scholarly journals SpoVG is an important regulator of sporulation and affects biofilm formation by regulating Spo0A transcription in Bacillus cereus 0–9

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Qiubin Huang ◽  
Zhen Zhang ◽  
Qing Liu ◽  
Fengying Liu ◽  
Yupeng Liu ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Biofilm Formation ◽  
Target Gene ◽  
Plant Diseases ◽  
Transcriptional Fusion ◽  
Transcription Level ◽  
Gram Positive ◽  
Qrt Pcr ◽  
Sporulation Process ◽  
Important Regulator

Abstract Background Bacillus cereus 0–9, a Gram-positive, endospore-forming bacterium isolated from healthy wheat roots in our previous research, is considered to be an effective biocontrol strain against several soil-borne plant diseases. SpoVG, a regulator that is broadly conserved among many Gram-positive bacteria, may help this organism coordinate environmental growth and virulence to survive. This study aimed to explore the multiple functions of SpoVG in B. cereus 0–9. Methods The gene knockout strains were constructed by homologous recombination, and the sporulation process of B. cereus 0–9 and its mutants were observed by fluorescence staining method. We further determined the spore yields and biofilm formation abilities of test strains. Transcriptional fusion strains were constructed by overlapping PCR technique, and the promoter activity of the target gene was detected by measuring its fluorescence intensity. The biofilm production and colonial morphology of B. cereus 0–9 and its mutants were determined to study the functions of the target genes, and the transcription level of the target gene was determined by qRT-PCR. Results According to observation of the sporulation process of B. cereus 0–9 in germination medium, SpoVG is crucial for regulating sporulation stage V of B. cereus 0–9, which is identical to that of Bacillus subtilis but differs from that of Bacillus anthracis. In addition, SpoVG could influence biofilm formation of B. cereus 0–9. The transcription levels of two genes closely related to biofilm-formation, sipW and calY, were downregulated in a ΔspoVG mutant. The role of SpoVG in regulating biofilm formation was further explored by deleting the genes abrB and sinR in the ΔspoVG mutant, respectively, generating the double mutant strains ΔspoVGΔabrB and ΔspoVGΔsinR. The phenotypes of these double mutants were congruent with those of the single abrB and sinR deletion strains, respectively, which showed increased biofilm formation. This indicated that spoVG was located upstream of abrB and sinR in the regulatory pathway of B. cereus biofilm formation. Further, the results of qRT-PCR and the luminescence intensity of transcriptional fusion strains indicated that spoVG gene deletion could inhibit the transcription of Spo0A. Conclusions SpoVG, an important regulator in the sporulation of B. cereus, is located upstream of Spo0A and participates in regulation of biofilm formation of B. cereus 0–9 through regulating the transcription level of spo0A. Sporulation and biofilm formation are crucial mechanisms by which bacteria respond to adverse conditions. SpoVG is therefore an important regulator of Spo0A and is crucial for both sporulation and biofilm formation of B. cereus 0–9. This study provides a new insight into the regulatory mechanism of environmental adaptation in bacteria and a foundation for future studies on biofilm formation of B. cereus.

scholarly journals GapB Is Involved in Biofilm Formation Dependent on LrgAB but Not the SinI/R System in Bacillus cereus 0-9

2020 ◽  
Vol 11 ◽  
Author(s):  
Juanmei Zhang ◽  
Li Meng ◽  
Yubing Zhang ◽  
Lidan Sang ◽  
Qing Liu ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Biofilm Formation ◽  
Living Cells ◽  
Bacterial Biofilm ◽  
Plant Diseases ◽  
Extracellular Dna ◽  
Formation Mechanisms ◽  
Wild Type ◽  
Mineral Salts ◽  
Sharp Eyespot

Bacillus cereus 0-9, a Gram-positive endospore-forming bacterium isolated from healthy wheat roots, has biological control capacity against several soil-borne plant diseases of wheat such as sharp eyespot and take-all. The bacterium can produce various biofilms that differ in their architecture and formation mechanisms, possibly for adapting to different environments. The gapB gene, encoding a glyceraldehyde-3-phosphate dehydrogenase (GAPDH), plays a key role in B. cereus 0-9 biofilm formation. We studied the function of GapB and the mechanism of its involvement in regulating B. cereus 0-9 biofilm formation. GapB has GAPDH activities for both NAD+- and NADP+-dependent dehydrogenases and is a key enzyme in gluconeogenesis. Biofilm yield of the ΔgapB strain decreased by 78.5% compared with that of wild-type B. cereus 0-9 in lysogeny broth supplemented with some mineral salts (LBS), and the ΔgapB::gapB mutants were recovered with gapB gene supplementation. Interestingly, supplementing the LBS medium with 0.1–0.5% glycerol restored the biofilm formation capacity of the ΔgapB mutants. Therefore, GapB regulates biofilm formation relative to its function in gluconeogenesis. To illustrate how GapB is involved in regulating biofilm formation through gluconeogenesis, we carried out further research. The results indicate that the GapB regulated the B. cereus 0-9 biofilm formation independently of the exopolysaccharides and regulatory proteins in the typical SinI/R system, likely owing to the release of extracellular DNA in the matrix. Transcriptome analysis showed that the gapB deletion caused changes in the expression levels of only 18 genes, among which, lrgAB was the most significantly increased by 6.17-fold. We confirmed this hypothesis by counting the dead and living cells in the biofilms and found the number of living cells in the biofilm formed by the ΔgapB strain was nearly 7.5 times than that of wild-type B. cereus 0-9. Therefore, we concluded that the GapB is involved in the extracellular DNA release and biofilm formation by regulating the expression or activities of LrgAB. These results provide a new insight into the regulatory mechanism of bacterial biofilm formation and a new foundation for further studying the stress resistance of B. cereus.

scholarly journals Isocitrate dehydrogenase of Bacillus cereus is involved in biofilm formation

2021 ◽  
Author(s):  
Linlin Zhao ◽  
Qing Liu ◽  
Qiubin Huang ◽  
Fengying Liu ◽  
Huiping Liu ◽  
...  
Keyword(s):  
Citric Acid ◽  
Bacillus Cereus ◽  
Isocitrate Dehydrogenase ◽  
Biofilm Formation ◽  
Gene Knockout ◽  
Tricarboxylic Acid Cycle ◽  
Tca Cycle ◽  
Stage I ◽  
Sporulation Process ◽  
Sporulation Rate

Abstract Tricarboxylic acid cycle (TCA cycle) is a central carbon metabolism pathway in prokaryotes and eukaryotes, and involved in matter metabolism and energy production. Isocitrate dehydrogenase (IDH), which is a key enzyme in the TCA cycle, participates in the formation of biofilms in Staphylococcus aureus by regulating the redox state inside the cell. At present, it remains to be clarified whether IDH is involved in the formation of Bacillus cereus biofilms. In this study, we found a gene icdH annotated as encoding IDH in the B. cereus genome, and cloned and expressed the protein encoded by this gene. The enzyme activity assay showed that the protein had IDH activity dependent on NADP+, indicating that this gene encoded an IDH. The mutant ΔicdH was obtained by gene knockout. Phenotypic analysis showed that the biofilm yield and sporulation rate of the mutant ΔicdH decreased. To reveal the role of IDH in biofilm formation, extracellular pH and citric acid content were measured. The results showed that a B.cereus 0–9 strain that lacked IDH exhibited accumulation of citric acid and acidification of the extracellular matrix. Given that citric acid is a metal chelator, the accumulation of citric acid may lead to a lack of metal ions in cells, resulting in reduced cell viability and affecting biofilm formation. Consistent with this hypothesis, the addition of excess Fe3+ restored biofilm formation in the mutant. These results suggest that IDH in B.cereus may regulate biofilm formation by modulating intracellular redox homeostasis. In addition, we found that the icdH deletion of B. cereus 0–9 resulted in the destruction of the stage I of sporulation process, and thus resulted in a reduced sporulation rate, which was significantly different from sporulation in B. subtilis caused by interruption of the stage I sporulation process due to icdH loss.

scholarly journals Discovery and characterization of a Gram-positive Pel polysaccharide biosynthetic gene cluster

2019 ◽  
Author(s):  
Gregory B. Whitfield ◽  
Lindsey S. Marmont ◽  
Cedoljub Bundalovic-Torma ◽  
Erum Razvi ◽  
Elyse J. Roach ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Gene Cluster ◽  
Biofilm Formation ◽  
Biosynthetic Gene Cluster ◽  
Diguanylate Cyclase ◽  
Biosynthetic Gene ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Biofilm Matrix

AbstractOur understanding of the biofilm matrix components utilized by Gram-positive bacteria, and the signalling pathways that regulate their production are largely unknown. In a companion study, we developed a computational pipeline for the unbiased identification of homologous bacterial operons and applied this algorithm to the analysis of synthase-dependent exopolysaccharide biosynthetic systems (https://doi.org/10.1101/769745). Here, we explore the finding that many species of Gram-positive bacteria have operons with similarity to the Pseudomonas aeruginosa pel locus. Our characterization of the pelDEADAFG operon from Bacillus cereus ATCC 10987, presented herein, demonstrates that this locus is required for biofilm formation and produces a polysaccharide structurally similar to Pel. We show that the degenerate GGDEF domain of the B. cereus PelD ortholog binds cyclic-3’,5’-dimeric guanosine monophosphate (c-di-GMP), and that this binding is required for biofilm formation. Finally, we identify a diguanylate cyclase, CdgF, and a c-di-GMP phosphodiesterase, CdgE, that reciprocally regulate the production of Pel. The discovery of this novel c-di-GMP regulatory circuit significantly contributes to our limited understanding of c-di-GMP signalling in Gram-positive organisms. Furthermore, conservation of the core pelDEADAFG locus amongst many species of Bacilli, Clostridia, Streptococci, and Actinobacteria suggests that Pel may be a common biofilm matrix component in many Gram-positive bacteria.Author summaryThe Pel polysaccharide is required for biofilm formation in P. aeruginosa and we have previously found that the genes necessary for biosynthesis of this polymer are broadly distributed across Gram-negative bacteria. Herein, we show that many species of Gram-positive bacteria also possess Pel biosynthetic genes and demonstrate that these genes are used Bacillus cereus for biofilm formation. We show that Pel production in B. cereus is regulated by c-di-GMP and have identified two enzymes, a diguanylate cyclase, CdgF, and a phosphodiesterase, CdgE, that control the levels of this bacterial signalling molecule. While Pel production in B. cereus also requires the binding of c-di-GMP to the receptor PelD, the divergence of this protein in Streptococci suggests a c-di-GMP independent mechanism of regulation is used in this species. The discovery of a Pel biosynthetic gene cluster in Gram-positive bacteria and our characterization of the pel operon in B. cereus suggests that Pel is a widespread biofilm component across all bacteria.

scholarly journals Thiazole Analogues of the Marine Alkaloid Nortopsentin as Inhibitors of Bacterial Biofilm Formation

Molecules ◽  
2020 ◽  
Vol 26 (1) ◽  
pp. 81
Author(s):  
Anna Carbone ◽  
Stella Cascioferro ◽  
Barbara Parrino ◽  
Daniela Carbone ◽  
Camilla Pecoraro ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Bacterial Biofilm ◽  
Thiazole Derivatives ◽  
Gram Positive ◽  
Lead Compounds ◽  
New Class ◽  
Marked Selectivity ◽  
Global Threat ◽  
New Compounds ◽  
Reference Strains

Anti-virulence strategy is currently considered a promising approach to overcome the global threat of the antibiotic resistance. Among different bacterial virulence factors, the biofilm formation is recognized as one of the most relevant. Considering the high and growing percentage of multi-drug resistant infections that are biofilm-mediated, new therapeutic agents capable of counteracting the formation of biofilms are urgently required. In this scenario, a new series of 18 thiazole derivatives was efficiently synthesized and evaluated for its ability to inhibit biofilm formation against the Gram-positive bacterial reference strains Staphylococcus aureus ATCC 25923 and S. aureus ATCC 6538 and the Gram-negative strain Pseudomonas aeruginosa ATCC 15442. Most of the new compounds showed a marked selectivity against the Gram-positive strains. Remarkably, five compounds exhibited BIC50 values against S. aureus ATCC 25923 ranging from 1.0 to 9.1 µM. The new compounds, affecting the biofilm formation without any interference on microbial growth, can be considered promising lead compounds for the development of a new class of anti-virulence agents.

Florid Bacillus cereus Infection of the Placenta Associated With Intrauterine Fetal Demise

2021 ◽  
pp. 109352662199902
Author(s):  
Stephanie Shea ◽  
Alberto Paniz-Mondolfi ◽  
Emilia Sordillo ◽  
Michael Nowak ◽  
Fumiko Dekio
Keyword(s):  
Bacillus Cereus ◽  
Placental Tissue ◽  
Maternal Blood ◽  
Gastrointestinal Infections ◽  
Gram Positive ◽  
Fetal Demise ◽  
Placental Infection ◽  
Intrauterine Fetal Demise ◽  
Acute Necrotizing ◽  
Poor Outcomes

Bacillus cereus is a gram-positive, rod-shaped bacterium that is commonly implicated in foodborne illness but has also become increasingly recognized as a source of serious non-gastrointestinal infections, including sepsis, meningitis, and pneumonia. Non-gastrointestinal B. cereus infections have been identified in children, especially in neonates; however, there are no previously described cases of fetal demise associated with B. cereus placental infection. We present a case of acute chorioamnionitis-related intrauterine fetal demise of twin A at 17 weeks gestation, noted two days after selective termination of twin B. Histological examination revealed numerous gram-positive bacilli in placental tissue, as well as fetal vasculature, in the setting of severe acute necrotizing chorioamnionitis and subchorionitis, intervillous abscesses, acute villitis, and peripheral acute funisitis. Cultures of maternal blood and placental tissue both yielded growth of B. cereus. This case underscores the importance of B. cereus as a human pathogen, and specifically demonstrates its potential as an agent of severe intraamniotic and placental infection with poor outcomes for the fetus.

scholarly journals Microrna-130a Downregulates HCV Replication through an atg5-Dependent Autophagy Pathway

Cells ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 338 ◽  
Author(s):  
Xiaoqiong Duan ◽  
Xiao Liu ◽  
Wenting Li ◽  
Jacinta A. Holmes ◽  
Annie J. Kruger ◽  
...  
Keyword(s):  
Immune Responses ◽  
Binding Sites ◽  
Target Gene ◽  
Target Genes ◽  
Pyruvate Metabolism ◽  
Host Immune Responses ◽  
Qrt Pcr ◽  
Antiviral Genes ◽  
Autophagy Pathway ◽  
Host Antiviral Response

We previously identified that miR-130a downregulates HCV replication through two independent pathways: restoration of host immune responses and regulation of pyruvate metabolism. In this study, we further sought to explore host antiviral target genes regulated by miR-130a. We performed a RT² Profiler™ PCR array to identify the host antiviral genes regulated by miR-130a. The putative binding sites between miR-130a and its downregulated genes were predicted by miRanda. miR-130a and predicted target genes were over-expressed or knocked down by siRNA or CRISPR/Cas9 gRNA. Selected gene mRNAs and their proteins, together with HCV replication in JFH1 HCV-infected Huh7.5.1 cells were monitored by qRT-PCR and Western blot. We identified 32 genes that were significantly differentially expressed more than 1.5-fold following miR-130a overexpression, 28 of which were upregulated and 4 downregulated. We found that ATG5, a target gene for miR-130a, significantly upregulated HCV replication and downregulated interferon stimulated gene expression. miR-130a downregulated ATG5 expression and its conjugation complex with ATG12. ATG5 and ATG5-ATG12 complex affected interferon stimulated gene (ISG) such as MX1 and OAS3 expression and subsequently HCV replication. We concluded that miR-130a regulates host antiviral response and HCV replication through targeting ATG5 via the ATG5-dependent autophagy pathway.

Effect of Thymus ciliatus oil-based disinfectant solutions against bio-films formed by Bacillus cereus strains isolated from pasteurized-milk processing lines in Algeria

2018 ◽  
Vol 8 (1) ◽  
pp. 01-12
Author(s):  
Amina Kalai ◽  
Fadila Malek ◽  
Leila Bousmaha-Marroki
Keyword(s):  
Essential Oil ◽  
Organic Acids ◽  
Bacillus Cereus ◽  
Biofilm Formation ◽  
Chemical Cleaning ◽  
Pasteurized Milk ◽  
Thyme Oil ◽  
Milk Processing ◽  
Planktonic Growth

Bacillus cereus is a foodborne pathogen that often persists in dairy environments and is associated with food poisoning and spoilage. This spore-forming bacterium has a high propensity to develop biofilms onto dairy processing equipment and resists to chemical cleaning and disinfecting. This study deals with the in vitro application of thyme oil-based sanitizer solutions against biofilms formed by B. cereus genotypes which persist in pasteurized-milk processing lines. The effect of Thymus ciliatus essential oil on B. cereus planktonic cells and biofilms was assessed. The oil was tested alone and in combination with organic acids or industrial cleaning agents, in order to improve the removal of B. cereus recurrent genotypes. Minimal inhibitory concentrations of planktonic growth (MICs), biofilm formation (MBIC) and biofilm eradication (MBEC) of oil and organic acids were evaluated by microdilution assays. Thyme oil was more effective than organic acids against B. cereus planktonic growth, biofilm formation and established bio-films. High values of MICs were obtained for the three organic acids tested (3.5-4.5%) in comparison with those of essential oil (0.082-0.088%). The combination of oil with other antimicrobials as acetic acid, NaOH or HNO3 improves their effectiveness against B. cereus biofilms. These oil-based sanitizer solutions allow complete B. cereus biofilm eradication and should be an attractive candidate for the control and removal of biofilms in the dairy envi-ronment.

scholarly journals miRNA-544a Regulates the Inflammation of Spinal Cord Injury by Inhibiting the Expression of NEUROD4

2018 ◽  
Vol 51 (4) ◽  
pp. 1921-1931 ◽  
Author(s):  
Lei Yang ◽  
Dawei Ge ◽  
Xi Chen ◽  
Chunzhi Jiang ◽  
Shengnai Zheng
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Grip Strength ◽  
Target Gene ◽  
Interleukin 1 ◽  
Luciferase Reporter ◽  
Interleukin 17 ◽  
Qrt Pcr ◽  
Gene Assay ◽  
Cord Injury

Background/Aims: To explore the potential role of miR-544a in spinal cord injury and the possible mechanism involved. Methods: We established a mouse model with spinal cord injury to examine the changes in grip force recovery of the forelimb or the posterior limb of the mouse. Microarray was performed to achieve differentiated miRNAs in the mice. The expressions of miR-544a, MCP-1, IL36B and IL17B after spinal cord injury were detected by qRT-PCR. Subsequently, miR-544a was overexpressed to observe changes in inflammation and grip strength after spinal cord injury. Target gene of miR-544a was then predicted using bioinformatics technology. Finally, dual luciferase reporter gene assay was used to verify the binding of miR-544a to its target gene. Results: Using mice models with spinal cord injury, we found that the strength of their four limbs began to recover 7 days after injury. The results of microarray and qRT-PCR confirmed that mir-544a level in mice with spinal cord injury decreased with increase of injury time, while the levels of inflammatory genes MCP-1 (monocyte chemoattractant protein-1), IL1 (interleukin-1) and TNF-α (tumor necrosis factor alpha) IL36B (interleukin-36 beta) and IL17B (interleukin-17 beta) were significantly increased. However, overexpression of miR-544a in the mice significantly reduced the level of inflammation and restored their grip strength in their four limbs. Finally, we found that miR-544a can bind to the NEUROD4 (Neurogenic differentiation 4) 3’UTR (Untranslated Region) region through bioinformatics website prediction, which was further confirmed by dual luciferase reporter assay. NEUROD4 level was significantly reduced following the overexpression of miR-544a. Conclusion: The expression of miR-544a was significantly decreased after spinal cord injury. High expression of miR-544a could alleviate the inflammation caused by spinal cord injury and promote the recovery of spinal cord via the inhibition of NEUROD4.

scholarly journals Strain variation in Bacillus cereus biofilms and their susceptibility to extracellular matrix-degrading enzymes

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0245708
Author(s):  
Eun Seob Lim ◽  
Seung-Youb Baek ◽  
Taeyoung Oh ◽  
Minseon Koo ◽  
Joo Young Lee ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Bacillus Cereus ◽  
Biofilm Formation ◽  
Dnase I ◽  
Extracellular Dna ◽  
Proteinase K ◽  
Strain Variation ◽  
Protein Ratio ◽  
Degrading Enzymes ◽  
Matrix Degrading Enzymes

Bacillus cereus is a foodborne pathogen and can form biofilms on food contact surfaces, which causes food hygiene problems. While it is necessary to understand strain-dependent variation to effectively control these biofilms, strain-to-strain variation in the structure of B. cereus biofilms is poorly understood. In this study, B. cereus strains from tatsoi (BC4, BC10, and BC72) and the ATCC 10987 reference strain were incubated at 30°C to form biofilms in the presence of the extracellular matrix-degrading enzymes DNase I, proteinase K, dispase II, cellulase, amyloglucosidase, and α-amylase to assess the susceptibility to these enzymes. The four strains exhibited four different patterns in terms of biofilm susceptibility to the enzymes as well as morphology of surface-attached biofilms or suspended cell aggregates. DNase I inhibited the biofilm formation of strains ATCC 10987 and BC4 but not of strains BC10 and BC72. This result suggests that some strains may not have extracellular DNA, or their extracellular DNA may be protected in their biofilms. In addition, the strains exhibited different patterns of susceptibility to protein- and carbohydrate-degrading enzymes. While other strains were resistant, strains ATCC 10987 and BC4 were susceptible to cellulase, suggesting that cellulose or its similar polysaccharides may exist and play an essential role in their biofilm formation. Our compositional and imaging analyses of strains ATCC 10987 and BC4 suggested that the physicochemical properties of their biofilms are distinct, as calculated by the carbohydrate to protein ratio. Taken together, our study suggests that the extracellular matrix of B. cereus biofilms may be highly diverse and provides insight into the diverse mechanisms of biofilm formation among B. cereus strains.

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