Comparative Genome Analyses Provide Insight into the Antimicrobial Activity of Endophytic Burkholderia.

Endophytic bacteria are endosymbionts that colonize a portion of plants without harming the plant for at least a part of its life cycle. Bacterial endophytes play an essential role in promoting plant growth using multiple mechanisms. The genus Burkholderia is an important member among endophytes and encompasses bacterial species with high genetic versatility and adaptability. In this study, the endophytic characteristics of Burkholderia species are investigated via comparative genomic analyses of several endophytic Burkholderia strains with pathogenic Burkholderia strains. A group of bacterial genes was identified and predicted as the putative endophytic behavior genes of Burkholderia. Multiple antimicrobial biosynthesis genes were observed in these endophytic bacteria; however, certain important pathogenic and virulence genes were absent. The majority of resistome genes were distributed relatively evenly among the endophytic and pathogenic bacteria. All known types of secretion systems were found in the studied bacteria. This includes T3SS and T4SS, which were previously thought to be disproportionately represented in endophytes. Additionally, questionable CRISPR-Cas systems with an orphan CRISPR array were prevalent, suggesting that intact CRISPR-Cas systems may not exist in symbiotes of Burkholderia. This research not only sheds light on the antimicrobial activities that contribute to biocontrol but also expands our understanding of genomic variations in Burkholderia's endophytic and pathogenic bacteria.

Development of a qPCR detection approach for pathogenic Burkholderia cenocepacia associated with fresh vegetables.

Natural environment serves as a reservoir for Burkholderia cepacia complex organisms, including the highly transmissible opportunistic human pathogen B. cenocepacia. Currently, there is a lack of an effective and quantitative method for B. cenocepacia detection in fresh food and other environmental niches. A quantitative real-time PCR (qPCR) detection method for B. cenocepacia bacteria was established in this study and validated using artificially inoculated fresh vegetable samples. Genome-wide comparative methods were applied to identify target regions for the design of species-specific primers. Assay specificity was measured with 12 strains of closely related Burkholderia bacteria and demonstrated the primer pair BCF6/R6 were 100% specific for detection of B. cenocepacia. The described qPCR assay evaluated B. cenocepacia with a 2 pg µl-1 limit of detection and appropriate linearity (R2 = 0.999). In 50 samples of experimentally infected produce (lettuce, onion, and celery), the assay could detect B. cenocepacia as low as 2.6 × 102 cells in each sample equal to 1 g. The established qPCR method quantitatively detects B. cenocepacia with high sensitivity and specificity, making it a promising technique for B. cenocepacia detection and epidemiological research on B. cepacia complex organisms from fresh vegetables.

A Polyketide Synthetase Gene Cluster Is Responsible for Antibacterial Activity of Burkholderia contaminans MS14.

Burkholderia contaminans MS14, isolated from a soil sample in Mississippi, is known for producing the novel antifungal compound occidiofungin. In addition, MS14 exhibits a broad range of antibacterial activities against common plant pathogens. Random mutagenesis and gene complementation indicate that four genes are required for antibacterial activity of strain MS14 against the fire blight pathogen Erwinia amylovora. With the aim of finding the biosynthetic gene cluster for the unknown antibacterial compound, we used RNA-seq to analyze the transcriptome of MS14 wild type and mutants lacking antibacterial activity. The twofold lower expressed genes in all mutants were studied, and a polyketide synthase (PKS) gene cluster was predicted to be directly involved in MS14 antibacterial activities. The nptII-resistance cassette and CRISPR-Cas9 systems were used to mutate the PKS gene cluster. Plate bioassays showed that either insertion or frame-shifting one of the PKS genes resulted in a loss of antibacterial activity. Considering that the antibacterial-defective mutants maintain the same antifungal activities as the wild-type strain, the results suggest that this PKS gene cluster is highly likely to be involved in or directly responsible for the production of MS14 antibacterial activity. Purification efforts revealed that the antibacterial activity of the compound synthesized by the gene cluster is sensitive to UV radiation. Nevertheless, these findings have provided more insights to understand the antibacterial activity of strain MS14.

PafS Containing GGDEF-Domain Regulates Life Activities of Pseudomonas glycinae MS82.

Cyclic dimeric guanosine monophosphate (c-di-GMP) is synthesized by diguanylate cyclase (DGC) with the GGDEF domain. As a ubiquitous bacterial second messenger, it regulates diverse life-activity phenotypes in some bacteria. Although 38 genes encoding GGDEF-domain-containing proteins have been identified in the genome of the Pseudomonas glycinae strain MS82, whether c-di-GMP functions as a facilitator or repressor of life-activity phenotypes is poorly understood. In this study, one of the 38 genes containing a GGDEF domain in MS82, PafS was investigated to explore its regulatory function in bacterial life activities. The PafS-deletion mutant ΔPafS and reversion mutant PafS-comp were constructed by the method of biparental conjugation and homologous recombination. The life activities of the mutants, such as antifungal activity, biofilm formation ability, polysaccharide content, and motor behavior, were explored. The results showed that all life-activity phenotypes were significantly reduced after knocking out PafS, whereas all were significantly restored to a similar level to that of MS82 after the complementation of PafS. These results suggested that PafS plays an important role in the regulation of a range of cellular activities by c-di-GMP in P. glycinae MS82.

Synthesis and characterization of semisynthetic analogs of the antifungal occidiofungin.

Occidiofungin is a broad-spectrum antifungal compound produced by Burkholderia contaminans MS14. It is a cyclic glycol-lipopeptide with a novel beta-amino acid (NAA2) containing a hydroxylated C18 fatty acid chain with a xylose sugar. This study reports a strategy to produce semisynthetic analogs of occidiofungin to further explore the structure activity relationships of this class of compounds. Oxidative cleavage of the diol present on carbons five C(5) and six C(6) removes the xylose and twelve carbons of the fatty acid chain. The resulting cyclic peptide product, occidiofungin aldehyde, is devoid of antifungal activity. However, the free aldehyde group on this product can be subjected to reductive amination reactions to provide interesting semisynthetic analogs. This chemistry allows the quick generation of analogs to study the structure activity relationships of this class of compounds. Despite restoring the length of the aliphatic side chain by reductive amination addition with undecylamine or dodecylamine to the free aldehyde group, the obtained analogs did not demonstrate any antifungal activity. The antifungal activity was partially restored by the addition of a DL-dihydrosphingosine. The dodecylamine analog was demonstrated to still bind to the cellular target actin, suggesting that the diol on the side chain of native occidiofungin is important for entry into the cell enabling access to cellular target F-actin. These results show that the alkyl side chain on NAA2 along with the diol present on this side chain is important for occidiofungin's antifungal activity.

Occidiofungin Is the Key Metabolite for Antifungal Activity of the Endophytic Bacterium Burkholderia sp. MS455 Against Aspergillus flavus.

Aflatoxin is a secondary metabolite produced by Aspergillus fungi and presents a major food safety concern globally. Among the available methods for prevention and control of aflatoxin, the application of antifungal bacteria has gained favor in recent years. An endophytic bacterium MS455, isolated from soybean, exhibited broad-spectrum antifungal activity against economically important pathogens, including Aspergillus flavus. MS455 was identified as a strain of Burkholderia based on genomic analysis. Random and site-specific mutations were used in discovery of the genes that share high homology to the ocf gene cluster of Burkholderia contaminans strain MS14, which is responsible for production of the antifungal compound occidiofungin. RNA sequencing analysis demonstrated that ORF1, a homolog to the ambR1 LuxR-type regulatory gene, regulates occidiofungin biosynthesis in MS455. Additionally, 284 differentially expressed genes, including 138 upregulated and 146 downregulated genes, suggesting that, in addition to its role in occidiofungin production, ORF1 is involved in expression of multiple genes, especially those involved in ornibactin biosynthesis. Plate bioassays showed the growth of A. flavus was significantly inhibited by the wild-type strain MS455 as compared with the ORF1 mutant. Similarly, corn kernel assays showed that growth of A. flavus and aflatoxin production were reduced significantly by MS455 as compared with buffer control and the ORF1 mutant. Collectively, the results demonstrated that production of occidiofungin is essential for antifungal activity of the endophytic bacterium MS455. This research has provided insights about antifungal mechanisms of MS455 and development of biological approaches to prevent aflatoxin contamination in plant production.

Phenazine-1-carboxylic Acid Produced by Pseudomonas chlororaphis YL-1 Is Effective against Acidovorax citrulli.

The bacterial pathogen Acidovorax citrulli causes the destructive fruit blotch (BFB) on cucurbit plants. Pseudomonas chlororaphis YL-1 is a bacterial strain isolated from Mississippi soil and its genome harbors some antimicrobial-related gene clusters, such as phenazine, pyrrolnitrin, and pyoverdine. Here, we evaluated the antimicrobial activity of strain YL-1 as compared with its deficient mutants of antimicrobial-related genes, which were obtained using a sacB-based site-specific mutagenesis strategy. We found that only phenazine-deficient mutants ΔphzE and ΔphzF almost lost the inhibitory effects against A. citrulli in LB plates compared with the wild-type strain YL-1, and that the main antibacterial compound produced by strain YL-1 in LB medium was phenazine-1-carboxylic acid (PCA) based on the liquid chromatography-mass spectrometry (LC-MS) analysis. Gene expression analyses revealed that PCA enhanced the accumulation of reactive oxygen species (ROS) and increased the activity of catalase (CAT) in A. citrulli. The inhibition effect of PCA against A. citrulli was lowered by adding exogenous CAT. PCA significantly upregulated the transcript level of katB from 6 to 10 h, which encodes CAT that helps to protect the bacteria against oxidative stress. Collectively, the findings of this research suggest PCA is one of the key antimicrobial metabolites of bacterial strain YL-1, a promising biocontrol agent for disease management of BFB of cucurbit plants.

Complete Genome Sequence Resource for the Endophytic Burkholderia sp. Strain MS389 Isolated from a Healthy Soybean Growing Adjacent to Charcoal Rot Disease Patch.

Burkholderia sp. strain MS389, an endophytic bacterium, was isolated from a healthy soybean plant growing adjacent to a patch of plants affected by charcoal rot disease, caused by the fungal pathogen Macrophomina phaseolina. Preliminary studies demonstrated that strain MS389 possesses antimicrobial activities against multiple plant pathogens. Burkholderia sp. strain MS389 was found to have three circular chromosomes of 3,563,380 bp, 3,002,449 bp, and 1,180,421 bp in size, respectively. The 7,746,250-bp genome, with 66.73% G+C content, harbors 6,756 protein coding genes in the predicted 6,985 genes. In total, 18 rRNAs, 68 tRNAs, and four ncRNAs were identified and 139 pseudogenes were annotated as well. The findings of this study will provide valuable data to explore the antimicrobial mechanisms of the endophytic bacterial strain.

Pyoverdines Are Essential for the Antibacterial Activity of Pseudomonas chlororaphis YL-1 under Low-Iron Conditions.

Pseudomonas chlororaphis YL-1 has extensive antimicrobial activities against phytopathogens, and its genome harbors a pyoverdine (PVD) biosynthesis gene cluster. The alternative sigma factor PvdS in Pseudomonas aeruginosa PAO1 acts as a critical regulator in response to iron starvation. The assembly of the PVD backbone starts with peptide synthetase enzyme PvdL. PvdF catalyzes formylation of l-OH-Orn to produce l-N5-hydroxyornithine. Here, we describe the characterization of PVD production in YL-1 and its antimicrobial activity in comparison with that of its PVD-deficient ΔpvdS, ΔpvdF, and ΔpvdL mutants, which were obtained using a sacB-based site-specific mutagenesis strategy. Using in vitro methods, we examined the effect of exogenous iron under low-iron conditions and an iron-chelating agent under iron-sufficient conditions on PVD production, antibacterial activity, and the relative expression of the PVD transcription factor gene pvdS in YL-1. We found that strain YL-1, the ΔpvdF mutant, and the ΔpvdS(pUCP26-pvdS) complemented strain produced visible PVDs and demonstrated a wide range of inhibitory effects against Gram-negative and Gram-positive bacteria in vitro under low-iron conditions and that with the increase of iron, its PVD production and antibacterial activity were reduced. The antibacterial compounds produced by strain YL-1 under low-iron conditions were PVDs based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Moreover, the antibacterial activity observed in vitro was correlated with in vivo control efficacies of strain YL-1 against rice bacterial leaf blight (BLB) disease caused by Xanthomonas oryzae pv. oryzae. Collectively, PVDs are responsible for the antibacterial activities of strain YL-1 under both natural and induced low-iron conditions.IMPORTANCE The results demonstrated that PVDs are essential for the broad-spectrum antibacterial activities of strain YL-1 against both Gram-positive and Gram-negative bacteria under low-iron conditions. Our findings also highlight the effect of exogenous iron on the production of PVD and the importance of this bacterial product in bacterial interactions. As a biocontrol agent, PVDs can directly inhibit the proliferation of the tested bacteria in addition to participating in iron competition.

Formulation, Pharmacological Evaluation, and Efficacy Studies of Occidiofungin, a Novel Antifungal.

Occidiofungin is a nonribosomally synthesized cyclic lipopeptide that possesses broad-spectrum antifungal properties at submicromolar concentrations. This report explores multiple routes of administration and formulations of occidiofungin, as well as its toxicity in mice. Further, infection studies were performed in mice to assess the application of occidiofungin for treating systemic and intravaginal yeast infections. Formulations for intravenous and intravaginal administration of occidiofungin were prepared. Pharmacokinetic analyses were performed in a murine model, and a liquid chromatography-mass spectrometry (LC-MS) method was developed and used to quantify occidiofungin in mouse plasma samples. Toxicological and histopathological analyses of two repeat-dose studies using occidiofungin were performed. In these animal models, following intravenous administration, a liposomal formulation of occidiofungin improved the half-life and peak plasma drug concentration over that with a liposome-free formulation. Two long-term repeat-dosing toxicity studies of occidiofungin indicated the absence of toxicity in organ tissues. Murine models of a systemic yeast infection and a vulvovaginal yeast infection were performed. The findings of the systemic infection study revealed limitations in the use of occidiofungin that may be alleviated with the development of novel structural analogs or with further formulation studies. The gel formulation of occidiofungin demonstrated improved efficacy over that of the commercial product Monistat 3 in a vulvovaginal candidiasis study. This report outlines the optimal routes of administration of occidiofungin and demonstrates minimal toxicity following chronic exposure. Further, the results of these studies provide a clear indication for the use of occidiofungin for the treatment of recurrent vulvovaginal candidiasis (RVVC), which is a serious and clinically relevant issue.

Pseudomonas glycinae sp. nov. isolated from the soybean rhizosphere.

Strains MS586T and MS82, which are aerobic, Gram-negative, rod-shaped, and polar-flagellated bacteria, were isolated from the soybean rhizosphere in Mississippi. Taxonomic positions of MS586T and MS82 were determined using a polyphasic approach. 16S rRNA gene sequence analyses of the two strains showed high pairwise sequence similarities (>98%) to some Pseudomonas species. Analysis of the concatenated 16S rRNA, rpoB, rpoD, and gyrB gene sequences indicated that the strains belonging to the Pseudomonas koreensis subgroup (SG) shared the highest similarity with Pseudomonas kribbensis strain 46-2T . Analyses of average nucleotide identity (ANI), genome-to-genome distance, delineated MS586T and MS82 from other species within the genus Pseudomonas. The predominant quinone system of the strain was ubiquinone 9 (Q-9), and the DNA G+C content was 60.48 mol%. The major fatty acids were C16:0 , C17:0 cyclo, and the summed features 3 and 8 consisting of C16:1 ω7c/C16:1 ω6c and C18:1 ω7c/C18:1 ω6c, respectively. The major polar lipids were phosphatidylglycerol, phosphatidylethanolamine, and diphosphatidylglycerol. Based on these data, it is proposed that strains MS586T and MS82 represent a novel species within the genus Pseudomonas. The proposed name for the new species is Pseudomonas glycinae, and the type strain is MS586T (accession NRRL B-65441 = accession LMG 30275).

Development of a qPCR Protocol to Detect the Cotton Bacterial Blight Pathogen, Xanthomonas citri pv. malvacearum, from Cotton Leaves and Seeds.

Bacterial blight, historically a seed-borne disease of cotton (Gossypium hirsutum) is caused by Xanthomonas citri pv. malvacearum, resulted in significant economic losses prior to development of resistant varieties and implementation of acid-delinting of planting seed. Periodic outbreaks have been associated with seed since the early twentieth century; of note, the disease has experienced a resurgence since 2011. Effective management of bacterial blight is dependent on accurate diagnosis and detection of the pathogen. Currently, detection of X. citri pv. malvacearum is performed by time-consuming microbiological methods. In this study, a novel and sensitive TaqMan-based qPCR protocol was developed to test for X. citri pv. malvacearum in cotton plant tissue. The primers developed are specific to five races of X. citri pv. malvacearum, but not to other Xanthomonas species or cotton-associated nonpathogenic bacteria. The efficiency of this assay was evaluated on artificially inoculated cotton leaves and seed, on naturally infected cotton leaves, and on bolls and seed originating from bacterial blight symptomatic bolls. The protocol's efficiency from artificially inoculated plant tissue was 102 copies g-1 and 37 copies from 1 g seed for leaves and seed, respectively. In addition, X. citri pv. malvacearum was detected from 94% of the seed samples originating from blight symptomatic bolls. The qPCR protocol provides a rapid and accurate method for diagnosis and detection of bacterial blight and offers a tool for monitoring X. citri pv. malvacearum and potentially reducing its spread in seed.

A Novel Actin Binding Drug with In Vivo Efficacy.

Occidiofungin is produced by the soil bacterium Burkolderia contaminans MS14 and is structurally similar or identical to the burkholdines, xylocandins, and cepacidines. This study identified the primary cellular target of occidiofungin, which was determined to be actin. The modification of occidiofungin with a functional alkyne group enabled affinity purification assays and localization studies in yeast. Occidiofungin has a subtle effect on actin dynamics that triggers apoptotic cell death. We demonstrate the highly specific localization of occidiofungin to cellular regions rich in actin in yeast and the binding of occidiofungin to purified actin in vitro Furthermore, a disruption of actin-mediated cellular processes, such as endocytosis, nuclear segregation, and hyphal formation, was observed. All of these processes require the formation of stable actin cables, which are disrupted following the addition of a subinhibitory concentration of occidiofungin. We were also able to demonstrate the effectiveness of occidiofungin in treating a vulvovaginal yeast infection in a murine model. The results of this study are important for the development of an efficacious novel class of actin binding drugs that may fill the existing gap in treatment options for fungal infections or different types of cancer.

The genome of the cotton bacterial blight pathogen Xanthomonas citri pv. malvacearum strain MSCT1.

Xanthomonas citri pv. malvacearum is a major pathogen of cotton, Gossypium hirsutum L.. In this study we report the complete genome of the X. citri pv. malvacearum strain MSCT1 assembled from long read DNA sequencing technology. The MSCT1 genome is the first X. citri pv. malvacearum genome with complete coding regions for X. citri pv. malvacearum transcriptional activator-like effectors. In addition functional and structural annotations are presented in this study that will provide a foundation for future pathogenesis studies with MSCT1.

The Siderophore Product Ornibactin Is Required for the Bactericidal Activity of Burkholderia contaminans MS14.

Burkholderia contaminans MS14 was isolated from soil in Mississippi. When it is cultivated on nutrient broth-yeast extract agar, the colonies exhibit bactericidal activity against a wide range of plant-pathogenic bacteria. A bacteriostatic compound with siderophore activity was successfully purified and was determined by nuclear magnetic resonance spectroscopy to be ornibactin. Isolation of the bactericidal compound has not yet been achieved; therefore, the exact nature of the bactericidal compound is still unknown. During an attempt to isolate the bactericidal compound, an interesting relationship between the production of ornibactin and the bactericidal activity of MS14 was characterized. Transposon mutagenesis resulted in two strains that lost bactericidal activity, with insertional mutations in a nonribosomal peptide synthetase (NRPS) gene for ornibactin biosynthesis and a luxR family transcriptional regulatory gene. Coculture of these two mutant strains resulted in restoration of the bactericidal activity. Furthermore, the addition of ornibactin to the NRPS mutant restored the bactericidal phenotype. It has been demonstrated that, in MS14, ornibactin has an alternative function, aside from iron sequestration. Comparison of the ornibactin biosynthesis genes in Burkholderia species shows diversity among the regulatory elements, while the gene products for ornibactin synthesis are conserved. This is an interesting observation, given that ornibactin is thought to have the same defined function within Burkholderia species. Ornibactin is produced by most Burkholderia species, and its role in regulating the production of secondary metabolites should be investigated.IMPORTANCE Identification of the antibacterial product from strain MS14 is not the key feature of this study. We present a series of experiments that demonstrate that ornibactin is directly involved in the bactericidal phenotype of MS14. This observation provides evidence for an alternative function for ornibactin, aside from iron sequestration. Ornibactin should be further evaluated for its role in regulating the biosynthesis of secondary metabolites in other Burkholderia species.

Draft Genome Sequence of Oral Bacterium Streptococcus mutans JH1140.

Streptococcus mutans JH1140 is an oral bacterium known to produce the bacteriocin mutacin 1140, and the strain has been genetically engineered to combat dental caries. Here, we report the 2.0-Mb draft genome of S. mutans JH1140. This genome provides new insights into the strain's superior colonization properties and its utility in replacement therapy.

Complete genome of Pseudomonas chlororaphis strain UFB2, a soil bacterium with antibacterial activity against bacterial canker pathogen of tomato.

Strain UFB2 was isolated from a soybean field soil in Mississippi and identified as a member of Pseudomonas chlororaphis. Strain UFB2 has a broad-spectrum antimicrobial activity against common soil-borne pathogens. Plate assays showed that strain UFB2 was especially efficient in inhibiting the growth of Clavibacter michiganensis 1-07, the causal agent of the devastating bacterial canker of tomato. Here, the complete genome sequence of P. chlororaphis strain UFB2 is reported and described. The strain UFB2 genome consists of a circular chromosome of 6,360,256 bp of which 87.86 % are protein-coding bases. Genome analysis revealed multiple gene islands encoding various secondary metabolites such as 2,4-diacetylphloroglucinol. Further genome analysis will provide more details about strain UFB2 antibacterial activities mechanisms and the use of this strain as a potential biocontrol agent.

SecG is required for antibiotic activities of Pseudomonas sp. YL23 against Erwinia amylovora and Dickeya chrysanthemi.

Strain YL23 was isolated from soybean root tips and identified to be Pseudomonas sp. This strain showed broad-spectrum antibacterial activity against bacterial pathogens that are economically important in agriculture. To characterize the genes dedicated to antibacterial activities against microbial phytopathogens, a Tn5-mutation library of YL23 was constructed. Plate bioassays revealed that the mutant YL23-93 lost its antibacterial activities against Erwinia amylovora and Dickeya chrysanthemi as compared with its wild type strain. Genetic and sequencing analyses localized the transposon in a homolog of the secG gene in the mutant YL23-93. Constitutive expression plasmid pUCP26-secG was constructed and electroporated into the mutant YL23-93. Introduction of the plasmid pUCP26-secG restored antibacterial activities of the mutant YL23-93 to E. amylovora and D. chrysanthemi. As expected, empty plasmid pUCP26 could not complement the phenotype of the antibacterial activity in the mutant. Thus the secG gene, belonging to the Sec protein translocation system, is required for antibacterial activity of strain YL23 against E. amylovora and D. chrysanthemi.

Draft Genome Sequence of Burkholderia pyrrocinia Lyc2, a Biological Control Strain That Can Suppress Multiple Plant Microbial Pathogens.

Burkholderia pyrrocinia strain Lyc2 was isolated from the tobacco rhizosphere in China. This bacterium exhibits a remarkable capacity to inhibit the growth of multiple pathogens and shows strong suppression of cotton seedling damping-off. Here, we present the draft genome sequence of Burkholderia pyrrocinia strain Lyc2.