Revisiting Fur Regulon Leads to a Comprehensive Understanding of Iron and Fur Regulation.

Iron is an essential element because it functions as a cofactor of many enzymes, but excess iron causes cell damage. Iron hemostasis in Escherichia coli was transcriptionally maintained by the ferric uptake regulator (Fur). Despite having been studied extensively, the comprehensive physiological roles and mechanisms of Fur-coordinated iron metabolism still remain obscure. In this work, by integrating a high-resolution transcriptomic study of the Fur wild-type and knockout Escherichia coli K-12 strains in the presence or absence of iron with high-throughput ChIP-seq assay and physiological studies, we revisited the regulatory roles of iron and Fur systematically and discovered several intriguing features of Fur regulation. The size of the Fur regulon was expanded greatly, and significant discrepancies were observed to exist between the regulations of Fur on the genes under its direct repression and activation. Fur showed stronger binding strength to the genes under its repression, and genes that were repressed by Fur were more sensitive to Fur and iron regulation as compared to the genes that were activated by Fur. Finally, we found that Fur linked iron metabolism to many essential processes, and the systemic regulations of Fur on carbon metabolism, respiration, and motility were further validated or discussed. These results highlight how Fur and Fur-controlled iron metabolism affect many cellular processes in a systematic way.

Effects of heavy metals and organic matter fractions on the fungal communities in mangrove sediments from Techeng Isle, South China.

Heavy metal pollution has become a serious environmental problem in mangrove ecosystems and has attracted more attention. Most of previous studies have mainly focused on the effects of heavy metals on bacterial communities in mangrove sediments. This study was the first to investigate the effects of heavy metals (e.g., As, Co, Cr, Cu, Mn, Ni, Pb, V and Zn) and organic matter fractions (including total organic carbon (TOC), total nitrogen (TN), and total sulfur (TS)) on the fungal communities in mangrove sediments from Techeng Isle, South China. The results of this study indicated that the average contents of Mn, Pb and V of 8.30-161.80 µg/g presented relatively higher pollution levels, while the concentrations of Zn, Cr, Cu and Ni of 0.80-21.93 µg/g were lower than those recorded in other mangrove ecosystems. Furthermore, the sediment fungal community structures responded differently to the nine heavy metals and three organic matter fractions. Heavy metals Cr, Pb and V displayed significant positive correlations with Eutypella (P < 0.05), whereas significant negative correlations with Cystobasidium, Lulworthia, Cladosporium, Lulwoana and Cephalotheca (P < 0.05). In addition, the effects of heavy metals and TS on many fungal genera were opposite to those of TOC and TN. Fungal genera that decreased with high TOC and TN contents may be increased with high heavy metal contents and TS, and vice versa, and the genera that increased with high TOC and TN contents may be decreased with high heavy metals and TS. Our results suggested that many heavy metals, such as Cr, Pb and V, were sensitive to several fungal genera in mangrove sediments, and heavy metals together with organic matter fractions may participate and shape the fungal communities in mangrove sediments.

Repurposing a bacterial prolidase for organophosphorus hydrolysis: Reshaped catalytic cavity switches substrate selectivity.

Enzyme promiscuity is critical to the acquisition of evolutionary plasticity in cells and can be recruited for high-value chemical synthesis or xenobiotic degradation. The molecular determinants of substrate ambiguity are essential to this activity; however, these details remain unknown. Here, we performed the directed evolution of a prolidase to enhance its initially weak paraoxonase activity. The in vitro evolution led to an unexpected 1,000,000-fold switch in substrate selectivity, with a 30-fold increase in paraoxon hydrolysis and 40,000-fold decrease in peptide hydrolysis. Structural and in silico analyses revealed enlarged catalytic cavities and substrate repositioning as responsible for rapid catalytic transitions between distinct chemical reactions.

In vivo immobilization of an organophosphorus hydrolyzing enzyme on bacterial polyhydroxyalkanoate nano-granules.

BACKGROUND: Polyhydroxyalkanoate (PHA) are nano-granules naturally produced by bacteria. Two types of proteins, PHA synthase (PhaC) and phasins (PhaPs), are attached to the PHA surface by covalent and hydrophobic interactions. Utilizing these anchored proteins, functionalized PHA nano-granules displaying proteins of interest can be easily prepared by fermentation. RESULTS: In this study, a one-step fabrication method was developed for stable and efficient immobilization of an organophosphorus degrading enzyme on PHA nano-granules. The nano-biocatalysts were produced in recombinant Escherichia coli cells into which the polyhydroxyalkanoate synthesis pathway from Cupriavidus necator had been introduced. Two different strategies, covalent attachment and hydrophobic binding, were investigated by fusing bacterial organophosphorus anhydride hydrolase (OPAA4301) with PhaC and PhaP, respectively. Using both methods, the tetrameric enzyme successfully self-assembled and was displayed on the PHA surface. The display density of the target fused enzyme was enhanced to 6.8% of total protein on decorated PHA by combination of covalent and non-covalent binding modes. Immobilization of the enzyme on PHA granules resulted in higher catalytic efficiency, increased stability and excellent reusability. The kcat values of the immobilized enzymes increased by threefold compared to that of the free enzyme. The pH stability under acidic conditions was significantly enhanced, and the immobilized enzyme was stable at pH 3.0-11.0. Furthermore, more than 80% of the initial enzyme activity retained after recycling ten times. CONCLUSIONS: This study provides a promising approach for cost-efficient in vivo immobilization of a tetrameric organophosphorus degrading enzyme. The immobilization process expands the utility of the enzyme, and may inspire further commercial developments of PHA nano-biocatalysts. As revealed by our results, combination of covalent and non-covalent binding is recommended for display of enzymes on PHA granules.

Role of two amino acid residues' insertion on thermal stability of thermophilic α-amylase AMY121 from a deep sea bacterium Bacillus sp. SCSIO 15121.

α-Amylases from Bacillus licheniformis (BLA) and Bacillus amyloliquefaciens (BAA) are both important industrial enzymes with high similarity in structure but significant differences in thermostability. The mechanisms underlying this discrepancy are still poorly understood. Here, we investigated the role of two amino acids' insertion on the thermostability of these two group amylases. A newly obtained thermophilic amylase AMY121 was found much closer to BLA in both primary structure and enzymological properties. Two amino acids' insertion widespread among BAA group α-amylases was identified as one of the key factors leading to the thermostability differences, since thermostability of insertion mutants (AMY121-EG and AMY121-AA) from AMY121 significantly decreased, while that of deletion mutant from BAA increased. Moreover, we proposed that conformational disturbance caused by insertion mutation might weaken the calcium-binding affinity and consequently decrease the enzyme thermostability.

Statistical optimization of alkaline protease production from Penicillium citrinum YL-1 under solid-state fermentation.

Proteases from halotolerant and halophilic microorganisms were found in traditional Chinese fish sauce. In this study, 30 fungi were isolated from fermented fish sauce in five growth media based on their morphology. However, only one strain, YL-1, which was identified as Penicillium citrinum by internal transcribed spacer (ITS) sequence analysis, can produce alkaline protease. This study is the first to report that a protease-producing fungus strain was isolated and identified in traditional Chinese fish sauce. Furthermore, the culture conditions of alkaline protease production by P. citrinum YL-1 in solid-state fermentation were optimized by response surface methodology. First, three variables including peptone, initial pH, and moisture content were selected by Plackett-Burman design as the significant variables for alkaline protease production. The Box-Behnken design was then adopted to further investigate the interaction effects between the three variables on alkaline protease production and determine the optimal values of the variables. The maximal production (94.30 U/mL) of alkaline protease by P. citrinum YL-1 took place under the optimal conditions of peptone, initial pH, and moisture content (v/w) of 35.5 g/L, 7.73, and 136%, respectively.

Small RNA GcvB Regulates Oxidative Stress Response of Escherichia coli.

Small non-translated regulatory RNAs control plenty of bacterial vital activities. The small RNA GcvB has been extensively studied, indicating the multifaceted roles of GcvB beyond amino acid metabolism. However, few reported GcvB-dependent regulation in minimal medium. Here, by applying a high-resolution RNA-seq assay, we compared the transcriptomes of a wild-type Escherichia coli K-12 strain and its gcvB deletion derivative grown in minimal medium and identified putative targets responding to GcvB, including flu, a determinant gene of auto-aggregation. The following molecular studies and the enhanced auto-aggregation ability of the gcvB knockout strain further substantiated the induced expression of these genes. Intriguingly, the reduced expression of OxyR (the oxidative stress regulator) in the gcvB knockout strain was identified to account for the increased expression of flu. Additionally, GcvB was characterized to up-regulate the expression of OxyR at the translational level. Accordingly, compared to the wild type, the GcvB deletion strain was more sensitive to oxidative stress and lost some its ability to eliminate endogenous reactive oxygen species. Taken together, we reveal that GcvB regulates oxidative stress response by up-regulating OxyR expression. Our findings provide an insight into the diversity of GcvB regulation and add an additional layer to the regulation of OxyR.

Gain of Spontaneous clpX Mutations Boosting Motility via Adaption to Environments in Escherichia coli.

Motility is finely regulated and is crucial to bacterial processes including colonization and biofilm formation. There is a trade-off between motility and growth in bacteria with molecular mechanisms not fully understood. Hypermotile Escherichia coli could be isolated by evolving non-motile cells on soft agar plates. Most of the isolates carried mutations located upstream of the flhDC promoter region, which upregulate the transcriptional expression of the master regulator of the flagellum biosynthesis, FlhDC. Here, we identified that spontaneous mutations in clpX boosted the motility of E. coli largely, inducing several folds of changes in swimming speed. Among the mutations identified, we further elucidated the molecular mechanism underlying the ClpXV78F mutation on the regulation of E. coli motility. We found that the V78F mutation affected ATP binding to ClpX, resulting in the inability of the mutated ClpXP protease to degrade FlhD as indicated by both structure modeling and in vitro protein degradation assays. Moreover, our proteomic data indicated that the ClpXV78F mutation elevated the stability of known ClpXP targets to various degrees with FlhD as one of the most affected. In addition, the specific tag at the C-terminus of FlhD being recognized for ClpXP degradation was identified. Finally, our transcriptome data characterized that the enhanced expression of the motility genes in the ClpXV78F mutations was intrinsically accompanied by the reduced expression of stress resistance genes relating to the reduced fitness of the hypermotile strains. A similar pattern was observed for previously isolated hypermotile E. coli strains showing high expression of flhDC at the transcriptional level. Hence, clpX appears to be a hot locus comparable to the upstream of the flhDC promoter region evolved to boost bacterial motility, and our finding provides insight into the reduced fitness of the hypermotile bacteria.

Escherichia coli segments its controls on carbon-dependent gene expression into global and specific regulations.

How bacteria adjust gene expression to cope with variable environments remains open to question. Here, we investigated the way global gene expression changes in E. coli correlated with the metabolism of seven carbon substrates chosen to trigger a large panel of metabolic pathways. Coarse-grained analysis of gene co-expression identified a novel regulation pattern: we established that the gene expression trend following immediately the reduction of growth rate (GR) was correlated to its initial expression level. Subsequent fine-grained analysis of co-expression demonstrated that the Crp regulator, coupled with a change in GR, governed the response of most GR-dependent genes. By contrast, the Cra, Mlc and Fur regulators governed the expression of genes responding to non-glycolytic substrates, glycolytic substrates or phosphotransferase system transported sugars following an idiosyncratic way. This work allowed us to expand additional genes in the panel of gene complement regulated by each regulator and to elucidate the regulatory functions of each regulator comprehensively. Interestingly, the bulk of genes controlled by Cra and Mlc were, respectively, co-regulated by Crp- or GR-related effect and our quantitative analysis showed that each factor took turns to work as the primary one or contributed equally depending on the conditions.

Deciphering global gene expression and regulation strategy in Escherichia coli during carbon limitation.

Despite decades of studies meant to analyse the bacterial response to carbon limitation, we still miss a high-resolution overview of the situation. All gene expression changes observed in such conditions cannot solely be accounted for by the global regulator Crp either free or bound to its effector, cyclic AMP. Here, for the first time, we evaluated the response of both CDS (protein-coding sequence) and ncRNA (non-coding RNA) genes to carbon limitation, revealed cellular functions of differentially expressed genes systematically, quantified the contribution of Crp-cAMP and other factors to regulation and deciphered regulation strategies at a genomewide scale. Approximately one-third of the differentially expressed genes we identified responded to Crp-cAMP via its direct or indirect control, while the remaining genes were subject to growth rate-dependent control or were controlled by other regulators, especially RpoS. Importantly, gene regulation mechanisms can be established by expression pattern studies. Here, we propose a comprehensive picture of how cells respond to carbon scarcity. The global regulation strategies thus exposed illustrate that the response of cell to carbon scarcity is not limited to maintaining sufficient carbon metabolism via cAMP signalling while the main response is to adjust metabolism to cope with a slow growth rate.

Cloning, Expression, and Characterization of a Cold-Active and Organic Solvent-Tolerant Lipase from Aeromicrobium sp. SCSIO 25071.

The gene encoding lipase (Lip98) from Aeromicrobium sp. SCSIO 25071 was cloned and functionally expressed in Escherichia coli. Lip98 amino acid sequence shares the highest (49%) identity to Rhodococcus jostii RHA1 lipase and contains a novel motif (GHSEG), which is different from other clusters in the lipase superfamily. The recombinant lipase was purified to homogeneity with Ni-NTA affinity chromatography. Lip98 showed an apparent molecular mass of 30 kDa on SDS gel. The optimal temperature and pH value for enzymatic activity were recorded at 30°C and 7.5, respectively. Lip98 exhibited high activity at low temperatures with 35% maximum activity at 0°C and good stability at temperatures below 35°C. Its calculated activation energy was 4.12 kcal/mol at the low temperature range of 15-30°C. Its activity was slightly affected by some metal ions such as K(+), Ca(2+), and Na(+). The activity of Lip98 was increased by various organic solvents such as DMSO, ethanol, acetone, and hexane with the concentration of 30% (v/v) and retained more than 30% residual activity in neat organic solvent. The unique characteristics of Lip98 imply that it is a promising candidate for industrial application as a nonaqueous biocatalyst and food additive.