Regulation of Class A β-Lactamase CzoA by CzoR and IscR in Comamonas testosteroni S44

A genomic analysis of Comamonas testosteroni S44 revealed a gene that encodes a LysR family transcriptional regulator (here named czoR, czo for cefazolin) located upstream of a putative class A β-lactamase encoding gene (here named czoA). A putative DNA-binding motif of the Fe–S cluster assembly regulator IscR was identified in the czoR–czoA intergenic region. Real-time RT-PCR and lacZ fusion expression assays indicated that transcription of czoA and czoR were induced by multiple β-lactams. CzoA expressed in Escherichia coli was shown to contribute to susceptibility to a wide range of β-lactams judged from minimum inhibitory concentrations. In vitro enzymatic assays showed that CzoA hydrolyzed seven β-lactams, including benzylpenicillin, ampicillin, cefalexin, cefazolin, cefuroxime, ceftriaxone, and cefepime. Deletion of either iscR or czoR increased susceptibility to cefalexin and cefazolin, while complemented strains restored their wild-type susceptibility levels. Electrophoretic mobility shift assays (EMSA) demonstrated that CzoR and IscR bind to different sites of the czoR–czoA intergenic region. Precise CzoR- and IscR-binding sites were confirmed via DNase I footprinting or short fragment EMSA. When cefalexin or cefazolin was added to cultures, czoR deletion completely inhibited czoA expression but did not affect iscR transcription, while iscR deletion decreased the expressions of both czoR and czoA. These results reveal that CzoR positively affects the expression of czoA with its own expression upregulated by IscR.

INTRODUCTION β-Lactam antibiotics are currently one of the most widely used antibacterial classes to treat infectious diseases, with cephalosporins (e.g., cefalexin, cefazolin, and cefradine) accounting for nearly half of all β-lactam antibiotic prescriptions (Bush and Bradford, 2016). Resistance of pathogenic bacteria to β-lactams has led to antibiotic treatment failures (Bush, 2013). Moreover, a number of environmental isolates have also shown a highly intrinsic or adaptive resistance to antibiotics. Production of β-lactamases is the primary mechanism of β-lactam resistance (Giwercman et al., 1992;Bush, 2013). Based on amino acid sequences, β-lactamases have been classified into four molecular classes. Classes A, C, and D β-lactamases hydrolyze their substrates through an active site serine, whereas Class B β-lactamases are metalloenzymes that require divalent zinc ions for their activities (Walsh et al., 2005).
A large number of β-lactamases have been reported, among which the Class A enzymes are the most abundant (Bush and Jacoby, 2010). The substrates of these class A β-lactamases are mostly penicillins, monobactams, early cephalosporins, and extended-spectrum cephalosporins, and these enzymes are inhibited by β-lactamase inhibitors, such as clavulanic acid, tazobactam, and sulbactam (Bush and Jacoby, 2010). Diverse class A β-lactamases have been studied for various genera, including GIL-1 from Citrobacter gillenii, AST-1 from Nocardia asteroids, PenA from Burkholderia cepacia, NmcA from Enterobacter cloacae, CumA from Proteus vulgaris, SmeA from Serratia marcescens, CdiA from Citrobacter diversus, Sed-1 from Citrobacter sedlakii, HugA from Proteus penneri, and BlaA from Yersinia enterocolitica (see review of Philippon et al., 2016).
Previous studies have shown that the [Fe-S] cluster biosynthesis-related genes are involved in antibiotic susceptibility in Escherichia coli. Bactericidal agents (quinolones, aminoglycosides, and β-lactams) were proposed to induce oxidative stress and produce reactive oxygen species (ROS), thereby destabilizing the Fe-S clusters and resulting in Fe(II)mediated Fenton reactions (Dwyer et al., 2009). Disruption of the Fe-S cluster biosynthesis genes iscS, iscU, hscA, hscB, and fdx increased susceptibility to various antibiotics (e.g., cephalosporins, penicillins, and glycopeptides) in E. coli (Liu et al., 2010). IscR was discovered as a negative regulator controlling the Fe-S biogenesis system (Schwartz et al., 2001). It is widely conserved in Proteobacteria and is proposed to be a member of the large Rrf2 family of winged helixturn-helix (wHTH) transcription factors (Schwartz et al., 2001). Recently, we showed that a transposon (Tn5) insertion in a gene encoding Fe-S cluster assembly regulator (iscR) affected selenite susceptibility and antimonite oxidation in Comamonas testosteroni S44 (Zheng et al., 2014;Liu H.L. et al., 2015). IscR is also reported to regulate more than 40 genes that are involved in various cellular processes in E. coli (Giel et al., 2006(Giel et al., , 2013Haines et al., 2015). Thus, IscR may be associated with the regulation of antibiotic susceptibility.
Comamonas testosteroni strains are primarily environmental bacteria that play an important role in environmental decontamination, having the ability to transform heavy metals and degrade a variety of toxic aromatic pollutants . Recently, C. testosteroni strains have also been recognized as human pathogens with potential to cause blood, endocardial, and abdominal infections (Duran et al., 2015;Parolin et al., 2016). Empiric therapy includes use of intravenous antibacterials of β-lactams and fluoroquinolones, especially cefoxitin and ciprofloxacin (Duran et al., 2015;Parolin et al., 2016). C. testosteroni S44 was isolated from the soil of an antimony (Sb) mine and is resistant to multiple heavy metals (Xiong et al., 2011) and some antibiotics, including cefalexin, cefazolin, benzylpenicillin, and ampicillin (unpublished data). The objective of this study was to elucidate the IscR-/CzoR-mediated regulatory mechanism of a newly identified Class A β-lactamase CzoA in C. testosteroni S44. Based on a gene knock-out and its complementation, electrophoretic mobility shift assay (EMSA), DNase I footprinting, and lacZ reporter gene assays, we found that the LysR-type transcriptional regulator CzoR positively regulates czoA expression and that IscR enhances this regulatory effect through binding with the czoR promoter region.

Bacterial Strains, Plasmids, and Culture Conditions
Bacterial strains, plasmids, and oligonucleotide primers used in this study are shown in Supplementary Table S1. All strains were grown at 37 • C in Luria-Bertani (LB, Oxoid, United Kingdom) broth unless otherwise stated. Mueller-Hinton (MH, Beijing Land Bridge Technology, China) broth dilution was used to determine the minimal inhibitory concentration (MIC) of antibiotics. Antibiotic disk (Hangzhou Microbial Reagent, China) diffusion tests were used for the antibiotic susceptibility assay (cephalexin and cefazolin). Appropriate antibiotic agents were added when preparing the seed liquid of all bacteria possessing a plasmid. Then, the seed liquid was used directly in relevant experiments.

Bioinformatic Analysis
Whole-genome shotgun sequencing was performed using a Roche 454 Genome Sequencer FLX instrument as described previously (Xiong et al., 2011). Multiple amino acid sequence alignments of CzoA with representative Class A β-lactamases and CzoR with its homologs were conducted using Clustal Omega 1 . The IscR-binding site was analyzed by the online program MEME 2 (Bailey and Elkan, 1994). The -35 and -10 sequences were predicted using Softberry BPROM Tool 3 (Solovyev and Salamov, 2011).

Construction of iscR and czoR Mutants and Complemented Strains
The iscR-mutant strain iscR-280 and its complemented strain iscR-280C were generated in our previous study (Liu H.L. et al., 2015). To create a czoR-mutant strain, the suicide allelic exchange vector pCM184-Cm was used as previously described (Chen et al., 2015). The upstream and downstream regions of czoR were amplified with the primer pairs M-czoR-up-F/M-czoR-up-R and M-czoR-down-F/M-czoR-down-R, respectively. Subsequently, the upstream and downstream PCR fragments were cloned into the AatII-BsrGI and ApaI-SacI sites of pCM184-Cm, respectively. The resulting czoR allelic exchange vector pCM184-czoR was introduced into the strain S44 via biparental conjugation with the E. coli strain S17-1 (λpir) (Simon et al., 1983), and the double crossover czoR mutant was selected using 50 µg/ml chloramphenicol and 25 µg/ml tetracycline. The tetracycline-sensitive and chloramphenicol-resistant mutant was then confirmed by PCR using primers czoR-inner-F/czoR-inner-R (Supplementary Table S1). For czoR complementation, the complete czoR-coding sequence was amplified via PCR and digested with XbaI and EcoRI. The fragment was subcloned into the broad host-range vector pCPP30, generating plasmid pCPP30::czoR. Then, the pCPP30::czoR plasmid was transferred into the czoR strain by biparental conjugation (Simon et al., 1983) to yield the complemented strain czoR-C.
Minimal inhibitory concentration profiles of the recombinant E. coli DH5α (Miller and Mekalanos, 1988) expressing czoA (pCT-Zori::czoA) and the isogenic strains of C. testosteroni S44 (S44, czoR, czoR-C, iscR, and iscR-C) were determined by the broth dilution method (Clinical and Laboratory Standards Institute, 2014) using 11 β-lactams. For the recombinant E. coli DH5α, two β-lactamase inhibitors, clavulanic acid, and tazobactam were also tested. E. coli DH5α (pCT-Zori) was used as a control. Clavulanic acid and tazobactam were generally fixed at a concentration of 2 and 4 µg/ml, respectively (Girlich et al., 2007;Naas et al., 2007;Lamoureaux et al., 2013;Clinical and Laboratory Standards Institute, 2014). Moreover, growth tendency of the isogenic strains of S44 under stress of 11 β-lactams with concentration below MIC was evaluated through OD 600 values. Isogenic strains of C. testosteroni S44 in exponential growth phase were inoculated with each of 11 β-lactams in MH medium at 37 • C and OD 600 values were determined using a spectrophotometer after growth for 24 h.

Disk Diffusion Susceptibility Testing
The disk diffusion method was used for an antibiotic susceptibility assay (Jorgensen et al., 1999). Antimicrobial disks impregnated with cefalexin or cefazolin were separately placed onto the inoculated MH agar plates. After being incubated at 37 • C for 36 h, an inhibition zones around each antibiotic disk was measured. In addition, a spotting dilution assay was performed to determine the susceptibility of strains to cefalexin and cefazolin. Overnight cultures of strains S44, iscR-280, iscR-280C, czoR, and czoR-C were grown in LB medium. Tenfold gradient dilutions of these strains (OD 600 = 1.0) were each plated (4 µl) onto solid LB medium containing 50 µg/ml of cefalexin or cefazolin. LB medium without antibiotics was used as a control. Agar plates were incubated at 37 • C and photographed daily until colonies formed.

DNase I Footprinting Assay
To identify exact CzoR-binding sites within the intergenic region of czoR-czoA, a DNase I footprinting experiment was performed as described previously (Shi et al., 2017). The binding reaction was carried out in a 30 µl system containing 0 or 0.12 nM of purified CzoR and 100 ng of 5 -FAM labeled DNA fragment. After an incubation at 28 • C for 30 min, DNase I (0.8 unit in 20 µl, Promega) was added to the binding mixture and incubated at 37 • C for 10 min. Then, the reaction was terminated by adding 10 µl of 50 mM EDTA and an incubation in a water bath at 65 • C for 10 min. Digested DNA fragments were purified using a NucleicSpin Gel and PCR Clean-up Kit (Macherey-Nagel, Germany) and analyzed with an Applied Biosystems 3730XL DNA Analyzer (Tsingke Biological Technology Company, Wuhan, China). Results were analyzed with GeneMarkerV1.6536 (Shi et al., 2017).

Real-Time Quantitative RT-PCR
Each strain of S44, iscR-280, iscR-280C, czoR, and czoR-C was each inoculated into LB medium and incubated at 37 • C for 8 h. Next, 0 or 25 µg/ml of cefalexin or cefazolin was added to the culture. After 1 h of induction, bacterial cells were harvested for total RNA extraction using TRIzol Reagent (Invitrogen, Grand Island, NY, United States) according to the manufacturer's instructions (Invitrogen, Grand Island, NY, United States). Real-time RT-PCR was carried out using an Applied Biosystems R ViiA TM 7 Real-Time PCR System (Life Technologies, Carlsbad, CA, United States) and primers listed in Supplementary Table S1. Gene expression was normalized by the CT method with an iQ5 Real-Time PCR Detection System (Bio-Rad, United States) (Pfaffl, 2001). An ATP-binding subunit encoding gene clpX (CTS44_RS19450) was used as a reference (Caille et al., 2014) and three technical and biological replicates were performed for each reaction. Statistically significant difference between control and treated samples was performed using Student's t-test with P < 0.01 as borderline and P < 0.01 as statistically significant level. Data are expressed as the average of three experiments.

Antibiotic Susceptibility
Considering C. testosteroni S44 contains multiple β-lactamases, we determined MICs of various β-lactam antibiotics against E. coli DH5α (pCT-Zori::czoA) expressing CzoA. MIC tests using a conventional broth dilution method indicated that the recombinant strain produced the β-lactamase and its activity was inhibited by clavulanic acid or tazobactam. Determination of MICs of β-lactams for E. coli DH5α (pCT-Zori::czoA) showed that it was resistant to benzylpenicillin, ampicillin, and some cephalosporins such as cefalexin, cefazolin, cefuroxime, ceftriaxone, cefepime, and imipenem, but it remained susceptible to cefoxitin, ceftazidime, and meropenem ( Table 1). The MICs of benzylpenicillin, ampicillin, ceftriaxone, and cefepime were significantly reduced by both tazobactam and clavulanic acid, while these of cefalexin, cefazolin, and cefuroxime were merely reduced by clavulanic acid (Table 1), and the others were not inhibited by tazobactam or clavulanic acid (data not shown in Table 1). It is interesting that MIC of ampicillin-tazobactam was 16-fold of that of ampicillin-clavulanic acid ( Table 1), which indicated clavulanic acid is a sound inhibitor for class A β-lactamases, while tazobactam is a good inhibitor for other class of β-lactamase.

CzoA Kinetics Analysis
Kinetic parameters of the CzoA β-lactamase obtained with the purified enzyme (without His 6 tag) (Supplementary Figure S3) showed that CzoA had strong activities (k cat values of 99-1085 s −1 ) to degrade benzylpenicillin, ampicillin, and cefazolin. Cefalexin, cefuroxime, ceftriaxone, and cefepime were hydrolyzed at low levels (k cat values of 2-12 s −1 ), whereas hydrolysis of cefoxitin, ceftazidime, meropenem, and imipenem was not detectable ( Table 2). These results are in agreement with  antibiotic susceptibility testing that CzoA did not show obvious hydrolyzing activity for cefoxitin, ceftazidime, and meropenem. However, for imipenem, the MIC difference was observed (1 vs. 0.125 µg/ml), which indicates imipenem may be a substrate of CzoA ( Table 1).

Effects of czoA/R and IscR under Different Antibiotics
Expression of czoA::lacZ was significantly induced by all 11 β-lactams tested (Figure 2A), although CzoA did not show obvious hydrolysis activity of cefoxitin, ceftazidime, meropenem, and imipenem in vitro ( Table 2). Substrate induction experiments indicated that CzoA was an inducible class A β-lactamase in presence of β-lactams. Transcription level of czoR was significantly induced by nine β-lactams, but not inducible by cefuroxime and cefoxitin ( Figure 2B); however, czoR still showed constitutive transcriptions without and with addition of cefuroxime and cefoxitin ( Figure 2B).
To further investigate effects of CzoR and IscR on cephalosporin susceptibility, a mutant strain czoR and its complemented strain czoR-C were constructed. Successful deletion and complementation of czoR were confirmed by diagnostic PCR, as shown in Supplementary Figure S4. An iscR-mutant strain iscR-280 ( iscR) and a complemented strain iscR-280C ( iscR-C) were obtained from our previous study (Liu H.L. et al., 2015). Both mutant strains czoR and iscR showed significantly inhibited growth compared to S44 under a certain concentration of different β-lactams ( Figure 2C). Antibiotic susceptibility phenotype of complemented strains czoR-C and iscR-C was mostly recovered (data not shown).

Effects of iscR and czoR on Cefalexin and Cefazolin Susceptibility
Real-time RT-PCR assays were performed in S44 with or without addition of cefalexin and cefazolin. Results showed that, similar to czoA and czoR, iscR expression was also significantly induced by cefalexin and cefazolin (Figure 3A), indicating iscR is also involved in cefalexin and cefazolin susceptibility. Kirby-Bauer disk diffusion assays showed that growth inhibition zones of czoR-and iscR-280-mutant strains were significantly larger than S44 in presence of cefalexin and cefazolin ( Figure 3B). Antibiotic susceptibility phenotypes of complemented strains were restored (Figure 3B). In addition, spotting assays showed that the iscR-280 strain, and to a greater extent for the czoR strain, was more susceptible to cefalexin and cefazolin relative to the wild-type strain S44 (Figure 3C). Phenotypes of the complemented strains were restored, and all strains showed a similar growth trend on LB plates without antibiotics ( Figure 3C). These results suggested that both IscR and CzoR are essential for cephalosporin susceptibility and that CzoR may play a more important role.

CzoR Binds to the Promoter Region of czoA
To examine interactions between CzoR and PczoA, EMSA was performed with a 259 bp fragment of PczoA and purified CzoR (Supplementary Figure S5). With an increasing CzoR concentration, free DNA substrates gradually disappeared, while intensity of shifted DNA bands increased ( Figure 4A). Reactions using heat-denatured CzoR and a non-specific DNA probe did not show any lagging bands. Moreover, unlabeled czoA DNA substrates could competitively inhibit CzoR binding to the FAMlabeled czoA (Figure 4A). These results indicated that CzoR could specifically bind to the czoA regulatory region.
Subsequently, a DNase I footprinting assay was conducted to determine exact binding sites of CzoR. Results showed that the -88 to -72 region in PczoA was obviously protected from DNase I digestion (Figure 4B), indicating that a 17 bp fragment (5 -TCTCAATCAAGATAAAA-3 ) upstream of czoA was a CzoR-binding box in S44 (Figure 4C). For further confirmation, interactions between CzoR and a 211 bp fragment of PczoA without CzoR-binding box were tested. EMSA results showed that there was no band shift without CzoR-binding box ( Figure 4D). These experiments demonstrated that the LysR family regulator CzoR can regulate the class A β-lactamase gene czoA in S44.

IscR Binds to the Regulatory Region of czoR-czoA
Based on IscR-binding motifs in C. testosteroni KF-1 obtained from the RegPrecise database (Novichkov et al., 2013), we found a putative IscR-binding site within czoR-czoA intergenic region. A putative IscR motif (TTTTCTAATGGATGGTGTCAATTAT) was located in the sense strand adjacent to the CzoR box ( Figure 5A). The interaction between IscR and czoR-czoA intergenic region was examined by EMSA. With an increasing IscR concentration, lagging bands were clearly observed. In contrast, negative controls (non-specific DNA probe or heatdenatured IscR) did not show any lagging bands ( Figure 5B). In addition, IscR was capable of binding to substrates containing a 30 bp sequence with a refined IscR-binding motif ( Figure 5C). These data suggest that IscR can directly bind to the czoR-czoA promoter region, and may regulate the expression of both czoR and czoA. Bacteria were cultured in LB medium with addition of 25 µg/ml benzylpenicillin, ampicillin, cephalexin, cefazolin, cefuroxime, cefoxitin, ceftazidime, ceftriaxone, cefepime, and 0.5 µg/ml meropenem and imipenem. A culture without β-lactams was used as a control. (B) Real-time RT-PCR results for czoR in S44. The relative czoR mRNA expression levels induced by 11 β-lactams (black bars) and in the control (without induction, white bars) were determined as described in the Section "Materials and Methods." Error bars represent standard deviations of triplicate tests. The selected induction concentrations, which did not affect normal growth, were well below the MIC for the β-lactams. * * Indicates a significant difference from the control (p < 0.01, Student's t-test). (C) The growth tendency of the isogenic strains of S44 under the stress of the 11 β-lactams [benzylpenicillin (2048 µg/ml), ampicillin (2048 µg/ml), cephalexin (1024 µg/ml), cefazolin (2048 µg/ml), cefuroxime (128 µg/ml), cefoxitin (128 µg/ml), ceftazidime (128 µg/ml), ceftriaxone (1024 µg/ml), cefepime (512 µg/ml), meropenem (1 µg/ml), and imipenem (4 µg/ml)] was evaluated through the OD 600 values.
FIGURE 3 | IscR and CzoR contribute to cephalosporin susceptibility. (A) Transcriptional levels of czoR, iscR, and czoA in S44 with or without (white bars) addition of 25 µg/ml of cefalexin (dark gray bars) and cefazolin (black bars). (B) Susceptibility tests of strains S44, iscR-280, iscR-280C, czoR, and czoR-C to different antibiotics (cefalexin and cefazolin) were determined using the Kirby-Bauer disk diffusion method. The inhibition zone of each disk was measured to the nearest millimeter. The diameter (0.7 cm) of susceptibility disks was counted in the inhibition zones. (C) Spotting assays for growths of strains S44, iscR-280, iscR-280C, czoR, and czoR-C on LB plates without antibiotics and those supplemented with 50 µg/ml cefalexin or cefazolin. Ten-fold serial dilutions of each culture were inoculated on the plates and incubated at 37 • C for 48 h. Error bars represent standard deviations of triplicate tests. * * Indicates a significant difference from the control (p < 0.01, Student's t-test).

CzoR Is Essential for czoA Expression and IscR Positively Regulates czoR Expression
To investigate how iscR and czoR influence each other and further affect czoA expression, real-time RT-PCR transcription analyses were performed using C. testosteroni S44 isogenic strains with or without addition of cefalexin or cefazolin. Deletion of iscR significantly decreased czoR expression, and czoR was not induced by cefalexin and cefazolin in iscR-280-mutant strain ( Figure 6A). czoR deletion did not affect transcription level of iscR ( Figure 6B) indicating that czoR induction by cefalexin and cefazolin was depend on IscR expression. Phenotypes of the complemented strain iscR-280C were recovered.
Subsequent efforts focused on czoA expression in the isogenic strains of C. testosteroni S44 using a lacZ reporter FIGURE 6 | Transcriptional analysis. (A,B) Represents real-time RT-PCR results for czoR and iscR. The relative czoR and iscR mRNA expression levels induced by cefalexin (gray bars) and cefazolin (black bars) and in the control (without induction, white bars) were determined as described in the section "Materials and Methods." Error bars represent standard deviations of triplicate tests. (C) Shows a lacZ reporter fusion of czoA. Bacteria were cultured in LB medium with or without (white bars) addition of 25 µg/ml cefalexin (gray bars) and cefazolin (black bars). * * Indicates a significant difference from the control (p < 0.01, Student's t-test).
gene assay. Results showed that expression of czoA::lacZ was significantly induced by cefalexin and cefazolin in S44, consistent with induction expressions of czoR and iscR in presence of cefalexin and cefazolin ( Figure 3A). Deletion of iscR (iscR-280) decreased expression of czoA::lacZ compared to S44 with addition of cefalexin and cefazolin (Figure 6C), possibly because transcription level of czoR was decreased in iscR-280 ( Figure 6A). However, in czoR, β-gal activity of CzoA::LacZ was almost non-existent, even at higher concentrations of cefalexin and cefazolin (50-100 µg/ml) or using different induction times (data not shown), indicating that czoR is essential for czoA expression. Although iscR expression was also induced by cefalexin and cefazolin in czoR, IscR could not directly regulate czoA expression without CzoR. Higher β-gal activities in iscR-280C and czoR-C compared to S44 may be attributable to a multicopy-based complementation used for both iscR and czoR. These results suggest that IscR may positively regulate czoR expression of and affect czoA expression, which provided a link between IscR and β-lactam susceptibility regulation.
In opposite orientation from the PczoA, a LysR-type transcriptional regulatory protein CzoR was identified (Figure 1). As is often the case for other LysR-regulated genes, genes encoding Class A β-lactamase and regulator were adjacent and opposite to one another, with overlapping and divergent promoters, which may provide tighter control of gene overexpression and prevent an inadvertent gene activation (Naas and Nordmann, 1994).
We observed that czoA expression was induced by 11 β-lactams and czoR was induced by 9 β-lactams and constitutively transcribed with or without cefuroxime and cefoxitin (Figures 2A,B). Such results are somehow in agreement with previous studies. For example, cdiAR operon encoding for Class A β-lactamase biosynthesis were also inducible by β-lactams in E. coli strains (Jones and Bennett, 1995). As for SmeR, β-lactams did not affect its expression (Naas et al., 1995). While in C. gillenii, no LysR-type regulatory gene was found upstream of the bla GIL−1 gene, which fits non-inducibility of β-lactamase expression . Although some β-lactams can induce AmpC, these β-lactams were not direct bind AmpR (Jones and Bennett, 1995).
In addition to the CzoR-binding motif, we also found a putative IscR-binding motif in the czoR-czoA intergenic region (Figure 5). Previous studies have shown that IscR is a global regulator involved in regulation of various physiological processes during growth and stress responses (Daung-nkern et al., 2010;Zheng et al., 2014;Liu H.L. et al., 2015), but little is known about the role of IscR in antibiotic susceptibility regulation interacting with β-lactamase. This study demonstrated that IscR indirectly influenced czoA expression through czoR regulation based on following observations (Figures 2, 3, 5, 6): (i) IscR/CzoR/CzoA was induced by cefalexin and cefazolin, and IscR could directly bind to the czoR-czoA promoter; (ii) iscR deletion decreased transcription level of czoR and czoA; (iii) czoR deletion had no effect on iscR transcription, although czoA expression was completely inhibited; and (iv) susceptibility to cefalexin and cefazolin was increased in iscR and further increased in czoR. CzoR, therefore, acted as a positive regulator for CzoA β-lactamase biosynthesis and IscR positive regulated czoR expression.
It has been shown that Fe-S cluster biosynthesis may also be involved in antibiotic susceptibility (Daung-nkern et al., 2010;Liu et al., 2010). Disruption of Fe-S cluster results in Fe(II)-mediated Fenton reactions and enhances oxidative stress. Our previous work showed that deletion of iscR significantly decreased cellular γ-glutamylcysteine ligase (γ-GCL) activity and glutathione (GSH) content (Liu H.L. et al., 2015), which play important roles in Fe-S cluster formation and H 2 O 2 consumption, respectively (Qi et al., 2012;Wang et al., 2012). To guard against oxidative stress resulting from bactericidal agents, such as β-lactams, IscR may respond to β-lactaminduced stress, such as cefalexin and cefazolin, faster than CzoR.
In summary, our results reveal a novel mechanism in which CzoR positively regulates czoA, and IscR enhances the regulation by CzoR. Since IscR is a global regulator for cellular oxidative stress response, it is reasonable that IscR regulates expression of some β-lactamases, such as CzoA expression which is related to bacterial cell wall stress remission. Our study provides a new insight into the regulatory mechanism of class A β-lactamases and demonstrates, for the first time, that IscR is involved in antibiotic susceptibility via the regulation of czoR-czoA.

AUTHOR CONTRIBUTIONS
WZ and HL designed and performed the experiments and wrote the manuscript. JL wrote and revised the draft of the manuscript. LC participated in the experiments. GW designed the study and revised the draft of the manuscript. All authors read and approved the final manuscript.