Expression of the pspABCDE operon of Escherichia coli is induced upon infection by filamentous phage and by many other stress conditions, including defects in protein export. Expression of the operon requires the alternative sigma factor 54 and the transcriptional activator PspF. In addition, PspA plays a negative regulatory role, and the integral-membrane proteins PspB and PspC play a positive one. In this study, we investigated whether the suggested protein-protein interactions implicated in this complex regulatory network can indeed be demonstrated. Antisera were raised against PspB, PspC, and PspD, which revealed, in Western blotting experiments, that PspC forms stable sodium dodecyl sulfate-resistant dimers and that the hypothetical pspD gene is indeed expressed in vivo. Fractionation experiments showed that PspD localizes as a peripherally bound inner membrane protein. Cross-linking studies with intact cells revealed specific interactions of PspA with PspB and PspC, but not with PspD. Furthermore, affinity-chromatography suggested that PspB could bind PspA only in the presence of PspC. These data indicate that regulation of the psp operon is mediated via protein-protein interactions.

The genes coding for the mature part of the lipases from Sfaphy1ococc~u.s uureuks NCTC8530 and Stup1iylococcu.s hyicus have been cloned and overexpressed in Escherichiu coli as fusion proteins with an N-terminal hexa-histidine tag. The enzymes accumulated in the cytoplasm and were purified using sequential precipitation with protamine sulphate and ammonium sulphate, followed by metal-affinity and hydroxyapatite chromatography. The yield of pure lipase was 4.5 mg/g wet cells for S. uureus lipase and 13 mglg for S. hyicus lipase. The purified enzymes need calcium for activity, albeit with different affinities, and a low residual activity was found in the absence of calcium. In contrast to S. hyicus lipase, not only strontium but also barium can replace calcium with full retention of activity of S. uureus lipase. Whereas S. hyicu.s lipase is optimally active at pH 8.5, the optimum pH for enzymatic activity for S. uureus lipase was found to be pH 6.5. The S. uureMs lipase has a narrow substrate specificity: short-chain triacylglycerols and acyl esters of both p-nitrophenol and umbelliferone are readily degraded, whereas medium-and long-chain lipids, as well as phospholipids, are poor substrates. In contrast, S. hyicus lipase prefers phospholipids as substrate and hydrolyses neutral lipids irrespective of their chain length. The results are discussed in view of the large sequence similarity between both lipases.Keywords: Stuphylococcus u u e u s lipase ; Sfaplzylococcw hyicus lipase ; overexpression ; purification ; substrate specificity.Lipases (glycerol ester hydrolase) are active at a lipid-water interface where they degrade water-insoluble triacylglycerols. These enzymes usually have a broad substrate specificity and also degrade acyl p-nitrophenyl esters, Tweens and phospholipids often with positional, stereo-and chain-length selectivity (Jaeger et al., 1994, and references therein).All lipases of known three-dimensional structure belong to the class of serine esterases (Brady et al., 1990; Grochulski et al., 1993: Schrag et al., 1991Winkler et al., 1990). The active site of these enzymes contains a catalytic triad consisting of Ser, His and an acidic residue which in the case of lipases is either Asp or Glu. This catalytic machinery is covered by at1 amphipathic surface loop, the so-called lid. The structures of a lipaseinhibitor complex and a lipase-micelle complex (Brzozowski et al., 1991 ; van Tilbeurgh et al., 1993) showed that the lid moves away, presumably due to the interaction with the substrate interCorr-[,sl)ondenc,r ro H. M. Verheij,

Pyoverdine I is the main siderophore secreted byPseudomonas aeruginosa PAO1 to obtain access to iron. After extracellular iron chelation, pyoverdine-Fe uptake into the bacteria involves a specific outer-membrane transporter, FpvA. Iron is then released in the periplasm by a mechanism involving no siderophore modification but probably iron reduction. The proteins involved in this dissociation step are currently unknown. The pyoverdine locus contains the fpvCDEF operon, which contains four genes. These genes encode an ABC transporter of unknown function with the distinguishing characteristic of encompassing two periplasmic binding proteins, FpvC and FpvF, associated with the ATPase, FpvE, and the permease, FpvD. Deletion of these four genes partially inhibited cytoplasmic uptake of (55)Fe in the presence of pyoverdine and markedly slowed down the in vivo kinetics of iron release from the siderophore. This transporter is therefore involved in iron acquisition by pyoverdine in P. aeruginosa. Sequence alignments clearly showed that FpvC and FpvF belong to two different subgroups of periplasmic binding proteins. FpvC appears to be a metal-binding protein, whereas FpvF has homology with ferrisiderophore binding proteins. In vivo cross-linking assays and incubation of purified FpvC and FpvF proteins showed formation of complexes between both proteins. These complexes were able to bind in vitro PVDI-Fe, PVDI-Ga, or apo PVDI. This is the first example of an ABC transporter involved in iron acquisition via siderophores, with two periplasmic binding proteins interacting with the ferrisiderophore. The possible roles of FpvCDEF in iron uptake by the PVDI pathway are discussed.

The psp (phage-shock protein) operon of Escherichia coli is induced when the bacteria are infected by filamentous phage and under several other stress conditions. The physiological role of the individual Psp proteins is still not known. We demonstrate here that the last gene of the operon, pspE, encodes a thiosulfate:cyanide sulfurtransferase (EC 2.8.1.1; rhodanese). Kinetic analysis revealed that catalysis occurs via a double displacement mechanism as described for other rhodaneses. The K m s for SSO 23 3 and CN 3 were 4.6 and 27 mM, respectively. ß