An allelic exchange system for compliant genetic manipulation of the select agents Burkholderia pseudomallei and Burkholderia mallei

Hamad, Mohamad A.; Zajdowicz, Sheryl L. W.; Holmes, Randall K.; Voskuil, Martin I.

doi:10.1016/j.gene.2008.10.011

Cited by 93 publications

(136 citation statements)

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“…Gene replacement experiments with B. pseudomallei were performed using the sacB-based vector pMo130, as previously described (24)(25)(26). Recombinant derivatives of pMo130 (Table 1) were electroporated into E. coli S17-1 (12.25 kV/cm) and conjugated with B. pseudomallei MSHR668 for 8 h. Pm was used to counterselect E. coli S17-1.…”

Section: Methodsmentioning

confidence: 99%

Proteomic Analysis of the Burkholderia pseudomallei Type II Secretome Reveals Hydrolytic Enzymes, Novel Proteins, and the Deubiquitinase TssM

2014

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bBurkholderia pseudomallei, the etiologic agent of melioidosis, is an opportunistic pathogen that harbors a wide array of secretion systems, including a type II secretion system (T2SS), three type III secretion systems (T3SS), and six type VI secretion systems (T6SS). The proteins exported by these systems provide B. pseudomallei with a growth advantage in vitro and in vivo, but relatively little is known about the full repertoire of exoproducts associated with each system. In this study, we constructed deletion mutations in gspD and gspE, T2SS genes encoding an outer membrane secretin and a cytoplasmic ATPase, respectively. The secretion profiles of B. pseudomallei MSHR668 and its T2SS mutants were noticeably different when analyzed by SDS-PAGE. We utilized liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify proteins present in the supernatants of B. pseudomallei MSHR668 and B. pseudomallei ⌬gspD grown in rich and minimal media. The MSHR668 supernatants contained 48 proteins that were either absent or substantially reduced in the supernatants of ⌬gspD strains. Many of these proteins were putative hydrolytic enzymes, including 12 proteases, two phospholipases, and a chitinase. Biochemical assays validated the LC-MS/MS results and demonstrated that the export of protease, phospholipase C, and chitinase activities is T2SS dependent. Previous studies had failed to identify the mechanism of secretion of TssM, a deubiquitinase that plays an integral role in regulating the innate immune response. Here we present evidence that TssM harbors an atypical signal sequence and that its secretion is mediated by the T2SS. This study provides the first in-depth characterization of the B. pseudomallei T2SS secretome.

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Section: Methodsmentioning

confidence: 99%

Proteomic Analysis of the Burkholderia pseudomallei Type II Secretome Reveals Hydrolytic Enzymes, Novel Proteins, and the Deubiquitinase TssM

2014

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“…For Xanthomonas cultures harboring plasmids, the medium was supplemented with Km (50 g/ml). Yeast tryptone broth (YTB) and yeast tryptone sucrose agar (YTSA) (YTB with 20 g/liter agar and sucrose at a final concentration of 15% [wt/vol]) were used for the resolution of mutants (10). Plasmid constructs were generated by using Escherichia coli DH5␣ MCR as the host.…”

Section: Methodsmentioning

confidence: 99%

“…Deletion of pilA in Xanthomonas strain EC-12 was performed as described previously by Hamad et al (10), except that cells were transformed using electroporation. Primers used are listed in Table S1 in the supplemental material.…”

Section: Methodsmentioning

confidence: 99%

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Characterization of Novel Virulent Broad-Host-Range Phages of Xylella fastidiosa and Xanthomonas

Ahern

Das

Bhowmick

et al. 2013

Journal of Bacteriology

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The xylem-limited bacterium Xylella fastidiosa is the causal agent of several plant diseases, most notably Pierce's disease of grape and citrus variegated chlorosis. We report the isolation and characterization of the first virulent phages for X. fastidiosa, siphophages Sano and Salvo and podophages Prado and Paz, with a host range that includes Xanthomonas spp. Phages propagated on homologous hosts had observed adsorption rate constants of ϳ4 ؋ 10 ؊12 ml cell ؊1 min ؊1 for X. fastidiosa strain Temecula 1 and ϳ5 ؋ 10 ؊10 to 7 ؋ 10 ؊10 ml cell ؊1 min ؊1 for Xanthomonas strain EC-12. Sano and Salvo exhibit >80% nucleotide identity to each other in aligned regions and are syntenic to phage BcepNazgul. We propose that phage BcepNazgul is the founding member of a novel phage type, to which Sano and Salvo belong. The lysis genes of the Nazgul-like phage type include a gene that encodes an outer membrane lipoprotein endolysin and also spanin gene families that provide insight into the evolution of the lysis pathway for phages of Gram-negative hosts. Prado and Paz, although exhibiting no significant DNA homology to each other, are new members of the phiKMV-like phage type, based on the position of the single-subunit RNA polymerase gene. The four phages are type IV pilus dependent for infection of both X. fastidiosa and Xanthomonas. The phages may be useful as agents for an effective and environmentally responsible strategy for the control of diseases caused by X. fastidiosa.T he plant-pathogenic bacterium Xylella fastidiosa is the causative agent of a number of economically important diseases, including Pierce's disease of grape, phony peach disease, periwinkle wilt, citrus variegated chlorosis, almond leaf scorch, oleander leaf scorch, and coffee leaf scorch (1). Existing disease control methods are often only partially successful and, in the case of systemic insecticides used to control vector populations, may be potentially harmful to the environment (2, 3). Recently, there has been renewed interest in the application of bacteriophages (phage) as an environmentally acceptable mode for the control of bacterial plant disease (4).To this end, our group isolated and characterized the first temperate (lysogenic) phage of X. fastidiosa, Xfas53 (5). However, temperate phage should not be used as biocontrol agents because of their capacity for lysogenic conversion, superinfection immunity, and risk of generalized transduction. Instead, virulent (lytic) phages are needed for an effective and sustainable phage-based control strategy (6). Here we report the isolation and characterization of the first virulent siphophages and podophages for X. fastidiosa that are members of the Nazgul-like phage and the phiKMV-like phage types, respectively. MATERIALS AND METHODSBacterial strains and culture conditions. Bacterial strains and plasmids used in this study are listed in Table 1. X. fastidiosa strains were cultured at 28°C in PW-M broth (PW-MB) (7) or on PW-M agar (PW-MA) plates (PW-MB with 20 g/liter plant cell culture-tested agar [...

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“…Most of these systems employed a strategy in which nonreplicative plasmids are integrated into specific locations in the chromosome via homologous recombination, resulting in polar (51), nonpolar (53), or conditional (122) mutations. Recently, five systems for the creation of unmarked gene deletions have been published for Burkholderia spp., including B. cenocepacia (12,33,52,61,98). These systems allow the creation of multiple deletions within a strain and can also be used to integrate DNA into heterologous locations in the chromosome for cis complementation experiments and other applications.…”

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confidence: 99%

A Decade of Burkholderia cenocepacia Virulence Determinant Research

2010

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The Burkholderia cepacia complex (Bcc) is a group of genetically related environmental bacteria that can cause chronic opportunistic infections in patients with cystic fibrosis (CF) and other underlying diseases. These infections are difficult to treat due to the inherent resistance of the bacteria to antibiotics. Bacteria can spread between CF patients through social contact and sometimes cause cepacia syndrome, a fatal pneumonia accompanied by septicemia. Burkholderia cenocepacia has been the focus of attention because initially it was the most common Bcc species isolated from patients with CF in North America and Europe. Today, B. cenocepacia, along with Burkholderia multivorans, is the most prevalent Bcc species in patients with CF. Given the progress that has been made in our understanding of B. cenocepacia over the past decade, we thought that it was an appropriate time to review our knowledge of the pathogenesis of B. cenocepacia, paying particular attention to the characterization of virulence determinants and the new tools that have been developed to study them. A common theme emerging from these studies is that B. cenocepacia establishes chronic infections in immunocompromised patients, which depend more on determinants mediating host niche adaptation than those involved directly in host cells and tissue damage.

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An allelic exchange system for compliant genetic manipulation of the select agents Burkholderia pseudomallei and Burkholderia mallei

Cited by 93 publications

References 43 publications

Proteomic Analysis of the Burkholderia pseudomallei Type II Secretome Reveals Hydrolytic Enzymes, Novel Proteins, and the Deubiquitinase TssM

Proteomic Analysis of the Burkholderia pseudomallei Type II Secretome Reveals Hydrolytic Enzymes, Novel Proteins, and the Deubiquitinase TssM

Characterization of Novel Virulent Broad-Host-Range Phages of Xylella fastidiosa and Xanthomonas

A Decade of Burkholderia cenocepacia Virulence Determinant Research

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