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Using the pressurized acoustic-resonance tube developed in this laboratory, the shift in the napier (relaxation) frequency of nitrogen at 168°C has been measured as a function of percent of added methane, both normal and fully deuterated. The shift is linear up to 2% of impurity in each case, but is by far the greater with the deuterated methane: 1350 Hz/1% vs 240 Hz/1% for the ordinary. The degenerate unsymmetrical stretching mode of vibration at 2259 cm-1 in deuterated methane differs by only 72 cm-1 from nitrogen's vibration at 2331 cm-1, and a resonance interchange of energy is clearly probable. The symmetrical stretch at 2109 cm-1 is also within 10% of nitrogen. In the case of ordinary methane and nitrogen, there are no such near approaches. Precisely the reverse obtains in the case of oxygen and the two methanes. [ABSTRACT FROM AUTHOR]

Management of widespread plant pathogens is challenging as climatic differences among crop-growing regions may alter key aspects of pathogen spread and disease severity. Xylella fastidiosa is a xylem-limited bacterial pathogen that is transmitted by xylem sap-feeding insects. Geographic distribution of X. fastidiosa is limited by winter climate, and vines infected with X. fastidiosa can recover from infection when held at cold temperatures. California has a long history of research on Pierce's disease and significant geographic and climatic diversity among grape-growing regions. This background in combination with experimental disease studies under controlled temperature conditions can inform risk assessment for X. fastidiosa spread and epidemic severity across different regions and under changing climate conditions. California's grape-growing regions have considerable differences in summer and winter climate. In northern and coastal regions, summers are mild and winters are cool, conditions which favor winter recovery of infected vines. In contrast, in inland and southern areas, summers are hot and winters mild, reducing likelihood of winter recovery. Here, winter recovery of three table grape cultivars (Flame, Scarlet Royal, and Thompson Seedless) and three wine grape cultivars (Sauvignon Blanc, Cabernet Sauvignon, and Zinfandel) were evaluated under temperature conditions representative of the San Joaquin Valley, an area with hot summers and mild winters that has been severely impacted by Pierce's disease and contains a large portion of California grape production. Mechanically inoculated vines were held in the greenhouse under one of three warming treatments to represent different seasonal inoculation dates prior to being moved into a cold chamber. Winter recovery under all treatments was generally limited but with some cultivar variation. Given hot summer temperatures of many grape-growing regions worldwide, as well as increasing global temperatures overall, winter recovery of grapevines should not be considered a key factor limiting X. fastidiosa spread and epidemic severity in the majority of cases., Competing Interests: The author(s) declare no conflict of interest.

Xylella fastidiosa is a xylem-limited bacterial pathogen that causes Pierce's disease (PD) of grapevine. In host plants, this bacterium exclusively colonizes the xylem, which is primarily non-living at maturity. Understanding how X. fastidiosa interfaces with this specialized conductive tissue is at the forefront of investigation for this pathosystem. Unlike many bacterial plant pathogens, X. fastidiosa lacks a type III secretion system and cognate effectors that aid in host colonization. Instead, X. fastidiosa utilizes plant cell-wall hydrolytic enzymes and lipases as part of its xylem colonization strategy. Several of these virulence factors are predicted to be secreted via the type II secretion system (T2SS), the main terminal branch of the Sec-dependent general secretory pathway. In this study, we constructed null mutants in xpsE and xpsG , which encode for the ATPase that drives the T2SS and the major structural pseudopilin of the T2SS, respectively. Both mutants were non-pathogenic and unable to effectively colonize Vitis vinifera grapevines, demonstrating that the T2SS is required for X. fastidiosa infection processes. Furthermore, we utilized mass spectrometry to identify type II-dependent proteins in the X. fastidiosa secretome. In vitro, we identified six type II-dependent proteins in the secretome that included three lipases, a β-1,4-cellobiohydrolase, a protease, and a conserved hypothetical protein. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license., Competing Interests: The author(s) declare no conflict of interest.

Bacterial cold shock-domain proteins are conserved nucleic acid binding chaperones that play important roles in stress adaptation and pathogenesis. Csp1 is a temperature-independent cold shock protein homolog in Xylella fastidiosa, a bacterial plant pathogen of grapevine and other economically important crops. Csp1 contributes to stress tolerance and virulence in X. fastidiosa. However, besides general single-stranded nucleic acid binding activity, little is known about the specific function(s) of Csp1. To further investigate the role(s) of Csp1, we compared phenotypic differences and transcriptome profiles between the wild type and a csp1 deletion mutant (Δ csp1 ). Csp1 contributes to attachment and long-term survival and influences gene expression. We observed reduced cell-to-cell attachment and reduced attachment to surfaces with the Δ csp1 strain compared to those with the wild type. Transmission electron microscopy imaging revealed that Δ csp1 was deficient in pili formation compared to the wild type and complemented strains. The Δ csp1 strain also showed reduced survival after long-term growth in vitro . Long-read nanopore transcriptome sequencing (RNA-Seq) analysis revealed changes in expression of several genes important for attachment and biofilm formation in Δ csp1 compared to that in the wild type. One gene of interest, pilA1 , which encodes a type IV pili subunit protein, was upregulated in Δ csp1 . Deleting pilA1 in X. fastidiosa strain Stag's Leap increased surface attachment in vitro and reduced virulence in grapevines. X. fastidiosa virulence depends on bacterial attachment to host tissue and movement within and between xylem vessels. Our results show that the impact of Csp1 on virulence may be due to changes in expression of attachment genes. IMPORTANCE Xylella fastidiosa is a major threat to the worldwide agriculture industry. Despite its global importance, many aspects of X. fastidiosa biology and pathogenicity are poorly understood. There are currently few effective solutions to suppress X. fastidiosa disease development or eliminate bacteria from infected plants. Recently, disease epidemics due to X. fastidiosa have greatly expanded, increasing the need for better disease prevention and control strategies. Our studies show a novel connection between cold shock protein Csp1 and pili abundance and attachment, which have not been reported for X. fastidiosa. Understanding how pathogenesis-related gene expression is regulated can aid in developing novel pathogen and disease control strategies. We also streamlined a bioinformatics protocol to process and analyze long-read nanopore bacterial RNA-Seq data, which will benefit the research community, particularly those working with non-model bacterial species.

Plasmid vectors are a valuable research tool for characterizing bacterial gene function, but there is a limited range of plasmids that are functional in nonmodel bacterial species. Described here is a set of broad-host-range plasmids modified for stability in the absence of antibiotic selection and for gene expression manipulation.

Iron is a key micronutrient for microbial growth but is often present in low concentrations or in biologically unavailable forms. Many microorganisms overcome this challenge by producing siderophores, which are ferric-iron chelating compounds that enable the solubilization and acquisition of iron in a bioactive form. Pantoea stewartii subsp. stewartii, the causal agent of Stewart's wilt of sweet corn, produces a siderophore under iron-limiting conditions. The proteins involved in the biosynthesis and export of this siderophore are encoded by the iucABCD-iutA operon, which is homologous to the aerobactin biosynthetic gene cluster found in a number of enteric pathogens. Mutations in iucA and iutA resulted in a decrease in surface-based motility that P. stewartii utilizes during the early stages of biofilm formation, indicating that active iron acquisition impacts surface motility for P. stewartii. Furthermore, bacterial movement in planta is also dependent on a functional siderophore biosynthesis and uptake pathway. Most notably, siderophore-mediated iron acquisition is required for full virulence in the sweet corn host, indicating that active iron acquisition is essential for pathogenic fitness for this important xylem-dwelling bacterial pathogen., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Reactive oxygen species (ROS) from a variety of sources are often encountered by invading plant pathogens during the infection process. Pantoea stewartii subsp. stewartii, the etiological agent of Stewart's wilt, is a serious bacterial pathogen of sweet corn that colonizes both the apoplast and xylem tissues in which ROS are produced. The P. stewartii genome predicts the presence of two redox-sensing transcriptional regulators, OxyR and SoxR, which both activate gene expression in response to oxidative stress. ROS exposure in the form of hydrogen peroxide and the superoxide-generating compound paraquat initiates an induced stress response through OxyR and SoxR that includes activation of the ROS-detoxifying enzymes alkyl hydroperoxide reductase and superoxide dismutase. P. stewartii ΔsoxR was more sensitive to paraquat and was compromised in the ability to form water-soaked lesions, while ΔoxyR was more sensitive to hydrogen peroxide treatment and was deficient in exopolysaccharide production and the elicitation of wilting symptoms. This demonstrates that both SoxR and OxyR play an important role in virulence in the different niches that P. stewartii colonize during the infection process.

Pantoea stewartii subsp. stewartii, the causal agent of Stewart's wilt of sweet corn, produces a yellow carotenoid pigment. A nonpigmented mutant was selected from a bank of mutants generated by random transposon mutagenesis. The transposon insertion site was mapped to the crtB gene, encoding a putative phytoene synthase, an enzyme involved in the early steps of carotenoid biosynthesis. We demonstrate here that the carotenoid pigment imparts protection against UV radiation and also contributes to the complete antioxidant pathway of P. stewartii. Moreover, production of this pigment is regulated by the EsaI/EsaR quorum-sensing system and significantly contributes to the virulence of the pathogen in planta.

Pantoea stewartii subsp. stewartii, a xylem-dwelling bacterium, is the causal agent of Stewart's wilt and blight of sweet corn. The goal of this study was to characterize the only gene in the P. stewartii subsp. stewartii genome predicted to encode an endoglucanase (EGase); this gene was designated engY. Culture supernatants from P. stewartii subsp. stewartii and Escherichia coli expressing recombinant EngY protein possessed both EGase and xylanase activities. Deletion of engY abolished EGase and xylanase activity, demonstrating that EngY appears to be the major EGase or xylanase produced by P. stewartii subsp. stewartii. Most importantly, our results show that EngY contributes to movement in the xylem and disease severity during the wilting phase of Stewart's wilt but is not required for water-soaked lesion formation.