New insights into the butyric acid metabolism of Clostridium acetobutylicum

Biosynthesis of acetone and n-butanol is naturally restricted to the group of solventogenic clostridia with Clostridium acetobutylicum being the model organism for acetone-butanol-ethanol (ABE) fermentation. According to limited genetic tools, only a few rational metabolic engineering approaches were conducted in the past to improve the production of butanol, an advanced biofuel. In this study, a phosphotransbutyrylase-(Ptb) negative mutant, C. acetobutylicum ptb::int(87), was generated using the ClosTron methodology for targeted gene knock-out and resulted in a distinct butyrate-negative phenotype. The major end products of fermentation experiments without pH control were acetate (3.2?g/l), lactate (4.0?g/l), and butanol (3.4?g/l). The product pattern of the ptb mutant was altered to high ethanol (12.1?g/l) and butanol (8.0?g/l) titers in pH?≥?5.0-regulated fermentations. Glucose fed-batch cultivation elevated the ethanol concentration to 32.4?g/l, yielding a more than fourfold increased alcohol to acetone ratio as compared to the wildtype. Although butyrate was never detected in cultures of C. acetobutylicum ptb::int(87), the mutant was still capable to take up butyrate when externally added during the late exponential growth phase. These findings suggest that alternative pathways of butyrate re-assimilation exist in C. acetobutylicum, supposably mediated by acetoacetyl-CoA:acyl-CoA transferase and acetoacetate decarboxylase, as well as reverse reactions of butyrate kinase and Ptb with respect to previous studies.     

The plug domain of yeast Sec61p is important for efficient protein translocation, but is not essential for cell viability

The Sec61/SecY translocon mediates translocation of proteins across the membrane and integration of membrane proteins into the lipid bilayer. The structure of the translocon revealed a plug domain blocking the pore on the lumenal side. It was proposed to be important for gating the protein conducting channel and for maintaining the permeability barrier in its unoccupied state. Here, we analyzed in yeast the effect of introducing destabilizing point mutations in the plug domain or of its partial or complete deletion. Unexpectedly, even when the entire plug domain was deleted, cells were viable without growth phenotype. They showed an effect on signal sequence orientation of diagnostic signal-anchor proteins, a minor defect in cotranslational and a significant deficiency in posttranslational translocation. Steady-state levels of the mutant protein were reduced, and when coexpressed with wild-type Sec61p, the mutant lacking the plug competed poorly for complex partners. The results suggest that the plug is unlikely to be important for sealing the translocation pore in yeast but that it plays a role in stabilizing Sec61p during translocon formation.     

Catheter colonization and abscess formation due to Staphylococcus epidermidis with normal and small-colony-variant phenotype is mouse strain dependent

Coagulase-negative staphylococci (CoNS) form a thick, multilayered biofilm on foreign bodies and are a major cause of nosocomial implant-associated infections. Although foreign body infection models are well-established, limited in vivo data are available for CoNS with small-colony-variant (SCV) phenotype described as causative agents in implant-associated infections. Therefore, we investigated the impact of the Staphylococcus epidermidis phenotype on colonization of implanted PVC catheters and abscess formation in three different mouse strains. Following introduction of a catheter subcutaneously in each flank of 8- to 12-week-old inbred C57BL/6JCrl (B6J), outbred Crl:CD1(ICR) (CD-1), and inbred BALB/cAnNCrl (BALB/c) male mice, doses of S. epidermidis O-47 wild type, its hemB mutant with stable SCV phenotype, or its complemented mutant at concentrations of 10(6) to 10(9) colony forming units (CFUs) were gently spread onto each catheter. On day 7, mice were sacrificed and the size of the abscesses as well as bacterial colonization was determined. A total of 11,500 CFUs of the complemented mutant adhered to the catheter in BALB/c followed by 9,960 CFUs and 9,900 CFUs from S. epidermidis wild type in BALB/c and CD-1, respectively. SCV colonization was highest in CD-1 with 9,500 CFUs, whereas SCVs were not detected in B6J. The minimum dose that led to colonization or abscess formation in all mouse strains was 10(7) or 10(8) CFUs of the normal phenotype, respectively. A minimum dose of 10(8) or 10(9) CFU of the hemB mutant with stable SCV phenotype led to colonization only or abscess formation, respectively. The largest abscesses were detected in BALB/c inoculated with wild type bacteria or SCV (64 mm(2) vs. 28 mm(2)). Our results indicate that colonization and abscess formation by different phenotypes of S. epidermidis in a foreign body infection model is most effective in inbred BALB/c followed by outbred CD-1 and inbred B6J mice.     

Heterologous production and characterisation of two distinct dihaem-containing membrane integral cytochrome b561 enzymes from Arabidopsis thaliana in Pichia pastoris and Escherichia coli cells

Cytochrome (cyt) b₅₆₁ proteins are dihaem-containing membrane proteins, belonging to the CYBASC (cytochrome-b₅₆₁-ascorbate-reducible) family, and are proposed to be involved in ascorbate recycling and/or the facilitation of iron absorption. Here, we present the heterologous production of two cyt b₅₆₁ paralogs from Arabidopsis thaliana (Acytb₅₆₁-A, Acytb₅₆₁-B) in Escherichia coli and Pichia pastoris, their purification, and initial characterisation. Spectra indicated that Acytb₅₆₁-A resembles the best characterised member of the CYBASC family, the cytochrome b₅₆₁ from adrenomedullary chromaffin vesicles, and that Acytb₅₆₁-B is atypical compared to other CYBASC proteins. Haem oxidation–reduction midpoint potential (E_M) values were found to be fully consistent with ascorbate oxidation activities and Fe^3 +-chelates reductase activities. The ascorbate dependent reduction and protein stability of both paralogs were found to be sensitive to alkaline pH values as reported for the cytochrome b₅₆₁ from chromaffin vesicles. For both paralogs, ascorbate-dependent reduction was inhibited and the low-potential haem E_M values were affected significantly by incubation with diethyl pyrocarbonate (DEPC) in the absence of ascorbate. Modification with DEPC in the presence of ascorbate left the haem E_M values unaltered compared to the unmodified proteins. However, ascorbate reduction was inhibited. We concluded that the ascorbate-binding site is located near the low-potential haem with the Fe^3 +-chelates reduction-site close to the high-potential haem. Furthermore, inhibition of ascorbate oxidation by DEPC treatment occurs not only by lowering the haem E_M values but also by an additional modification affecting ascorbate binding and/or electron transfer. Analytical gel filtration experiments suggest that both cyt b₅₆₁ paralogs exist as homodimers.     

Large-scale bioreactor production of the herbicide-degrading Aminobacter sp. strain MSH1

The Aminobacter sp. strain MSH1 has potential for pesticide bioremediation because it degrades the herbicide metabolite 2,6-dichlorobenzamide (BAM). Production of the BAM-degrading bacterium using aerobic bioreactor fermentation was investigated. A mineral salt medium limited for carbon and with an element composition similar to the strain was generated. The optimal pH and temperature for strain growth were determined using shaker flasks and verified in bioreactors. Glucose, fructose, and glycerol were suitable carbon sources for MSH1 (μ?=?0.1 h^?1); slower growth was observed on succinate and acetic acid (μ?=?0.01 h^?1). Standard conditions for growth of the MSH1 strain were defined at pH 7 and 25 °C, with glucose as the carbon source. In bioreactors (1 and 5 L), the specific growth rate of MSH1 increased from μ?=?0.1 h^?1 on traditional mineral salt medium to μ?=?0.18 h^?1 on the optimized mineral salt medium. The biomass yield under standard conditions was 0.47 g dry weight biomass/g glucose consumed. An investigation of the catabolic capacity of MSH1 cells harvested in exponential and stationary growth phases showed a degradation activity per cell of about 3?×?10^?9 μg BAM h^?1. Thus, fast, efficient, large-scale production of herbicide-degrading Aminobacter was possible, bringing the use of this bacterium in bioaugmentation field remediation closer to reality.     

Crystallographic studies of the Escherichia coli quinol-fumarate reductase with inhibitors bound to the quinol-binding site

The quinol-fumarate reductase (QFR) respiratory complex of Escherichia coli is a four-subunit integral-membrane complex that catalyzes the final step of anaerobic respiration when fumarate is the terminal electron acceptor. The membrane-soluble redox-active molecule menaquinol (MQH(2)) transfers electrons to QFR by binding directly to the membrane-spanning region. The crystal structure of QFR contains two quinone species, presumably MQH(2), bound to the transmembrane-spanning region. The binding sites for the two quinone molecules are termed Q(P) and Q(D), indicating their positions proximal (Q(P)) or distal (Q(D)) to the site of fumarate reduction in the hydrophilic flavoprotein and iron-sulfur protein subunits. It has not been established whether both of these sites are mechanistically significant. Co-crystallization studies of the E. coli QFR with the known quinol-binding site inhibitors 2-heptyl-4-hydroxyquinoline-N-oxide and 2-[1-(p-chlorophenyl)ethyl] 4,6-dinitrophenol establish that both inhibitors block the binding of MQH(2) at the Q(P) site. In the structures with the inhibitor bound at Q(P), no density is observed at Q(D), which suggests that the occupancy of this site can vary and argues against a structurally obligatory role for quinol binding to Q(D). A comparison of the Q(P) site of the E. coli enzyme with quinone-binding sites in other respiratory enzymes shows that an acidic residue is structurally conserved. This acidic residue, Glu-C29, in the E. coli enzyme may act as a proton shuttle from the quinol during enzyme turnover.     

Specificity and reactive loop length requirements for crmA inhibition of serine proteases

The viral serpin, crmA, is distinguished by its small size and ability to inhibit both serine and cysteine proteases utilizing a reactive loop shorter than most other serpins. Here, we characterize the mechanism of crmA inhibition of serine proteases and probe the reactive loop length requirements for inhibition with two crmA reactive loop variants. P1 Arg crmA inhibited the trypsin-like proteases, thrombin, and factor Xa, with moderate efficiencies (approximately 10(2)-10(4) M(-1)sec(-1)), near equimolar inhibition stoichiometries, and formation of SDS-stable complexes which were resistant to dissociation (k(diss) approximately 10(-7) sec(-1)), consistent with a serpin-type inhibition mechanism. Trypsin was not inhibited, but efficiently cleaved the variant crmA as a substrate (k(cat)/K(M) of approximately 10(6) M(-1) sec(-1)). N-terminal sequencing confirmed that the P1 Arg-P1'Cys bond was the site of cleavage. Altering the placement of the Arg in a double mutant P1 Gly-P1'Arg crmA resulted in minimal ability to inhibit any of the trypsin family proteases. This variant was cleaved by the proteases approximately 10-fold less efficiently than P1 Arg crmA. Surprisingly, pancreatic elastase was rapidly inhibited by wild-type and P1 Arg crmAs (10(5)-10(6) M(-1)sec(-1)), although with elevated inhibition stoichiometries and higher rates of complex dissociation. N-terminal sequencing showed that elastase attacked the P1'Cys-P2'Ala bond, indicating that crmA can inhibit proteases using a reactive loop length similar to that used by other serpins, but with variations in this inhibition arising from different effective P2 residues. These results indicate that crmA inhibits serine proteases by the established serpin conformational trapping mechanism, but is unusual in inhibiting through either of two adjacent reactive sites.     

Adiposity Is Associated with Gender-Specific Reductions in Left Ventricular Myocardial Perfusion during Dobutamine Stress

Background

Obesity and visceral adiposity are increasingly recognized risk factors for cardiovascular disease. Visceral fat may reduce myocardial perfusion by impairing vascular endothelial function. Women experience more anginal symptoms compared to men despite less severe coronary artery stenosis, as assessed by angiography. Women and men have different fat storage patterns which may account for the observed differences in cardiovascular disease. Therefore, our objective was to evaluate the relationship between visceral adipose tissue distributions and myocardial perfusion in men and women.

Methods

Visceral and subcutaneous fat distributions and myocardial perfusion were measured in 69 men and women without coronary artery disease using magnetic resonance imaging techniques. Myocardial perfusion index was quantified after first-pass perfusion with gadolinium contrast at peak dose dobutamine stress.

Results

We observed inverse relationships between female gender (r = -0.35, p = 0.003), pericardial fat (r = -0.36, p = 0.03), intraperitoneal fat (r = -0.37, p = 0.001), and retroperitoneal fat (r = -0.36, p = 0.002) and myocardial perfusion index. Visceral fat depots were not associated with reduced myocardial perfusion at peak dose dobutamine in men. However, in women, BMI (r = -0.33, p = 0.04), pericardial fat (r = -0.53, p = 0.02), subcutaneous fat (r = -0.39, p = 0.01) and intraperitoneal fat (r = -0.30, p = 0.05) were associated with reduced myocardial perfusion during dobutamine stress.

Conclusions

Higher visceral fat volumes are associated with reduced left ventricular myocardial perfusion at peak dose dobutamine stress in women but not in men. These findings suggest that visceral fat may contribute to abnormal microcirculatory coronary artery perfusion syndromes, explaining why some women exhibit more anginal symptoms despite typically lower grade epicardial coronary artery stenoses than men.