Acetylation coordinates the crosstalk between carbon metabolism and ammonium assimilation in Salmonella enterica.

Enteric bacteria use up to 15% of their cellular energy for ammonium assimilation via glutamine synthetase (GS)/glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH) in response to varying ammonium availability. However, the sensory mechanisms for effective and appropriate coordination between carbon metabolism and ammonium assimilation have not been fully elucidated. Here, we report that in Salmonella enterica, carbon metabolism coordinates the activities of GS/GDH via functionally reversible protein lysine acetylation. Glucose promotes Pat acetyltransferase-mediated acetylation and activation of adenylylated GS. Simultaneously, glucose induces GDH acetylation to inactivate the enzyme by impeding its catalytic centre, which is reversed upon GDH deacetylation by deacetylase CobB. Molecular dynamics (MD) simulations indicate that adenylylation is required for acetylation-dependent activation of GS. We show that acetylation and deacetylation occur within minutes of "glucose shock" to promptly adapt to ammonium/carbon variation and finely balance glutamine/glutamate synthesis. Finally, in a mouse infection model, reduced S. enterica growth caused by the expression of adenylylation-mimetic GS is rescued by acetylation-mimicking mutations. Thus, glucose-driven acetylation integrates signals from ammonium assimilation and carbon metabolism to fine-tune bacterial growth control.

Zymograph profiling reveals a divergent evolution of sirtuin that may originate from class III enzymes.

Sirtuins are a group of NAD+-dependent deacylases that conserved in three domains of life and comprehensively involved in the regulation of gene transcription, chromosome segregation, RNA splicing, apoptosis, and aging. Previous studies in mammalian cells have revealed that sirtuins not only exist as multiple copies, but also show distinct deacylase activities in addition to deacetylation. However, the understanding of sirtuin zymographs in other organisms with respect to molecular evolution remains at an early stage. Here, we systematically analyze the sirtuin activities in representative species from archaea, bacteria, and eukaryotes, using both the HPLC assay and a 7-amino-4-methylcoumarin-based fluorogenic method. Global profiling suggests that the deacylase activities of sirtuins could be divided into three categories and reveals undifferentiated zymographs of class III sirtuins, especially for those from bacteria and archaea. Nevertheless, initial differentiation of enzymatic activity was also observed for the class III sirtuins at both paralog and ortholog levels. Further phylogenetic analyses support a divergent evolution of sirtuin that may originate from class III sirtuins. Together, this work demonstrates a comprehensive panorama of sirtuin zymographs and provides new insights into the cellular specific regulation and molecular evolution of sirtuins.

A Noncoding A-to-U Kozak Site Change Related to the High Transmissibility of Alpha, Delta, and Omicron VOCs.

Three prevalent SARS-CoV-2 variants of concern (VOCs) emerged and caused epidemic waves. It is essential to uncover advantageous mutations that cause the high transmissibility of VOCs. However, viral mutations are tightly linked, so traditional population genetic methods, including machine learning-based methods, cannot reliably detect mutations conferring a fitness advantage. In this study, we developed an approach based on the sequential occurrence order of mutations and the accelerated furcation rate in the pandemic-scale phylogenomic tree. We analyzed 3,777,753 high-quality SARS-CoV-2 genomic sequences and the epidemiology metadata using the Coronavirus GenBrowser. We found that two noncoding mutations at the same position (g.a28271-/u) may be crucial to the high transmissibility of Alpha, Delta, and Omicron VOCs although the noncoding mutations alone cannot increase viral transmissibility. Both mutations cause an A-to-U change at the core position -3 of the Kozak sequence of the N gene and significantly reduce the protein expression ratio of ORF9b to N. Using a convergent evolutionary analysis, we found that g.a28271-/u, S:p.P681H/R, and N:p.R203K/M occur independently on three VOC lineages, suggesting that coordinated changes of S, N, and ORF9b proteins are crucial to high viral transmissibility. Our results provide new insights into high viral transmissibility co-modulated by advantageous noncoding and nonsynonymous changes.

Mycobacterial fatty acid catabolism is repressed by FdmR to sustain lipogenesis and virulence.

Host-derived fatty acids are an important carbon source for pathogenic mycobacteria during infection. How mycobacterial cells regulate the catabolism of fatty acids to serve the pathogenicity, however, remains unknown. Here, we identified a TetR-family transcriptional factor, FdmR, as the key regulator of fatty acid catabolism in the pathogen Mycobacterium marinum by combining use of transcriptomics, chromatin immunoprecipitation followed by sequencing, dynamic 13C-based flux analysis, metabolomics, and lipidomics. An M. marinum mutant deficient in FdmR was severely attenuated in zebrafish larvae and adult zebrafish. The mutant showed defective growth but high substrate consumption on fatty acids. FdmR was identified as a long-chain acyl-coenzyme A (acyl-CoA)-responsive repressor of genes involved in fatty acid degradation and modification. We demonstrated that FdmR functions as a valve to direct the flux of exogenously derived fatty acids away from ß-oxidation toward lipid biosynthesis, thereby avoiding the overactive catabolism and accumulation of biologically toxic intermediates. Moreover, we found that FdmR suppresses degradation of long-chain acyl-CoAs endogenously synthesized through the type I fatty acid synthase. By modulating the supply of long-chain acyl-CoAs for lipogenesis, FdmR controls the abundance and chain length of virulence-associated lipids and mycolates and plays an important role in the impermeability of the cell envelope. These results reveal that despite the fact that host-derived fatty acids are used as an important carbon source, overactive catabolism of fatty acids is detrimental to mycobacterial cell growth and pathogenicity. This study thus presents FdmR as a potentially attractive target for chemotherapy.

The use of amino acids and their derivates to mitigate against pesticide-induced toxicity.

Exposure to pesticides induces oxidative stress and deleterious effects on various tissues in non-target organisms. Numerous models investigating pesticide exposure have demonstrated metabolic disturbances such as imbalances in amino acid levels within the organism. One potentially effective strategy to mitigate pesticide toxicity involves dietary intervention by supplementing exogenous amino acids and their derivates to augment the body's antioxidant capacity and mitigate pesticide-induced oxidative harm, whose mechanism including bolstering glutathione synthesis, regulating arginine-NO metabolism, mitochondria-related oxidative stress, and the open of ion channels, as well as enhancing intestinal microecology. Enhancing glutathione synthesis through supplementation of substrates N-acetylcysteine and glycine is regarded as a potent mechanism to achieve this. Selection of appropriate amino acids or their derivates for supplementation, and determining an appropriate dosage, are of the utmost importance for effective mitigation of pesticide-induced oxidative harm. More experimentation is required that involves large population samples to validate the efficacy of dietary intervention strategies, as well as to determine the effects of amino acids and their derivates on long-term and low-dose pesticide exposure. This review provides insights to guide future research aimed at preventing and alleviating pesticide toxicity through dietary intervention of amino acids and their derivates.

Ginsenoside Rb1, Compound K and 20(S)-Protopanaxadiol Attenuate High-Fat Diet-Induced Hyperlipidemia in Rats via Modulation of Gut Microbiota and Bile Acid Metabolism.

Hyperlipidemia, characterized by elevated serum lipid concentrations resulting from lipid metabolism dysfunction, represents a prevalent global health concern. Ginsenoside Rb1, compound K (CK), and 20(S)-protopanaxadiol (PPD), bioactive constituents derived from Panax ginseng, have shown promise in mitigating lipid metabolism disorders. However, the comparative efficacy and underlying mechanisms of these compounds in hyperlipidemia prevention remain inadequately explored. This study investigates the impact of ginsenoside Rb1, CK, and PPD supplementation on hyperlipidemia in rats induced by a high-fat diet. Our findings demonstrate that ginsenoside Rb1 significantly decreased body weight and body weight gain, ameliorated hepatic steatosis, and improved dyslipidemia in HFD-fed rats, outperforming CK and PPD. Moreover, ginsenoside Rb1, CK, and PPD distinctly modified gut microbiota composition and function. Ginsenoside Rb1 increased the relative abundance of Blautia and Eubacterium, while PPD elevated Akkermansia levels. Both CK and PPD increased Prevotella and Bacteroides, whereas Clostridium-sensu-stricto and Lactobacillus were reduced following treatment with all three compounds. Notably, only ginsenoside Rb1 enhanced lipid metabolism by modulating the PPARγ/ACC/FAS signaling pathway and promoting fatty acid ß-oxidation. Additionally, all three ginsenosides markedly improved bile acid enterohepatic circulation via the FXR/CYP7A1 pathway, reducing hepatic and serum total bile acids and modulating bile acid pool composition by decreasing primary/unconjugated bile acids (CA, CDCA, and ß-MCA) and increasing conjugated bile acids (TCDCA, GCDCA, GDCA, and TUDCA), correlated with gut microbiota changes. In conclusion, our results suggest that ginsenoside Rb1, CK, and PPD supplementation offer promising prebiotic interventions for managing HFD-induced hyperlipidemia in rats, with ginsenoside Rb1 demonstrating superior efficacy.

RT-RPA-Cas12a-based assay facilitates the discrimination of SARS-CoV-2 variants of concern.

Timely and accurate detection of SARS-CoV-2 variants of concern (VOCs) is urgently needed for pandemic surveillance and control. Great efforts have been made from a mass of scientists in increasing the detection sensitivity and operability, and reducing the turn-around time and cost. Here, we report a nucleic acid testing-based method aiming to detect and discriminate SARS-CoV-2 mutations by combining RT-RPA and CRISPR-Cas12a detecting assays (RRCd). With a detection limit of 10 copies RNA/reaction, RRCd was validated in 194 clinical samples, showing 89% positive predictive agreement and 100% negative predictive agreement, respectively. Critically, using specific crRNAs, representatives of single nucleotide polymorphisms and small deletions in SARS-CoV-2 VOCs including N501Y, T478K and ΔH69-V70 were discriminated by RRCd, demonstrating 100% specificity in clinical samples with C t < 33. The method completes within 65 min and could offer visible results without using any electrical devices, which probably facilitate point-of-care testing of SARS-CoV-2 variants and other epidemic viruses.

Global Insights Into Lysine Acylomes Reveal Crosstalk Between Lysine Acetylation and Succinylation in Streptomyces coelicolor Metabolic Pathways.

Lysine acylations are reversible and ubiquitous post-translational modifications that play critical roles in regulating multiple cellular processes. In the current study, highly abundant and dynamic acetylation, besides succinylation, was uncovered in a soil bacterium, Streptomyces coelicolor. By affinity enrichment using anti-acetyl-lysine antibody and the following LC-MS/MS analysis, a total of 1298 acetylation sites among 601 proteins were identified. Bioinformatics analyses suggested that these acetylated proteins have diverse subcellular localization and were enriched in a wide range of biological functions. Specifically, a majority of the acetylated proteins were also succinylated in the tricarboxylic acid cycle and protein translation pathways, and the bimodification occurred at the same sites in some proteins. The acetylation and succinylation sites were quantified by knocking out either the deacetylase ScCobB1 or the desuccinylase ScCobB2, demonstrating a possible competitive relationship between the two acylations. Moreover, in vitro experiments using synthetically modified peptides confirmed the regulatory crosstalk between the two sirtuins, which may be involved in the collaborative regulation of cell physiology. Collectively, these results provided global insights into the S. coelicolor acylomes and laid a foundation for characterizing the regulatory roles of the crosstalk between lysine acetylation and succinylation in the future.

Deacetylation enhances ParB-DNA interactions affecting chromosome segregation in Streptomyces coelicolor.

Reversible lysine acetylation plays regulatory roles in diverse biological processes, including cell metabolism, gene transcription, cell apoptosis and ageing. Here, we show that lysine acetylation is involved in the regulation of chromosome segregation, a pivotal step during cell division in Streptomyces coelicolor. Specifically, deacetylation increases the DNA-binding affinity of the chromosome segregation protein ParB to the centromere-like sequence parS. Both biochemical and genetic experiments suggest that the deacetylation process is mainly modulated by a sirtuin-like deacetylase ScCobB1. The Lys-183 residue in the helix-turn-helix region of ParB is the major deacetylation site responsible for the regulation of ParB-parS binding. In-frame deletion of SccobB1 represses formation of ParB segregation complexes and leads to generation of abnormal spores. Taken together, these observations provide direct evidence that deacetylation participates in the regulation of chromosome segregation by targeting ParB in S. coelicolor.

A recently evolved diflavin-containing monomeric nitrate reductase is responsible for highly efficient bacterial nitrate assimilation.

Nitrate is one of the major inorganic nitrogen sources for microbes. Many bacterial and archaeal lineages have the capacity to express assimilatory nitrate reductase (NAS), which catalyzes the rate-limiting reduction of nitrate to nitrite. Although a nitrate assimilatory pathway in mycobacteria has been proposed and validated physiologically and genetically, the putative NAS enzyme has yet to be identified. Here, we report the characterization of a novel NAS encoded by Mycolicibacterium smegmatis Msmeg_4206, designated NasN, which differs from the canonical NASs in its structure, electron transfer mechanism, enzymatic properties, and phylogenetic distribution. Using sequence analysis and biochemical characterization, we found that NasN is an NADPH-dependent, diflavin-containing monomeric enzyme composed of a canonical molybdopterin cofactor-binding catalytic domain and an FMN-FAD/NAD-binding, electron-receiving/transferring domain, making it unique among all previously reported hetero-oligomeric NASs. Genetic studies revealed that NasN is essential for aerobic M. smegmatis growth on nitrate as the sole nitrogen source and that the global transcriptional regulator GlnR regulates nasN expression. Moreover, unlike the NADH-dependent heterodimeric NAS enzyme, NasN efficiently supports bacterial growth under nitrate-limiting conditions, likely due to its significantly greater catalytic activity and oxygen tolerance. Results from a phylogenetic analysis suggested that the nasN gene is more recently evolved than those encoding other NASs and that its distribution is limited mainly to Actinobacteria and Proteobacteria. We observed that among mycobacterial species, most fast-growing environmental mycobacteria carry nasN, but that it is largely lacking in slow-growing pathogenic mycobacteria because of multiple independent genomic deletion events along their evolution.

Epidemiology and Ecology of Severe Fever With Thrombocytopenia Syndrome in China, 2010‒2018.

BACKGROUND: The growing epidemics of severe fever with thrombocytopenia syndrome (SFTS), an emerging tick-borne disease in East Asia, and its high case fatality rate have raised serious public health concerns. METHODS: Surveillance data on laboratory-confirmed SFTS cases in China were collected. The spatiotemporal dynamics and epidemiological features were explored. The socioeconomic and environmental drivers were identified for SFTS diffusion using survival analysis and for SFTS persistence using a two-stage generalized boosted regression tree model. RESULTS: During 2010‒2018, a total of 7721 laboratory-confirmed SFTS cases were reported in China, with an overall case fatality rate (CFR) of 10.5%. The average annual incidence increased >20 times and endemic areas expanded from 27 to 1574 townships, whereas the CFR declined from 19% to 10% during this period. Four geographical clusters-the Changbai Mountain area, the Jiaodong Peninsula, the Taishan Mountain area, and the Huaiyangshan Mountain area-were identified. Diffusion and persistence of the disease were both driven by elevation, high coverages of woods, crops, and shrubs, and the vicinity of habitats of migratory birds but had different meteorological drivers. Residents ≥60 years old in rural areas with crop fields and tea farms were at increased risk to SFTS. CONCLUSIONS: Surveillance of SFTS and intervention programs need to be targeted at areas ecologically suitability for vector ticks and in the vicinity of migratory birds to curb the growing epidemic.

Oxidation of dCTP contributes to antibiotic lethality in stationary-phase mycobacteria.

Growing evidence shows that generation of reactive oxygen species (ROS) derived from antibiotic-induced metabolic perturbation contribute to antibiotic lethality. However, our knowledge of the mechanisms by which antibiotic-induced oxidative stress actually kills cells remains elusive. Here, we show that oxidation of dCTP underlies ROS-mediated antibiotic lethality via induction of DNA double-strand breaks (DSBs). Deletion of mazG-encoded 5-OH-dCTP-specific pyrophosphohydrolase potentiates antibiotic killing of stationary-phase mycobacteria, but did not affect antibiotic efficacy in exponentially growing cultures. Critically, the effect of mazG deletion on potentiating antibiotic killing is associated with antibiotic-induced ROS and accumulation of 5-OH-dCTP. Independent lines of evidence presented here indicate that the increased level of DSBs observed in the ΔmazG mutant is a dead-end event accounting for enhanced antibiotic killing. Moreover, we provided genetic evidence that 5-OH-dCTP is incorporated into genomic DNA via error-prone DNA polymerase DnaE2 and repair of 5-OH-dC lesions via the endonuclease Nth leads to the generation of lethal DSBs. This work provides a mechanistic view of ROS-mediated antibiotic lethality in stationary phase and may have broad implications not only with respect to antibiotic lethality but also to the mechanism of stress-induced mutagenesis in bacteria.

Imidacloprid increases intestinal permeability by disrupting tight junctions.

The neonicotinoid pesticide, imidacloprid (IMI), is frequently detected in the environment and in foods. It is absorbed and metabolized by the intestine; however, its effects on intestinal barrier integrity are not well studied. We investigated whether IMI disrupts the permeability of the intestinal epithelial barrier via in vivo tests on male Wistar rats, in vitro assays using the human intestinal epithelial cell line, Caco-2, and in silico analyses. A repeated oral dose 90-day toxicity study was performed (0.06 mg/kg body weight/day). IMI exposure significantly increased intestinal permeability, which led to significantly elevated serum levels of endotoxin and inflammatory biomarkers (tumor necrosis factor-alpha and interleukin-1 beta) without any variation in body weight. Decreased transepithelial electrical resistance with increased permeability was also observed in 100 nM and 100 µM IMI-treated Caco-2 cell monolayers. Amounts of tight junction proteins in IMI-treated colon tissues and between IMI-treated Caco-2 cells were significantly lower than those of controls. Increased levels of myosin light chain phosphorylation, myosin light chain kinase (MLCK), and p65 subunit of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB p65) phosphorylation were found in IMI-exposed cells compared with control cells. Furthermore, the barrier loss caused by IMI was rescued by the MLCK inhibitor, ML-7, and cycloheximide. Pregnane X receptor (PXR, NR1I2) was inhibited by low-dose IMI treatment. In silico analysis indicated potent binding sites between PXR and IMI. Together, these data illustrate that IMI induces intestinal epithelial barrier disruption and produces an inflammatory response, involving the down-regulation of tight junctions and disturbance of the PXR-NF-κB p65-MLCK signaling pathway. The intestinal barrier disruption caused by IMI deserves attention in assessing the safety of this neonicotinoid pesticide.

The cyanobacterial ornithine-ammonia cycle involves an arginine dihydrolase.

Living organisms have evolved mechanisms for adjusting their metabolism to adapt to environmental nutrient availability. Terrestrial animals utilize the ornithine-urea cycle to dispose of excess nitrogen derived from dietary protein. Here, we identified an active ornithine-ammonia cycle (OAC) in cyanobacteria through an approach combining dynamic 15N and 13C tracers, metabolomics, and mathematical modeling. The pathway starts with carbamoyl phosphate synthesis by the bacterial- and plant-type glutamine-dependent enzyme and ends with conversion of arginine to ornithine and ammonia by a novel arginine dihydrolase. An arginine dihydrolase-deficient mutant showed disruption of OAC and severely impaired cell growth when nitrogen availability oscillated. We demonstrated that the OAC allows for rapid remobilization of nitrogen reserves under starvation and a high rate of nitrogen assimilation and storage after the nutrient becomes available. Thus, the OAC serves as a conduit in the nitrogen storage-and-remobilization machinery in cyanobacteria and enables cellular adaptation to nitrogen fluctuations.

The CCTL (Cpf1-assisted Cutting and Taq DNA ligase-assisted Ligation) method for efficient editing of large DNA constructs in vitro.

As Cpf1 cleaves double-stranded DNA in a staggered way, it can be used in DNA assembly. However, the Cpf1 cleavage was found to be inaccurate, which may cause errors in DNA assembly. Here, the Cpf1 cleavage sites were precisely characterized, where the cleavage site on the target strand was around the 22nd base relative to the protospacer adjacent motif site, but the cleavage on the non-target strand was affected by the spacer length. When the spacer length was 20 nt or longer, Cpf1 mainly cleaved around the 14th and the 18th bases on the non-target strand; otherwise, with a shorter spacer (i.e. 17-19 nt), Cpf1 mainly cleaved after the 14th base, generating 8-nt sticky ends. With this finding, Cpf1 with a 17-nt spacer crRNA were employed for in vitro substitution of the actII-orf4 promoter in the actinorhodin biosynthetic cluster with a constitutively expressing promoter. The engineered cluster yielded more actinorhodin and produced actinorhodin from an earlier phase. Moreover, Taq DNA ligase was further employed to increase both the ligation efficiency and the ligation accuracy of the method. We expect this CCTL (Cpf1-assisted Cutting and Taq DNA ligase-mediated Ligation) method can be widely used in in vitro editing of large DNA constructs.

The draft genomes and investigation of serotype distribution, antimicrobial resistance of group B Streptococcus strains isolated from urine in Suzhou, China.

BACKGROUND: The group B Streptococcus (GBS) is a human commensal bacterium, which is capable of causing several infectious diseases in infants, and people with chronic diseases. GBS has been the most common cause of infections in urinary tract of the elders, but relatively few studies reported the urine-isolated GBS and their antimicrobial susceptibilities. Hence, we decided to investigate GBS specially isolated from urine in Suzhou, China. METHODS: 27 GBS samples were isolated from urine in Suzhou, China. The PCR and agarose gel electrophoresis were used to identify the serotype distribution. Susceptibility tests were based on MIC test and Kirby-Bauer test. Genome were sequenced via Illumina Hiseq platform and assembled by SPAdes. Genomes of five isolates were sequenced and submitted to NCBI genome database. The sequencing files in fastq format were submitted to NCBI SRA database. RESULTS: Five serotypes were identified. The resistant rates measured for tetracycline, erythromycin, clindamycin and fluoroquinolones were 74.1, 63.0, 44.4 and 48.1%, respectively. 18.5% of the isolates were nonsusceptible to nitrofurantoin. The resistance to tetracycline was mainly associated with the gene tetM. The erythromycin resistance was mainly associated with the genes ermB and mefE. The genes ermB and lnuB were the prevalent genes in cMLSB type. No known nitrofurantoin resistance gene was found in nitrofurantoin-nonsusceptible GBS. CONCLUSIONS: Five serotypes were identified in our study. High rates of GBS isolates were resistant to tetracycline, erythromycin, clindamycin and fluoroquinolones. The genes ermB and lnuB occupied high rates in cMLSB phenotype.

Direct Involvement of the Master Nitrogen Metabolism Regulator GlnR in Antibiotic Biosynthesis in Streptomyces.

GlnR, an OmpR-like orphan two-component system response regulator, is a master regulator of nitrogen metabolism in the genus Streptomyces In this work, evidence that GlnR is also directly involved in the regulation of antibiotic biosynthesis is provided. In the model strain Streptomyces coelicolor M145, an in-frame deletion of glnR resulted in markedly increased actinorhodin (ACT) production but reduced undecylprodigiosin (RED) biosynthesis when exposed to R2YE culture medium. Transcriptional analysis coupled with DNA binding studies revealed that GlnR represses ACT but activates RED production directly via the pathway-specific activator genes actII-ORF4 and redZ, respectively. The precise GlnR-binding sites upstream of these two target genes were defined. In addition, the direct involvement of GlnR in antibiotic biosynthesis was further identified in Streptomyces avermitilis, which produces the important anthelmintic agent avermectin. We found that S. avermitilis GlnR (GlnRsav) could stimulate avermectin but repress oligomycin production directly through the respective pathway-specific activator genes, aveR and olmRI/RII To the best of our knowledge, this report describes the first experimental evidence demonstrating that GlnR regulates antibiotic biosynthesis directly through pathway-specific regulators in Streptomyces Our results suggest that GlnR-mediated regulation of antibiotic biosynthesis is likely to be universal in streptomycetes. These findings also indicate that GlnR is not only a master nitrogen regulator but also an important controller of secondary metabolism, which may help to balance nitrogen metabolism and antibiotic biosynthesis in streptomycetes.

An efficient system for deletion of large DNA fragments in Escherichia coli via introduction of both Cas9 and the non-homologous end joining system from Mycobacterium smegmatis.

Accompanied with the internal non-homologous end joining (NHEJ) system, Cas9 can be used to easily inactivate a gene or delete a fragment through introduction of DNA double-stranded breaks (DSBs) in eukaryotic cells. While in most prokaryotes (e.g. Escherichia coli), due to the lack of NHEJ, homologous recombination (HR) is required for repair of DSBs, which is less convenient. Here, a markerless system was developed for rapid gene inactivation or fragment deletion in E. coli via introduction of both Cas9 and a bacterial NHEJ system. Three bacterial NHEJ systems, i.e. Mycobacterium smegmatis (Msm), Mycobacterium tuberculosis (Mtb) and Bacillus subtilis (Bs), were tested in E. coli, and the MsmNHEJ system showed the best efficiency. With the employment of Cas9 and MsmNHEJ, we efficiently mutated lacZ gene, deleted glnALG operon and two large DNA fragments (67 kb and 123 kb) in E. coli, respectively. Moreover, the system was further designed to allow for continuous inactivation of genes or deletion of DNA fragments in E. coli. We envision this system can be extended to other bacteria, especially those with low HR efficiency.

Molecular evidence for the coordination of nitrogen and carbon metabolisms, revealed by a study on the transcriptional regulation of the agl3EFG operon that encodes a putative carbohydrate transporter in Streptomyces coelicolor.

In the agl3EFGXYZ operon (SCO7167-SCO7162, abbreviated as agl3 operon) of Streptomyces coelicolor M145, agl3EFG genes encode a putative ABC-type carbohydrate transporter. The transcription of this operon has been proved to be repressed by Agl3R (SCO7168), a neighboring GntR-family regulator, and this repression can be released by growth on poor carbon sources. Here in this study, we prove that the transcription of agl3 operon is also directly repressed by GlnR, a central regulator governing the nitrogen metabolism in S. coelicolor. The electrophoretic mobility shift assay (EMSA) employing the agl3 promoter and mixtures of purified recombinant GlnR and Agl3R indicates that GlnR and Agl3R bind to different DNA sequences within the promoter region of agl3 operon, which is further confirmed by the DNase I footprinting assay. As Agl3R and GlnR have been demonstrated to sense the extracellular carbon and nitrogen supplies, respectively, it is hypothesized that the transcription of agl3 operon is stringently governed by the availabilities of extracellular carbon and nitrogen sources. Consistent with the hypothesis, the agl3 operon is further found to be derepressed only under the condition of poor carbon and rich nitrogen supplies, when both regulators are inactivated. It is believed that activation of the expression of agl3 operon may facilitate the absorption of extracellular carbohydrates to balance the ratio of intracellular carbon to nitrogen.

Effects of glycinin basic peptide on physicochemical characteristics and microbial inactivation of pasteurized milk.

The effects of glycinin basic peptide (GBP) on physicochemical characteristics and microbial inactivation of pasteurized milk were investigated over 21d of storage at 4°C. Sensory properties, total bacterial count, pH, alcohol levels, lactose content, and protein changes of pasteurized milk differentially treated with GBP were analyzed periodically during refrigerated storage. Compared with the control, reductions for total bacterial count and specific bacterium (Staphylococcus aureus) in pasteurized milk treated with GBP during storage were found. However, sensory scores, pH, lactose, and protein contents of pasteurized milk treated with GBP were much higher than those of the control. A concentration of 0.015% (wt/vol) GBP could effectively inhibit the growth and reproduction of bacteria in pasteurized milk, enhance its sensory and physicochemical properties, and extend its shelf life to 15d. Thus, GBP has good potential to be a natural milk preservative.