The Serratia marcescens hemolysin represents the prototype of a growing family of pore forming toxins. The available bacterial genome sequences reveal Serratia hemolysin homologues in additional species. However, only S. marcescens hemolysin has been studied in great molecular detail. This family of toxins has nothing in common with the pore forming toxins of E. coli type (RTX toxins), the Staphylococcus aureus alpha-toxin or the thiol activated toxin of group A beta-hemolytic streptococci (Streptolysin O). Studies on erythrocytes, eukaryotic cells and artificial black lipid membranes, have shown that the mechanism of pore formation of ShlA is different form other pore forming toxins. The S. marcescens hemolysin proteins ShlB and ShlA, exhibit protein sequence homologues in Proteus mirabilis, Haemophilus ducreyi, Yersinia pestis, Yersinia enterocolitica, Edwardsiella tarda, Photorhabdus luminescens and Xylella fastidiosa . The family of Serratia type pore forming toxins show a unique secretory mechanism which has been described as a two partner secretion system (TPSS) or type V-secretion system. Not only Serratia type pore forming toxins are secreted via TPSS but also adhesins from Bordetella pertussis, Erwinia chrysanthemi and Haemophilus influenzae. The uniqueness of the Serratia family is underlined by the fact that activation of ShlA by ShlB strictly requires phosphatidylethanolamine as a cofactor. And, quite unusual, ShlA undergoes a conformational change during activation.

Background: Serratia marcescens, a frequent agent of catheterization-associated bacteriuria, strongly adheres to human bladder epithelial cells in culture. The epithelium normally provides a barrier between lumal organisms and the interstitium; the tight adhesion of bacteria to the epithelial cells can lead to internalization and subsequent lysis. However, internalisation was not shown yet for S. marcescens strains.

Studies on the chemical nature of the microbial biosynthesis of vitamin B12[1.141 have revealed that the organization of the individual reactions in the aerobe Pseudomonas denitriJicans and the anaerobe Propionibacterium shermanii differs significantly, and that these differences could be evolutionary in their origins. The work of Blanche, Crouzet et al.lla-cl has provided deep insight into the molecular nature of vitamin B, , synthesis. Their construction and efficient application of recombinant strains of P. denitrificans led to the discovery and isolation of new and spectacular vitamin B,, precursors with the responsible cloned enzymes.It was shown that the tetrapyrrolic vitamin B,, ligand hydrogenobyrinic acid 7I2] is synthesized without cobalt via the precorrins 2(1) and 3(2)r2a, l 4 I as well as the newly discovered ring-contracted precorrins 4(3)[31, 6~( 4 ) , [~] 6y(5),I5' and 8~( 6 ) [~J (Scheme 1). After two obligatory amidations leading to 7-a,c-diamide,['I cobalt is inserted by the cobaltchelatase responsible for vitamin B,, formation."' In the case of P. shermanii neither 7 nor 7-apdiamide are precursors of vitamin B,, . [ 2 9 9 a 1 Instead cobyrinic acid @), the cobaltcontaining analogue of 7, and 8-a,c-diamide appear in the biosynthetic pathway.['". O1 Enzyme preparations from P. shermanii are able to transform in vitro not only the oxidized and aromatized precorrins 2(1 a) and 3(2a)r2".'41 (known as factors I1 and 111, respectively) into 8, but also the cobalt complexes of 1 a and 2a.[91 In contrast, the enzyme systems from P. denitrificans that produce 7 are able to

Incubation of human epithelial cells with nanomolar concentrations of chromatographically purified Serratia marcescenshemolysin (ShlA) caused irreversible vacuolation and subsequent lysis of the cells. Vacuolation differed from vacuole formation byHelicobacter pylori VacA. Sublytic doses of ShlA led to a reversible depletion of intracellular ATP. Restoration to the initial ATP level was presumably due to the repair of the toxin damage and was inhibited by cycloheximide. Pores formed in epithelial cells and fibroblasts without disruption of the plasma membrane, and the pores appeared to be considerably smaller than those observed in artificial lipid membranes and in erythrocytes and did not allow the influx of propidium iodide or trypan blue. All cytotoxic effects induced by isolated recombinant ShlA were also obtained with exponentially growingS. marcescens cells. The previously suggested role of the hemolysin in the pathogenicity of S. marcescens is supported by these data.

At high bacterial cell density the gene expression program of Pseudomonas aeruginosa is regulated by quorum sensing. Among the gene products highly up-regulated by this system is an exoprotease, leucine aminopeptidase (PA-LAP), which is coexpressed with several known virulence factors and secreted as a proenzyme. We undertook a study of its activation by expressing the full-length proform of PA-LAP recombinantly in Escherichia coli (here termed, rLAP55) and characterizing individual steps in its conversion to an active enzyme. Activation is initiated with the proteolytic removal of a C-terminal prosequence. Removal of ϳ20 amino acids is accomplished by Pseudomonas elastase, which is also positively regulated by quorum sensing. Activation is also mediated by other proteases that cleave rLAP55 near its C terminus. The importance of the C terminus was confirmed by showing that C-terminal deletions of 1-24 amino acids produce a fully active enzyme. The removal of C-terminal prosequences either by proteolysis or deletion leads to an unusual autoprocessing event at the N terminus. Autoprocessing is apparently an intramolecular event, requires the active site of LAP, and results in the removal of 12 N-terminal amino acids. Furthermore, a detailed analysis of the C-terminal prosequence suggests that the proenzyme state is dependent on the presence of a basic side chain contributed by the last amino acid, lysine 536. Our data support a model whereby full-length PA-LAP is activated in a two-step process; proteolytic cleavage at the C terminus is followed by an intramolecular autocatalytic removal of a 12-amino acid propeptide at the N terminus.