Comparative genome analysis of rice-pathogenic Burkholderia provides insight into capacity to adapt to different environments and hosts

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.Background In addition to human and animal diseases, bacteria of the genus Burkholderia can cause plant diseases. The representative species of rice-pathogenic Burkholderia are Burkholderia glumae, B. gladioli, and B. plantarii, which primarily cause grain rot, sheath rot, and seedling blight, respectively, resulting in severe reductions in rice production. Though Burkholderia rice pathogens cause problems in rice-growing countries, comprehensive studies of these rice-pathogenic species aiming to control Burkholderia-mediated diseases are only in the early stages. Results We first sequenced the complete genome of B. plantarii ATCC 43733T. Second, we conducted comparative analysis of the newly sequenced B. plantarii ATCC 43733T genome with eleven complete or draft genomes of B. glumae and B. gladioli strains. Furthermore, we compared the genome of three rice Burkholderia pathogens with those of other Burkholderia species such as those found in environmental habitats and those known as animal/human pathogens. These B. glumae, B. gladioli, and B. plantarii strains have unique genes involved in toxoflavin or tropolone toxin production and the clustered regularly interspaced short palindromic repeats (CRISPR)-mediated bacterial immune system. Although the genome of B. plantarii ATCC 43733T has many common features with those of B. glumae and B. gladioli, this B. plantarii strain has several unique features, including quorum sensing and CRISPR/CRISPR-associated protein (Cas) systems. Conclusions The complete genome sequence of B. plantarii ATCC 43733T and publicly available genomes of B. glumae BGR1 and B. gladioli BSR3 enabled comprehensive comparative genome analyses among three rice-pathogenic Burkholderia species responsible for tissue rotting and seedling blight. Our results suggest that B. glumae has evolved rapidly, or has undergone rapid genome rearrangements or deletions, in response to the hosts. It also, clarifies the unique features of rice pathogenic Burkholderia species relative to other animal and human Burkholderia species

Prediction of Host-Specific Genes by Pan-Genome Analyses of the Korean Ralstonia solanacearum Species Complex

The soil-borne pathogenic Ralstonia solanacearum species complex (RSSC) is a group of plant pathogens that is economically destructive worldwide and has a broad host range, including various solanaceae plants, banana, ginger, sesame, and clove. Previously, Korean RSSC strains isolated from samples of potato bacterial wilt were grouped into four pathotypes based on virulence tests against potato, tomato, eggplant, and pepper. In this study, we sequenced the genomes of 25 Korean RSSC strains selected based on these pathotypes. The newly sequenced genomes were analyzed to determine the phylogenetic relationships between the strains with average nucleotide identity values, and structurally compared via multiple genome alignment using Mauve software. To identify candidate genes responsible for the host specificity of the pathotypes, functional genome comparisons were conducted by analyzing pan-genome orthologous group (POG) and type III secretion system effectors (T3es). POG analyses revealed that a total of 128 genes were shared only in tomato-non-pathogenic strains, 8 genes in tomato-pathogenic strains, 5 genes in eggplant-non-pathogenic strains, 7 genes in eggplant-pathogenic strains, 1 gene in pepper-non-pathogenic strains, and 34 genes in pepper-pathogenic strains. When we analyzed T3es, three host-specific effectors were predicted: RipS3 (SKWP3) and RipH3 (HLK3) were found only in tomato-pathogenic strains, and RipAC (PopC) were found only in eggplant-pathogenic strains. Overall, we identified host-specific genes and effectors that may be responsible for virulence functions in RSSC in silico. The expected characters of those genes suggest that the host range of RSSC is determined by the comprehensive actions of various virulence factors, including effectors, secretion systems, and metabolic enzymes

Structural and Functional Analysis of Phytotoxin Toxoflavin-Degrading Enzyme

Pathogenic bacteria synthesize and secrete toxic low molecular weight compounds as virulence factors. These microbial toxins play essential roles in the pathogenicity of bacteria in various hosts, and are emerging as targets for antivirulence strategies. Toxoflavin, a phytotoxin produced by Burkholderia glumae BGR1, has been known to be the key factor in rice grain rot and wilt in many field crops. Recently, toxoflavin-degrading enzyme (TxDE) was identified from Paenibacillus polymyxa JH2, thereby providing a possible antivirulence strategy for toxoflavin-mediated plant diseases. Here, we report the crystal structure of TxDE in the substrate-free form and in complex with toxoflavin, along with the results of a functional analysis. The overall structure of TxDE is similar to those of the vicinal oxygen chelate superfamily of metalloenzymes, despite the lack of apparent sequence identity. The active site is located at the end of the hydrophobic channel, 9 Å in length, and contains a Mn(II) ion interacting with one histidine residue, two glutamate residues, and three water molecules in an octahedral coordination. In the complex, toxoflavin binds in the hydrophobic active site, specifically the Mn(II)-coordination shell by replacing a ligating water molecule. A functional analysis indicated that TxDE catalyzes the degradation of toxoflavin in a manner dependent on oxygen, Mn(II), and the reducing agent dithiothreitol. These results provide the structural features of TxDE and the early events in catalysis

Xanthomonas campestris pv. glycines: Pathogenicity genes, races, and soybean yield

Sixteen nonpathogenic mutants of X. campestris pv. glycines strain 8ra were generated with N-methyl-N-nitro-N\sp\prime-nitrosoguanidine to identify and characterize pathogenicity genes of the bacterium. A rapid bioassay technique using soybean cotyledons was developed to evaluate the large number of mutants for their pathogenicity. One nonpathogenic mutant, NP1, did not multiply in soybean cotyledons. One cosmid clone with a 31 kb insert, pIH1, that complemented mutant NP1 was isolated from the genomic library of the strain 8ra. When this cosmid clone was introduced into the other fifteen mutants, pathogenicity was restored in three. A restriction map of pIH1 was constructed, and deletion analyses identified a 10 kb HindIII fragment that restored pathogenicity to NP1. Three regions responsible for restoring pathogenicity have been identified by Tn3-HoHo1 mutagenesis. Two are in a 2.7 kb ClaI fragment and one in a 2.1 kb XbaI/BamHI fragment. Two possible open reading frames (ORF1 and ORF2) that encode proteins of about 19 kd and 23 kd, respectively, were found in the 2.7 kb ClaI fragment. A promoterless CAT cassette and lacZ fusions in ORF1 and ORF2 indicated that ORF2 but not ORF1 may be expressed in E. coli and in X. campestris pv. glycines. The 10 kb hindIII fragment were conserved among other X. campestris pathovars tested but not in Pseudomonas syringae pvs. glycinea and tabaci. The functions of two putative polypeptides encoded by ORF1 and ORF2 are unknown. However, the carboxy terminus of the potential polypeptide encoded by ORF2 shows considerable homology with the gamma subunit of oxaloacetate decarboxylase of Klebsiella pneumoniae. Five races were identified from nine isolates of X. campestris pv. glycines, and five cultivars, 'Chippewa', 'Harosoy', 'Mukden', 'Pella', and 'Williams' were selected to differentiate five races. Development of bacterial blight, bacterial pustule, and wildfire resulting from individual and multiple infections was not affected by mixed inoculations with other pathogens. Effects of individual and multiple diseases on yield were not observed in 1986 and 1987.U of I OnlyETDs are only available to UIUC Users without author permissio

Glutamate uptake is important for osmoregulation and survival in the rice pathogen <i>Burkholderia glumae</i>

<div><p>Bacteria exhibit an optimal growth rate in culture media with sufficient nutrients at an optimal temperature and pH. In addition, the concentration of solutes plays a critical role in bacterial growth and survival. Glutamate is known to be a major anionic solute involved in osmoregulation and the bacterial cell’s response to changes in solute concentration. To determine how glutamate uptake is involved in osmoregulation in the rice bacterial pathogen <i>Burkholderia glumae</i> BGR1, we mutated the <i>gltI</i> gene encoding a periplasmic substrate binding protein of a glutamate transport system to abolish glutamate uptake, and monitored the growth of the <i>gltI</i> null mutant in Luria-Bertani medium. We found that the <i>gltI</i> null mutant showed a slower growth rate than the wild-type strain and experienced hyperosmotic stress resulting in water loss from the cytoplasm in stationary phase. When the incubation time was extended, the mutant population collapsed due to the hyperosmotic stress. The <i>gltI</i> null mutant exhibited loss of adaptability under both hypoosmotic and hyperosmotic stresses. The growth rate of the <i>gltI</i> null mutant was restored to the level of wild-type growth by exogenous addition of glycine betaine to the culture medium, indicating that glycine betaine is a compatible solute in <i>B</i>. <i>glumae</i>. These results indicate that glutamate uptake from the environment plays a key role in osmoregulation in <i>B</i>. <i>glumae</i>.</p></div

The loss of adaptability in the <i>gltI</i> mutant under hypoosmotic and hyperosmotic conditions.

<p>The <i>B</i>. <i>glumae</i> wild-type strain BGR1, the <i>gltI</i> mutant (BGLT1), and the <i>gltI</i> mutant complementation with pGLT1 were grown in LB medium with various concentrations of NaCl (0–5%). Error bars indicate the SE ranges of three independent experiments.</p

Expression levels of genes involved in the <i>de novo</i> synthesis of glutamate in the <i>B</i>. <i>glumae</i> strains.

<p>The gene expression levels of the <i>gltB</i>, <i>glnA</i>, and <i>gdhA</i> genes encoding for glutamine oxoglutarate aminotransferase, glutamine synthetase, and glutamate dehydrogenase, respectively, were quantified in the wild-type strain, <i>gltI</i> mutant, and <i>gltI</i> mutant complementation after 10 and 16 h of incubation by quantitative reverse transcription polymerase chain reaction (qRT-PCR) with three biological replicates. The bars indicate ± SE. The asterisks (*) represent a significant difference in expression level (p < 0.05) between the <i>gltI</i> mutant and the wild-type strain or the complementation as determined by ANOVA/Tukey’s correction for multiple comparisons.</p

Slow increase in cellular osmolality in the <i>gltI</i> mutant.

<p>The cellular osmolality levels in the wild-type strain (BGR1), the <i>gltI</i> mutant (BGLT1), and <i>gltI</i> mutant complementation with pGLT1 in LB medium. The levels of cellular osmolality were measured from <i>B</i>. <i>glumae</i> strains cultured for 6, 12, 24, and 36 h. All samples were normalized to weight of cells. Error bars indicate the SE ranges of three independent experiments. The asterisks (*) indicate a significant difference (p < 0.05) in osmolality among the <i>B</i>. <i>glumae</i> strains as determined by ANOVA/Tukey’s correction for multiple comparisons.</p

Effect of the addition of glycine betaine on the growth of the <i>gltI</i> mutant.

<p>Growth of the <i>B</i>. <i>glumae</i> wild-type strain BGR1, the <i>gltI</i> mutant (BGLT1), and <i>gltI</i> mutant complementation with pGLT1 [BGRT1(pBGLT1)] with various concentration of glycine betaine as a compatible solute in LB media. Error bars indicate the SE ranges of three independent experiments.</p