Improving protein secretion by engineering components of the bacterial translocation machinery.

The increased insight into the mechanism of bacterial protein translocation has resulted in new concepts for the production of heterologous proteins. The periplasm of gram-negative bacteria is revealed to have a role as a 'protein construction compartment', which can be used to fold complex proteins. Passage across the outer membrane, however, remains a challenge due to the high selectivity of the outer membrane translocase. In gram-positive bacteria, slow folding at the membrane-cell-wall interface can make heterologous proteins vulnerable to degradation by wall-associated proteases. The recent identification of thiol-disulfide oxidoreductases in Bacillus subtilis might open the possibility of secreting proteins containing multiple disulfide bonds from this host.

Molecular organization of the xcp gene cluster in Pseudomonas putida: absence of an xcpX (gspK) homologue.

A DNA fragment containing xcp (gsp) gene homologues, required for extracellular protein secretion by the general secretory pathway (GSP) in various Gram-negative bacteria, was cloned from Pseudomonas putida (Pp) strain WCS358 and sequenced. The results presented here and those previously reported (de Groot, A., Krijger, J.-J., Filloux, A., Tommassen, J., 1996. Characterization of type II protein secretion (xcp) genes in the plant growth-stimulating Pseudomonas putida, strain WCS358 Mol. Gen. Genet. 250, 491-504) complete the sequence of the xcp gene cluster of Pp. Unlike that of Pseudomonas aeruginosa (Pa), the xcp gene cluster of Pp contains a gspN homologue. More surprisingly, in contrast to all known gsp gene clusters, the xcpX (gspK) homologue is not found. In addition, genes flanking the xcp cluster of Pp are not related to those flanking the xcp genes of Pa. Overall, the xcp gene products of Pp are as much related to those of Pa as to gsp gene products of enterobacterial species, suggesting that the xcp clusters of Pp and Pa have evolved separately.

The phenotype enhancement method identifies the Xcp outer membrane secretion machinery from Pseudomonas alcaligenes as a bottleneck for lipase production.

Pseudomonas alcaligenes M-1 has been selected from an intensive screening for micro-organisms that can naturally produce a lipase active in detergent formulations. The lipase expression has been increased to allow high level secretion from Pseudomonas alcaligenes, via the introduction of multi-copy plasmids. In order to improve the lipase yield further, the phenotype enhancement method has been developed. This idea comprises the reintroduction of a cosmid library with random chromosomal fragments in a P. alcaligenes strain with already high lipase productivity. One of the strains which showed an enhanced lipase production appeared to contain a cosmid encoding the outer membrane secretion genes. These xcp-genes are clustered in two divergently transcribed operons similar to the situation in Pseudomonas aeruginosa. Remarkably and dissimilar to P. aeruginosa, in between the two xcp gene clusters, two reading frames of unknown function--OrfV and OrfX--are present. For OrfX no equivalent can be found in the known protein data bases. On the other hand, OrfV shows homology to the regulatory proteins MalT and AcoK. Some evidence is provided that suggests that OrfV acts as a regulator of the xcp operons. A model is proposed for the regulation of the secretion system from P. alcaligenes.

Characterization of the promoter and upstream activating sequence from the Pseudomonas alcaligenes lipase gene.

Pseudomonas alcaligenes secretes a lipase with a high pH optimum, which has interesting properties for application in detergents. The expression of the lipase is strongly dependent on the presence of lipids in the growth medium such as soybean oil. The promoter of the gene was characterized and found to have resemblance to sigma54 controlled promoters, which are known to be tightly regulated. The transcription start was mapped precisely downstream of a sequence with close similarity to the -12/-24 consensus sequence of sigma54 controlled promoters. Interestingly, a hyperproducer mutant strain was isolated and found to have a C to T mutation in the -12/-24 promoter consensus region. In addition an Upstream Activating Sequence (UAS) with homology to sigma54 UAS consensus sequences was identified. It was demonstrated that an increase of the distance from the UAS to the transcription start or the deletion of the UAS results in significantly lower expression levels of lipase. A systematic mutational analysis of the UAS sequence has resulted in a variant with an increased lipase expression.

Development of a lipase fermentation process that uses a recombinant Pseudomonas alcaligenes strain.

Pseudomonas alcaligenes M-1 secretes an alkaline lipase, which has excellent characteristics for the removal of fatty stains under modern washing conditions. A fed-batch fermentation process based on the secretion of the alkaline lipase from P. alcaligenes was developed. Due to the inability of P. alcaligenes to grow on glucose, citric acid and soybean oil were applied as substrates in the batch phase and feed phase, respectively. The gene encoding the high-alkaline lipase from P. alcaligenes was isolated and characterized. Amplification of lipase gene copies in P. alcaligenes with the aid of low- and high-copy-number plasmids resulted in an increase of lipase expression that was apparently colinear with the gene copy number. It was found that overexpression of the lipase helper gene, lipB, produced a stimulating effect in strains with high copy numbers (> 20) of the lipase structural gene, lipA. In strains with lipA on a low-copy-number vector, the lipB gene did not show any effect, suggesting that LipB is required in a low ratio to LipA only. During scaling up of the fermentation process to 100 m3, severe losses in lipase productivity were observed. Simulations have identified an increased level of dissolved carbon dioxide as the most probable cause for the scale-up losses. A large-scale fermentation protocol with a reduced dissolved carbon dioxide concentration resulted in a substantial elimination of the scale-up loss.

Cloning, characterization, and multiple chromosomal integration of a Bacillus alkaline protease gene.

Extracellular Bacillus proteases are used as additives in detergent powders. We identified a Bacillus strain that produces a protease with an extremely alkaline pH optimum; this protease is suitable for use in modern alkaline detergent powders. The alkalophilic strain Bacillus alcalophilus PB92 gene encoding this high-alkaline serine protease was cloned and characterized. Sequence analysis revealed an open reading frame of 380 amino acids composed of a signal peptide (27 amino acids), a prosequence (84 amino acids), and a mature protein of 269 amino acids. Amino acid comparison with other serine proteases shows good homology with protease YaB, which is also produced by an alkalophilic Bacillus strain. Both show moderate homology with subtilisins but show some remarkable differences from subtilisins produced by neutrophilic bacilli. The prosequence of PB92 protease has no significant homology with prosequences of subtilisins. The abundance of negatively charged residues in the prosequences of PB92 protease is especially remarkable. The cloned gene was used to increase the production level of the protease. For this purpose the strategy of gene amplification in the original alkalophilic Bacillus strain was chosen. When introduced on a multicopy plasmid, the recombinant strain was unstable; under production conditions, plasmid segregation occurred. More stable ways of gene amplification were obtained by chromosomal integration. This was achieved by (i) homologous recombination, resulting in a strain with two tandemly arranged genes, and (ii) illegitimate recombination, resulting in a strain with a second copy of the protease gene on a locus not adjacent to the originally present gene. Both strains showed increased production and were more stable than the plasmid-containing strain. Absolute stability was only found when nontandem duplication occurred. This method of gene amplification circumvents stability problems often encountered in gene amplification in Bacillus species when plasmids or tandemly arranged genes in the chromosome are used.

Exchange of Xcp (Gsp) secretion machineries between Pseudomonas aeruginosa and Pseudomonas alcaligenes: species specificity unrelated to substrate recognition.

Pseudomonas aeruginosa and Pseudomonas alcaligenes are gram-negative bacteria that secrete proteins using the type II or general secretory pathway, which requires at least 12 xcp gene products (XcpA and XcpP to -Z). Despite strong conservation of this secretion pathway, gram-negative bacteria usually cannot secrete exoproteins from other species. Based on results obtained with Erwinia, it has been proposed that the XcpP and/or XcpQ homologs determine this secretion specificity (M. Linderberg, G. P. Salmond, and A. Collmer, Mol. Microbiol. 20:175-190, 1996). In the present study, we report that XcpP and XcpQ of P. alcaligenes could not substitute for their respective P. aeruginosa counterparts. However, these complementation failures could not be correlated to species-specific recognition of exoproteins, since these bacteria could secrete exoproteins of each other. Moreover, when P. alcaligenes xcpP and xcpQ were expressed simultaneously in a P. aeruginosa xcpPQ deletion mutant, complementation was observed, albeit only on agar plates and not in liquid cultures. After growth in liquid culture the heat-stable P. alcaligenes XcpQ multimers were not detected, whereas monomers were clearly visible. Together, our results indicate that the assembly of a functional Xcp machinery requires species-specific interactions between XcpP and XcpQ and between XcpP or XcpQ and another, as yet uncharacterized component(s).

The structure-function relationship of the lipases from Pseudomonas aeruginosa and Bacillus subtilis.

Within the BRIDGE T-project on lipases we investigate the structure-function relationships of the lipases from Bacillus subtilis and Pseudomonas aeruginosa. Construction of an overproducing Bacillus strain allowed the purification of > 100 mg lipase from 30 l culture supernatant. After testing a large variety of crystallization conditions, the Bacillus lipase gave crystals of reasonable quality in PEG-4000 (38-45%), Na2SO4 and octyl-beta-glucoside at 22 degrees C, pH 9.0. A 2.5 A dataset has been obtained which is complete from 15 to 2.5 A resolution. P.aeruginosa wild-type strain PAC1R was fermented using conditions of maximum lipase production. More than 90% of the lipase was cell bound and could be solubilized by treatment of the cells with Triton X-100. This permitted the purification of approximately 50 mg lipase. So far, no crystals of sufficient quality were obtained. Comparison of the model we built for the Pseudomonas lipase, on the basis of sequences and structures of various hydrolases which were found to possess a common folding pattern (alpha/beta hydrolase fold), with the X-ray structure of the P.glumae lipase revealed that it is possible to correctly build the structure of the core of a protein even in the absence of obvious sequence homology with a protein of known 3-D structure.