Antibacterial MccM as the Major Microcin in Escherichia coli Nissle 1917 against Pathogenic Enterobacteria

Probiotic Escherichia coli Nissle 1917 (EcN) possesses excellent antibacterial effects on pathogenic enterobacteria. The microcins MccM and MccH47 produced in EcN played critical roles, but they are understudied and poorly characterized, and the individual antibacterial mechanisms are still unclear. In this study, three EcN mutants (ΔmcmA, ΔmchB, and ΔmcmAΔmchB) were constructed and compared with wild-type EcN (EcN wt) to test for inhibitory effects on the growth of Escherichia coli O157: H7, Salmonella enterica (SE), and Salmonella typhimurium (ST). The antibacterial effects on O157: H7 were not affected by the knockout of mcmA (MccM) and mchB (MccH47) in EcN. However, the antibacterial effect on Salmonella declined sharply in EcN mutants ΔmcmA. The overexpressed mcmA gene in EcN::mcmA showed more efficient antibacterial activity on Salmonella than that of EcN wt. Furthermore, the EcN::mcmA strain significantly reduced the abilities of adhesion and invasion of Salmonella to intestinal epithelial cells, decreasing the invasion ability of ST by 56.31% (62.57 times more than that of EcN wt) while reducing the adhesion ability of ST by 50.14% (2.41 times more than that of EcN wt). In addition, the supernatant of EcN::mcmA culture significantly decreased the mRNA expression and secretion of IL-1β, TNF-α, and IL-6 on macrophages induced by LPS. The EcN::mcmA strain generated twice as much orange halo as EcN wt by CAS agar diffusion assay by producing more siderophores. MccM was more closely related to the activity of EcN against Salmonella, and MccM-overproducing EcN inhibited Salmonella growth by producing more siderophores-MccM to compete for iron, which was critical to pathogen growth. Based on the above, EcN::mcmA can be developed as engineered probiotics to fight against pathogenic enterobacteria colonization in the gut.


Introduction
Pathogenic enterobacteria and related diseases have attracted increasing attention from consumers and food safety regulators because of their undeniable impact on health and the economy [1]. Enterohaemorrhagic Escherichia coli (EHEC) O157: H7 and Salmonella are major pathogenic enterobacteria that cause potentially fatal diseases such as hemorrhagic colitis, acute gastroenteritis, and salmonellosis [2]. Shiga toxin 1 (Stx1) and 2 (Stx2) are among the virulence factors that contribute to the pathogenesis of EHEC [3]. Salmonella enterica (SE) and Salmonella Typhimurium (ST) accounted for the majority of Salmonella and were considered to be responsible for most of the salmonellosis infections [4]. Salmonella can even thrive in the inflamed gut using unique carbon and energy [5] and have a partial nutritional immune response to the antibacterial proteins secreted by the host [6]. The pathogenesis and colonization of host tissues by Salmonella depend on special systems called secretory systems type III (T3SS), which contain SPI1 and SPI2, which play different

Effect of Probiotic EcN on EHEC O157: H7 Growth and Stx Gene Expression
The bacterial concentrations of EHEC O157: H7 monocultures as well as co-cultures with EcN wt or mutants were determined at the beginning of the experiment (0 h) and at 4 h, 8 h, 12 h, and 24 h. In LB co-culture experiments, the growth of EHEC O157: H7 was significantly inhibited by EcN after 8 h. The EcN ∆mcmA∆mchB strain had the best inhibition effect, reaching 99% in 24 h compared with monoculture as a control; there was no significant difference from EcN wt (Figure 1a). Of course, the number of EHEC O157: H7 inhibited by EcN wt was reduced by 92% at 4 h. In the fluorescence microscope image, it could be seen that the green fluorescence was significantly less than the red fluorescence ( Figure 2a). Under iron-rich conditions, the growth inhibition of EcN on EHEC O157: H7 was weakened, indicating that ions had an impact on the antibacterial activity of EcN ( Figure 1b). Unexpectedly, no matter whether in iron-limited or iron-rich conditions, the growth of EcN was not influenced by EHEC O157: H7 (Figure 1c,d). The expression of stx mRNA was detected after 2 h, 4 h, and 24 h co-culture of EHEC O157: H7 with EcN (Supplementary Figure S3). RT-PCR data showed that stx expression of EHEC O157: H7 incubated with EcN was significantly reduced compared with EHEC O157: H7 alone in LB medium or iron-rich medium. The strain EcN ∆mcmA ∆mchB showed the best effect. there was no significant difference from EcN wt (Figure 1a). Of course, the number of EHEC O157: H7 inhibited by EcN wt was reduced by 92% at 4 hrs. In the fluorescence microscope image, it could be seen that the green fluorescence was significantly less than the red fluorescence ( Figure 2a). Under iron-rich conditions, the growth inhibition of EcN on EHEC O157: H7 was weakened, indicating that ions had an impact on the antibacterial activity of EcN ( Figure 1b). Unexpectedly, no matter whether in iron-limited or iron-rich conditions, the growth of EcN was not influenced by EHEC O157: H7 (Figure 1c,d). The expression of stx mRNA was detected after 2 hrs, 4 hrs, and 24 hrs co-culture of EHEC O157: H7 with EcN (Supplementary Figure S3). RT-PCR data showed that stx expression of EHEC O157: H7 incubated with EcN was significantly reduced compared with EHEC O157: H7 alone in LB medium or iron-rich medium. The strain EcN ΔmcmA ΔmchB showed the best effect.  on EHEC O157: H7 was weakened, indicating that ions had an impact on the antiba activity of EcN ( Figure 1b). Unexpectedly, no matter whether in iron-limited or iro conditions, the growth of EcN was not influenced by EHEC O157: H7 (Figure 1c,d expression of stx mRNA was detected after 2 hrs, 4 hrs, and 24 hrs co-culture of O157: H7 with EcN (Supplementary Figure S3). RT-PCR data showed that stx expr of EHEC O157: H7 incubated with EcN was significantly reduced compared with O157: H7 alone in LB medium or iron-rich medium. The strain EcN ΔmcmA showed the best effect.

E. coli Nissle 1917 Significantly Reduced the Growth of Salmonella in Iron-Limited Media
Growth inhibition of Salmonella by EcN increased over time after 4 hrs of co-culture ( Figure 3). The antagonistic effect of EcN on Salmonella in LB medium is equivalent to that in iron-rich medium supplemented with 4 mM iron citrate, so LB medium was regarded as an iron-rich condition. Under the iron-limiting condition, compared with SE monoculture as a control, the number of SE co-cultured with EcN wt decreased by 99% (~100-fold) in 12 hrs and 99.9% (~1000-fold) in 24 hrs, whereas co-cultured with EcN ΔmchB decreased by 98.7% (~100-fold) in 12 hrs and 99.2% (~100-fold) in 24 hrs ( Figure  3a,b). Similarly, the number of ST was significantly reduced at 8 hrs, and the EcN wt strain had the best inhibitory effect: 99.69% at 8 h, 99.88% (~1000-fold) at 12 hrs, and 99.97% at 24 hrs. When co-cultured with EcN ΔmchB, the number of ST was suppressed at 99.22% in 8 hrs, 99.70% in 12 hrs, and 99.92% (~1000-fold) in 24 hrs (Figure 3e,f). Similar results were found in fluorescence microscope images (Figure 2b), where the green fluorescence number decreased sharply when EcN wt and EcN ΔmchB were added to co-culture compared with the control (Supplementary Figure S4).
On Salmonella, the best effect was reached at 24 hrs. In addition, the growth inhibitory effect of the mutant strain EcN ΔmcmA on Salmonella was similar to that of EcN ΔmcmAΔmchB and significantly lower than that of EcN wt (Figure 3b,f). The results indicated that MccM influenced SE/ST growth more effectively under iron-deficiency stress. However, when four EcN strains were co-cultured with Salmonella, the number of EcN strains was unaffected compared to EcN alone (Figure 3c,d,g,h).

E. coli Nissle 1917 Significantly Reduced the Growth of Salmonella in Iron-Limited Media
Growth inhibition of Salmonella by EcN increased over time after 4 h of co-culture ( Figure 3). The antagonistic effect of EcN on Salmonella in LB medium is equivalent to that in iron-rich medium supplemented with 4 mM iron citrate, so LB medium was regarded as an iron-rich condition. Under the iron-limiting condition, compared with SE monoculture as a control, the number of SE co-cultured with EcN wt decreased by 99% (~100-fold) in 12 h and 99.9% (~1000-fold) in 24 h, whereas co-cultured with EcN ∆mchB decreased by 98.7% (~100-fold) in 12 h and 99.2% (~100-fold) in 24 h (Figure 3a,b). Similarly, the number of ST was significantly reduced at 8 h, and the EcN wt strain had the best inhibitory effect: 99.69% at 8 h, 99.88% (~1000-fold) at 12 h, and 99.97% at 24 h. When co-cultured with EcN ∆mchB, the number of ST was suppressed at 99.22% in 8 h, 99.70% in 12 h, and 99.92% (~1000-fold) in 24 h (Figure 3e,f). Similar results were found in fluorescence microscope images (Figure 2b), where the green fluorescence number decreased sharply when EcN wt and EcN ∆mchB were added to co-culture compared with the control (Supplementary Figure S4).
On Salmonella, the best effect was reached at 24 h. In addition, the growth inhibitory effect of the mutant strain EcN ∆mcmA on Salmonella was similar to that of EcN ∆mcmA∆mchB and significantly lower than that of EcN wt (Figure 3b    Antibacterial experiments in vitro showed that engineered strains EcN::mcmA and EcN wt could inhibit the growth of Salmonella in LB medium or in iron-limiting conditions, and the inhibition effect was particularly significant in an iron-limited environment. Among them, the antibacterial effect of the engineered strain on Salmonella was stronger than that of EcN wt (Figure 4). In LB medium, the engineered strain reached its maximum effect at 8 h, reducing the number of SE by 75.8% and ST by 61.7%. However, the inhibition rate of EcN wt on SE was 36.4%, and there was no effect on ST (Figure 4a   After recombinant plasmids were transformed into EcN wt, the whole cell lysates of recombinant EcN were detected by SDS-PAGE for confirmation of MccM overexpression. The recombinant protein MccM could hardly be detected by Coomassie blue staining due to its low expression. Therefore, Western blot (WB) analysis was carried out using HRP-conjugated 6*His antibody for the immunodetection of the recombinant protein.
The molecular weight of MccM was 11.4 kDa, calculated from the amino acid sequence. The bands of WB results corresponding to the 11.4 kDa protein were detected in wholecell lysates of the recombinant EcN harboring pUC19-mcmA, suggesting that MccM was successfully expressed (Figure 5a).

EER REVIEW 8 o
The recombinant protein MccM could hardly be detected by Coomassie blue staining to its low expression. Therefore, Western blot (WB) analysis was carried out using H conjugated 6*His antibody for the immunodetection of the recombinant protein. molecular weight of MccM was 11.4 kDa, calculated from the amino acid sequence. bands of WB results corresponding to the 11.4 kDa protein were detected in wholelysates of the recombinant EcN harboring pUC19-mcmA, suggesting that MccM successfully expressed (Figure 5a).

Antibacterial Mechanism of the Engineered Strain EcN::mcmA
At present, the detection method commonly used for siderophores is the general C agar plate method. When the siderophores produced by microorganisms combine w the blue complex composed of CAS, cetyltrimethylammonium bromide (HDTMA), iron, it will take away the iron and change color from blue to orange, producing a dist siderophore halo. The CAS agar diffusion assay found that the engineered st EcN::mcmA could produce an orange halo of 0.6 mm, which was twice the orange h (0.3 mm) produced by EcN wt (Figure 5b). It could be seen from the CAS liq measurement that the absorbance at 695 nm decreased with the increase in the amoun supernatant added to the bacterial liquid, and the absorbance at 425 nm increased. W diluted 7-fold, the decrease in absorbance at 695 nm of the engineered strain EcN::mc

Antibacterial Mechanism of the Engineered Strain EcN::mcmA
At present, the detection method commonly used for siderophores is the general CAS agar plate method. When the siderophores produced by microorganisms combine with the blue complex composed of CAS, cetyltrimethylammonium bromide (HDTMA), and iron, it will take away the iron and change color from blue to orange, producing a distinct siderophore halo. The CAS agar diffusion assay found that the engineered strain EcN::mcmA could produce an orange halo of 0.6 mm, which was twice the orange halo (0.3 mm) produced by EcN wt (Figure 5b). It could be seen from the CAS liquid measurement that the absorbance at 695 nm decreased with the increase in the amount of supernatant added to the bacterial liquid, and the absorbance at 425 nm increased. When diluted 7-fold, the decrease in absorbance at 695 nm of the engineered strain EcN::mcmA was 1.7 times that of the EcN wt (Figure 6a-d), which indicated that the engineered strain produced more siderophores than EcN wt. Figure 6. Determination of various components in the UV visible absorption s CAS+Fe, and CAS+HDTMA complexes all have absorption peaks near 425 nm. W a ternary detection complex CAS+Fe+HDTMA, the absorption peak shifted to 69 siderophores chelate iron, the peak at 695 nm linearly decreases, and the peak at The supernatant of the EcN wt (E) and engineered strain EcN::mcmA (M) cultur added to CAS+Fe+ HDTMA according to the dilution times, and the absorbance 695 nm and 425 nm, respectively (b,c). The absorbance of the strains supernata was measured (d).

The Engineered Strain EcN::mcmA Significantly Inhibited the Adhesion an Salmonella to Intestinal Epithelial Cells
After the co-infection of engineered strain EcN::mcmA and Salmonell epithelial cells HT-29, compared with Salmonella infection alone, the adh SE and ST decreased by 60.01% and 50.14%, respectively, 1.23 times and co-infection with EcN wt (Figure 7a). Similarly, the invasion numbe decreased by 56.16% and 56.31%, respectively, 2.44 times and 62.57 times with the wild-type EcN strain (Figure 7c). However, Salmonella had litt adhesion and invasion of EcN strains (Figure 7b,d). The above results in EcN strain, after overexpressing the mcmA gene, had an enhanced inhibit adhesion and invasion of Salmonella into HT-29 cells.

The Engineered Strain EcN::mcmA Significantly Inhibited the Expressio Cytokines
To investigate the anti-inflammatory effect of the engineered stra PCR and ELISA were used to detect the expression of TNF-α, IL-1β treatment of CT26-mediated RAW264.7 cells with the supernatan EcN::mcmA. The results indicated that the mRNA expression of IL-1 TNF-α and IL-1β induced by LPS were significantly inhibited after th of EcN wt treatment (p < 0.05, Figure 8a,d,e), while the effects of it on th 6 were not significantly altered (p > 0.05, Figure 8c,f). Moreover, comp engineered strain EcN::mcmA has a more obvious inhibitory effect on inflammatory cytokines, and 1.5% supernatant significantly red

Discussion
The overuse and misuse of antibiotics, coupled with the lack of new antibiotics, induced the emergence and spread of multi-resistant bacteria [29]. This phenomenon constitutes an enormous risk of global morbidity and mortality [30]. Microcins appeared to be a potential antimicrobial alternative to conventional antibiotics. The main challenge in using Mccs was delivering them to the infection site in sufficient quantities [31]. Thus, engineered probiotics were considered in situ producers of Mccs to combat pathogenic enterobacteria or reduce the colonization of multidrug-resistant bacteria [32]. EcN has been used as a probiotic for more than 100 years, and several different health advantages have been reported [33]. Therefore, we constructed an engineered EcN strain as an in situ producer, producing a large amount of MccM to antagonize pathogenic enterobacteria and provide the possibility for new antibacterial agents.
The EHEC pathogen is most commonly transmitted through contaminated food and water, and the Shiga toxin contributes to the pathogenicity of the organism [34]. Our research found that probiotic EcN reduces the growth and Shiga toxin expression of EHEC O157: H7, which was consistent with previous studies, and that the growth of EcN was not affected by EHEC O157: H7. However, this specific inhibition mechanism has not been studied. The EcN ΔmcmA ΔmchB strain had the best inhibition effect, reaching 99% in 24 hrs, indicating that microcins are not associated with this inhibitory effect in our study. This was consistent with previous research [35]. Some studies show that EcN may inhibit the expression of adhesin in EHEC or induce the expression of human β-defensin 2 through the flagellum to kill EHEC [36]. In our experiment, we found that EcN more significantly inhibited the growth of EHEC O157: H7 in iron-limiting conditions. EcN has a competitive advantage in the uptake of iron in cases of iron deficiency. There are some

Discussion
The overuse and misuse of antibiotics, coupled with the lack of new antibiotics, induced the emergence and spread of multi-resistant bacteria [29]. This phenomenon constitutes an enormous risk of global morbidity and mortality [30]. Microcins appeared to be a potential antimicrobial alternative to conventional antibiotics. The main challenge in using Mccs was delivering them to the infection site in sufficient quantities [31]. Thus, engineered probiotics were considered in situ producers of Mccs to combat pathogenic enterobacteria or reduce the colonization of multidrug-resistant bacteria [32]. EcN has been used as a probiotic for more than 100 years, and several different health advantages have been reported [33]. Therefore, we constructed an engineered EcN strain as an in situ producer, producing a large amount of MccM to antagonize pathogenic enterobacteria and provide the possibility for new antibacterial agents.
The EHEC pathogen is most commonly transmitted through contaminated food and water, and the Shiga toxin contributes to the pathogenicity of the organism [34]. Our research found that probiotic EcN reduces the growth and Shiga toxin expression of EHEC O157: H7, which was consistent with previous studies, and that the growth of EcN was not affected by EHEC O157: H7. However, this specific inhibition mechanism has not been studied. The EcN ∆mcmA ∆mchB strain had the best inhibition effect, reaching 99% in 24 h, indicating that microcins are not associated with this inhibitory effect in our study. This was consistent with previous research [35]. Some studies show that EcN may inhibit the expression of adhesin in EHEC or induce the expression of human β-defensin 2 through the flagellum to kill EHEC [36]. In our experiment, we found that EcN more significantly inhibited the growth of EHEC O157: H7 in iron-limiting conditions. EcN has a competitive advantage in the uptake of iron in cases of iron deficiency. There are some proteins connected with the iron uptake system that can bring advantages to EcN in outgrowing and competing with pathogens using similar siderophores [37]. Only a few studies have researched the role of microcins on the Shiga toxin production of EHEC O157: H7. According to our study, Shiga toxin gene expression was downregulated in the absence of microcins, which suggested that microcins might play a role in promoting Shiga toxin production. One study found that microcins of EcN increased Shiga toxin production through an SOS-dependent pathway, similar to how ciprofloxacin produces Shiga toxin [38]. So, the mechanism of EcN's effects on the growth of EHEC O157: H7 needs to be investigated in the future.
EcN microcins were shown to have antibacterial activity against Salmonella in ironlimited environments [27]. Salmonella was the main cause of acute gastroenteritis, which was characterized by inflammatory diarrhea. Some mechanisms were utilized by the host to limit the availability of iron during inflammation [39,40]. When Salmonella was co-cultured with EcN wt, the number of Salmonella decreased significantly in iron-limiting (~1000-fold) and less in iron-rich (~7-fold) conditions at our experiments, which was probably because of microcins, and siderophores were only expressed when iron deficiency occurred [25]. Interestingly, the growth of four EcN was not affected by Salmonella. Research showed that the single deletion of MccM structural gene mcmA or MccH47 structural gene mchB did not weaken the inhibitory effect of EcN on Escherichia coli LF82 but weakened it after the combined deletion of mcmA and mchB [41]. In contrast, in our study, the antibacterial activity of EcN against Salmonella was affected by a single deletion of the MccM structural gene mcmA. Therefore, we deduced that MccM was more important than MccH47 in EcN against Salmonella.
Next, MccM was successfully overexpressed in EcN in our study. We showed that overexpression of the precursor of MccM in the engineered probiotic bacteria EcN::mcmA had a more effective antibacterial effect against Salmonella and produced more siderophores compared with EcN wt. It is a common strategy for pathogens to survive in iron-limited environments where they produce siderophores, which are small, potent iron-chelating molecules. Similarly, EcN produces a variety of siderophores, which makes it superior in competing with other pathogenic enterobacteria for iron, thus promoting its colonization in the intestine. A recent study demonstrated that multiple siderophores enable EcN to outcompete S. Typhimurium for iron acquisition [25]. Therefore, overexpression of MccM in our study may promote siderophores production, which can improve the competitive advantage of iron to outcompete Salmonella. Enterobactin is a catechol-type siderophore that is used to modify microcins. In the MccH47 genetic system carried by strains, mchS4 was identified as the single gene responsible for enterobactin overproduction. The MccH47 genetic system promotes enterobactin production, and the enterobactin synthesis pathway plays a role in MccH47 synthesis [42]. MccH47 was widely heterologously expressed in EcN or other Escherichia coli strains [43]. Due to the post-translational modification of the enterobactin moiety [44], MccH47 enters the pathogenic bacteria via the catecholate siderophore receptors (FepA, Fiu, or Cir), which are then applied to the F0 proton channel, triggering the unregulated entry of protons and thereby dissipating the membrane potential. The mechanism of MccM is not clear, but it may be consistent with MccH47 [45]. Therefore, the future of research should focus on MccM, including antibacterial mechanisms, the construction of engineered bacteria, heterologous expression, etc.
As Salmonella colonization and pathogenicity depend on adhesion to the intestinal epithelium, inhibiting its adhesion ability can prevent bacterial invasion and reduce intestinal colonization. The bacterial adhesion to intestinal epithelial cells has long been considered an ideal prerequisite for probiotic strains [46]. Therefore, a lot of probiotics have the ability to adhere to the intestine [47]. It has been demonstrated in our study that the engineered strain EcN::mcmA prevents Salmonella from colonizing intestinal epithelial cells. The repression on adhesion and invasion of SE and ST was higher than that of EcN wt, respectively. EcN has been proven to possess anti-inflammatory properties in several cell types, including human colonic epithelial cells [48]. It has been suggested that intestinal in-fections may be triggered by lipopolysaccharides (LPS) released by Gram-negative bacteria such as Salmonella [49]. Macrophages play a dual role in the process of Salmonella invading the host. In addition to their role in the immune response, macrophages are also host cells for Salmonella, facilitating its systemic spread and causing infection [50]. In this study, we found that the engineered strain EcN::mcmA significantly inhibited the LPS-induced expression of IL-1β, TNF-α, and IL-6 in RAW264.7 macrophages compared with EcN wt.  Table 1. Plasmid pUC19 with chloramphenicol or ampicillin resistance was electroporated into probiotic EcN and pathogenic enterobacteria for screening. The plasmids and partial primers used in this research are listed in Table 1. All strains were cultured in LB broth or agar plates with antibiotics at 37 • C. The antibiotic concentrations are chloramphenicol (Cm + , 25 µg/mL), spectinomycin (Sm + , 25 µg/mL), ampicillin (Amp + , 100 µg/mL), or kanamycin (Kan + , 50 µg/mL).

Materials and Methods
Lowercase letters indicate homologous arm of restriction-free cloning (RF-cloning) and enzyme digestion sites HindIII and Xhol.

Generation of Bacterial Mutants
Mutants in EcN (EcN ∆mcmA, EcN ∆mchB, and EcN ∆mcmA ∆mchB) were constructed using CRISPR/Cas9 gene targeting. The gene-specific single guide RNAs (sgRNA) were designed using the software sgRNAcas9_3.0.5 (http://biootools.com/col.jsp?id=140) on 12 June 2021. The program was written in Perl (http://www.perl.org). The DNA sequences of the target genes mcmA (UniProtKB-Q2WEK3) and mchB (UniProtKB-P62530) were found on the EcN genome (GenBank: CP022686.1), and the sgRNA primers A20-F/A20-R and B20-F/B20-R (respective upper case N20 sequence of 20 bp) were designed using the procedures mentioned in this method. The selected homology arms H1 and H2 were about 500 bp upstream and downstream of the target genes mcmA and mchB, and the designed amplification primers were AH1-F and AH1-R, AH2-F and AH2-R, BH1-F and BH1-R, and BH2-F and BH2-R. The strain names of the mutants and primers are listed in Table 1. Construction of sgRNA recombinant plasmids by double digestion, restriction-free cloning technology (RF cloning) [51], and seamless cloning [52]. Subsequently, the primers ∆ mcmA-F/∆ mcmA-R and ∆ mchB-F/∆ mchB-R were used for knockout verification and sent to the company (Sangon Biotech, Shanghai, China) for sequencing. Subsequently, the plasmid pUC19-mCherry containing chloramphenicol resistance and the plasmid pUC19-sGFP containing ampicillin resistance were introduced into the constructed EcN and Salmonella for resistance screening.

Quantitative Real-Time PCR
Bacterial co-cultured liquid with four EcN strains and EHEC O157: H7 for 24 h was collected for analysis of stx gene mRNA expression by real-time PCR (RT-PCR), and total RNA was extracted from the bacterial cells with a total RNA extraction kit (Tiangen, Beijing, China). All RNA samples were reverse transcribed using reagents (Novizan, Nanjing, China). RT-PCR was performed with SYBR Green (Yisheng, Shanghai, China) and a Roche LightCycler ® 96 instrumentation system (Roche, Basel, Switzerland). Analyses were conducted using the comparative 2 ∆∆Ct method.

Spectrofluorometric Assays
In this experiment, the pUC19 plasmid was used as the expression vector for red fluorescent protein (mCherry) and green fluorescent protein (GFP). Strains were inoculated in an iron-limited medium or an iron-rich medium, as mentioned above. Then the cultured cells were centrifuged at 4000× g for 15 mins. The cells were collected, and the supernatant was discarded. After washing with sterile water three times, 200 µL of resuspension was placed on a 96-well glass bottom plate (a special culture plate for laser confocal) with a specification of 1.5 high-level glass slides (thickness: 0.17 ± 0.005 mm, Cellvis) and observed by a confocal laser scanning microscope (Leica, Wetzlar, Germany). The excitation wavelengths were 570 and 480 nm, respectively.

Protein Overexpression and Western Blot Analysis
The overexpression vector pUC19-mcmA was constructed by seamless cloning, and then MccM was overexpressed in EcN. Using pUC-mcmA-F/pUC-mcmA-R and pUC19-F/pUC19-R as primers (Table 1), the mcmA gene fragment was amplified from the EcN genome, and the linearized pUC19 plasmid was from the pUC19 plasmid with the chloramphenicol resistance gene, respectively. Then, they were connected by ligase 2 × Seamless Master Mix (Sangon Biotech, China) and electrically transferred to EcN to construct the engineered strain EcN::mcmA. The strains were cultured overnight in a selective medium containing 25 µg/mL chloramphenicol, then diluted at 1: 50 and cultured at 37 • C (220 rpm) for 4-8 h to fully express the protein. Overexpression of microcin M was determined by Western blotting [53].

CAS Agar Diffusion Assay (CASDA)
Due to the chelation effect of siderophores on iron, chromium azurol S (CAS) agar analysis detected that the color of the CAS-iron complex changed from blue to orange. The CAS agar formula used in this experiment was modified [54]. Firstly, 60.5 mg of CAS solution was dissolved in 50 mL of water, and then the deionized water was mixed with 10 mL of iron III solutions. With stirring, the solution was mixed slowly with 72.9 mg of detergent (HDTMA) in 40 mL of water. Afterward, the obtained dark blue solution was mixed with 900 mL of water, including 9000 mg agar, 295.25 mg NaH 2 PO 4 ·2H 2 O, 1213.5 mg Na 2 HPO 4 ·12H 2 O, 125 mg NH 4 Cl, 37.5 mg KH 2 PO 4 , and 62.5 mg NaCl, pH (6.8). Subsequently, holes with a diameter of 6 mm were punched and sealed on the improved CAS agar plate and then stored at 4 • C. Every hole was filled with 100 µL of iron-limited medium and the supernatant of EcN wt or the engineered strain EcN::mcmA. The test strains were grown in iron-limited conditions at 37 • C and 220 rpm for 24 h. After centrifugation at 4 • C for 10 min at 4000× g, the culture supernatant was collected and filtered for disinfection (pore size 0.22 µm) and then analyzed for siderophores production with the previously described improved CAS agar diffusion method.

CAS Assay Methods
CAS colorimetric analysis was used to assess iron removal from a CAS-HDTMA-Fe ternary complex. This blue complex was composed of organic dyes, metals, and surfactants and had an absorption peak at 695 nm. When siderophores replaced iron in the analytical complex, the color changed to yellow, and the absorption peak shifted to 425 nm. The hexadentate siderophores removed iron from the analytical complex in a 1:1 ratio, so the reduced absorption at 695 nm due to iron removal was proportional to the siderophores concentration in the sample. The detailed operation was described in a previous study [54]. The supernatant of EcN wt or engineered strain EcN::mcmA was added in diluted proportions based on CAS+HDTMA+Fe.

Adhesion and Invasion Inhibition Assays
HT29 cells were seeded on a 24-well cell culture plate (JET, Guangzhou, China), with 4 × 10 5 cells per well, cultured for 20 h, and the cell monolayers were washed three times with phosphate-buffered saline (PBS, pH = 7.2). Every single molecular layer was infected with multiple infections (MOI) of 100 bacteria per epithelial cell in 1 mL of cell culture DMEM.
After infection at 37 • C, 5% CO 2 for 1 h, the infected cell monolayers were washed with PBS three times. Cells were lysed in deionized water using 1% Triton X-100 (Sigma), which does not affect the vitality of bacteria for at least 30 mins. To determine invasiveness, fresh DMEM containing 1% penicillin and streptomycin was added to kill the bacteria on the surface of the cells and determine the bacteria in the cells after incubating for 2 h. Subsequently, the bacteria were counted using resistant agar plates. Repeat three to five experiments for each measurement.
4.10. Anti-Inflammatory Activity of E. coli Nissle 1917 E. coli Nissle1917 was cultured overnight at 37 • C, and then the overnight culture of 1 × 10 6 CFU mL −1 was inoculated in 10 mL of iron-limited conditions for 24 h at 37 • C. The culture was then centrifuged at 10,000× g for 10 min at 4 • C to create a supernatant, and then Tris-HCL was used to adjust the pH value to 7.2~7.4. Finally, they were filtered through 0.22 µm filters.
The CT26 and RAW246.7 were cultured in DMEM medium with 10% FBS and 1% penicillin and streptomycin at 37 • C in 5% CO 2 . For transwell experiments, CT26 cells (1 × 10 4 cells/well) in the logarithmic growth phase were inoculated in the inserts of 24-well plates in a transwell system (1 µM nucleopore size; Costar, Corning Inc, New York, NY, USA) and placed above the 600 µL medium for two days. For determining proinflammatory cytokine production, RAW246.7 cells (5 × 10 4 cells/well) were seeded into 24-well cell culture plates (JET, Guangzhou, China) and cultured at 37 • C until confluence was reached. Monolayers of RAW246.7 cells were initially stimulated for 20 h with 1 µg/mL lipopolysaccharide (LPS, Sigma-Aldrich). Then, transwell inserts containing adherent monolayers of CT26 cells were transferred to 24-well plates containing RAW246.7 cells in the lower chamber. One percent supernatant of EcN wt and the engineered strain EcN::mcmA (MOI = 10) were added to transwell inserts, and the cells were cultured for 5 h. At last, the mRNA expression and secretion of IL-1β, TNF-α, and IL-6 on macrophages induced by LPS were measured by RT-PCR and ELISA.

Conclusions
To sum up, we discovered that the microcins appeared not to be responsible for the inhibition of EHEC O157: H7, but microcin MccM was more closely related to the activity of EcN against Salmonella than expected. The growth, adhesion, and invasion of SE and ST were decreased more by EcN::mcmA. We developed an engineered probiotic, EcN, as the producer of overexpressing MccM to antagonize pathogenic enterobacteria, which opens the way to providing substitutes for antibiotics.