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ABSTRACT Protein production strategies in bacteria are often limited due to the need for cell lysis and complicated purification schemes. To avoid these challenges, researchers have developed bacterial strains capable of secreting heterologous protein products outside the cell, but secretion titers often remain too low for commercial applicability. Improved understanding of the link between secretion system structure and its secretory abilities can help overcome the barrier to engineering higher secretion titers. Here, we investigated this link with the PrgI protein, the monomer of the secretory channel of the type 3 secretion system (T3SS) of Salmonella enterica. Despite detailed knowledge of the PrgI needle’s assembly and structure, little is known about how its structure influences its secretory capabilities. To study this, we recently constructed a comprehensive codon mutagenesis library of the PrgI protein utilizing a novel one-pot recombineering approach. We then screened this library for functional T3SS assembly and secretion titer by measuring the secretion of alkaline phosphatase using a high-throughput activity assay. This allowed us to construct a first-of-its-kind secretion fitness landscape to characterize the PrgI needle’s mutability at each position as well as the mutations which lead to enhanced T3SS secretion. We discovered new design rules for building a functional T3SS as well as identified hypersecreting mutants. This work can be used to increase understanding of the T3SS’s assembly and identify further targets for engineering. This work also provides a blueprint for future efforts to engineer other complex protein assemblies through the construction of fitness landscapes.IMPORTANCEProtein secretion offers a simplified alternative method for protein purification from bacterial hosts. However, the current state-of-the-art methods for protein secretion in bacteria are still hindered by low yields relative to traditional protein purification strategies. Engineers are now seeking strategies to enhance protein secretion titers from bacterial hosts, often through genetic manipulations. In this study, we demonstrate that protein engineering strategies focused on altering the secretion apparatus can be a fruitful avenue toward this goal. Specifically, this study focuses on how changes to the PrgI needle protein from the type 3 secretion system from Salmonella enterica can impact secretion titer. We demonstrate that this complex is amenable to comprehensive mutagenesis studies and that this can yield both PrgI variants with increased secretory capabilities and insight into the normal functioning of the type 3 secretion system.

Ralstonia solanacearum RSc0454 is predicted as a FAD-linked oxidase based on protein homologies, while it contains distinct domains of lactate dehydrogenase and succinate dehydrogenase. A previous study demonstrated that RSc0454 exhibits lactate dehydrogenase activity using pyruvate and NADH as substrates, and is essential for pathogenicity of R. solanacearum. Here, we genetically characterized involvement of RSc0454 on bacterial growth and expression of genes for the type III secretion system (T3SS, a pathogenicity determinant) in R. solanacearum. The RSc0454 mutant grew normally in rich medium but grew faintly in host plants, and failed to grow in minimal medium. Supplementary succinate but not lactate could substantially restore some phenotypes of RSc0454 mutants, including faint growth in host plants, diminished growth in the minimal medium, and lost pathogenicity toward host plants. Expression of T3SS genes is directly controlled by a master regulator, HrpB, and hrpB expression is positively regulated by HrpG and PrhG in parallel ways. Deletion of RSc0454 substantially reduced expression levels of hrpB and T3SS both in vitro and in planta. Moreover, RSc0454 is revealed to be required for the T3SS expression via HrpG and PrhG, although through some novel pathway, and impaired expression of these genes was not due to growth deficiency of RSc0454 mutants. RSc0454 is suggested to be important for redox balance inside cells, and supplementary NADH partially restored diminished growth of the RSc0454 mutant in the minimal medium only in the presence of succinate at some moderate concentrations, indicating that the unbalanced redox in the RSc0454 mutant might be responsible for its diminished growth in the minimal medium. Taken together, these results provide novel insights into the understanding of various biological functions of this FAD-linked oxidase RSc0454 and involvement of the redox balance on expression of the T3SS in R. solanacearum.[Graphic: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Microbes have been coevolving with their host for millions of years, exploiting host resources to their own benefit. We show that viral and bacterial pathogens convergently evolved to hijack cellular mitogen-activated protein kinase (MAPK) p90-ribosomal S6-kinases (RSKs). Theiler's virus leader (L) protein binds RSKs and prevents their dephosphorylation, thus maintaining the kinases active. Recruitment of RSKs enables L-protein-mediated inhibition of eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2 or PKR) and stress granule formation. Strikingly, ORF45 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) and YopM protein of Yersinia use the same peptide motif as L to recruit and activate RSKs. All three proteins interact with a conserved surface-located loop of RSKs, likely acting as an allosteric regulation site. Some unrelated viruses and bacteria thus evolved to harness RSKs in a common fashion, yet to target distinct aspects of innate immunity. As documented for Varicella zoster virus ORF11, additional pathogens likely evolved to hijack RSKs, using a similar short linear motif. [ABSTRACT FROM AUTHOR]

Protein production strategies in bacteria are often limited due to the need for cell lysis and complicated purification schemes. To avoid these challenges, researchers have developed bacterial strains capable of secreting heterologous protein products outside the cell, but secretion titers often remain too low for commercial applicability. Improved understanding of the link between secretion system structure and its secretory abilities can help overcome the barrier to engineering higher secretion titers. Here, we investigated this link with the PrgI protein, the monomer of the secretory channel of the type 3 secretion system (T3SS) of Salmonella enterica . Despite detailed knowledge of the PrgI needle's assembly and structure, little is known about how its structure influences its secretory capabilities. To study this, we recently constructed a comprehensive codon mutagenesis library of the PrgI protein utilizing a novel one-pot recombineering approach. We then screened this library for functional T3SS assembly and secretion titer by measuring the secretion of alkaline phosphatase using a high-throughput activity assay. This allowed us to construct a first-of-its-kind secretion fitness landscape to characterize the PrgI needle's mutability at each position as well as the mutations which lead to enhanced T3SS secretion. We discovered new design rules for building a functional T3SS as well as identified hypersecreting mutants. This work can be used to increase understanding of the T3SS's assembly and identify further targets for engineering. This work also provides a blueprint for future efforts to engineer other complex protein assemblies through the construction of fitness landscapes.IMPORTANCEProtein secretion offers a simplified alternative method for protein purification from bacterial hosts. However, the current state-of-the-art methods for protein secretion in bacteria are still hindered by low yields relative to traditional protein purification strategies. Engineers are now seeking strategies to enhance protein secretion titers from bacterial hosts, often through genetic manipulations. In this study, we demonstrate that protein engineering strategies focused on altering the secretion apparatus can be a fruitful avenue toward this goal. Specifically, this study focuses on how changes to the PrgI needle protein from the type 3 secretion system from Salmonella enterica can impact secretion titer. We demonstrate that this complex is amenable to comprehensive mutagenesis studies and that this can yield both PrgI variants with increased secretory capabilities and insight into the normal functioning of the type 3 secretion system., Competing Interests: D.T.-E. is affiliated with Opera Bioscience, which utilizes technology reported in this article.

Xanthomonas oryzae pv. oryzae, the causal bacterium of bacterial blight limits rice production globally. Currently, genome sequences for only a few X. oryzae pv. oryzae isolates are available from India. Based on the next-generation sequencing and single-molecule sequencing in real-time technologies, we present here the complete genome sequence of X. oryzae pv. oryzae race 4, a highly virulent member of the Indian X. oryzae pv. oryzae population that has been extensively used in different research studies. The genome data will contribute to our understanding of X. oryzae pv. oryzae genomic features and pave the way for research on rice–X. oryzae pv. oryzae interactions.

Type-3 secretion systems are sophisticated syringe-like nanomachines present in many animal and plant Gram-negative pathogens. They are capable of translocating an arsenal of specific bacterial toxins (effector proteins) from the prokaryotic cytoplasm across the three biological membranes directly into the eukaryotic cytosol, some of which modulate host cell mechanisms for the benefit of the pathogen. They populate a particular biological niche, which is maintained by specific, pathogen-dependent effectors. In contrast, the needle complex, which is the central component of this specialized protein delivery machine, is structurally well-conserved. It is a large supramolecular cylindrical structure composed of multiple copies of a relatively small subset of proteins, is embedded in the bacterial membranes and protrudes from the pathogen’s surface with a needle filament. A central channel traverses the entire needle complex, and serves as a hollow conduit for proteins destined to travel this secretion pathway. In the past few years, there has been a tremendous increase in an understanding on both the structural and the mechanistic level. This review will thus focus on new insights of this remarkable molecular machine. [ABSTRACT FROM AUTHOR]

Ralstonia solanacearum RSc0454 is predicted as a FAD-linked oxidase based on protein homologies, while it contains distinct domains of lactate dehydrogenase and succinate dehydrogenase. A previous study demonstrated that RSc0454 exhibits lactate dehydrogenase activity using pyruvate and NADH as substrates, and is essential for pathogenicity of R. solanacearum . Here, we genetically characterized involvement of RSc0454 on bacterial growth and expression of genes for the type III secretion system (T3SS, a pathogenicity determinant) in R. solanacearum . The RSc0454 mutant grew normally in rich medium but grew faintly in host plants, and failed to grow in minimal medium. Supplementary succinate but not lactate could substantially restore some phenotypes of RSc0454 mutants, including faint growth in host plants, diminished growth in the minimal medium, and lost pathogenicity toward host plants. Expression of T3SS genes is directly controlled by a master regulator, HrpB, and hrpB expression is positively regulated by HrpG and PrhG in parallel ways. Deletion of RSc0454 substantially reduced expression levels of hrpB and T3SS both in vitro and in planta. Moreover, RSc0454 is revealed to be required for the T3SS expression via HrpG and PrhG, although through some novel pathway, and impaired expression of these genes was not due to growth deficiency of RSc0454 mutants. RSc0454 is suggested to be important for redox balance inside cells, and supplementary NADH partially restored diminished growth of the RSc0454 mutant in the minimal medium only in the presence of succinate at some moderate concentrations, indicating that the unbalanced redox in the RSc0454 mutant might be responsible for its diminished growth in the minimal medium. Taken together, these results provide novel insights into the understanding of various biological functions of this FAD-linked oxidase RSc0454 and involvement of the redox balance on expression of the T3SS in R. solanacearum .[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Xanthomonas oryzae pv. oryzae , the causal bacterium of bacterial blight limits rice production globally. Currently, genome sequences for only a few X. oryzae pv. oryzae isolates are available from India. Based on the next-generation sequencing and single-molecule sequencing in real-time technologies, we present here the complete genome sequence of X. oryzae pv. oryzae race 4, a highly virulent member of the Indian X. oryzae pv. oryzae population that has been extensively used in different research studies. The genome data will contribute to our understanding of X. oryzae pv. oryzae genomic features and pave the way for research on rice- X. oryzae pv. oryzae interactions.

An understanding of how biological diversity affects plant-microbe interactions is becoming increasingly important, particularly with respect to components of the pathogen effector arsenal and the plant immune system. Although technological improvements have greatly advanced our ability to examine molecular sequences and interactions, relatively few advances have been made that facilitate high-throughput, in vivo pathology screens. Here, we present a high-throughput, microplate-based, nondestructive seedling pathology assay, and apply it to identify Arabidopsis thaliana effector-triggered immunity (ETI) responses against Pseudomonas syringae type III secreted effectors. The assay was carried out in a 48-well microplate format with spray inoculation, and disease symptoms were quantitatively recorded in a semiautomated manner, thereby greatly reducing both time and costs. The assay requires only slight modifications of common labware and uses no proprietary software. We validated the assay by recapitulating known ETI responses induced by P. syringae in Arabidopsis . We also demonstrated that we can quantitatively differentiate responses from a diversity of plant genotypes grown in the same microplate. Finally, we showed that the results obtained from our assay can be used to perform genome-wide association studies to identify host immunity genes, recapitulating results that have been independently obtained with mature plants.

Vascular wilt bacteria such as Pantoea stewartii , the causal agent of Stewart's bacterial wilt of maize (SW), are destructive pathogens that are difficult to control. These bacteria colonize the xylem, where they form biofilms that block sap flow leading to characteristic wilting symptoms. Heritable forms of SW resistance exist and are used in maize breeding programs but the underlying genes and mechanisms are mostly unknown. Here, we show that seedlings of maize inbred lines with pan1 mutations are highly resistant to SW. However, current evidence suggests that other genes introgressed along with pan1 are responsible for resistance. Genomic analyses of pan1 lines were used to identify candidate resistance genes. In-depth comparison of P. stewartii interaction with susceptible and resistant maize lines revealed an enhanced vascular defense response in pan1 lines characterized by accumulation of electron-dense materials in xylem conduits visible by electron microscopy. We propose that this vascular defense response restricts P. stewartii spread through the vasculature, reducing both systemic bacterial colonization of the xylem network and consequent wilting. Though apparently unrelated to the resistance phenotype of pan1 lines, we also demonstrate that the effector WtsE is essential for P. stewartii xylem dissemination, show evidence for a nutritional immunity response to P. stewartii that alters xylem sap composition, and present the first analysis of maize transcriptional responses to P. stewartii infection.