CRISPR-Cas12a-Assisted Recombineering in Bacteria.

Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a (Cpf1) has emerged as an effective genome editing tool in many organisms. Here, we developed and optimized a CRISPR-Cas12a-assisted recombineering system to facilitate genetic manipulation in bacteria. Using this system, point mutations, deletions, insertions, and gene replacements can be easily generated on the chromosome or native plasmids in Escherichia coli, Yersinia pestis, and Mycobacterium smegmatis Because CRISPR-Cas12a-assisted recombineering does not require introduction of an antibiotic resistance gene into the chromosome to select for recombinants, it is an efficient approach for generating markerless and scarless mutations in bacteria.IMPORTANCE The CRISPR-Cas9 system has been widely used to facilitate genome editing in many bacteria. CRISPR-Cas12a (Cpf1), a new type of CRISPR-Cas system, allows efficient genome editing in bacteria when combined with recombineering. Cas12a and Cas9 recognize different target sites, which allows for more precise selection of the cleavage target and introduction of the desired mutation. In addition, CRISPR-Cas12a-assisted recombineering can be used for genetic manipulation of plasmids and plasmid curing. Finally, Cas12a-assisted recombineering in the generation of point mutations, deletions, insertions, and replacements in bacteria has been systematically analyzed. Taken together, our findings will guide efficient Cas12a-mediated genome editing in bacteria.

HmsC, a periplasmic protein, controls biofilm formation via repression of HmsD, a diguanylate cyclase in Yersinia pestis.

Yersinia pestis, the cause of plague, forms a biofilm in the foregut of its flea vector to enhance transmission. Biofilm formation in Y. pestis is controlled by the intracellular levels of the second messenger molecule cyclic diguanylate (c-di-GMP). HmsT and Y3730, the two diguanylate cyclases (DGC) in Y. pestis, are responsible for the synthesis of c-di-GMP. Y3730, which we name here as HmsD, has little effect on in vitro biofilms, but has a major effect on biofilm formation in the flea. The mechanism by which HmsD plays differential roles in vivo and in vitro is not understood. In this study, we show that hmsD is part of a three-gene operon (y3729-31), which we designate as hmsCDE. Deletion of hmsC resulted in increased, hmsD-dependent biofilm formation, while deletion or overexpression of hmsE did not affect biofilm formation. Localization experiments suggest that HmsC resides in the periplasmic space. In addition, we provide evidence that HmsC might interact directly with the periplasmic domain of HmsD and cause the proteolysis of HmsD. We propose that HmsC senses the environmental signals, which in turn regulates HmsD, and controls the c-di-GMP synthesis and biofilm formation in Y. pestis.

Toxin-Antitoxin Systems Alter Adaptation of Mycobacterium smegmatis to Environmental Stress.

Toxin-antitoxin (TA) systems are ubiquitous genetic elements in prokaryotes, but their biological importance is poorly understood. Mycobacterium smegmatis contains eight putative TA systems. Previously, seven TAs have been studied, with five of them being verified as functional. Here, we show that Ms0251-0252 is a novel TA system in that expression of the toxin Ms0251 leads to growth inhibition that can be rescued by the antitoxin Ms0252. To investigate the functional roles of TA systems in M. smegmatis, we deleted the eight putative TA loci and assayed the mutants for resistance to various stresses. Deletion of all eight TA loci resulted in decreased survival under starvation conditions and altered fitness when exposed to environmental stresses. Furthermore, we showed that deletion of the eight TA loci decreased resistance to phage infection in Sauton medium compared with the results using 7H10 medium, suggesting that TA systems might have different contributions depending on the nutrient environment. Furthermore, we found that MazEF specifically played a dominant role in resistance to phage infection. Finally, transcriptome analysis revealed that MazEF overexpression led to differential expression of multiple genes, including those related to iron acquisition. Altogether, we demonstrate that TA systems coordinately function to allow M. smegmatis to adapt to changing environmental conditions. IMPORTANCE Toxin-antitoxin (TA) systems are mechanisms for rapid adaptation of bacteria to environmental changes. Mycobacterium smegmatis, a model bacterium for studying Mycobacterium tuberculosis, encodes eight putative TA systems. Here, we constructed an M. smegmatis mutant with deletions of all eight TA-encoding genes and evaluated the resistance of these mutants to environmental stresses. Our results showed that different TA systems have overlapping and, in some cases, opposing functions in adaptation to various stresses. We suggest that complementary TA modules may function together to regulate the bacterial stress response, enabling adaptation to changing environments. Together, this study provides key insights into the roles of TA systems in resistance to various environmental stresses, drug tolerance, and defense against phage infection.

Sodium fluoride induces apoptosis and alters bcl-2 family protein expression in MC3T3-E1 osteoblastic cells.

Chronic excessive fluoride intake is known to be toxic and can lead to fluorosis and bone pathologies. However, the cellular mechanisms underlying NaF-induced cytotoxicity in osteoblasts are not well understood. The objectives of this study were to determine the effects of fluoride treatment on MC3T3-E1 osteoblastic cell viability, cell cycle analysis, apoptosis and the expression levels of bcl-2 family members: bcl-2 and bax. MC3T3-E1 cells were treated with 10(-5); 5 × 10(-5); 10(-4); 5 × 10(-4) and 10(-3)M NaF for up to 48 h. NaF was found to reduce cell viability in a temporal and concentration dependent manner and promote apoptosis even at low concentrations (10(-5)M). This increased apoptosis was due to alterations in the expression of both pro-apoptotic bax and anti-apoptotic bcl-2. The net result was a decrease in the bcl-2/bax ratio which was found at both the mRNA and protein levels. Furthermore, we also noted that NaF-induced S-phase arrest during the cell cycle of MC3T3-E1 cells. These data suggest that fluoride-induced osteoblast apoptosis is mediated by direct effects of fluoride on the expression of bcl-2 family members.

Sodium fluoride suppress proliferation and induce apoptosis through decreased insulin-like growth factor-I expression and oxidative stress in primary cultured mouse osteoblasts.

It has been reported that sodium fluoride suppressed proliferation and induced apoptosis in osteoblasts. However, the details about the mechanism at work in bone metabolism are limited. In this study, we further investigated the mechanisms of NaF on proliferation and apoptosis in the primary cultured mouse osteoblasts, which were exposed to different concentration of NaF (10(-6)-5 × 10(-4) M). We examined the effect of NaF on proliferation, cell cycle, apoptosis, oxidative stress, and the protein level of insulin-like growth factor-I (IGF-I) in osteoblasts. All the tested NaF inhibited proliferation and arrested cell cycle at S phase in osteoblasts, and further demonstrated to induce apoptosis in osteoblasts. On the other hand, we found that NaF increased oxidative stress and decreased protein expression of IGF-I. Our study herein suggested that NaF caused proliferation suppression, and apoptosis may contribute to decrease IGF-I expression and increased oxidative stress damage by NaF in the primary mouse osteoblasts.

Corrigendum: HmsC Controls Yersinia pestis Biofilm Formation in Response to Redox Environment.

[This corrects the article on p. 355 in vol. 7, PMID: 28848715.].

HmsC Controls Yersinia pestis Biofilm Formation in Response to Redox Environment.

Yersinia pestis biofilm formation, controlled by intracellular levels of the second messenger molecule cyclic diguanylate (c-di-GMP), is important for blockage-dependent plague transmission from fleas to mammals. HmsCDE is a tripartite signaling system that modulates intracellular c-di-GMP levels to regulate biofilm formation in Y. pestis. Previously, we found that Y. pestis biofilm formation is stimulated in reducing environments in an hmsCDE-dependent manner. However, the mechanism by which HmsCDE senses the redox state remains elusive. Using a dsbA mutant and the addition of Cu2+ to simulate reducing and oxidizing periplasmic environments, we found that HmsC protein levels are decreased and the HmsC-HmsD protein-protein interaction is weakened in a reducing environment. In addition, we revealed that intraprotein disulphide bonds are critical for HmsC since breakage lowers protein stability and diminishes the interaction with HmsD. Our results suggest that HmsC might play a major role in sensing the environmental changes.

Regulatory 3' Untranslated Regions of Bacterial mRNAs.

The untranslated regions (UTRs) of mRNA contain important features that are relevant to the post-transcriptional and translational regulation of gene expression. Most studies of bacterial UTRs have focused on the 5'regions; however, 3'UTRs have recently emerged as a new class of post-transcriptional regulatory elements. 3'UTRs were found to regulate the decay and translation initiation in their own mRNAs. In addition, 3'UTRs constitute a rich reservoir of small regulatory RNAs, regulating target gene expression. In the current review, we describe several recently discovered examples of bacterial regulatory 3'UTRs, discuss their modes of action, and illustrate how they facilitate gene regulation in various environments.

Differential Regulation of c-di-GMP Metabolic Enzymes by Environmental Signals Modulates Biofilm Formation in Yersinia pestis.

Cyclic diguanylate (c-di-GMP) is essential for Yersinia pestis biofilm formation, which is important for flea-borne blockage-dependent plague transmission. Two diguanylate cyclases (DGCs), HmsT and HmsD and one phosphodiesterase (PDE), HmsP are responsible for the synthesis and degradation of c-di-GMP in Y. pestis. Here, we systematically analyzed the effect of various environmental signals on regulation of the biofilm phenotype, the c-di-GMP levels, and expression of HmsT, HmsD, and HmsP in Y. pestis. Biofilm formation was higher in the presence of non-lethal high concentration of CaCl2, MgCl2, CuSO4, sucrose, sodium dodecyl sulfate, or dithiothreitol, and was lower in the presence of FeCl2 or NaCl. In addition, we found that HmsD plays a major role in biofilm formation in acidic or redox environments. These environmental signals differentially regulated expression of HmsT, HmsP and HmsD, resulting in changes in the intracellular levels of c-di-GMP in Y. pestis. Our results suggest that bacteria can sense various environmental signals, and differentially regulate activity of DGCs and PDEs to coordinately regulate and adapt metabolism of c-di-GMP and biofilm formation to changing environments.

The role of selenium in insulin-like growth factor I receptor (IGF-IR) expression and regulation of apoptosis in mouse osteoblasts.

Selenium (Se) is an essential component for animals and human beings. The chemoprotective role of Se, via the regulation of the cell cycle, stimulation of apoptosis and activation of some cytokines among others, is well known; however, the comprehensive effects of Se on the expression of IGF-IR and its regulation of apoptosis have not been investigated. Thus the aim of this study was to report on the effects that different concentrations of Se extert on body weight, blood serum IGF-IR levels and histopathology in mice; and on IGF-IR expression, proliferation and apoptosis in mouse osteoblasts. In vivo experiments showed a significant decrease in body weight, serum levels of IGF-IR and prominent toxicant effects on the liver, kidney, heart and spleen following the administration of defined concentrations of Se for 30 d. However, moderate levels (0.1 mg/kg) of Se gradually improved weight and serum IGF-IR. In vitro osteoblast experiments revealed that at concentrations of 5 × 10(-6) and 10(-5) mol/L Se, MTT activity decreased in comparison with control cells. Cell cycle, TEM and caspase-3 activity supported these observations including an increase in the sub-G1 phase and notable apoptosis in osteoblasts, along with a decrease in the expression of mRNA and protein levels of IGF-IR. Moreover, the MTT activity, mRNA and protein levels of IGF-IR in osteoblasts were decreased and caspase-3 activity was increased in siRNA groups as compared with non-siRNA groups. These data suggest that Se significantly affects IGF-IR expression, and that it contributes to the proliferation and regulation of apoptosis in osteoblasts.

Differential regulation of the hmsCDE operon in Yersinia pestis and Yersinia pseudotuberculosis by the Rcs phosphorelay system.

Yersinia pestis, the agent of plague, forms a biofilm in its flea vector to enhance transmission. Y. pestis biofilm development is positively regulated by hmsT and hmsD, encoding diguanylate cyclases (DGCs) involved in synthesis of the bacterial second messenger c-di-GMP. rcsA, encoding an auxiliary protein in Rcs phosphorelay, is nonfunctional in Y. pestis, while in Yersinia pseudotuberculosis, rcsA is functional and represses biofilms. Previously we showed that Rcs phosphorelay negatively regulates transcription of hmsT in Y. pestis and its ancestor Yersinia pseudotuberculosis. In this study, we show that Rcs positively regulates hmsCDE operon (encoding HmsD) in Y. pestis; while in the presence of functional rcsA, Rcs represses hmsCDE operon in Y. pseudotuberculosis. Loss of rcsA's function in Y. pestis not only causes derepression of hmsT but also causes activation of hmsD, which may account for the increased biofilm formation in Y. pestis. In addition, differential regulation of the two DGCs, HmsT and HmsD by Rcs may help Y. pestis to adapt to different environment.

Sodium fluoride affects proliferation and apoptosis through insulin-like growth factor I receptor in primary cultured mouse osteoblasts.

Insulin-like growth factor-I receptor (IGF-IR) is important for maintaining proliferation and apoptosis in osteoblasts. However, the details about the effect of sodium fluoride supplementation on primary osteoblast cultures on mediating IGF-IR expression are still not well-known. In this study, we used mouse osteoblasts to examine the impact of sodium fluoride on the proliferation and apoptosis; the cells were treated with IGF-IR small interfering RNA (or left untreated) and subsequently divided into a control group and six experimental groups, which were exposed to different concentrations of NaF (10(-6), 10(-5), 10(-4), 10(-3), 5 × 10(-3), and 10(-2) mol/L) for analysis at 48 h. In particular, we examined cell proliferation, apoptosis, IGF-IR messenger RNA, and protein expression levels of the various cell groups. In summary, our findings suggest that the administration of NaF affects the expression of IGF-IR in mouse osteoblasts, contributing to the proliferation and apoptosis induced by fluoride.

Simultaneous administration of fluoride and selenite regulates proliferation and apoptosis in murine osteoblast-like MC3T3-E1 cells by altering osteoprotegerin.

The receptor activator nuclear factor kappa-B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG), are important for maintaining the balance between bone formation and resorption. However, the regulation of microelements on these factors remains unclear. In this study, we used murine osteoblast-like MC3T3-E1 cells to examine the impact of sodium fluoride (NaF) and/or sodium selenite (Na2SeO3) on the OPG/RANKL system. MC3T3-E1 cells were treated with OPG or RANKL siRNA (or left untreated), and subsequently divided into a control group and five experimental groups, which were exposed to different concentrations of NaF and/or Na2SeO3, and subsequently analysed at 24 h. In particular, we examined cell viability, OPG and RANKL mRNA and protein expression, caspase-3 activity, and the cell cycle of the various cell groups. In summary, our findings suggest that the administration of NaF and/or Na2SeO3 affects the expression of OPG in osteoblast-like MC3T3-E1 cells, thereby contributing to the proliferation and apoptosis induced by the OPG.

Isolation and characterization of a reovirus causing spleen necrosis in Pekin ducklings.

High rates of mortality for Pekin ducklings have been recorded in several duck farms in China since 2006. Dead ducklings were characterized by spleen necrosis, suggesting microbial infection as a cause of disease. Laboratory investigations led to the isolation of a virus strain from the spleen tissues of dead ducklings, designated DRV-HC. Subsequent experimental infections with DRV-HC resulted in marked spleen necrosis in the ducklings similar to those observed in the natural outbreaks. Electron microscopy of the cultured DRV-HC revealed viral particles that were non-enveloped and icosahedral with a mean diameter of approximately 72 nm. Agar gel precipitating tests showed that the isolate shared a common group-specific antigen with chicken reovirus S1133. DNA sequencing revealed that this isolate was closely related to Muscovy duck reoviruses. Experimental infection with DRV-HC resulted in death of young chicks with necrotic foci in the liver and spleen. To the best of our knowledge, this is the first report of the isolation of a duck reovirus with high virulence in Pekin ducklings and SPF chickens.