Subcellular location and unique secretion of the hemolysin of Serratia marcescens

It is shown that Serratia marcescens exports a hemolysin to the cell surface and secretes it to the extracellular space. Escherichia coli containing the cloned hemolysin genes shlA and shlB exported and secreted the S. marcescens hemolysin. A nonhemolytic secretion-incompetent precursor of the hemolysin, designated ShlA*, was synthesized in a shlB deletion mutant and accumulated in the periplasmic space of E. coli. Immunogold-labeled ultrathin sections revealed ShlA* bound to the outer face of the cytoplasmic membrane and to the inner face of the outer membrane. A number of mutants carrying 3' deletions in the shlA gene secreted truncated polypeptides, the smallest of which contained only 261 of the 1578 amino acids of the mature ShlA hemolysin, showing that the information for export to the cell surface of E. coli and secretion into the culture medium is located in the NH2-terminal segment of the hemolysin. We propose a secretion pathway in which ShlA and ShlB are exported across the cytoplasmic membrane via a signal sequence-dependent mechanism. ShlB is integrated into the outer membrane. ShlA is translocated across the outer membrane with the help of ShlB. During the latter export process or at the cell surface, ShlA acquires the hemolytically active conformation and is released to the extracellular space. The hemolysin secretion pathway appears to be different from any other secretion system hitherto reported and involves only a single specific export protein.     

Serratia marcescens forms a new type of cytolysin

Most Serratia marcescens strains produce a new type of cytolysin (hemolysin) which is also found in other Serratia species. The hemolytic polypeptide ShlA (M(r) 162 101) is secreted across the outer membrane through the help of the ShlB protein which also involves conversion of an inactive precursor in an hemolytically active form. Both proteins are synthesized with signal sequences which are released during export across the cytoplasmic membrane. Mutants expressing inactive ShlB derivatives are impaired in activation and secretion suggesting a tight coupling between both processes. The region of ShlA for activation and secretion is confined to the N-terminal 16% of the polypeptide which contains the sequence NPNG which is also found in the Proteus hemolysin, the Bordetella pertussis filamentous hemagglutinin and two highly expressed outer membrane proteins of Haemophilus influenzae. Substitution of the first asparagine (N) residue by isoleucine converts the Serratia hemolysin into an inactive secretion incompetent form. It is concluded that this region is recognized by ShlB for activation and secretion of ShlA. The Serratia hemolysin forms defined pores in erythrocyte membranes.     

Superlytic hemolysin mutants of Serratia marcescens.

Hemolysis by Serratia marcescens is caused by two proteins, ShlA and ShlB. ShlA is the hemolysin proper, and ShlB transports ShlA through the outer membrane, whereby ShlA is converted into a hemolysin. Superhemolytic ShlA derivatives that displayed 7- to 20-fold higher activities than wild-type ShlA were isolated. ShlA80 carried the single amino acid replacement of G to D at position 326 (G326D), ShlA87 carried S386N, and ShlA80III carried G326D and N236D. Superhemolysis was attributed to the greater stability of the mutant ShlA derivatives because they aggregated less than the wild-type hemolysin, which lost activity within 3 min at 20 degrees C. In contrast to the highly hemolytic wild-type ShlA at 0 degrees C, the hyperlytic hemolysins were nonhemolytic at 0 degrees C, suggesting that the hyperlytic derivatives differed from wild-type ShlA in adsorption to and insertion into the erythrocyte membrane. However, the size of the pores formed at 20 degrees C by superhemolytic hemolysins could not be distinguished from that of wild-type ShlA. In addition to the N-terminal sequence up to residue 238, previously identified to be important for activation and secretion, sites 326 and 386 contribute to hemolysin activity since they are contained in regions that participate in hemolysin inactivation through aggregation.     

In vitro activation of the Serratia marcescens hemolysin through modification and complementation.

The hemolytic activity of Serratia marcescens is determined by two polypeptides, termed ShlA and ShlB. ShlA is synthesized as an inactive precursor (ShlA*) and secreted with the help of ShlB, which is located in the outer membrane. In this study, it is shown that a cell lysate containing ShlB as well as partially purified ShlB converted ShlA* to the active ShlA hemolysin. ShlA remained active after removal of ShlB by column chromatography. In contrast to the stable modification of ShlA* by ShlB, a reversible activation was achieved by adding to ShlA* an N-terminal fragment of ShlA (ShlA16), consisting of 269 amino acid residues of ShlA and 18 residues of the vector. The nonhemolytic ShlA16 complemented ShlA* only when it was synthesized in an ShlB-producing cell. A deletion derivative of ShlA*, lacking residues 4 to 117, was complemented by ShlA16 but not activated by ShlB. Activation of ShlA* by ShlB at 4 degrees C proceeded at a much slower rate than complementation by ShlA16. It is concluded that ShlA* is modified by ShlB. ShlA16 modified by ShlB complements the missing modification of ShlA* in trans. Modification by ShlB occurs in the N-terminal part of ShlA*, which is also the reaction in vivo which results in active ShlA hemolysin in the culture supernatant. The HpmA hemolysin of Proteus mirabilis, which is very similar to ShlA, was also activated in vitro by ShlB and complemented by ShlA16.     

The haemolysin-secreting ShlB protein of the outer membrane of Serratia marcescens : determination of surface-exposed residues and formation of ion-permeable pores by ShlB mutants in artificial lipid bilayer membranes

The ShlB protein in the outer membrane of Serratia marcescens is the only protein known to be involved in secretion of the ShlA protein across the outer membrane. At the same time, ShlB converts ShlA into a haemolytic and a cytolytic toxin. Surface-exposed residues of ShlB were determined by reaction of an M2 monoclonal antibody with the M2 epitope DYKDDDDK inserted at 25 sites along the entire ShlB polypeptide. The antibody bound to the M2 epitope at 17 sites in intact cells, which indicated surface exposure of the epitope, and to 23 sites in isolated outer membranes. Two insertion mutants contained no ShlB(M2) protein in the outer membrane. The ShlB derivatives activated and/or secreted ShlA. To gain insights into the secretion mechanism, we studied whether highly purified ShlB and ShlB deletion derivatives formed pores in artificial lipid bilayer membranes. Wild-type ShlB formed channels with very low single channel conductance that rarely assumed an open channel configuration. In contrast, open channels with a considerably higher single channel conductance were observed with the deletion mutants ShlB(Delta65-186), ShlB(Delta87-153), and ShlB(Delta126-200). ShlB(Delta126-200) frequently formed permanently open channels, whereas the conductance caused by ShlB(Delta65-186) and ShlB(Delta87-153) did not assume a stationary value, but fluctuated rapidly between open and closed configurations. The results demonstrate the orientation of large portions of ShlB in the outer membrane and suggest that ShlB may function as a specialized pore through which ShlA is secreted.     

Integration of the Serratia marcescens haemolysin into human erythrocyte membranes

The haemolytic activity of Serratia marcescens is determined by two proteins, ShlA and ShlB. ShlA integrates into the erythrocyte membrane and causes osmotic lysis through channel formation. The conformation of ShlA and its interaction with erythrocyte membranes were studied by determining the cleavage of ShlA by added trypsin. Our results suggest that the conformation of inactive ShlA (from an ShlB- strain) differs from the active ShlA, and that in a hydrophobic environment (detergent or membrane) active ShlA assumes a conformation distinct from that in buffer. Only active haemolysin adsorbed to erythrocytes. ShlA was firmly integrated into the erythrocyte membrane since it was only released under conditions which also dissolved the integral erythrocyte membrane proteins. Moreover, ShlA integrated into 'ghosts' remained there and was not haemolytic when incubated with erythrocytes. From the trypsin cleavage pattern obtained with haemolysin and C-terminally truncated, but still active, haemolysin derivatives integrated into erythrocytes, and sealed and unsealed erythrocyte 'ghosts', we conclude that ShlA is preferentially cleaved by trypsin at a few sites but only from the inside of the erythrocyte. Haemolysin in the erythrocyte membrane forms a water-filled channel and is resistant to trypsin and other proteases.     

The Serratia marcescens hemolysin is secreted but not activated by stable protoplast-type L-forms of Proteus mirabilis

The outer-membrane protein ShlB of Serratia marcescens activates and secretes hemolytic ShlA into the culture medium. Without ShlB, inactive ShlA (termed ShlA*) remains in the periplasm. Since Proteus mirabilis L-form cells lack an outer membrane and a periplasm, it was of interest to determine in which compartment recombinant ShlA* and ShlB are localized and whether ShlB activates ShlA*. The cloned shlB and shlA genes were transcribed in P. mirabilis stable L-form cells by the temperature-inducible phage T7 RNA polymerase. Radiolabeling, Western blotting, and complementation with C-terminally truncated ShlA (ShlA255) identified inactive ShlA* in the culture supernatant. ShlB remained cell-bound and did not activate ShlA without integration in an outer membrane. Although hemolytic ShlA added to L-form cells had access to the cytoplasmic membrane, it did not affect L-form cells. Synthesis of the large ShlA protein (165 kDa) in P. mirabilis L-form cells under phage T7 promoter control demonstrates that L-form cells are suitable for the synthesis and secretion of large recombinant proteins. This property and the easy isolation of released proteins make L-form cells suitable for the biotechnological production of proteins. Received: 17 February 1998 / Accepted: 30 June 1998     

Two-step fast protein liquid chromatographic purification of the Serratia marcescens hemolysin and peptide mapping with mass spectrometry

The pore forming toxin of Serratia marcescens (ShlA) is secreted and activated by an outer membrane protein (ShlB). Activation of inactive ShlA (termed ShlA*) by ShlB is dependent on phosphatidylethanolamine (PE). Activation may be a covalent modification of ShlA. To test this hypothesis, the responsible activation domain (in the N-terminal 255 amino acids of ShlA) was isolated from whole bacteria with 8 M urea in an inactive form (ShlA-255*) and from the culture supernatant in an active form (ShlA-255), followed by a two-step purification by anion-exchange chromatography and gel permeation chromatography. Comparison of a tryptic peptide map of both forms with subsequent electrospray mass spectrometry (ES-MS) and sequencing by tandem ES-MS revealed no modification. These data imply that ShlB presumably imposes a conformation on ShlA-255 that triggers activity.     

Specific phosphatidylethanolamine dependence of Serratia marcescens cytotoxin activity

The cytolytic and haemolytic activity of Serratia marcescens is determined by the ShlA protein, which is secreted across the outer membrane with the aid of the ShlB protein. In the absence of ShlB, inactive ShlA* remains in the periplasm of Escherichia coli transformed with an shlA-encoding plasmid, which indicates that ShlB converts ShlA* to active ShlA. ShlA* in a periplasmic extract and partially purified ShlA* were activated in vitro by partially purified ShlB. When both proteins were highly purified, ShlA* was only activated by ShlB when phosphatidylethanolamine (PE) or phosphatidylserine was added to the assay, while phosphatidylglycerol contributed little to ShlA* activation. Lyso-PE, cardiolipin, phosphatidylcholine, phosphatidic acid, lipopolysaccharide and various detergents could not substitute for PE. Although radioactively labelled PE was so tightly associated with ShlA that it remained bound to ShlA after heating and SDS–PAGE, it was not covalently linked to ShlA as PE could be removed by thin-layer chromatography with organic solvents. The number of PE molecules associated per molecule of ShlA was 3.9 ± 2.2. Active ShlA was inactivated by treatment with phospholipase A₂, which indicated that PE is also required for ShlA activity. ShlA-255 (containing the 255 N-terminal amino acids of ShlA) reversibly complemented ShlA* to active ShlA and was inactivated by phospholipase A₂, which demonstrated that PE binds to the N-terminal portion of ShlA; this region has previously been found to be involved in ShlA secretion and activation. Electrospray mass spectroscopy of ShlA-255 determined a molar mass that corresponded to that of unmodified ShlA-255. An E. coli mutant that synthesized only minute amounts of PE did not secrete ShlA but contained residual cell-bound haemolytic activity. Since PE binds strongly to ShlA* in the absence of ShlB without converting ShlA* to haemolytic ShlA, ShlB presumably imposes a conformation on ShlA that brings PE into a position to mediate interaction of the hydrophilic haemolysin with the lipid bilayer of the eukaryotic membrane.     

Influence of growth temperature and lipopolysaccharide on hemolytic activity of Serratia marcescens.

Log-phase cells of Serratia marcescens cultured at 30 degrees C were approximately 10-fold more hemolytic than those grown at 37 degrees C. By using a cloned gene fusion of the promoter-proximal part of the hemolysin gene (shlA) to the Escherichia coli alkaline phosphatase gene (phoA), hemolysin gene expression as a function of alkaline phosphatase activity was measured at 30 and 37 degrees C. No difference in alkaline phosphatase activity was observed as a function of growth temperature, although more hemolysin was detectable immunologically in whole-cell extracts of cells grown at 30 degrees C. The influence of temperature was, however, growth phase dependent, because the hemolytic activities of cells cultured to early log phase at 30 and 37 degrees C were comparable. Given the outer membrane location of the hemolysin, lipopolysaccharide (LPS) was examined as a candidate for mediating the temperature effect on hemolytic activity. Silver staining of LPS in polyacrylamide gels revealed a shift towards shorter O-antigen molecules at 37 degrees C relative to 30 degrees C. Moreover, there was less binding of O-antigen-specific bacteriophage to S. marcescens with increasing growth temperature, a finding consistent with temperature-mediated changes in LPS structure. Smooth strains of S. marcescens were 20- to 30-fold more hemolytic than rough derivatives, a result confirming that changes in LPS structure can influence hemolytic activity. The alkaline phosphatase activity of rough strains harboring the shlA-phoA fusion was threefold lower than that of smooth strains harboring the fusion plasmids, a result consistent with a decrease in hemolysin gene expression in rough strains. The absence of a similar effect of temperature on gene expression may be related to less-marked changes in LPS structure as a function of temperature compared with a smooth-to-rough mutational change.     

Nucleotide sequence of the Serratia marcescens threonine operon and analysis of the threonine operon mutations which alter feedback inhibition of both aspartokinase I and homoserine dehydrogenase I.

The nucleotide sequence of the Serratia marcescens threonine operon (thrA1A2BC) was determined. Three long open reading frames were identified; these open reading frames code for aspartokinase I (AKI)-homoserine dehydrogenase I (HDI), homoserine kinase, and threonine synthase, in that order. The predicted amino acid sequences of these enzymes were similar to the amino acid sequences of the corresponding enzymes in Escherichia coli. The AKI-HDI protein is apparently a tetramer composed of monomer polypeptides that are 819 amino acids long. A deletion analysis revealed that the central and C-terminal region was responsible for threonine-resistant HDI activity, a monomeric fragment extending from the N terminus to residue 306 was responsible for threonine-resistant AKI activity, and an N-terminal portion containing 468 residues was responsible for threonine-sensitive AKI activity. The thrA(1)1A(2)1 and thrA(1)5A(2)5 mutations of threonine-excreting strains HNr21 and TLr156, which result in the loss of threonine-mediated feedback inhibition of both AKI activity and HDI activity, cause single amino acid substitutions (Gly to Asp at position 330 and Ser to Phe at position 352, respectively) in the central region of the AKI-HDI protein. The thrA1+A(2)2 mutation of strain HNr59, which results in a threonine-sensitive AKI and a threonine-resistant HDI, also causes a single amino acid substitution (Ala to Thr at position 479).     

Identification and nucleotide sequence of the gene determining the adhesion capacity of Serratia marcescens.

Three open reading frames, designated smfE, smfF, and smfG, within the mannose-resistant fimbria gene cluster of Serratia marcescens were identified. smfG, which is responsible for determining the receptor binding of S. marcescens, encodes a 280-amino-acid polypeptide with a typical prokaryotic signal sequence.     

Two-step purification of the outer membrane transporter and activator protein ShlB from Escherichia coli using internally His6-tagged constructs

ShlB from Serratia marcescens was isolated and purified from a porin-deficient Escherichia coli BL21 strain using a combination of detergent extraction, affinity and ion-exchange chromatography. An internal histidine affinity tag was introduced that did not interfere with activity. At each stage of the purification scheme biological activity of the ShlB protein was assessed. Using this scheme, several His(6)-tagged mutants of ShlB were purified to electrophoretic homogeneity.     

粘质沙雷氏菌氯霉素抗性基因的克隆及其性质分析

通过构建粘质沙雷氏菌KMR-3菌株的基因组DNA文库, 克隆到了与该菌的氯霉素抗性相关基因, 并对其部分特性进行了初步研究。结果表明: 克隆到的氯霉素抗性基因所编码的蛋白属于PRK10473蛋白, 由397个氨基酸编码, 与变形斑沙雷氏菌(Serratia proteamaculans 568) Bcr/CflA亚家族药物抗性转运蛋白同源性最高, 达到92%, 并对该基因的调控序列(启动子、终止子、SD序列及转录起始位点) 进行了分析。     

A novel cold-adapted phospholipase A(1) from Serratia sp. xjF1: Gene cloning, expression and characterization

The gene encoding a cold-adapted phospholipase A(1) (PLA(1)) from a psychrotrophic, glacier soil bacterium Serratia sp. xjF1 was cloned by two-step PCR (general PCR and TAIL-PCR). The full-length fragment comprised two open reading frames plA and plS. The gene product of plA encoding 320 amino acids with a molecular weight of 33.8kDa was identified as a phospholipase A(1). Its amino acid sequence exhibited the highest homology to PLA(1) of Serratia marcescens (71%). plS encoded a protein of 251 amino acids, which showed no enzymatic activity. The result of plA expression in Escherichia coli indicated that plS might improve the efficient expression of PLA(1) in E. coli. Furthermore, PLA(1) was functionally expressed in Pichia pastoris, yielding 41.8U/mL in a 3.7L fermentor. The purified recombinant phospholipase A(1) (rPLA(1)) had features typical of cold-adapted enzymes with a temperature optimum of 35°C and a maximum activity of 70% at 10°C. The rate of catalysis was optimal at pH 9.0 and the enzyme could be slightly activated by Ca(2+). This is the first report on gene isolation and expression of cold-adapted PLA(1).     

Amino acid replacements in the Serratia marcescens haemolysin ShlA define sites involved in activation and secretion

The haemolysin of Serratia marcescens (ShlA) is translocated through the cytoplasmic membrane by the signal peptide-dependent export apparatus. Translocation across the outer membrane (secretion) is mediated by the ShIB protein. Only the secreted form of ShlA is haemolytic. ShIB also converts in vitro inactive ShlA (ShlA*), synthesized in the absence of ShIB, into the haemolytic form (a process termed activation). To define regions in ShlA involved in both processes, ShlA derivatives were isolated and tested for secretion and activation. Analysis of C-terminally truncated proteins (ShlA) assigned the secretion signal to the amino-terminal 238 residues of ShlA. Trypsin cleavage of a secreted ShlA' derivative yielded a 15kDa N-terminal fragment, by which a haemolytically inactive ShlA* protein could be activated in vitro. It is suggested that the haemolysin activation site is located in this N-terminal fragment. Replacement of asparagine-69 and asparagine-109 by isoleucine yielded inactive haemolysin derivatives. Both asparagine residues are part of two short sequence motifs, reading Ala-Asn-Pro-Asn, which are critical to both activation and secretion. These point mutants as well as N-terminal deletion derivatives which were not activated by ShIB were activated by adding a non-haemolytic N-terminal fragment synthesized in an ShIB⁺ strain (complementation). Apparently the activated N-terminal fragment substituted for the missing activation of the ShlA derivatives and directed them into the erythrocyte membrane, where they formed pores. It is concluded that activation is only required for initiation of pore formation, and that in vivo activation and secretion are tightly coupled processes. Complementation may also indicate that haemolysin oligomers form the pores.     

The hemolytic and cytolytic activities of Serratia marcescens phospholipase A (PhlA) depend on lysophospholipid production by PhlA

Background  

Serratia marcescens is a gram-negative bacterium and often causes nosocomial infections. There have been few studies of the virulence factors of this bacterium. The only S. marcescens hemolytic and cytotoxic factor reported, thus far, is the hemolysin ShlA.     

Escherichia coli hemolysin is released extracellularly without cleavage of a signal peptide.

A 110-kilodalton polypeptide isolated from cell-free culture supernatants of hemolytic Escherichia coli was shown to be associated with hemolytic activity. The relative amount of the extracellular 110-kilodalton species detected directly reflects the extracellular hemolysin activity associated with Escherichia coli strains harboring different hemolysin recombinant plasmids. The predicted molecular mass of the hemolysin structural gene (hlyA) based on DNA sequence analysis was 109,858 daltons. Amino-terminal amino acid sequence analysis of the 110-kilodalton polypeptide provided direct evidence that it was encoded by hlyA. Based on this information, it was also demonstrated that the HlyA polypeptide was released extracellularly without signal peptidase-like cleavage. An examination of hemolysin-specific polypeptides detected by use of recombinant plasmids in a minicell-producing strain of Escherichia coli was performed. These studies demonstrated how hemolysin-associated 110- and 58-kilodalton polypeptides detected in the minicell background could be misinterpreted as a precursor-product relationship.