RESUMO
BACKGROUND: In recent years, the industrial workhorse Corynebacterium glutamicum has gained increasing interest as a host organism for the secretory production of heterologous proteins. Generally, the yield of a target protein in the culture supernatant depends on a multitude of interdependent biological and bioprocess parameters which have to be optimized. So far, the monitoring of such optimization processes depends on the availability of a direct assay for the respective target protein that can be handled also in high throughput approaches. Since simple assays, such as standard enzymatic activity assays, are not always at hand, the availability of a general protein secretion biosensor is highly desirable. RESULTS: High level secretion of proteins via the Sec protein export pathway leads to secretion stress, a phenomenon that is thought to be caused by the accumulation of incompletely or misfolded proteins at the membrane-cell envelope interface. We have analyzed the transcriptional responses of C. glutamicum to the secretory production of two different heterologous proteins and found that, in both cases, the expression of the gene encoding a homologue of the extracytosolic HtrA protease was highly upregulated. Based on this finding, a C. glutamicum Sec secretion biosensor strain was constructed in which the htrA gene on the chromosome was replaced by the eyfp gene. The fluorescence of the resulting reporter strain responded to the secretion of different heterologous proteins (cutinase from Fusarium solani pisi and alkaline phosphatase PhoA from Escherichia coli) in a dose-dependent manner. In addition, three differently efficient signal peptides for the secretory production of the cutinase could be differentiated by the biosensor signal. Furthermore, we have shown that an efficient signal peptide can be separated from a poor signal peptide by using the biosensor signal of the respective cells in fluorescence activated cell sorting experiments. CONCLUSIONS: We have succeeded in the construction of a C. glutamicum biosensor strain that allows for the monitoring of Sec-dependent secretion of heterologous proteins in a dose-dependent manner, independent of a direct assay for the desired target protein.
Assuntos
Proteínas de Bactérias/biossíntese , Técnicas Biossensoriais , Corynebacterium glutamicum/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas de Bactérias/metabolismo , Biotecnologia/métodos , Engenharia Genética , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/metabolismo , Via Secretória , Proteínas de Transporte Vesicular/metabolismoRESUMO
When the cell envelope integrity is compromised, bacteria trigger signaling cascades resulting in the production of proteins that counteract these extracytoplasmic stresses. Here, we show that the two-component system EsrSR regulates a cell envelope stress response in the Actinobacterium Corynebacterium glutamicum. The sensor kinase EsrS possesses an amino-terminal phage shock protein C (PspC) domain, a property that sets EsrSR apart from all other two-component systems characterized so far. An integral membrane protein, EsrI, whose gene is divergently transcribed to the esrSR gene locus and which interestingly also possesses a PspC domain, acts as an inhibitor of EsrSR under non-stress conditions. The resulting EsrISR three-component system is activated among others by antibiotics inhibiting the lipid II cycle, such as bacitracin and vancomycin, and it orchestrates a broad regulon including the esrI-esrSR gene locus itself, genes encoding heat shock proteins, ABC transporters, and several putative membrane-associated or secreted proteins of unknown function. Among those, the ABC transporter encoded by cg3322-3320 was shown to be directly involved in bacitracin resistance of C. glutamicum. Since similar esrI-esrSR loci are present in a large number of actinobacterial genomes, EsrISR represents a novel type of stress-responsive system whose components are highly conserved in the phylum Actinobacteria.
Assuntos
Proteínas de Bactérias/metabolismo , Corynebacterium glutamicum/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Membrana/metabolismo , Antibacterianos/farmacologia , Bacitracina/farmacologia , Sequência de Bases , Sítios de Ligação , Parede Celular/metabolismo , Corynebacterium glutamicum/genética , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , Fator sigma/metabolismo , Estresse Fisiológico , Transcrição Gênica/efeitos dos fármacos , Vancomicina/metabolismoRESUMO
BACKGROUND: Technical bulk enzymes represent a huge market, and the extracellular production of such enzymes is favorable due to lowered cost for product recovery. Protein secretion can be achieved via general secretion (Sec) pathway. Specific sequences, signal peptides (SPs), are necessary to direct the target protein into the translocation machinery. For example, >150 Sec-specific SPs have been identified for Bacillus subtilis alone. As the best SP for a target protein of choice cannot be predicted a priori, screening of homologous SPs has been shown to be a powerful tool for different expression organisms. While SP libraries between closely related species were successfully applied to optimize recombinant protein secretion, this was not investigated for distantly related species. Therefore, in this study a Sec SP library from low-GC firmicutes B. subtilis is investigated to optimize protein secretion in high-GC actinobacterium Corynebacterium glutamicum using cutinase from Fusarium solani pisi as model protein. RESULTS: A homologous SP library (~150 SP) for recombinant cutinase secretion in B. subtilis was successfully transferred to C. glutamicum as alternative secretion host. Cutinase secretion in C. glutamicum was quantified using an automated micro scale cultivation system for online growth monitoring, cell separation and cutinase activity determination. Secretion phenotyping results were correlated to those from a previous study, in which the same SP library was used to optimize secretion of the same cutinase but using B. subtilis as host. Strikingly, behavior of specific SP-cutinase combinations was changed dramatically between B. subtilis and C. glutamicum. Some SPs showed comparable cutinase secretion performances in both hosts, whereas other SPs caused diametrical extracellular cutinase activities. CONCLUSION: The optimal production strain for a specific target protein of choice still cannot be designed in silico. Not only the best SP for a target protein has to be evaluated each time from scratch, the expression host also affects which SP is best. Thus, (heterologous) SP library screening using high-throughput methods is considered to be crucial to construct an optimal production strain for a target protein.
Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Corynebacterium glutamicum/metabolismo , Sinais Direcionadores de Proteínas/fisiologia , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Hidrolases de Éster Carboxílico/genética , Corynebacterium glutamicum/enzimologia , Corynebacterium glutamicum/genética , Biblioteca de Peptídeos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
BACKGROUND: High-throughput methods are widely-used for strain screening effectively resulting in binary information regarding high or low productivity. Nevertheless achieving quantitative and scalable parameters for fast bioprocess development is much more challenging, especially for heterologous protein production. Here, the nature of the foreign protein makes it impossible to predict the, e.g. best expression construct, secretion signal peptide, inductor concentration, induction time, temperature and substrate feed rate in fed-batch operation to name only a few. Therefore, a high number of systematic experiments are necessary to elucidate the best conditions for heterologous expression of each new protein of interest. RESULTS: To increase the throughput in bioprocess development, we used a microtiter plate based cultivation system (Biolector) which was fully integrated into a liquid-handling platform enclosed in laminar airflow housing. This automated cultivation platform was used for optimization of the secretory production of a cutinase from Fusarium solani pisi with Corynebacterium glutamicum. The online monitoring of biomass, dissolved oxygen and pH in each of the microtiter plate wells enables to trigger sampling or dosing events with the pipetting robot used for a reliable selection of best performing cutinase producers. In addition to this, further automated methods like media optimization and induction profiling were developed and validated. All biological and bioprocess parameters were exclusively optimized at microtiter plate scale and showed perfect scalable results to 1 L and 20 L stirred tank bioreactor scale. CONCLUSIONS: The optimization of heterologous protein expression in microbial systems currently requires extensive testing of biological and bioprocess engineering parameters. This can be efficiently boosted by using a microtiter plate cultivation setup embedded into a liquid-handling system, providing more throughput by parallelization and automation. Due to improved statistics by replicate cultivations, automated downstream analysis, and scalable process information, this setup has superior performance compared to standard microtiter plate cultivation.
Assuntos
Reatores Biológicos/microbiologia , Automação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biomassa , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Corynebacterium glutamicum/crescimento & desenvolvimento , Corynebacterium glutamicum/metabolismo , Fusarium/enzimologia , Concentração de Íons de Hidrogênio , Oxigênio/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
Corynebacterium glutamicum is the major host for the industrial production of amino acids and has become one of the best studied model organisms in microbial biotechnology. Rational strain construction has led to an improvement of producer strains and to a variety of novel producer strains with a broad substrate and product spectrum. A key factor for the success of these approaches is detailed knowledge of transcriptional regulation in C. glutamicum. Here, we present a large compendium of 927 manually curated microarray-based transcriptional profiles for wild-type and engineered strains detecting genome-wide expression changes of the 3,047 annotated genes in response to various environmental conditions or in response to genetic modifications. The replicates within the 927 experiments were combined to 304 microarray sets ordered into six categories that were used for differential gene expression analysis. Hierarchical clustering confirmed that no outliers were present in the sets. The compendium provides a valuable resource for future fundamental and applied research with C. glutamicum and contributes to a systemic understanding of this microbial cell factory. Measurement(s) Gene Expression Analysis Technology Type(s) Two Color Microarray Factor Type(s) WT condition A vs. WT condition B ⢠Plasmid-based gene overexpression in parental strain vs. parental strain with empty vector control ⢠Deletion mutant vs. parental strain Sample Characteristic - Organism Corynebacterium glutamicum Sample Characteristic - Environment laboratory environment Sample Characteristic - Location Germany.
Assuntos
Corynebacterium glutamicum , Aminoácidos , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , AlemanhaRESUMO
Invasion of bacteria into nonphagocytic host cells is an important pathogenicity factor for escaping the host defence system. Gram-positive organisms, for example Staphylococcus aureus and Listeria monocytogenes, are invasive in nonphagocytic cells, and this mechanism is discussed as an important part of the infection process. Uropathogenic Escherichia coli and Staphylococcus saprophyticus can cause acute and recurrent urinary tract infections as well as bloodstream infections. Staphylococcus saprophyticus shows strong adhesion to human urinary bladder carcinoma and Hep2 cells and expresses the 'Microbial Surface Components Recognizing Adhesive Matrix molecule' (MSCRAMM)-protein SdrI with collagen-binding activity. MSCRAMMs are responsible for adhesion and collagen binding in S. aureus and are discussed as an important pathogenicity factor for invasion. To investigate internalization in S. aureus, several fluorescence activated cell sorting (FACS) assays have been described recently. We used a previously described FACS assay, with slight modifications, in addition to an antibiotic protection assay and transmission electron microscopy to show that S. saprophyticus ATCC 15305 and the wild-type strain 7108 were internalized into the human urinary bladder carcinoma cell line 5637. The discovery of the internalization of S. saprophyticus may be an important step for understanding the pathogenicity of recurrent infections caused by this organism.
Assuntos
Células Epiteliais/microbiologia , Staphylococcus/patogenicidade , Bexiga Urinária/microbiologia , Antibacterianos/farmacologia , Linhagem Celular Tumoral , Contagem de Colônia Microbiana , Citoplasma/microbiologia , Citometria de Fluxo , Humanos , Viabilidade Microbiana , Microscopia Eletrônica de TransmissãoRESUMO
Staphylococcus saprophyticus, an important cause of urinary tract infections, produces a surface-associated lipase, Ssp. In contrast to other lipases, Ssp is a protein that is present in high amounts on the surface of the bacteria and it was shown that it is a true lipase. Characterization of S. saprophyticus lipase (Ssp) showed that it is more similar to Staphylococcus aureus lipase and Staphylococcus epidermidis lipase than to Staphylococcus hyicus lipase and Staphylococcus simulans lipase. Ssp showed an optimum of lipolytic activity at pH 6 and lost its activity at pH>8 or pH<5. The present results show that Ssp activity is dependent on Ca(2+). Consequently, activity increased c. 10-fold in the presence of 2 mM Ca(2+). Optimal activity was reached at 30 degrees C. It was also observed that the enzymatic activity of Ssp depends strongly on the acyl chain length of the substrate molecule.
Assuntos
Lipase/metabolismo , Staphylococcus/enzimologia , Concentração de Íons de Hidrogênio , Cinética , Lipase/química , Staphylococcus/genética , Staphylococcus/metabolismo , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genética , Staphylococcus epidermidis/enzimologia , Staphylococcus epidermidis/genética , Especificidade por Substrato , TemperaturaRESUMO
Staphylococcus saprophyticus is an important cause of urinary tract infection, and its cell surface hydrophobicity may contribute to virulence by facilitating adherence of the organism to uroepithelia. S. saprophyticus expresses the surface protein SdrI, a member of the serine-aspartate repeat (SD) protein family, which has multifunctional properties. The SdrI knock out mutant has a reduced hydrophobicity index (HPI) of 25%, and expressed in the non-hydrophobic Staphylococcus carnosus strain TM300 causes hydrophobicity. Using hydrophobic interaction chromatography (HIC), we confined the hydrophobic site of SdrI to the N-terminal repeat region. S. saprophyticus strains carrying different plasmid constructs lacking either the N-terminal repeats, both B or SD-repeats were less hydrophobic than wild type and fully complemented SdrI mutant (HPI: 51%). The surface hydrophobicity and HPI of both wild type and the complemented strain were also influenced by calcium (Ca(2+)) and were reduced from 81.3% and 82.4% to 10.9% and 12.3%, respectively. This study confirms that the SdrI protein of S. saprophyticus is a crucial factor for surface hydrophobicity and also gives a first significant functional description of the N-terminal repeats, which in conjunction with the B-repeats form an optimal hydrophobic conformation.
Assuntos
Proteínas de Bactérias/química , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/química , Estrutura Terciária de Proteína , Staphylococcus saprophyticus/química , Proteínas de Bactérias/genética , Expressão Gênica , Técnicas de Inativação de Genes , Teste de Complementação Genética , Proteínas de Membrana/genética , Staphylococcus saprophyticus/genéticaRESUMO
Staphylococcal lipases have been proposed as pathogenicity factors. In Staphylococcus saprophyticus the surface-associated protein (Ssp) has been previously characterized as a cell wall-associated true lipase. A S. saprophyticus Δssp::ermB mutant has been described as less virulent in an in vivo model of urinary tract infection compared with its wild-type. This is the first report showing that S. saprophyticus induced a lifespan reduction in Caenorhabditis elegans similar to that of S. aureus RN4220. In two S. saprophyticus Δssp::ermB mutants lifespan reduction in C. elegans was partly abolished. In order to attribute virulence to the lipase activity itself and distinguish this phenomenon from the presence of the Ssp-protein, the conserved active site of the lipase was modified by site-directed ligase-independent mutagenesis and lipase activity-deficient mutants were constructed. These results indicate that the Ssp is associated with pathogenicity in C. elegans and one could speculate that the lipase activity itself is responsible for this virulence.
Assuntos
Caenorhabditis elegans/microbiologia , Caenorhabditis elegans/fisiologia , Lipase/metabolismo , Proteínas de Membrana/metabolismo , Staphylococcus saprophyticus/enzimologia , Staphylococcus saprophyticus/patogenicidade , Fatores de Virulência/metabolismo , Animais , Domínio Catalítico , Análise Mutacional de DNA , Deleção de Genes , Lipase/genética , Longevidade , Proteínas de Membrana/genética , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Staphylococcus saprophyticus/genética , Virulência , Fatores de Virulência/genéticaRESUMO
BACKGROUND: The main aim of this study was to examine the genotypic and phenotypic diversity of Staphylococcus saprophyticus isolates from human and animal origin. FINDINGS: In total, 236 clinical isolates and 15 animal isolates of S. saprophyticus were characterized in respect of the occurrence of 9 potential virulence genes and four surface properties. All strains were PCR positive for the regulatory genes agr, sar >it>A and rot as well as for the surface proteins UafA and Aas. Nearly 90% of the clinical isolates were found to possess the gene for the surface-associated lipase Ssp and 10% for the collagen binding MSCRAMM SdrI. All animal isolates were negative forsdrI. Lipolytic activity could be detected in 66% of the clinical and 46% of the animal isolates. Adherence to collagen type I was shown of 20% of the clinical strains and 6% of the strains of animal origin. Most S. saprophyticus strains showed hydrophobic properties and only few could agglutinate sheep erythrocytes. CONCLUSIONS: We described a broad analysis of animal and human S. saprophyticus isolates regarding virulence genes and phenotypic properties such as lipase activity, hydrophobicity, and adherence. While S. saprophyticus strains from animal sources have prerequisites for colonization of the urinary tract like the D-serine-deaminase, out findings suggested that they need to acquire new genes e.g. MSCRAMMS for adherence like sdrI and to modulate their existing properties e.g. increasing the lipase activity or reducing hydrophobicity. These apparently important new genes or properties for virulence have to be further analyzed.
RESUMO
Human urine contains a relatively high concentration of d-serine, which is toxic to several nonuropathogenic bacteria, but can be utilized or detoxified by uropathogenic Escherichia coli (UPEC). The sequenced genome of uropathogenic Staphylococcus saprophyticus contains a gene with homology to the d-serine deaminase gene (dsdA) of UPEC. We found the gene in several clinical isolates of S. saprophyticus; however, the gene was absent in Staphylococcus xylosus and Staphylococcus cohnii, phylogenetically close relatives of S. saprophyticus, and could also not be detected in isolates of Staphylococcus aureus, Staphylococcus epidermidis and 13 other staphylococcal species. In addition, the genomes of other sequenced staphylococci do not harbor homologues of this operon. Interestingly, S. saprophyticus could grow in media supplemented with relatively high concentrations of d-serine, whereas S. aureus, S. epidermidis and other staphylococcal species could not. The association of the dsdA gene with growth in media including d-serine was proved by introducing the gene into S. aureus Newman. Given the fact that UPEC and S. saprophyticus tolerate this compound, d-serine utilization and detoxification may be a general property of uropathogenic bacteria.
Assuntos
Serina/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus saprophyticus/metabolismo , Infecções Urinárias/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Humanos , Hidroliases/genética , Hidroliases/metabolismo , Infecções Estafilocócicas/metabolismo , Staphylococcus saprophyticus/enzimologia , Staphylococcus saprophyticus/genética , Infecções Urinárias/metabolismoRESUMO
Staphylococcus saprophyticus, an important cause of urinary tract infections in young women, expresses the surface protein SdrI, a member of the serine-aspartate repeat (SD) protein family. Here we analyse the fibronectin-binding ability of SdrI, as S. saprophyticus is known to bind fibronectin and there is no known SD protein with this function. This protein does not contain the binding motif typical for fibronectin-binding proteins. Using recombinant fragments of SdrI, we localized the binding domain in the A region and show that SdrI bound to the N-terminal 30-kDa fragment of fibronectin. The fibronectin-binding function was shown in the natural host using an SdrI knockout mutant that showed decreased binding to fibronectin compared with wild-type strain 7108.
Assuntos
Adesinas Bacterianas/metabolismo , Fibronectinas/metabolismo , Staphylococcus/metabolismo , Adesinas Bacterianas/química , Motivos de Aminoácidos , Animais , Sítios de Ligação , Feminino , Fibronectinas/química , Técnicas de Inativação de Genes , Genes Bacterianos , Interações Hospedeiro-Patógeno , Humanos , Ligação Proteica , Coelhos , Proteínas Recombinantes/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus/química , Staphylococcus/genética , Infecções Urinárias/microbiologiaRESUMO
A gene encoding a serine-aspartate repeat protein of Staphylococcus saprophyticus, an important cause of urinary tract infections in young women, has been cloned and sequenced. In contrast to other SD repeat proteins, SdrI carries 21 additional N-terminal repeats with a consensus sequence of (P/A)ATKE(K/E)A(A/V)(T/I)(A/T/S)EE and has the longest SD(AD)(1-5) repetitive region (854 amino acids) described so far. This highly repetitive sequence contains only the amino acids serine, asparagine, and a distinctly greater amount of alanine (37%) than all other known SD repeat proteins (2.3 to 4.4%). In addition, it is a collagen-binding protein of S. saprophyticus and the second example in this organism of a surface protein carrying the LPXTG motif. We constructed an isogenic sdrI knockout mutant that showed decreased binding to immobilized collagen compared with wild-type S. saprophyticus strain 7108. Binding could be reconstituted by complementation. Collagen binding is specifically caused by SdrI, and the recently described UafA protein, the only LPXTG-containing protein in the genome sequence of the type strain, is not involved in this trait. Our experiments suggest that, as in other staphylococci, the presence of different LPXTG-anchored cell wall proteins is common in S. saprophyticus and support the notion that the presence of matrix-binding surface proteins is common in staphylococci.