Journal: Applied and Environmental Microbiology

Article Title: A Histone-Like Nucleoid Structuring Protein Regulates Several Virulence Traits in Burkholderia multivorans

doi: 10.1128/AEM.00369-21

Figure Lengend Snippet: Loss of the mucoid phenotype by a B. multivorans ATCC 17616 evolved colony is caused by reduction of bce gene expression. (A) Images of the mucoid colony morphologies of the ATCC 17616 ancestor and evolved 17616nmv colony grown in yeast extract-mannitol agar medium for 72 h. (B) Exopolysaccharide production in salts-mannitol medium by the ancestor and evolved colony estimated by recovery of the ethanol precipitate from culture supernatants. (C) Quantitative RT-PCR analysis of transcript levels of bceB , bceF , bceQ , and bceR genes in evolved 17616nmv compared to B. multivorans ATCC 17616, during growth in salts-mannitol medium up to 48 h at 37°C. Data are the average result from at least three independent growth cultures. Error bars indicate standard deviations. WT, wild type.

Article Snippet: To find these potential regulatory targets of the Bmul_0158 gene product, transcriptomic data were overlaid with the results of BLASTP comparisons of ATCC 17616 with other B. multivorans genomes (BAA-247, AU1185, and FDAARGOS_263), on the circular representation of the ATCC 17616 three chromosomes ( ).

Techniques: Gene Expression, Quantitative RT-PCR

Journal: Applied and Environmental Microbiology

Article Title: A Histone-Like Nucleoid Structuring Protein Regulates Several Virulence Traits in Burkholderia multivorans

doi: 10.1128/AEM.00369-21

Figure Lengend Snippet: Insertion of the IS 406 element in an intergenic region leads to increased expression of gene Bmul_0158 encoding a histone-like protein. (A) Using whole-genome sequencing of the 17616nmv strain, an insertion sequence of the IS 406 family was identified in the chromosome 1 intergenic region between genes Bmul_0157 and Bmul_0158. The nucleotide sequence of the intergenic region where the IS element was inserted is shown; the target sites of this IS element are shown in red, and partial sequences of the inverted repeats of the IS element are underlined. The two inverted orange triangles indicate the distance from IS 406 to the beginning of Bmul_0157 or Bmul_0158 coding sequences. nt, nucleotides. (B) Electrophoretic separation of the PCR products amplified from the genome of the ancestor ATCC 17616 (lane 1) and the evolved strain 17616nmv (lane 2) with expected sizes of 699 and 2,066 bp, respectively (red arrows in panel A indicate primer-binding sites). (C) qRT-PCR analysis of transcript levels of genes Bmul_0157 and Bmul_0158 and other hns -like genes in the evolved strain compared with the ancestor ATCC 17616 grown for 8 h in SM. (D) Amino acid sequence alignments of the C-terminal DNA-binding region of Mycobacterium tuberculosis Lsr2 ( ALB20845.1 ), Escherichia coli K-12 H-NS ( NP_415753.1 ), Salmonella enterica H-NS ( AUO51906.1 ), Acinetobacter baumannii ATCC 17978 H-NS ( QDQ68268.1 ), Burkholderia vietnamiensis Bv3F ( A4JS72.1 ), and B. multivorans ATCC 17616 Bmul_0158 ( ABX13853 ) are displayed. The conserved motif T/SXQ/RGRXPA implicated in DNA binding is highlighted in red. Asterisks indicate the amino acid residues that are identical in all proteins; one or two dots indicate semiconserved or conserved substitutions, respectively.

Article Snippet: To find these potential regulatory targets of the Bmul_0158 gene product, transcriptomic data were overlaid with the results of BLASTP comparisons of ATCC 17616 with other B. multivorans genomes (BAA-247, AU1185, and FDAARGOS_263), on the circular representation of the ATCC 17616 three chromosomes ( ).

Techniques: Expressing, Sequencing, Amplification, Binding Assay, Quantitative RT-PCR

Journal: Applied and Environmental Microbiology

Article Title: A Histone-Like Nucleoid Structuring Protein Regulates Several Virulence Traits in Burkholderia multivorans

doi: 10.1128/AEM.00369-21

Figure Lengend Snippet: Overexpression of the Bmul_0158 gene in wild-type Burkholderia strains reduces the mucoid phenotype of the colonies. (A) qRT-PCR analysis of transcript levels of genes Bmul_0158 and bceB in B. multivorans ATCC 17616 harboring the vector expressing Bmul_0158 from its own promoter (pLM20-4) compared with ATCC harboring the empty vector pBBR1MCS. (B) Mobilization of the vector (left panel) or pLM20-4 (right panel) to strains of B. multivorans , B. anthina , B. contaminans , and B. vietnamiensis followed by incubation in YEM agar medium supplemented with appropriate antibiotics for 3 days. Bar, 1 mm. (C) Quantification of the diameter of at least 20 randomly chosen colonies using the software Zen 3.1 from Zeiss. Colony diameter of strains overexpressing Bmul_0158 was significantly smaller than that for the ones carrying the empty vector. ***, P < 0.001 by Tukey’s honestly significant difference (HSD) multiple-comparison test.

Article Snippet: To find these potential regulatory targets of the Bmul_0158 gene product, transcriptomic data were overlaid with the results of BLASTP comparisons of ATCC 17616 with other B. multivorans genomes (BAA-247, AU1185, and FDAARGOS_263), on the circular representation of the ATCC 17616 three chromosomes ( ).

Techniques: Over Expression, Quantitative RT-PCR, Plasmid Preparation, Expressing, Incubation, Software, Comparison

Journal: Applied and Environmental Microbiology

Article Title: A Histone-Like Nucleoid Structuring Protein Regulates Several Virulence Traits in Burkholderia multivorans

doi: 10.1128/AEM.00369-21

Figure Lengend Snippet: The evolved variant has decreased surface hydrophobicity and forms smaller cellular aggregates. (A) Growth curves of B. multivorans ATCC 17616 and evolved 17616nmv in SM at 37°C as measured by optical density and counting of CFU. The evolved variant differed significantly from its ancestor regarding the optical density at 640 nm and CFU at the indicated time points. ***, P < 0.001 by Tukey’s HSD multiple-comparison test. (B to D) Light microscopy images of B. multivorans ATCC 17616 and evolved 17616nmv grown in SM at 37°C for 48 h (B), followed by dry-weight biomass determination (C) and by quantification of the percentage of aggregates and free cells (D). The evolved variant differed significantly from its ancestor regarding the percentage of cells in the form of aggregates. ***, P < 0.001 by Tukey’s HSD multiple-comparison test; ns, nonsignificant. (E) Relative surface hydrophobicity of B. multivorans ATCC 17616 and evolved 17616nmv. Bacterial suspensions cultured in SM were adjusted to an OD 640 of 0.6 (OD initial ). After the addition of n -hexadecane, the OD aq of the aqueous phase was measured. The hydrophobic activity (HP) was calculated from the formula % HP = [1 − (OD aq /OD initial ) × 100]. Values are the means of three independent experiments conducted in triplicates; error bars represent standard deviations. Cell surface hydrophobicity of the evolved variant was significantly lower than that of the ancestor. **, P < 0.01 by Tukey’s HSD multiple-comparison test.

Article Snippet: To find these potential regulatory targets of the Bmul_0158 gene product, transcriptomic data were overlaid with the results of BLASTP comparisons of ATCC 17616 with other B. multivorans genomes (BAA-247, AU1185, and FDAARGOS_263), on the circular representation of the ATCC 17616 three chromosomes ( ).

Techniques: Variant Assay, Comparison, Light Microscopy, Cell Culture, Activity Assay, Cell Surface Hydrophobicity

Journal: Applied and Environmental Microbiology

Article Title: A Histone-Like Nucleoid Structuring Protein Regulates Several Virulence Traits in Burkholderia multivorans

doi: 10.1128/AEM.00369-21

Figure Lengend Snippet: The evolved variant has increased motility but lower adhesion to a CF lung epithelial cell line. (A) Swimming motility assayed in 1% tryptone, 0.5% NaCl medium with 0.3% agar incubated at 37°C for 24 h and swarming motility assayed in Broomfield medium with 0.6% agar incubated at 37°C for 48 h were significantly higher for the strains overexpressing Bmul_0158 compared to the ancestors. ***, P < 0.001 by Tukey’s HSD multiple-comparison test. (B) Adhesion to CF lung epithelial cells by B. multivorans ATCC 17616 and evolved 17616nmv using an MOI of 10. The evolved variant differed significantly from its ancestor regarding the percentage of cells that adhere to the CF lung cell line. ***, P < 0.001 by Tukey’s HSD multiple-comparison test. (C) Susceptibility to the indicated antibiotics determined at 37°C after 24 h of incubation by measuring the diameter of cell growth inhibition. The evolved variant differed significantly from the ancestors only for resistance to kanamycin. ***, P < 0.001 by Tukey’s HSD multiple-comparison test.

Article Snippet: To find these potential regulatory targets of the Bmul_0158 gene product, transcriptomic data were overlaid with the results of BLASTP comparisons of ATCC 17616 with other B. multivorans genomes (BAA-247, AU1185, and FDAARGOS_263), on the circular representation of the ATCC 17616 three chromosomes ( ).

Techniques: Variant Assay, Incubation, Comparison, Inhibition

Journal: Applied and Environmental Microbiology

Article Title: A Histone-Like Nucleoid Structuring Protein Regulates Several Virulence Traits in Burkholderia multivorans

doi: 10.1128/AEM.00369-21

Figure Lengend Snippet: Functional distribution into COGs of genes that are differentially expressed shows enrichment of genes implicated in motility and intracellular trafficking, secretion, and vesicular transport. (A) qRT-PCR analysis of transcript levels of gene Bmul_0158 in B. multivorans ATCC 17616 grown in SM. Comparison for the different time points (t x ) was made against the expression level at 4 h (t 4 ). (B) qRT-PCR analysis performed in 17616nmv and B. multivorans ATCC 17616 and comparison with the microarray expression data. LB-FC, lower bound of fold change. (C) Clustering, based on biological function, of the differentially expressed genes with COG attributed. The percentage of genes differentially expressed within each category was calculated from the total number of B. multivorans genes attributed to each COG category. The cumulative distribution function (CDF) of the hypergeometric distribution was used to calculate enrichment of genes by COG. ***, P < 0.001; *, P < 0.05.

Article Snippet: To find these potential regulatory targets of the Bmul_0158 gene product, transcriptomic data were overlaid with the results of BLASTP comparisons of ATCC 17616 with other B. multivorans genomes (BAA-247, AU1185, and FDAARGOS_263), on the circular representation of the ATCC 17616 three chromosomes ( ).

Techniques: Functional Assay, Quantitative RT-PCR, Comparison, Expressing, Microarray

Journal: Applied and Environmental Microbiology

Article Title: A Histone-Like Nucleoid Structuring Protein Regulates Several Virulence Traits in Burkholderia multivorans

doi: 10.1128/AEM.00369-21

Figure Lengend Snippet: Selection of a set of genes differentially expressed between 17616nmv and B. multivorans ATCC 17616 grown in SM for 10 h, separated by functional groups

Article Snippet: To find these potential regulatory targets of the Bmul_0158 gene product, transcriptomic data were overlaid with the results of BLASTP comparisons of ATCC 17616 with other B. multivorans genomes (BAA-247, AU1185, and FDAARGOS_263), on the circular representation of the ATCC 17616 three chromosomes ( ).

Techniques: Selection, Functional Assay, Chemotaxis Assay, Membrane, Histone Deacetylase Assay, Transduction

Journal: Applied and Environmental Microbiology

Article Title: A Histone-Like Nucleoid Structuring Protein Regulates Several Virulence Traits in Burkholderia multivorans

doi: 10.1128/AEM.00369-21

Figure Lengend Snippet: Mapping of the putative Bmul_0158 H-NS protein targets in the ATCC 17616 genome. Gene expression data were mapped onto circular representation of B. multivorans ATCC 17616 chromosomes using the software CGView. Downregulated genes are shown in red and upregulated genes in blue, located in the rings between FDAARGOS_623 and the GC content. To identify potentially horizontally acquired genomic regions, comparative BLASTP analyses between ATCC 17616 (outer rings) and B. multivorans AU1185 (light blue), FDAARGOS_623 (green), and BAA-247 (red) are included. Various downregulated genes or gene clusters not fully conserved between ATCC 17616 and other genomes were identified (blue boxes), being putative H-NS target sites. CDS, coding sequence.

Article Snippet: To find these potential regulatory targets of the Bmul_0158 gene product, transcriptomic data were overlaid with the results of BLASTP comparisons of ATCC 17616 with other B. multivorans genomes (BAA-247, AU1185, and FDAARGOS_263), on the circular representation of the ATCC 17616 three chromosomes ( ).

Techniques: Gene Expression, Software, Sequencing

Journal: Applied and Environmental Microbiology

Article Title: A Histone-Like Nucleoid Structuring Protein Regulates Several Virulence Traits in Burkholderia multivorans

doi: 10.1128/AEM.00369-21

Figure Lengend Snippet: The majority of the downregulated genes have upstream intergenic regions of low GC content. (A) Representation of the GC percentage of the upstream intergenic region of each differentially expressed gene obtained from the microarray data set. The average GC percentage of the B. multivorans ATCC 17616 genomic-wide intergenic regions was determined as being 63.4%. The cumulative distribution function of the hypergeometric distribution was used to calculate enrichment of genes above or below 63.4% GC content. (B) Schematic representation of the bce gene clusters directing the biosynthesis of cepacian, with indication of the GC percentage of three intergenic regions containing putative promoters for genes bceA and bceB to - K , bceO to - M , bceP to - R , and bceS - T as indicated by black boxes. The GC content plots were derived from the B. multivorans ATCC 17616 genome using the GC content calculator at Biologics International Corp.

Article Snippet: To find these potential regulatory targets of the Bmul_0158 gene product, transcriptomic data were overlaid with the results of BLASTP comparisons of ATCC 17616 with other B. multivorans genomes (BAA-247, AU1185, and FDAARGOS_263), on the circular representation of the ATCC 17616 three chromosomes ( ).

Techniques: Microarray, Derivative Assay

Journal: Applied and Environmental Microbiology

Article Title: A Histone-Like Nucleoid Structuring Protein Regulates Several Virulence Traits in Burkholderia multivorans

doi: 10.1128/AEM.00369-21

Figure Lengend Snippet: Strains and plasmids used in this work

Article Snippet: To find these potential regulatory targets of the Bmul_0158 gene product, transcriptomic data were overlaid with the results of BLASTP comparisons of ATCC 17616 with other B. multivorans genomes (BAA-247, AU1185, and FDAARGOS_263), on the circular representation of the ATCC 17616 three chromosomes ( ).

Techniques: Plasmid Preparation, Variant Assay, Cloning, Sequencing