Abstract Background The transcription regulator PhoP has been shown to be important for Y. pestis survival in macrophages and under various in vitro stresses. However, the mechanism by which PhoP promotes bacterial intracellular survival is not fully understood. Our previous microarray analysis suggested that PhoP governed a wide set of cellular pathways in Y. pestis . A series of biochemical experiments were done herein to study members of the PhoP regulon of Y. pestis biovar Microtus . Results By using gel mobility shift assay and quantitative RT-PCR, a total of 30 putative transcription units were characterized as direct PhoP targets. The primer extension assay was further used to determine the transcription start sites of 18 PhoP-dependent promoters and to localize the -10 and -35 elements. The DNase I footprinting was used to identify the PhoP-binding sites within 17 PhoP-dependent promoters, enabling the identification of PhoP box and matrix that both represented the conserved signals for PhoP recognition in Y. pestis . Data presented here providing a good basis for modeling PhoP-promoter DNA interactions that is crucial to the PhoP-mediated transcriptional regulation. Conclusion The proven direct PhoP targets include nine genes encoding regulators and 21 genes or operons with functions of detoxification, protection against DNA damages, resistance to antimicrobial peptides, and adaptation to magnesium limitation. We can presume that PhoP is a global regulator that controls a complex regulatory cascade by a mechanism of not only directly controlling the expression of specific genes, but also indirectly regulating various cellular pathways by acting on a set of dedicated regulators. These results help us gain insights into the PhoP-dependent mechanisms by which Y. pestis survives the antibacterial strategies employed by host macrophages.
Regulatory noncoding RNAs (ncRNAs) play important roles in bacterial gene regulation, primarily at the post-transcriptional level. There are four broad categories of regulatory ncRNAs including trans-encoded ncRNAs, cis-encoded ncRNAs, RNA thermometers and riboswitches, and they can influence the translation and/or stability of mRNAs by binding to the base-pairing sites in their target transcripts. In pathogenic bacteria, numerous ncRNAs are involved in the coordinated expression of virulence determinants to facilitate the pathogenicity in a concerted manner. This review discusses the modes of action of different regulatory ncRNAs and, furthermore, exemplifies their roles in regulating bacterial pathogenicity.
Our previous work have shown that certain subpopulations of Klebsiella pneumoniae exhibit significant phenotypic changes under simulated microgravity (SMG), including enhanced biofilm formation and cellulose synthesis, which may be evoked by changes in gene expression patterns. It is well known that prokaryotic cells genomic DNA can be hierarchically organized into different higher-order three-dimensional structures, which can highly influence gene expression. It is remain elusive whether phenotypic changes induced by SMG in the subpopulations of K. pneumoniae are driven by genome higher-order structural changes. Here, we investigated the above-mentioned issue using the wild-type (WT) K. pneumoniae (WT was used as a control strain and continuously cultivated for 2 weeks under standard culture conditions of normal gravity) and two previous identified subpopulations (M1 and M2) obtained after 2 weeks of continuous incubation in a SMG device. By the combination of genome-wide chromosome conformation capture (Hi-C), RNA-seq and whole-genome methylation (WGS) analyses, we found that the along with the global chromosome interactions change, the compacting extent of M1, M2 subpopulations were much looser under SMG and even with an increase in active, open chromosome regions. In addition, transcriptome data showed that most differentially expressed genes (DEGs) were upregulated, whereas a few DEGs were downregulated in M1 and M2. The functions of both types DEGs were mainly associated with membrane fractions. Additionally, WGS analysis revealed that methylation levels were lower in M1 and M2. Using combined analysis of multi-omics data, we discovered that most upregulated DEGs were significantly enriched in the boundary regions of the variable chromosomal interaction domains (CIDs), in which genes regulating biofilm formation were mainly located. These results suggest that K. pneumoniae may regulate gene expression patterns through DNA methylation and changes in genome structure, thus resulting in new phenotypes in response to altered gravity.
Abstract Background The cAMP receptor protein (CRP) is a global bacterial regulator that controls many target genes. The CRP-cAMP complex regulates the ompR-envZ operon in E. coli directly, involving both positive and negative regulations of multiple target promoters; further, it controls the production of porins indirectly through its direct action on ompR-envZ . Auto-regulation of CRP has also been established in E. coli . However, the regulation of porin genes and its own gene by CRP remains unclear in Y. pestis . Results Y. pestis employs a distinct mechanism indicating that CRP has no regulatory effect on the ompR-envZ operon; however, it stimulates ompC and ompF directly, while repressing ompX . No transcriptional regulatory association between CRP and its own gene can be detected in Y. pestis , which is also in contrast to the fact that CRP acts as both repressor and activator for its own gene in E. coli . It is likely that Y. pestis OmpR and CRP respectively sense different signals (medium osmolarity, and cellular cAMP levels) to regulate porin genes independently. Conclusion Although the CRP of Y. pestis shows a very high homology to that of E. coli , and the consensus DNA sequence recognized by CRP is shared by the two bacteria, the Y. pestis CRP can recognize the promoters of ompC , F , and X directly rather than that of its own gene, which is different from the relevant regulatory circuit of E. coli . Data presented here indicate a remarkable remodeling of the CRP-mediated regulation of porin genes and of its own one between these two bacteria.
Gene modification is an important technique to understand gene function. We firstly constructed Δhfq::Spe and Δrne-710::Spe mutant strains of Escherichia coli MG1655. The fragment lacking of hfq and rne-710 was ligated to the auxiliary plasmid and separately replace the spectinomycin box by homologous recombinase system to obtain the Δhfq and Δrne-710 mutant strains. The combination of two-plasmid scarless genetic modification and fusion PCR led to a new way for the long DNA fragment gene deletions.基因敲除技术是了解基因功能的重要技术手段。以大肠杆菌K-12 MG1655 基因组hfq (309 bp) 和rne-710 (1056 bp) 基因为模型，首先构建Δhfq∷Spe 和Δrne-710∷Spe 菌株，通过融合PCR 方法分别构建缺失hfq (309 bp) 和rne-710 (1056 bp) 的融合片段并连接至辅助质粒，缺失hfq 和rne-710 的片段经重组分别替换壮观霉素抗性盒，得到无痕敲除株Δhfq 和Δrne-710。双质粒无痕敲除和融合PCR 方法相结合为大片段基因缺失开辟了新的途径。.
Abstract In our previous studies, we found that plague vaccines can induce long‐term antibody response, but no significant antibody boost was observed when the immunized mice were challenged with virulent Yersinia pestis . However, a booster vaccination of subunit vaccine on week 3 after primary immunization elicited a significantly higher antibody titre than a single dose, whereas no significant antibody titre difference was observed between a single dose and two doses of EV 76 vaccination. To address these issues, in this study, we first investigated the kinetics of memory B cells and plasma cells in the mice immunized with EV 76 or F1 protein by flow cytometry and then determined antibody titre in five groups of mice immunized with various vaccination strategy. The results showed that memory B cells dropped to a low level at day 56 after primary immunization. In contrast, plasma cells were maintained for more than 98 days. The group with primary immunization of EV 76 and booster of F1 antigen developed a higher antibody titre than the group with immunization of F1 antigen and booster of EV 76. This result supports a hypothesis that an excess of antigens can neutralize pre‐existing antibodies, and then the redundant antigen induces antibody boost. Taken together, a boost of antibody titre after revaccination may be dependent on the existence of memory B cells and an excess of antigen vaccination. In addition, this study showed an ideal immunization strategy that involves first immunization with a live attenuated vaccine, such as EV 76, and then with a subunit vaccine.
Abstract Edible bird's nest (EBN) is a traditional food which was nourishing and functional. Particularly, there is the epidermal growth factor (EGF) in EBN, which is thought to play an important role in promoting skin repair. However, the type and content of EGF in EBN were not determined yet. In this study, the type of EGF in EBN was identified as bird EGF by enzyme-linked immunosorbent assay and this method was validated to be accurate and precise. Moreover, it was found that the content of EGF in raw-unclean EBN, raw-clean EBN and stewed EBN was 3000 pg/g–4000 pg/g and there were no significant differences, which suggested that the batches, origins, forms, stewing temperatures and stewing times of EBN had no effect on the content of EGF in EBN. However, it was due to that enzyme destroyed the primary structure of EGF, the EGF content of neutral protease and trypsin hydrolysates of EBN was lower than that of flavor enzymes, alkaline protease and pepsin hydrolysates of EGF. This study was the first to determine the type and content of EGF in EBN, and provided a theoretical basis for the selection and processing of EBN and using EBN as a source of EGF.
The genus Yersinia includes 11 species, 3 of which ( Y. pestis , Y. pseudotuberculosis , and Y. enterocolitica ) are pathogenic for humans. The remaining 8 species ( Y. frederiksenii , Y. intermedia , Y. kristensenii , Y. bercovieri , Y. mollaretii , Y. rohdei , Y. ruckeri , and Y. aldovae ) are merely opportunistic pathogens found mostly in the environment. In this work, the genomic differences among Yersinia were determined using a Y. pestis-specific DNA microarray. The results revealed 292 chromosomal genes that were shared by all Yersinia species tested, constituting the conserved gene pool of the genus Yersinia. Hierarchical clustering analysis of the microarray data revealed the genetic relationships among all 11 species in this genus. The microarray analysis in conjunction with PCR screening greatly reduced the number of chromosomal genes (32) specific for Y. pestis to 16 genes and uncovered a high level of genomic plasticity within Y. pseudotuberculosis, indicating that its different serotypes have undergone an extensively parallel loss or acquisition of genetic content, which is likely to be important for its adaptation to changes in environmental niches.
In this study, we revealed the regulatory role of the AraC-family transcriptional regulator BfvR (YPO1737) in biofilm formation and virulence of Yersinia pestis. Phenotypic assays showed that BfvR enhanced Y. pestis biofilm formation and repressed its virulence in mice. Further molecular biological assays demonstrated that BfvR directly stimulated the expression of hmsHFRS, waaAE-coaD, and hmsCDE, which in turn affected the production of exopolysaccharide, LPS and c-di-GMP, respectively. In addition, BfvR directly and indirectly repressed psaABC and psaEF transcription, respectively, which affected virulence by possibly interfering with bacterial entry into pulmonary epithelial cells. We speculated that the modulation of biofilm- and virulence-related genes by BfvR leads to biofilm formation and the reduced virulence of Y. pestis. Biofilm formation is critical for blocking flea foregut and hence for transmission of Y. pestis by flea biting. The AraC-family transcriptional regulator BfvR is found to regulate biofilm-associated genes is important for understanding the biofilm formation of Y. pestis in flea foregut. This paper also revealed that BfvR reduced Y. pestis virulence through regulating virulence-associated genes. These findings demonstrated the balanced role of the regulator BfvR in biofilm formation and virulence of Y. pestis.
Both zhenbao pills II and III are Mongolian medicine of a kind. Zhenbao pill II was the artificial syntheses and zhenbao pill III was made from natural materials. In this paper, the flavonoids of Mongolian medicine zhenbao pills II and III were extracted by Soxhlet apparatus with methyl alcohol as extracting agent, the colorimetric method was applied to the determination of flavonoids, and the experimental procedure was studied with zhenbao pill II as the test sample. The result showed that the linear range of quantitative determination was 8.05-48.28 microg x mg(-1). The standard addition recovery (SAR) was 99.49%-100.50%. The RSD (n = 3) was 0.54%. The range of contents of flavonoids was 1.47-1.55 mg x g(-1) in zhenbao pill II and was 2.88-3.00 mg x g(-1) in zhenbao pill III. This method was simple and accurate with good reproducibility, and is suitable for the determination of flavonoids in all kinds of pills. The contents of flavonoids can be used to prove whether zhenbao pill is artificial syntheses or natural material.
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