pei, Caixia; Zhou, Hui; Lü, Yang; He, Yun; Li, Yunsen; Han, Jing; Xiang, Hua; Eichler, Jerry; Jin, Cheng

doi: 10.1111/mmi.14577pmid: 32706435

Like both eukaryotes and bacteria, archaea can decorate proteins with N‐ and O‐linked glycans. Whereas pathways and roles of N‐glycosylation have been studied in several model archaeal organisms, little is known of O‐glycosylation. To explore commonalities and variations of these two versions of glycosylation, we used Haloarcula hispanica as a model. Our previous work showed that H. hispanica S‐layer glycoproteins are modified by an N‐linked glucose‐α‐(1, 2)‐[sulfoquinovosamine‐β‐(1, 6)‐]galactose trisaccharide and an O‐linked glucose‐α‐(1, 4)‐galactose disaccharide. Here, we found that H. hispanica membrane contains C60 dolichol phosphate (DolP) as a lipid carrier for glycosylation. As revealed by bioinformatics, gene deletion and phenotype analysis, gene HAH_1571, renamed agl22, encodes a predicted glucosyltransferase that transfers glucose from glucose‐DolP onto galactose‐DolP to form the glucose‐α‐(1, 4)‐galactose‐DolP precursor of the N‐glycosylation. Gene HAH_2016, renamed agl23, encodes a putative flippase‐associated protein responsible for flipping of hexose‐DolPs across the membrane to face the exterior. Our results also suggested that the synthesis of the N‐ and O‐linked glycans onto target protein occurs on the outer surface of the cell using hexose‐DolPs as sugar donors. Deletion mutant showed that N‐ and O‐glycosylation are required for growth in the defined medium mimicking the natural habitat of H. hispanica.

Cid, Nicolas G.; Puca, Gervasio; Nudel, Clara B.; Nusblat, Alejandro D.

doi: 10.1111/mmi.14578pmid: 32713049

Sphingolipids are bioactive lipids present in all eukaryotes. Tetrahymena thermophila is a ciliate that displays remarkable sphingolipid moieties, that is, the unusual phosphonate‐linked headgroup ceramides, present in membranes. To date, no identification has been made in this organism of the functions or related genes implicated in sphingolipid metabolism. By gathering information from the T. thermophila genome database together with sphingolipid moieties and enzymatic activities reported in other Tetrahymena species, we were able to reconstruct the putative de novo sphingolipid metabolic pathway in T. thermophila. Orthologous genes of 11 enzymatic steps involved in the biosynthesis and degradation pathways were retrieved. No genes related to glycosphingolipid or phosphonosphingolipid headgroup transfer were found, suggesting that both conserved and innovative mechanisms are used in ciliate. The knockout of gene TTHERM_00463850 allowed to identify the gene encoding a putative fatty acid 2‐hydroxylase, which is involved in the biosynthesis pathway. Knockout cells have shown several impairments in the sexual stage of conjugation since different mating types of knockout strains failed to form cell pairs and complete the conjugation process. This fatty acid 2‐hydroxylase gene is the first gene of a sphingolipid metabolic pathway to be identified in ciliates and have a critical role in their sexual stage.

Rocha, Raquel O.; Wilson, Richard A.

doi: 10.1111/mmi.14580pmid: 32936940

The fungus Magnaporthe oryzae causes blast, the most devastating disease of cultivated rice. After penetrating the leaf cuticle, M. oryzae grows as a biotroph in intimate contact with living rice epidermal cells before necrotic lesions develop. Biotrophic growth requires maintaining metabolic homeostasis while suppressing plant defenses, but the metabolic connections and requirements involved are largely unknown. Here, we characterized the M. oryzae nucleoside diphosphate kinase‐encoding gene NDK1 and discovered it was essential for facilitating biotrophic growth by suppressing the host oxidative burst—the first line of plant defense. NDK enzymes reversibly transfer phosphate groups from tri‐ to diphosphate nucleosides. Correspondingly, intracellular nucleotide pools were perturbed in M. oryzae strains lacking NDK1 through targeted gene deletion, compared to WT. This affected metabolic homeostasis: TCA, purine and pyrimidine intermediates, and oxidized NADP+, accumulated in Δndk1. cAMP and glutathione were depleted. ROS accumulated in Δndk1 hyphae. Functional appressoria developed on rice leaf sheath surfaces, but Δndk1 invasive hyphal growth was restricted and redox homeostasis was perturbed, resulting in unsuppressed host oxidative bursts that triggered immunity. We conclude Ndk1 modulates intracellular nucleotide pools to maintain redox balance via metabolic homeostasis, thus quenching the host oxidative burst and suppressing rice innate immunity during biotrophy.