Genomic Diversity of Burkholderia pseudomallei Clinical Isolates: Subtractive Hybridization Reveals a Burkholderia mallei-Specific Prophage in B. pseudomallei 1026b

ABSTRACT Burkholderia pseudomallei is the etiologic agent of the disease melioidosis and is a category B biological threat agent. The genomic sequence of B. pseudomallei K96243 was recently determined, but little is known about the overall genetic diversity of this species. Suppression subtractive hybridization was employed to assess the genetic variability between two distinct clinical isolates of B. pseudomallei, 1026b and K96243. Numerous mobile genetic elements, including a temperate bacteriophage designated φ1026b, were identified among the 1026b-specific suppression subtractive hybridization products. Bacteriophage φ1026b was spontaneously produced by 1026b, and it had a restricted host range, infecting only Burkholderia mallei. It possessed a noncontractile tail, an isometric head, and a linear 54,865-bp genome. The mosaic nature of the φ1026b genome was revealed by comparison with bacteriophage φE125, a B. mallei-specific bacteriophage produced by Burkholderia thailandensis. The φ1026b genes for DNA packaging, tail morphogenesis, host lysis, integration, and DNA replication were nearly identical to the corresponding genes in φE125. On the other hand, φ1026b genes involved in head morphogenesis were similar to head morphogenesis genes encoded by Pseudomonas putida and Pseudomonas aeruginosa bacteriophages. Consistent with this observation, immunogold electron microscopy demonstrated that polyclonal antiserum against φE125 reacted with the tail of φ1026b but not with the head. The results presented here suggest that B. pseudomallei strains are genetically heterogeneous and that bacteriophages are major contributors to the genomic diversity of this species. The bacteriophage characterized in this study may be a useful diagnostic tool for differentiating B. pseudomallei and B. mallei, two closely related biological threat agents.

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Genomic Markers for Differentiation of Francisella tularensis subsp. tularensis A.I and A.II Strains

Applied and Environmental Microbiology ◽

10.1128/aem.01522-07 ◽

2007 ◽

Vol 74 (1) ◽

pp. 336-341 ◽

Cited By ~ 15

Author(s):

Claudia R. Molins-Schneekloth ◽

John T. Belisle ◽

Jeannine M. Petersen

Keyword(s):

Suppression Subtractive Hybridization ◽

Francisella Tularensis ◽

Geographical Location ◽

Subtractive Hybridization ◽

Clinical Isolates ◽

Type B ◽

Tularensis Subsp ◽

Genomic Markers ◽

Pcr Assays ◽

Genomic Regions

ABSTRACT Tularemia is caused by two subspecies of Francisella tularensis, F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B). F. tularensis subsp. tularensis is further subdivided into two genetically distinct populations (A.I and A.II) that differ with respect to geographical location, anatomical source of recovered isolates, and disease outcome. Using two human clinical isolates, suppression subtractive hybridization was performed to identify 13 genomic regions of difference between A.I and A.II strains. Two PCR assays, one to identify A.I and A.II as well as to discriminate between F. tularensis subsp. holarctica and F. novicida and another specific for A.I, were developed. This is the first report to identify and characterize conserved genomic differences between A.I and A.II.

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Identification of genetic differences between two clinical isolates of Streptococcus mutans by suppression subtractive hybridization

Oral Microbiology and Immunology ◽

10.1111/j.1399-302x.2006.00306.x ◽

2006 ◽

Vol 21 (6) ◽

pp. 372-380 ◽

Cited By ~ 13

Author(s):

L. H. Guo ◽

J. N. Shi ◽

Y. Zhang ◽

X. D. Liu ◽

J. Duan ◽

...

Keyword(s):

Streptococcus Mutans ◽

Suppression Subtractive Hybridization ◽

Subtractive Hybridization ◽

Genetic Differences ◽

Clinical Isolates

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Suppression subtractive hybridization as a basis to assess Mycoplasma agalactiae and Mycoplasma bovis genomic diversity and species-specific sequences

Microbiology ◽

10.1099/mic.0.27590-0 ◽

2005 ◽

Vol 151 (2) ◽

pp. 475-489 ◽

Cited By ~ 52

Author(s):

Marc S. Marenda ◽

Evelyne Sagné ◽

François Poumarat ◽

Christine Citti

Keyword(s):

Suppression Subtractive Hybridization ◽

Type Strain ◽

Subtractive Hybridization ◽

Genomic Diversity ◽

Mycoplasma Species ◽

Mycoplasma Bovis ◽

Dna Fragments ◽

Mycoplasma Agalactiae ◽

Field Isolates ◽

Species Specific

The phylogenically related Mycoplasma agalactiae and Mycoplasma bovis species are two ruminant pathogens difficult to differentiate and for which a limited amount of sequence data are available. To assess the degree of genomic diversity existing between and within these mycoplasma species, sets of DNA fragments specific for M. bovis type-strain PG45 or for M. agalactiae type-strain PG2 were isolated by suppression subtractive hybridization and used as probes on a panel of M. agalactiae and M. bovis field isolates. Results indicated that approximately 70 % of the DNA fragments specific to one or the other type strain are represented in all field isolates of the corresponding species. Only one M. bovis isolate, which was first classified as M. agalactiae, reacted with 15 % of the PG2-specific probes, while several M. agalactiae isolates reacted with 15 % of the PG45-specific probes. Sequence analyses indicated that most of the genomic diversity observed within one species is related to ORFs with (i) no homologies to proteins recorded in the databases or (ii) homologies to proteins encoded by restriction modification systems. Reminiscent of gene transfer as a means for genomic diversity, a PG45-specific DNA fragment with significant homologies to a central protein of an integrative conjugative element of Mycoplasma fermentans (ICEF) was found in most M. bovis field isolates and in a few M. agalactiae isolates. Finally, sequences encoding part of DNA polymerase III were found in both sets of M. agalactiae- and M. bovis-specific DNA fragments and were used to design a species-specific PCR assay for the identification and differentiation of M. agalactiae and M. bovis.

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