Null mutation in sspA of Cronobacter sakazakii influences its tolerance to environmental stress

2021 ◽  
Author(s):  
Jie Zhan ◽  
Xin Tan ◽  
Xiaoyuan Wang
Keyword(s):  
Stress Tolerance ◽  
Protein A ◽  
Bacterial Chemotaxis ◽  
Water Concentration ◽  
Acid Tolerance ◽  
Opportunistic Pathogen ◽  
Cronobacter Sakazakii ◽  
Knockout Mutant ◽  
Cellular Survival ◽  
E Coli

Cronobacter sakazakii is a known foodborne opportunistic pathogen that can affect the intestinal health of infants. Despite undergoing complex manufacturing processes and low water concentration in the finished product, infant formula has been associated with Cronobacter infections, suggesting that C. sakazakii’s pathogenicity may be related to its tolerance to stress. In this study, the effect of the stringent starvation protein A (SspA), which plays an important role in E. coli cellular survival under environmental stresses, on the stress tolerance of C. sakazakii BAA894 was investigated by creating an sspA-knockout mutant. The effects of this mutation on the acid, desiccation and drug tolerance were assessed, and results showed that acid tolerance decreased, while desiccation tolerance increased in LB and decreased in M9. Moreover, the MICs of 10 antibiotics in LB medium and 8 antibiotics in M9 medium were determined and compared of the wild-type and ΔsspA. Transcriptome analysis showed that 27.21% or 37.78% of the genes in ΔsspA were significantly differentially expressed in LB or M9 media, the genes relevant to microbial metabolism in diverse environments and bacterial chemotaxis were detailed analyzed. The current study contributes towards an improved understanding of the role of SspA in C. sakazakii BAA894 stress tolerance.

scholarly journals Structural basis for transcription inhibition by E. coli SspA

2020 ◽  
Vol 48 (17) ◽  
pp. 9931-9942 ◽  
Author(s):  
Fulin Wang ◽  
Jing Shi ◽  
Dingwei He ◽  
Bei Tong ◽  
Chao Zhang ◽  
...  
Keyword(s):  
Structural Information ◽  
Protein A ◽  
Acid Tolerance ◽  
In Vitro Transcription ◽  
Nucleotide Metabolism ◽  
Structural Basis ◽  
E Coli ◽  
Promoter Escape ◽  
Zinc Binding Domain

Abstract Stringent starvation protein A (SspA) is an RNA polymerase (RNAP)-associated protein involved in nucleotide metabolism, acid tolerance and virulence of bacteria. Despite extensive biochemical and genetic analyses, the precise regulatory role of SspA in transcription is still unknown, in part, because of a lack of structural information for bacterial RNAP in complex with SspA. Here, we report a 3.68 Å cryo-EM structure of an Escherichia coli RNAP-promoter open complex (RPo) with SspA. Unexpectedly, the structure reveals that SspA binds to the E. coli σ70-RNAP holoenzyme as a homodimer, interacting with σ70 region 4 and the zinc binding domain of EcoRNAP β′ subunit simultaneously. Results from fluorescent polarization assays indicate the specific interactions between SspA and σ70 region 4 confer its σ selectivity, thereby avoiding its interactions with σs or other alternative σ factors. In addition, results from in vitro transcription assays verify that SspA inhibits transcription probably through suppressing promoter escape. Together, the results here provide a foundation for understanding the unique physiological function of SspA in transcription regulation in bacteria.

scholarly journals Functional Overlap but Lack of Complete Cross-Complementation of Streptococcus mutans and Escherichia coli YidC Orthologs

2008 ◽  
Vol 190 (7) ◽  
pp. 2458-2469 ◽  
Author(s):  
Yuxia Dong ◽  
Sara R. Palmer ◽  
Adnan Hasona ◽  
Shushi Nagamori ◽  
H. Ronald Kaback ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Streptococcus Mutans ◽  
Protein A ◽  
Membrane Integrity ◽  
Acid Tolerance ◽  
Growth Conditions ◽  
Stress Sensitivity ◽  
Protein Insertion ◽  
E Coli ◽  
Membrane Protein Insertion

ABSTRACT Oxa/YidC/Alb family proteins are chaperones involved in membrane protein insertion and assembly. Streptococcus mutans has two YidC paralogs. Elimination of yidC2, but not yidC1, results in stress sensitivity with decreased membrane-associated F1Fo ATPase activity and an inability to initiate growth at low pH or high salt concentrations (A. Hasona, P. J. Crowley, C. M. Levesque, R. W. Mair, D. G. Cvitkovitch, A. S. Bleiweis, and L. J. Brady, Proc. Natl. Acad. Sci. USA 102:17466-17471, 2005). We now show that Escherichia coli YidC complements for acid tolerance, and partially for salt tolerance, in S. mutans lacking yidC2 and that S. mutans YidC1 or YidC2 complements growth in liquid medium, restores the proton motive force, and functions to assemble the F1Fo ATPase in a previously engineered E. coli YidC depletion strain (J. C. Samuelson, M. Chen, F. Jiang, I. Moller, M. Wiedmann, A. Kuhn, G. J. Phillips, and R. E. Dalbey, Nature 406:637-641, 2000). Both YidC1 and YidC2 also promote membrane insertion of known YidC substrates in E. coli; however, complete membrane integrity is not fully replicated, as evidenced by induction of phage shock protein A. While both function to rescue E. coli growth in broth, a different result is observed on agar plates: growth of the YidC depletion strain is largely restored by 247YidC2, a hybrid S. mutans YidC2 fused to the YidC targeting region, but not by a similar chimera, 247YidC1, nor by YidC1 or YidC2. Simultaneous expression of YidC1 and YidC2 improves complementation on plates. This study demonstrates functional redundancy between YidC orthologs in gram-negative and gram-positive organisms but also highlights differences in their activity depending on growth conditions and species background, suggesting that the complete functional spectrum of each is optimized for the specific bacteria and environment in which they reside.

scholarly journals Contribution of dps to Acid Stress Tolerance and Oxidative Stress Tolerance in Escherichia coli O157:H7

2000 ◽  
Vol 66 (9) ◽  
pp. 3911-3916 ◽  
Author(s):  
Sang Ho Choi ◽  
David J. Baumler ◽  
Charles W. Kaspar
Keyword(s):  
Oxidative Stress ◽  
Escherichia Coli ◽  
Hydrogen Peroxide ◽  
Stationary Phase ◽  
Stress Tolerance ◽  
Parent Strain ◽  
Acid Stress ◽  
Acid Tolerance ◽  
Gastric Fluid ◽  
Acid Challenge

ABSTRACT An Escherichia coli O157:H7dps::nptI mutant (FRIK 47991) was generated, and its survival was compared to that of the parent in HCl (synthetic gastric fluid, pH 1.8) and hydrogen peroxide (15 mM) challenges. The survival of the mutant in log phase (5-h culture) was significantly impaired (4-log10-CFU/ml reduction) compared to that of the parent strain (ca. 1.0-log10-CFU/ml reduction) after a standard 3-h acid challenge. Early-stationary-phase cells (12-h culture) of the mutant decreased by ca. 4 log10CFU/ml while the parent strain decreased by approximately 2 log10 CFU/ml. No significant differences in the survival of late-stationary-phase cells (24-h culture) between the parent strain and the mutant were observed, although numbers of the parent strain declined less in the initial 1 h of acid challenge. FRIK 47991 was more sensitive to hydrogen peroxide challenge than was the parent strain, although survival improved in stationary phase. Complementation of the mutant with a functional dps gene restored acid and hydrogen peroxide tolerance to levels equal to or greater than those exhibited by the parent strain. These results demonstrate that decreases in survival were from the absence of Dps or a protein regulated by Dps. The results from this study establish that Dps contributes to acid tolerance in E. coli O157:H7 and confirm the importance of Dps in oxidative stress protection.

Effect offurmutation on acid-tolerance response and in vivo virulence of avian septicemicEscherichia coli

2002 ◽  
Vol 48 (5) ◽  
pp. 458-462 ◽  
Author(s):  
Chengru Zhu ◽  
Musangu Ngeleka ◽  
Andrew A Potter ◽  
Brenda J Allan
Keyword(s):  
Parent Strain ◽  
Acid Tolerance ◽  
Wild Type ◽  
Acid Tolerance Response ◽  
E Coli ◽  
Regulator Protein ◽  
In The Wild ◽  
Tolerance Response ◽  
Wild Type Parent

The Fur (ferric uptake regulator) protein is a master regulator of iron metabolism in gram-negative bacteria. In the present study, the effect of a partial deletion of the fur gene on the acid-tolerance response and in vivo virulence of avian Escherichia coli was examined. The fur mutant was unable to trigger the acid-tolerance response as observed in the wild-type parent strain. However, the mutant was as virulent as the wild-type parent strain when tested in 1-day-old chickens by subcutaneous inoculation. These data indicate that the fur gene is involved in the acid-tolerance response but not involved in the virulence of E. coli, as detected by the ability to cause septicemia in our experimental infection.Key words: E. coli, fur, acid-tolerance response.

Viability of Acid-Adapted Escherichia coli O157:H7 in Ground Beef Treated with Acidic Calcium Sulfate

2004 ◽  
Vol 67 (3) ◽  
pp. 591-595 ◽  
Author(s):  
LARRY R. BEUCHAT ◽  
ALAN J. SCOUTEN
Keyword(s):  
Escherichia Coli ◽  
Acetic Acid ◽  
Calcium Sulfate ◽  
Ground Beef ◽  
Storage Period ◽  
Acid Tolerance ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Acidic Environments ◽  
Subsequent Acid

The effects of lactic acid, acetic acid, and acidic calcium sulfate (ACS) on viability and subsequent acid tolerance of three strains of Escherichia coli O157:H7 were determined. Differences in tolerance to acidic environments were observed among strains, but the level of tolerance was not affected by the acidulant to which cells had been exposed. Cells of E. coli O157:H7 adapted to grow on tryptic soy agar acidified to pH 4.5 with ACS were compared to cells grown at pH 7.2 in the absence of ACS for their ability to survive after inoculation into ground beef treated with ACS, as well as untreated beef. The number of ACS-adapted cells recovered from ACS-treated beef was significantly (α = 0.05) higher than the number of control cells recovered from ACS-treated beef during the first 3 days of a 10-day storage period at 4°C, suggesting that ACS-adapted cells might be initially more tolerant than unadapted cells to reduced pH in ACS-treated beef. Regardless of treatment of ground beef with ACS or adaptation of E. coli O157:H7 to ACS before inoculating ground beef, the pathogen survived in high numbers.

scholarly journals AOA-2 Derivatives as Outer Membrane Protein A Inhibitors for Treatment of Gram-Negative Bacilli Infections

2021 ◽  
Vol 12 ◽  
Author(s):  
Rafael Ayerbe-Algaba ◽  
Nuria Bayó ◽  
Ester Verdú ◽  
Raquel Parra-Millán ◽  
Jesús Seco ◽  
...  
Keyword(s):  
Protein A ◽  
A549 Cells ◽  
Lung Epithelial Cells ◽  
Host Cells ◽  
Gram Negative ◽  
E Coli ◽  
Cyclic Hexapeptide ◽  
Diphenyltetrazolium Bromide ◽  
Peptide Derivatives

Previously, we identified that a cyclic hexapeptide AOA-2 inhibited the interaction of Gram-negative bacilli (GNB) like Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli to host cells thereby preventing the development of infection in vitro and in a murine sepsis peritoneal model. In this work, we aimed to evaluate in vitro a library of AOA-2 derivatives in order to improve the effect of AOA-2 against GNB infections. Ten AOA-2 derivatives were synthetized for the in vitro assays. Their toxicities to human lung epithelial cells (A549 cells) for 24 h were evaluated by determining the A549 cells viability using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The effect of these peptide derivatives and AOA-2 at 250, 125, 62.5, and 31.25 μg/mL on the attachment of A. baumannii ATCC 17978, P. aeruginosa PAO1 and E. coli ATCC 25922 strains to A549 cells was characterized by adherence and viability assays. None of the 10 derivatives showed toxicity to A549 cells. RW01 and RW06 have reduced more the adherence of ATCC 17978, PAO1 and ATCC 2599 strains to A549 cells when compared with the original compound AOA-2. Moreover, both peptides have increased slightly the viability of infected A549 cells by PAO1 and ATCC 25922 than those observed with AOA-2. Finally, RW01 and RW06 have potentiated the activity of colistin against ATCC 17978 strain in the same level with AOA-2. The optimization program of AOA-2 has generated two derivatives (RW01 and RW06) with best effect against interaction of GNB with host cells, specifically against P. aeruginosa and E. coli.

scholarly journals Improved CUT&RUN chromatin profiling and analysis tools

2019 ◽  
Author(s):  
Michael P. Meers ◽  
Terri Bryson ◽  
Steven Henikoff
Keyword(s):  
Fusion Protein ◽  
High Efficiency ◽  
Low Cost ◽  
Protein A ◽  
Micrococcal Nuclease ◽  
Permeabilized Cells ◽  
E Coli ◽  
Carry Over ◽  
Chromatin Profiling ◽  
Premature Release

AbstractWe previously described a novel alternative to Chromatin Immunoprecipitation, Cleavage Under Targets & Release Using Nuclease (CUT&RUN), in which unfixed permeabilized cells are incubated with antibody, followed by binding of a Protein A-Micrococcal Nuclease (pA/MNase) fusion protein (1). Upon activation of tethered MNase, the bound complex is excised and released into the supernatant for DNA extraction and sequencing. Here we introduce four enhancements to CUT&RUN: 1) a hybrid Protein A-Protein G-MNase construct that expands antibody compatibility and simplifies purification; 2) a modified digestion protocol that inhibits premature release of the nuclease-bound complex; 3) a calibration strategy based on carry-over of E. coli DNA introduced with the fusion protein; and 4) a novel peak-calling strategy customized for the low-background profiles obtained using CUT&RUN. These new features, coupled with the previously described low-cost, high efficiency, high reproducibility and high-throughput capability of CUT&RUN make it the method of choice for routine epigenomic profiling.

scholarly journals Identification and Probiotic Potency Screening of Cellulolytic Bacilli Isolated from Fecal Pellets and Gastrointestinal Tract of Nypha Worm (Namalycastis rhodochorde), West Kalimantan, Indonesia

2021 ◽  
Vol 17 (2) ◽  
pp. 189-195
Author(s):  
TR Setyawati ◽  
AH Yanti ◽  
R. Kurniatuhadi
Keyword(s):  
Escherichia Coli ◽  
Gastrointestinal Tract ◽  
Organic Acids ◽  
Acid Tolerance ◽  
Distilled Water ◽  
Bacterial Identification ◽  
Bacterial Isolates ◽  
Fecal Pellets ◽  
West Kalimantan ◽  
E Coli

The bacterial isolates NrLtF1, NrLtF4, NrLtF5, and NrLtG2 isolated from fecal pellets and gastrointestinal tract of nypha worms (Namalycastis rhodochorde) have cellulolytic, proteolytic activity and produce organic acids. The four isolates have the potency to be developed as probiotics in nypha worm cultivation feed. This study aims to determine the probiotics potency and identify the species of NrLtF1, NrLtF4, NrLtF5, and NrLtG2 isolate based on 16srDNA sequence. The probiotic potency was carried out by the acid tolerance assays on distilled water and 0.3% acid bile media, and the antimicrobial testing against Escherichia coli (MF exp21.12). Bacterial identification was carried out by sequencing of 16sDNA sequence based on GeneBank data. The results showed that the bacterial isolates of NrLtF1, NrLtF4, NrLtF5, and NrLtG2 were able to grow on 0.3% distilled water and acid bile media. However, only the NrLtF4 and NrLtF5 inhibited E. coli (MF exp21.12) with halo zones 30 mm and 18 mm, respectively. Blasting results of the 16srDNA sequences showed that the NrLtF1, NrLtF4, NrLtF5, and NrLtG2 were closely related to Bacillus wiedmannii, Brevibacterium sediminis, Bacillus proteolyticus, and Bacillus paramycoides. The nypha worm bacterial isolates have the potency to be developed as probiotics in nypha worm culture.

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