Versatile Dual-Technology System for Markerless Allele Replacement in Burkholderia pseudomallei

Burkholderia pseudomallei is the etiologic agent of melioidosis, a rare but serious tropical disease. In the United States, genetic research with this select agent bacterium is strictly regulated. Although several select agent compliant methods have been developed for allelic replacement, all of them suffer from some drawbacks, such as a need for specific host backgrounds or use of minimal media. Here we describe a versatile select agent compliant allele replacement system for B. pseudomallei based on a mobilizable vector, pEXKm5, which contains (i) a multiple cloning site within a lacZα gene for facile cloning of recombinant DNA fragments, (ii) a constitutively expressed gusA indicator gene for visual detection of merodiploid formation and resolution, and (iii) elements required for resolution of merodiploids using either I-SceI homing endonuclease-stimulated recombination or sacB-based counterselection. The homing endonuclease-based allele replacement system is completed by pBADSce, which contains an araC-P_BAD-I-sceI expression cassette for arabinose-inducible I-SceI expression and a temperature-sensitive pRO1600 replicon for facile plasmid curing. Complementing these systems is the improved Δasd Escherichia coli mobilizer strain RHO3. This strain is susceptible to commonly used antibiotics and allows nutritional counterselection on rich media because of its diaminopimelic acid auxotrophy. The versatility of the I-SceI- and sacB-based methods afforded by pEXKm5 in conjunction with E. coli RHO3 was demonstrated by isolation of diverse deletion mutants in several clinical, environmental, and laboratory B. pseudomallei strains. Finally, sacB-based counterselection was employed to isolate a defined chromosomal fabD(Ts) allele that causes synthesis of a temperature-sensitive FabD, an essential fatty acid biosynthesis enzyme.Burkholderia pseudomallei is the etiologic agent of melioidosis (3, 35). While the bacterium and disease are typically endemic to tropical and subtropical regions of the world (5), historical precedent for use in bioweapon development programs, low infectious doses, high morbidity and mortality, and arduous therapy caused B. pseudomallei to be listed as a category B select agent by the Centers for Disease Control and Prevention. In the United States, transport, possession, and use of select agents is regulated by strict federal guidelines. These guidelines restrict the use of antibiotic resistance markers in research to those that do not compromise the use of the respective drugs in humans, veterinary medicine, or agriculture (27). The paucity of selection markers approved for this bacterium has led to development of genetic manipulation strategies that allow the isolation of unmarked mutants. These include fragment mutagenesis, where a linear DNA fragment containing the mutation, assembled in vitro by PCR, is transferred to the host strain and selection for the antibiotic resistance encoded by the fragment results in gene replacement in the homologous region of the chromosome (4, 32). When the selection markers are flanked by Cre or Flp recombinase target sites, they can be removed in vivo by temporary expression of the respective site-specific recombinase, resulting in markerless mutants (4).Additionally, allelic replacement schemes driven by genetically engineered pheS- (1, 20), sacB- (9, 15), and rpsL- (19, 30) based counterselection markers have been developed for use in B. pseudomallei. With these technologies, regions of homology containing markerless mutations are cloned into a nonreplicative plasmid. Transfer of the recombinant plasmid into the bacterial host followed by selection of an antibiotic resistance encoded by a gene located on the plasmid backbone leads to integration of the nonreplicative plasmid by regions of homology. Loss of plasmid sequences by homologous recombination results in a population in which a significant portion of the survivors of the appropriate counterselection will have undergone the desired gene replacement (Fig. ​(Fig.1,1, lower right).Open in a separate windowFIG. 1.Schematic of allele exchange procedures. For plasmid-based allelic exchange, a PCR-assembled chromosomal segment containing a deletion of orfY with flanking orfX and orfZ sequences is cloned into an appropriate vector, e.g., pEXKm5. The nonreplicative plasmid is delivered to the host strain by conjugation (or electroporation), followed by kanamycin resistance selection. This step results in integration of the allelic replacement construct into the chromosome by homologous recombination between cloned and chromosomal sequences and can be visualized by the appearance of blue colonies on Km- and X-Gluc-containing medium. The two different merodiploid resolution strategies enabled by pEXKm5 are illustrated. For I-SceI-catalyzed resolution (illustrated on the left side), the merodiploid is transformed with the I-SceI expression construct, which results in double-stranded cleavage of the chromosome and release of most of the plasmid backbone. This event can be monitored by the appearance of white colonies on X-Gluc-containing medium. Repair of the double-stranded break by homologous flanking repeat sequences leads to formation of a wild-type strain (event denoted by the circled number 1) or a mutant strain (event denoted by the circled number 2). The two events are distinguished by phenotypic analyses and/or PCR. In a final step that is not illustrated in this figure, purification of colonies with the desired mutant genotype/phenotype at 42°C leads to loss of the pBADSce expression vector in 100% of the colonies. For sacB-mediated counterselection (illustrated on the right side), the merodiploid strain is plated on medium containing sucrose. This counterselection will either result in a wild-type strain (event denoted by Δ1) or in a mutant strain (event denoted by Δ2). These events can be monitored by the appearance of white colonies on X-Gluc- and sucrose-containing medium and are distinguished by phenotypic analyses and/or PCR. Abbreviations: gusA, Escherichia coli glucuronidase-encoding gene; ori, pMB9-derived narrow-range origin of replication; sacB, Bacillus subtilis levansucrase-encoding gene optimized for expression and localization in B. pseudomallei (9).An alternative to counterselection schemes involves the use of the intron-encoded homing endonuclease I-SceI (18). This enzyme recognizes a specific 18-bp sequence which is absent from all eukaryotic genomes (except the source, Saccharomyces cerevisiae) and prokaryotic genomes sequenced to date. The basic principle of the method is that cleavage of the bacterial chromosome at an artificially introduced I-SceI site(s) stimulates recombination (21). In this scheme, a nonreplicative plasmid containing cloned regions of homology and an I-SceI site(s) is integrated into the chromosome by homologous recombination between cloned and chromosomal sequences. This integration event is selected by an antibiotic resistance encoded by a gene on the plasmid backbone and results in merodiploid formation. Next, expression of the I-SceI enzyme, either encoded by the integrated plasmid or by a separately introduced plasmid, results in cleavage at the I-SceI site(s) within the integrated vector sequences (Fig. ​(Fig.1,1, lower left). The resulting double-strand break is repaired by the host recombination machinery by recombination of the regions of sequence homology flanking the break in the merodiploid. Loss of plasmid sequences by homologous recombination results in a mixed population in which a certain percentage will have undergone the desired gene replacement, given the gene is nonessential under the experimental conditions. Although cleavage of the chromosome induces the host''s SOS response, this does not result in an increased mutation rate (21). The use of I-SceI for promoting allelic exchange in Escherichia coli (21), Bacillus anthracis (11), Burkholderia cenocepacia (8), Corynebacterium glutamicum (31), and Pseudomonas aeruginosa (36) has been reported.Biparental or triparental mating is the most efficient way to introduce nonreplicative plasmids into B. pseudomallei for purposes of allele replacement. An obvious disadvantage of this method is that a donor strain must be available that is compatible with the conjugative plasmid, e.g., it cannot contain antibiotic resistance markers that interfere with those encoded by the conjugative plasmid. And herein lie the problems. The most commonly used E. coli mobilizer strains, S17-1 (29) and SM10 (29) and its derivatives, e.g., SM10(λpir) (17), contain chromosomally integrated RP4 sequences and, therefore, different resistance markers, depending on how they were engineered. For example, the most versatile mobilizer strain, SM10(λpir), is kanamycin resistant (Km^r) and can therefore not be used with genetic elements containing an nptII Km^r-encoding gene, one of the few approved selection markers that work with all clinical and environmental B. pseudomallei strains tested to date. Conjugation experiments require counterselection against the donor and untransformed recipient strains. This is usually achieved by utilizing an antibiotic or growth medium that precludes growth of the donor strain but does not affect the recipient strain. For instance, E. coli is highly susceptible to polymyxin B but Burkholderia spp. are naturally resistant to this antimicrobial. Similarly, Pseudomonas aeruginosa can utilize citrate but E. coli cannot. To avoid the use of antibiotics or for situations where other intrinsic properties cannot be exploited, donor strains have been engineered that require nutritional supplements for growth, but they either still contain antibiotic resistance markers or are based on nutritional requirements that preclude the use of rich media (1, 6).Here we describe a versatile select agent compliant allele replacement system for B. pseudomallei based on a single vector which contains the features required for resolution of merodiploids using either I-SceI-driven recombination or sacB-based counterselection. Complementing this system is an improved E. coli mobilizer strain susceptible to all antibiotics and counterselectable on rich media. The versatility of these methods was demonstrated by isolation of deletion mutants as well as a temperature-sensitive allele in an essential fatty acid biosynthesis gene.     

Neurotoxin Gene Clusters in Clostridium botulinum Type Ab Strains

There is limited knowledge of the neurotoxin gene diversity among Clostridium botulinum type Ab strains. Only the sequences of the bont/A and bont/B genes in C. botulinum type Ab strain CDC1436 and the sequence of the bont/B gene in C. botulinum type Ab strain CDC588 have been reported. In this study, we sequenced the entire bont/A- and bont/B-associated neurotoxin gene clusters of C. botulinum type Ab strain {"type":"entrez-protein","attrs":{"text":"CDC41370","term_id":"524503451","term_text":"CDC41370"}}CDC41370 and the bont/A gene of strain CDC588. In addition, we analyzed the organization of the neurotoxin gene clusters in strains CDC588 and CDC1436. The bont/A nucleotide sequence of strain {"type":"entrez-protein","attrs":{"text":"CDC41370","term_id":"524503451","term_text":"CDC41370"}}CDC41370 differed from those of the known bont/A subtypes A1 to A4 by 2 to 7%, and the predicted amino acid sequence differed by 4% to 14%. The bont/B nucleotide sequence in strain {"type":"entrez-protein","attrs":{"text":"CDC41370","term_id":"524503451","term_text":"CDC41370"}}CDC41370 showed 99.7% identity to the sequence of subtype B1. The bont/A nucleotide sequence of strain CDC588 was 99.9% identical to that of subtype A1. Although all of the C. botulinum type Ab strains analyzed contained the two sets of neurotoxin clusters, similar to what has been found in other bivalent strains, the intergenic spacing of p21-orfX1 and orfX2-orfX3 varied among these strains. The type Ab strains examined in this study had differences in their toxin gene cluster compositions and bont/A and bont/B nucleotide sequences, suggesting that they may have arisen from separate recombination events.Clostridium botulinum is a gram-positive anaerobic bacterium that produces an extremely potent toxin, the botulinum neurotoxin (BoNT). There are seven serologically distinct types of BoNT (serotypes A through G). Although most strains of C. botulinum express a single toxin serotype, some isolates have been shown to produce more than one, namely, Ab, Af, Ba, and Bf (11). In addition, many strains designated type A by mouse bioassay harbor nucleotide sequences for both type A and B toxins (6). These strains have been designated A(B) to indicate the presence of the bont/B gene without type B-specific toxicity.Based on phylogenetic analysis of the neurotoxin gene sequences, four subtypes have been identified within serotype A and five subtypes within serotype B (12). The neurotoxin gene nucleotide sequences of these subtypes differ by up to 8%, and the predicted amino acid sequences differ by up to 16%. In addition, the genes encoding components of the toxin complexes are arranged in clusters that differ in composition and organization (14) (Fig. ​(Fig.1).1). The toxin gene cluster of subtype A1 (termed ha cluster) includes the gene encoding the nontoxic nonhemagglutinin (ntnh), a regulatory gene (botR), and an operon encoding three hemagglutinins (ha70, ha33, and ha17). The toxin gene clusters containing bont/A2 or bont/A3 (termed orfX cluster) include the ntnh and p21 (analogous to botR) genes and several genes of unknown function (orfX1, orfX2, orfX3, and p47). Type Ba and A(B) strains contain two sets of neurotoxin cluster genes in which ha70, ha33, and ha17 are associated with the bont/B gene, and orfX1, orfX2, orfX3, and p47 are associated with the bont/A gene. In addition, some A1 strains contain a neurotoxin gene cluster that is similar to those in A2 and A3, but the bont/A nucleotide sequence is 99.9% identical to that in other A1 strains. These strains have been designated HA⁻ Orfx⁺ A1 (14). The neurotoxin gene cluster in type B strains includes the ntnh, botR, ha70, ha33, and ha17 genes. Notably, no differences in the neurotoxin gene cluster arrangements among the subtypes within serotype B have been reported.Open in a separate windowFIG. 1.Toxin gene cluster arrangements for BoNT type A-producing strains, including Ab, A(B), and Ba strains.Although several studies have described the organization and the nucleotide sequences of the neurotoxin gene cluster components among type A and B strains [including type Ba and A(B) strains], there is limited information regarding the diversity of the neurotoxin cluster genes among C. botulinum type Ab strains. The nucleotide sequences of the bont/A and bont/B genes in C. botulinum type Ab strain CDC1436 and the sequence of the bont/B gene of C. botulinum type Ab strain CDC588 have been previously reported; strain CDC1436 harbors a bont/A2 gene, and both strains CDC1436 and CDC588 harbor a bont/bvB gene (12, 15). Four additional type Ab strains from Italy have been analyzed by a restriction fragment length polymorphism method to determine the bont/A and bont/B subtypes (7, 9). To the best of our knowledge, the complete nucleotide sequences of the neurotoxin gene clusters in C. botulinum type Ab strains have not been reported. Thus, the objective of this study was to analyze the neurotoxin gene cluster composition in three C. botulinum type Ab strains ({"type":"entrez-protein","attrs":{"text":"CDC41370","term_id":"524503451","term_text":"CDC41370"}}CDC41370, CDC588, and CDC1436) available in the CDC strain collection. We report differences in bont/A gene sequence among type Ab strains, including the identification of a novel bont/A nucleotide sequence in strain {"type":"entrez-protein","attrs":{"text":"CDC41370","term_id":"524503451","term_text":"CDC41370"}}CDC41370, and describe differences in the organization of the neurotoxin gene clusters among these strains.     

Differential ultrastructural changes in tomato hormonal mutants exposed to cadmium

Cadmium (Cd) is a toxic heavy metal, which can cause severe damage to plant development. The aim of this work was to characterize ultrastructural changes induced by Cd in miniature tomato cultivar Micro-Tom (MT) mutants and their wild-type counterpart. Leaves of diageotropica (dgt) and Never ripe (Nr) tomato hormonal mutants and wild-type MT were analysed by light, scanning and transmission electron microscopy in order to characterize the structural changes caused by the exposure to 1 mM CdCl₂. The effect of Cd on leaf ultrastructure was observed most noticeably in the chloroplasts, which exhibited changes in organelle shape and internal organization, of the thylakoid membranes and stroma. Cd caused an increase in the intercellular spaces in Nr leaves, but a decrease in the intercellular spaces in dgt leaves, as well as a decrease in the size of mesophyll cells in the mutants. Roots of the tomato hormonal mutants, when analysed by light microscopy, exhibited alterations in root diameter and disintegration of the epidermis and the external layers of the cortex. A comparative analysis has allowed the identification of specific Cd-induced ultrastructural changes in wild-type tomato, the pattern of which was not always exhibited by the mutants.     

Microenvironmental pH Is a Key Factor for Exosome Traffic in Tumor Cells

Exosomes secreted by normal and cancer cells carry and deliver a variety of molecules. To date, mechanisms referring to tumor exosome trafficking, including release and cell-cell transmission, have not been described. To gain insight into this, exosomes purified from metastatic melanoma cell medium were labeled with a lipid fluorescent probe, R18, and analyzed by spectrofluorometry and confocal microscopy. A low pH condition is a hallmark of tumor malignancy, potentially influencing exosome release and uptake by cancer cells. Using different pH conditions as a modifier of exosome traffic, we showed (i) an increased exosome release and uptake at low pH when compared with a buffered condition and (ii) exosome uptake by melanoma cells occurred by fusion. Membrane biophysical analysis, such as fluidity and lipid composition, indicated a high rigidity and sphingomyelin/ganglioside GM3 (N-acetylneuraminylgalactosylglucosylceramide) content in exosomes released at low pH. This was likely responsible for the increased fusion efficiency. Consistent with these results, pretreatment with proton pump inhibitors led to an inhibition of exosome uptake by melanoma cells. Fusion efficiency of tumor exosomes resulted in being higher in cells of metastatic origin than in those derived from primary tumors or normal cells. Furthermore, we found that caveolin-1, a protein involved in melanoma progression, is highly delivered through exosomes released in an acidic condition. The results of our study provide the evidence that exosomes may be used as a delivery system for paracrine diffusion of tumor malignancy, in turn supporting the importance of both exosomes and tumor pH as key targets for future anti-cancer strategies.     

Genomic and functional characterization of StCDPK1

StCDPK1 is a calcium dependent protein kinase expressed in tuberizing potato stolons and in sprouting tubers. StCDPK1 genomic sequence contains eight exons and seven introns, the gene structure is similar to Arabidopsis, rice and wheat CDPKs belonging to subgroup IIa. There is one copy of the gene per genome and it is located in the distal portion of chromosome 12. Western blot and immunolocalization assays (using confocal and transmission electron microscopy) performed with a specific antibody against StCDPK1 indicate that this kinase is mainly located in the plasma membrane of swelling stolons and sprouting tubers. Sucrose (4–8%) increased StCDPK1 protein content in non-induced stolons, however the amount detected in swelling stolons was higher. Transgenic lines with reduced expression of StCDPK1 (β7) did not differ from controls when cultured under multiplication conditions, but when grown under tuber inducing conditions some significant differences were observed: the β7 line tuberized earlier than controls without the addition of CCC (GA inhibitor), developed more tubers than wild type plants in the presence of hormones that promote tuberization in potato (ABA and BAP) and was more insensitive to GA action (stolons were significantly shorter than those of control plants). StCDPK1 expression was induced by GA, ABA and BAP. Our results suggest that StCDPK1 plays a role in GA-signalling and that this kinase could be a converging point for the inhibitory and promoting signals that influence the onset of potato tuberization.     

Crystal Structures of Cif from Bacterial Pathogens Photorhabdus luminescens and Burkholderia pseudomallei

A pre-requisite for bacterial pathogenesis is the successful interaction of a pathogen with a host. One mechanism used by a broad range of Gram negative bacterial pathogens is to deliver effector proteins directly into host cells through a dedicated type III secretion system where they modulate host cell function. The cycle inhibiting factor (Cif) family of effector proteins, identified in a growing number of pathogens that harbour functional type III secretion systems and have a wide host range, arrest the eukaryotic cell cycle. Here, the crystal structures of Cifs from the insect pathogen/nematode symbiont Photorhabdus luminescens (a γ-proteobacterium) and human pathogen Burkholderia pseudomallei (a β-proteobacterium) are presented. Both of these proteins adopt an overall fold similar to the papain sub-family of cysteine proteases, as originally identified in the structure of a truncated form of Cif from Enteropathogenic E. coli (EPEC), despite sharing only limited sequence identity. The structure of an N-terminal region, referred to here as the ‘tail-domain’ (absent in the EPEC Cif structure), suggests a surface likely to be involved in host-cell substrate recognition. The conformation of the Cys-His-Gln catalytic triad is retained, and the essential cysteine is exposed to solvent and addressable by small molecule reagents. These structures and biochemical work contribute to the rapidly expanding literature on Cifs, and direct further studies to better understand the molecular details of the activity of these proteins.     

Aislamiento y evaluación de la actividad enzimática de hongos descomponedores de madera (Quindío,Colombia)

White rot fungi (Ascomycota and Basidiomycota) were collected on fallen trunks with different decay stages, in a subandean forest (La Montaña del Ocaso nature reserve), and it was evaluated their ligninolitic activity. They were cultured on malt extract agar. Then it was performed semiquantitative tests for laccase and cellobiose dehydrogenase (CDH) activity using ABTS and DCPIP as enzymatic inducers. Based on the results of these tests, the fungi with higher activities from trunks with different decay stages were selected: Cookeina sulcipes (for stage 1), a fungus from the family Corticiaceae (for stage 2), Xylaria polymorpha (for stage 3) and Earliella sp. (for stage 4). A fermentation was performed at 28 °C, during 11 days, in a rotatory shaker at 150 rpm. Biomass, glucose, proteins and enzyme activities measurements were performed daily. The fungi that were in the trunks with decay states from 1 to 3, showed higher laccase activity as the state of decay increased. A higher DCH activity was also associated with a higher. Also, there was a positive relationship between both enzymes' activities. Erliella was the fungus which presented the highest biomass production (1140,19 g/l), laccase activity (157 UL^?1) and CDH activity (43,50 UL^?1). This work is the first report of laccase and CDH activity for Cookeina sulcipes and Earliella sp. Moreover, it gives basis for the use of these native fungi in biotechnological applications and the acknowledgment of their function in the wood decay process in native forest.     

A toxic Pseudo-nitzschia bloom in Todos Santos Bay,northwestern Baja California,Mexico

A toxic Pseudo-nitzschia spp. bloom in the Todos Santos Bay area (31.8°N), Mexico, is described. This is the southernmost report of the presence of domoic acid (DA) in the California Current System and it is also the first report of the distribution of toxic Pseudo-nitzschia species and DA on the Baja California west coast. The maximum cell abundance of Pseudo-nitzschia was 3.02 × 10⁵ cells L^?1 and the maximum concentration of DA in particulate matter (pDA) was 0.86 μg L^?1. P. australis constituted the major proportion of cells identified as Pseudo-nitzschia. The environmental conditions associated with wind-driven upwelling were the cause for the accumulation of toxic cells. Maximum pDA and cell concentration were detected around 14 °C. The ratio of the concentration of macronutrients seemed to be the important factor for the accumulation of P. australis. The highest cell abundance was detected in areas with a high Si(OH)₄ to N ratio in the entire water column. Therefore, the relative increase of silicate concentration related to upwelling conditions was the probable cause for the accumulation of P. australis. Maximum photosystem II (PSII) quantum efficiency of charge separation (F_v/F_m) was negatively correlated to the pDA to fucoxanthin ratio. This ratio was used in this work as an index of cellular DA content. Therefore, the photosynthetic competence of the cells might be an important factor that affected their DA cellular content.