Decontamination Efficacy and Skin Toxicity of Two Decontaminants against Bacillus anthracis

Decontamination of bacterial endospores such as Bacillus anthracis has traditionally required the use of harsh or caustic chemicals. The aim of this study was to evaluate the efficacy of a chlorine dioxide decontaminant in killing Bacillus anthracis spores in solution and on a human skin simulant (porcine cadaver skin), compared to that of commonly used sodium hypochlorite or soapy water decontamination procedures. In addition, the relative toxicities of these decontaminants were compared in human skin keratinocyte primary cultures. The chlorine dioxide decontaminant was similarly effective to sodium hypochlorite in reducing spore numbers of Bacillus anthracis Ames in liquid suspension after a 10 minute exposure. After five minutes, the chlorine dioxide product was significantly more efficacious. Decontamination of isolated swine skin contaminated with Bacillus anthracis Sterne with the chlorine dioxide product resulted in no viable spores sampled. The toxicity of the chlorine dioxide decontaminant was up to two orders of magnitude less than that of sodium hypochlorite in human skin keratinocyte cultures. In summary, the chlorine dioxide based decontaminant efficiently killed Bacillus anthracis spores in liquid suspension, as well as on isolated swine skin, and was less toxic than sodium hypochlorite in cultures of human skin keratinocytes.     

Novel System for Efficient Isolation of Clostridium Double-Crossover Allelic Exchange Mutants Enabling Markerless Chromosomal Gene Deletions and DNA Integration

Isolation of Clostridium mutants based on gene replacement via allelic exchange remains a major limitation for this important genus. Use of a heterologous counterselection marker can facilitate the identification of the generally rare allelic exchange events. We report on the development of an inducible counterselection marker and describe its utility and broad potential in quickly and efficiently generating markerless DNA deletions and integrations at any genomic locus without the need for auxotrophic mutants or the use of the mobile group II introns. This system is based on a codon-optimized mazF toxin gene from Escherichia coli under the control of a lactose-inducible promoter from Clostridium perfringens. This system is potentially applicable to almost all members of the genus Clostridium due to their similarly low genomic GC content and comparable codon usage. We isolated all allelic-exchange-based gene deletions (ca_p0167, sigF, and sigK) or disruptions (ca_p0157 and sigF) we attempted and integrated a 3.6-kb heterologous DNA sequence (made up of a Clostridium ljungdahlii 2.1-kb formate dehydrogenase [fdh] gene plus a FLP recombination target [FRT]-flanked thiamphenicol resistance marker) into the Clostridium acetobutylicum chromosome. Furthermore, we report on the development of a plasmid system with inducible segregational instability, thus enabling efficient deployment of the FLP-FRT system to generate markerless deletion or integration mutants. This enabled expeditious deletion of the thiamphenicol resistance marker from the fdh integrant strain as well as the sigK deletion strain. More generally, our system can potentially be applied to other organisms with underdeveloped genetic tools.     

Panning of a Phage Display Library against a Synthetic Capsule for Peptide Ligands That Bind to the Native Capsule of Bacillus anthracis

Bacillus anthracis is the causative agent of anthrax with the ability to not only produce a tripartite toxin, but also an enveloping capsule comprised primarily of γ-D-glutamic acid residues. The purpose of this study was to isolate peptide ligands capable of binding to the native capsule of B. anthracis from a commercial phage display peptide library using a synthetic form of the capsule consisting of 12 γ-D-glutamic acid residues. Following four rounds of selection, 80 clones were selected randomly and analysed by DNA sequencing. Four clones, each containing a unique consensus sequence, were identified by sequence alignment analysis. Phage particles were prepared and their derived 12-mer peptides were also chemically synthesized and conjugated to BSA. Both the phage particles and free peptide-BSA conjugates were evaluated by ELISA for binding to encapsulated cells of B. anthracis as well as a B. anthracis capsule extract. All the phage particles tested except one were able to bind to both the encapsulated cells and the capsule extract. However, the peptide-BSA conjugates could only bind to the encapsulated cells. One of the peptide-BSA conjugates, with the sequence DSSRIPMQWHPQ (termed G1), was fluorescently labelled and its binding to the encapsulated cells was further confirmed by confocal microscopy. The results demonstrated that the synthetic capsule was effective in isolating phage-displayed peptides with binding affinity for the native capsule of B. anthracis.     

Microevolution of Anthrax from a Young Ancestor (M.A.Y.A.) Suggests a Soil-Borne Life Cycle of Bacillus anthracis

During an anthrax outbreak at the Pollino National Park (Basilicata, Italy) in 2004, diseased cattle were buried and from these anthrax-foci Bacillus anthracis endospores still diffuse to the surface resulting in local accumulations. Recent data suggest that B. anthracis multiplies in soil outside the animal-host body. This notion is supported by the frequent isolation of B. anthracis from soil lacking one or both virulence plasmids. Such strains represent an evolutionary dead end, as they are likely no longer able to successfully infect new hosts. This loss of virulence plasmids is explained most simply by postulating a soil-borne life cycle of the pathogen. To test this hypothesis we investigated possible microevolution at two natural anthrax foci from the 2004 outbreak. If valid, then genotypes of strains isolated from near the surface at these foci should be on a different evolutionary trajectory from those below residing in deeper-laying horizons close to the carcass. Thus, the genetic diversity of B. anthracis isolates was compared conducting Progressive Hierarchical Resolving Assays using Nucleic Acids (PHRANA) and next generation Whole Genome Sequencing (WGS). PHRANA was not discriminatory enough to resolve the fine genetic relationships between the isolates. Conversely, WGS of nine isolates from near-surface and nine from near-carcass revealed five isolate specific SNPs, four of which were found only in different near-surface isolates. In support of our hypothesis, one surface-isolate lacked plasmid pXO1 and also harbored one of the unique SNPs. Taken together, our results suggest a limited soil-borne life cycle of B. anthracis.     

Establishment of a Markerless Mutation Delivery System in Bacillus subtilis Stimulated by a Double-Strand Break in the Chromosome

Bacillus subtilis has been a model for gram-positive bacteria and it has long been exploited for industrial and biotechnological applications. However, the availability of facile genetic tools for physiological analysis has generally lagged substantially behind traditional genetic models such as Escherichia coli and Saccharomyces cerevisiae. In this work, we have developed an efficient, precise and scarless method for rapid multiple genetic modifications without altering the chromosome of B. subtilis. This method employs upp gene as a counter-selectable marker, double-strand break (DSB) repair caused by exogenous endonuclease I-SceI and comK overexpression for fast preparation of competent cell. Foreign dsDNA can be simply and efficiently integrated into the chromosome by double-crossover homologous recombination. The DSB repair is a potent inducement for stimulating the second intramolecular homologous recombination, which not only enhances the frequency of resolution by one to two orders of magnitude, but also selects for the resolved product. This method has been successfully and reiteratively used in B. subtilis to deliver point mutations, to generate in-frame deletions, and to construct large-scale deletions. Experimental results proved that it allowed repeated use of the selectable marker gene for multiple modifications and could be a useful technique for B. subtilis.     

Development of a Markerless Genetic Exchange System for Desulfovibrio vulgaris Hildenborough and Its Use in Generating a Strain with Increased Transformation Efficiency

In recent years, the genetic manipulation of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough has seen enormous progress. In spite of this progress, the current marker exchange deletion method does not allow for easy selection of multiple sequential gene deletions in a single strain because of the limited number of selectable markers available in D. vulgaris. To broaden the repertoire of genetic tools for manipulation, an in-frame, markerless deletion system has been developed. The counterselectable marker that makes this deletion system possible is the pyrimidine salvage enzyme, uracil phosphoribosyltransferase, encoded by upp. In wild-type D. vulgaris, growth was shown to be inhibited by the toxic pyrimidine analog 5-fluorouracil (5-FU), whereas a mutant bearing a deletion of the upp gene was resistant to 5-FU. When a plasmid containing the wild-type upp gene expressed constitutively from the aph(3′)-II promoter (promoter for the kanamycin resistance gene in Tn5) was introduced into the upp deletion strain, sensitivity to 5-FU was restored. This observation allowed us to develop a two-step integration and excision strategy for the deletion of genes of interest. Since this in-frame deletion strategy does not retain an antibiotic cassette, multiple deletions can be generated in a single strain without the accumulation of genes conferring antibiotic resistances. We used this strategy to generate a deletion strain lacking the endonuclease (hsdR, DVU1703) of a type I restriction-modification system that we designated JW7035. The transformation efficiency of the JW7035 strain was found to be 100 to 1,000 times greater than that of the wild-type strain when stable plasmids were introduced via electroporation.The anaerobic sulfate-reducing bacteria (SRB) are found in a remarkable variety of habitats. These bacteria have received attention recently because they have a potential role in toxic metal bioremediation (23, 26). To fully understand the potential benefits and to maximize opportunities for successful manipulation of the SRB, it would be useful to create deletions in critically important genes. Several activities of particular interest are represented by multiple isozymes, suggesting that compensation may occur upon elimination of one or more of these genes. To fully elucidate alternative pathways, genetic approaches allowing the construction of multiple mutations are needed. The genetic manipulation of the SRB Desulfovibrio vulgaris Hildenborough has seen significant improvements in recent years (reviewed in reference 3). Chloramphenicol and kanamycin marker exchange mutagenesis methods have been developed (2, 10). Although gene deletions can be constructed, the necessary retention of antibiotic resistance limits sequential deletions, since each deletion would require an additional antibiotic cassette. To eliminate the necessity of marker retention, an in-frame markerless deletion system has been developed.A two-step method for marker exchange/deletion that used the counterselectable marker sacB (13) was used by Fu and Voordouw (10) to generate the first deletion by marker exchange in D. vulgaris. The sacB gene from Bacillus subtilis encodes levansucrase and confers sensitivity to sucrose in many gram-negative bacteria (7, 32, 35), including D. vulgaris (8, 10, 15, 18, 19, 21). In the first step of the process, a suicide plasmid carrying DNA regions from up- and downstream of a target gene flanking a chloramphenicol resistance (Cm^r) cassette was introduced into D. vulgaris by conjugation and single recombinants were selected as Cm^r colonies (10). After confirmation of the integration of this plasmid, the double-recombination event was selected on medium containing chloramphenicol and sucrose. This method, with some variation, has been used to make several mutants by the Voordouw group (8, 10, 15, 18, 19, 21). One unexpected complication was the observation that 50% of sucrose resistant colonies were due to events other than the removal of the sacB gene and plasmid through a second recombination as desired (11). Also, sensitivity to sucrose is apparently strongly affected by medium composition, initial culture density, and the time of exposure (11). This method involved a large time investment, but it ultimately resulted in a marker exchange mutant (Cm^r) and established the effectiveness of a two-step recombination process in D. vulgaris.Among alternative counterselectable markers are the purine and pyrimidine salvage enzymes, phosphoribosyl transferases (PRTases). These enzymes allow the recycling of free bases from internal or environmental sources, as well as the incorporation of base analogs into nucleoside monophosphates. Importantly, the incorporation of base analogs can be lethal and are the reason these nucleotide salvage pathways have been widely used as counterselectable markers for gene knockout systems in bacteria, archaea, and eukaryotes (4, 5, 8, 9, 12, 16, 22, 24, 27, 29, 34). Specifically, the incorporation of the pyrimidine analog 5-fluorouracil (5-FU) is lethal in a number of bacteria (8, 16, 22). Mutants whose genes encoding the pertinent PRTases have been deleted are resistant to the toxic base analogs (4, 5, 8, 9, 12, 16, 22, 24, 27, 29, 34). Reintroduction of these genes restores sensitivity. In order to utilize the genes for PRTases as counterselectable markers, a deletion of the endogenous PRTase gene must be created in the host strain. We have previously shown that wild-type D. vulgaris is extremely sensitive to low levels of 5-FU, as little as 0.1 μg/ml (3). In the present study, we deleted the upp gene (DVU1025) encoding the putative uracil phosphoribosyl transferase in D. vulgaris creating strain JW710 and showed that it was resistant to 5-FU. When the upp gene was reintroduced into JW710 (Δupp), it restored sensitivity to wild-type levels of 5-FU. These phenotypic observations indicate that the loss of the upp provides a selectable marker for a two-step integration and excision strategy for the deletion of target genes without a residual marker exchange. A second advantage of using this markerless method is the facile ability to generate in-frame deletions, eliminating potential polarity.To test the effectiveness of using the upp as a counterselectable marker in D. vulgaris, we deleted the gene encoding the endonuclease of a type I restriction-modification system, hsdR (DVU1703), and the downstream conserved hypothetical gene (CHP; DVU1702), creating strain JW7035 [Δupp Δ(hsdR-CHP)]. The type I restriction-modification system was targeted for deletion in hopes of increasing the transformation efficiency of D. vulgaris and facilitating the construction of future deletions. As anticipated, electroporation experiments with stable plasmids revealed an improvement in transformation efficiency for stable plasmids compared to wild-type D. vulgaris. Finally, Gateway Technology (Invitrogen) was applied to generate a destination vector (pMO727) containing the constitutively expressed wild-type upp gene. This vector will expedite the process of creating the required suicide deletion vectors for future markerless deletions.     

Development of a Markerless Deletion System for the Fish-Pathogenic Bacterium Flavobacterium psychrophilum

Flavobacterium psychrophilum is a Gram-negative fish pathogen that causes important economic losses in aquaculture worldwide. Although the genome of this bacterium has been determined, the function and relative importance of genes in relation to virulence remain to be established. To investigate their respective contribution to the bacterial pathogenesis, effective tools for gene inactivation are required. In the present study, a markerless gene deletion system has been successfully developed for the first time in this bacterium. Using this method, the F. psychrophilum fcpB gene, encoding a predicted cysteine protease homologous to Streptococcus pyogenes streptopain, was deleted. The developed system involved the construction of a conjugative plasmid that harbors the flanking sequences of the fcpB gene and an I-SceI meganuclease restriction site. Once this plasmid was integrated in the genome by homologous recombination, the merodiploid was resolved by the introduction of a plasmid expressing I-SceI under the control of the fpp2 F. psychrophilum inducible promoter. The resulting deleted fcpB mutant presented a decrease in extracellular proteolytic activity compared to the parental strain. However, there were not significant differences between their LD₅₀ in an intramuscularly challenged rainbow trout infection model. The mutagenesis approach developed in this work represents an improvement over the gene inactivation tools existing hitherto for this “fastidious” bacterium. Unlike transposon mutagenesis and gene disruption, gene markerless deletion has less potential for polar effects and allows the mutation of virtually any non-essential gene or gene clusters.     

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.     

Development of a Method for Markerless Genetic Exchange in Enterococcus faecalis and Its Use in Construction of a srtA Mutant

Enterococcus faecalis is a gram-positive commensal bacterium of the gastrointestinal tract and an important opportunistic pathogen. Despite the increasing clinical significance of the enterococci, genetic analysis of these organisms has thus far been limited in scope due to the lack of advanced genetic tools. To broaden the repertoire of genetic tools available for manipulation of E.faecalis, we investigated the use of phosphoribosyl transferases as elements of a counterselection strategy. We report here the development of a counterselectable markerless genetic exchange system based on the upp-encoded uracil phosphoribosyl transferase of E. faecalis. Whereas wild-type E. faecalis is sensitive to growth inhibition by the toxic base analog 5-fluorouracil (5-FU), a mutant bearing an in-frame deletion of upp is resistant to 5-FU. When a cloned version of upp was ectopically introduced into the deletion mutant, sensitivity to 5-FU growth inhibition was restored, thereby providing the basis for a two-step integration and excision strategy for the transfer of mutant alleles to the enterococcal chromosome by recombination. This method was validated by the construction of a ΔsrtA mutant of E. faecalis and by the exchange of the surface protein Asc10, encoded on the pheromone-responsive conjugative plasmid pCF10, with a previously isolated mutant allele. Analysis of the ΔsrtA mutant indicated that SrtA anchors Asc10 to the enterococcal cell wall, facilitating the pheromone-induced aggregation of E. faecalis cells required for high-frequency conjugative plasmid transfer in liquid matings. The system of markerless exchange reported here will facilitate detailed genetic analysis of these important pathogens.     

A Markerless 3D Computerized Motion Capture System Incorporating a Skeleton Model for Monkeys

In this study, we propose a novel markerless motion capture system (MCS) for monkeys, in which 3D surface images of monkeys were reconstructed by integrating data from four depth cameras, and a skeleton model of the monkey was fitted onto 3D images of monkeys in each frame of the video. To validate the MCS, first, estimated 3D positions of body parts were compared between the 3D MCS-assisted estimation and manual estimation based on visual inspection when a monkey performed a shuttling behavior in which it had to avoid obstacles in various positions. The mean estimation error of the positions of body parts (3–14 cm) and of head rotation (35–43°) between the 3D MCS-assisted and manual estimation were comparable to the errors between two different experimenters performing manual estimation. Furthermore, the MCS could identify specific monkey actions, and there was no false positive nor false negative detection of actions compared with those in manual estimation. Second, to check the reproducibility of MCS-assisted estimation, the same analyses of the above experiments were repeated by a different user. The estimation errors of positions of most body parts between the two experimenters were significantly smaller in the MCS-assisted estimation than in the manual estimation. Third, effects of methamphetamine (MAP) administration on the spontaneous behaviors of four monkeys were analyzed using the MCS. MAP significantly increased head movements, tended to decrease locomotion speed, and had no significant effect on total path length. The results were comparable to previous human clinical data. Furthermore, estimated data following MAP injection (total path length, walking speed, and speed of head rotation) correlated significantly between the two experimenters in the MCS-assisted estimation (r = 0.863 to 0.999). The results suggest that the presented MCS in monkeys is useful in investigating neural mechanisms underlying various psychiatric disorders and developing pharmacological interventions.     

Development of a Markerless Knockout Method for Actinobacillus succinogenes

Actinobacillus succinogenes is one of the best natural succinate-producing organisms, but it still needs engineering to further increase succinate yield and productivity. In this study, we developed a markerless knockout method for A. succinogenes using natural transformation or electroporation. The Escherichia coli isocitrate dehydrogenase gene with flanking flippase recognition target sites was used as the positive selection marker, making use of A. succinogenes''s auxotrophy for glutamate to select for growth on isocitrate. The Saccharomyces cerevisiae flippase recombinase (Flp) was used to remove the selection marker, allowing its reuse. Finally, the plasmid expressing flp was cured using acridine orange. We demonstrate that at least two consecutive deletions can be introduced into the same strain using this approach, that no more than a total of 1 kb of DNA is needed on each side of the selection cassette to protect from exonuclease activity during transformation, and that no more than 200 bp of homologous DNA is needed on each side for efficient recombination. We also demonstrate that electroporation can be used as an alternative transformation method to obtain knockout mutants and that an enriched defined medium can be used for direct selection of knockout mutants on agar plates with high efficiency. Single-knockout mutants of the fumarate reductase and of the pyruvate formate lyase-encoding genes were obtained using this knockout strategy. Double-knockout mutants were also obtained by deleting the citrate lyase-, β-galactosidase-, and aconitase-encoding genes in the pyruvate formate lyase knockout mutant strain.     

Extensive Phenotypic Variation among Allelic T-DNA Inserts in Arabidopsis thaliana

T-DNA insertion mutants are a tool used widely in Arabidopsis thaliana to disrupt gene function. We phenotyped multiple homozygous T-DNA A. thaliana mutants at each of two loci (AT1G11060 and AT4G00210). We measured life history traits, including germination, size at reproduction and fruit production. Allelic T-DNA lines differed for most traits at AT1G11060 but not at AT4G00210. However, insertions in exons differed from other insertion positions in AT4G00210 but not in AT1G11060. We found evidence for additional insertions in approximately half of the lines, but found few phenotypic consequences. In general, our results suggest that a cautious interpretation of T-DNA phenotypes is warranted.     

Establishment of a Successive Markerless Mutation System in Haemophilus parasuis through Natural Transformation

Haemophilus parasuis, belonging to the family Pasteurellaceae, is the causative agent of Glässer’s disease leading to serious economic losses. In this study, a successive markerless mutation system for H. parasuis using two sequential steps of natural transformation was developed. By the first homologous recombination, the target genes were replaced by a cassette carrying kanamycin resistance gene and sacB (which confers sensitivity to sucrose) gene using kanamycin selection, followed by the second reconstruction to remove the selection cassette, with application of sucrose to further screen unmarked mutants. To improve DNA transformation frequency, several parameters have been analyzed further in this work. With this method, two unmarked deletions in one strain have been generated successfully. It is demonstrated that this system can be employed to construct multi-gene scarless deletions, which is of great help for developing live attenuated vaccines for H. parasuis.     

Use of glnQ as a Counterselectable Marker for Creation of Allelic Exchange Mutations in Group B Streptococci

Efficient allelic exchange mutagenesis in group B streptococci (GBS) has been hampered by the lack of a counterselectable marker system. Growth inhibition of GBS by the glutamine analog gamma-glutamyl hydrazide requires glnQ. We have used this phenomenon to create a counterselectable marker system for efficient selection of allelic exchange mutants in GBS.     

Phylogenetic Analysis of Bacillus subtilis Strains Applicable to Natto (Fermented Soybean) Production

Spore-forming Bacillus strains that produce extracellular poly-γ-glutamic acid were screened for their application to natto (fermented soybean food) fermentation. Among the 424 strains, including Bacillus subtilis and B. amyloliquefaciens, which we isolated from rice straw, 59 were capable of fermenting natto. Biotin auxotrophism was tightly linked to natto fermentation. A multilocus nucleotide sequence of six genes (rpoB, purH, gyrA, groEL, polC, and 16S rRNA) was used for phylogenetic analysis, and amplified fragment length polymorphism (AFLP) analysis was also conducted on the natto-fermenting strains. The ability to ferment natto was inferred from the two principal components of the AFLP banding pattern, and natto-fermenting strains formed a tight cluster within the B. subtilis subsp. subtilis group.     

Thermodynamic Analysis Reveals a Temperature-dependent Change in the Catalytic Mechanism of Bacillus stearothermophilus Tyrosyl-tRNA Synthetase

Catalysis of tRNA^Tyr aminoacylation by tyrosyl-tRNA synthetase can be divided into two steps. In the first step, tyrosine is activated by ATP to form the tyrosyl-adenylate intermediate. In the second step, the tyrosyl moiety is transferred to the 3′ end of tRNA. To investigate the roles that enthalpic and entropic contributions play in catalysis by Bacillus stearothermophilus tyrosyl-tRNA synthetase (TyrRS), the temperature dependence for the activation of tyrosine and subsequent transfer to tRNA^Tyr has been determined using single turnover kinetic methods. A van''t Hoff plot for binding of ATP to the TyrRS·Tyr complex reveals three distinct regions. Particularly striking is the change occurring at 25 °C, where the values of ΔH⁰ and ΔS⁰ go from –144 kJ/mol and –438 J/mol K below 25 °C to +137.9 kJ/mol and +507 J/mol K above 25 °C. Nonlinear Eyring and van''t Hoff plots are also observed for formation of the TyrRS·[Tyr-ATP]^‡ and TyrRS·Tyr-AMP complexes. Comparing the van''t Hoff plots for the binding of ATP to tyrosyl-tRNA synthetase in the absence and presence of saturating tyrosine concentrations indicates that the temperature-dependent changes in ΔH⁰ and ΔS⁰ for the binding of ATP only occur when tyrosine is bound to the enzyme. Previous investigations revealed a similar synergistic interaction between the tyrosine and ATP substrates when the “KMSKS” signature sequence is deleted or replaced by a nonfunctional sequence. We propose that the temperature-dependent changes in ΔH⁰ and ΔS⁰ are because of the KMSKS signature sequence being conformationally constrained and unable to disrupt this synergistic interaction below 25 °C.Aminoacyl-tRNA synthetases catalyze the transfer of amino acids to the 3′ end of tRNA in a two-step reaction shown as Reactions 1 and 2, REACTION 1 REACTION 2 where aaRS,² AA, and tRNA^AA represent the aminoacyl-tRNA synthetase, its amino acid substrate, and the cognate tRNA, respectively, and “·” and “–” represent noncovalent and covalent interactions, respectively. In general, the first step of the reaction (the activation of the amino acid) does not require the binding of tRNA to the enzyme (1–5). This allows the two steps in the tRNA aminoacylation reaction to be run independently of each other.The aminoacyl-tRNA synthetases can be separated into two classes that are structurally distinct (6–10). Class I aminoacyl-tRNA synthetases are characterized by an amino-terminal Rossmann fold domain containing the active site and two signature sequences, “HIGH” and “KMSKS” (6, 7, 10–15). These sequences stabilize the transition state for the amino acid activation step of the reaction (16–24). In class II aminoacyl-tRNA synthetases, the active site domain consists of a seven-stranded β-sheet surrounded by three α-helices (25–33). With the exception of the tyrosyl- and tryptophanyl-tRNA synthetases, which are functional homodimers, all of the class I aminoacyl-tRNA synthetases are functional monomers (34). In contrast, all of the class II aminoacyl-tRNA synthetases are functional dimers (34).The class I aminoacyl-tRNA synthetases contain an insertion domain, known as the CP1 domain, between the two halves of the Rossmann fold (10, 11, 35). In tyrosyl-tRNA synthetase (and the structurally related tryptophanyl-tRNA synthetase), the CP1 domains of the two monomers form the dimer interface. Although tyrosyl-tRNA synthetase (TyrRS) is composed of two identical monomers, in solution it displays an extreme form of negative cooperativity, known as “half-of-the-sites” reactivity, with respect to tyrosine binding and tyrosyl-adenylate formation (36, 37).Kinetic analysis of the tyrosine activation reaction supports a random order mechanism for the binding of tyrosine and ATP to tyrosyl-tRNA synthetase (38–40). In contrast, initial analysis of the Bacillus stearothermophilus tyrosyl-tRNA synthetase crystal structure suggested that the tyrosine binding pocket is blocked when ATP is bound to the enzyme (6, 7). This apparent contradiction between the kinetic and structural results was initially resolved by invoking a virtual equilibrium for the binding of tyrosine to the TyrRS·ATP complex (41). More recently, analysis of the structurally related tryptophanyl-tRNA synthetase and molecular dynamics simulations of tyrosyl-tRNA synthetase suggest that the enzyme·ATP complex exists in an open conformation that allows access to the amino acid binding pocket (42, 43). This model is consistent with a random order mechanism for substrate binding to tyrosyl-tRNA synthetase.In general, interactions between tyrosyl-tRNA synthetase and the tyrosine substrate form on the initial binding of tyrosine and do not change in strength throughout the course of the reaction (41). In contrast, the initial binding of ATP is relatively weak (K′ ^ATP_d = 4.7 mm for B. stearothermophilus tyrosyl-tRNA synthetase; where K′ ^ATP_d indicates the dissociation of ATP from the TyrRS·Tyr·ATP complex), with most of the interactions between the enzyme and ATP being formed in the transition state of the reaction (41). In other words, tyrosyl-tRNA synthetase uses tyrosine binding energy to increase the specificity of the active site and ATP binding energy to catalyze the activation of tyrosine. In addition, tyrosyl-tRNA synthetase variants, in which the KMSKS signature sequence has been deleted or made nonfunctional through mutagenesis, display a 20-fold increase in ATP binding affinity relative to that of the wild-type enzyme (19, 22). This increased affinity for ATP is dependent on the binding of tyrosine to the enzyme (22). These observations indicate that there is a synergistic interaction between the tyrosine and ATP substrates that occurs in the TyrRS·Tyr·ATP complex when the KMSKS sequence is absent or nonfunctional. One of the functions of the KMSKS sequence is to disrupt this synergistic interaction during the initial binding of ATP, allowing it to instead be used to stabilize the transition state of the amino acid activation step of the reaction (22).In this study, we investigate the role that enthalpy and entropy play in catalysis of tRNA^Tyr aminoacylation by B. stearothermophilus tyrosyl-tRNA synthetase using single turnover conditions. Although the standard free energy for this reaction is not significantly affected by increasing temperatures, there is a dramatic shift in both the van''t Hoff and Eyring plots at ∼25 °C for the tyrosine activation reaction. The hypothesis that a synergistic interaction between tyrosine and ATP is responsible for this temperature-dependent change is tested.     

Development of a Novel Genetic System To Create Markerless Deletion Mutants of Bdellovibrio bacteriovorus

Bdellovibrio bacteriovorus is a species of unique obligate predatory bacteria that utilize gram-negative bacteria as prey. Their life cycle alternates between a motile extracellular phase and a growth phase within the prey cell periplasm. The mechanism of prey cell invasion and the genetic networks and regulation during the life cycle have not been elucidated. The obligate predatory nature of the B. bacteriovorus life cycle suggests the use of this bacterium in potential applications involving pathogen control but adds complexity to the development of practical genetic systems that can be used to determine gene function. This work reports the development of a genetic technique for allelic exchange or gene inactivation by construction of in-frame markerless deletion mutants including the use of a counterselectable marker in B. bacteriovorus. A suicide plasmid carrying the sacB gene for counterselection was used to inactivate the strB gene in B. bacteriovorus HD100 by an in-frame deletion. Despite the inactivation of the strB gene, B. bacteriovorus was found to retain resistance to high concentrations of streptomycin. The stability of a plasmid for use in complementation experiments was also investigated, and it was determined that pMMB206 replicates autonomously in B. bacteriovorus. Development of this practical genetic system now facilitates the study of B. bacteriovorus at the molecular level and will aid in understanding the regulatory networks and gene function in this fascinating predatory bacterium.     

In-Frame and Unmarked Gene Deletions in Burkholderia cenocepacia via an Allelic Exchange System Compatible with Gateway Technology

Burkholderia cenocepacia is an emerging opportunistic pathogen causing life-threatening infections in immunocompromised individuals and in patients with cystic fibrosis, which are often difficult, if not impossible, to treat. Understanding the genetic basis of virulence in this emerging pathogen is important for the development of novel treatment regimes. Generation of deletion mutations in genes predicted to encode virulence determinants is fundamental to investigating the mechanisms of pathogenesis. However, there is a lack of appropriate selectable and counterselectable markers for use in B. cenocepacia, making its genetic manipulation problematic. Here we describe a Gateway-compatible allelic exchange system based on the counterselectable pheS gene and the I-SceI homing endonuclease. This system provides efficiency in cloning homology regions of target genes and allows the generation of precise and unmarked gene deletions in B. cenocepacia. As a proof of concept, we demonstrate its utility by deleting the Bcam1349 gene, encoding a cyclic di-GMP (c-di-GMP)-responsive regulator protein important for biofilm formation.