BackgroundThe Gram-positive bacterium Bacillus subtilis has been widely used as a cell factory for the production of proteins due to its generally regarded as safe (GRAS) nature and secretion capability. Of the known secretory pathways in B. subtilis, the majority of proteins are exported from the cytoplasm by Sec pathway, Tat pathway and ABC transporters, etc. However, the production of heterologous proteins by B. subtilis is unfortunately not that straight forward because of the bottlenecks in classical secretion pathways. The aim of this work is to explore a new method for protein production based on non-classical secretion pathway.ResultsOne d-psicose 3-epimerase (RDPE) which converts d-fructose into d-psicose from Ruminococcus sp. 5_1_39BFAA was successfully and substantially secreted into the extracellular milieu without the direction of signal peptide. Subsequently, we demonstrated that RDPE contained no native signal peptide, and the secretion of RDPE was not dependent on Sec or Tat pathway or due to cell lysis, which indicated that RDPE is a non-classically secreted protein. Then, we attempted to evaluate the possibility of using RDPE as a signal to export eighteen reporter proteins into the culture medium. Five of eleven homologous proteins, two of five heterologous proteins from other bacterium and two heterologous proteins of eukaryotic source were successfully secreted into the extracellular milieu at different secretion levels when they were fused to RDPE mediated by a flexible 21-bp linker to keep a distance between two single proteins. Furthermore, the secretion rates of two fusion proteins (RDPE-DnaK and RDPE-RFP) reached more than 50 %. In addition, most of the fusion proteins retained enzyme or biological activity of their corresponding target proteins, and all of the fusions still had the activity of RDPE.ConclusionsWe found and identified a heterologous non-classically secreted protein RDPE, and showed that RDPE could direct proteins of various types into the culture medium, and thus non-classical protein secretion pathway can be used as a novel secretion pathway for recombinant proteins. This novel strategy for recombinant protein production is helpful to make B. subtilis as a more ideal cell factory for protein production.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0469-8) contains supplementary material, which is available to authorized users.

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The signal recognition particle (SRP) is conserved in all living organisms, and it cotranslationally delivers proteins to the inner membrane or endoplasmic reticulum. Recently, SRP loss was found not to be lethal in either the eukaryote Saccharomyces cerevisiae or the prokaryote Streptococcus mutans. In Escherichia coli, the role of SRP in mediating inner membrane protein (IMP) targeting has long been studied. However, the essentiality of SRP remains a controversial topic, partly hindered by the lack of strains in which SRP is completely absent. Here we show that the SRP was nonessential in E. coli by suppressor screening. We identified two classes of extragenic suppressors—two translation initiation factors and a ribosomal protein—all of which are involved in translation initiation. The translation rate and inner membrane proteomic analyses were combined to define the mechanism that compensates for the lack of SRP. The primary factor that contributes to the efficiency of IMP targeting is the extension of the time window for targeting by pausing the initiation of translation, which further reduces translation initiation and elongation rates. Furthermore, we found that easily predictable features in the nascent chain determine the specificity of protein targeting. Our results show why the loss of the SRP pathway does not lead to lethality. We report a new paradigm in which the time delay in translation initiation is beneficial during protein targeting in the absence of SRP. IMPORTANCE Inner membrane proteins (IMPs) are cotranslationally inserted into the inner membrane or endoplasmic reticulum by the signal recognition particle (SRP). Generally, the deletion of SRP can result in protein targeting defects in Escherichia coli. Suppressor screening for loss of SRP reveals that pausing at the translation start site is likely to be critical in allowing IMP targeting and avoiding aggregation. In this work, we found for the first time that SRP is nonessential in E. coli. The time delay in initiation is different from the previous mechanism that only slows down the elongation rate. It not only maximizes the opportunity for untranslated ribosomes to be near the inner membrane but also extends the time window for targeting translating ribosomes by decreasing the speed of translation. We anticipate that our work will be a starting point for a more delicate regulatory mechanism of protein targeting.

Non-classical protein secretion in bacteria is a common phenomenon. However, the selection principle for non-classical secretion pathways remains unclear. Here, our experimental data, to our knowledge, are the first to show that folded multimeric proteins can be recognized and excreted by a non-classical secretion pathway in Bacillus subtilis. We explored the secretion pattern of a typical cytoplasmic protein D-psicose 3-epimerase from Ruminococcus sp. 5_1_39BFAA (RDPE), and showed that its non-classical secretion is not simply due to cell lysis. Analysis of truncation variants revealed that the C- and N-terminus, and two hydrophobic domains, are required for structural stability and non-classical secretion of RDPE. Alanine scanning mutagenesis of the hydrophobic segments of RDPE revealed that hydrophobic residues mediated the equilibrium between its folded and unfolded forms. Reporter mCherry and GFP fusions with RDPE regions show that its secretion requires an intact tetrameric protein complex. Using cross-linked tetramers, we show that folded tetrameric RDPE can be secreted as a single unit. Finally, we provide evidence that the non-classical secretion pathway has a strong preference for multimeric substrates, which accumulate at the poles and septum region. Altogether, these data show that a multimer recognition mechanism is likely applicable across the non-classical secretion pathway.

The growth response, non-specific immune activities and disease resistance were measured in sea cucumber, Apostichopus japonicus Selenka (initial average weight 6.80 ± 0.10 g), when fed diets supplemented with graded levels of guanosine from the guanosine-5'-monophosphate disodium (GMP) at 0 (control), 0.3, 0.6 and 1.2 g/kg for 8 weeks. The results showed that GMP supplemented at 0.6 and 1.2 g/kg significantly enhanced the growth of sea cucumber. Sea cucumber fed a diet with 0.6 g/kg of GMP had significantly higher intracellular superoxide anion production, nitric oxide synthase activity, lysozyme activity and the total superoxide dismutase (T-SOD) activity than those in control group (p < .05). Increased lysozyme activity and T-SOD activity were also found in sea cucumber fed GMP at 1.2 g/kg. Moreover, there was significantly lower cumulative mortality after the disease challenge in sea cucumber fed the diets with 0.6 and 1.2 g/kg GMP than that in control and 0.3 g/kg GMP groups (p < .05), and no significant difference was observed between 0.6 and 1.2 g/kg GMP groups. These results suggested that feeding GMP at a dose of 0.6 g/kg could enhance growth, non-specific immunity of sea cucumber as well as its resistance against Vibrio splendidus. K E Y W O R D Sgrowth, immunity, immunostimulant, nucleotide, sea cucumber | INTRODUCTIONSea cucumber Apostichopus japonicus Selenka is a common echinoderm in Japan, Korea and China and believed to have hygienical and curative properties, which results in its high commercial value (Liao, 1997). The rapid increase in market demand has resulted in the overexploitation of natural sea cucumber populations all over the world. As a result, sustainable industries with modern hatchery techniques have been established in several countries (Conand, 2004). However, the quick spread of intensified farming has led to an increase in various pathogenic diseases, which has become a major limiting factor in the industry. For example, the acute peristome oedema disease and the highly contagious skin ulceration disease in A. japonicus Selenka had broken out in China and caused great economic loss (Wang, Chang, Yu, Li, &Rong, 2004). In addition, the highly contagious skin ulceration disease has also been recorded on Isostichopus fuscus in Equator, on Holothuria scabra in Madagascar, in Australia and in New Caledonia (Becker et al., 2004). Artificial infection test proved that the causative pathogen associated with this disease was V. splendidus (Zhang, Wang, & Rong, 2006 antibiotic-resistant bacteria, thus posing potential threats to human beings and environment (Smith, 2008). Searching for an alternative for antibiotics has become urgent. Non-specific immune system acts to fight against disease in most aquatic animals (Zhang et al., 2014).Immunostimulants, which increase resistance to infectious disease by enhancing non-specific defence mechanisms, are considered to be safe and effective against various pathogens in aquaculture (Sakai, 1999). Dietary nucleotides have been shown to bene...

Signal recognition particle (SRP) is critical for delivering co-translational proteins to the bacterial inner membrane. Previously, we identified SRP suppressors in Escherichia coli that inhibit translation initiation and elongation, which provided insights into the mechanism of bypassing the requirement of SRP. Suppressor mutations tended to be located in regions that govern protein translation under evolutionary pressure. To test this hypothesis, we re-executed the suppressor screening of SRP. Here, we isolated a novel SRP suppressor mutation located in the Shine–Dalgarno sequence of the S10 operon, which partially offset the targeting defects of SRP-dependent proteins. We found that the suppressor mutation decreased the protein translation rate, which extended the time window of protein targeting. This increased the possibility of the correct localization of inner membrane proteins. Furthermore, the fidelity of translation was decreased in suppressor cells, suggesting that the quality control of translation was inactivated to provide an advantage in tolerating toxicity caused by the loss of SRP. Our results demonstrated that the inefficient protein targeting due to SRP deletion can be rescued through modulating translational speed and accuracy.

Signal recognition particle (SRP) is critical for delivering co-translational proteins to the bacterial inner membrane. Previously, we identified SRP suppressors in Escherichia coli that inhibit translation initiation and elongation, which provides an insight into the mechanism of bypassing the requirement of SRP. Suppressor mutations tend to be located in regions that govern protein translation under evolutionary pressure. To verify this hypothesis, we re-executed the suppressor screening of SRP. Here we isolated a novel SRP suppressor mutation located in the Shine-Dalgarno sequence of S10 operon, which partially offset the targeting defects of SRP-dependent proteins. We found that the suppressor mutation slowed the translation rate of proteins, especially of inner membrane proteins, which could compensate for the targeting defects of inner membrane proteins via extending the time window of protein targeting. Furthermore, the fidelity of translation was decreased in suppressor cells, suggesting that the quality control of translation was inactivated to provide a survival advantage under the loss of SRP stress. Our results demonstrate that the inefficient protein targeting due to SRP deletion can be rescued through modulating translational speed and accuracy.SUMMARYThe signal recognition particle (SRP)-dependent delivery pathway is essential for membrane protein biogenesis. Previously, we reported that SRP is nonessential in Escherichia coli and slowing translation speed plays a critical role in membrane protein targeting. Here we identified a novel SRP suppressor that is also involved in translation. We found that translation speed and accuracy regulate membrane protein targeting. A slowdown of translation speed extends the time window for protein targeting. Meanwhile, a moderate decrease in translation fidelity ensures a suitable translation speed for better cell growth. These results provide that translation control could be a practical way to compensate for the loss of SRP.