Paper-based sensor from pyrrolidinyl peptide nucleic acid for the efficient detection of Bacillus cereus.

Bacillus cereus is one of the most common foodborne pathogens found in various kinds of staple foods such as rice and wheat. A rapid and accurate detection method for this pathogen is highly desirable for the sustainable production of relevant food products. While several classical and molecular-based detection methods are available for the identification of B. cereus, they suffered one or more limitations such as the requirement for a tedious and time-consuming process, less than ideal specificity, and the lack of portability. Herein, we developed the first paper-based sensing device that exhibits high species specificity with sufficiently low limit of detection for the visual detection of specific DNA sequences of B. cereus. The success is attributed to the strategic planning of fabrication in various dimensions including thorough bioinformatics search for highly specific genes, the use of the pyrrolidinyl peptide nucleic acid (PNA) probe whose selectivity advantage is well documented, and an effective PNA immobilization and DNA-binding visualization method with an internal cross-checking system for validating the results. Testing in rice matrices indicates that the sensor is capable of detecting and distinguishing B. cereus from other bacterial species. Hence, this paper-based sensor has potential to be adopted as a practical means to detect B. cereus in food industries.

Insights into the phylogeny and transcriptional response of serine proteases in a halotolerant cyanobacterium Halothece sp. PCC7418.

Serine proteases are a class of versatile proteolytic enzymes. They are necessary for protein catabolism, intracellular amino acid turnover, and regulation of proteins involved in diverse molecular and cellular processes across taxa. In this study, bioinformatic analyses revealed a significantly large number of serine proteases in the halotolerant cyanobacterium Halothece sp. PCC7418 (hereafter referred to as Halothece 7418) compared to the model freshwater cyanobacterium Synechococcus elongatus PCC7942 (hereafter referred to as S. elongatus 7942). The cyanobacterial serine proteases are likely derived from different linages since no conserved motifs were detected. The presence of highly diverse serine proteases in Halothece 7418 implicated an evolutionary-mediated modification of several proteases, which may play numerous physiological roles. We also examined the gene expression patterns of 34 serine protease encoding genes in Halothece 7418 exposed to salt stress. Our results revealed that several serine protease genes were drastically up-regulated under salt with high concentration but remained unchanged under salt with low concentration. All four clp genes (H1996, H1997, H0950, and H3375) and H3553 gene (which encodes a putative HtrA protease) were significantly induced upon salt stress. These responses support the roles of the housekeeping pathways in both the degradation of damaged proteins induced by salt stress and regulation of proteins involved in the molecular recovery from salt stress. Since serine proteases share several biochemical features and physiological functions, the results from this study provide an insight into diversification of serine proteases in cyanobacteria. Further, these results will increase our understanding of several mechanisms at the subcellular level.

The Effects of Salts and Osmoprotectants on Enzyme Activities of Fructose-1,6-biphosphate Aldolases in a Halotolerant Cyanobacterium, Halothece sp. PCC 7418.

The halotolerant cyanobacterium, Halothece sp. PCC 7418, possesses two classes of fructose-1,6-bisphosphate aldolase (FBA): H2846 and H2847. Though class I (CI)-FBA H2846 is thought to be associated with salt tolerance, the regulatory mechanisms, molecular characteristics, and expression profiles between H2846 and class II (CII)-FBA H2847 have scarcely been investigated. Here, we show that the accumulation of the H2846 protein is highly responsive to both up- and down-shock with NaCl, whereas H2847 is constitutively expressed. The activity of CI- and CII-FBA in cyanobacterial extracts is correlated with the accumulation patterns of H2846 and H2847, respectively. In addition, it was found that these activities were inhibited by NaCl and KCl, with CII-FBA activity strikingly inhibited. It was also found that the CI-FBA activity of recombinant H2846 was hindered by salts and that this hindrance could be moderated by the addition of glycine betaine (GB), whereas no moderation occurred with other potential osmoprotectant molecules (proline, sucrose, and glycerol). In addition, a phylogenetic analysis showed that CI-FBAs with higher similarities to H2846 tended to be distributed among potential GB-synthesizing cyanobacteria. Taken together, our results provide insights into the independent evolution of the CI- and CII-FBA gene families, which show distinct expression profiles and functions following salt stress.

The evolutionarily conserved HtrA is associated with stress tolerance and protein homeostasis in the halotolerant cyanobacterium Halothece sp. PCC7418.

The HtrA protein family represents an important class of serine proteases that are widely distributed across taxa. These evolutionarily conserved proteins are crucial for survival and function as monitors of protein synthesis during various stresses. Here, we performed gene expression analysis of the entire set of putative serine protease genes in Halothece sp. PCC7418 under salt stress conditions. The gene-encoding HtrA2 (H3553) was highly upregulated. This gene was cloned and functionally characterized, and its sub-cellular localization was determined. The recombinant H3553 protein (rH3553) displayed a pH optimum of 8.0, remained stable at 45 °C, and its proteolytic activity was not affected by salts. H3553 completely degraded the unfolded model protein, ß-casein. In contrast, the folded model substrates (lysozyme or BSA) were not degraded by rH3553. Denaturation of BSA at a high temperature significantly increased its degradation by rH3553. H3553 was detected in the soluble protein fraction as well as the plasma membrane and thylakoid membrane fractions. Interestingly, the majority of H3553 was present in the plasma membrane under salt and heat stress conditions. Thus, H3553 resides in multiple sub-cellular locations and its localization drastically changes after exposure to stresses. Taken together, H3553 underpins protein quality-control process and is involved in the response and adaptation to salinity and heat stresses.

A class I fructose-1,6-bisphosphate aldolase is associated with salt stress tolerance in a halotolerant cyanobacterium Halothece sp. PCC 7418.

Fructose-1,6-bisphosphate aldolase (FBA) is a key metabolic enzyme, which is involved in glycolysis, gluconeogenesis and the Calvin cycle. The distinct physiological roles of FBAs in various organisms have been reported; however, in cyanobacteria, the functional characterization of FBAs and investigation of the intracellular dynamics of FBAs largely remains unknown. Here, we utilized a two-step chromatographic technique to identify a class I FBA (CI-FBA), which we named H2846. H2846 was induced by salt stress in the halotolerant cyanobacterium Halothece sp. PCC 7418 (hereafter referred to as Halothece 7418). Phylogenetic analysis showed that H2846-like CI-FBAs existed mainly in cyanobacterial species that inhabit hypersaline environments. Subcellular fractionation revealed that H2846 localized in the cytosolic and periplasmic spaces and size-exclusion chromatography suggested that H2846 formed a homohexamer. The CI-FBA activity of recombinant H2846-mediated cleavage of fructose bisphosphate (FBP) was characterized using a coupled enzymatic assay. This analysis allowed us to determine the Km and Vmax values of recombinant H2846, which were then compared to previously reported Km and Vmax values of several FBAs. Our data suggested that H2846 was likely responsible for the salt stress-induced CI-FBA activity from the total soluble protein extracts derived from Halothece 7418 cells. Moreover, heterologous expression of H2846 but not H2847, a class II FBA (CII-FBA), conferred salt stress tolerance to the salt-sensitive freshwater cyanobacterium, Synechococcus elongatus PCC 7942, which only contains the CII-FBA, S1443. S. elongatus PCC 7942 with a S1443 gene deletion was complemented by H2847 expression, but was not complemented by expression of H2846. Taken together, these results indicate the functional differences between two distinct sets of FBAs in cyanobacteria. H2846 is an active CI-FBA that contributes to the mechanism of salt stress tolerance in Halothece 7418.

Enzymatic hydrolysis of tropical weed xylans using xylanase from Aureobasidium melanogenum PBUAP46 for xylooligosaccharide production.

The maximum yield of xylanase from Aureobasidium melanogenum PBUAP46 was 5.19 ± 0.08 U ml-1 when cultured in a production medium containing 3.89% (w/v) rice straw and 0.75% (w/v) NaNO3 as carbon and nitrogen sources, respectively, for 72 h. This enzyme catalyzed well and was relatively stable at pH 7.0 and room temperature (28 ± 2 °C). The produced xylanase was used to hydrolyze xylans from four tropical weeds, whereupon it was found that the highest amounts of reducing sugars in the xylan hydrolysates of cogon grass (Imperata cylindrical), Napier grass (Pennisetum purpureum), and vetiver grass (Vetiveria zizanioides) were at 20.44 ± 0.84, 17.50 ± 0.29, and 19.44 ± 0.40 mg 100 mg xylan-1, respectively, but it was not detectable in water hyacinth (Eichhornia crassipes) hydrolysate. The highest combined amount of xylobiose and xylotriose was obtained from vetiver grass; thus, it was selected for further optimization. After optimization, xylanase digestion of vetiver grass xylan at 27.94 U g xylan-1 for 92 h 19 min gave the highest amount of reducing sugars (23.65 ± 1.34 mg 100 mg xylan-1), which were principally xylobiose and xylotriose. The enriched XOs exhibited a prebiotic property, significantly stimulating the growth of Lactobacillus brevis and L. casei by a factor of up to 3.5- and 6.5-fold, respectively, compared to glucose.