38 research outputs found
Response mechanism of the docosahexaenoic acid producer Aurantiochytrium under cold stress
No full textAurantiochytrium is a commercial docosahexaenoic acid (DHA) producer, and its DHA content can be significantly increased under cold stress. Given this response to low temperature, we examined proteomics changes in Aurantiochytrium under cold stress. We detected approximately 700 protein spots using twodimensional gels, whereas using iTRAQ technology, we detected 4650 types of proteins and successfully identified> 53%. The results indicated that cold stress inhibits the cellular energy supply from glycolysis and the TCA cycle, to ensure a sufficient supply of NADPH and ribose for anabolism. In contrast, the pentose phosphate pathway was not affected. With respect to lipid synthesis, low temperature led to a significant downregulation and up-regulation of fatty acid synthase and polyunsaturated fatty acid synthase, respectively, and restricted the protein synthesis of diacylglycerol O-acyltransferase and phospholipid: diacylglycerol acyltransferase. These results show the preferential biosynthesis of polyunsaturated fatty acids and phospholipids by Aurantiochytrium, which collectively serve to increase cell survival rates in cold environments
Multiplexed CRISPR-Cpf1-Mediated Genome Editing in <i>Clostridium difficile</i> toward the Understanding of Pathogenesis of <i>C. difficile</i> Infection
No full textAssociation Between Biofilm Formation and Structure and Antibiotic Resistance in H. pylori
No full textXiaojuan Wu,1,2,* Daoyan Wu,1,2,* Guzhen Cui,1 Khui Hung Lee,3 Tingxiu Yang,1,2 Zhengrong Zhang,1,2 Qi Liu,4 Jinbao Zhang,1 Eng Guan Chua,3 Zhenghong Chen1,2 1Key Laboratory of Microbiology and Parasitology of Education Department of Guizhou & Joint Laboratory of Helicobacter Pylori and Intestinal Microecology of Affiliated Hospital of Guizhou Medical University, Guizhou Medical University, Guiyang, People’s Republic of China; 2Key Laboratory of Endemic and Ethnic Diseases of Ministry of Education, Guizhou Medical University, Guiyang, People’s Republic of China; 3Helicobacter Research Laboratory, the Marshall Centre for Infectious Disease Research and Training, School of Biomedical Sciences, University of Western Australia, Perth, Western Australia, Australia; 4Department of Gastroenterology, Affiliated Hospital of Guizhou Medical University, Guiyang, People’s Republic of China*These authors contributed equally to this workCorrespondence: Zhenghong Chen, Department of Microbiology, School of Basic Medical Science, Guizhou Medical University, Guiyang, People’s Republic of China, Tel + 86-13985006815, Email [email protected]: Persistent infections caused by Helicobacter pylori (H. pylori), which are resistant to antibiotic treatment, pose a growing global public health concern. Biofilm formation is known to be associated with persistent infections due to its role in enhancing antimicrobial resistance and the tolerance of many pathogenic bacteria.Objective: This study aims to evaluate the biofilm formation of clinical isolates of H. pylori and its impact on antibiotic eradication.Methods: The thickness, morphology, and structure of biofilms derived from nine H. pylori strains were examined using confocal laser scanning microscopy, scanning electron microscopy, and transmission electron microscopy. Subsequently, the susceptibility of both planktonic and biofilm bacteria was assessed through the determination of minimum inhibitory concentration and minimum biofilm eradication concentration for amoxicillin, clarithromycin, levofloxacin, and tetracycline.Results: The results revealed varying biofilm thicknesses and densities among the strains, characterised by the presence of numerous filaments intertwining and connecting bacterial cells. Additionally, several cases exhibited susceptibility based on MIC measurements but resistance according to MBEC measurements, with MBEC indicating a higher resistance rate. Pearson Correlation analysis demonstrated a positive correlation between biofilm thickness and MBEC results (0 < r < 1), notably significant for amoxicillin (r = 0.801, P = 0.009) and tetracycline (r = 0.696, P = 0.037).Conclusion: Different strains of H. pylori exhibit variations in their capacity to release outer membrane vesicles (OMVs) and form biofilms. Biofilm formation can influence the effectiveness of amoxicillin and tetracycline in eradicating susceptible bacterial strains.Keywords: Helicobacter pylori, antibiotic resistance, biofilm formation, biofilm-specific resistanc
Multiplexed CRISPR-Cpf1-Mediated Genome Editing in <i>Clostridium difficile</i> toward the Understanding of Pathogenesis of <i>C. difficile</i> Infection
No full textClostridium difficile is often the primary cause
of nosocomial diarrhea, leading to thousands of deaths annually worldwide.
The availability of an efficient genome editing tool for C.
difficile is essential to understanding its pathogenic mechanism
and physiological behavior. Although CRISPR-Cas9 has been extensively
employed for genome engineering in various organisms, large gene deletion
and multiplex genome editing is still challenging in microorganisms
with underdeveloped genetic engineering tools. Here, we describe a
streamlined CRISPR-Cpf1-based toolkit to achieve precise deletions
of fur, tetM, and ermB1/2 in C. difficile with high efficiencies. All of
these genes are relevant to important phenotypes (including iron uptake,
antibiotics resistance, and toxin production) as related to the pathogenesis
of C. difficile infection (CDI). Furthermore, we
were able to delete an extremely large locus of 49.2-kb comprising
a phage genome (phiCD630-2) and realized multiplex
genome editing in a single conjugation with high efficiencies (simultaneous
deletion of cwp66 and tcdA). Our
work highlighted the first application of CRISPR-Cpf1 for multiplexed
genome editing and extremely large gene deletion in C. difficile, which are both crucial for understanding the pathogenic mechanism
of C. difficile and developing strategies to fight
against CDI. In addition, for the DNA cloning, we developed a one-step-assembly
protocol along with a Python-based algorithm for automatic primer
design, shortening the time for plasmid construction to half that
of conventional procedures. The approaches we developed herein are
easily and broadly applicable to other microorganisms. Our results
provide valuable guidance for establishing CRISPR-Cpf1 as a versatile
genome engineering tool in prokaryotic cells
Molecular Basis of TcdR-Dependent Promoter Activity for Toxin Production by Clostridioides difficile Studied by a Heterologous Reporter System
No full textThe alternative σ factor TcdR controls the synthesis of two major enterotoxins: TcdA and TcdB in Clostridioides difficile. Four potential TcdR-dependent promoters in the pathogenicity locus of C. difficile showed different activities. In this study, we constructed a heterologous system in Bacillus subtilis to investigate the molecular basis of TcdR-dependent promoter activity. The promoters of the two major enterotoxins showed strong TcdR-dependent activity, while the two putative TcdR-dependent promoters in the upstream region of the tcdR gene did not show detectable activity, suggesting that the autoregulation of TcdR may need other unknown factors involved. Mutation analysis indicated that the divergent -10 region is the key determinant for different activities of the TcdR-dependent promoters. Analysis of the TcdR model predicted by AlphaFold2 suggested that TcdR should be classified into group 4, i.e., extracytoplasmic function, σ70 factors. The results of this study provide the molecular basis of the TcdR-dependent promoter recognition for toxin production. This study also suggests the feasibility of the heterologous system in analyzing σ factor functions and possibly in drug development targeting these factors
Multiplexed CRISPR-Cpf1-Mediated Genome Editing in <i>Clostridium difficile</i> toward the Understanding of Pathogenesis of <i>C. difficile</i> Infection
No full textClostridium difficile is often the primary cause
of nosocomial diarrhea, leading to thousands of deaths annually worldwide.
The availability of an efficient genome editing tool for C.
difficile is essential to understanding its pathogenic mechanism
and physiological behavior. Although CRISPR-Cas9 has been extensively
employed for genome engineering in various organisms, large gene deletion
and multiplex genome editing is still challenging in microorganisms
with underdeveloped genetic engineering tools. Here, we describe a
streamlined CRISPR-Cpf1-based toolkit to achieve precise deletions
of fur, tetM, and ermB1/2 in C. difficile with high efficiencies. All of
these genes are relevant to important phenotypes (including iron uptake,
antibiotics resistance, and toxin production) as related to the pathogenesis
of C. difficile infection (CDI). Furthermore, we
were able to delete an extremely large locus of 49.2-kb comprising
a phage genome (phiCD630-2) and realized multiplex
genome editing in a single conjugation with high efficiencies (simultaneous
deletion of cwp66 and tcdA). Our
work highlighted the first application of CRISPR-Cpf1 for multiplexed
genome editing and extremely large gene deletion in C. difficile, which are both crucial for understanding the pathogenic mechanism
of C. difficile and developing strategies to fight
against CDI. In addition, for the DNA cloning, we developed a one-step-assembly
protocol along with a Python-based algorithm for automatic primer
design, shortening the time for plasmid construction to half that
of conventional procedures. The approaches we developed herein are
easily and broadly applicable to other microorganisms. Our results
provide valuable guidance for establishing CRISPR-Cpf1 as a versatile
genome engineering tool in prokaryotic cells
Cellulosome stoichiometry in Clostridium cellulolyticum is regulated by selective RNA processing and stabilization
No full textThe mechanism, physiological relevance and evolutionary implication of selective RNA processing and stabilization (SRPS) remain elusive. Here we report the genome-wide maps of transcriptional start sites (TSs) and post-transcriptional processed sites (PSs) for Clostridium cellulolyticum. The PS-associated genes are preferably associated with subunits of heteromultimeric protein complexes, and the intergenic PSs (iPSs) are enriched in operons exhibiting highly skewed transcript-abundance landscape. Stem-loop structures associated with those iPSs located at 3' termini of highly transcribed genes exhibit folding free energy negatively correlated with transcript-abundance ratio of flanking genes. In the cellulosome-encoding cip-cel operon, iPSs and stem-loops precisely regulate structure and abundance of the subunit-encoding transcripts processed from a primary polycistronic RNA, quantitatively specifying cellulosome stoichiometry. Moreover, cellulosome evolution is shaped by the number, position and biophysical nature of TSs, iPSs and stem-loops. Our findings unveil a genome-wide RNA-encoded strategy controlling in vivo stoichiometry of protein complexes
Multiplexed CRISPR-Cpf1-Mediated Genome Editing in <i>Clostridium difficile</i> toward the Understanding of Pathogenesis of <i>C. difficile</i> Infection
No full textClostridium difficile is often the primary cause
of nosocomial diarrhea, leading to thousands of deaths annually worldwide.
The availability of an efficient genome editing tool for C.
difficile is essential to understanding its pathogenic mechanism
and physiological behavior. Although CRISPR-Cas9 has been extensively
employed for genome engineering in various organisms, large gene deletion
and multiplex genome editing is still challenging in microorganisms
with underdeveloped genetic engineering tools. Here, we describe a
streamlined CRISPR-Cpf1-based toolkit to achieve precise deletions
of fur, tetM, and ermB1/2 in C. difficile with high efficiencies. All of
these genes are relevant to important phenotypes (including iron uptake,
antibiotics resistance, and toxin production) as related to the pathogenesis
of C. difficile infection (CDI). Furthermore, we
were able to delete an extremely large locus of 49.2-kb comprising
a phage genome (phiCD630-2) and realized multiplex
genome editing in a single conjugation with high efficiencies (simultaneous
deletion of cwp66 and tcdA). Our
work highlighted the first application of CRISPR-Cpf1 for multiplexed
genome editing and extremely large gene deletion in C. difficile, which are both crucial for understanding the pathogenic mechanism
of C. difficile and developing strategies to fight
against CDI. In addition, for the DNA cloning, we developed a one-step-assembly
protocol along with a Python-based algorithm for automatic primer
design, shortening the time for plasmid construction to half that
of conventional procedures. The approaches we developed herein are
easily and broadly applicable to other microorganisms. Our results
provide valuable guidance for establishing CRISPR-Cpf1 as a versatile
genome engineering tool in prokaryotic cells
Multiplexed CRISPR-Cpf1-Mediated Genome Editing in <i>Clostridium difficile</i> toward the Understanding of Pathogenesis of <i>C. difficile</i> Infection
No full textClostridium difficile is often the primary cause
of nosocomial diarrhea, leading to thousands of deaths annually worldwide.
The availability of an efficient genome editing tool for C.
difficile is essential to understanding its pathogenic mechanism
and physiological behavior. Although CRISPR-Cas9 has been extensively
employed for genome engineering in various organisms, large gene deletion
and multiplex genome editing is still challenging in microorganisms
with underdeveloped genetic engineering tools. Here, we describe a
streamlined CRISPR-Cpf1-based toolkit to achieve precise deletions
of fur, tetM, and ermB1/2 in C. difficile with high efficiencies. All of
these genes are relevant to important phenotypes (including iron uptake,
antibiotics resistance, and toxin production) as related to the pathogenesis
of C. difficile infection (CDI). Furthermore, we
were able to delete an extremely large locus of 49.2-kb comprising
a phage genome (phiCD630-2) and realized multiplex
genome editing in a single conjugation with high efficiencies (simultaneous
deletion of cwp66 and tcdA). Our
work highlighted the first application of CRISPR-Cpf1 for multiplexed
genome editing and extremely large gene deletion in C. difficile, which are both crucial for understanding the pathogenic mechanism
of C. difficile and developing strategies to fight
against CDI. In addition, for the DNA cloning, we developed a one-step-assembly
protocol along with a Python-based algorithm for automatic primer
design, shortening the time for plasmid construction to half that
of conventional procedures. The approaches we developed herein are
easily and broadly applicable to other microorganisms. Our results
provide valuable guidance for establishing CRISPR-Cpf1 as a versatile
genome engineering tool in prokaryotic cells
Homology Modeling and Activity Analysis of the HMG-CoA Reductase from Streptococcus Pneumoniae
No full text
