Regulation of corA, the Magnesium, Nickel, Cobalt Transporter, and Its Role in the Virulence of the Soft Rot Pathogen, Pectobacterium versatile Strain Ecc71

Pectobacterium versatile (formally P. carotovorum) causes disease on diverse plant species by synthesizing and secreting copious amount of plant-cell-wall-degrading exoenzymes including pectate lyases, polygalacturonases, cellulases, and proteases. Exoenzyme production and virulence are controlled by many factors of bacterial, host, and environmental origin. The ion channel forming the magnesium, nickel, and cobalt transporter CorA is required for exoenzyme production and full virulence in strain Ecc71. We investigated CorA’s role as a virulence factor and its expression in P. versatile. Inhibiting the transport function of CorA by growing a CorA+ strain in the presence of specific CorA inhibitor, cobalt (III) hexaammine (Co (III)Hex), has no effect on exoenzyme production. Transcription of pel-1, encoding a pectate lyase isozyme, is decreased in the absence of CorA, suggesting that CorA influences exoenzyme production at the transcriptional level, although apparently not through its transport function. CorA− and CorA+ strains grown in the presence of Co (III)Hex transcriptionally express corA at higher levels than CorA+ strains in the absence of an inhibitor, suggesting the transport role of corA contributes to autorepression. The expression of corA is about four-fold lower in HrpL− strains lacking the hrp-specific extracytoplasmic sigma factor. The corA promoter region contains a sequence with a high similarity to the consensus Hrp box, suggesting that corA is part of Hrp regulon. Our data suggest a complex role, possibly requiring the physical presence of the CorA protein in the virulence of the Pectobacterium versatile strain Ecc71

Development of PCR-Based Detection System for Soft Rot Pectobacteriaceae Pathogens Using Molecular Signatures

Pectobacterium and Dickeya species, usually referred to as soft rot Enterobacteriaceae, are phytopathogenic genera of bacteria that cause soft rot and blackleg diseases and are responsible for significant yield losses in many crops across the globe. Diagnosis of soft rot disease is difficult through visual disease symptoms. Pathogen detection and identification methods based on cultural and morphological identification are time-consuming and not always reliable. A polymerase chain reaction (PCR)-based detection method with the species-specific primers is fast and reliable for detecting soft rot pathogens. We have developed a specific and sensitive detection system for some species of soft rot Pectobacteriaceae pathogens in the Pectobacterium and Dickeya genera based on the use of species-specific primers to amplify unique genomic segments. The specificities of primers were verified by PCR analysis of genomic DNA from 14 strains of Pectobacterium, 8 strains of Dickeya, and 6 strains of non-soft rot bacteria. This PCR assay provides a quick, simple, powerful, and reliable method for detection of soft rot bacteria

A mini-Tn5-derived transposon with reportable and selectable markers enables rapid generation and screening of insertional mutants in Gram-negative bacteria

We re-engineered a classic tool for mutagenesis and gene expression studies in Gram-negative bacteria. Our modified Tn5-based transposon contains multiple features that allow rapid selection for mutants, direct quantification of gene expression and straightforward cloning of the inactivated gene. The promoter-less gfp-km cassette provides selection and reporter assay depending on the activity of the promoter upstream of the transposon insertion site. The cat gene facilitates positive antibiotic selection for mutants, while the narrow R6Kγ replication origin forces transposition in recipient strains lacking the pir gene and enables cloning of the transposon flanked with the disrupted gene from the chromosome. The suicide vector pCKD100, a plasmid that could be delivered into recipient cells through biparental mating or electroporation, harbours the modified transposon. We used the transposon to mutagenize Pectobacterium versatile KD100, Pseudumonas coronafaciens PC27R and Escherichia coli 35150N. The fluorescence intensities of mutants expressing high GFP could be quantified and detected qualitatively. Transformation efficiency from conjugation ranged from 1600 to 1900 CFU per ml. We sequenced the upstream flanking regions, identified the putative truncated genes and demonstrated the restoration of the GFP phenotype through marker exchange. The mini-Tn5 transposon was also utilized to construct mutant a library of P. versatile for forward genetic screens

The Gene Encoding NAD-Dependent Epimerase/Dehydratase, wcaG, Affects Cell Surface Properties, Virulence, and Extracellular Enzyme Production in the Soft Rot Phytopathogen, Pectobacterium carotovorum

Pectobacterium carotovorum is a gram-negative bacterium that, together with other soft rot Enterobacteriaceae causes soft rot disease in vegetables, fruits, and ornamental plants through the action of exoproteins including plant cell wall-degrading enzymes (PCWDEs). Although pathogenicity in these bacteria is complex, virulence levels are proportional to the levels of plant cell wall-degrading exoenzymes (PCWDEs) secreted. Two low enzyme-producing transposon Tn5 mutants were isolated, and compared to their parent KD100, the mutants were less virulent on celery petioles and carrot disks. The inactivated gene responsible for the reduced virulence phenotype in both mutants was identified as wcaG. The gene, wcaG (previously denoted fcl) encodes NAD-dependent epimerase/dehydratase, a homologue of GDP-fucose synthetase of Escherichia coli. In Escherichia coli, GDP-fucose synthetase is involved in the biosynthesis of the exopolysaccharide, colanic acid (CA). The wcaG mutants of P. carotovorum formed an enhanced level of biofilm in comparison to their parent. In the hydrophobicity test the mutants showed more hydrophobicity than the parent in hexane and hexadecane as solvents. Complementation of the mutants with extrachromosomal copies of the wild type gene restored these functions to parental levels. These data indicate that NAD-dependent epimerase/dehydratase plays a vital rule in cell surface properties, exoenzyme production, and virulence in P. carotovorum

Antibacterial Properties of Citric Acid/β-Alanine Carbon Dots against Gram-Negative Bacteria

While multi-drug resistance in bacteria is an emerging concern in public health, using carbon dots (CDs) as a new source of antimicrobial activity is gaining popularity due to their antimicrobial and non-toxic properties. Here we prepared carbon dots from citric acid and β-alanine and demonstrated their ability to inhibit the growth of diverse groups of Gram-negative bacteria, including E. coli, Salmonella, Pseudomonas, Agrobacterium, and Pectobacterium species. Carbon dots were prepared using a one-pot, three-minute synthesis process in a commercial microwave oven (700 W). The antibacterial activity of these CDs was studied using the well-diffusion method, and their minimal inhibitory concentration was determined by exposing bacterial cells for 20 h to different concentrations of CDs ranging from 0.5 to 10 mg/mL. Our finding indicates that these CDs can be an effective alternative to commercially available antibiotics. We also demonstrated the minimum incubation time required for complete inhibition of bacterial growth, which varied depending on bacterial species. With 15-min incubation time, A. tumefaciens and P. aeruginosa were the most sensitive strains, whereas E. coli and S. enterica were the most resistant bacterial strains requiring over 20 h incubation with CDs

The Bacterial Soft Rot Pathogens, Pectobacterium carotovorum and P. atrosepticum, Respond to Different Classes of Virulence-Inducing Host Chemical Signals

Soft rot bacteria of the Pectobacterium and Dickeya genera are Gram-negative phytopathogens that produce and secrete plant cell wall-degrading enzymes (PCWDE), the actions of which lead to rotting and decay of their hosts in the field and in storage. Host chemical signals are among the factors that induce the bacteria into extracellular enzyme production and virulence. A class of compounds (Class I) made up of intermediate products of cell wall (pectin) degradation induce exoenzyme synthesis through KdgR, a global negative regulator of exoenzyme production. While the KdgR− mutant of P. carotovorum is no longer inducible by Class I inducers, we demonstrated that exoenzyme production is induced in this strain in the presence of extracts from hosts including celery, potato, carrot, and tomato, suggesting that host plants contain another class of compounds (Class II inducers) different from the plant cell wall-degradative products that work through KdgR. The Class II inducers are thermostable, water-soluble, diffusible, and dialysable through 1 kDa molecular weight cut off pore size membranes, and could be a target for soft rot disease management strategies

Citric acid/β-alanine carbon dots as a novel tool for delivery of plasmid DNA into E. coli cells

Successful delivery of plasmid DNA into the microbial cells is fundamental in recombinant DNA technology. Natural bacterial transformation is limited to only certain species due in part to the repulsive forces between negatively charged DNA and bacterial membranes. Most common method of DNA delivery into bacteria is artificial transformation through heat shock and electroporation. These methods require sophisticated instruments and tedious steps in preparation of competent cells. Transformation by conjugation is also not applicable to all plasmids. Nanoparticles have been used successfully in therapeutics for drug delivery into animal cells. They are starting to gain popularity in plant sciences as novel DNA nano carriers. Despite their promise as tool for DNA delivery, their use in microbial cell transformation has not been reported yet. Here we report the synthesis of carbon dots (CDs) from citric acid and β-alanine and their use in DNA delivery into E. coli cells. CDs were fabricated using microwave assisted synthesis. Plasmids carrying RFP reporter and ampicillin resistance genes were transferred to bacterial cells and further confirmed using polymerase chain reaction. Our findings indicate that CDs can be used successfully for delivery of foreign DNA of up to 10 kb into E. coli. We have demonstrated the use of β-alanine/citric acid carbon dots as nanocarriers of DNA into E. coli cells and identified their limitation in terms of the size of plasmid DNA they could carry. Use of these carbon dots is a novel method in foreign DNA delivery into bacterial cells and have a potential for the transformation of resistant organism for which there is still no reliable DNA delivery systems

Identification of Bacterial Wilt (Erwinia tracheiphila) Resistances in USDA Melon Collection

Bacterial wilt (BW) caused by the Gram-negative bacterium, Erwinia tracheiphila (Et.), is an important disease in melon (Cucumis melo L.). BW-resistant commercial melon varieties are not widely available. There are also no effective pathogen-based disease management strategies as BW-infected plants ultimately die. The purpose of this study is to identify BW-resistant melon accessions in the United States Department of Agriculture (USDA) collection. We tested 118 melon accessions in two inoculation trials under controlled environments. Four-week-old seedlings of test materials were mechanically inoculated with the fluorescently (GFP) labeled or unlabeled E. tracheiphila strain, Hca1-5N. We recorded the number of days to wilting of inoculated leaf (DWIL), days to wilting of whole plant (DWWP) and days to death of the plant (DDP). We identified four melon lines with high resistance to BW inoculation based on all three parameters. Fluorescent microscopy was used to visualize the host colonization dynamics of labeled bacteria from the point of inoculation into petioles, stem and roots in resistant and susceptible melon accessions, which provides an insight into possible mechanisms of BW resistance in melon. The resistant melon lines identified from this study could be valuable resistance sources for breeding of BW resistance as well as the study of cucurbit—E. tracheiphila interactions

Rapid detection of pathogenic E. coli based on CRISPR Cas system

Access to safe and nutritious food is critical for maintaining life and supporting good health. Eating food that is contaminated with pathogens leads to serious diseases ranging from diarrhea to cancer. Many foodborne infections can cause long-term impairment or even death. Hence, early detection of foodborne pathogens such as pathogenic Escherichia coli strains is essential for public safety. Conventional methods for detecting these bacteria are based on culturing on selective media and following standard biochemical identification. Despite their accuracy, these methods are time-consuming. PCR-based detection of pathogens relies on sophisticated equipment and specialized technicians which are difficult to find in areas with limited resources. Whereas CRISPR technology is more specific and sensitive for identifying pathogenic bacteria because it employs programmable CRISPR-Cas systems that target particular DNA sequences, minimizing non-specific binding and cross-reactivity. In this project, a robust detection method based on CRISPR-Cas12a sensing was developed, which is rapid, sensitive and specific for detection of pathogenic E. coli isolates that were collected from the fecal samples from adult goats from 17 farms in Tennessee. Detection reaction contained amplified PCR products for the pathogenic regions, reporter probe, Cas12a enzyme, and crRNA specific to three pathogenic genes—stx1, stx2, and hlyA. The CRISPR reaction with the pathogenic bacteria emitted fluorescence when excited under UV light. To evaluate the detection sensitivity and specificity of this assay, its results were compared with PCR based detection assay. Both methods resulted in similar results for the same samples. This technique is very precise, highly sensitive, quick, cost effective, and easy to use, and can easily overcome the limitations of the present detection methods. This project can result in a versatile detection method that is easily adaptable for rapid response in the detection and surveillance of diseases that pose large-scale biosecurity threats to human health, and plant and animal production