Genome sequence analysis of the emerging human pathogenic acetic acid bacterium Granulibacter bethesdensis - PubMed (original) (raw)

Genome sequence analysis of the emerging human pathogenic acetic acid bacterium Granulibacter bethesdensis

David E Greenberg et al. J Bacteriol. 2007 Dec.

Abstract

Chronic granulomatous disease (CGD) is an inherited immune deficiency characterized by increased susceptibility to infection with Staphylococcus, certain gram-negative bacteria, and fungi. Granulibacter bethesdensis, a newly described genus and species within the family Acetobacteraceae, was recently isolated from four CGD patients residing in geographically distinct locales who presented with fever and lymphadenitis. We sequenced the genome of the reference strain of Granulibacter bethesdensis, which was isolated from lymph nodes of the original patient. The genome contains 2,708,355 base pairs in a single circular chromosome, in which 2,437 putative open reading frames (ORFs) were identified, 1,470 of which share sequence similarity with ORFs in the nonpathogenic but related Gluconobacter oxydans genome. Included in the 967 ORFs that are unique to G. bethesdensis are ORFs potentially important for virulence, adherence, DNA uptake, and methanol utilization. GC% values and best BLAST analysis suggested that some of these unique ORFs were recently acquired. Comparison of G. bethesdensis to other known CGD pathogens demonstrated conservation of some putative virulence factors, suggesting possible common mechanisms involved in pathogenesis in CGD. Genotyping of the four patient isolates by use of a custom microarray demonstrated genome-wide variations in regions encoding DNA uptake systems and transcriptional regulators and in hypothetical ORFs. G. bethesdensis is a genetically diverse emerging human pathogen that may have recently acquired virulence factors new to this family of organisms.

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Figures

FIG. 1.

FIG. 1.

Genome circle of Granulibacter bethesdensis. The genome is 2,708,355 base pairs long, and the rings, starting inside and moving out, show the following: GC content with sliding 20-kb window; GC skew; ORFs colored by metabolic category (virulence = turquoise, cell wall metabolism = light yellow, stress = light orange, phage = orange, signal transduction = purple, cellular processing = yellow, secondary metabolism = blue, membrane transport = cyan, secretion = teal, motility and chemotaxis = pink, information processing = sage, phosphorus metabolism = olive, nitrogen metabolism = tangerine, coenzyme and cofactor metabolism = saffron, sulfur metabolism = light green, bioenergetics = lime, one-carbon metabolism = beige, aromatic compound degradation = light purple, nucleotide metabolism = light blue, lipid metabolism = lemon, carbohydrate metabolism = rust, amino acid metabolism = peach); RNAs (tRNAs are shown in green, and rRNAs are shown in red); ORFs colored and positioned by orientation (orange, positive strand; and blue, negative strand); yellow shaded areas representing regions undergoing DNA changes across the four isolates, based on chip hybridization data; ORFs present in G. bethesdensis that are unique compared to G. oxydans (black bars); and numeric markers.

FIG. 2.

FIG. 2.

Comparative genomic context of Granulibacter bethesdensis methanol dehydrogenase genes. A comparative region analysis of two methanol dehydrogenase subunit 1 ORFs in G. bethesdensis compared to other best-hit genes and genomes is shown. (A) GbCGDNIH1_0344 and surrounding genes and their relationships to those in other methylotrophic organisms. (B) GbCGDNIH1_1922 and surrounding genes needed for methanol utilization and their relationship to the methylotrophic bacterium Methylobacterium extorquens AM1.

FIG. 3.

FIG. 3.

Quantitative PCR analysis of six different Granulibacter bethesdensis gene mRNAs at 35°C in the stationary phase of growth. Bacteria were cultured in YPG broth at 35°C to stationary phase. Multiplex quantitative PCR was performed to measure the transcripts from six different G. bethesdensis genes, and their signals were normalized to the mRNA signal from the transcript for the protein translation elongation factor TU (tufA) gene. The y axis shows relative mRNA levels for each of the six genes compared to tufA, and the x axis shows the six individual genes tested.

FIG. 4.

FIG. 4.

Ultrastructural evidence of extracellular polysaccharide capsule and conjugative pilus-like structures. (A and B) Transmission electron micrographs of G. bethesdensis NIH1.1 prepared with (A) and without (B) ruthenium red for preserving and contrasting extracellular polysaccharides. The arrow in panel A points out the capsular material on the surface. (C and D) Transmission (C) and scanning (D) electron micrographs of structures resembling conjugative pili. Bars, 100 nm.

FIG. 5.

FIG. 5.

Dendrogram of DNA hybridization of seven isolates with NIH1.1. A DNA-DNA hybridization microarray-based investigation of gene distributions among G. bethesdensis isolates was performed. The absence of genes is shown in blue, and the presence of genes is shown in yellow and red. A dendrogram is shown with the strain names listed. The analysis was performed with GeneSpring software.

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