Yersinia enterocolitica, a member of Enterobacteriaceae, is a zoonotic pathogen widely distributed in nature (Wang et al., 2011; Liang et al., 2012). Most Y. enterocolitica exhibits intrinsic resistance to β-lactm antibiotics by the production of chromosomally encoded β-lactamases called BlaA (a class A enzyme showing constitutive expression) and BlaB (an inducible AmpC-type β-lactamase), respectively (Cornelis and Abraham, 1975; Bent and Young, 2010).

The process of ampC (blaB) regulation is tightly linked to the peptidoglycan recycling and controlled by AmpG, AmpD, AmpR, and NagZ (Vollmer et al., 2008; Zeng and Lin, 2013). Briefly, peptidoglycan degradation products including GlcNAc-1,6-anhydromuropeptide is transported into the cytoplasm by AmpG and further hydrolyzedcosaminidase) to yielding 1,6-anhydromuropeptides, which is the AmpR activator ligand for ampC derepression (Zamorano et al., 2010; Huang et al., 2012; Yang et al., 2014). On the other hand, the stem peptides of GlcNAc-1,6-anhydromuropeptide and 1,6-anhydromuropeptides can be removed by AmpD (N-acetylmuramyl-l-alanine amidase) and eventually recycled into UDP-MurNAc-pentapeptide, which is the AmpR repressor ligand to repress ampC expression level (Juan et al., 2006; Balasubramanian et al., 2015; Liu et al., 2016). Penicillin-binding proteins (PBPs) also play an important role in ampC regulation (Sanders et al., 1997; Pfeifle et al., 2000). Recent studies have found that in P. aeruginosa, PBP4 (DacB), PBP5 (DacC), and PBP7 (PbpG) are involved in ampC regulation, and PBP4 is the major cause of ampC derepressed in clinical strains (Moya et al., 2009; Ropy et al., 2015).

Theoretically, NagZ is indispensable in chromosomal ampC derepression. In P. aeruginosa, nagZ inactivation dramatically reduces the β-lactam resistance of both PAOΔampD (ampD inactivation) and PAOΔdacB (pbp4 inactivation; Zamorano et al., 2010). However, although nagZ inactivation nearly abolished the basal-level derepressed β-lactamase activity of KJΔampDI (ampD inactivation), it did not affect the β-lactamase activity of KJΔmrcA (pbp1a inactivation) in Stenotrophomonas maltophilia (Huang et al., 2012).

Since the effects of the above-mentioned genes in Y. enterocolitica were seldom reported, we elucidated the role of low-molecular-mass penicillin-binding proteins (LMM PBPs) (PBP4, PBP5, PBP6, and PBP7), NagZ and AmpR in the Y. enterocolitica ampC regulation. Firstly, we investigated the effects of each LMM PBP on the expression of AmpC β-lactamase by monitoring the ampC promoter activity from a series of LMM PBPs mutant strains and confirmed by quantitative reverse transcription-PCR (qRT-PCR). Secondly, nagZ gene was deleted in two ampC derepressed strains YEΔD123 and YEΔ4Δ6Δ5Δ7 to determine the role for ampC expression.

The ampR-ampC system from Citrobacter freundii and Enterobacter cloacae has been well studied in the early 1990s (Lindberg et al., 1987; Peter et al., 1988). However, newly discovered ampC regulators such as, PBP4 (DacB) or NagZ in Enterobacteriaceae was not yet understood. A deep study in Y. enterocolitica ampR-ampC system would be helpful to improve the comprehensive understanding of Enterobacteriaceae ampC regulation.

PBPs are a group of enzymes involved in cell-wall recycling and the processes of AmpC β-lactamases regulation. In E. coli model, deletion of three or four PBPs and the concomitant inhibition of PBP 1a, 1b, and/or 2 results in an increased level of β-lactamase induction (Pfeifle et al., 2000). However, since E. coli lacks the chromosomal ampR gene, the result may be inconsistent with other members of the Gram-negative bacteria which have a chromosome encoding the ampR-ampC system. In 2009, Moya et al. demonstrated the inactivation of DacB (PBP4), a nonessential low-molecular mass PBPs is the principal reason for one-step high-level ampC expression in clinical strains of P. aeruginosa (Moya et al., 2009). Interestingly, inactivation of PBP4 in E. cloacae triggered a significant increase of β-lactams resistance, but without an obvious upregulation of ampC gene, it may be suggested that PBP4 regulates AmpC at a post-transcriptional level (Guerin et al., 2015). In this study, we found deletion of pbp4 did not elevate the ampC expression level, this result is accordance with E. cloacae. After that, we deleted all four LMM PBPs one after another, and found that PBP5 is the most effective PBP involved in the regulation of ampC in Y. enterocolitica. Of the single-mutation strains, only the pbp5 deletion strain YEΔ5 showed an obvious rise in ampC expression level. Likewise, for multi-mutation strains, the function of PBP4, PBP6, and PBP7 in ampC regulation were detected only if in Δpbp5 background. According to the results shown in Figure 1 and Table 2, we deduced the hierarchy of the role of PBPs genes in ampC derepression: PBP5 > PBP4 > PBP7 > PBP6. Although DacB may regulates AmpC at a post-transcriptional level (Guerin et al., 2015), but no trace of post-transcriptional mechanism has been found in Y. enterocolitica.

Along with the popular research of ampC regulation, there is growing evidence that some bacteria may regulate the expression of ampC through at least two different ways, one of which was NagZ-dependent, while the other worked without the participation of NagZ (Huang et al., 2012; Guerin et al., 2015). In the study on P. aeruginosa, nagZ inactivation was shown to attenuate ampC expression and was critical for basal-level ampC derepression in both PAΔD (ampD inactivation) and PAΔdB (pbp4 inactivation) mutants (Asgarali et al., 2009; Zamorano et al., 2010). However, ΔnagZ had little effect on the cefoxitin-induced ampC expression level in both PAΔD and PAΔdB, which indicated that an unidentified non-NagZ product at work in this induction process. Furthermore, two different regulation ways of β-lactamase have been found in S. maltophilia, on one hand NagZ was essential for KJΔDI (ampD inactivation) ampC overexpression, on the other hand, nagZ inactivation hardly influenced the ampC expression level of KJΔmrcA (pbp1a inactivation; Huang et al., 2012). In this study, we also found two different ampC regulation ways exist in Y. enterocolitica, the patterns of which were just the reverse of that in S. maltophilia (Huang et al., 2012). The β-lactamase activity of YEΔD123 was not affected by the inactivation of the nagZ gene, whereas the introduction of ΔnagZ into the PBP mutation strain YEΔ4Δ5Δ6Δ7 dramatically reduced the β-lactamase activities at both the basal and induced level (Figure 2). As shown in Table 3, the antibiotic resistance of YEΔ4Δ5Δ6Δ7 and YEΔD123 were marked improved compare with wild-type strain, the MIC value of these two strains in TZP, PIP, CFZ, CAZ, CRO, and ATM is rising sharply. While after inactivation of nagZ gene simultaneously, only a marginal distinction between YEΔD123 and YEΔD123ΔZ was found, but the MIC values of YEΔ4Δ5Δ6Δ7ΔZ has shifted down significantly, far from its parent strain YEΔ4Δ5Δ6Δ7 for almost all tested β-lactams. To further confirm the function of NagZ, we constructed a nagZ deletion strain YEΔZ, and detected the N-acetyl-β-glucosaminidase activity of it to compare with the wild-type strain Y. enterocolitica 105.5R(r), the results showed that the ability of hydrolysis chromogenic substrate was completely lost in nagZ mutation strain YEΔZ (Figure 3), suggesting that NagZ (YE105_RS06670) was the only enzyme that possessed N-acetyl-β-glucosaminidase activity in Y. enterocolitica 105.5R(r). However, even though there is no readable N-acetyl-β-glucosaminidase activity in YEΔZ, we also did the bioinformatic search to look for possible NagZ homologs in genome to find the protein worked in YEΔD123ΔZ. According to the gene function annotation of 105.5R(r), we considered the YE105_RS13000 may have similar function with NagZ, but it was not clear if this protein participated the ampC regulation or not. Therefore, further studies needed to performed to elucidate the function of YE105_RS13000 in Y. enterocolitica ampC regulation.

In Y. enterocolitica, the function of AmpR was roughly the same as other members of Enterobacteriaceae or P. aeruginosa. The introduction of ΔampR into the AmpC hyperproduction strains YEΔD123 and YEΔ4Δ5Δ6Δ7 resulted in a sharp decline in the ampC expression (Figure 2). The inducibility of YEΔD123ΔR and YEΔ4Δ5Δ6Δ7ΔR also disappeared completely (Lindberg et al., 1985; Lindberg and Normark, 1987).

In conclusion, in terms of AmpC β-lactamase regulation, Y. enterocolitica shared some common characteristics with P. aerugiosa and other members of Enterobacteriaceae, but it also had its own features. This was the first investigation to the characterization of Y. enterocolitica ampC regulation. It provided a more comprehensive understanding of the AmpC β-lactamase regulation in Gram-negative bacteria.

CL, CCL, SS, HJ, and XW designed the experiment together. YC and HH performed data analysis. JL and RD participated in the manuscript translation. ZG, JZ, and ZZ contributed to finish the work. All authors contributed to writing of the manuscript.