Extracellular Protease Secretion Research Articles

DUF1127-containing protein and ProQ had opposite effects on biofilm formation in Vibrio alginolyticus

The RNA binding protein is crucial for gene regulation at the post transcription level. In this study, functions of the DUF1127-containing protein and ProQ, which are RNA-binding proteins, were revealed in Vibrio alginolyticus. DUF1127 deletion increased the ability of biofilm formation, whereas ProQ deletion reduced the amount of biofilm. Moreover, extracellular proteinase secretion was significantly reduced in the DUF1127 deletion strain. ProQ, not DUF1127-containing protein, can help the cell to defense oxidative stress. Deletion of DUF1127 resulted in a higher ROS level in the cell, however, ProQ deletion showed no difference. RNA-seq unveiled the expression of genes involved in extracellular protease secretion were significantly downregulated and biofilm synthesis-related genes, such as rbsB and alsS, were differentially expressed in the DUF1127 deletion strain. ProQ affected the expression of genes involved in biofilm synthesis (flgC and flgE), virulence (betB and hutG), and oxidative stress. Moreover, the DUF1127-containing and ProQ affected the mRNA levels of various regulators, such as LysR and BetI. Overall, our study revealed that the DUF1127-containing protein and ProQ have crucial functions on biofilm formation in V. alginolyticus.

Bacillus spp. Isolated from Miang as Potential Probiotics in Nile Tilapia Culture-In Vitro Research.

Among 79 Bacillus spp. isolated from Miang, a fermented tea in north Thailand, 17 Bacillus strains were selected with probiotic potential in Nile tilapia culture based on the capabilities of bacteriocin production and associated antimicrobial activities against fish pathogens, Aeromonas hydrophila and Streptococcus agalactiae. However, only six isolates were selected for further extensive studies based on the strength of their antimicrobial activities and their tolerance against simulated gastrointestinal conditions. The molecular identification by 16S rRNA gene sequence analysis revealed that five isolates, K2.1, K6.1, K7.1, K15.4, and K22.6, were Bacillus tequilensis, and the isolate K29.2 was Bacillus siamensis. B. siamensis K29.2 showed complete susceptibility to antibiotics tested in this study, while B. tequilensis K 15.4 showed moderate resistance to some antibiotics; therefore, both strains were selected as potential probiotic bacteria. B. tequilensis K15.4 and B. siamensis K29.2 were capable of the production and secretion of extracellular protease and polysaccharide degrading enzymes, including cellulase, xylanase, and β-mannanase. The tannin tolerant test also demonstrated their ability to grow on selective agar plates and secrete cellulase and β-mannanase in the presence of hydrolyzable tannin. In addition, in vitro digestion of commercial fish substrate revealed that the extracellular enzymes produced by both strains efficiently reacted with feed protein and polysaccharides. Based on the results from this study, B. siamensis K29.2 was deemed to have the highest potential multifunctional probiotic qualities for application in Nile tilapia culture, while the antibiotic-resistant gene in B. tequilensis K15.4 must be clarified before field application.

Antagonistic and Plant Growth-Promoting Properties of Streptomyces F2 Isolated from Vineyard Soil

Streptomyces can produce secondary metabolites with a wide range of activities and is often used in agriculture as a biocontrol strain to control soil-borne diseases. Screening and isolation from infected soil is an effective method to obtain active strains. In this study, the best antagonistic inter-root growth-promoting bacteria were isolated from grapevine inter-root soil samples, and strain F2 was identified as Streptomyces sp. based on morphological, physiological, and biochemical characteristics as well as 16S rDNA sequencing results. The results showed that the fermentation broth/liquid and sterile filtrate of strain F2 exhibited antagonistic effects against 10 plant pathogens, with an inhibition rate reaching up to 80%. Notably, two of them exhibited remarkable inhibitory effects against Phytophthora capsici with inhibition rates of 80.58% and 87.71%, respectively. The P. capsici leaf control experiment revealed that the control effect of strain F2 fermentation liquid on P. capsici filaments was 61.09%. Furthermore, indoor pot experiments demonstrated that the fermentation liquid of strain F2 had a significant inhibitory effect on pepper blight, with a maximum inhibition of 83.31%. Antagonistic factor analysis indicated that strain F2 had specific organophosphorus hydrolysis, nitrogen fixation, extracellular protease secretion, and IAA production capabilities. Additionally, root treatment with strain F2’s fermentation liquid significantly enhanced capsicum growth. Taking together, Streptomyces F2 not only exhibits a wide-spectrum antagonistic effect against plant pathogens but also promotes plant growth, which suggests that Streptomyces F2 can be used as an effective biological control resource and provides important theoretical support for the application of Streptomyces F2.

Role of sodium pyruvate in maintaining the survival and cytotoxicity of Staphylococcus aureus under high glucose conditions.

Glucose is a crucial carbon source for the growth of Staphylococcus aureus, but an excess of glucose is detrimental and even leads to cell death. Pyruvate, the central metabolite of glycolysis, has been shown to have anti-inflammatory and antioxidant properties. This study aimed to investigate the protective effect of pyruvate on S. aureus under high glucose conditions. Sodium pyruvate greatly increased the cytotoxicity of S. aureus strain BAA-1717 to human erythrocytes and neutrophils in vitro. However, the cytotoxicity and survival of S. aureus were significantly reduced by high glucose, which was restored to normal levels by the addition of sodium pyruvate. The expression of hlg and lukS in S. aureus was higher in the LB-GP cultures than that in LB-G cultures, but there was no significant difference in cytotoxicity between LB-GP and LB-G cultures. Furthermore, the hemolytic activity of S. aureus supernatants could be inhibited by the cell-free culture medium (CFCM) of LB-G cultures, suggesting that high levels of extracellular proteases were presence in the CFCM of LB-G cultures, resulting in degradation of the hemolytic factors. The expression of sarA, which negatively regulates extracellular protease secretion, was higher in LB-GP cultures than that in LB-G cultures. Additionally, sodium pyruvate increased acetate production in S. aureus, which helps maintain cell viability under acidic environment. In conclusion, pyruvate plays an important role in the survival and cytotoxicity of S. aureus under high glucose conditions. This finding may aid in the development of effective treatments for diabetic foot infections.

Effect and regulation of fatty acids on bacillomycin D synthesis.

Bacillomycin D is a cyclic antimicrobial lipopeptide that has excellent antifungal effects, but its application is limited due to its low yield. At present, it is not clear whether fatty acids regulate the synthesis of bacillomycin D. Therefore, the effects of nine fatty acids on the yield of bacillomycin D produced by Bacillus amyloliquefaciens fmbJ were studied. The results showed that sodium propionate, propionic acid, and butyric acid could increase the yield of bacillomycin D by 44, 40, and 10%, respectively. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression levels of bacillomycin D synthesis gene, signaling factors and genes related to fatty acid metabolism, so as to explore the mechanism of sodium propionate regulating bacillomycin D synthesis. In conclusion, sodium propionate could accelerate the tricarboxylic acid cycle and promoted spore formation, cell movement, the secretion of extracellular protease and the transcription of bacillomycin D synthesis gene by upregulating the expression of signal factors degU, degQ, sigH, sigM and spo0A and ultimately promoted the synthesis of bacillomycin D. In this study, the mechanism of sodium propionate increasing bacillomycin D production was explored from multiple perspectives, which provided theoretical support for the large-scale production of bacillomycin D and was expected to promote its wide application in food, agriculture and medicine fields.

The global role of Lrp in Vibrio alginolyticus and its response to diverse physicochemical factors

Leucine-responsive regulatory protein (Lrp) is an essential transcriptional regulator in prokaryotes. However, the regulatory role of lrp in Vibrio alginolyticus has still not been studied in detail. In this study, an lrp mutant strain was constructed to gain insight into the role of lrp in Vibrio alginolyticus. The absence of lrp significantly enhances swarming motility, biofilm formation, extracellular protease secretion activity, and tolerance to copper ions. The cumulative mortality of zebrafish (Danio rerio) challenged by intraperitoneal injection against the lrp mutant strain reached 68.89%, significantly higher than the 40.00% suffered by fish injected with the wild-type strain. The expression levels of lrp decreased gradually with increasing culture time under the influence of various physicochemical factors. The expression level of lrp was significantly increased after two hours of culture at pH 5, 22°C, 5% NaCl, the presence of 1 mM Cu2+, 1/4/7 mM ferric citrate, 0.1 mg/L NaNO3, and 0.1 mg/L KH2PO4. The mRNA level of lrp decreased significantly after six hours of culture at 37°C, 1% and 5% NaCl, and the presence of 1/7 mM ferric citrate, 0.1/5 mg/L NaNO3, and 0.1/0.5/2.0 mg/L KH2PO4. The expression of lrp increased after ten hours of culture at pH 5/9, 22°C, 1% NaCl, and the presence of 1 mM Cu2+, 7 mM ferric citrate, 5 mg/L NaNO3, and 0.1 mg/L KH2PO4. Overall, this study indicates that lrp negatively controls the virulence of V. alginolyticus, probably by reducing its swarming motility, biofilm formation, extracellular protease secretion activity, and tolerance to copper ions, and that the expression of lrp is affected by numerous physicochemical factors, and is especially up-regulated after 2 hours of culture in bacterial growth.

ToxR modulates biofilm formation in fish pathogen Vibrio harveyi

Vibrio harveyi is a common aquaculture pathogen causing diseases in a variety of aquatic animals. toxR, a conserved virulence-associated gene in vibrios, is identified in V. harveyi 345, a pathogenic strain isolated from diseased fish. In this study, to gain insight into function of ToxR in V. harveyi, an in-frame deletion of the toxR gene was constructed to reveal the role of ToxR in the physiology and virulence of V. harveyi. The statistical analysis showed no significant differences in the growth ability, motility, extracellular protease secretion, antibiotic susceptibility, virulence by intraperitoneal injection and the ability of V. harveyi to colonize the spleen and liver tissues of the pearl gentian grouper between the wild-type (WT) and the toxR mutant. However, the deletion of toxR increased the biofilm formation. The structure of the V. harveyi biofilm was further analysed by using scanning electron microscopy (SEM) and confocal laser scanning microscopy, and the results showed that deletion of toxR increased the number and density of V. harveyi biofilm. Since biofilm production is flagella, exopolysaccharide (EPS) and lipopolysaccharide dependent, 16 of V. harveyi biofilm-related genes were selected for further analysis. Based on quantitative real-time reverse transcription-PCR, the expression levels of these genes, including genes flrB, motY and mshA, flaE, flrA and gmhD, were significantly up-regulated in the ΔtoxR+ strain as compared with the WT+ and C-ΔtoxR strains during the early and mid-exponential, while epsG, flaA, flaE, flgD, flgE, flrB, flrC, lpxB, motY, mshA and scrG genes were inhibited because of deletion of the toxR gene in the stationary growth phase. Our results indicate that ToxR plays an important role in controlling the biofilm in V. harveyi.

LuxS modulates motility and secretion of extracellular protease in fish pathogen Vibrio harveyi.

Vibrio harveyi can cause infections and diseases in a variety of marine vertebrates and invertebrates, which are harmful to the aquaculture industry. The LuxS quorum-sensing system regulates the expression of virulence factors in a wide variety of pathogenic bacteria. In this study, an in-frame deletion of the luxS gene was constructed to reveal the role of LuxS in the physiology and virulence of V. harveyi. Statistical analysis showed no significant differences in the growth ability, biofilm formation, antibiotic susceptibility, virulence by intraperitoneal injection, and ability of V. harveyi to colonize the spleen and liver of the pearl gentian grouper between the wild-type (WT) and luxS mutant. However, deletion of luxS decreased the secretion of extracellular protease, while increasing swimming and swarming abilities. Simultaneously, a luxS-deleted mutant showed overproduction of lateral flagella, and an intact luxS complemented this defect. Since motility is flagella dependent, 16 V. harveyi flagella biogenesis related genes were selected for further analysis. Based on quantitative real-time reverse transcription-PCR (qRT-PCR), the expression levels of these genes, including the polar flagella genes flaB, flhA, flhF, flhB, flhF, fliS, and flrA and the lateral flagella genes flgA, flgB, fliE, fliF, lafA, lafK, and motY, were significantly upregulated in the ΔluxS: pMMB207 (ΔluxS+) strain as compared with the V. harveyi 345: pMMB207 (WT+) and C-ΔluxS strains during the early, mid-exponential, and stationary growth phases. Our results indicate that LuxS plays an important role in controlling motility, flagella biogenesis, and extracellular protease secretion in V. harveyi.

Functional characterization of VscCD, an important component of the type Ⅲ secretion system of Vibrio harveyi

Vibrio harveyi is a Gram-negative bacterium that occurs widely in the ocean and a kind of pathogenic bacteria associated with vibriosis in grouper. We investigated whether the VscCD protein of the type Ⅲ secretion system (T3SS) was important for pathogenicity of V. harveyi. Mutations to the vscC and vscD genes (ΔvscCD) and complementation of the ΔvscCD mutant (C-ΔvscCD) were created. Moreover, the biological characteristics of the wild-type (WT) and mutant strains of V. harveyi 345 were compared. The results showed that deletion of the vscCD genes had no effect on bacterial growth, swimming/swarming ability, secretion of extracellular protease, or biofilm formation. However, as compared with the V. harveyi 345: pMMB207 (WT+) and complementary (C-ΔvscCD) strains, the ΔvscCD: pMMB207 (ΔvscCD+) mutant displayed decreased resistance to acid stress, H2O2, and antibiotics. In addition, infection of the pearl gentian grouper (♀Epinephelus fuscoguttatus × ♂Epinephelus lanceolatu) showed that as compared with the WT+ and C-ΔvscCD strains, the ΔvscCD+ strain significantly reduced cumulative mortality of the host. The colonization ability of the ΔvscCD+ mutant in the spleen and liver tissues of the pearl gentian grouper was significantly lower than that of the WT+ and C-ΔvscCD strains. In the early stage of infection with the ΔvscCD+ strain, the expression levels of IL-1β, IL-16, TLR3, TNF-α, MHC-Iα, and CD8α were up-regulated to varying degrees. As compared with the WT+ and C-ΔvscCD strains, luxR expression was significantly up-regulated in the ΔvscCD+ strain, while the expression of vcrH and vp1668 was significantly down-regulated. As an important component of the T3SS, VscCD seemed to play a significant role in the pathogenesis of V. harveyi.

Enhancement of Cellulase Production in Trichoderma reesei via Disruption of Multiple Protease Genes Identified by Comparative Secretomics.

The filamentous fungus Trichoderma reesei is one of the most studied cellulolytic organisms and the major producer of cellulases for industrial applications. However, undesired degradation of cellulases often happens in culture filtrates and commercial enzyme preparations. Even studies have been reported about describing proteolytic degradation of heterologous proteins in T. reesei, there are few systematic explorations concerning the extracellular proteases responsible for degradation of cellulases. In this study, the cellulase activity was observed to rapidly decrease at late cultivation stages using corn steep liquor (CSL) as the nitrogen source in T. reesei. It was discovered that this decrease may be caused by proteases. To identify the proteases, comparative secretomics was performed to analyze the concomitant proteases during the cellulase production. 12 candidate proteases from the secretome of T. reesei were identified and their encoding genes were individually deleted via homologous recombination. Furthermore, three target proteases (tre81070, tre120998, and tre123234) were simultaneously deleted by one-step genetic transformation. The triple deletion strain ΔP70 showed a 78% decrease in protease activity and a six-fold increase in cellulase activity at late fermentation stages. These results demonstrated the feasibility of improvement of cellulase production by genetically disrupting the potential protease genes to construct the T. reesei strains with low extracellular protease secretion. This dataset also provides an efficient approach for strain improvement by precise genetic engineering combined with “omics” strategy for high-production of industrial enzymes to reduce the cost of lignocellulose bioconversion.

Understanding the regulation of extracellular protease gene expression in fungi: a key step towards their biotechnological applications.

The secretion of proteases by certain species of yeast and filamentous fungi is of importance not only for their biological function and survival, but also for their biotechnological application to various processes in the food, beverage, and bioprocessing industries. A key step towards understanding the role that these organisms play in their environment, and how their protease-secreting ability may be optimally utilised through industrial applications, involves an evaluation of those factors which influence protease production. The objective of this review is to provide an overview of the findings from investigations directed at elucidating the regulatory mechanisms underlying extracellular protease secretion in yeast and filamentous fungi, and the environmental stimuli that elicit these responses. The influence of nitrogen-, carbon-, and sulphur-containing compounds, as well as proteins, temperature, and pH, on extracellular protease regulation, which is frequently exerted at the transcriptional level, is discussed in particular depth. Protease-secreting organisms of biotechnological interest are also presented in this context, in an effort to explore the areas of industrial significance that could possibly benefit from such knowledge. In this way, the establishment of a platform of existing knowledge regarding fungal protease regulation is attempted, with the particular goal of aiding in the practical application of these organisms to processes that require secretion of this enzyme.

Genome-Wide Screening for Novel Candidate Virulence Related Response Regulator Genes in Xanthomonas oryzae pv. oryzicola.

Two-component regulatory system (TCS), a major type of cellular signal transduction system, is widely used by bacteria to adapt to different conditions and to colonize certain ecological niches in response to environmental stimuli. TCSs are of distinct functional diversity, genetic diversity, and species specificity (pathovar specificity, even strain specificity) across bacterial groups. Although TCSs have been demonstrated to be crucial to the virulence of Xanthomonas, only a few researches have been reported about the studies of TCSs in Xanthomonas oryzae pathovar oryzicola (hereafter Xoc), the pathogen of rice bacterial streak disease. In the genome of Xoc strain GX01, it has been annotated 110 TCSs genes encoding 54 response regulators (RRs), 36 orthodox histidine kinase (HKs) and 20 hybrid histidine kinase (HyHKs). To evaluate the involvement of TCSs in the stress adaptation and virulence of Xoc, we mutated 50 annotated RR genes in Xoc GX01 by homologous vector integration mutagenesis and assessed their phenotypes in given conditions and tested their virulence on host rice. 17 RR genes were identified to be likely involved in virulence of Xoc, of which 10 RR genes are novel virulence genes in Xanthomonas, including three novel virulence genes for bacteria. Of the novel candidate virulence genes, some of which may be involved in the general stress adaptation, exopolysaccharide production, extracellular protease secretion and swarming motility of Xoc. Our results will facilitate further studies on revealing the biological functions of TCS genes in this phytopathogenic bacterium.

A common evolutionary pathway for maintaining quorum sensing in Pseudomonas aeruginosa.

In the bacterium Pseudomonas aeruginosa, the synthesis and secretion of extracellular protease is a typical cooperative behavior regulated by quorum sensing. However, this type of cooperative behavior is easily exploited by other individuals who do not synthesize public goods, which is known as the "tragedy of the commons". Here P. aeruginosa was inoculated into casein media with different nitrogen salts added. In casein broth, protease (a type of public good) is necessary for bacterial growth. After 30 days of sequential transfer, some groups propagated stably and avoided "tragedy of the commons". The evolved cooperators who continued to synthesize protease were isolated from these stable groups. By comparing the characteristics of quorum sensing in these cooperators, an identical evolutionary pattern was found. A variety of cooperative behaviors regulated by quorum sensing, such as the synthesis and secretion of protease and signals, were significantly reduced during the process of evolution. Such reductions improved the efficiency of cooperation, helping to prevent cheating. In addition, the production of pyocyanin, which is regulated by the RhlIR system, increased during the process of evolution, possibly due to its role in stabilizing the cooperation. This study contributes towards our understanding of the evolution of quorum sensing of P. aeruginosa.

Improving Production of Protease from Pseudoalteromonas sp. CSN423 by Random Mutagenesis

Pseudoalteromonas sp. CSN423, a marine strain, can express a major protease designated as E423 and it was secreted into the supernatant. To improve the protease E423 yield, Pseudoalteromonas sp. CSN423 was subjected to mutagenesis using UV irradiation. Mutant strain with 5.1-fold higher protease yield was isolated and named as Pseudoalteromonas sp. CSN423-M. Three protease bands were detected by zymography with casein as substrate, and results of mass spectrometry (MS) showed that two lower molecular weight protein bands were the same protease but with different mature forms. The entire protease operon was sequenced and no mutation was found. Mutant strain-associated changes of expression levels of protease synthesis and secretion-related genes were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Mutant strain had higher expression of e423 than wild-type strain. Such result was consistent with protease activity profiles. Moreover, the mutant strain had higher transcriptional levels of citrate synthase (cs), α-ketoglutarate decarboxylase (kgd), cytochrome c oxidase subunit I (coxI), tolC, hlyD (membrane protein), luxR3, luxO, and luxT (transcriptional regulator). However, hexokinase (hk), pyruvate dehydrogenase E1 (pd-e1), epsD (membrane protein), and luxR1 remained unchanged, and luxR2 decreased sharply in the mutant. These results suggested that the redox pathway was promoted in the mutant strain, and LuxR family transcriptional regulators in Pseudoalteromonas spp. may play some role in regulating protease expression. Meanwhile, the secretion of extracellular protease was closely related to ABC transport system. These results may shed some light on the molecular mechanism underlying higher yield of protease E423 from Pseudoalteromonas sp. CSN423-M.

Within-host evolution decreases virulence in an opportunistic bacterial pathogen.

BackgroundPathogens evolve in a close antagonistic relationship with their hosts. The conventional theory proposes that evolution of virulence is highly dependent on the efficiency of direct host-to-host transmission. Many opportunistic pathogens, however, are not strictly dependent on the hosts due to their ability to reproduce in the free-living environment. Therefore it is likely that conflicting selection pressures for growth and survival outside versus within the host, rather than transmission potential, shape the evolution of virulence in opportunists. We tested the role of within-host selection in evolution of virulence by letting a pathogen Serratia marcescens db11 sequentially infect Drosophila melanogaster hosts and then compared the virulence to strains that evolved only in the outside-host environment.ResultsWe found that the pathogen adapted to both Drosophila melanogaster host and novel outside-host environment, leading to rapid evolutionary changes in the bacterial life-history traits including motility, in vitro growth rate, biomass yield, and secretion of extracellular proteases. Most significantly, selection within the host led to decreased virulence without decreased bacterial load while the selection lines in the outside-host environment maintained the same level of virulence with ancestral bacteria.ConclusionsThis experimental evidence supports the idea that increased virulence is not an inevitable consequence of within-host adaptation even when the epidemiological restrictions are removed. Evolution of attenuated virulence could occur because of immune evasion within the host. Alternatively, rapid fluctuation between outside-host and within-host environments, which is typical for the life cycle of opportunistic bacterial pathogens, could lead to trade-offs that lower pathogen virulence.

Comparative sensitivity to zinc sulphate of Shigella isolates recovered from Nigerian children with low and marginal plasma zinc concentrations

This study aims to determine the ability of zinc sulphate to inhibit growth of <i>Shigella in vitro</i> and test the hypothesis that the isolates recovered from Nigerian children with low plasma zinc concentration are more sensitive than the marginal plasma zinc isolates. The effect of zinc sulphate on extracellular protease secretion was also investigated. A total of 15 <i>Shigella</i> isolates recovered from the stool samples of 117 diarrheic children at various health centers in Lagos, Nigeria were tested for sensitivity to zinc sulphate <i>in vitro</i> by agar dilution method. Plasma zinc concentration was determined spectrophotometrically to enable stratification of the patients into two zinc arms: low (plasma zinc <9.2 μmol/L) and marginal (plasma zinc: 9.2–4 μmol/L). The in vitro effect of zinc sulphate on extracellular protease secretion based on azocasein hydrolysis in these isolates was also determined. Isolates of <i>Shigella dysenteriae</i> and <i>Shigella flexneri</i> were found to be more sensitive to zinc sulphate (minimum inhibitory concentration = 0.89–0.98 ± 0.03–0.06 mg/mL) than the isolates of <i>Shigella sonnei</i> and <i>Shigella boydii</i> (minimum inhibitory concentration = 1.13–1.4 ± 0.06–0.1 mg/mL (<i>P</i> < 0.05). The late logarithmic growth kinetics of these isolates also produced similar response to zinc sulphate <i>in vitro</i>. The observed zinc sulphate-induced growth inhibition of the <i>Shigella isolates</i> tested was further found to be associated with a decline in their extracellular protease secretion in both zinc arms. Zinc sulphate elicits <i>in vitro</i> inhibition of growth and serogroups-dependent decline in extracellular protease activity among <i>Shigella</i> isolates from Nigerian children irrespective of their systemic zinc levels. We conclude that zinc sulphate may have potentials to be used as a shigellocidal and anti-virulent agent in the management of shigellosis in children.

Role of extracellular protease in nitrogen substrate management during antibiotic fermentation: a process model and experimental validation

Kinetics of extracellular protease (ECP) production has typically been studied for processes that involve protease as a product. We argue that ECP is equally important in fermentations where protease is not a product of interest. Industrial fermentations typically use complex nitrogen substrates, which are proteolytically hydrolyzed to amino acids (AA) by ECP before assimilation. However, high AA concentrations may lead to nitrogen catabolite repression (NCR) of the products such as antibiotics. Thus, ECP plays a crucial role in managing the nitrogen substrate supply thereby affecting the antibiotic productivity. Here, we have studied the induction of ECP and its effect on the antibiotic productivity for a rifamycin B overproducer strain Amycolatopsis meditterranei S699. This organism produces ECP at the level of 14 U mL(-1) in complex media, which is sufficient for hydrolysis of proteins in the media but low compared to other ECP overproducers. We find ECP secretion to be repressed by ammonia, AA, and under conditions that support high growth rate. We propose a structured kinetic model which accounts for the kinetics of ECP secretion, amino acid availability, growth, and antibiotic production. In addition to the quantity, the timing of ECP induction was critical in achieving higher rifamycin productivity. We artificially created conditions that led to delayed protease secretion, which in turn led to premature termination of batch and lower productivity. The predictive value of the model can be useful in better management of the available nitrogen supply, minimization of NCR, and in the monitoring of fermentation batches.

Decolorizing Activity of Malachite Green and Its Mechanisms Involved in Dye Biodegradation by Achromobacter xylosoxidans MG1

An Achromobacter xylosoxidans MG1 strainisolated from the effluent treatment plant of a textile and dyeing factory from Yunnan Province in China was found capable of decolorizing the malachite green dye at a high efficacy. Strain MG1 reduced 86% malachite green at the concentration of 2,000 mg/l within 1 h, representing a greater ability for decolorizing and a higher tolerance of this compound than all previously reported bacteria. Color removal was optimal at pH 6 and 38°C. Further experimental evidences demonstrated that both cytoplasmic and extracellular biodegradation contributed to the decolorization of malachite green. Nested PCR was employed to identify the candidate genes responsible for malachite green decolorization, and we identified a cytoplasmic triphenylmethane reductase gene with 100% amino acid similarity to the corresponding gene in Citrobacter sp. strain. In contrast to our expectation, the addition of metyrapone had little effect on the cytoplasmic biodegradation, suggesting that cytochrome P450 was not involved in the high-performance reduction. The extracellular biodegradation was likely attributable to the secretion of extracellular proteases and some heat-resistant compounds.

Control of extracellular cleavage of ProBDNF by high frequency neuronal activity

Pro- and mature neurotrophins often elicit opposing biological effects. For example, mature brain-derived neurotrophic factor (mBDNF) is critical for long-term potentiation induced by high-frequency stimulation, whereas proBDNF facilitate long-term depression induced by low-frequency stimulation. Because mBDNF is derived from proBDNF by endoproteolytic cleavage, mechanisms regulating the cleavage of proBDNF may control the direction of BDNF regulation. Using methods that selectively detect proBDNF or mBDNF, we show that low-frequency stimulation induced predominant proBDNF secretion in cultured hippocampal neurons. In contrast, high-frequency stimulation preferentially increased extracellular mBDNF. Inhibition of extracellular, but not intracellular cleavage of proBDNF greatly reduced high-frequency stimulation-induced extracellular mBDNF. Moreover, high-frequency, but not low-frequency stimulation selectively induced the secretion of tissue plasminogen activator, a key protease involved in extracellular proBDNF to mBDNF conversion. Thus, high-frequency neuronal activity controls the ratio of extracellular proBDNF/mBDNF by regulating the secretion of extracellular proteases. Our study demonstrates activity-dependent control of extracellular proteolytic cleavage of a secretory protein, and reveals an important mechanism that controls diametrically opposed functions of BDNF isoforms.

Twitching Motility Is Essential for Virulence in Dichelobacter nodosus

Type IV fimbriae are essential virulence factors of Dichelobacter nodosus, the principal causative agent of ovine foot rot. The fimA fimbrial subunit gene is required for virulence, but fimA mutants exhibit several phenotypic changes and it is not certain if the effects on virulence result from the loss of type IV fimbria-mediated twitching motility, cell adherence, or reduced protease secretion. We showed that mutation of either the pilT or pilU gene eliminated the ability to carry out twitching motility. However, the pilT mutants displayed decreased adhesion to epithelial cells and reduced protease secretion, whereas the pilU mutants had wild-type levels of extracellular protease secretion and adherence. These data provided evidence that PilT is required for the type IV fimbria-dependent protease secretion pathway in D. nodosus. It was postulated that sufficient fimbrial retraction must occur in the pilU mutants to allow protease secretion, but not twitching motility, to take place. Although no cell movement was detected in a pilU mutant of D. nodosus, aberrant motion was detected in an equivalent mutant of Pseudomonas aeruginosa. These observations explain how in D. nodosus protease secretion can occur in a pilU mutant but not in a pilT mutant. In addition, virulence studies with sheep showed that both the pilT and pilU mutants were avirulent, providing evidence that mutation of the type IV fimbrial system affects virulence by eliminating twitching motility, not by altering cell adherence or protease secretion.