Homologous Expression of the Lipase and ABC Transporter Gene Cluster, tliDEFA, Enhances Lipase Secretion in Pseudomonas spp.

The ABC transporter TliDEF was found to be an efficient secretory apparatus for extracellular lipase TliA in Pseudomonas fluorescens. For the enhanced secretion of the lipase, we tried to coexpress tliA and tliDEF in various Pseudomonas species. Whereas the coexpression of tliA and tliDEF was required for the lipase secretion in P. fragi, the expression of tliA was sufficient for the lipase secretion in P. fluorescens, P. syringae, and P. putida, indicating the existence of compatible ABC transporter in these species. However, P. fluorescens harboring tliDEFA secreted much more lipase than P. fluorescens harboring only tliA, but the tliDEF was functional only at temperatures below 30°C. The recombinant P. fluorescens overexpressing tliDEFA showed the highest secretion level, 217 U/ml · OD (optical density) (28 μg/ml · OD) of lipase in Luria-Bertani medium under microaerated conditions. With the increase of aeration, the lipase production was decreased and the lipase seemed to be degraded as the cells entered the cell death phase. These results demonstrate that P. fluorescens can be used as a host system for the secretory production of the lipase using the ABC transporter, thus producing lipase in over 14% of the total protein.     

Export of recombinant proteins in Escherichia coli using ABC transporter with an attached lipase ABC transporter recognition domain (LARD)

Background  

ATP binding cassette (ABC) transporter secretes the protein through inner and outer membranes simultaneously in gram negative bacteria. Thermostable lipase (TliA) of Pseudomonas fluorescens SIK W1 is secreted through the ABC transporter. TliA has four glycine-rich repeats (GGXGXD) in its C-terminus, which appear in many ABC transporter-secreted proteins. From a homology model of TliA derived from the structure of P. aeruginosa alkaline protease (AprA), lipase ABC transporter domains (LARDs) were designed for the secretion of fusion proteins.     

Efficient extracellular production of type I secretion pathway-dependent Pseudomonas fluorescens lipase in recombinant Escherichia coli by heterologous ABC protein exporters

Heterologous ABC protein exporters, the apparatus of type I secretion pathway in Gram-negative bacteria, were used for extracellular production of Pseudomonas fluorescens lipase (TliA) in recombinant Escherichia coli. The effect of the expression of different ABC protein exporter gene clusters (P. fluorescens tliDEF, Pseudomonas aeruginosa aprDEF, Erwinia chrysanthemi prtDEF, and Serratia marcescens lipBCD genes) was examined on the secretion of TliA at growth temperatures of 20, 25, 30 and 35 °C. TliA secretion in recombinant E. coli XL10-Gold varied depending upon type of ABC protein exporter and culture temperature. E. coli expressing S. marcescens lipBCD genes showed the highest secretion level of TliA (122.8 U ml^?1) when cultured at 25 °C. Thus, optimized culture conditions for efficient extracellular production of lipase in recombinant E. coli can be designed by changing the type of ABC protein exporter and the growth temperature.     

Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System

The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.     

Streptococcus pneumoniae Serine Protease HtrA,but Not SFP or PrtA,Is a Major Virulence Factor in Pneumonia

Streptococcus (S.) pneumoniae is a common causative pathogen in pneumonia. Serine protease orthologs expressed by a variety of bacteria have been found of importance for virulence. Previous studies have identified two serine proteases in S. pneumoniae, HtrA (high-temperature requirement A) and PrtA (cell wall-associated serine protease A), that contributed to virulence in models of pneumonia and intraperitoneal infection respectively. We here sought to identify additional S. pneumoniae serine proteases and determine their role in virulence. The S. pneumoniae D39 genome contains five putative serine proteases, of which HtrA, Subtilase Family Protein (SFP) and PrtA were selected for insertional mutagenesis because they are predicted to be secreted and surface exposed. Mutant D39 strains lacking serine proteases were constructed by in-frame insertion deletion mutagenesis. Pneumonia was induced by intranasal infection of mice with wild-type or mutant D39. After high dose infection, only D39ΔhtrA showed reduced virulence, as reflected by strongly reduced bacterial loads, diminished dissemination and decreased lung inflammation. D39ΔprtA induced significantly less lung inflammation together with smaller infiltrated lung surface, but without influencing bacterial loads. After low dose infection, D39ΔhtrA again showed strongly reduced bacterial loads; notably, pneumococcal burdens were also modestly lower in lungs after infection with D39Δsfp. These data confirm the important role for HtrA in S. pneumoniae virulence. PrtA contributes to lung damage in high dose pneumonia; it does not however contribute to bacterial outgrowth in pneumococcal pneumonia. SFP may facilitate S. pneumoniae growth after low dose infection.     

Enhanced production of ATP-binding cassette protein exporter-dependent lipase by modifying the growth medium components of Pseudomonas fluorescens

The industrially-important thermostable lipase, TliA, was extracellularly produced in the recombinant Pseudomonas fluorescens by the homologous expression of TliA and its cognate ABC protein exporter, TliDEF. To increase the secretory production of TliA, we optimized the growth temperature and the culture medium of P. fluorescens. The total amount and the specific productivity of lipase was highest at 25 °C of cell growth temperature, although maximal cell growth was observed at 30 °C. Using the culture medium composed of 20 g dextrin l^?1, 40 g Tween 80 l^?1 and 30 g peptone l^?1, TliA was produced at a level of 2,200 U ml^?1 in a flask culture. The TliA production increased about 3.8-fold (8,450 U ml^?1) in batch fermentation using a 2.5 l fermentor, which was about 7.7-fold higher than that of previously reported TliA production.     

High-level production of Serratia proteamaculans metalloprotease using a recombinant ABC protein exporter-mediated secretion system in Pseudomonas fluorescens

Serratia proteamaculans metalloprotease (SPP) was successfully secreted by a heterologous ABC protein exporter, the Pseudomonas fluorescens TliDEF, in recombinant host strains. Escherichia coli and P. fluorescens cells containing the SPP-encoding gene showed the extracellular protease activity only when the TliDEF-encoding gene cluster was coexpressed. Recombinant P. fluorescens produced an approximately 34.8-fold higher amount of extracellular SPP than did E. coli. The use of a more nutrient-rich medium and controlled dissolved oxygen conditions was effective in increasing SPP secretion in P. fluorescens batch fermentation (an 8.7-fold increase from 41.8 U/mL to 365.2 U/mL). Therefore, SPP, which could not be secreted without an ABC protein exporter, was produced in large quantities by applying the heterologous TliDEF exporter in P. fluorescens. The results also suggest that the use of the ABC protein exporter in P. fluorescens could be an efficient production platform for an industrially promising type I secretion pathway-dependent enzyme.     

Tissue-specific position effects on alcohol dehydrogenase expression in Drosophila melanogaster

Summary Erwinia chrysanthemi, a phytopathogenic enterobacterium, secretes three proteases (PrtA, PrtB and PrtC) into the extracellular medium. The gene encoding the 50 kDa protease, prtA, was subcloned from a recombinant cosmid carrying a fragment of the E. chrysanthemi B374 chromosome. prtA was shown to be located immediately 3 to the structural genes for the other two extracellular proteases. The amino acid sequence of PrtA, as predicted from the prtA nucleotide sequence, showed a high level of homology with a family of metalloproteases that are all secreted via a signal peptide-independent pathway, including PrtB and PrtC of E. chrysanthemi B374, PrtC of E. chrysanthemi EC16, PrtSM of Serratia marcescens and AprA of Pseudomonas aeruginosa. PrtA secretion requires the E. chrysanthemi protease secretion factors PrtD, PrtE and PrtF. The secretion signal of PrtA is near to the carboxy-terminal end of the protein, as was previously shown to be the case for PrtB and PrtSM and for Escherichia coli -hemolysin. The C-termini of these four proteins do not show extensive primary sequence homology, but PrtA, PrtB and PrtSM each have a potential amphipathic -helix located close to the C-terminus.     

Genomic and proteomic analyses of the coral pathogen Vibrio coralliilyticus reveal a diverse virulence repertoire

Vibrio coralliilyticus has been implicated as an important pathogen of coral species worldwide. In this study, the nearly complete genome of Vibrio coralliilyticus strain P1 (LMG23696) was sequenced and proteases implicated in virulence of the strain were specifically investigated. The genome sequence of P1 (5 513 256 bp in size) consisted of 5222 coding sequences and 58 RNA genes (53 tRNAs and at least 5 rRNAs). Seventeen metalloprotease and effector (vgrG, hlyA and hcp) genes were identified in the genome and expressed proteases were also detected in the secretome of P1. As the VcpA zinc-metalloprotease has been considered an important virulence factor of V. coralliilyticus, a vcpA deletion mutant was constructed to evaluate the effect of this gene in animal pathogenesis. Both wild-type and mutant (ΔvcpA) strains exhibited similar virulence characteristics that resulted in high mortality in Artemia and Drosophila pathogenicity bioassays and strong photosystem II inactivation of the coral dinoflagellate endosymbiont (Symbiodinium). In contrast, the ΔvcpA mutant demonstrated higher hemolytic activity and secreted 18 proteins not secreted by the wild type. These proteins included four types of metalloproteases, a chitinase, a hemolysin-related protein RbmC, the Hcp protein and 12 hypothetical proteins. Overall, the results of this study indicate that V. coralliilyticus strain P1 has a diverse virulence repertoire that possibly enables this bacterium to be an efficient animal pathogen.     

Synthetic Effect between Envelope Stress and Lack of Outer Membrane Vesicle Production in Escherichia coli

Outer membrane vesicles (OMVs) are composed of outer membrane and periplasmic components and are ubiquitously secreted by Gram-negative bacteria. OMVs can disseminate virulence factors for pathogenic bacteria as well as serve as an envelope stress response. From a transposon mutant screen for OMV phenotypes, it was discovered that an nlpA mutant of Escherichia coli produces fewer OMVs than the wild type, whereas a degP mutant produces higher levels of OMVs. NlpA is an inner-membrane-anchored lipoprotein that has a minor role in methionine import. DegP is a periplasmic chaperone/protease for misfolded envelope proteins that is critical when cells are heat shocked. To reveal how these proteins contribute to OMV production, the mutations were combined and the double mutant analyzed. The ΔnlpA ΔdegP strain displayed a high-temperature growth defect that corresponded to the production of fewer OMVs than produced by the ΔdegP strain. This phenotype also pertained to other undervesiculation mutations in a ΔdegP background. The hypovesiculation phenotype of ΔnlpA in the wild-type strain as well as in the degP deletion strain was found to be a stationary-phase phenomenon. The periplasm of the ΔnlpA ΔdegP strain was determined to contain significantly more protein in stationary phase than the wild type. Additionally, misfolded DegP substrate outer membrane porins were detected in ΔdegP mutant-derived OMVs. These data suggest that an accumulation of envelope proteins resulting from decreased vesiculation was toxic and contributed to the growth defect. We conclude that OMV production contributes to relieve the envelope of accumulated toxic proteins and that NlpA plays an important role in the production of vesicles in stationary phase.     

Modulation of telomere terminal structure by telomerase components in Candida albicans

The telomerase ribonucleoprotein in Candida albicans is presumed to contain at least three Est proteins: CaEst1p, CaEst2p/TERT and CaEst3p. We constructed mutants missing each of the protein subunit of telomerase and analyzed overall telomere dynamics and single-stranded telomere overhangs over the course of many generations. The est1-ΔΔ mutant manifested abrupt telomere loss and recovery, consistent with heightened recombination. Both the est2-ΔΔ and est3-ΔΔ mutant exhibited progressive telomere loss, followed by the gradual emergence of survivors with long telomeres. In no case was telomere loss accompanied by severe growth defects, suggesting that cells with short telomeres can continue to proliferate. Furthermore, the amount of G-strand terminal overhangs was greatly increased in the est2-ΔΔ mutant, but not others. Our results suggest that in addition to their well-characterized function in telomere elongation, both CaEst1p and CaEst2p mediate some aspects of telomere protection in Candida, with the former suppressing excessive recombination, and the latter preventing excessive C-strand degradation.     

Construction of heterologous gene expression cassettes for the development of recombinant <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis>

Gene-expression cassettes for the construction of recombinant Clostridium beijerinckii were developed as potential tools for metabolic engineering of C. beijerinckii. Gene expression cassettes containing ColE1 origin and pAMB origin along with the erythromycin resistance gene were constructed, in which promoters from Escherichia coli, Lactococcus lactis, Ralstonia eutropha, C. acetobutylicum, and C. beijerinckii are examined as potential promoters in C. beijerinckii. Zymogram analysis of the cell extracts and comparison of lipase activities of the recombinant C. beijerinckii strains expressing Pseudomonas fluorescens tliA gene suggested that the tliA gene was functionally expressed by all the examined promoters with different expression level. Also, recombinant C. beijerinckii expressing C. beijerinckii secondary alcohol dehydrogenase by the constructed expression cassettes successfully produced 2-propanol from glucose. The best promoter for TliA expression was the R. eutropha phaP promoter while that for 2-propanol production was the putative C. beijerinckii pta promoter. Gene expression cassettes developed in this study may be useful tools for the construction of recombinant C. beijerinckii strains as host strains for the valuable chemicals and fuels from renewable resources.     

Yeast Pah1p Phosphatidate Phosphatase Is Regulated by Proteasome-mediated Degradation

Yeast PAH1-encoded phosphatidate phosphatase is the enzyme responsible for the production of the diacylglycerol used for the synthesis of triacylglycerol that accumulates in the stationary phase of growth. Paradoxically, the growth phase-mediated inductions of PAH1 and phosphatidate phosphatase activity do not correlate with the amount of Pah1p; enzyme abundance declined in a growth phase-dependent manner. Pah1p from exponential phase cells was a relatively stable protein, and its abundance was not affected by incubation with an extract from stationary phase cells. Recombinant Pah1p was degraded upon incubation with the 100,000 × g pellet fraction of stationary phase cells, although the enzyme was stable when incubated with the same fraction of exponential phase cells. MG132, an inhibitor of proteasome function, prevented degradation of the recombinant enzyme. Endogenously expressed and plasmid-mediated overexpressed levels of Pah1p were more abundant in the stationary phase of cells treated with MG132. Pah1p was stabilized in mutants with impaired proteasome (rpn4Δ, blm10Δ, ump1Δ, and pre1 pre2) and ubiquitination (hrd1Δ, ubc4Δ, ubc7Δ, ubc8Δ, and doa4Δ) functions. The pre1 pre2 mutations that eliminate nearly all chymotrypsin-like activity of the 20 S proteasome had the greatest stabilizing effect on enzyme levels. Taken together, these results supported the conclusion that Pah1p is subject to proteasome-mediated degradation in the stationary phase. That Pah1p abundance was stabilized in pah1Δ mutant cells expressing catalytically inactive forms of Pah1p and dgk1Δ mutant cells with induced expression of DGK1-encoded diacylglycerol kinase indicated that alteration in phosphatidate and/or diacylglycerol levels might be the signal that triggers Pah1p degradation.     

Identification and Functional Characterization of Glycosylation of Recombinant Human Platelet-Derived Growth Factor-BB in Pichia pastoris

Yeast Pichia pastoris is a widely used system for heterologous protein expression. However, post-translational modifications, especially glycosylation, usually impede pharmaceutical application of recombinant proteins because of unexpected alterations in protein structure and function. The aim of this study was to identify glycosylation sites on recombinant human platelet-derived growth factor-BB (rhPDGF-BB) secreted by P. pastoris, and investigate possible effects of O-linked glycans on PDGF-BB functional activity. PDGF-BB secreted by P. pastoris is very heterogeneous and contains multiple isoforms. We demonstrated that PDGF-BB was O-glycosylated during the secretion process and detected putative O-glycosylation sites using glycosylation staining and immunoblotting. By site-directed mutagenesis and high-resolution LC/MS analysis, we, for the first time, identified two threonine residues at the C-terminus as the major O-glycosylation sites on rhPDGF-BB produced in P. pastoris. Although O-glycosylation resulted in heterogeneous protein expression, the removal of glycosylation sites did not affect rhPDGF-BB mitogenic activity. In addition, the unglycosylated PDGF-BB^ΔGly mutant exhibited the immunogenicity comparable to that of the wild-type form. Furthermore, antiserum against PDGF-BB^ΔGly also recognized glycosylated PDGF-BB, indicating that protein immunogenicity was unaltered by glycosylation. These findings elucidate the effect of glycosylation on PDGF-BB structure and biological activity, and can potentially contribute to the design and production of homogeneously expressed unglycosylated or human-type glycosylated PDGF-BB in P. pastoris for pharmaceutical applications.     

The Expression of Superoxide Dismutase (SOD) and a Putative ABC Transporter Permease Is Inversely Correlated during Biofilm Formation in Listeria monocytogenes 4b G

Little is known about the molecular basis of biofilm formation in Listeria monocytogenes. The superoxide dismutase (SOD) of the deletion mutant of lm.G_1771 gene, which encodes for a putative ABC transporter permease, is highly expressed in biofilm. In this study, the sod gene deletion mutant Δsod, and double deletion mutant of the sod and lm. G_1771 genes Δ1771Δsod were used to investigate the role of SOD and its relationship to the expression of the putative ABC transporter permease in biofilm formation. Our results showed that the ability to form a biofilm was significantly reduced in the Δsod mutant and the Δ1771Δsod double mutant. Both Δsod and Δ1771Δsod mutants exhibited slow growth phenotypes and produced more reactive oxygen species (ROS). The growth was inhibited in the mutants by methyl viologen (MV, internal oxygen radical generator) treatment. In addition, the expression of one oxidation resistance gene (kat), two stress regulators encoding genes (perR and sigB), and one DNA repair gene (recA) were analyzed in both the wild-type L. monocytogenes 4b G and the deletion mutants by RT-qPCR. The expression levels of the four genes were increased in the deletion mutants when biofilms were formed. Taken together, our data indicated that SOD played an important role in biofilm formation through coping with the oxidant burden in deficient antioxidant defenses.     

Sap6, a secreted aspartyl proteinase,participates in maintenance the cell surface integrity of Candida albicans

Background

The polymorphic species Candida albicans is the major cause of candidiasis in humans. The secreted aspartyl proteinases (Saps) of C. albicans, encoded by a family of 10 SAP genes, have been investigated as the virulent factors during candidiasis. However, the biological functions of most Sap proteins are still uncertain. In this study, we applied co-culture system of C. albicans and THP-1 human monocytes to explore the pathogenic roles and biological functions of Sap proteinases.

Results

After 1 hr of co-culture of C. albicans strains and THP-1 human monocytes at 37°C, more than 60% of the THP-1-engulfed wild type and Δsap5 Candida cells were developing long hyphae. However, about 50% of THP-1-engulfed Δsap6 Candida cells were generating short hyphae, and more dead Candida cells were found in Δsap6 strain that was ingested by THP-1 cells (about 15% in Δsap6 strain vs. 2 ~ 2.5% in SC5314 and Δsap5 strains). The immunofluorescence staining demonstrated that the Sap6 is the major hyphal tip located Sap protein under THP-1 phagocytosis. The sap6-deleted strains (Δsap6, Δsap4/6, and Δsap5/6) appeared slower growth on Congo red containing solid medium at 25°C, and the growth defect was exacerbated when cultured at 37°C in Congo red or SDS containing medium. In addition, more proteins were secreted from Δsap6 strain and the β-mercaptoethanol (β-ME) extractable surface proteins from Δsap6 mutant were more abundant than that of extracted from wild type strain, which included the plasma membrane protein (Pma1p), the ER-chaperone protein (Kar2p), the protein transport-related protein (Arf1p), the cytoskeleton protein (Act1), and the mitochondrial outer membrane protein (porin 1). Moreover, the cell surface accessibility was increased in sap6-deleted strains.

Conclusion

From these results, we speculated that the cell surface constitution of C. albicans Δsap6 strain was defect. This may cause the more accessible of β-ME to disulfide-bridged cell surface components and may weaken the resistance of Δsap6 strain encountering phagocytosis of THP-1 cells. Sap6 protein displays a significant function involving in maintenance the cell surface integrity.     

Rhomboids of Mycobacteria: Characterization Using an aarA Mutant of Providencia stuartii and Gene Deletion in Mycobacterium smegmatis

Background

Rhomboids are ubiquitous proteins with unknown roles in mycobacteria. However, bioinformatics suggested putative roles in DNA replication pathways and metabolite transport. Here, mycobacterial rhomboid-encoding genes were characterized; first, using the Providencia stuartii null-rhomboid mutant and then deleted from Mycobacterium smegmatis for additional insight in mycobacteria.

Methodology/Principal Findings

Using in silico analysis we identified in M. tuberculosis genome the genes encoding two putative rhomboid proteins; Rv0110 (referred to as “rhomboid protease 1”) and Rv1337 (“rhomboid protease 2”). Genes encoding orthologs of these proteins are widely represented in all mycobacterial species. When transformed into P. stuartii null-rhomboid mutant (ΔaarA), genes encoding mycobacterial orthologs of “rhomboid protease 2” fully restored AarA activity (AarA is the rhomboid protein of P. stuartii). However, most genes encoding mycobacterial “rhomboid protease 1” orthologs did not. Furthermore, upon gene deletion in M. smegmatis, the ΔMSMEG_4904 single mutant (which lost the gene encoding MSMEG_4904, orthologous to Rv1337, “rhomboid protease 2”) formed the least biofilms and was also more susceptible to ciprofloxacin and novobiocin, antimicrobials that inhibit DNA gyrase. However, the ΔMSMEG_5036 single mutant (which lost the gene encoding MSMEG_5036, orthologous to Rv0110, “rhomboid protease 1”) was not as susceptible. Surprisingly, the double rhomboid mutant ΔMSMEG_4904–ΔMSMEG_5036 (which lost genes encoding both homologs) was also not as susceptible suggesting compensatory effects following deletion of both rhomboid-encoding genes. Indeed, transforming the double mutant with a plasmid encoding MSMEG_5036 produced phenotypes of the ΔMSMEG_4904 single mutant (i.e. susceptibility to ciprofloxacin and novobiocin).

Conclusions/Significance

Mycobacterial rhomboid-encoding genes exhibit differences in complementing aarA whereby it''s only genes encoding “rhomboid protease 2” orthologs that fully restore AarA activity. Additionally, gene deletion data suggests inhibition of DNA gyrase by MSMEG_4904; however, the ameliorated effect in the double mutant suggests occurrence of compensatory mechanisms following deletion of genes encoding both rhomboids.