We characterized a new quorum-sensing regulator, PlcRa, which is present in various members of the B. cereus group and identified a signaling heptapeptide for PlcRa activity: PapRa7. We demonstrated that PlcRa is a 3D structural paralog of PlcR using sequence analysis and homology modeling. A comparison of the transcriptomes at the onset of stationary phase of a ΔplcRa mutant and the wild-type B. cereus ATCC 14579 strain showed that 68 genes were upregulated and 49 genes were downregulated in the ΔplcRa mutant strain (>3-fold change). Genes involved in the cysteine metabolism (putative CymR regulon) were downregulated in the ΔplcRa mutant strain. We focused on the gene with the largest difference in expression level between the two conditions, which encoded -AbrB2- a new regulator of the AbrB family. We demonstrated that purified PlcRa bound specifically to the abrB2 promoter in the presence of synthetic PapRa7, in an electrophoretic mobility shift assay. We further showed that the AbrB2 regulator controlled the expression of the yrrT operon involved in methionine to cysteine conversion. We found that the ΔplcRa mutant strain was more sensitive to hydrogen peroxide- and disulfide-induced stresses than the wild type. When cystine was added to the culture of the ΔplcRa mutant, challenged with hydrogen peroxide, growth inhibition was abolished. In conclusion, we identified a new RNPP transcriptional regulator in B. cereus that activated the oxidative stress response and cysteine metabolism in transition state cells.
Background: Mild cognitive impairment (MCI) and Alzheimer's disease (AD) are common in older adults. Much recent work has implicated the connection between the gut and the brain via bidirectional communication of the gastrointestinal tract and the central nervous system through biochemical signaling. Altered gut microbiota composition has shown controversial results based on geographic location, age, diet, physical activity, psychological status, underlying diseases, medication, and drug use. Objectives: This study aimed to investigate the relationships of gut microbiota with MCI and AD. Methods: 16S metagenome profiles from stool collection of participant groups (normal; n=20, MCI; n= 12, AD; n=20) were analyzed. The diagnosis of cognitive conditions was made by standard criteria consisting of clinical interviews, physical examinations, cognitive assessments, laboratory examinations, and neuroimaging by both structural neuroimaging and amyloid positron emission tomography scans. Correlations between medical factors with food frequency and the fecal microbiome were elucidated. Results: A significant difference at the operational taxonomic unit level was observed. The significantly higher abundance of bacteria in nondementia patients belonged to the Clostridiales order, including Clostridium sensu stricto 1 (p<0.0001), Fusicatenibacter (p=0.0007), Lachnospiraceae (p=0.001), Agathobacter (p=0.021), and Fecalibacterium (p<0.0001). In contrast, Escherichia-Shigella (p=0.0002), Bacteroides (p=0.0014), Holdemanella (p<0.0001), Romboutsia (p=0.001), and Megamonas (p=0.047) were the dominant genera in the AD group. Left and right-hippocampus and right amygdala volumes were significantly decreased in the AD group (p < 0.001) and significantly correlated with the groups of bacteria that were significantly different between groups. Conclusion: There was a relationship between the composition of the gut microbiome and neurodegenerative disorders, including MCI and AD. Reduction of Clostidiaceae and increases in Enterobactericeae and Bacteroides were associated with persons with MCI and AD, consistent with previous studies. The altered gut microbiome could be potentially targeted for the early diagnosis of dementia and the reduction of AD risk.
In this study, plant-root-associated Bacillus species were evaluated as antifungal biocontrol agents by analyzing the production of surface bioactive molecules known as lipopeptide biosurfactants. This study aimed to isolate and characterize antifungal biosurfactant-producing Bacillus bacterium. Bacillusvelezensis PW192 was isolated from the rhizosphere of Lagerstroemia macrocarpa var macrocarpa and identified based on phylogenetic analysis of the 16S rRNA gene. The biosurfactant was excreted to cultured supernatant and exhibited emulsification power up to 60% and a decrease in surface tension from 72 in distilled water to 21 mN/m. The surface tension properties were stable in a broad range of pH from 6 to 10, in high temperatures up to 100 °C, and in salinities with a NaCl concentration up to 12% (w/v). Starting from 0.5 mg of acid, precipitated crude biosurfactant exhibited antifungal activity toward Anthracnose, caused by the phytopathogens Colletotrichum gloeosporioides and C. musae. The chemical structures of the biosurfactant were structurally characterized as lipopeptides fengycin A and fengycin B. The stability of the biosurfactant, as well as the antifungal properties of B. velezensis PW192, can potentially make them useful as agricultural biocontrol agents, as well as in other biotechnological applications.
Aims To increase the Cry1Da production in Bacillus thuringiensis by enhancing BtI promoter activity and fusion with upstream sequence from cry1Ab. Methods and Results The effects of joining the upstream sequence of cry1Ab that contains E2 subunit pyruvate dehydrogenase (PDH) recognition site to the cry1Da promoter as well as the effects of substitution mutation of conserved sequences of its BtI promoter on cry1Da expression was monitored by constructing cry1Da promoter‐lacZ fusions. Changing the −35 region of the cry1Da BtI promoter to that of cry1Ab enhanced β‐galactosidase activity about three fold as comparing to that of the wild‐type promoter with its own upstream sequence. In contrast, the same cry1Da mutated promoter linked to the above upstream sequence of cry1Ab enhanced enzyme activity up to seven fold, but was five fold lower than that of the full‐length cry1Ab promoter. The cry1Ab‐cry1Da hybrid promoter with the −35 BtI mutation efficiently increased Cry1Da synthesis by 133% and resulted in a 2·3‐fold increase in insect larval toxicity when comparing to the wild type. Conclusions The cry1Ab promoter as well as mutation of −35 region of BtI promoter together with fusion with E2 subunit PDH recognition site efficiently enhanced Cry1Da production in B. thuringiensis. Significance and Impact of the study The results provide useful information to construct an efficient cry1Da gene expression in B. thuringiensis.
The Gram-positive pathogen Bacillus cereus is able to grow in chains of rod-shaped cells, but the regulation of chaining remains largely unknown. Here, we observe that glucose-grown cells of B. cereus ATCC 14579 form longer chains than those grown in the absence of glucose during the late exponential and transition growth phases, and identify that the clhAB2 operon is required for this chain lengthening phenotype. The clhAB2 operon is specific to the B. cereus group (i.e., B. thuringiensis, B. anthracis and B. cereus) and encodes two membrane proteins of unknown function, which are homologous to the Staphylococcus aureus CidA and CidB proteins involved in cell death control within glucose-grown cells. A deletion mutant (ΔclhAB2) was constructed and our quantitative image analyses show that ΔclhAB2 cells formed abnormal short chains regardless of the presence of glucose. We also found that glucose-grown cells of ΔclhAB2 were significantly wider than wild-type cells (1.47 μm ±CI95% 0.04 vs 1.19 μm ±CI95% 0.03, respectively), suggesting an alteration of the bacterial cell wall. Remarkably, ΔclhAB2 cells showed accelerated autolysis under autolysis-inducing conditions, compared to wild-type cells. Overall, our data suggest that the B. cereus clhAB2 operon modulates peptidoglycan hydrolase activity, which is required for proper cell shape and chain length during cell growth, and down-regulates autolysin activity. Lastly, we studied the transcription of clhAB2 using a lacZ transcriptional reporter in wild-type, ccpA and codY deletion-mutant strains. We found that the global transcriptional regulatory protein CodY is required for the basal level of clhAB2 expression under all conditions tested, including the transition growth phase while CcpA, the major global carbon regulator, is needed for the high-level expression of clhAB2 in glucose-grown cells.
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