Edited by Mike Shipston Many bacteria and some archaea produce the second messenger cyclic diadenosine monophosphate (c-di-AMP). c-di-AMP controls the uptake of osmolytes in Firmicutes, including the human pathogen Listeria monocytogenes, making it essential for growth. c-di-AMP is known to directly regulate several potassium channels involved in osmolyte transport in species such as Bacillus subtilis and Streptococcus pneumoniae, but whether this same mechanism is involved in L. monocytogenes, or even whether similar ion channels were present, was not known. Here, we have identified and characterized the putative L. monocytogenes' potassium transporters KimA, KtrCD, and KdpABC. We demonstrate that Escherichia coli expressing KimA and KtrCD, but not KdpABC, transport potassium into the cell, and both KimA and KtrCD are inhibited by c-di-AMP in vivo. For KimA, c-di-AMP-dependent regulation requires the C-terminal domain. In vitro assays demonstrated that the dinucleotide binds to the cytoplasmic regulatory subunit KtrC and to the KdpD sensor kinase of the KdpDE two-component system, which in Staphylococcus aureus regulates the corresponding KdpABC transporter. Finally, we also show that S. aureus contains a homolog of KimA, which mediates potassium transport. Thus, the c-di-AMP-dependent control of systems involved in potassium homeostasis seems to be conserved in phylogenetically related bacteria. Surprisingly, the growth of an L. monocytogenes mutant lacking the c-di-AMP-synthesizing enzyme cdaA is only weakly inhibited by potassium. Thus, the physiological impact of the c-di-AMP-dependent control of potassium uptake seems to be less pronounced in L. monocytogenes than in other Firmicutes. This work was supported by Grant CO 1139/2-1 from the Deutsche Forschungsgemeinschaft via Priority Program SPP1879, the Fonds der Chemischen Industrie, and the Max-Buchner-Forschungsstiftung (MBFSt-Kennziffer 3381) (to F. M. C.

A focused library comprising nine residues of the active site of P450cam monooxygenase resulting in ∼ 300,000 protein variants was screened for activity on diphenylmethane (DPM). The assay was based on the depletion of NADH by an in vitro reconstituted P450cam system in a 96-well scale. The throughput was increased by the parallel cultivation, purification and analysis of 20 variants per well (cluster screening). Thus ∼ 20,000 protein variants could be screened in summary of which five were found to transform DPM with a specific activity of up to 75% of the wild-type activity on d-camphor and a coupling rate of 7-18%. One variant converting DPM to 4-hydroxydiphenylmethane (4HDPM) was subjected to site-directed mutagenesis and saturation mutagenesis, which revealed the particular importance of positions F87, Y96 and L244 for substrate selectivity and the possibility for further improvements of this variant. Moreover, a reduction in size of the amino acid at position 396 decreased specific activity dramatically but increased coupling and switched the main product formation from 4HDPM towards diphenylmethanol.

We show that the turbidimetric method of Ziegenhorn et al. (Clin. Chem. 25: 1067, 1979) for determination of pancreatic lipase is not influenced by lipoprotein lipase. This improved specificity as compared to standard lipase methods is explained by the presence of purified colipase and the high concentration of bile acids in the substrate emulsion.

An enzymatic method for the calibration of a turbidimetric lipase assay is described, based on measurement of free fatty acids liberated by the action of lipase. The substrate of the turbidimetric assay is a colipase-containing triolein emulsion. For determination of the free fatty acids a commercial test kit including acyl-CoA synthetase, acyl-CoA oxidase, and peroxidase is used. Intra- and interassay imprecision (CV) is about 5% at above-normal lipase activities, about 10% at normal values. Temperature coefficients are 1.24 and 1.45, respectively, for measurements at 30 and 37 degrees C vs 25 degrees C.

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