Role of the Na(+)-translocating NADH:quinone oxidoreductase in voltage generation and Na(+) extrusion in Vibrio cholerae.

For Vibrio cholerae, the coordinated import and export of Na(+) is crucial for adaptation to habitats with different osmolarities. We investigated the Na(+)-extruding branch of the sodium cycle in this human pathogen by in vivo (23)Na-NMR spectroscopy. The Na(+) extrusion activity of cells was monitored after adding glucose which stimulated respiration via the Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR). In a V. cholerae deletion mutant devoid of the Na(+)-NQR encoding genes (nqrA-F), rates of respiratory Na(+) extrusion were decreased by a factor of four, but the cytoplasmic Na(+) concentration was essentially unchanged. Furthermore, the mutant was impaired in formation of transmembrane voltage (ΔΨ, inside negative) and did not grow under hypoosmotic conditions at pH8.2 or above. This growth defect could be complemented by transformation with the plasmid encoded nqr operon. In an alkaline environment, Na(+)/H(+) antiporters acidify the cytoplasm at the expense of the transmembrane voltage. It is proposed that, at alkaline pH and limiting Na(+) concentrations, the Na(+)-NQR is crucial for generation of a transmembrane voltage to drive the import of H(+) by electrogenic Na(+)/H(+) antiporters. Our study provides the basis to understand the role of the Na(+)-NQR in pathogenicity of V. cholerae and other pathogens relying on this primary Na(+) pump for respiration.

The Staphylococcus aureus NuoL-like protein MpsA contributes to the generation of membrane potential.

In aerobic microorganisms, the entry point of respiratory electron transfer is represented by the NADH:quinone oxidoreductase. The enzyme couples the oxidation of NADH with the reduction of quinone. In the type 1 NADH:quinone oxidoreductase (Ndh1), this reaction is accompanied by the translocation of cations, such as H(+) or Na(+). In Escherichia coli, cation translocation is accomplished by the subunit NuoL, thus generating membrane potential (Δψ). Some microorganisms achieve NADH oxidation by the alternative, nonelectrogenic type 2 NADH:quinone oxidoreductase (Ndh2), which is not cation translocating. Since these enzymes had not been described in Staphylococcus aureus, the goal of this study was to identify proteins operating in the NADH:quinone segment of its respiratory chain. We demonstrated that Ndh2 represents a NADH:quinone oxidoreductase in S. aureus. Additionally, we identified a hypothetical protein in S. aureus showing sequence similarity to the proton-translocating subunit NuoL of complex I in E. coli: the NuoL-like protein MpsA. Mutants with deletion of the nuoL-like gene mpsA and its corresponding operon, mpsABC (mps for membrane potential-generating system), exhibited a small-colony-variant-like phenotype and were severely affected in Δψ and oxygen consumption rates. The MpsABC proteins did not confer NADH oxidation activity. Using an Na(+)/H(+) antiporter-deficient E. coli strain, we could show that MpsABC constitute a cation-translocating system capable of Na(+) transport. Our study demonstrates that MpsABC represent an important functional system of the respiratory chain of S. aureus that acts as an electrogenic unit responsible for the generation of Δψ.

Lactose autoinduction with enzymatic glucose release: characterization of the cultivation system in bioreactor.

The lactose autoinduction system for recombinant protein production was combined with enzymatic glucose release as a method to provide a constant feed of glucose instead of using glycerol as a carbon substrate. Bioreactor cultivation confirmed that the slow glucose feed does not prevent the induction by lactose. HPLC studies showed that with successful recombinant protein production only a very low amount of lactose was metabolized during glucose-limited fed-batch conditions by the Escherichia coli strain BL21(DE3)pLysS in well-aerated conditions, which are problematic for glycerol-based autoinduction systems. We propose that slow enzymatic glucose feed does not cause a full activation of the lactose operon. However recombinant PDI-A protein (A-domain of human disulfide isomerase) was steadily produced until the end of the cultivation. The results of the cultivations confirmed our earlier observations with shaken cultures showing that lactose autoinduction cultures based on enzymatic glucose feed have good scalability, and that this system can be applied also to bioreactor cultivations.

Use of slow glucose feeding as supporting carbon source in lactose autoinduction medium improves the robustness of protein expression at different aeration conditions.

Recombinant protein expression from lac derived promoters by the autoinduction regime is based on diauxic growth of Escherichia coli on glucose and lactose. Glycerol is used as a supporting carbon source during the lactose-induced expression. While this glycerol-based formulation usually provides high cell densities, successful protein expression by autoinduction is often very dependent on correct aeration level. This complicates the reproducibility and scalability of the cultures. In this study we investigate the use of an alternative autoinduction formulation, in which the supporting carbon source is provided by fed-batch-like slow glucose feed from a biocatalytically degraded polysaccharide. The glucose feed as supporting carbon source allowed for high level of autoinduced target protein expression from T7lac promoter in E. coli BL21(DE3) and from T5lac promoter in E. coli K-12 RB791(lacI(q)) with lactose concentrations of 0.5-2gl(-1). Cell densities and protein yields per culture volume were similar to or higher than in the glycerol-based ZYM-5052 medium. In the glycerol-based medium, protein production was adversely influenced by high aeration level, resulting in 75-90% reduction in protein yield per cell compared to more moderately aerated conditions. The glucose fed-batch medium attenuated this oxygen-sensitivity and provided robust high-yield expression also under high aeration rates. It is concluded that the slow glucose feed as supporting carbon source mitigates aeration-related scale differences in autoinduced protein expression, and combined with the benefit of high product yields this makes the fed-batch autoinduction medium ideal for high-throughput screening and scale-up of the production process.

Vectors for improved Tet repressor-dependent gradual gene induction or silencing in Staphylococcus aureus.

A set of vectors for improved tetracycline-dependent gene regulation in Staphylococcus aureus is presented. Plasmid pRAB11 was generated from pRMC2 by adding a second tet operator within the TetR-regulated promoter P(xyl/tet). Pronounced repression was observed in the absence of anhydrotetracycline (ATc) combined with high induction in the presence of the drug, as demonstrated for pRAB11 bearing staphylococcal nuclease nuc1, lacZ or gfp. Also, in plasmid pCG261, the pRAB11 tetR-P(xyl/tet) regulatory architecture permitted tight repression and a stepwise increase in transcript amounts of the target gene rny (putative RNase) correlated with rising ATc concentrations. Additionally, pRAB11-derived vectors harbouring semi-rationally designed P(xyl/tet)-like fragments, mutated at up to six defined positions, were constructed. Sixteen mutant sequences with single to quadruple exchanges were analysed for transcriptional strength and ATc-dependent inducibility. A set of promoters with gradually decreased activities and improved repression is presented. Finally, the implementation of reverse TetR revtetR-r2, which exhibits three amino acid exchanges and binds to tetO in the presence of ATc, yielded an efficiently co-repressible vector within the pRAB11 system. Intriguingly, revtetR was found to contain a fourth mutation only after propagation in S. aureus. We predict that the described vectors constitute valuable tools for staphylococcal genetics.

Inhibition of microbial production of the malodorous substance isovaleric acid by 4,4' dichloro 2-hydroxydiphenyl ether (DCPP).

Human body malodour is a complex phenomenon. Several types of sweat glands produce odorless secretions that are metabolized by a consortium of skin-resident microorganisms to a diverse set of malodorous substances. Isovaleric acid, a sweaty-smelling compound, is one major malodorous component produced by staphylococci with the skin-derived amino acid L-leucine as a substrate. During wearing, fabrics are contaminated with sweat and microorganisms and high humidity propagates growth and microbial malodour production. Incomplete removal of sweat residues and microorganisms from fabrics during laundry with bleach-free detergents and at low temperatures elevate the problem of textile malodour. This study aimed to analyze the inhibitory effect of the antimicrobial 4,4' dichloro 2-hydroxydiphenyl ether (DCPP) on the formation of isovaleric acid on fabrics. Therefore, GC-FID- and GC-MS-based methods for the analysis of isovaleric acid in an artificial human sweat-mimicking medium and in textile extracts were established. Here, we show that antimicrobials capable to deposit on fabrics during laundry, such as DCPP, are effective in growth inhibition of typical malodour-generating bacteria and prevent the staphylococcal formation of isovaleric acid on fabrics in a simple experimental setup. This can contribute to increased hygiene for mild laundry care approaches, where bacterial contamination and malodour production represent a considerable consumer problem.

Electro-mechanical (dys-)function in long QT syndrome type 1.

BACKGROUND: Prolonged repolarization is the hallmark of long QT syndrome (LQTS), which is associated with subclinical mechanical dysfunction. We aimed at elucidating mechanical cardiac function in LQTS type 1 (loss of IKs) and its modification upon further prolongation of the action potential (AP) by IKr-blockade (E-4031). METHODS: Transgenic LQT1 and wild type (WT) rabbits (n = 12/10) were subjected to tissue phase mapping MRI, ECG, and epicardial AP recording. Protein and mRNA levels of ion channels and Ca2+ handling proteins (n = 4/4) were determined. In silico single cell AP and tension modeling was performed. RESULTS: At baseline, QT intervals were longer in LQT1 compared to WT rabbits, but baseline systolic and diastolic myocardial peak velocities were similar in LQT1 and WT. E-4031 prolonged QT more pronouncedly in LQT1. Additionally, E-4031 increased systolic and decreased diastolic peak velocities more markedly in LQT1 - unmasking systolic and diastolic LQT1-specific mechanical alterations. E-4031-induced alterations of diastolic peak velocities correlated with the extent of QT prolongation. CONCLUSION: While baseline mechanical function is normal in LQT1 despite a distinct QT prolongation, further prolongation of repolarization by IKr-blocker E-4031 unmasks mechanical differences between LQT1 and WT with enhanced systolic and impaired diastolic function only in LQT1. These data indicate an importance of the extent of QT prolongation and the contribution of different impaired ion currents for conveying mechanical dysfunction.

Both terminal oxidases contribute to fitness and virulence during organ-specific Staphylococcus aureus colonization.

In their recent article, Hammer et al. (N. D. Hammer, M. L. Reniere, J. E. Cassat, Y. Zhang, A. O. Hirsch, M. Indriati Hood, and E. P. Skaar, mBio 4:e00241-13, 2013) described the dual functions of the two terminal oxidases encoded by cydBA and qoxABCD in Staphylococcus aureus. The aerobic growth of cydB or qoxB single mutant bacteria was barely affected. However, a cydB qoxB double mutant was completely unable to respire and exhibited the small-colony variant phenotype that is typical of menaquinone and heme biosynthesis mutants. The authors found that the two terminal oxidases play a role in pathogenesis. In a systemic mouse infection model, it turned out that in the cydB mutant the bacterial burden was significantly decreased in the heart, kidneys, and liver, while in the qoxB mutant it was decreased only in the liver. These results illustrate that both terminal oxidases contribute to fitness and virulence, representing promising candidates for the development of antimicrobials.

Intracellular monitoring of target protein production in Staphylococcus aureus by peptide tag-induced reporter fluorescence.

An intracellular approach for monitoring protein production in Staphylococcus aureus is described. mCherry, fused to the dodecapeptide Tip, was capable of inducing tetracycline repressor (TetR). Time- and concentration-dependent production of mCherry could be correlated to TetR-controlled GFPmut2 activity. This approach can potentially be extended to native S. aureus proteins.