Breah LaSarre

SUMMARY: Cucurbit yellow vine disease (CYVD) is a phloem disease caused by a group of Serratia ureilytica bacteria within the S. marcescens complex. The CYVD pathogen is distinct from other phloem pathogens in two fundamental ways: i) unlike other bacterial phloem pathogens, the CYVD pathogen readily grows on standard laboratory media; and ii) whereas most bacterial phloem diseases are transmitted solely by hemipteran insects, CYVD can be transmitted by both hemipteran (squash bug) and non-hemipteran (cucumber beetle) vectors. The phloem of cucurbits is also distinct, with cucurbits having a unique secondary phloem system, the extrafascicular phloem (EFP), for which a role in CYVD has not been investigated. The distinctive features of this pathosystem prompted a re-evaluation of pathogen localization in planta using a green fluorescent protein-tagged CYVD strain during infection of squash ( Cucurbita pepo ). In epifluorescence and confocal microscopy images of transverse and longitudinal sections of stem and petiole tissues, the fluorescence patterns of the CYVD pathogen were consistent with pathogen colonization of all four known EFP tissues, namely the peripheral, entocyclic, ectocyclic, and commissural sieve tubes. The CYVD pathogen may enter the EFP during herbivory by pathogen-infected vectors that cause mechanical damage, as the EFP exhibits much slower blockage following injury than the conventional fascicular (bundle) phloem. Moreover, although EFP has been proposed to have an antimicrobial function, EFP exudate collected from the stem and petioles of C. pepo supported rapid pathogen growth. These results provide evidence for a unique tissue tropism of the newly emerged plant pathogen S. ureilytica . SIGNIFICANCE STATEMENT: Bacterial phloem pathogens generally colonize the conventional fascicular (bundle) phloem of their host plant. We demonstrate that the bacterial pathogen causing cucurbit yellow vine disease is atypical in that it colonizes the extrafascicular phloem, a unique secondary phloem system of cucurbits that is thought to function beyond photosynthate transport. These findings reshape our understanding of phloem pathogenicity and indicate that the extrafascicular phloem is more physiologically accessible and biologically consequential than previously recognized.

Characterizing community composition is fundamental to understanding microbial community function. Recent advances in Oxford Nanopore Technology (ONT) long-read sequencing now allow community profiling using full-length gene amplicons, affording better taxonomic resolution than standard short-amplicon Illumina sequencing. However, robust ONT-compatible profiling workflows are lacking. To address this, we have created the Amplicon Consensus Taxonomy (ACT) pipeline for classifying long-read amplicons. ACT combines output from three existing pipelines – Emu, Sintax, and LACA – to leverage the strengths of each while offsetting their individual limitations. We also developed the ACT database (ACT-DB), a sequence-similarity-aware reference database that clusters highly similar sequences into multi-taxa groups to reduce overclassification. We benchmarked ACT performance against Emu and Sintax using a defined simple mock community, simulated datasets, and a complex rhizosphere community supplemented with novel species. While ACT exhibited generally comparable or superior performance across datasets, ACT demonstrated a marked advantage over Emu and Sintax in identifying novel and low-abundance taxa in both simple and complex communities, resulting in significantly higher species-richness estimates that better reflected those observed in prior Illumina amplicon studies. Furthermore, by clustering ambiguous reference sequences, ACT-DB allowed ACT to resolve reads to meaningful multi-species groups, improving resolution without coercing artificial precision. Together, ACT and ACT-DB form a robust long-read amplicon profiling workflow that confidently identifies known species while reducing overclassification and preserving low-abundance and unknown taxa. IMPORTANCE: Microbial communities are frequently characterized by amplicon sequencing of marker genes, such as the bacterial 16S rRNA gene and fungal ITS region. Historically, the standard profiling method has been Illumina sequencing of 200-300 bp amplicons, but improved accuracy of ONT long-read sequencing means it is now possible to sequence amplicons spanning full genes of any size, prompting the need for tools optimized for long amplicons. Here, we describe the ACT bioinformatic pipeline for assigning taxonomy to amplicons of any length. We evaluated ACT performance using full-length 16S amplicon data relative to that of two commonly used pipelines. Additionally, we developed a sequence ambiguity-aware ACT database (ACT-DB) of 16S rRNA sequences to further improve classification accuracy and resolution.

Cucurbit yellow vine disease (CYVD) is an emerging phloem disease in the United States that causes yield losses of up to 100% in squash, pumpkin, and watermelon. CYVD is caused by a group of closely related strains of Serratia ureilytica, a species comprising entomopathogenic, phytopathogenic, clinical, and environmental strains. One genomic trait distinguishing CYVD-causing S. ureilytica strains from non-CYVD S. ureilytica strains is the presence of a specific type I fimbrial locus, which we found to be uniquely plasmid-borne in CYVD strains. We investigated the contribution of this fim locus to CYVD by comparing the virulence of the wild-type CYVD strain Z07 to a deletion mutant Z07Δ fim that lacked the entire seven-gene fim locus. Infection assays with these strains showed that this locus contributes to virulence on squash ( Cucurbita pepo) and does so by contributing to a higher probability of developing large populations in planta. Moreover, the Z07Δ fim mutant formed larger biofilms than the wild type in vitro, suggesting that these type I fimbriae interfere with biofilm formation. These results support a model in which biofilm development following S. ureilytica plant infection negatively impacts virulence, such as by restricting the number of sites accessed in the phloem tissue, thereby impeding spread and limiting colonization.

Cucurbit yellow vine disease (CYVD) is an emerging disease that can cause up to 100% cucurbit crop losses. CYVD is transmitted by squash bugs ( Anasa tristis), but anecdotal reports indicate the presence of CYVD in cucurbit fields in the apparent absence of squash bugs and presence of cucumber beetles. This study tested the vector competence of two cucumber beetle species ( Acalymma vittatum and Diabrotica undecimpunctata howardi) for CYVD and explored the phylogenetic relatedness of CYVD-causing isolates (i.e., CYVD strains) from cucurbits to those from wild cucumber beetles from fields with endemic CYVD. CYVD strains have heretofore been classified as Serratia marcescens; however, pairwise genomic comparisons and phylogenomic analyses using complete genome assemblies of five CYVD strains, including two from wild cucumber beetles, indicate that CYVD strains form a clade in the S. ureilytica species, which is a species within the S. marcescens complex. The S. ureilytica isolates from both cucumber beetle species were pathogenic based on their ability to induce CYVD when injected into squash ( Cucurbita pepo) plants. Moreover, field-collected cucumber beetles of both species harboring the CYVD pathogen induced CYVD in squash plants in laboratory transmission tests, demonstrating that cucumber beetles can transmit S. ureilytica to plants. Our results support a nomenclature change for the causal agent of CYVD to S. ureilytica and demonstrate that cucumber beetles can both harbor and transmit S. ureilytica CYVD strains. S. ureilytica is thus distinct among phytopathogenic bacteria in its ability to use both hemipterans and coleopterans as vectors for transmission to plants.

Formate is a single-carbon compound that is challenging to assimilate, including when assimilation involves a CO ₂ intermediate that can diffuse from the cell. Mutations that overcome such challenges can be identified through adaptive laboratory evolution. We evolved the anoxygenic phototrophic bacterium Rhodopseudomonas palustris to use formate as the sole carbon source. Through gene deletions, we determined that formate is assimilated via oxidation to CO ₂ by formate dehydrogenase followed by CO ₂ fixation by the Calvin cycle. However, this pathway had no clear link to three genes that were commonly mutated in evolved isolates: (i) ribB , a flavin synthesis enzyme, (ii) ppsR2 , a repressor of light-harvesting genes, and (iii) RPA0893, a regulator of unknown function. A RibB mutation was necessary and sufficient for formate assimilation and improved formate oxidation. PpsR2 mutations occurred early, facilitated formate assimilation, and caused elevated pigmentation. Pigment production generates CO ₂ and alkaline conditions that, along with intracellular chromatophore membranes, could represent a rudimentary but important CO ₂ -concentrating mechanism. RPA0983 mutations emerged late and facilitated formate assimilation. RPA0983’s proximity to a CO ₂ -liberating pigment synthesis gene suggests a similar effect as PpsR2 mutations. Our findings reveal unintuitive pathway intersections that could have broad implications for formate and CO ₂ -utilizing organisms.

Algimonas porphyrae is a dimorphic, prosthecate member of the family Robigintomaculaceae , order Caulobacterales, in the class Alphaproteobacteria, originally isolated from the red alga Porphyra yezoensis . Here we report the complete genome of type strain LMG 26424 ^T (0 C-2-2 ^T ) obtained by sequencing with Oxford Nanopore technology.

Glycocaulis abyssi is a dimorphic, prosthecate member of the family Maricaulaceae , order Caulobacterales, in the class Alphaproteobacteria, originally isolated from an abyssal hydrothermal vent. Here, we report the complete genome sequence of type strain LMG 27140 ^T (MCS33 ^T ) obtained using Oxford Nanopore technology.

Fretibacter rubidus is a dimorphic, prosthecate member of the family Maricaulaceae , order Caulobacterales, in the class Alphaproteobacteria. Here, we report the genome of type strain JCM 15585 ^T (JC2236 ^T ), sequenced with Oxford Nanopore Technology.

The ecological significance of light perception in nonphotosynthetic bacteria remains largely elusive. In terrestrial environments, diurnal oscillations in light are often temporally coupled to other environmental changes, including increased temperature and evaporation. Here, we report that light functions as an anticipatory cue that triggers protective adaptations to tolerate a future rapid loss of environmental water. We demonstrate this photo-anticipatory stress tolerance in leaf-associated Pseudomonas syringae pv. syringae ( Pss ) and other plant- and soil-associated pseudomonads. We found that light influences the expression of 30% of the Pss genome, indicating that light is a global regulatory signal, and this signaling occurs almost entirely via a bacteriophytochrome photoreceptor that senses red, far-red, and blue wavelengths. Bacteriophytochrome-mediated light control disproportionally up-regulates water-stress adaptation functions and confers enhanced fitness when cells encounter light prior to water limitation. Given the rapid speed at which water can evaporate from leaf surfaces, such anticipatory activation of a protective response enhances fitness beyond that of a reactive stress response alone, with recurring diurnal wet–dry cycles likely further amplifying the fitness advantage over time. These findings demonstrate that nonphotosynthetic bacteria can use light as a cue to mount an adaptive anticipatory response against a physiologically unrelated but ecologically coupled stress.

Denitrification is a form of anaerobic respiration wherein nitrate (NO ₃ ^- ) is sequentially reduced via nitrite (NO ₂ ^- ), nitric oxide, and nitrous oxide (N ₂ O) to dinitrogen gas (N ₂ ) by four reductase enzymes. Partial denitrifying bacteria possess only one, or some, of these four reductases and use them as independent respiratory modules. However, it is unclear if partial denitrifiers sense and respond to denitrification intermediates outside of their reductase repertoire. Here we tested the denitrifying capabilities of two purple nonsulfur bacteria, Rhodopseudomonas palustris CGA0092 and Rhodobacter capsulatus SB1003. Each had denitrifying capabilities that matched their genome annotation; CGA0092 reduced NO ₂ ^- to N ₂ and SB1003 reduced N ₂ O to N ₂ . For each bacterium, N ₂ O reduction could be used for both electron balance during growth on electron-rich organic compounds in light and for energy transformation via respiration in the dark. However, N ₂ O reduction required supplementation with a denitrification intermediate, including those for which there was no associated denitrification enzyme. For CGA0092, NO ₃ ^- served as a stable, non-catalyzable molecule that was sufficient to activate N ₂ O reduction. Using a β-galactosidase reporter we found that NO ₃ ^- acted, at least in part, by stimulating N ₂ O reductase gene expression. In SB1003, NO ₂ ^- , but not NO ₃ ^- , activated N ₂ O reduction but NO ₂ ^- was slowly removed, likely by a promiscuous enzyme activity. Our findings reveal that partial denitrifiers can still be subject to regulation by denitrification intermediates that they cannot use. Importance: Denitrification is a form of microbial respiration wherein nitrate is converted via several nitrogen oxide intermediates into harmless dinitrogen gas. Partial denitrifying bacteria, which individually have some but not all denitrifying enzymes, can achieve complete denitrification as a community by cross-feeding nitrogen oxide intermediates. However, the last intermediate, nitrous oxide (N ₂ O), is a potent greenhouse gas that often escapes, motivating efforts to understand and improve the efficiency of denitrification. Here we found that at least some partial denitrifying N ₂ O reducers can sense and respond to nitrogen oxide intermediates that they cannot otherwise use. The regulatory effects of nitrogen oxides on partial denitrifiers are thus an important consideration in understanding and applying denitrifying bacterial communities to combat greenhouse gas emissions.