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Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, State Key Laboratory of Applied Microbiology Southern China, Guangdong Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou, 510070, China
Bile salts is one of essential components of bile secreted into the intestine to confer antibacterial protection. Cronobacter species are associated with necrotizing enterocolitis in newborns and show a strong tolerance to bile salts. However, little attempt has been made to focus on the molecular basis of the tolerance to bile salts. In this study, we investigated the roles of tolC on growth, cell morphology, motility, and biofilm formation ability in Cronobacter malonaticus under bile salt stress. The results indicated that the absence of tolC significantly affected the colony morphology and outer membrane structure in a normal situation, compared with those of the wild type strain. The deletion of tolC caused the decline in resistance to bile salt stress, inhibition of growth, and observable reduction in relative growth rate and motility. Moreover, the bacterial stress response promoted the biofilm formation ability of the mutant strain. The expression of the AcrAB-TolC system (acrA, acrB, and tolC) was effectively upregulated compared with the control sample when exposed to different bile salt concentrations. The findings provide valuable information for deeply understanding molecular mechanisms about the roles of tolC under bile salt stress and the prevention and control of C. malonaticus.
The Cronobacter genus, which is a group of opportunistic pathogens, causes rare but life-threatening cases of necrotizing enterocolitis, meningitis, cyst formation, intracerebral infarctions, bacteremia, and sepsis in premature neonates and infants with underlying chronic conditions (
). Seven different species of Cronobacter have been identified, namely Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter muytjensii, Cronobacter dublinensis, Cronobacter universalis, and Cronobacter condimenti (
During the manufacture, transport, and storage of PIF and within the infected host, Cronobacter encounters various environmental stresses including desiccation, heat, acid, and hyperosmosis (
). Therefore, the resistance of C. malonaticus to unfavorable environmental conditions is a critical factor in its ability to survive and proliferate in PIF. For the food industry, highly efficient precautions against and control of C. malonaticus contamination are an even more severe challenge.
Pathogenic bacteria must resist antimicrobial substances such as bile salts to survive in the intestinal tract (
). Bacteria that have adapted to life in the intestine have developed mechanisms of resistance to bile, including modified membrane structures that reduce bile permeability and efflux pumps that can transport bile out of the cell (
). The bile salt resistance mechanism in the model enterobacterium Escherichia coli is the restriction of intracellular accumulation by employing diverse, active efflux pumps (
A single-component multidrug transporter of the major facilitator superfamily is part of a network that protects Escherichia coli from bile salt stress.
). The primary machinery involved is a tripartite multidrug efflux system called AcrAB-TolC. It belongs to the RND family (resistance nodulation cell division family), which has a wide substrate spectrum encompassing antibiotics, dyes, detergents, bile salts, toxins, and environmental compounds (
). However, little attention has been focused on the influence of AcrAB-TolC in Cronobacter species when exposed to bile salt stress.
In this study, we compared the growth, motility, and biofilm formation of tolC mutant and parental strains to better understand the roles of tolC on bacterial phenotypes in C. malonaticus when exposed to bile salt stress. Also, quantitative real-time PCR (qRT-PCR) was used to quantify the expression of the AcrAB-TolC system in wild type (WT) to explore the potential molecular mechanism for the tolC gene under bile salt stress.
MATERIALS AND METHODS
Bacterial Strains, Growth Curves, and Colony Morphology
Cronobacter malonaticus WT strain was from Guangdong Microbiology Culture Center. The tolC was deleted by the insertion of the Cmr cassette according to the protocol described by
. The WT and ΔtolC strain were routinely cultured in Luria-Bertani (LB; Huankai) broth medium for incubation at 37°C for 24 h with shaking at 180 rpm. The optical density at 600 nm (OD600) was measured every 2 h, and a growth curve was drawn. The bacterial colony morphology was observed after marking 3 zones on the Trypticase Soy Agar (Huankai) medium at 37°C for 24 h.
Effect of TolC on C. Malonaticus Growth Under Bile Salt Stress
Two strains were incubated into LB medium without and with bile salts (0.005%, 0.01%, and 0.02%) for 2 h. The number of C. malonaticus under different media was counted using a colony counting method. Relative growth rates were calculated as the number of C. malonaticus cells in the control sample divided by the number of cells in different concentrations of bile salt samples. Each experiment was done in triplicate.
Biofilm Formation of C. Malonaticus When Exposed to Bile Salts
The biofilm formation on 96-well polystyrene microplates was quantified with the crystal violet staining (CVS) method (
). Overnight cultures of 2 strains were diluted 100-fold into fresh LB broth medium and LB with different bile salts. Two hundred microliters of diluted cultures were added to 96-well plates, and the plates were incubated at 37°C for 24, 48, and 72 h. The subsequent treatment was done as described by
Overnight cultures with 1% (vol/vol) were transferred to fresh LB medium with or without bile salt concentration (0.005%, 0.01%, and 0.02%). Then, the glass coverslips were immersed into the above medium in 24-well plates for incubation at 37°C for 24, 48, and 72 h, respectively. Subsequently, cells immobilized on the cell climbing slice were examined by scanning electron microscopy (Hitachi) as described previously by
. Samples were prepared for scanning electron microscopy by fixation in 3% glutaraldehyde at 4°C for 5 h, then dehydration in ethanol followed by tertiary butanol. Dehydrated samples were dried with a CO2-critical point dryer, coated with gold, and imaged by scanning electron microscopy at 20 kV.
To better visualize the architecture of the biofilms, biofilms were stained with the Live/Dead BacLight Bacterial Viability Kit (Thermo Fisher Scientific) and observed by confocal laser scanning microscopy (CLSM). For CLSM, the glass coverslips were prepared as the same as for scanning electron microscopy detection. In BacLight, propidium iodide and Syto9 were added to stain nucleic acids (
A 96-well plate-based optical method for the quantitative and qualitative evaluation of Pseudomonas aeruginosa biofilm formation and its application to susceptibility testing.
). Swarming medium was prepared with bile salt concentrations of 0.005%, 0.01%, and 0.02% (wt/vol). To determine swarming motility, WT and ΔtolC strains were grown to the mid-exponential phase. Then single colonies were picked onto soft agar motility plates (LB containing 0.5% agar) incubated at 37°C for 16 h. After incubation, Vernier caliper can be used to measure the bacterial movement diameter. Each experiment was done in triplicate.
Quantitative Expression of AcrAB-tolC Efflux System
Overnight cultures of WT bacteria were transferred to LB medium without bile salts and bile salts concentration (0.05%, 0.05%, and 5.00%). Total RNA was isolated and purified using the Bacterial RNA extraction kit (Meiji) according to the manufacturer's instructions with RNA eluted in 50 µL of RNase-free water. The RNA concentration was quantified using a nucleic acid protein analyzer, and quality was detected by electrophoresis. Up to 1 µL of RNA per sample was reverse transcribed to cDNA using the PrimeScript RT reagent kit with gDNA Eraser (Takara) according to the manufacturer's protocol. We obtained the gene sequence of C. malonaticus AcrAB-TolC and gyrB from the National Center for Biotechnology Information and used Primer-BLAST software (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome) for primer design (Table 1). The qRT-PCR was performed in optical tubes and caps in a fluorescence quantitative PCR instrument (LightCycler 96) and a qRT-PCR program, which consisted of an initial 30-s denaturation step at 95°C followed by 40 cycles of repeated denaturation at 95°C for 5 s, annealing at 52°C for 30 s, and elongation at 72°C for 45 s. The 2−ΔΔCT method was used to determine the relative changes in gene expression.
Table 1Primers used for quantitative real-time PCR in this study
The experimental determinations were performed at least in triplicate with independent microbial cultures. Data in the figure correspond to the average and the mean standard deviation (error bars). t-Tests were carried out using the GraphPad PRISM 5 software (GraphPad Software Inc.).
RESULTS AND DISCUSSION
Growth Curve, Colony Morphology, and Outer Membrane Structure
According to Figure 1A, the growth curves of bacteria strains (WT and ΔtolC) were similar. However, there were visible differences in colony morphology and outer membrane structure between WT and mutant strains. The colony size of ΔtolC was larger than that of WT. In addition, the colony of WT was full and round with a neat edge and smooth surface. Conversely, ΔtolC was irregular with a rough, irregular edge and uneven surface (Figure 1B). To a certain extent, colony morphology is a macroscopic response of cell morphology. The AcrAB-TolC efflux system plays a vital role in the pathogenesis of Salmonella typhimurium, and ΔtolC weakens the adhesion and invasion of human embryonic intestinal cells and mouse monocyte macrophages (
). Therefore, changes in colony morphology may affect the resistance and pathogenicity of C. malonaticus in certain adverse environments.
Figure 1Growth curves, colony morphology, and outer membrane structure of wild type (WT) and mutant type (ΔtolC) under normal Luria-Bertani broth condition. (A) The growth curves of WT and ΔtolC; (B) comparison of colony morphology between WT and ΔtolC; (C) outer membrane structure changes of WT and ΔtolC using transmission electron microscopy. Arrows indicate the outer membranes of the bacterial cells. OD600 = optical density at 600 nm.
Furthermore, we found that the ability producing yellow color in WT strain is stronger than that in the ΔtolC strain (Figure 1B). Cronobacter is considered an opportunistic foodborne pathogen that is characterized by the formation of yellow-pigmented colonies. In the previous study, the carotenoid nature of pigment was identified in Cronobacter strain ES5 on a molecular and chemical level (
Knockout of crtB or crtI gene blocks the carotenoid biosynthetic pathway in Deinococcus radiodurans R1 and influences its resistance to oxidative DNA-damaging agents due to change of free radicals scavenging ability.
). Moreover, pigments play a role in the survival of bacteria in harmful environments and have been found to increase the virulence of pathogens such as Staphylococcus aureus and Erwinia chrysanthemi (
). Therefore, the color change of colonies indicated that the deletion of tolC may destroy the yellow pigment synthesis pathway in C. malonaticus, affecting environmental resistance and toxicity.
Next, the outer membrane structures of bacteria (WT and ΔtolC) were observed and compared by transmission electron microscopy. As showed in Figure 1C, it was found that the outer membrane of WT was smooth and round with clear periplasmic space. However, the outer membrane of ΔtolC was relatively wrinkled and irregular, and the periplasmic space became a little fuzzy. These results indicated that the deletion of the tolC gene significantly changed the outer membrane structure of C. malonticus. The role of TolC in E. coli physiology has been previously examined; the pleiotropic phenotypes of tolC, which are involved in drug resistance, outer membrane composition, virulence regulation, and acid tolerance, may be linked to the complex network of tolC and other regulatory factors (
), outer membrane structure might be disrupted in the absence of TolC, resulting in disturbance membrane permeability. Similarly, the outer membrane structure of tolC mutant in C. malonaticus has changed, affecting some functions.
Growth, Cell Morphology, and Motility of C. Malonaticus
As shown in Figure 2A, when the concentration of bile salts exceeded 0.005%, the relative growth rate of ΔtolC decreased sharply and was almost zero when the concentration exceeded 0.02%. In addition, ΔtolC decreased markedly in a time-dependent manner. However, the relative growth rate of WT reduced gradually when the concentration exceeded 0.02% and did not change much under bile salt stress over time. Next, we measured the growth curve of WT and ΔtolC after determining the selected bile salt concentration (0.005%, 0.01%, and 0.02%). According to Figure 2B, when the concentration was 0.005%, the growth stages of ΔtolC were significantly backward, and the bacterial biomass decreased more dramatically in the stable phase than that in WT. The ΔtolC almost stopped increasing when the concentration of bile salts exceeded 0.01%. For WT, the growth curve did not change with the increase of bile salt concentration. The results showed that the absence of tolC gene in C. malonaticus significantly weakened the bile salt tolerance and affected the growth period of bacteria when exposed to bile salt stress. Furthermore, the outer membrane channel protein TolC is the main component for the bile salt excretion system of C. malonaticus. In S. typhimurium, the tolC mutation affects the bacteria susceptibility to various antibacterial substances, including bile salts (
). Bile salt is an effective antibacterial agent that can damage the cell membranes, and cause protein misfolding and DNA oxidative damage, thus protecting the body from invasion of microorganisms (
). Therefore, it can reduce the relative growth rate of C. malonaticus (WT and ΔtolC). In addition, the relative growth rate of ΔtolC decreases significantly with time. The reason for this may be that the mutant strain lacks TolC protein to eliminate bile salts in time, which resulted in bile salts accumulating, destroying cells, and causing bacterial death. The MnhF mediates the efflux of bile salts in Staphylococcus aureus, and the absence of MnhF reduces the survival rate of Staph. aureus (
Figure 2The relative growth rate and growth curve of wild type (WT) and mutant-type (ΔtolC) when exposed to bile salts. (A) The relative growth rate of WT and ΔtolC with different bile salt concentrations; (B) comparison of growth curve under bile salt (0.005%, 0.01%, and 0.02%) stress. Error bars indicate SD. Asterisks indicate difference between WT and mutant at *P < 0.05 and ****P < 0.0001. OD600 = optical density at 600 nm; LB = Luria-Bertani.
The morphology of pathogenic bacteria is easily affected by the external environment. In this study, C. malonaticus (WT and ΔtolC) were cultured when exposed to bile salt stress for 2 h and it was observed whether the bacteria morphology had changed. The observation was made that WT and ΔtolC bacteria had equivalent morphology in normal LB medium (Figure 3A). However, the ΔtolC strains became clustered and longer at a concentration of 0.005% bile salts; the bacterial deformation of the ΔtolC intensified, and the cells grew more obvious with 0.01% bile salts. As the bile salt concentration increased to 0.02%, ΔtolC strains were shortened significantly, most of them became round, and the outer membrane structure was destroyed (Figure 3A). Bacterial deformation is usually associated with DNA damage, interference with cell wall synthesis, and cell membrane composition changes. Bacteria often deform in the face of adversity that is not conducive to survival, to adapt to the environment to survive. Pseudomonas aeruginosa adapts to the nutrient deficiency environment by growing and increasing its size (
). Therefore, this dynamic change of aberrant morphology may be associated with an adaptive mechanism to strengthen defense against stress and maintain vital movement by modifying the length, thickness, and size of bacteria.
Figure 3Differences of cell morphology and motility under bile salt stress. (A) Cell morphology of wild type (WT) and mutant-type (ΔtolC) by scanning electron microscopy. Arrows indicate sites of morphological injury to the bacteria. (B) Movability of WT and ΔtolC. Error bars indicate SD. Asterisks indicate difference between WT and mutant at ****P < 0.0001.
Movability was similar between WT and ΔtolC under normal conditions (Figure 3B). After the addition of 0.005%, 0.01%, and 0.02% bile salts, the movement ability of WT decreased slightly. Accompanied by increasing bile salts, on the contrary, the movability of ΔtolC decreased markedly (Figure 3B). This result meant that the deletion of the tolC gene caused a significant decrease in the movability of C. malonaticus to tolerate bile salt stress. Flagella-mediated motility not only plays an essential role in reaching the ideal infection site in Salmonella enterica, Campylobacter jejuni, P. aeruginosa, and E. coli, but also has other functions in the pathogenicity process, particularly adherence, biofilm formation, and immune modulation (
Adherence of coagulase-negative Staphylococci to plastic tissue culture plates: A quantitative model for the adherence of Staphylococci to medical devices.
). Bacterial cells can modify their motility velocity and modes for physiological needs confronting environmental stimuli (e.g., nutrients, pollutants, and light) and reposition themselves dynamically toward optimal niches (
Multidrug efflux pumps have been recognized as playing a central role in the biology of bacteria and have roles in cell division, drug resistance, pathogenicity, and as recently described, the formation of biofilms (
). In this study, the biofilm formation of the 2 strains was detected by CVS, scanning electron microscopy, and CLSM. The biofilm formation ability of ΔtolC is significantly lower than that of WT through the above 3 methods under normal LB culture (Figure 4). Mutants, such as the E. coli K-12 strain, lacking functional multidrug efflux pump-related genes such as emrD, emrE, emrK acrD, acrE, or mdtE exhibit decreased biofilm formation (
). In addition, research demonstrated the inability of mutants lacking any of the 9 multidrug resistance efflux pumps of Salmonella to form a mature biofilm (
Loss of or inhibition of all multidrug resistance efflux pumps of Salmonella enterica serovar Typhimurium results in impaired ability to form a biofilm.
J. Antimicrob. Chemother.2012; 67 (22733653): 2409-2517
). Similarly, our results also showed that the tolC gene mutation weakened biofilm formation in C. malonaticus.
Figure 4Difference of biofilm formation ability when exposed to bile salt stress. (A) Biofilm formation measured by crystal violet staining under bile salt stress for 24, 48, and 72 h; (B) Biofilm formation of Cronobacter malonaticus wild type (WT) and mutant type (ΔtolC) under bile salt stress for 24, 48, and 72 h using confocal laser scanning microscopy (200 μm). (C) Biofilm formation of C. malonaticus WT and ΔtolC under bile salt stress for 24, 48, and 72 h using scanning electron microscopy (100 μm). OD590 = optical density at 590 nm. Error bars indicate SD. Asterisks indicate difference between WT and mutant at *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
The quantity and surface structure of biofilms of WT and ΔtolC mutant were compared by CLSM and scanning electron microscopy. For CLSM (Figure 4B), the biofilm was initially formed at 24 h and scattered on the cell climbing sheet full of live bacteria. At 48 h, the biofilm was basically mature and was gradually connected into a piece, spread on the cell climbing sheet, and the thickness of the biofilm increased significantly and became dense. The thickness of biofilm decreased and gradually became dispersed with more and more dead bacteria at 72 h. For scanning electron microscopy, the 2 strains could not form biofilms after 24 h of incubation, as shown in Figure 4C, and cells were loosely adhered to the glass coverslips. With 48 h of incubation, biofilms with the mature and spatial structures were observed in the 2 strains, and disassembly of biofilms occurred after 72 h.
Overall, the trend of biofilm formation of 2 strains (WT and ΔtolC) under bile salt stress observed with CLSM and scanning electron microscopy was consistent with the quantification by CVS (Figure 4). The biofilm-forming ability of WT decreased slightly when exposed to bile salt stress (0.005%, 0.01%, and 0.02%). The reason may be that the concentration of bile salts was too small to affect WT. In addition, studies have shown that efflux pumps play critical roles in the biofilm growth of E. coli, Klebsiella pneumoniae, Staph. aureus, and S. typhimurium (
). In agreement, the WT strain contains a complete AcrAB-TolC system, which can eliminate bile salts in time to avoid being affected.
Interestingly, biofilm formation was promoted in ΔtolC when the bile salt concentration was 0.005% and 0.01%. Biofilm formation emerges as an adaptive trait of microorganisms under harsh conditions, as it protects the community from external threats (
). The ΔtolC may be caused by a stressful environment due to the inability to excrete bile salts in time. In several pathogenic bacteria, such as Shigella flexneri, Vibrio cholerae, C. jejuni, and Listeria monocytogenes, induction of biofilm formation in the presence of bile salts is generally viewed as an adaptive response contributing to virulence and bacterial survival during colonic infection (
). A recent study by Dubois et al. also indicated that deoxycholate sublethal concentrations stimulate biofilm formation, protecting Clostridium difficile from antimicrobial compounds (
). Therefore, low concentrations of bile salts may induce stress response and promote biofilm formation, which is conducive to the survival of ΔtolC. However, when the concentration increased to 0.02%, the biofilm formation ability decreased. The reason may be that bile salt concentration of 0.02% is too high for ΔtolC, which is more harmful to bacteria, and the bacteria have died basically. A previous study confirmed that inactivation of TolC compromised extraintestinal pathogenic E. coli biofilm formation and curli production in response to high osmolarity (
). When confronting high bile salt conditions, the bacterial cells lacking TolC became unable to carry out proper osmolarity regulations and, therefore, were sensitive to these stresses. In addition, most efflux pump genes require an associated outer membrane protein in the tol family (TolA and TolC) to protect against membrane destruction following bile salt exposure (
). However, the mechanisms by which TolC regulates biofilm formation in C. malonaticus have not been fully understood.
Expression of AcrAB-TolC System Under Bile Salt Stress
Finally, we studied the changes in the gene expression of AcrAB-TolC system (acrA, acrB, and tolC) in WT under different bile salts concentrations. The 23S, 16S, and 5S electrophoresis bands of total RNA were uniform and complete, as shown in Figure 5A. It can be seen from Figure 5B that the gene expression of the AcrAB-TolC system showed a significant increase with different bile salt concentrations. Furthermore, the AcrAB-TolC system had the highest gene expression when the bile salt concentration was 0.5%. Previous research has shown that bile salts can regulate the expression of acrAB and tolC multidrug efflux genes in E. coli, S. typhimurium, and intestinal serous pullorum (
Bile salts and fatty acids induce the expression of Escherichia coli AcrAB multidrug efflux pump through their interaction with Rob regulatory protein.
Bile-mediated activation of the acrAB and tolC multidrug efflux genes occurs mainly through transcriptional derepression of ramA in Salmonella enterica serovar Typhimurium..
J. Antimicrob. Chemother.2014; 69 (24816212): 2400-2417
). Our experimental results indicated that the effect of bile salts could significantly increase the gene expression of the AcrAB-TolC system in C. malonaticus, which confirmed that the AcrAB-TolC system in C. malonaticus is an essential system for the excretion of bile salts.
Figure 5Expression changes of AcrAB-TolC system under bile salt stress. (A) Gel chart of total RNA mass of Cronobacter malonaticus (wild type, WT; M: RNA marker; 1: control; 2: 0.05% bile salts; 3: 0.5% bile salts; 4: 5% bile salts; 5: negative control). The 3 bands from top to bottom refer to 23 (2,900 bp), 16 (1,540 bp), and 5 s (120 bp), respectively. (B) Expression changes of AcrAB-tolC system (acrA, acrB, tolC) under different bile salt concentrations. Error bars indicate SD. Asterisks indicate difference among acrA, acrB, tolC, and C. malonaticus (WT) at ****P < 0.0001.
The present study provided valuable evidence about the roles of tolC in C. malonaticus when exposed to bile salts. The deletion of the tolC gene led to a decrease of bile salt resistance, including inhibition of growth, deformation, and reduction of motility. The bacterial stress response promoted the biofilm formation of ΔtolC at a low bile salt concentration. These results highlighted the importance of TolC protein in tolerance to bile salt stress. Moreover, the expression of the AcrAB-TolC system (acrA, acrB, and tolC) upregulated when exposed to bile salts. This means that the AcrAB-TolC system is a vital system for the efflux of C. malonaticus bile salts. Further work on the functions of tolC and its detailed regulation mechanism needs to be clarified urgently. Understanding the mechanisms of bile salt stress tolerance will play a guiding role in developing strategies to control and prevent C. malonaticus infections.
ACKNOWLEDGMENTS
We gratefully acknowledge the financial support of the National Key Research and Development program, China (2017YFC1601200, 2017YFC1601200), the financial support of the National Natural Science Foundation of China, China (31671951), Local Innovative and Research Teams Project of Guangdong PEARL River Talents Program (2017BT01S174), the Science and Technology Planning Project of Guangdong Province, Guangdong Province (2016A050502033), the Project of Science and Technology in Guangzhou (201604020036), and the Fundamental Research Funds for the Central Universities (JZ2020HGQA0151). The authors have not stated any conflicts of interest.
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Comparative outer membrane protein analysis of high and low-invasive strains of Cronobacter malonaticus..
Bile-mediated activation of the acrAB and tolC multidrug efflux genes occurs mainly through transcriptional derepression of ramA in Salmonella enterica serovar Typhimurium..
J. Antimicrob. Chemother.2014; 69 (24816212): 2400-2417
Loss of or inhibition of all multidrug resistance efflux pumps of Salmonella enterica serovar Typhimurium results in impaired ability to form a biofilm.
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Adherence of coagulase-negative Staphylococci to plastic tissue culture plates: A quantitative model for the adherence of Staphylococci to medical devices.
A 96-well plate-based optical method for the quantitative and qualitative evaluation of Pseudomonas aeruginosa biofilm formation and its application to susceptibility testing.
A single-component multidrug transporter of the major facilitator superfamily is part of a network that protects Escherichia coli from bile salt stress.
Bile salts and fatty acids induce the expression of Escherichia coli AcrAB multidrug efflux pump through their interaction with Rob regulatory protein.
Knockout of crtB or crtI gene blocks the carotenoid biosynthetic pathway in Deinococcus radiodurans R1 and influences its resistance to oxidative DNA-damaging agents due to change of free radicals scavenging ability.
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