Effects and molecular mechanism of sugar transporter ESA_RS15745 on desiccation resistance, motility, and biofilm formation of Cronobacter sakazakii.

Cronobacter sakazakii, an important Gram-negative foodborne pathogen, can cause neonatal meningitis and sepsis with high rates of infection and death. Gene ESA_RS15745 encodes a sugar transporter protein, which is not only essential for osmotic pressure maintenance during bacterial growth and reproduction but also associated with their desiccation tolerance, motility, and biofilm formation. Here, a mutant strain of ESA_RS15745 (ΔESA_RS15745) and the complementation strain (cpESA_RS15745) were constructed using a suicide vector knockout and gene complementation. ΔESA_RS15745 was found to have a decrease in its ability to transport maltose and trehalose and resist desiccation, whereas an increase in the ability of motility and biofilm formation, implying that ESA_RS15745 may positively regulate sugar transport and desiccation tolerance and negatively regulate motility and biofilm formation. To further investigate the molecular mechanisms underlying the function of related genes, RNA-seq was performed to explore the differentially expressed genes in the mutants. RNA-seq results showed the upregulation of 114 genes (mainly including those regulating chemotaxis and flagellar motility) and the downregulation of 22 genes (mainly including those regulating sugar transport). qRT-PCR analysis supported the RNA-seq results and showed that ESA_RS15745 may influence the dehydration tolerance though decreasing the intracellular trehalose content and negatively regulate the motility though the chemotactic signaling pathway. In addition, the biofilm formation of C. sakazakii should also be speculated to negatively regulate by ESA_RS15745 by consuming the extracellular carbohydrates concentration and then downregulating the intracellular cyclic diguanosine monophosphate. This study offers a reference for comprehending the molecular mechanism of gene ESA_RS15745 in C. sakazakii.

Direct interactions between molecular chaperones heat-shock protein (Hsp) 70 and Hsp40: yeast Hsp70 Ssa1 binds the extreme C-terminal region of yeast Hsp40 Sis1.

Heat-shock protein 40 (Hsp40) enables Hsp70 to play critical roles in a number of cellular processes, such as protein folding, assembly, degradation and translocation in vivo. Hsp40 recognizes and binds non-native polypeptides and delivers them to Hsp70. Then Hsp40 stimulates the ATPase activity of Hsp70 to fold the polypeptides. By using yeast Hsp40 Sis1 and yeast Hsp70 Ssa1 as our model proteins, we found that the Sis1 peptide-binding fragment interacts directly with the full-length Ssa1 in vitro. Further studies showed that the C-terminal lid domain of Ssa1 could interact with Sis1 peptide-binding domain physically in vitro. The Sis1 peptide-binding fragment forms a stable complex with the Ssa1 C-terminal lid domain in solution. The interactions between these two proteins appear to be charge-charge interactions because high-ionic-strength buffer can dissociate the complex. Further mapping studies showed that the Sis1 peptide-binding fragment binds the extreme C-terminal 15 amino acid residues of Ssa1. A flexible glycine-rich region is followed by these 15 residues in the Ssa1 primary sequence. Atomic force microscopy of the Sis1-Ssa1 complex showed that only one end of the Ssa1 lid domain binds the Sis1 peptide-binding-fragment dimer at the upper level of the huge groove within the Sis1 dimer. Based on the data, we propose an "anchoring and docking" model to illustrate the mechanisms by which Hsp40 interacts with Hsp70 and delivers the non-native polypeptide to Hsp70.

Effect of extraneous sugar on the reactive oxygen species metabolism of strept ococcus mutans / 实用口腔医学杂志

Objective:To study the effect of extraneous sugar on rea ct ive oxygen species metabolism of Streptococcus mutans. Methods: Electron spin resonance (ESR) method was used to measure reactive oxygen spe cies of Streptococcus mutans incubated in the different sugar incubation con ditions. Results:With the increase of glucose at 0.0062~0.05 mo l/L, the quantities of reactive oxygen species produced by Streptococcus mutan s increased and increased to the maximum when Streptococcus mutans were in cubated in different sugar culture fluids within 2 h. All kinds of sugar promote d reactive oxygen species metabolism of Streptococcus mutans. Conclu sion:Extraneous sugar promotes reactive oxygen species metabolism of Streptococcus mutans.

The effect of methylene blue on the metabolism of Streptococcus mutans / 实用口腔医学杂志

Objective: To study the effect of methylene blue on the metabolism of reactive oxygen and acid production of Streptococcus mutans . Methods: Streptococcus of cricetusa, rattusb, mutansc, sobrinsd and mutans e,f and g were exposed to methylene blue at 2.5?10 -4 g/L for 1 h. The metabolism of reactive oxygen and acid production were detected by electron spin trapping method and gas chromatography respectively. Results: Electron spin resonance (ESR) spectrums of DMPO-O - ? 2 and DMPO-OH ? were observed in Streptococcus mutans cultured in glucose fluid, but were not found in those exposed to methylene. The total of acid production by Streptococcus mutans incubated with glucose + methylene blue were significantly lower than that with single glucose. Conclusion: Methylene blue may inhibit oxygen species metabolism and acid production of Streptococcus mutans , it may be used as a kind of dental caries prevention agent.