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.

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.

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.

HIV p17 enhances T cell proliferation by suppressing autophagy through the p17-OLA1-GSK3ß axis under nutrient starvation.

Nutrient starvation is a common phenomenon that occurs during T cell activation. Upon pathogen infection, large amounts of immune cells migrate to infection sites, and antigen-specific T cells are activated; this is followed by rapid proliferation through clonal expansion. The dramatic expansion of cells will commonly lead to nutrient shortage. Cellular autophagy is often upregulated as a way to sustain the body's energy requirements. During infection, human immunodeficiency virus (HIV) co-opts a series of host cell metabolic pathways for replication. Several HIV proteins, such as Env, Nef, and Vpr, have already been reported as being involved in autophagy-related processes. In this report, we identified that the HIV p17 protein acts as a major factor in suppressing the autophagic process in T cells, especially under glucose starvation condition. HIV p17 interacts with Obg-like ATPase 1 (OLA1) and disrupts OLA1-glycogen synthase kinase-3 beta (GSK3ß) complex, leading to GSK3ß hyperactivation. Consequently, a prior proliferation of HIV-infected T cells under glucose starvation will occur. The inhibition of autophagy also aids HIV replication by antagonizing the antiviral effect of autophagy. Our study shows a new cellular pathway that HIV can hijack for viral spreading by a prior proliferation of HIV-loaded T cells and may provide new therapeutic targets for acquired immunodeficiency syndrome intervention.

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).

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.