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Abstract Base editors (BEs) are widely used as revolutionary genome manipulation tools for cell evolution. To screen the targeted individuals, it is often necessary to expand the editing window to ensure highly diverse variant library. However, current BEs suffer from a limited editing window of 5–6 bases, corresponding to only 2–3 amino acids. Here, by engineering the CRISPR‒Cas12b, the study develops dCas12b‐based CRISPRi system, which can efficiently repress gene expression by blocking the initiation and elongation of gene transcription. Further, based on dCas12b, a new‐generation of BEs with an expanded editing window is established, covering the entire protospacer or more. The expanded editing window results from the smaller steric hindrance compared with other Cas proteins. The universality of the new BE is successfully validated in Bacillus subtilis and Escherichia coli. As a proof of concept, a spectinomycin‐resistant E. coli strain (BL21) and a 6.49‐fold increased protein secretion efficiency in E. coli JM109 are successfully obtained by using the new BE. The study, by tremendously expanding the editing window of BEs, increased the capacity of the variant library exponentially, greatly increasing the screening efficiency for microbial cell evolution.

Abstract Early-life human gut microbiome is a pivotal driver of gut homeostasis and infant health. However, the viral component (known as “virome”) remains mostly unexplored. Here, we establish the Early-Life Gut Virome (ELGV), a catalog of 160,478 non-redundant DNA and RNA viral sequences from 8130 gut virus-like particles (VLPs) enriched or bulk metagenomes in the first three years of life. By clustering, 82,141 viral species are identified, 68.3% of which are absent in existing databases built mainly from adults, and 64 and 8 viral species based on VLPs-enriched and bulk metagenomes, respectively, exhibit potentials as biomarkers to distinguish infants from adults. With the largest longitudinal population of infants profiled by either VLPs-enriched or bulk metagenomic sequencing, we track the inherent instability and temporal development of the early-life human gut virome, and identify differential viruses associated with multiple clinical factors. The mother-infant shared virome and interactions between gut virome and bacteriome early in life are further expanded. Together, the ELGV catalog provides the most comprehensive and complete metagenomic blueprint of the early-life human gut virome, facilitating the discovery of pediatric disease-virome associations in future.

l-Tyrosine, an aromatic non-essential amino acid, is the raw material for many important chemical products, including levodopa, resveratrol, and hydroxytyrosol. It is widely used in the food, drug, and chemical industries. There are many studies on the synthesis of l-tyrosine by microorganisms, however, the low titer of l-tyrosine limited the industrial large-scale production. In order to enhance l-tyrosine production in Escherichia coli, the expression of key enzymes in the shikimate pathway was up- or down-regulated. The l-tyrosine transport system and the acetic acid biosynthesis pathway were modified to further enhance l-tyrosine production. In addition, the phosphoketolase pathway was introduced in combination with cofactor engineering to redirect carbon flux to the shikimate pathway. Finally, after adaptive laboratory evolution to low pH an optimal strain was obtained. The strain can produce 92.5 g/L of l-tyrosine in a 5-L fermenter in 62 h, with a yield of 0.266 g/g glucose.

Abstract Core genome multilocus sequence typing (cgMLST) is commonly used to classify bacterial strains into different types, for taxonomical and epidemiological applications. However, cgMLST schemes require central databases for the nomenclature of new alleles and sequence types, which must be synchronized worldwide and involve increasingly intensive calculation and storage demands. Here, we describe a distributed cgMLST (dcgMLST) scheme that does not require a central database of allelic sequences and apply it to study evolutionary patterns of epidemic and endemic strains of the genus Neisseria. We classify 69,994 worldwide Neisseria strains into multi-level clusters that assign species, lineages, and local disease outbreaks. We divide Neisseria meningitidis into 168 endemic lineages and three epidemic lineages responsible for at least 9 epidemics in the past century. According to our analyses, the epidemic and endemic lineages experienced very different population dynamics in the past 100 years. Epidemic lineages repetitively emerged from endemic lineages, disseminated worldwide, and apparently disappeared rapidly afterward. We propose a stepwise model for the evolutionary trajectory of epidemic lineages in Neisseria, and expect that the development of similar dcgMLST schemes will facilitate epidemiological studies of other bacterial pathogens.

Abstract The present study investigated the composition, abundance, and diversity of gut microbes in full-term and late-preterm infants from a medical center in eastern China. A total of 144 genomes of stool samples were captured for 16S rRNA metagenomic analyses. A high abundance of commensal intestinal bacteria was detected in these samples such as Phocaeicola vulgatus, Escherichia coli, and Faecalibacterium prausnitzii, indicating a relatively consistent diversity of gut microbes in the present full-term infants aged 38–40 weeks. However, late preterm infants (n = 50) with mandatory antimicrobials feeding exhibited lower diversity but a higher composition of opportunistic pathogens such as Enterococcus species. Centralized on the situation, we explored the regulatory effect of Clostridium butyricum as probiotics on these late preterm infants. The consumption of C. butyricum did not restore the composition of gut microbes altered by antimicrobials to normal levels, although several opportunistic pathogens decreased significantly after probiotic therapy including Staphylococcus aureus, Sphingomonas echinoides, and Pseudomonas putida. We also compared the effects of day-fed versus night-fed probiotics. Intriguingly, the nighttime feeding showed a higher proportion of C. butyricum compared with probiotic day-feeding. Finally, fecal metabolome and metabolites were analyzed in late preterm infants with (n = 20) or without probiotic therapy (n = 20). The KEGG enrichment analysis demonstrated that vitamin digestion and absorption, synaptic vesicle cycle, and biotin metabolism were significantly increased in the probiotic-treated group, while MSEA indicated that a series of metabolism were significantly enriched in probiotic-treated infants including glycerolipid, biotin, and lysine, indicating the complex effects of probiotic therapy on glutathione metabolism and nutrients digestion and absorption in late preterm infants. Overall, this study provided metagenomic and metabolomic profile of the gut microbes in full-term newborns and late preterm infants in eastern China. Further studies are needed to support and elucidate the role of probiotic feeding in late preterm infants with mandatory antimicrobial treatment.

Salmonella, especially antimicrobial resistant strains, remains one of the leading causes of foodborne bacterial disease. Retail chicken is a major source of human salmonellosis. Here, we investigated the prevalence, antimicrobial resistance (AMR), and genomic characteristics of Salmonella in 88 out of 360 (24.4%) chilled chicken carcasses, together with 86 Salmonella from humans with diarrhea in Qingdao, China in 2020. The most common serotypes were Enteritidis and Typhimurium (including the serotype I 4,[5],12:i:-) among Salmonella from both chicken and humans. The sequence types were consistent with serotypes, with ST11, ST34 and ST19 the most dominantly identified. Resistance to nalidixic acid, ampicillin, tetracycline and chloramphenicol were the top four detected in Salmonella from both chicken and human sources. High multi-drug resistance (MDR) and resistance to third-generation cephalosporins resistance were found in Salmonella from chicken (53.4%) and humans (75.6%). In total, 149 of 174 (85.6%) Salmonella isolates could be categorized into 60 known SNP clusters, with 8 SNP clusters detected in both sources. Furthermore, high prevalence of plasmid replicons and prophages were observed among the studied isolates. A total of 79 antimicrobial resistant genes (ARGs) were found, with aac(6′)-Iaa, blaTEM-1B, tet(A), aph(6)-Id, aph(3″)-Ib, sul2, floR and qnrS1 being the dominant ARGs. Moreover, nine CTX-M-type ESBL genes and the genes blaNMD-1, mcr-1.1, and mcr-9.1 were detected. The high incidence of MDR Salmonella, especially possessing lots of mobile genetic elements (MGEs) in this study posed a severe risk to food safety and public health, highlighting the importance of improving food hygiene measures to reduce the contamination and transmission of this bacterium. Overall, it is essential to continue monitoring the Salmonella serotypes, implement the necessary prevention and strategic control plans, and conduct an epidemiological surveillance system based on whole-genome sequencing.

Staphylococcus aureus is an opportunistic foodborne pathogen occasionally isolated from diarrhea patients. In recent years, increasing studies have reported the detection of S. aureus in food poisoning incidents due to food contamination in the North and South of China. However, the epidemiology and genetic characteristics of S. aureus from food poisoning incidents in Eastern China remain unknown. The present study examined the genetic characteristics, antimicrobial resistance, and virulent factors of multidrug-resistant S. aureus isolated from 22 food poisoning incidents reported by the hospitals and health centers in Eastern China from 2011 to 2021. A total of 117 resistant and enterotoxigenic S. aureus isolates were collected and sequenced, among which 20 isolates were identified as methicillin resistant. Genetic analysis revealed 19 distinct CC/ST types, with CC6, CC22, CC59, CC88, and CC398 being the most frequent variants in methicillin-resistant S. aureus (MRSA). A considerable shift in CC types from CC1 to CC398 between 2011 and 2021 was observed in this study, indicating that CC398 may be the main epidemic strain circulating in the current food poisoning incidents. Additionally, genes for enterotoxins were detected in 55 isolates, with a prevalence of 27.8% (27/97) for methicillin-sensitive variants and 35.0% (7/20) for MRSA. The scn gene was detected in 59.0% of the isolates, demonstrating diverse contaminations of S. aureus among livestock-to-human transmission. Of the 117 isolates, only ten isolates displayed multi-drug resistance (MDR) to penicillin, tetracycline, and macrolides. None of the 117 foodborne S. aureus isolates tested positive for vanA in this study. Together, the present study provided phylogenetic characteristics of S. aureus from food poisoning incidents that emerged in Eastern China from 2011 to 2021. Our results suggested that these diarrhea episodes were hypotonic and merely transient low-MDR infections, however, further research for continued surveillance given the detection of virulence and antimicrobial resistance determinants is required to elucidate the genomic characteristics of pathogenic S. aureus in food poisoning incidents in the context of public health.

L-aspartate α-decarboxylase (ADC) is a pyruvoyl-dependent decarboxylase that catalyzes the conversion of L-aspartate to β-alanine in the pantothenate pathway. The enzyme has been extensively used in the biosynthesis of β-alanine and D-pantothenic acid. However, the broad application of ADCs is hindered by low specific activity. To address this issue, we explored 412 sequences and discovered a novel ADC from Corynebacterium jeikeium (CjADC). CjADC exhibited specific activity of 10.7 U/mg and Km of 3.6 mM, which were better than the commonly used ADC from Bacillus subtilis. CjADC was then engineered leveraging structure-guided evolution and generated a mutant, C26V/I88M/Y90F/R3V. The specific activity of the mutant is 28.8 U/mg, which is the highest among the unknown ADCs. Furthermore, the mutant displayed lower Km than the wild-type enzyme. Moreover, we revealed that the introduced mutations increased the structural stability of the mutant by promoting the frequency of hydrogen-bond formation and creating a more hydrophobic region around the active center, thereby facilitating the binding of L-aspartate to the active center and stabilizing the substrate orientation. Finally, the whole-cell bioconversion showed that C26V/I88M/Y90F/R3V completely transformed 1-molar L-aspartate in 12 h and produced 88.6 g/L β-alanine. Our study not only identified a high-performance ADC but also established a research framework for rapidly screening novel enzymes using a protein database.

The present study investigated the water quality index, microbial composition and antimicrobial resistance genes in urban water habitats. Combined chemicals testing, metagenomic analyses and qualitative PCR (qPCR) were conducted on 20 locations, including rivers from hospital surrounds (n = 7), community surrounds (n = 7), and natural wetlands (n = 6). Results showed that the indexes of total nitrogen, phosphorus, and ammonia nitrogen of hospital waters were 2–3 folds high than that of water from wetlands. Bioinformatics analysis revealed a total of 1,594 bacterial species from 479 genera from the three groups of water samples. The hospital-related samples had the greatest number of unique genera, followed by those from wetlands and communities. The hospital-related samples contained a large number of bacteria associated with the gut microbiome, including Alistipes, Prevotella, Klebsiella, Escherichia, Bacteroides, and Faecalibacterium, which were all significantly enriched compared to samples from the wetlands. Nevertheless, the wetland waters enriched bacteria from Nanopelagicus, Mycolicibacterium and Gemmatimonas, which are typically associated with aquatic environments. The presence of antimicrobial resistance genes (ARGs) that were associated with different species origins in each water sample was observed. The majority of ARGs from hospital-related samples were carried by bacteria from Acinetobacter, Aeromonas and various genera from Enterobacteriaceae, which each was associated with multiple ARGs. In contrast, the ARGs that were exclusively in samples from communities and wetlands were carried by species that encoded only 1 to 2 ARGs each and were not normally associated with human infections. The qPCR showed that water samples of hospital surrounds had higher concentrations of intI1 and antimicrobial resistance genes such as tetA, ermA, ermB, qnrB, sul1, sul2 and other beta-lactam genes. Further genes of functional metabolism reported that the enrichment of genes associated with the degradation/utilization of nitrate and organic phosphodiester were detected in water samples around hospitals and communities compared to those from wetlands. Finally, correlations between the water quality indicators and the number of ARGs were evaluated. The presence of total nitrogen, phosphorus, and ammonia nitrogen were significantly correlated with the presence of ermA and sul1. Furthermore, intI1 exhibited a significant correlation with ermB, sul1, and blaSHV, indicating a prevalence of ARGs in urban water environments might be due to the integron intI1’s diffusion-promoting effect. However, the high abundance of ARGs was limited to the waters around the hospital, and we did not observe the geographical transfer of ARGs along with the river flow. This may be related to water purifying capacity of natural riverine wetlands. Taken together, continued surveillance is required to assess the risk of bacterial horizontal transmission and its potential impact on public health in the current region.

Highlights • The global lineage of M. caseolyticus strains were divided into four clades from A to D. • MLST typing revealed novel alleles in M. caseolyticus strains isolated in China. • Global clades carried significantly less AMR genes and more virulence factors than present isolates. • As the close relative of the genus Staphylococcus , Macrococcus caseolyticus has a larger diameter and thicker cell wall compared with S.aureus ATCC 25923. • Macrococcus caseolyticus may serve as a vector for methicillin resistance habitats in foodborne microorganisms.

Here the authors present a large-scale resource of the early-life human gut microbiome from children under three years old, which comprises 32,277 metagenome-assembled gut genomes, representing 2172 species, and more than 80 million gut proteins representing >4 million protein clusters, spanning multiple clinical factors including age, delivery mode, gestational age, and feeding patterns.

Abstract Background The construction of protein expression systems is mainly focused on carbon catabolite repression and quorum-sensing systems. However, each of these regulatory modes has an inherent flaw, which is difficult to overcome. Organisms also prioritize using different nitrogen sources, which is called nitrogen catabolite repression. To date, few gene regulatory systems based on nitrogen catabolite repression have been reported. Results In this study, we constructed a nitrogen switching auto-inducible expression system (NSAES) based on nitrogen catabolite regulation and nitrogen utilization in Aspergillus nidulans. The PniaD promoter that is highly induced by nitrate and inhibition by ammonia was used as the promoter. Glucuronidase was the reporter protein. Glucuronidase expression occurred after ammonium was consumed in an ammonium and nitrate compounding medium, achieving stage auto-switching for cell growth and gene expression. This system maintained a balance between cell growth and protein production to maximize stress products. Expressions of glycosylated and secretory proteins were successfully achieved using this auto-inducible system. Conclusions We described an efficient auto-inducible protein expression system based on nitrogen catabolite regulation. The system could be useful for protein production in the laboratory and industrial applications. Simultaneously, NSAES provides a new auto-inducible expression regulation mode for other filamentous fungi.

Abstract NADPH provides the reducing power for decomposition of reactive oxygen species (ROS), making it an indispensable part during ROS defense. It remains uncertain, however, if living cells respond to the ROS challenge with an elevated intracellular NADPH level or a more complex NADPH-mediated manner. Herein, we employed a model fungus Aspergillus nidulans to probe this issue. A conditional expression of glucose-6-phosphate dehydrogenase (G6PD)-strain was constructed to manipulate intracellular NADPH levels. As expected, turning down the cellular NADPH concentration drastically lowered the ROS response of the strain; it was interesting to note that increasing NADPH levels also impaired fungal H2O2 resistance. Further analysis showed that excess NADPH promoted the assembly of the CCAAT-binding factor AnCF, which in turn suppressed NapA, a transcriptional activator of PrxA (the key NADPH-dependent ROS scavenger), leading to low antioxidant ability. In natural cell response to oxidative stress, we noticed that the intracellular NADPH level fluctuated “down then up” in the presence of H2O2. This might be the result of a co-action of the PrxA-dependent NADPH consumption and NADPH-dependent feedback of G6PD. The fluctuation of NADPH is well correlated to the formation of AnCF assembly and expression of NapA, thus modulating the ROS defense. Our research elucidated how A. nidulans precisely controls NADPH levels for ROS defense. Graphical Abstract

ABSTRACT The development of efficient, low-cost, and robust expression systems is important for the mass production of proteins and natural products in large amounts using cell factories. Glycerol is an ideal carbon source for large-scale fermentation due to its low cost and favorable maintenance of the fermentation process. Here, we used the antiterminator protein GlpP and its target promoter PglpD to construct a highly efficient glycerol-inducible expression system (GIES) in Bacillus subtilis. This system was able to express heterologous genes in an autoinducible manner based on the sequential utilization of glucose and glycerol under the regulation of carbon catabolite repression. In such a system, the concentration of glycerol regulated the strength of gene expression, and the concentration of glucose affected both the timing of induction and the strength of gene expression. By enhancing GlpP, the GIES was further strengthened for high-level intracellular expression of aspartase and secretory expression of nattokinase. High yields of nattokinase in a 5-L fermenter through batch and fed-batch fermentation demonstrated the potential to apply the GIES for large-scale enzyme production. Through the evolution of the −10 box of PglpD, mutants with gradient activities were obtained. In addition, hybrid glycerol-inducible promoters were successfully constructed by combining the constitutive promoters and the 5′ untranslated region of PglpD. Collectively, this study developed a GIES to obtain high-value products from inexpensive glycerol. More importantly, the great potential of the pair of inherent terminator and antiterminator protein as a portable biological tool for various purposes in synthetic biology is proposed. IMPORTANCE In this study, a GIES was constructed in B. subtilis by employing the antiterminator protein GlpP and the GlpP-regulated promoter PglpD. Based on the sequential utilization of glucose and glycerol by B. subtilis, the GIES was able to express genes in an autoinducible manner. The amounts and ratio of glucose and glycerol can regulate the gene induction timing and expression strength. The GIES was further applied for high yields of nattokinase, and its robustness in production scale-up was confirmed in a 5-L fermenter. The high-level expression of heterologous proteins demonstrated the huge application potential of the GIES. Furthermore, mutants of PglpD with gradient activities and hybrid glycerol-inducible promoters were obtained through the evolution of the −10 box of PglpD and the combination of the constitutive promoters and the 5′ untranslated region of PglpD, respectively. These results demonstrated the use of the antiterminator protein as a regulator for various purposes in synthetic biology.

This study described the knowledge, attitude, practice regarding antimicrobial resistance (AMR) among medical students between 2017 and 2022 in East China. A questionnaire-based survey was conducted with a total of 1,066 respondents. We highlighted that the undergraduates had a significant increase in the knowledge of antimicrobial resistance during the 5 years from 2017 to 2022 (p < 0.001). The majority of the assertions about the AMR were correctly identified by respondents. However, gaps were still observed in the issues of antimicrobial targets and bacterial transmission. In addition, overconfident attitudes and inappropriate behaviors of antimicrobial overuse and misuse were observed in the respondents. A number of 30.2% to 45.2% of the respondents asserted that there is no risk of AMR as long as the antimicrobials are taken correctly, and a proportion of the students (25.3% in 2022; 69.3% in 2017, p < 0.001) declared to buy antimicrobials from friends or family members to treat the same illness. Finally, spearman correlation coefficient was enrolled to compare the correlation of the student's KAP. Results showed that the students' knowledge of antimicrobials had a correlation with attitude (p = 0.0126) and practice (p < 0.001), suggesting that public education on knowledge could influence the behaviors among the medical students. Taken all together, our findings show a need to strengthen the medical students' cogitation on antimicrobial attitude and practice of appropriate usage as an essential strategy to reduce intractable public health problems. Additional curriculum reforms will be needed to add more specific AMR-related lectures to raise awareness amongst medical students in China.

Staphylococcus aureus is an opportunistic pathogen that causes invasive infections in humans. In recent years, increasing studies have focused on the prevalence of S. aureus infections in adults; however, the epidemiology and molecular characteristics of S. aureus from Chinese pediatric patients remain unknown. The present study examined the population structure, antimicrobial resistance, and virulent factors of methicillin-resistant and -susceptible S. aureus isolated from Chinese pediatric patients from one medical center in eastern China. A total of 81 cases were screened with positive S. aureus infections among 864 pediatric patients between 2016 and 2022 in eastern China. Molecular analysis showed that ST22 (28.4%) and ST59 (13.6%) were the most typical strains, and associations between different clonal complex (CC) types/serotype types (ST) and the age of pediatric patients were observed in this study. CC398 was the predominant type in neonates under 1 month of age, while CC22 was mainly found in term-infant (under 1 year of age) and toddlers (over 1 year of age). Additionally, 17 S. aureus isolates were resistant to at least three antimicrobials and majority of them belonged to CC59. The blaZ gene was found in 59 isolates and mecA gene was present in 26 strains identified as methicillin-resistant. Numerous virulent factors were detected in S. aureus isolated from present pediatric patients. Remarkably, lukF-PV and lukS-PV were dominantly carried by CC22, tsst-1 genes were detected in CC188, CC7, and CC15, while exfoliative toxin genes were found only in CC121. Only 41.98% of the S. aureus isolates possessed scn gene, indicating that the sources of infections in pediatric patients may include both human-to-human transmissions as well as environmental and nosocomial infections. Together, the present study provided a phylogenetic and genotypic comparison of S. aureus from Chinese pediatric patients in Suzhou city. Our results suggested that the colonization of multi-drug resistant isolates of S. aureus may raise concern among pediatric patients, at least from the present medical center in eastern China.

Background: Large amounts of β-alanine are required in fine chemical and pharmaceutical synthesis and other fields. Profitable and green methods are required for the industrial production of β-alanine. Results: Replacing endogenous panD of Escherichia coli with heterologous CgpanD from Corynebacterium glutamicum enabled β-alanine synthesis of 0.67 g/L by strain B0016-082BB. Overexpressing CgpanD on both plasmids and chromosomes to enhance the rate-limiting step improved the β-alanine titer to 4.25 g/L in strain B0016-083BB/pPL451-panD with a slighter metabolic burden. Growth factors were introduced by addition of yeast extract, and 6.65 g/L of β-alanine was synthesized by strain B0016-083BB/pPL451-panD in the M9-3Y medium. Conclusions: Enhancement of the rate-limiting steps in the β-alanine biosynthetic pathway, recruitment of the temperature-sensitive inducible pL promoter, and optimization of the fermentation process could efficiently increase β-alanine production in E. coli.How to cite: Xua J, Zhua Y, Zhou Z. Systematic engineering of the rate-limiting step of β-alanine biosynthesis in Escherichia col. Electron J Biotechnol 2021;51. https://doi.org/10.1016/j.ejbt.2021.03.002.

Salmonella enterica serovar Typhimurium strain ATCC14028s is commercially available from multiple national type culture collections, and has been widely used since 1960 for quality control of growth media and experiments on fitness ("laboratory evolution"). ATCC14028s has been implicated in multiple cross-contaminations in the laboratory, and has also caused multiple laboratory infections and one known attempt at bioterrorism. According to hierarchical clustering of 3002 core gene sequences, ATCC14028s belongs to HierCC cluster HC20_373 in which most internal branch lengths are only one to three SNPs long. Many natural Typhimurium isolates from humans, domesticated animals and the environment also belong to HC20_373, and their core genomes are almost indistinguishable from those of laboratory strains. These natural isolates have infected humans in Ireland and Taiwan for decades, and are common in the British Isles as well as the Americas. The isolation history of some of the natural isolates confirms the conclusion that they do not represent recent contamination by the laboratory strain, and 10% carry plasmids or bacteriophages which have been acquired in nature by HGT from unrelated bacteria. We propose that ATCC14028s has repeatedly escaped from the laboratory environment into nature via laboratory accidents or infections, but the escaped micro-lineages have only a limited life span. As a result, there is a genetic gap separating HC20_373 from its closest natural relatives due to a divergence between them in the late 19th century followed by repeated extinction events of escaped HC20_373.

Intracellular pH (pHi) is a crucial parameter in cell biology; thus, a series of pH probes have been developed to determine pHi changes in living cells. However, more sensitive and non-perturbing ratiometric pH probes are needed for accurate pHi measurements. While the fluorescence of circular permutated YFP (cpYFP) is hypersensitive to pH changes due to its intrinsic properties, the single excitation peak of this protein restricts its capacity of becoming a rational type of pH sensor. Herein, we collected several cpYFP-based probes with dual excitation peaks and constructed their corresponding loss-of-function mutants to screen for a potential competent pH probe. The most sensitive probe was named NocPer. NocPer consists of cpYFP inserted into inactive-mutated GAF and AAA+, which are two regulatory domains of E. coli NorR, a nitric oxide (NO)-specific transcription factor. Fluorescence emission of NocPer peaks at 517 nm while exhibiting dual excitation peaks at 420 and 495 nm, which can be used for ratiometric imaging. This new pH sensor has a large ratio response dynamic (pH range of 7.0–11.0), which covers the physiological pH range (pH 7.0–8.0), and exhibits an approximately 3-fold higher fluorescent signal in response to a pH increase from 7.0 to 8.0 than that of pHluorin. Using NocPer, we discovered a new biological phenomenon in which NO exposure decreases the E. coli pHi, which led to the hypothesis that pathogens decrease their own pHi during infection. Further, we elucidated that the NO-induced inhibition of cytochrome c oxidase in the respiratory chain is responsible for the decline in pHi, which might represent a protective strategy of E. coli under NO stress conditions. Our results demonstrated that NocPer is a ratiometric pH probe with high sensitivity for the physiological pH range.

Abstract Background Promoter evolution by synthetic promoter library (SPL) is a powerful approach to development of functional synthetic promoters to synthetic biology. However, it requires much tedious and time-consuming screenings because of the plethora of different variants in SPL. Actually, a large proportion of mutants in the SPL are significantly lower in strength, which contributes only to fabrication of a promoter library with a continuum of strength. Thus, to effectively obtain the evolved synthetic promoter exhibiting higher strength, it is essential to develop novel strategies to construct mutant library targeting the pivotal region rather than the arbitrary region of the template promoter. In this study, a strategy termed stepwise evolution targeting the spacer of core promoter (SETarSCoP) was established in Bacillus subtilis to effectively evolve the strength of bacterial promoter. Results The native promoter, PsrfA, from B. subtilis, which exhibits higher strength than the strong promoter P43, was set as the parental template. According to the comparison of conservation of the spacer sequences between − 35 box and − 10 box among a set of strong and weak native promoter, it revealed that 7-bp sequence immediately upstream of the − 10 box featured in the regulation of promoter strength. Based on the conservative feature, two rounds of consecutive evolution were performed targeting the hot region of PsrfA. In the first round, a primary promoter mutation library (pPML) was constructed by mutagenesis targeting the 3-bp sequence immediately upstream of the − 10 box of the PsrfA. Subsequently, four evolved mutants from pPML were selected to construction of four secondary promoter mutation libraries (sPMLs) based on mutagenesis of the 4-bp sequence upstream of the first-round target. After the consecutive two-step evolution, the mutant PBH4 was identified and verified to be a highly evolved synthetic promoter. The strength of PBH4 was higher than PsrfA by approximately 3 times. Moreover, PBH4 also exhibited broad suitability for different cargo proteins, such as β-glucuronidase and nattokinase. The proof-of-principle test showed that SETarSCoP successfully evolved both constitutive and inducible promoters. Conclusion Comparing with the commonly used SPL strategy, SETarSCoP facilitates the evolution process to obtain strength-evolved synthetic bacterial promoter through fabrication and screening of small-scale mutation libraries. This strategy will be a promising method to evolve diverse bacterial promoters to expand the toolbox for synthetic biology.

Abstract Background Nattokinase (NK), which is a member of the subtilisin family, is a potent fibrinolytic enzyme that might be useful for thrombosis therapy. Extensive work has been done to improve its production for the food industry. The aim of our study was to enhance NK production by tandem promoters in Bacillus subtilis WB800. Results Six recombinant strains harboring different plasmids with a single promoter (P P43 , P HpaII , P BcaprE , P gsiB , P yxiE or P luxS ) were constructed, and the analysis of the fibrinolytic activity showed that P P43 and P HpaII exhibited a higher expression activity than that of the others. The NK yield that was mediated by P P43 and P HpaII reached 140.5 ± 3.9 FU/ml and 110.8 ± 3.6 FU/ml, respectively. These promoters were arranged in tandem to enhance the expression level of NK, and our results indicated that the arrangement of promoters in tandem has intrinsic effects on the NK expression level. As the number of repetitive P P43 or P HpaII increased, the expression level of NK was enhanced up to the triple-promoter, but did not increase unconditionally. In addition, the repetitive core region of P P43 or P HpaII could effectively enhance NK production. Eight triple-promoters with P P43 and P HpaII in different orders were constructed, and the highest yield of NK finally reached 264.2 ± 7.0 FU/ml, which was mediated by the promoter P HpaII -P HpaII -P P43 . The scale-up production of NK that was promoted by P HpaII -P HpaII -P P43 was also carried out in a 5-L fermenter, and the NK activity reached 816.7 ± 30.0 FU/mL. Conclusions Our studies demonstrated that NK was efficiently overproduced by tandem promoters in Bacillus subtilis. The highest fibrinolytic activity was promoted by P HpaII -P HpaII -P P43 , which was much higher than that had been reported in previous studies. These multiple tandem promoters were used successfully to control NK expression and might be useful for improving the expression level of the other genes.

Background: Most publicly available genomes of Salmonella enterica are from human disease in the US and the UK, or from domesticated animals in the US. Methods: Here we describe a historical collection of 10,000 strains isolated between 1891-2010 in 73 different countries. They encompass a broad range of sources, ranging from rivers through reptiles to the diversity of all S. enterica isolated on the island of Ireland between 2000 and 2005. Genomic DNA was isolated, and sequenced by Illumina short read sequencing. Results: The short reads are publicly available in the Short Reads Archive. They were also uploaded to EnteroBase, which assembled and annotated draft genomes. 9769 draft genomes which passed quality control were genotyped with multiple levels of multilocus sequence typing, and used to predict serovars. Genomes were assigned to hierarchical clusters on the basis of numbers of pair-wise allelic differences in core genes, which were mapped to genetic Lineages within phylogenetic trees. Conclusions: The University of Warwick/University College Cork (UoWUCC) project greatly extends the geographic sources, dates and core genomic diversity of publicly available S. enterica genomes. We illustrate these features by an overview of core genomic Lineages within 33,000 publicly available Salmonella genomes whose strains were isolated before 2011. We also present detailed examinations of HC400, HC900 and HC2000 hierarchical clusters within exemplar Lineages, including serovars Typhimurium, Enteritidis and Mbandaka. These analyses confirm the polyphyletic nature of multiple serovars while showing that discrete clusters with geographical specificity can be reliably recognized by hierarchical clustering approaches. The results also demonstrate that the genomes sequenced here provide an important counterbalance to the sampling bias which is so dominant in current genomic sequencing.

Tc toxins were originally identified in entomopathogenic bacteria, which are important as biological pest control agents. Tc toxins are heteromeric exotoxins composed of three subunit types, TcA, TcB, and TcC. The C-terminal portion of the TcC protein encodes the actual toxic domain, which is translocated into host cells by an injectosome nanomachine comprising the other subunits. Currently the pathogenic roles and distribution of Tc toxins among different bacterial genera remain unclear. Here we have performed a comprehensive genome-wide analysis, and established a database that includes 1,608 identified Tc loci containing 2,528 TcC proteins in 1,421 Gram-negative and positive bacterial genomes. Our findings indicate that TcCs conform to the architecture of typical polymorphic toxins, with C-terminal hypervariable regions (HVR) encoding more than 100 different classes of putative toxic domains, most of which have not been previously recognized. Based on further analysis of Tc loci in the genomes of all Salmonella and Yersinia strains in EnteroBase, a "two-level" evolutionary dynamics scenario is proposed for TcC homologues. This scenario implies that the conserved TcC RHS core domain plays a critical role in the taxonomical specific distribution of TcC HVRs. This study provides an extensive resource for the future development of Tc toxins as valuable agrochemical tools. It furthermore implies that Tc proteins, which are encoded by a wide range of pathogens, represent an important versatile toxin superfamily with diverse pathogenic mechanisms.

[This corrects the article DOI: 10.1371/journal.ppat.1002776.].

Background: Most publicly available genomes of Salmonella enterica are from human disease in the US and the UK, or from domesticated animals in the US. Methods: Here we describe a historical collection of 10,000 strains isolated between 1891-2010 in 73 different countries. They encompass a broad range of sources, ranging from rivers through reptiles to the diversity of all S. enterica isolated on the island of Ireland between 2000 and 2005. Genomic DNA was isolated, and sequenced by Illumina short read sequencing. Results: The short reads are publicly available in the Short Reads Archive. They were also uploaded to EnteroBase, which assembled and annotated draft genomes. 9769 draft genomes which passed quality control were genotyped with multiple levels of multilocus sequence typing, and used to predict serovars. Genomes were assigned to hierarchical clusters on the basis of numbers of pair-wise allelic differences in core genes, which were mapped to genetic Lineages within phylogenetic trees. Conclusions: The University of Warwick/University College Cork (UoWUCC) project greatly extends the geographic sources, dates and core genomic diversity of publicly available S. enterica genomes. We illustrate these features by an overview of core genomic Lineages within 33,000 publicly available Salmonella genomes whose strains were isolated before 2011. We also present detailed examinations of HC400, HC900 and HC2000 hierarchical clusters within exemplar Lineages, including serovars Typhimurium, Enteritidis and Mbandaka. These analyses confirm the polyphyletic nature of multiple serovars while showing that discrete clusters with geographical specificity can be reliably recognized by hierarchical clustering approaches. The results also demonstrate that the genomes sequenced here provide an important counterbalance to the sampling bias which is so dominant in current genomic sequencing.

Bacillus subtilis is an important Gram-positive bacterium for industrial biotechnology, which has been widely used to produce diverse high-value added chemicals and industrially and pharmaceutically relevant proteins. Robust and versatile toolkits for genome editing in B. subtilis are highly demanding to design higher version chassis. Although the Streptococcus pyogenes (Sp) CRISPR-Cas9 has been extensively adapted for genome engineering of multiple bacteria, it has many defects, such as higher molecular weight which leads to higher carrier load, low deletion efficiency and complexity of sgRNA construction for multiplex genome editing. Here, we designed a CRISPR-Cpf1-based toolkit employing a type V Cas protein, Cpf1 from Francisella novicida. Using this platform, we precisely deleted single gene and gene cluster in B. subtilis with high editing efficiency, such as sacA, ganA, ligD & ligV, and bac operon. Especially, an extremely large gene cluster of 38 kb in B. subtilis genome was accurately deleted from the genome without introducing any unexpected mutations. Meanwhile, the synthetic platform was further upgraded to a version for multiplex genome editing, upon which two genes sacA and aprE were precisely and efficiently deleted using only one plasmid harboring two targeting sequences. In addition, we successfully inserted foreign genes into the genome of the chassis using the CRISPR-Cpf1 platform. Our work highlighted the availability of CRISPR-Cpf1 to gene manipulation in B. subtilis, including the flexible deletion of a single gene and multiple genes or a gene cluster, and gene knock-in. The designed genome-editing platform was easily and broadly applicable to other microorganisms. The novel platforms we constructed in this study provide a promising tool for efficient genome editing in diverse bacteria.

Nitrile hydratase (NHase, EC 4.2.1.84) is one type of metalloenzyme participating in the biotransformation of nitriles into amides. Given its catalytic specificity in amide production and eco-friendliness, NHase has overwhelmed its chemical counterpart during the past few decades. However, unclear catalytic mechanism, low thermostablity, and narrow substrate specificity limit the further application of NHase. During the past few years, numerous studies on the theoretical and industrial aspects of NHase have advanced the development of this green catalyst. This review critically focuses on NHase research from recent years, including the natural distribution, gene types, posttranslational modifications, expression, proposed catalytic mechanism, biochemical properties, and potential applications of NHase. The developments of NHase described here are not only useful for further application of NHase, but also beneficial for the development of the fields of biocatalysis and biotransformation.

A robust and portable expression system is of great importance in enzyme production, metabolic engineering, and synthetic biology, which maximizes the performance of the engineered system. In this study, a tailor-made cobalt-induced expression system (CIES) was developed for low-cost and eco-friendly nitrile hydratase (NHase) production. First, the strong promoter Pveg from Bacillus subtilis, the Ni(II)/Co(II) responsive repressor RcnR, and its operator were reorganized to construct a CIES. In this system, the expression of reporter green fluorescent protein (GFP) was specifically triggered by Co(II) over a broad range of concentration. The performance of the cobalt-induced system was evolved to version 2.0 (CIES 2.0) for adaptation to different concentrations of Co(II) through programming a homeostasis system that rebalances cobalt efflux and influx with RcnA and NiCoT, respectively. Harnessing these synthetic platforms, the induced expression of NHase was coupled with enzyme maturation by Co(II) in a synchronizable manner without requiring additional inducers, which is a unique feature relative to other induced systems for production of NHase. The yield of NHase was 111.2 ± 17.9 U/ml using CIES and 114.9 ± 1.4 U/ml using CIES 2.0, which has a producing capability equivalent to that of commonly used isopropyl thiogalactoside (IPTG)-induced systems. In a scale-up system using a 5-L fermenter, the yielded enzymatic activity reached 542.2 ± 42.8 U/ml, suggesting that the designer platform for NHase is readily applied to the industry. The design of CIES in this study not only provided a low-cost and eco-friendly platform to overproduce NHase but also proposed a promising pipeline for development of synthetic platforms for expression of metalloenzymes.

Precise regulation of gene expression is fundamental for tailor-made gene circuit design in synthetic biology. Current strategies for this type of development are mainly based on directed evolution beginning with a native promoter template. The performances of engineered promoters are usually limited by the growth phase because only one promoter is recognized by one type of sigma factor (σ). Here, we constructed multiple-σ recognizable artificial hybrid promoters (AHPs) composed of tandems of dual and triple natural minimal promoters (NMPs). These NMPs, which use σA, σH and σW, had stable functions in different growth phases. The functions of these NMPs resulted from an effect called transcription compensation, in which AHPs sequentially use one type of σ in the corresponding growth phase. The strength of the AHPs was influenced by the combinatorial order of each NMP and the length of the spacers between the NMPs. More importantly, the output of the precise regulation was achieved by equipping AHPs with synthetic ribosome binding sites and by redesigning them for induced systems. This strategy might offer promising applications to rationally design robust synthetic promoters in diverse chassis to spur the construction of more complex gene circuits, which will further the development of synthetic biology.

Summary: Several phage-tail-like nanomachines were shown to play an important role in the interactions between bacteria and their eukaryotic hosts. These apparatuses appear to represent a new injection paradigm. Here, with three verified extracellular contractile injection systems (eCISs), a protein profile and genomic context-based iterative approach was applied to identify 631 eCIS-like loci from the 11,699 publicly available complete bacterial genomes. The eCIS superfamily, which is phylogenetically diverse and sub-divided into six families, is distributed among Gram-negative and -positive bacteria in addition to archaea. Our results show that very few bacteria are seen to possess intact operons of both eCIS and type VI secretion systems (T6SSs). An open access online database of all detected eCIS-like loci is presented to facilitate future studies. The presence of this bacterial injection machine in a multitude of organisms suggests that it may play an important ecological role in the life cycles of many bacteria. : Based on genetic information from three recently identified extracellular contractile injection systems (eCISs), Chen et al. identify 631 eCIS-like loci from 11,699 publicly available complete bacterial genomes, including archaea, and Gram-negative and Gram-positive bacteria. These loci may represent a class of protein delivery systems to serve a variety of roles in these microorganisms. Keywords: Secretion system, bacteria pathogenicity, eCIS, T6SS, database

β-alanine is widely used as an intermediate in industrial production. However, the low production of microbial cell factories limits its further application. Here, to improve the biosynthesis production of β-alanine in Escherichia coli, multivariate modular metabolic engineering was recruited to manipulate the β-alanine biosynthesis pathway through keeping the balance of metabolic flux among the whole metabolic network. The β-alanine biosynthesis pathway was separated into three modules: the β-alanine biosynthesis module, TCA module, and glycolysis module. Global regulation was performed throughout the entire β-alanine biosynthesis pathway rationally and systematically by optimizing metabolic flux, overcoming metabolic bottlenecks and weakening branch pathways. As a result, metabolic flux was channeled in the direction of β-alanine biosynthesis without huge metabolic burden, and 37.9 g/L β-alanine was generated by engineered Escherichia coli strain B0016-07 in fed-batch fermentation. This study was meaningful to the synthetic biology of β-alanine industrial production.

The strategy of anaerobic biosynthesis of β-alanine by Escherichia coli (E. coli) has been reported. However, the low energy production under anaerobic condition limited cell growth and then affected the production efficiency of β-alanine. Here, the adaptive laboratory evolution was carried out to improve energy production of E. coli lacking phosphoenolpyruvate carboxylase under anaerobic condition. Five mutants were isolated and analyzed. Sequence analysis showed that most of the consistent genetic mutations among the mutants were related with pyruvate accumulation, indicating that pyruvate accumulation enabled the growth of the lethal parent. It is possible that the accumulated pyruvate provides sufficient precursors for energy generation and CO2 fixing reaction catalyzed by phosphoenolpyruvate carboxykinase. B0016-100BB (B0016-090BB, recE::FRT, mhpF::FRT, ykgF::FRT, mhpB:: mhpB *, mhpD:: mhpD *, rcsA:: rcsA *) was engineered based on the analysis of the genetic mutations among the mutants for the biosynthesis of β-alanine. Along with the recruitment of glycerol as the sole carbon source, 1.07 g/L β-alanine was generated by B0016-200BB (B0016-100BB, aspA::FRT) harboring pET24a-panD-AspDH, which was used for overexpression of two key enzymes in β-alanine fermentation process. Compared with the starting strain, which can hardly generate β-alanine under anaerobic condition, the production efficiency of β-alanine of the engineered cell factory was significantly improved.

High thermostability and catalytic activity are key properties for nitrile hydratase (NHase, EC 4.2.1.84) as a well-industrialized catalyst. In this study, rational design was applied to tailor the thermostability of NHase from Pseudonocardia thermophila JCM3095 (PtNHase) by combining FireProt server prediction and molecular dynamics (MD) simulation. Site-directed mutagenesis of non-catalytic residues provided by the rational design was subsequentially performed. The positive multiple-point mutant, namely, M10 (αI5P/αT18Y/αQ31L/αD92H/βA20P/βP38L/βF118W/βS130Y/βC189N/βC218V), was obtained and further analyzed. The Melting temperature (Tm) of the M10 mutant showed an increase by 3.2 °C and a substantial increase in residual activity of the enzyme at elevated temperatures was also observed. Moreover, the M10 mutant also showed a 2.1-fold increase in catalytic activity compared with the wild-type PtNHase. Molecular docking and MD simulations demonstrated better substrate affinity and improved thermostability for the mutant.

For many decades, Salmonella enterica has been subdivided by serological properties into serovars or further subdivided for epidemiological tracing by a variety of diagnostic tests with higher resolution. Recently, it has been proposed that so-called eBurst groups (eBGs) based on the alleles of seven housekeeping genes (legacy multilocus sequence typing [MLST]) corresponded to natural populations and could replace serotyping. However, this approach lacks the resolution needed for epidemiological tracing and the existence of natural populations had not been independently validated by independent criteria. Here, we describe EnteroBase, a web-based platform that assembles draft genomes from Illumina short reads in the public domain or that are uploaded by users. EnteroBase implements legacy MLST as well as ribosomal gene MLST (rMLST), core genome MLST (cgMLST), and whole genome MLST (wgMLST) and currently contains over 100,000 assembled genomes from Salmonella. It also provides graphical tools for visual interrogation of these genotypes and those based on core single nucleotide polymorphisms (SNPs). eBGs based on legacy MLST are largely consistent with eBGs based on rMLST, thus demonstrating that these correspond to natural populations. rMLST also facilitated the selection of representative genotypes for SNP analyses of the entire breadth of diversity within Salmonella. In contrast, cgMLST provides the resolution needed for epidemiological investigations. These observations show that genomic genotyping, with the assistance of EnteroBase, can be applied at all levels of diversity within the Salmonella genus.

Phenylalanine ammonia-lyase (RgPAL) from Rhodotorula glutinis JN-1 stereoselectively catalyzes the conversion of the l-phenylalanine into trans-cinnamic acid and ammonia, and was used in chiral resolution of dl-phenylalanine to produce the d-phenylalanine under acidic condition. However, the optimum pH of RgPAL is 9 and the RgPAL exhibits low catalytic efficiency at acidic side. Therefore, a mutant RgPAL with a lower optimum pH is expected. Based on catalytic mechanism and structure analysis, we constructed a mutant RgPAL-Q137E by site-directed mutagenesis, and found that this mutant had an extended optimum pH 7–9 with activity of 1.8-fold higher than that of the wild type at pH 7. As revealed by Friedel–Crafts-type mechanism of RgPAL, the improvement of the RgPAL-Q137E might be due to the negative charge of Glu137 which could stabilize the intermediate transition states through electrostatic interaction. The RgPAL-Q137E mutant was used to resolve the racemic dl-phenylalanine, and the conversion rate and the eeD value of d-phenylalanine using RgPAL-Q137E at pH 7 were increased by 29% and 48%, and achieved 93% and 86%, respectively. This work provides an effective strategy to shift the optimum pH which is favorable to further applications of RgPAL.

Nitrile hydratase (NHase) from Rhodococcus rhodochrous J1 is widely used for industrial production of acrylamide and nicotinamide. However, the two types of NHases (L-NHase and H-NHase) from R. rhodochrous J1 were only slightly expressed in E. coli by routine methods, which limits the comprehensive and systematic characterization of the enzyme properties. We successfully expressed the two types of recombinant NHases in E. coli by codon-optimization, engineering of Ribosome Binding Site (RBS) and spacer sequences. The specific activity of the purified L-NHase and H-NHase were 400 U/mg and 234 U/mg, respectively. The molecular mass of L-NHase and H-NHase was identified to be 94 kDa and 504 kDa, respectively, indicating that the quaternary structure of the two types of NHases was the same as those in R. rhodochrous J1. H-NHase exhibited higher substrate and product tolerance than L-NHase. Moreover, higher activity and shorter culture time were achieved in recombinant E. coli, and the whole cell catalyst of recombinant E. coli harboring H-NHase has equivalent efficiency in tolerance to the high-concentration product relative to that in R. rhodochrous J1. These results indicate that biotransformation of nitrile by R. rhodochrous J1 represents a potential alternative to NHase-producing E. coli.

ABSTRACT For 100 years, it has been obvious that Salmonella enterica strains sharing the serotype with the formula 1,4,[5],12:b:1,2—now known as Paratyphi B—can cause diseases ranging from serious systemic infections to self-limiting gastroenteritis. Despite considerable predicted diversity between strains carrying the common Paratyphi B serotype, there remain few methods that subdivide the group into groups that are congruent with their disease phenotypes. Paratyphi B therefore represents one of the canonical examples in Salmonella where serotyping combined with classical microbiological tests fails to provide clinically informative information. Here, we use genomics to provide the first high-resolution view of this serotype, placing it into a wider genomic context of the Salmonella enterica species. These analyses reveal why it has been impossible to subdivide this serotype based upon phenotypic and limited molecular approaches. By examining the genomic data in detail, we are able to identify common features that correlate with strains of clinical importance. The results presented here provide new diagnostic targets, as well as posing important new questions about the basis for the invasive disease phenotype observed in a subset of strains. IMPORTANCE Salmonella enterica strains carrying the serotype Paratyphi B have long been known to possess Jekyll and Hyde characteristics; some cause gastroenteritis, while others cause serious invasive disease. Understanding what makes up the population of strains carrying this serotype, as well as the source of their invasive disease, is a 100-year-old puzzle that we address here using genomics. Our analysis provides the first high-resolution view of this serotype, placing strains carrying serotype Paratyphi B into the wider genomic context of the Salmonella enterica species. This work reveals a history of disease dating back to the middle ages, caused by a group of distinct lineages with various abilities to cause invasive disease. By quantifying the key genomic differences between the invasive and noninvasive populations, we are able to identify key virulence-related targets that can form the basis of simple, rapid, point-of-care tests.

Vibrio cholerae has caused massive outbreaks and even trans-continental epidemics. In 2008 and 2010, at least 3 remarkable cholera outbreaks occurred in Hainan, Anhui and Jiangsu provinces of China. To address the possible transmissions and the relationships to the 7th pandemic strains of those 3 outbreaks, we sequenced the whole genomes of the outbreak isolates and compared with the global isolates from the 7th pandemic. The three outbreaks in this study were caused by a cluster of V. cholerae in clade 3.B which is parallel to the clade 3.C that was transmitted from Nepal to Haiti and caused an outbreak in 2010. Pan-genome analysis provided additional evolution information on the mobile element and acquired multiple antibiotic resistance genes. We suggested that clade 3.B should be monitored because the multiple antibiotic resistant characteristics of this clade and the 'amplifier' function of China in the global transmission of current Cholera pandemic. We also show that dedicated whole genome sequencing analysis provided more information than the previous techniques and should be applied in the disease surveillance networks.

In 2013 Zhou et al. concluded that Salmonella enterica serovar Agona represents a genetically monomorphic lineage of recent ancestry, whose most recent common ancestor existed in 1932, or earlier. The Abstract stated 'Agona consists of three lineages with minimal mutational diversity: only 846 single nucleotide polymorphisms (SNPs) have accumulated in the non-repetitive, core genome since Agona evolved in 1932 and subsequently underwent a major population expansion in the 1960s.' These conclusions have now been criticized by Pettengill, who claims that the evolutionary models used to date Agona may not have been appropriate, the dating estimates were inaccurate, and the age of emergence of Agona should have been qualified by an upper limit reflecting the date of its divergence from an outgroup, serovar Soerenga. We dispute these claims. Firstly, Pettengill's analysis of Agona is not justifiable on technical grounds. Secondly, an upper limit for divergence from an outgroup would only be meaningful if the outgroup were closely related to Agona, but close relatives of Agona are yet to be identified. Thirdly, it is not possible to reliably date the time of divergence between Agona and Soerenga. We conclude that Pettengill's criticism is comparable to a tempest in a teapot.

An efficient enzymatic process was developed to produce optically pure D-phenylalanine through asymmetric resolution of the racemic DL-phenylalanine using immobilized phenylalanine ammonia-lyase (RgPAL) from Rhodotorula glutinis JN-1. RgPAL was immobilized on a modified mesoporous silica support (MCM-41-NH-GA). The resulting MCM-41-NH-GA-RgPAL showed high activity and stability. The resolution efficiency using MCM-41-NH-GA-RgPAL in a recirculating packed-bed reactor (RPBR) was higher than that in a stirred-tank reactor. Under optimal operational conditions, the volumetric conversion rate of L-phenylalanine and the productivity of D-phenylalanine reached 96.7 mM h⁻¹ and 0.32 g L⁻¹ h⁻¹, respectively. The optical purity (eeD) of D-phenylalanine exceeded 99%. The RPBR ran continuously for 16 batches, the conversion ratio did not decrease. The reactor was scaled up 25-fold, and the productivity of D-phenylalanine (eeD>99%) in the scaled-up reactor reached 7.2 g L⁻¹ h⁻¹. These results suggest that the resolution process is an alternative method to produce highly pure D-phenylalanine.

Besides the catalytic ability, many enzymes contain conserved domains to perform some other physiological functions. However, sometimes these conserved domains were unnecessary or even detrimental to the catalytic process for industrial application of the enzymes. In this study, based on homology modeling and molecular dynamics simulations, we found that Bacillus subtilis aminopeptidase contained a thermal sensitive domain (protease-associated domain) in the non-catalytic region, and predicted that deletion of this flexible domain can enhance the structure stability. This prediction was then verified by the deletion of protease-associated domain from the wild-type enzyme. The thermal stability analysis showed that deletion of this domain improved the T50 (the temperature required to reduce initial activity by 50% in 30 min) of the enzyme from 71 °C to 77 °C. The melting temperature (Tm) of the enzyme also increased, which was measured by thermal denaturation experiments using circular dichroism spectroscopy. Further studies indicated that this deletion did not affect the activity and specificity of the enzyme toward aminoacyl-p-nitroanilines, but improved its hydrolytic ability toward a 12-aa-long peptide (LKRLKRFLKRLK) and soybean protein. These findings suggested the possibility of a simple technique for enzyme modification and the artificial enzyme obtained here was more suitable for the protein hydrolysis in food industry than the wild-type enzyme.

Salmonella enterica serovar Agona has caused multiple food-borne outbreaks of gastroenteritis since it was first isolated in 1952. We analyzed the genomes of 73 isolates from global sources, comparing five distinct outbreaks with sporadic infections as well as food contamination and the environment. Agona consists of three lineages with minimal mutational diversity: only 846 single nucleotide polymorphisms (SNPs) have accumulated in the non-repetitive, core genome since Agona evolved in 1932 and subsequently underwent a major population expansion in the 1960s. Homologous recombination with other serovars of S. enterica imported 42 recombinational tracts (360 kb) in 5/143 nodes within the genealogy, which resulted in 3,164 additional SNPs. In contrast to this paucity of genetic diversity, Agona is highly diverse according to pulsed-field gel electrophoresis (PFGE), which is used to assign isolates to outbreaks. PFGE diversity reflects a highly dynamic accessory genome associated with the gain or loss (indels) of 51 bacteriophages, 10 plasmids, and 6 integrative conjugational elements (ICE/IMEs), but did not correlate uniquely with outbreaks. Unlike the core genome, indels occurred repeatedly in independent nodes (homoplasies), resulting in inaccurate PFGE genealogies. The accessory genome contained only few cargo genes relevant to infection, other than antibiotic resistance. Thus, most of the genetic diversity within this recently emerged pathogen reflects changes in the accessory genome, or is due to recombination, but these changes seemed to reflect neutral processes rather than Darwinian selection. Each outbreak was caused by an independent clade, without universal, outbreak-associated genomic features, and none of the variable genes in the pan-genome seemed to be associated with an ability to cause outbreaks.

An Escherichia coli O157:H7 outbreak in China in 1999 caused 177 deaths due to hemolytic uremic syndrome. Sixteen outbreak associated isolates were found to belong to a new clone, sequence type 96 (ST96), based on multilocus sequence typing of 15 housekeeping genes. Whole genome sequencing of an outbreak isolate, Xuzhou21, showed that the isolate is phylogenetically closely related to the Japan 1996 outbreak isolate Sakai, both of which share the most recent common ancestor with the US outbreak isolate EDL933. The levels of IL-6 and IL-8 of peripheral blood mononuclear cells induced by Xuzhou21 and Sakai were significantly higher than that induced by EDL933. Xuzhou21 also induced a significantly higher level of IL-8 than Sakai while both induced similar levels of IL-6. The expression level of Shiga toxin 2 in Xuzhou21 induced by mitomycin C was 68.6 times of that under non-inducing conditions, twice of that induced in Sakai (32.7 times) and 15 times higher than that induced in EDL933 (4.5 times). Our study shows that ST96 is a novel clone and provided significant new insights into the evolution of virulence of E. coli O157:H7.

Salmonella enterica subspecies enterica is traditionally subdivided into serovars by serological and nutritional characteristics. We used Multilocus Sequence Typing (MLST) to assign 4,257 isolates from 554 serovars to 1092 sequence types (STs). The majority of the isolates and many STs were grouped into 138 genetically closely related clusters called eBurstGroups (eBGs). Many eBGs correspond to a serovar, for example most Typhimurium are in eBG1 and most Enteritidis are in eBG4, but many eBGs contained more than one serovar. Furthermore, most serovars were polyphyletic and are distributed across multiple unrelated eBGs. Thus, serovar designations confounded genetically unrelated isolates and failed to recognize natural evolutionary groupings. An inability of serotyping to correctly group isolates was most apparent for Paratyphi B and its variant Java. Most Paratyphi B were included within a sub-cluster of STs belonging to eBG5, which also encompasses a separate sub-cluster of Java STs. However, diphasic Java variants were also found in two other eBGs and monophasic Java variants were in four other eBGs or STs, one of which is in subspecies salamae and a second of which includes isolates assigned to Enteritidis, Dublin and monophasic Paratyphi B. Similarly, Choleraesuis was found in eBG6 and is closely related to Paratyphi C, which is in eBG20. However, Choleraesuis var. Decatur consists of isolates from seven other, unrelated eBGs or STs. The serological assignment of these Decatur isolates to Choleraesuis likely reflects lateral gene transfer of flagellar genes between unrelated bacteria plus purifying selection. By confounding multiple evolutionary groups, serotyping can be misleading about the disease potential of S. enterica. Unlike serotyping, MLST recognizes evolutionary groupings and we recommend that Salmonella classification by serotyping should be replaced by MLST or its equivalents.

BACKGROUND: The ciliated protozoan Tetrahymena thermophila is a well-studied single-celled eukaryote model organism for cellular and molecular biology. However, the lack of extensive T. thermophila cDNA libraries or a large expressed sequence tag (EST) database limited the quality of the original genome annotation. METHODOLOGY/PRINCIPAL FINDINGS: This RNA-seq study describes the first deep sequencing analysis of the T. thermophila transcriptome during the three major stages of the life cycle: growth, starvation and conjugation. Uniquely mapped reads covered more than 96% of the 24,725 predicted gene models in the somatic genome. More than 1,000 new transcribed regions were identified. The great dynamic range of RNA-seq allowed detection of a nearly six order-of-magnitude range of measurable gene expression orchestrated by this cell. RNA-seq also allowed the first prediction of transcript untranslated regions (UTRs) and an updated (larger) size estimate of the T. thermophila transcriptome: 57 Mb, or about 55% of the somatic genome. Our study identified nearly 1,500 alternative splicing (AS) events distributed over 5.2% of T. thermophila genes. This percentage represents a two order-of-magnitude increase over previous EST-based estimates in Tetrahymena. Evidence of stage-specific regulation of alternative splicing was also obtained. Finally, our study allowed us to completely confirm about 26.8% of the genes originally predicted by the gene finder, to correct coding sequence boundaries and intron-exon junctions for about a third, and to reassign microarray probes and correct earlier microarray data. CONCLUSIONS/SIGNIFICANCE: RNA-seq data significantly improve the genome annotation and provide a fully comprehensive view of the global transcriptome of T. thermophila. To our knowledge, 5.2% of T. thermophila genes with AS is the highest percentage of genes showing AS reported in a unicellular eukaryote. Tetrahymena thus becomes an excellent unicellular model eukaryote in which to investigate mechanisms of alternative splicing.

Self-subunit swapping is one of the post-translational maturation of the cobalt-containing nitrile hydratase (Co-NHase) family of enzymes. All of these NHases possess a gene organization of , which allows the activator protein to easily form a mediatory complex with the α-subunit of the NHase after translation. Here, we discovered that the incorporation of cobalt into another type of Co-NHase, with a gene organization of , was also dependent on self-subunit swapping. We successfully isolated a recombinant NHase activator protein (P14K) of Pseudomonas putida NRRL-18668 by adding a Strep-tag N-terminal to the P14K gene. P14K was found to form a complex [α(StrepP14K)(2)] with the α-subunit of the NHase. The incorporation of cobalt into the NHase of P. putida was confirmed to be dependent on the α-subunit substitution between the cobalt-containing α(StrepP14K)(2) and the cobalt-free NHase. Cobalt was inserted into cobalt-free α(StrepP14K)(2) but not into cobalt-free NHase, suggesting that P14K functions not only as a self-subunit swapping chaperone but also as a metallochaperone. In addition, NHase from P. putida was also expressed by a mutant gene that was designed with a order. Our findings expand the general features of self-subunit swapping maturation.

Mycoplasma, the smallest self-replicating organism with a minimal metabolism and little genomic redundancy, is expected to be a close approximation to the minimal set of genes needed to sustain bacterial life. This study employs comparative evolutionary analysis of twenty Mycoplasma genomes to gain an improved understanding of essential genes. By analyzing the core genome of mycoplasmas, we finally revealed the conserved essential genes set for mycoplasma survival. Further analysis showed that the core genome set has many characteristics in common with experimentally identified essential genes. Several key genes, which are related to DNA replication and repair and can be disrupted in transposon mutagenesis studies, may be critical for bacteria survival especially over long period natural selection. Phylogenomic reconstructions based on 3,355 homologous groups allowed robust estimation of phylogenetic relatedness among mycoplasma strains. To obtain deeper insight into the relative roles of molecular evolution in pathogen adaptation to their hosts, we also analyzed the positive selection pressures on particular sites and lineages. There appears to be an approximate correlation between the divergence of species and the level of positive selection detected in corresponding lineages.

Although over 50 complete Escherichia coli/Shigella genome sequences are available, it is only for closely related strains, for example the O55:H7 and O157:H7 clones of E. coli, that we can assign differences to individual evolutionary events along specific lineages. Here we sequence the genomes of 14 isolates of a uropathogenic E. coli clone that persisted for 3 years within a household, including a dog, causing a urinary tract infection (UTI) in the dog after 2 years. The 20 mutations observed fit a single tree that allows us to estimate the mutation rate to be about 1.1 per genome per year, with minimal evidence for adaptive change, including in relation to the UTI episode. The host data also imply at least 6 host transfer events over the 3 years, with 2 lineages present over much of that period. To our knowledge, these are the first direct measurements for a clone in a well-defined host community that includes rates of mutation and host transmission. There is a concentration of non-synonymous mutations associated with 2 transfers to the dog, suggesting some selection pressure from the change of host. However, there are no changes to which we can attribute the UTI event in the dog, which suggests that this occurrence after 2 years of the clone being in the household may have been due to chance, or some unknown change in the host or environment. The ability of a UTI strain to persist for 2 years and also to transfer readily within a household has implications for epidemiology, diagnosis, and clinical intervention.

There are 29 E. coli genome sequences available, mostly related to studies of species diversity or mode of pathogenicity, including two genomes of the well-known O157:H7 clone. However, there have been no genome studies of closely related clones aimed at exposing the details of evolutionary change. Here we sequenced the genome of an O55:H7 strain, closely related to the major pathogenic O157:H7 clone, with published genome sequences, and undertook comparative genomic and proteomic analysis. We were able to allocate most differences between the genomes to individual mutations, recombination events, or lateral gene transfer events, in specific lineages. Major differences include a type II secretion system present only in the O55:H7 chromosome, fewer type III secretion system effectors in O55:H7, and 19 phage genomes or phagelike elements in O55:H7 compared to 23 in O157:H7, with only three common to both. Many other changes were found in both O55:H7 and O157:H7 lineages, but in general there has been more change in the O157:H7 lineages. For example, we found 50% more synonymous mutational substitutions in O157:H7 compared to O55:H7. The two strains also diverged at the proteomic level. Mutational synonymous SNPs were used to estimate a divergence time of 400 years using a new clock rate, in contrast to 14,000 to 70,000 years using the traditional clock rates. The same approaches were applied to three closely related extraintestinal pathogenic E. coli genomes, and similar levels of mutation and recombination were found. This study revealed for the first time the full range of events involved in the evolution of the O157:H7 clone from its O55:H7 ancestor, and suggested that O157:H7 arose quite recently. Our findings also suggest that E. coli has a much lower frequency of recombination relative to mutation than was observed in a comparable study of a Vibrio cholerae lineage.