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.     

Alpha-lactalbumin unfolding is not sufficient to cause apoptosis, but is required for the conversion to HAMLET (human alpha-lactalbumin made lethal to tumor cells)

HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a complex of human alpha-lactalbumin and oleic acid (C18:1:9 cis) that kills tumor cells by an apoptosis-like mechanism. Previous studies have shown that a conformational change is required to form HAMLET from alpha-lactalbumin, and that a partially unfolded conformation is maintained in the HAMLET complex. This study examined if unfolding of alpha-lactalbumin is sufficient to induce cell death. We used the bovine alpha-lactalbumin Ca(2+) site mutant D87A, which is unable to bind Ca(2+), and thus remains partially unfolded regardless of solvent conditions. The D87A mutant protein was found to be inactive in the apoptosis assay, but could readily be converted to a HAMLET-like complex in the presence of oleic acid. BAMLET (bovine alpha-lactalbumin made lethal to tumor cells) and D87A-BAMLET complexes were both able to kill tumor cells. This activity was independent of the Ca(2+)site, as HAMLET maintained a high affinity for Ca(2+) but D87A-BAMLET was active with no Ca(2+) bound. We conclude that partial unfolding of alpha-lactalbumin is necessary but not sufficient to trigger cell death, and that the activity of HAMLET is defined both by the protein and the lipid cofactor. Furthermore, a functional Ca(2+)-binding site is not required for conversion of alpha-lactalbumin to the active complex or to cause cell death. This suggests that the lipid cofactor stabilizes the altered fold without interfering with the Ca(2+)site.     

An extended hydrophobic core induces EF-hand swapping

The structure of calbindin D(9k) with two substitutions was determined by X-ray crystallography at 1.8-A resolution. Unlike wild-type calbindin D(9k), which is a monomeric protein with two EF-hands, the structure of the mutated calbindin D(9k) reveals an intertwined dimer. In the dimer, two EF-hands of the monomers have exchanged places, and thus a 3D domain-swapped dimer has been formed. EF-hand I of molecule A is packed toward EF-hand II of molecule B and vice versa. The formation of a hydrophobic cluster, in a region linking the EF-hands, promotes the conversion of monomers to 3D domain-swapped dimers. We propose a mechanism by which domain swapping takes place via the apo form of calbindin D(9k). Once formed, the calbindin D(9k) dimers are remarkably stable, as with even larger misfolded aggregates like amyloids. Thus calbindin D(9k) dimers cannot be converted to monomers by dilution. However, heating can be used for conversion, indicating high energy barriers separating monomers from dimers.     

Characterization of a transient covalent adduct formed during dimethylarginine dimethylaminohydrolase catalysis

Dimethylarginine dimethylaminohydrolase (DDAH) regulates the concentrations of human endogenous inhibitors of nitric oxide synthase, N(omega)-methyl-l-arginine (NMMA), and asymmetric N(omega),N(omega)-dimethyl-l-arginine (ADMA). Pharmacological regulation of nitric oxide synthesis is an important goal, but the catalytic mechanism of DDAH remains largely unexplored. A DDAH from Pseudomonas aeruginosa was cloned, and asymmetrically methylated arginine analogues were shown to be the preferred substrates, with ADMA displaying a slightly higher k(cat)/K(M) value than NMMA. DDAH is similar to members of a larger superfamily of guanidino-modifying enzymes, some of which have been shown to use an S-alkylthiouronium intermediate during catalysis. No covalent intermediates were found to accumulate during steady-state turnover reactions of DDAH with NMMA or ADMA. However, identification of a new substrate with an activated leaving group, S-methyl-l-thiocitrulline (SMTC), enabled acid trapping and ESI-MS characterization of a transient covalent adduct with a mass of 158 +/- 10 Da that accumulates during steady-state turnover. Subsequent trapping, proteolysis, peptide mapping and fragmentation by mass spectrometry, and site-directed mutagenesis demonstrated that this covalent adduct was attached to an active site residue and implicates Cys249 as the catalytic nucleophile required for intermediate formation. The use of covalent catalysis clearly links DDAH to this superfamily of enzymes and suggests that an S-alkylthiouronium intermediate may be a conserved feature in their mechanisms.     

The quorum-quenching metallo-gamma-lactonase from Bacillus thuringiensis exhibits a leaving group thio effect

Lactone-hydrolyzing enzymes derived from some Bacillus species are capable of disrupting quorum sensing in bacteria that use N-acyl-l-homoserine lactones (AHLs) as intercellular signaling molecules. Despite the promise of these quorum-quenching enzymes as therapeutic and anti-biofouling agents, the ring opening mechanism and the role of metal ions in catalysis have not been elucidated. Labeling studies using (18)O, (2)H, and the AHL lactonase from Bacillus thuringiensis implicate an addition-elimination pathway for ring opening in which a solvent-derived oxygen is incorporated into the product carboxylate, identifying the alcohol as the leaving group. (1)H NMR is used to show that metal binding is required to maintain proper folding. A thio effect is measured for hydrolysis of N-hexanoyl-l-homoserine lactone and the corresponding thiolactone by AHL lactonase disubstituted with alternative metal ions, including Mn(2+), Co(2+), Zn(2+), and Cd(2+). The magnitude of the thio effect on k(cat) values and the thiophilicity of the metal ion substitutions vary in parallel and are consistent with a kinetically significant interaction between the leaving group and the active site metal center during turnover. X-ray absorption spectroscopy confirms that dicobalt substitution does not result in large structural perturbations at the active site. Finally, substitution of the dinuclear metal site with Cd(2+) results in a greatly enhanced catalyst that can hydrolyze AHLs 1600-24000-fold faster than other reported quorum-quenching enzymes.     

The effects of Lepeophtheirus salmonis infections on the stress response and immunological status of Atlantic salmon (Salmo salar)

This study was conducted to determine the effects of a high level of infection of the parasitic copepod L. salmonis on the stress response and immunological status of Atlantic salmon. An initial low-level initial infection was carried out 14d prior to a second infection in which twice as many parasites were introduced. Plasma cortisol and prostaglandin E(2) (PGE(2)) levels were monitored concurrent to the expression of six immune-related genes over five sample times (9, 21, 26, 33 and 40days post initial infection, dpii). The mean lice counts on the infected fish increased significantly from the first infection (16.3+/-1.89 at 9dpii) to the second (142.8+/-12.8 at 26dpii). Plasma cortisol levels increased significantly at 26, 33 and 40dpii in infected fish compared to controls. Plasma PGE(2) levels were significantly higher in infected fish at 9, 33 and 40dpii, when compared to controls. At 9dpii, expression of interleukin-1beta (IL-1beta), tumour necrosis factor-alpha (TNFalpha)-like cytokine, major histocompatibility class II (MH II), transforming growth factor-beta (TGFbeta)-like cytokine and cyclooxygenase-2 genes were increased in infected fish compared to controls. The expression of most of these genes returned to control levels at 21dpii when the highest expression of the MH class I gene was observed in infected fish (significantly higher than controls). Major histocompatibility class I gene expression remained higher in infected fish at 26 and 33dpii compared to controls and this was observed for the TNFalpha-like gene. By 33dpii, MH class II and TGFbeta-like genes had higher expression in infected fish compared to controls. Interleukin-1beta and TNFalpha-like gene were the only genes that showed significantly higher expression in infected fish compared to controls at 40dpii, while MH class I gene expression was significantly depressed in infected fish at this time. The expression of nearly all immune-related genes studied here increased following initial infection with L. salmonis, however, immunological stimulation did not reduce parasite numbers or protect against re-infection.     

Intramural optical mapping of V(m) and Ca(i)2+ during long-duration ventricular fibrillation in canine hearts

Intramural gradients of intracellular Ca(2+) (Ca(i)(2+)) Ca(i)(2+) handling, Ca(i)(2+) oscillations, and Ca(i)(2+) transient (CaT) alternans may be important in long-duration ventricular fibrillation (LDVF). However, previous studies of Ca(i)(2+) handling have been limited to recordings from the heart surface during short-duration ventricular fibrillation. To examine whether abnormalities of intramural Ca(i)(2+) handling contribute to LDVF, we measured membrane voltage (V(m)) and Ca(i)(2+) during pacing and LDVF in six perfused canine hearts using five eight-fiber optrodes. Measurements were grouped into epicardial, midwall, and endocardial layers. We found that during pacing at 350-ms cycle length, CaT duration was slightly longer (by ?10%) in endocardial layers than in epicardial layers, whereas action potential duration (APD) exhibited no difference. Rapid pacing at 150-ms cycle length caused alternans in both APD (APD-ALT) and CaT amplitude (CaA-ALT) without significant transmural differences. For 93% of optrode recordings, CaA-ALT was transmurally concordant, whereas APD-ALT was either concordant (36%) or discordant (54%), suggesting that APD-ALT was not caused by CaA-ALT. During LDVF, V(m) and Ca(i)(2+) progressively desynchronized when not every action potential was followed by a CaT. Such desynchronization developed faster in the epicardium than in the other layers. In addition, CaT duration strongly increased (by ～240% at 5 min of LDVF), whereas APD shortened (by ～17%). CaT rises always followed V(m) upstrokes during pacing and LDVF. In conclusion, the fact that V(m) upstrokes always preceded CaTs indicates that spontaneous Ca(i)(2+) oscillations in the working myocardium were not likely the reason for LDVF maintenance. Strong V(m)-Ca(i)(2+) desynchronization and the occurrence of long CaTs during LDVF indicate severely impaired Ca(i)(2+) handling and may potentially contribute to LDVF maintenance.     

Quantifying personality in the terrestrial hermit crab: Different measures, different inferences

There is much interest in studying animal personalities but considerable debate as to how to define and evaluate them. We assessed the utility of one proposed framework while studying personality in terrestrial hermit crabs (Coenobita clypeatus). We recorded the latency of individuals to emerge from their shells over multiple trials in four unique manipulations. We used the specific testing situations within these manipulations to define two temperament categories (shyness-boldness and exploration-avoidance). Our results identified individual behavioral consistency (i.e., personality) across repeated trials of the same situations, within both categories. Additionally, we found correlations between behaviors across contexts (traits) that suggested that the crabs had behavioral syndromes. While we found some correlations between behaviors that are supposed to measure the same temperament trait, these correlations were not inevitable. Furthermore, a principal component analysis (PCA) of our data revealed new relationships between behaviors and provided the foundation for an alternate interpretation: measured behaviors may be situation-specific, and may not reflect general personality traits at all. These results suggest that more attention must be placed on how we infer personalities from standardized methods, and that we must be careful to not force our data to fit our frameworks.     

Clostridia: the importance of their exceptional substrate and metabolite diversity for biofuel and biorefinery applications

Clostridia are anaerobic Firmicutes producing a large array of metabolites by utilizing simple and complex carbohydrates, such as cellulose, as well as CO2/H2 or CO. Their exceptional substrate diversity is enhanced by their ability to produce a broad spectrum of chemicals that can be used as precursors to or directly as biofuels and industrial chemicals. Genetic and genomic tools are under intense development, and recent efforts to metabolically engineer clostridia demonstrate their potential for biofuel and biorefinery applications. Pathway engineering to combine established substrate-utilization programs, such as for cellulose, CO2/H2 or CO, with desirable metabolic programs could lead to modular design of strains suitable for many applications. Engineering complex phenotypes--aerotolerance, abolished sporulation, and tolerance to toxic chemicals--could lead to superior bioprocessing strains.