Quantitative Mass Spectrometry by SILAC in Haloferax volcanii.

The development of mass spectrometry (MS)-based proteomics methods has been critical in providing new insight about cellular processes and adaptations in all domains of life. While traditional MS-based methods are not inherently quantitative, technologies are now available to overcome this limitation. Of note, stable isotope labeling of amino acids in cell culture (SILAC) is reported as a reliable tool to label proteomes for quantitative MS-based proteomics that is accurate and flexible for multiplexing. The isotopically labeled lysine and arginine are easily incorporated into the proteome of cells auxotrophic for these amino acids. Microorganisms of the domain Archaea provide a fascinating alternative to understanding cellular adaptations and responses to environmental stresses. However, the availability of preferred SILAC-based quantitative analyses is limited. This protocol describes the use of SILAC to quantitatively analyze the proteome of Haloferax volcanii, a mesophilic halophilic archaeon that is easy to grow and requires no special equipment to maintain.

Expression and tandem affinity purification of 20S proteasomes and other multisubunit complexes in Haloferax volcanii.

Tandem affinity purification is a useful strategy to isolate multisubunit complexes of high yield and purity but can be limited when working with halophilic proteins that are not properly expressed in Escherichia coli. Halophilic proteins are desirable for bioindustrial applications as they are often stable and active in organic solvents; however, these proteins can be difficult to express, fold, and purify by traditional technologies. Haloarchaea provide a useful alternative for expression of halophilic proteins. These microorganisms use a salt-in strategy to maintain homeostasis and express most of their proteins with halophilic properties and low pI. Here, we provide detailed protocols for the genetic modification, expression and tandem affinity purification of "salt-loving" multisubunit complexes from the haloarchaeon Haloferax volcanii. The strategy for isolation of affinity tagged 20S proteasomes that form cylindrical proteolytic nanomachines of α1, α2 and ß subunits is described.

High-level synthesis and secretion of laccase, a metalloenzyme biocatalyst, by the halophilic archaeon Haloferax volcanii.

Haloarchaea and their enzymes have extremophilic properties desirable for use as platform organisms and biocatalysts in the bioindustry. These GRAS (generally regarded as safe) designated microbes thrive in hypersaline environments and use a salt-in strategy to maintain osmotic homeostasis. This unusual strategy has resulted in the evolution of most of the intracellular and extracellular enzymes of haloarchaea to be active and stable not only in high salt (2-5M) but also in low salt (0.2M). This salt tolerance is correlated with a resilience to low water activity, thus, rendering the haloarchaeal enzymes active and stable in organic solvent and temperatures of 50-60°C used in the enzymatic biodelignification and saccharification of lignocellulosic materials. High-level secretion of haloarchaeal enzymes to the extracellular milieu is useful for many applications, including enzymes that deconstruct biomass to allow for lignin depolymerization and simultaneous fermentation of sugars released from hemicellulose and cellulose fractions of lignocellulosics. Here we detail strategies and methods useful for high-level secretion of a laccase, HvLccA, that mediates oxidation of various phenolics by engineering a recombinant strain of the haloarchaeon Haloferax volcanii.

Identification of melioidosis outbreak by multilocus variable number tandem repeat analysis.

Endemic melioidosis is caused by genetically diverse Burkholderia pseudomallei strains. However, clonal outbreaks (multiple cases caused by 1 strain) have occurred, such as from contaminated potable water. B. pseudomallei is designated a group B bioterrorism agent, which necessitates rapidly recognizing point-source outbreaks. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) can identify genetically related isolates, but results take several days to obtain. We developed a simplified 4-locus multilocus variable number tandem repeat analysis (MLVA-4) for rapid typing and compared results with PFGE and MLST for a large number of well-characterized B. pseudomallei isolates. MLVA-4 compared favorably with MLST and PFGE for the same isolates; it discriminated between 65 multilocus sequence types and showed relatedness between epidemiologically linked isolates from outbreak clusters and between isolates from individual patients. MLVA-4 can establish or refute that a clonal outbreak of melioidosis has occurred within 8 hours of receipt of bacterial strains.

Genetic diversity of Burkholderia pseudomallei isolates in Australia.

Melioidosis is caused by the gram-negative saprophytic bacterium Burkholderia pseudomallei, which is endemic to southeast Asia and northern Australia. We have previously found evidence of geographic localization of strains based on multilocus sequence typing (MLST). In this study, we examined the diversity of 277 isolates from northern Australia, which were resolved into 159 different sequence types. No sequence types were common to both Queensland and the Northern Territory, and there was significant differentiation between the alleles present in the two regions. The considerable diversity in sequence types contrasts with the limited diversity of alleles at MLST loci, supporting previous work suggesting a high rate of recombination relative to mutation in B. pseudomallei, where new sequence types are primarily generated by reassortment of existing alleles.

Recovery of a Burkholderia thailandensis-like isolate from an Australian water source.

BACKGROUND: Burkholderia thailandensis, a close relative of Burkholderia pseudomallei, has previously been reported only from Southeast Asia and North America. It is biochemically differentiated from B. pseudomallei by the ability to utilize arabinose. During the course of environmental sampling for B. pseudomallei in the Northern Territory of Australia, an isolate, MSMB 43, was recovered that is arabinose positive. RESULTS: Genetic analysis using 16S rDNA sequencing and DNA/DNA hybridization indicates that MSMB 43 is most similar to B. thailandensis although multi-locus sequence typing indicates that this isolate is divergent from both B. pseudomallei and other described B. thailandensis. CONCLUSION: We report the isolation and initial characterization of strain MSMB 43, which is a B. thailandensis-like isolate recovered in Australia.

Tandem repeat regions within the Burkholderia pseudomallei genome and their application for high resolution genotyping.

BACKGROUND: The facultative, intracellular bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a serious infectious disease of humans and animals. We identified and categorized tandem repeat arrays and their distribution throughout the genome of B. pseudomallei strain K96243 in order to develop a genetic typing method for B. pseudomallei. We then screened 104 of the potentially polymorphic loci across a diverse panel of 31 isolates including B. pseudomallei, B. mallei and B. thailandensis in order to identify loci with varying degrees of polymorphism. A subset of these tandem repeat arrays were subsequently developed into a multiple-locus VNTR analysis to examine 66 B. pseudomallei and 21 B. mallei isolates from around the world, as well as 95 lineages from a serial transfer experiment encompassing ~18,000 generations. RESULTS: B. pseudomallei contains a preponderance of tandem repeat loci throughout its genome, many of which are duplicated elsewhere in the genome. The majority of these loci are composed of repeat motif lengths of 6 to 9 bp with 4 to 10 repeat units and are predominately located in intergenic regions of the genome. Across geographically diverse B. pseudomallei and B.mallei isolates, the 32 VNTR loci displayed between 7 and 28 alleles, with Nei's diversity values ranging from 0.47 and 0.94. Mutation rates for these loci are comparable (>10-5 per locus per generation) to that of the most diverse tandemly repeated regions found in other less diverse bacteria. CONCLUSION: The frequency, location and duplicate nature of tandemly repeated regions within the B. pseudomallei genome indicate that these tandem repeat regions may play a role in generating and maintaining adaptive genomic variation. Multiple-locus VNTR analysis revealed extensive diversity within the global isolate set containing B. pseudomallei and B. mallei, and it detected genotypic differences within clonal lineages of both species that were identical using previous typing methods. Given the health threat to humans and livestock and the potential for B. pseudomallei to be released intentionally, MLVA could prove to be an important tool for fine-scale epidemiological or forensic tracking of this increasingly important environmental pathogen.

VNTR analysis of selected outbreaks of Burkholderia pseudomallei in Australia.

Molecular typing methods for Burkholderia pseudomallei have been successful at assigning isolates into epidemiologically related groups, but have not been able to detect differences and define evolutionary patterns within groups. Our variable number tandem repeat (VNTR) analysis of a set of 121 Australian B. pseudomallei isolates, 104 of which were associated with nine epidemiological groups, provides fine scale differentiation even among very closely related isolates. We used a Bayesian model based upon mutation accumulation patterns to define the close phylogenetic relationships within these epidemiological groups. Our results reveal that genetic diversity can exist within a very small geographic area, and that low levels of diversity can exist even within a single infection. These methods provide the ability to generate robust evolutionary hypotheses that enable tracking of B. pseudomallei in forensic and epidemiological outbreaks at fine phylogenetic scales.

Using BOX-PCR to exclude a clonal outbreak of melioidosis.

BACKGROUND: Although melioidosis in endemic regions is usually caused by a diverse range of Burkholderia pseudomallei strains, clonal outbreaks from contaminated potable water have been described. Furthermore B. pseudomallei is classified as a CDC Group B bioterrorism agent. Ribotyping, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) have been used to identify genetically related B. pseudomallei isolates, but they are time consuming and technically challenging for many laboratories. METHODS: We have adapted repetitive sequence typing using a BOX A1R primer for typing B. pseudomallei and compared BOX-PCR fingerprinting results on a wide range of well-characterized B. pseudomallei isolates with MLST and PFGE performed on the same isolates. RESULTS: BOX-PCR typing compared favourably with MLST and PFGE performed on the same isolates, both discriminating between the majority of multilocus sequence types and showing relatedness between epidemiologically linked isolates from various outbreak clusters. CONCLUSION: Our results suggest that BOX-PCR can be used to exclude a clonal outbreak of melioidosis within 10 hours of receiving the bacterial strains.

Clinical variation in melioidosis in pigs with clonal infection following possible environmental contamination from bore water.

An outbreak of melioidosis occurred in pigs on a rural property in the tropical north of the Northern Territory of Australia. The pigs were mostly asymptomatic but lesions in the parotid glands suggested an oral route of infection. Skin lesions were also common and one piglet had disseminated infection. Pulsed-field gel electrophoresis showed an identical pattern amongst Burkholderia pseudomallei isolates from the pigs, with similarity to an isolate from the unchlorinated bore water supplying the property.

Extreme weather events and environmental contamination are associated with case-clusters of melioidosis in the Northern Territory of Australia.

BACKGROUND: Melioidosis, the infection due to the environmental organism Burkholderia pseudomallei, is endemic to northern Australia and South East Asia. It is associated with exposure to mud and pooled surface water, but environmental determinants of this disease are poorly understood. We defined case-clusters in northern Australia, determined their contribution to the observed rate of melioidosis, and explored clinical features and associated environmental factors. METHODS: Using geographical information systems data, we examined clustering of melioidosis cases in time and geographical space in the Top End of the Northern Territory of Australia between 1990 and 2002 using a scan statistic. DNA macrorestriction analysis, resolved by pulsed field gel electrophoresis, was performed on isolates from patients. RESULTS: We defined five case-clusters involving 27 patients that occurred within 7-28 days and/or a radius of 100-300 km. Clustered cases were associated with extreme weather events or environmental contamination; no difference in the clinical pattern of disease was noted from other patients not involved in clusters. Isolates from patients linked to environmental contamination were caused by isolates with similar DNA macrorestriction patterns, but isolates from patients linked to severe weather events had more diverse DNA macrorestriction patterns. CONCLUSION: Case-clusters of melioidosis where isolates exhibit diverse DNA macrorestriction patterns in our region are linked to extreme weather events and outbreaks where isolates are predominantly of the same DNA macrorestriction pattern are linked with contamination of an environmental source.

Burkholderia pseudomallei strain type, based on pulsed-field gel electrophoresis, does not determine disease presentation in melioidosis.

Melioidosis, the infection due to Burkholderia pseudomallei, may present with a spectrum of severity and may affect any site in the body. Differential strain virulence and tropism suggested by previous studies would have implications for virulence and vaccine work. We explored clinical correlations using pulsed-field gel electrophoresis (PFGE) typing in a well-characterised clinical collection. Two methods of analysis were used based on band-based similarity values: first, a conventional cluster analysis formed by the unweighted paired group mean analysis, and second, an analysis of the distribution of the "within-group" and "between-group" Dice coefficient. Clinical isolates from 114 cases of melioidosis occurring in the Northern Territory, Australia were studied; 71 strain types were defined with a Simpson's index of 0.91. No correlation was found between strain type and disease severity or site of melioidosis on presentation, with no differences in similarity values found when comparing within and between-groups. In particular, isolates from patients with neurological melioidosis were not clustered. There was evidence of geographical localisation. This study suggests that the variation in strain type may not be as important as host and environmental factors in determining the pattern of disease.

Short report: application of a polymerase chain reaction to detect Burkholderia pseudomallei in clinical specimens from patients with suspected melioidosis.

The diagnostic potential of a Burkholderia pseudomallei type three secretion system (TTS1) polymerase chain reaction (PCR) was examined on clinical specimens from 27 patients with sepsis in the Northern Territory of Australia, a region endemic for melioidosis. The TTS1 PCR was conducted on DNA extracted from a range of clinical specimens (blood, sputum, urine, joint, pericardial and pleural fluid, and swabs from skin lesions, throat, nose, and rectum). The PCR sensitivity in culture-positive clinical specimens from the nine confirmed patients with melioidosis was 65% and the specificity was 100%, with no PCR-positive results in specimens from 18 patients without melioidosis. The PCR based on the B. pseudomallei TTS1 has the potential to substantially improve the timeliness of diagnosis of melioidosis.

Contamination of hand wash detergent linked to occupationally acquired melioidosis.

Two mechanics working at a garage in tropical northern Australia simultaneously developed upper limb melioidosis ulcers. Both patients had Burkholderia pseudomallei of identical pulsed-field gel electrophoresis (PFGE) type (Spe I). Environmental sampling identified B. pseudomallei in a container of commercial hand wash detergent as the likely source of infection, although there were multiple isolates of different PFGE types to the clinical isolates.

Chlorination and pH of drinking water do not correlate with rates of melioidosis in the Northern Territory, Australia.

Prompted by 2 outbreaks of melioidosis with fatalities linked to culture-positive drinking water, we theorized that there may be a correlation between low drinking water pH or lack of chlorination and the rate of melioidosis in rural communities in tropical Australia. However, following adjustment for rainfall, such associations were not apparent in a multivariate regression model.

Sensitive and specific molecular detection of Burkholderia pseudomallei, the causative agent of melioidosis, in the soil of tropical northern Australia.

Burkholderia pseudomallei, the cause of the severe disease melioidosis in humans and animals, is a gram-negative saprophyte living in soil and water of areas of endemicity such as tropical northern Australia and Southeast Asia. Infection occurs mainly by contact with wet contaminated soil. The environmental distribution of B. pseudomallei in northern Australia is still unclear. We developed and evaluated a direct soil B. pseudomallei DNA detection method based on the recently published real-time PCR targeting the B. pseudomallei type III secretion system. The method was evaluated by inoculating different soil types with B. pseudomallei dilution series and by comparing B. pseudomallei detection rate with culture-based detection rate for 104 randomly collected soil samples from the Darwin rural area in northern Australia. We found that direct soil B. pseudomallei DNA detection not only was substantially faster than culture but also proved to be more sensitive with no evident false-positive results. This assay provides a new tool to detect B. pseudomallei in soil samples in a fast and highly sensitive and specific manner and is applicable for large-scale B. pseudomallei environmental screening studies or in outbreak situations. Furthermore, analysis of the 104 collected soil samples revealed a significant association between B. pseudomallei-positive sites and the presence of animals at these locations and also with moist, reddish brown-to-reddish gray soils.

Use of a variable amplicon typing scheme reveals considerable variation in the accessory genomes of isolates of Burkholderia pseudomallei.

Melioidosis, a disease caused by the bacterium Burkholderia pseudomallei, is endemic in southeast Asia and northern Australia. We used suppression subtractive hybridization (SSH) to identify sequences that varied between two B. pseudomallei isolates from Australia and determined the distribution of 45 SSH-derived sequences among a panel of B. pseudomallei and B. thailandensis isolates. Sequences exhibiting variable prevalence were included in a variable amplicon typing (VAT) scheme designed to score the presence or absence of 14 PCR amplicons. VAT analysis was carried out with 48 isolates from Thailand, which were typed by multilocus sequence typing (MLST), and 44 isolates from Australia of known MLST type. The VAT scheme could be used to divide the 48 isolates from Thailand into 23 VAT types and the 44 isolates from Australia into 36 VAT types. Some of the sequences included in the VAT scheme were more commonly PCR positive among isolates from Australia than among isolates from Thailand, and vice versa. No isolate from Australia was PCR positive for genomic island 11 or a putative transposase sequence, whereas four SSH-derived sequences were far more prevalent among the Australian isolates. Analysis based on the VAT scheme indicated that the isolates clustered into groups, some of which were mainly or exclusively from one geographical origin. One cluster included Australian isolates that were mostly associated with severe disease, including rare neurological melioidosis, suggesting that the content of the accessory genome may play an important role in determining the clinical manifestation of the disease.

Development and evaluation of a real-time PCR assay targeting the type III secretion system of Burkholderia pseudomallei.

Here we report on the development of a discriminatory real-time assay for the rapid identification of Burkholderia pseudomallei isolates and the evaluation of this assay for sensitivity against related species and detection in spiked human blood samples. The assay targets a 115-base-pair region within orf2 of the B. pseudomallei type III secretion system gene cluster and distinguishes B. pseudomallei from other microbial species. Assay performance was evaluated with 224 geographically, temporally, and clinically diverse B. pseudomallei isolates from the Centers for Disease Control and Prevention strain collection. This represents the first real-time PCR for rapid and sensitive identification of B. pseudomallei that has been tested for cross-reactivity with 23 Burkholderia mallei, 5 Burkholderia thailandensis, and 35 Burkholderia and 76 non-Burkholderia organisms which have historically presented diagnostic challenges. The assay performed with 100% specificity. The limit of detection was found to be 76 femtograms of DNA (equivalent to 5.2 x 10(3) genome equivalents per ml) in a single PCR. In spiked human blood, the assay could detect as few as 8.4 x 10(3) CFU per ml. This rapid assay is a valuable tool for identification of B. pseudomallei and may improve diagnosis in regions endemic for melioidosis.

Clinical evaluation of a type III secretion system real-time PCR assay for diagnosing melioidosis.

A Burkholderia pseudomallei type III secretion system real-time PCR assay was evaluated on clinical specimens in a region where melioidosis is endemic. The PCR was positive in 30/33 (91%) patients with culture-confirmed melioidosis. All six patients with melioidosis septic shock were blood PCR positive, suggesting potential for rapid diagnosis and commencement of appropriate therapy.

Isolates of Burkholderia pseudomallei from Northern Australia are distinct by multilocus sequence typing, but strain types do not correlate with clinical presentation.

Melioidosis is the disease caused by the saprophytic organism Burkholderia pseudomallei. Previous studies have suggested some strain tropism and differential virulence. In this study, we defined strains by multilocus sequence typing (MLST) of isolates taken from the Top End of Australia's Northern Territory and compared the results with those of other strains typed worldwide. We specifically sought clinical and geographical correlates of strain types. Among 87 Australian isolates, 48 sequence types were defined. None of the sequence types in this study has been found elsewhere in the world. Strains were distributed widely throughout the region, and the different presentations of disease, including neurological and prostatic infection, were associated with many different strains. There was excellent congruence between pulsed-field gel electrophoresis and MLST, and the two typing methods had a similar level of strain discrimination. The work suggests that host and environmental factors may be more important in determining disease presentation than infecting strain type. It is possible that the distinct but diverse strain types found in this study reflect Australia's geographical isolation over many millions of years.