Protein export by a gram-negative bacterium: production of aerolysin by Aeromonas hydrophila.

The synthesis and export of aerolysin, an extracellular protein toxin released by the gram-negative bacterium Aeromonas hydrophila, was studied by pulse-labeling with [35S]methionine. The toxin was synthesized as a higher-molecular-weight precursor. This was processed cotranslationally, resulting in the appearance within the cell of the mature protein, which was then exported to the supernatant. Precursor aerolysin accumulated in cells incubated in the presence of carbonyl cyanide m-chlorophenyl hydrazone, a substance which also inhibited the export of mature aerolysin from the cell. The entrapped mature toxin could not be shocked from the cells, although it could be digested by protease applied to shocked cells. The toxin was processed and translocated across the inner membrane of pleiotropic export mutants and accumulated in the periplasm. The results indicate that more than one step is required for the export of the protein and that aerolysin does not cross the inner and outer membranes simultaneously.     

Purification of cloned proaerolysin released by a low protease mutant of Aeromonas salmonicida

The precursor to the hole-forming toxin aerolysin has been purified in high yield from culture supernatants of a mutant of Aeromonas salmonicida containing the cloned structural gene. The mutant strain was generated by Tn5 mutagenesis. It released little or no protease or other extracellular proteins, including phospholipase, suggesting that it is a regulatory mutant. The absence of protease allowed the isolation of protoxin free from contaminating aerolysin. Typically, more than 50 mg of pure proaerolysin was obtained from 2 L of culture supernatant. The purified protein was completely unable to lyse human erythrocytes without prior activation with trypsin.     

Site-directed mutagenesis of a single tryptophan near the middle of the channel-forming toxin aerolysin inhibits its transfer across the outer membrane of Aeromonas salmonicida

The channel-forming protein aerolysin must cross both the inner and outer bacterial membranes during its secretion from Aeromonas hydrophila or from Aeromonas salmonicida containing the cloned structural gene. We examined the fate of three mutant proteins in which Trp-227, near the middle of the amino acid chain, was replaced with glycine, leucine, or phenylalanine by site-directed mutagenesis. All three proteins crossed the inner membrane and entered the periplasm in the same way as wild-type, and in each case the signal sequence was removed correctly. Little or none of the proaerolysin substituted with glycine or leucine was released into the culture supernatant. Instead, significant amounts became associated with the outer membrane. The Phe-227 protoxin was secreted by the bacteria but at a reduced rate. The leucine and phenylalanine mutant proteins were purified and compared with native proaerolysin. They were processed correctly to the mature forms by treatment with trypsin, and like native aerolysin, both were resistant to further proteolysis. In each case, processing was followed by the formation of oligomers similar to those produced by native toxin. The hemolytic activity of the processed Phe-227 mutant was one-quarter that of wild-type toxin whereas Leu-227 aerolysin had less than one-hundredth the wild-type activity. These results are further evidence that aerolysin is secreted in at least two steps. As well, they show that the last step, crossing the outer membrane, can be blocked by an apparently small change in the structure of the protein.     

Production in Escherichia coli and one-step purification of bifunctional hybrid proteins which bind maltose. Export of the Klenow polymerase into the periplasmic space

Two enzymes, the secreted Staphylococcus aureus nuclease A and the Klenow fragment of the cytoplasmic Escherichia coli DNA polymerase I, were fused, at the genetic level, to MalE, the periplasmic maltose-binding protein of E. coli, or to a signal-sequence mutant. The hybrid proteins were synthesized in large amounts by E. coli under control of promoter malEp. The synthesis was repressed with glucose and could be totally switched off in a malT mutant strain. The hybrid between MalE and the nuclease was exported into the periplasmic space. Several criteria demonstrated that a fraction of the hybrid chains with the Klenow polymerase was exported to the periplasm in a signal-sequence-specific manner and ruled out the possibility of a membrane leakage. The hybrid with the Klenow polymerase was not exported and remained in the cytoplasm when carrying a tight signal-sequence mutation in its MalE portion. The hybrid proteins were purified in one step by affinity chromatography on cross-linked amylose. Most of the hybrid chains in the periplasm but only a fraction of those in the other cell compartments had their MalE portion correctly folded. The nuclease and the Klenow polymerase had their full specific activities in the purified hybrids. The potential of MalE as a vector for the production, export and purification of desirable proteins in E. coli is discussed.     

The superoxide dismutase SodA is targeted to the periplasm in a SecA-dependent manner by a novel mechanism

The manganese/iron-type superoxide dismutase (SodA) of Rhizobium leguminosarum bv. viciae 3841 is exported to the periplasm of R. l. bv. viciae and Escherichia coli. However, it does not possess a hydrophobic cleaved N-terminal signal peptide typically present in soluble proteins exported by the Sec-dependent (Sec) pathway or the twin-arginine translocation (TAT) pathway. A tatC mutant of R. l. bv. viciae exported SodA to the periplasm, ruling out export of SodA as a complex with a TAT substrate as a chaperone. The export of SodA was unaffected in a secB mutant of E. coli, but its export from R. l. bv. viciae was inhibited by azide, an inhibitor of SecA ATPase activity. A temperature-sensitive secA mutant of E. coli was strongly reduced for SodA export. The 10 N-terminal amino acid residues of SodA were sufficient to target the reporter protein alkaline phosphatase to the periplasm. Our results demonstrate the export of a protein lacking a classical signal peptide to the periplasm by a SecA-dependent, but SecB-independent targeting mechanism. Export of the R. l. bv. viciae SodA to the periplasm was not limited to the genus Rhizobium, but was also observed in other proteobacteria.     

Expression and properties of an aerolysin--Clostridium septicum alpha toxin hybrid protein

Aerolysin is a bilobal channel-forming toxin secreted by Aeromonas hydrophila. The alpha toxin produced by Clostridium septicum is homologous to the large lobe of aerolysin. However, it does not contain a region corresponding to the small lobe of the Aeromonas toxin, leading us to ask what the function of the small lobe is. We fused the small lobe of aerolysin to alpha toxin, producing a hybrid protein that should structurally resemble aerolysin. Unlike aerolysin, the hybrid was not secreted when expressed in Aeromonas salmonicida. The purified hybrid was activated by proteolytic processing in the same way as both parent proteins and, after activation, it formed oligomers that corresponded to the aerolysin heptamer. Like aerolysin, the hybrid was far more active than alpha toxin against human erythrocytes and mouse T lymphocytes. Both aerolysin and the hybrid bound to human glycophorin, and both were inhibited by preincubation with this erythrocyte glycoprotein, whereas alpha toxin was unaffected. We conclude that aerolysin contains two receptor binding sites, one for glycosyl-phosphatidylinositol-anchored proteins that is located in the large lobe and is also found in alpha toxin, and a second site, located in the small lobe, that binds a surface carbohydrate determinant.     

Cloned aerolysin of Aeromonas hydrophila is exported by a wild-type marine Vibrio strain but remains periplasmic in pleiotropic export mutants.

With a wide host range vector, the structural gene aerA for the hole-forming extracellular protein aerolysin of Aeromonas hydrophila was cloned into the marine Vibrio sp. strain 60 and into three pleiotropic export mutants (epr mutants). The parent strain and all of the mutants were able to express the protein with the aerA promoter in the plasmid. The parent strain exported proaerolysin into the medium, while all of the mutants accumulated the protoxin in their periplasms. Two of the mutants also accumulated protease; however, as we have found earlier with A. hydrophila, the periplasmic form of proaerolysin in the Vibrio sp. must somehow be protected from proteolysis because it was not converted to active toxin until the cells were shocked. Conversion could be prevented by adding o-phenanthroline to the solutions used in shocking. These results show that the export pathway in the marine Vibrio sp. is very similar to the pathway in A. hydrophila.     

Secretion of an Aeromonas hydrophila aerolysin by a mutant strain of Escherichia coli

Abstract Expression of the Aeromonas hydrophila AH2 aerolysin was analysed in 2 mutant derivatives of Escherichia coli 5K that overproduce E. coli haemolysin, encoded by the multicopy plasmid pANN202–312. When plasmid pHPC3–700 carrying the A. hydrophila aerolysin genes was transformed into one of the mutants, Hha-2T, the transformants produced external aerolysin. Neither the parental 5K strain or the other mutant, Hha-1T, showed extracellular aerolysin activity. For strain Hha-2T, the kinetics of external aerolysin production was similar to that previously reported for A. hydrophila AH2. No cell lysis or release of other proteins to the culture medium could be detected for the period of time that strain Hha-2T exported aerolysin into the external medium.     

Cloning, expression and characterization of aerolysin from Aeromonas hydrophila in Escherichia coli

Aerolysin is a toxin (protein in nature) secreted by the strains of Aeromonas spp. and plays an important role in the virulence of Aeromonas strains. It has also found several applications such as for detection of glycosylphosphatidylinositol (GPI)-anchored proteins etc. A. hydrophila is a ubiquitous Gram-negative bacterium which causes frequent harm to the aquaculture. To obtain a significant amount of recombinant aerolysin in the active form, in this study, we expressed the aerolysin in E. coli under the control of T7 RNase promoter. The coding region (AerA-W) of the aerA gene of A. hydrophila XS91-4-1, excluding partial coding region of the signal peptide was cloned into the vector pET32a and then transformed into E. coli b121. After optimizing the expression conditions, the recombinant protein AerA-W was expressed in a soluble form and purified using His.Bind resin affinity chromatography. Recombinant aerolysin showed hemolytic activity in the agar diffusive hemolysis test. Western blot analysis demonstrated good antigenicity of the recombinant protein.     

A signal sequence is not required for protein export in prlA mutants of Escherichia coli.

The prlA/secY gene, which codes for an integral membrane protein component of the Escherichia coli protein export machinery, is the locus of the strongest suppressors of signal sequence mutations. We demonstrate that two exported proteins of E.coli, maltose-binding protein and alkaline phosphatase, each lacking its entire signal sequence, are exported to the periplasm in several prlA mutants. The export efficiency can be substantial; in a strain carrying the prlA4 allele, 30% of signal-sequenceless alkaline phosphatase is exported to the periplasm. Other components of the E.coli export machinery, including SecA, are required for this export. SecB is required for the export of signal-sequenceless alkaline phosphatase even though the normal export of alkaline phosphatase does not require this chaperonin. Our findings indicate that signal sequences confer speed and efficiency upon the export process, but that they are not always essential for export. Entry into the export pathway may involve components that so overlap in function that the absence of a signal sequence can be compensated for, or there may exist one or more means of entry that do not require signal sequences at all.     

A short N-proximal region of prochymosin inhibits the secretion of hybrid proteins from Escherichia coli

A gene encoding bovine prochymosin (PC) was fused to the coding sequence (phoA) for the Escherichia coli alkaline phosphatase (AP) signal peptide and expressed in E. coli under the control of the phoA promoter. Upon induction, an AP-PC fusion protein was produced which was neither processed nor exported into the periplasm. We investigated this lack of secretion by constructing a series of gene fusions in which different regions of the PC gene were inserted between the coding regions of the AP leader and mature protein. Analysis of the cellular location of the proteins encoded by these fusions revealed that a region of PC (between amino acids 6 and 29) prevented processing and secretion of an AP-PC fusion when inserted near to the AP signal peptide. In contrast, when this 'blocking sequence' was inserted elsewhere in AP the hybrid proteins were efficiently processed and translocation was initiated.     

Activation of the hole-forming toxin aerolysin by extracellular processing.

A precursor-product relationship between aerolysin and a protein with a higher molecular weight was observed in culture supernatants of Aeromonas hydrophila. The larger protein was isolated by ammonium sulfate precipitation and ion-exchange and hydroxyapatite chromatography and compared with purified aerolysin. It was at least 250 times less hemolytic than aerolysin. Both proteins had the same amino acid sequence at the amino terminus. Cyanogen bromide fragments obtained from the two were identical except that each protein contained one unique fragment, and the fragment from the larger protein was 2,500 daltons larger than the fragment obtained from aerolysin. Treatment with trypsin or with an extracellular Aeromonas protease resulted in rapid conversion of the larger protein to a form corresponding in molecular weight and activity to aerolysin. The results indicate that aerolysin is exported to the culture supernatant as a protoxin which is later activated by proteolytic removal of a peptide from the C terminus.     

The leucine zipper of Aeromonas salmonicida AbcA is required for the transcriptional activation of the P2 promoter of the surface-layer structural gene, vapA, in Escherichia coli

The Aeromonas salmonicida AbcA protein is involved in the synthesis of the O-polysaccharide side-chains on the lipopolysaccharide and is also capable of enhancing the expression of the structural gene for the A-layer, vapA , when cloned into Escherichia coli . The P2 promoter of the vapA gene of A. salmonicida was cloned into a promoter probe vector and expression in E. coli was monitored. The expression of P2:: lacZ was shown to be increased when abcA was provided in trans . AbcA contains an N-terminal ATP-binding domain as well as a C-terminal leucine zipper domain. Site-directed mutagenesis has been used to show that the ATP-binding domain is required for the synthesis of the O-polysaccharide side-chains, but not for the enhancement of vapA expression. Conversely, the leucine zipper is needed for the increase in vapA expression, but not for O-polysaccharide side-chain synthesis. This indicates that AbcA is a bifunctional protein that can influence the synthesis of the two principle antigenic components of the A. salmonicida cell surface.     

Escherichia coli “TatExpress” strains super‐secrete human growth hormone into the bacterial periplasm by the Tat pathway

Numerous high‐value proteins are secreted into the Escherichia coli periplasm by the General Secretory (Sec) pathway, but Sec‐based production chassis cannot handle many potential target proteins. The Tat pathway offers a promising alternative because it transports fully folded proteins; however, yields have been too low for commercial use. To facilitate Tat export, we have engineered the TatExpress series of super‐secreting strains by introducing the strong inducible bacterial promoter, ptac , upstream of the chromosomal tatABCD operon, to drive its expression in E. coli strains commonly used by industry (e.g., W3110 and BL21). This modification significantly improves the Tat‐dependent secretion of human growth hormone (hGH) into the bacterial periplasm, to the extent that secreted hGH is the dominant periplasmic protein after only 1 hr induction. TatExpress strains accumulate in excess of 30 mg L^?1 periplasmic recombinant hGH, even in shake flask cultures. A second target protein, an scFv, is also shown to be exported at much higher rates in TatExpress strains.

     

Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli

The twin-arginine translocation (Tat) system targets cofactor-containing proteins across the Escherichia coli cytoplasmic membrane via distinct signal peptides bearing a twin-arginine motif. In this study, we have analysed the mechanism and capabilities of the E. coli Tat system using green fluorescent protein (GFP) fused to the twin-arginine signal peptide of TMAO reductase (TorA). Fractionation studies and fluorescence measurements demonstrate that GFP is exported to the periplasm where it is fully active. Export is almost totally blocked in tat deletion mutants, indicating that the observed export in wild-type cells occurs predominantly, if not exclusively, by the Tat pathway. Imaging studies reveal a halo of fluorescence in wild-type cells corresponding to the exported periplasmic form; the GFP is distributed uniformly throughout the cytoplasm in a tat mutant. Because previous work has shown GFP to be incapable of folding in the periplasm, we propose that GFP is exported in a fully folded, active state. These data also show for the first time that heterologous proteins can be exported in an active form by the Tat pathway.     

An approach to the production of soluble protein from a fungal gene encoding an aggregation-prone xylanase in Escherichia coli

The development of new procedures and protocols that allow researchers to obtain recombinant proteins is of fundamental importance in the biotechnology field. A strategy was explored to overcome inclusion-body formation observed when expressing an aggregation-prone fungal xylanase in Escherichia coli. pHsh is an expression plasmid that uses a synthetic heat-shock (Hsh) promoter, in which gene expression is regulated by an alternative sigma factor (σ(32)). A derivative of pHsh was constructed by fusing a signal peptide to xynA2 gene to facilitate export of the recombinant protein to the periplasm. The xylanase was produced in a soluble form. Three factors were essential to achieving such soluble expression of the xylanase: 1) the target gene was under the control of the Hsh promoter, 2) the gene product was exported into the periplasm, and 3) gene expression was induced by a temperature upshift. For the first time we report the expression of periplasmic proteins under the control of an Hsh promoter regulated by σ(32). One unique feature of this approach was that over 200 copies of the Hsh promoter in an E. coli cell significantly increased the concentration of σ(32). The growth inhibition of the recombinant cells corresponded to an increase in the levels of soluble periplasmic protein. Therefore, an alternative protocol was designed to induce gene expression from pHsh-ex to obtain high levels of active soluble enzymes.     

Identification and molecular characterization of two tandemly located flagellin genes from Aeromonas salmonicida A449.

Two tandemly located flagellin genes, flaA and flaB, with 79% nucleotide sequence identity were identified in Aeromonas salmonicida A449. The fla genes are conserved in typical and atypical strains of A. salmonicida, and they display significant divergence at the nucleotide level from the fla genes of the motile species Aeromonas hydrophila and Aeromonas veronii biotype sobria. flaA and flaB encode unprocessed flagellins with predicted Mrs of 32,351 and 32,056, respectively. When cloned under the control of the Ptac promoter, flaB was highly expressed when induced in Escherichia coli DH5alpha, and the FlaB protein was detectable even in the uninduced state. In flaA clones containing intact upstream sequence, FlaA was barely detectable when uninduced and poorly expressed on induction. The A. salmonicida flagellins are antigenically cross-reactive with the A. hydrophila TF7 flagellin(s) and evolutionarily closely related to the flagellins of Pseudomonas aeruginosa and Vibrio anguillarum. Electron microscopy showed that A. salmonicida A449 expresses unsheathed polar flagella at an extremely low frequency under normal laboratory growth conditions, suggesting the presence of a full complement of genes whose products are required to make flagella; e.g., immediately downstream of flaA and flaB are open reading frames encoding FlaG and FlaH homologs.     

渗压震扰法释放Aeromonas sp.2016菌株外周间质中几丁质酶的研究

气单胞菌Aeromonassp．2016菌株能产生多种几丁质酶,其中的胞外酶C可能聚集于细胞外周胞质。为了避免破碎菌体而产生过多的杂蛋白,探索了用渗压震扰法(osmoticshock)来释放这部分酶。主要步骤是:先将菌体悬浮在20％蔗糖-0．03mol／LTris-HCI(pH8．0)高渗透压的溶液中,再快速转移到纯水低渗透压溶液中,产生瞬间渗压震荡,释放细胞外周胞质中的酶。结果表明,通过渗压震扰法释放出的酶纯度最高,比活力达到142．79U／g,比培养液上清液的54．46U／g和菌体破碎样品的14．66U／g分别高1．6倍和8．7倍,可用于纯化目的蛋白。