The Escherichia coli yiiP gene encodes an iron transporter, ferrous iron efflux (FieF), which belongs to the cation diffusion facilitator family (CDF). Transcription of fieF correlated with iron concentration; however, expression appeared to be independent of the ferrous iron uptake regulator Fur. Absence of FieF led to decreased growth of E. coli cells in complex growth medium but only if fur was additionally deleted. The presence of EDTA was partially able to relieve this growth deficiency. Expression of fieF in trans rendered the double deletion strain more tolerant to iron. Furthermore, E. coli cells exhibited reduced accumulation of (55)Fe when FieF was expressed in trans. FieF catalyzed active efflux of Zn(II) in antiport with protons energized by NADH via the transmembrane pH gradient in everted membrane vesicles. Using the iron-sensitive fluorescent indicator PhenGreen-SK encapsulated in proteoliposomes, transmembrane fluxes of iron cations were measured with purified and reconstituted FieF by fluorescence quenching. This suggests that FieF is an iron and zinc efflux system, which would be the first example of iron detoxification by efflux in any organism.

There is accumulating evidence that transenvelope efflux pumps of the resistance, nodulation, cell division protein family (RND) are excreting toxic substances from the periplasm across the outer membrane directly to the outside. This would mean that resistance of Gram-negative bacteria to organic toxins and heavy metals is in fact a two-step process: one set of resistance factors control the concentration of a toxic substance in the periplasm, another one that in the cytoplasm. Efficient periplasmic detoxification requires periplasmic toxin sensing and transduction of this signal into the cytoplasm to control expression of the periplasmic detoxification system. Such a signal transduction system was analyzed using the Cnr nickel resistance system from Cupriavidus (Wautersia, Ralstonia, Alcaligenes) metallidurans strain CH34. Resistance is based on nickel efflux mediated by the CnrCBA efflux pump encoded by the cnrYHXCBAT metal resistance determinant. The products of the three genes cnrYXH transcriptionally regulate expression of cnr. CnrY and CnrX are membrane-bound proteins probably functioning as anti sigma factors while CnrH is a cnr-specific extracytoplasmic functions (ECF) sigma factors. Experimental data provided here indicate a signal transduction chain leading from nickel in the periplasm to transcription initiation at the cnr promoters cnrYp and cnrCp, which control synthesis of the nickel efflux pump CnrCBA.

A novel membrane proteinase of the nosocomial important bacteria species Bacillus cereus (synonyms: camelysin, CCMP) was purified up to homogeneity as was shown by mass spectrometry in its amphiphilic form. Camelysin is a neutral metalloprotease with a molecular mass of 19 kDa. Its unique N-terminus Phe-Phe-Ser-Asp-Lys-Glu-Val-Ser-Asn-Asn-Thr-Phe-Ala-Ala-Gly-Thr-Leu-Asp-Leu-Thr-Leu-Asn-Pro-Lys-Thr-Leu-Val-Asp-(Ile-Lys-Asp)- was not detected in the protein data bases during BLAST searches, but in the partially sequenced genome of Bacillus anthracis, coding for an unknown protein. Cleavage sites of the membrane proteinase for the insulin A- and B-chains were determined by mass spectrometry and N-terminal sequencing. Camelysin prefers cleavage sites in front of aliphatic and hydrophilic amino acid residues (-OH, -SO3H, amido group), avoiding bulky aromatic residues. The internally quenched fluorogenic substrates of the matrix metalloproteases 2 and 7 were cleaved with the highest efficiency at the Leu-decrease-Gly or Leu-decrease-Ala bond with the smaller residue in the P1' position. The protein specificity is broad--all various kinds of casein were cleaved as well as acid-soluble collagen, globin and ovalbumin; intact insulin was destroyed only to a low extent. Actin, collagen type I, fibrinogen, fibrin, alpha2-antiplasmin and alpha1-antitrypsin were cleaved. The protease formed SDS-stable complexes with Glu-plasminogen and antithrombin III, visible after SDS electrophoresis by gold staining and Western blot. The CCMP-plasminogen complex caused a partial activation of plasminogen to plasmin. Camelysin interacts with proteins of the blood coagulation cascade and could facilitate the penetration of fibrin clots and of the extracellular matrix during bacterial invasion.

Bacillus cereus, a gram-positive, ubiquitous soil bacterial species, is closely related to Bacillus anthracis and Bacillus thuringiensis (20,25,27,48). There are remarkable morphological and biochemical similarities between these species, for example, in the structures of their rRNAs and their cell wall composition (59). The main divergence between the species is the occurrence of different toxins, causing a variable spectrum of disease symptoms (20). B. cereus was described as a foodpoisoning organism, causing illness due to production of a heat-stable emetic toxin (4, 43) and diarrheal enterotoxins (20,23).B. cereus strains, when detected in clinical specimens, were earlier mistaken for accidentally occurring contaminating germs. However, during the last decade they have been identified to an increasing extent as pathogenic agents themselves (5, 32, 57). B. cereus can be the cause of severe, even lethal infections such as sepsis, pneumonia, meningitis, endocarditis, or wound infections, especially for patients in an immunocompromised state. Additionally, B. cereus is of great importance as the common pathogen for the highly fulminant posttraumatic endophthalmitis (7,12).B. cereus strains secrete a wide spectrum of extracellular virulence factors and exoenzymes (18,20,32), including an ADP-ribosylating enzyme, phospholipase C, enterotoxins, subtilisin-like proteases, and neutral metalloproteases (bacillolysin) with high homology to thermolysin. Expression of those exoproteins is under the control of the PlcR regulator in the stationary growth phase (3, 18). Hitherto, the participation of the different pathogenic factors and their interactions in nongastrointestinal infections caused by B. cereus have not been well-understood, and they remain a subject of intensive investigations.During the search for bacterial surface proteases, a highly active, cell envelope-bound azocaseinolytic activity was detected in B. cereus. The protease was purified in its detergent form. It was homogeneous in mass spectrometry (MS), with a molecular mass of 19,073.1 Ϯ 15 Da, and was called caseincleaving metalloproteinase or camelysin (16, 17). The protein differs from known extra-and intracellular Bacillus proteinases in its N-terminal sequence, substrate specificity, and inhibition pattern. Camelysin preferentially cleaves peptide bonds in front of aliphatic hydrophobic amino acids and hydrophilic amino acid residues, avoiding bulky aromatic residues in the P 1 Ј position, and is therefore almost completely unable to release chromogenic and fluorogenic groups from this position (17). Camelysin belongs to the neutral metalloproteases, showing their typical strong inhibition by metal chelators (16), but it is insensitive to phosphoramidon or zincov, which are the strongest inhibitors of neutral metalloproteinases of the thermolysin-type (clan MA) (47).Bacterial surface proteases were detected and characterized for some gram-positive species (8, 40) and gram-negative species (26, 54), often playing a role as important virulence factors...

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