Biology subjects

Helmann, T. C.

Publications and source records attributed to Helmann, T. C..

7 recordsLinked to original sources

bioRxiv2026

Comparative functional genomics of six Pectobacterium species reveals core and unique fitness determinants in potato tubers

Pectobacterium, a member of the soft rot Pectobacteriaceae (SRP), can cause highly destructive soft rot and blackleg diseases on many important crop and other host plants, particularly potato (Solanum tuberosum). Pectobacterium and its sister genus Dickeya have common virulence strategies, yet a highly diverse "open" pangenome containing many unique and hypothetical proteins. Here, we used randomly-barcoded transposon insertion-site sequencing (RB-TnSeq) on six Pectobacterium species to identify genes involved in bacterial survival and growth in potato tubers, as well as in minimal media conditions containing different single carbon sources. The strains evaluated varied broadly in genomic content as well as host plant and year of isolation. We focused on identifying conditionally essential genes across all strains tested, as well as genes that were important for growth in one or few of the tested strains. These results highlight the importance of many common genes involved in metabolism and maintaining cellular homeostasis, as well as shared and rare virulence traits including polygalacturonate catabolism, genes involved in polysaccharide biosynthesis, immunity proteins, and (for P. brasiliense) self-tolerance of the antibiotic carbapenem. This large dataset of conditionally essential genes, generated across Pectobacterium species during soft rot infection of the agriculturally important plant host potato, can serve as a rich dataset for further hypothesis generation and identification of important and/or novel in planta fitness factors.

microbiology↗

bioRxiv2021

Genome-wide identification of genes important for growth of Dickeya dadantii and D. dianthicola in potato (Solanum tuberosum) tubers

Dickeya species are causal agents of soft rot diseases in many economically important crops, including soft rot disease of potato (Solanum tuberosum). Using random barcode transposon-site sequencing (RB-TnSeq), we generated genome-wide mutant fitness profiles of Dickeya dadantii 3937, Dickeya dianthicola ME23, and Dickeya dianthicola 67-19 isolates collected after passage through several in vitro and in vivo conditions. Tubers from the potato cultivars "Atlantic", "Dark Red Norland", and "Upstate Abundance" provided highly similar conditions for bacterial growth. Using the homolog detection software PyParanoid, we matched fitness values for orthologous genes in the three bacterial strains. Direct comparison of fitness among the strains highlighted shared and variable traits important for growth. Bacterial growth in minimal medium required many metabolic traits that were also essential for competitive growth in planta, such as amino acid, carbohydrate, and nucleotide biosynthesis. Growth in tubers specifically required the pectin degradation gene kduD. Disruption in three putative DNA-binding proteins had strain-specific effects on competitive fitness in tubers. Though the Soft Rot Pectobacteriaceae can cause disease with little host specificity, it remains to be seen the extent to which strain-level variation impacts virulence.

microbiology↗

bioRxiv2020

Genome-wide identification of tomato xylem sap fitness factors for Ralstonia pseudosolanacearum and Ralstonia syzygii

Plant pathogenic Ralstonia spp. colonize plant xylem and cause wilt diseases on a broad range of host plants. To identify genes that promote growth of diverse Ralstonia strains in xylem sap from tomato plants, we performed genome-scale genetic screens (random barcoded transposon mutant sequencing screens; RB-TnSeq) in three strains spanning the genetic, geographical, and physiological range of plant pathogenic Ralstonia: R. solanacearum IBSBF1503, R. pseudosolanacearum GMI1000, and R. syzygii PSI07. Contrasting mutant fitness phenotypes in culture media versus in xylem sap suggest that Ralstonia strains are adapted to ex vivo xylem sap and that culture media impose foreign selective pressures. Although wild-type Ralstonia grew in sap and in rich medium with similar doubling times and to a similar carrying capacity, more genes were essential for growth in sap than in rich medium. Each strain required many genes associated with envelope remodeling and repair processes for full fitness in xylem sap. These genes were associated with peptidoglycan peptide formation (murI), secretion of periplasmic proteins (tatC), periplasmic protein folding (dsbA), synthesis of osmoregulated periplasmic glucans (mdoGH), and LPS biosynthesis. Mutants in four genes had strong, sap-specific fitness defects in all strain backgrounds: murI, thiC, purU, and a lipoprotein (RSc2007). Many amino acid biosynthesis genes were required for fitness in both minimal medium and xylem sap. Multiple mutants with insertions in virulence regulators had gains-of-fitness in culture media and neutral fitness in sap. Our genome-scale genetic screen identified Ralstonia fitness factors that promote growth in xylem sap, an ecologically relevant condition. ImportanceTraditional transposon mutagenesis genetic screens pioneered molecular plant pathology and identified core virulence traits like the type III secretion system. TnSeq approaches that leverage next-generation sequencing to rapidly quantify transposon mutant phenotypes are ushering in a new wave of biological discovery. Here we have adapted a genome-scale approach, random barcoded transposon mutant sequencing (RB-TnSeq), to discover fitness factors that promote growth of three related bacterial strains in a common niche, tomato xylem sap. Fitness of wild-type and mutants show that Ralstonia spp. are adapted to grow well in xylem sap from their natural host plant, tomato. Our screen identified multiple sap-specific fitness factors with roles in maintaining the bacterial envelope. These factors include putative adaptations to resist plant defenses that may include antimicrobial proteins and specialized metabolites that damage bacterial membranes.

microbiology↗

bioRxiv2020

Distinctiveness of genes contributing to growth of Pseudomonas syringae in diverse host plant species

A variety of traits are necessary for bacterial colonization of the interior of plant hosts, including well-studied virulence effectors as well as other phenotypes contributing to bacterial growth and survival within the apoplast. High-throughput methods such as transposon sequencing (TnSeq) are powerful tools to identify such genes in bacterial pathogens. However, there is little information as to the distinctiveness of traits required for bacterial colonization of different hosts. Here, we utilize randomly barcoded TnSeq (RB-TnSeq) to identify the genes that contribute to the ability of Pseudomonas syringae strain B728a to grow within common bean (Phaseolus vulgaris), lima bean (Phaseolus lunatus), and pepper (Capsicum annuum); species representing two different plant families. The magnitude of contribution of most genes to apoplastic fitness in each of the plant hosts was similar. However, 50 genes significantly differed in their fitness contributions to growth within these species. These genes encoded proteins in various functional categories including polysaccharide synthesis and transport, amino acid metabolism and transport, cofactor metabolism, and phytotoxin synthesis and transport. Six genes that encoded unannotated, hypothetical proteins also contributed differentially to growth in these hosts. The genetic repertoire of a relatively promiscuous pathogen such as P. syringae may thus be shaped, at least in part, by the conditional contribution of some fitness determinants.

microbiology↗

bioRxiv2020

Differential virulence contributions of the efflux transporter MexAB-OprM in Pseudomonas syringae infecting a variety of host plants

Efflux transporters such as MexAB-OprM contribute to bacterial resistance to diverse antimicrobial compounds. Here, we show that MexB contributes to epiphytic and apoplastic growth of Pseudomonas syringae strain B728a, as well as lesion formation in common bean (Phaseolus vulgaris). While a mexB deletion mutant formed fewer lesions after topical application to common bean, these lesions contain the same number of cells (105 to 107 cells) as those caused by the wild-type strain. The internalized population size of both the WT and the MexB mutant within small segments of surface-sterilized asymptomatic portions of the leaves varied from undetectably low to as high as 105 cells/cm2. Localized populations of bacteria within the leaf must apparently exceed a threshold size of about 105 cells/cm2 in order for a visible lesion to form. Strain B728a was capable of moderate to extensive apoplastic growth in diverse host plants including lima bean (P. lunatus), fava bean (Vicia faba), pepper (Capsicum annuum), Nicotiana benthamiana, sunflower (Helianthus annuus), and tomato (Solanum lycopersicum). MexB was not required for growth in a subset of these plant species, and in which the duration of growth after inoculation was longer, indicating apparent variation in onset time or magnitude of plant chemical defenses among those hosts. The use of a hyper-susceptible efflux pump mutant strain is an informative strategy to explore the diversity of host chemical immune responses.

microbiology↗

bioRxiv2019

Genome-wide transposon screen of a Pseudomonas syringae mexB mutant reveals the substrates of efflux transporters

Bacteria express numerous efflux transporters that confer resistance to diverse toxicants present in their environment. Due to a high level of functional redundancy of these transporters, it is difficult to identify those that are of most importance in conferring resistance to specific compounds. The resistance-nodulation-division (RND) protein family is one such example of redundant transporters that are widespread among Gram-negative bacteria. Within this family, the MexAB-OprM protein complex is highly-expressed and conserved among Pseudomonas species. We exposed barcoded transposon mutant libraries in isogenic wild-type and {Delta}mexB backgrounds in P. syringae B728a to diverse toxic compounds in vitro to identify mutants with increased susceptibility to these compounds. Mutants in genes encoding both known and novel redundant transporters, but with partially overlapping substrate specificities were observed in a {Delta}mexB background. Psyr_0228, an uncharacterized member of the Major Facilitator Superfamily of transporters, preferentially contributes to tolerance of acridine orange and acriflavine. Another transporter located in the inner membrane, Psyr_0541, contributes to tolerance to acriflavine and berberine. The presence of multiple redundant, genomically encoded, efflux transporters appears to enable bacterial strains to tolerate a diversity of environmental toxins. This genome-wide screen in a hyper-susceptible mutant strain revealed numerous transporters that would otherwise be dispensable in these conditions. Bacterial strains such as P. syringae that likely encounter diverse toxins in their environment such as in association with many different plant species, probably benefit from possessing multiple redundant transporters that enable versatility to tolerate novel toxicants.

microbiology↗

bioRxiv2019

Genome-wide identification of Pseudomonas syringae genes required for competitive fitness during colonization of the leaf surface and apoplast

The foliar plant pathogen Pseudomonas syringae can establish large epiphytic populations on leaf surfaces before infection. However, the bacterial genes that contribute to these lifestyles have not been completely defined. The fitness contributions of most genes in P. syringae pv. syringae B728a were determined by genome-wide fitness profiling with a randomly barcoded transposon mutant library that was grown on the leaf surface and in the apoplast of the susceptible plant Phaseolus vulgaris. Genes within the functional categories of amino acid and polysaccharide (including alginate) biosynthesis contributed most to fitness both on the leaf surface (epiphytic) or in the leaf interior (apoplast), while genes in the type III secretion system and syringomycin synthesis were primarily important in the apoplast. Numerous other genes that had not been previously associated with in planta growth were also required for maximum epiphytic or apoplastic fitness. Many hypothetical proteins and uncategorized glycosyltransferases were also required for maximum competitive fitness in and on leaves. For most genes, no relationship was seen between fitness in planta and either the magnitude of their expression in planta or degree of induction in planta compared to in vitro conditions measured in other studies. A lack of association of gene expression and fitness has important implications for the interpretation of transcriptional information and our broad understanding of plant-microbe interactions.\n\nSignificance StatementMany plant pathogenic bacteria can extensively colonize leaf surfaces before entry and multiplication within the leaf to cause disease. While these habitats presumably require distinct adaptations, the genes required in these habitats and how they would differ was unknown. Using a genome-wide library of barcoded insertional mutants in the plant pathogen Pseudomonas syringae, we ascertained the common and unique genes required to colonize these habitats. A lack of association between gene expression and contribution to fitness suggests that many genes that are highly expressed or induced in planta are dispensable or redundant. As a model bacterium for plant pathogenesis and colonization, our comprehensive genetic dataset allows us to better understand the traits needed for association with leaves.

microbiology↗