*Article* **Identification and Characterization of Genes Related to Ampicillin Antibiotic Resistance in** *Zymomonas mobilis*

**Binan Geng † , Xingyu Huang † , Yalun Wu, Qiaoning He \* and Shihui Yang \***

> State Key Laboratory of Biocatalysis and Enzyme Engineering, Environmental Microbial Technology Center of Hubei Province, School of Life Sciences, Hubei University, Wuhan 430062, China

**\*** Correspondence: qiaoninghe@hubu.edu.cn (Q.H.); shihui.yang@hubu.edu.cn (S.Y.)

† These authors contributed equally to this work.

**Abstract:** Antibiotics can inhibit or kill microorganisms, while microorganisms have evolved antibiotic resistance strategies to survive antibiotics. *Zymomonas mobilis* is an ideal industrial microbial chassis and can tolerate multiple antibiotics. However, the mechanisms of antibiotic resistance and genes associated with antibiotic resistance have not been fully analyzed and characterized. In this study, we investigated genes associated with antibiotic resistance using bioinformatic approaches and examined genes associated with ampicillin resistance using CRISPR/Cas12a−based genome−editing technology. Six ampicillin−resistant genes (*ZMO0103*, *ZMO0893*, *ZMO1094*, *ZMO1650*, *ZMO1866*, and *ZMO1967*) were identified, and five mutant strains ZM4∆0103, ZM4∆0893, ZM4∆1094, ZM4∆1650, and ZM4∆1866 were constructed. Additionally, a four−gene mutant ZM4∆ARs was constructed by knocking out *ZMO0103*, *ZMO0893*, *ZMO1094*, and *ZMO1650* continuously. Cell growth, morphology, and transformation efficiency of mutant strains were examined. Our results show that the cell growth of ZM4∆0103 and ZM4∆ARs was significantly inhibited with 150 µg/mL ampicillin, and cells changed to a long filament shape from a short rod shape. Moreover, the transformation efficiencies of ZM4∆0103 and ZM4∆ARs were decreased. Our results indicate that ZMO0103 is the key to ampicillin resistance in *Z. mobilis*, and other ampicillin−resistant genes may have a synergetic effect with it. In summary, this study identified and characterized genes related to ampicillin resistance in *Z. mobilis* and laid a foundation for further study of other antibiotic resistance mechanisms.

**Keywords:** *Zymomonas mobilis*; antibiotic resistance; ampicillin; genome editing; CRISPR−Cas12a; resistance selection markers

#### **1. Introduction**

Antibiotics are natural secondary metabolites or artificially synthesized analogs produced by organisms, such as bacteria, animals, and plants during the metabolic process to kill pathogens [1]. They are commonly used during plasmid and strain construction in genetic engineering and are usually used as the feed additive for the growth and disease resistance of plants and animals [2]. In the environment, competition exists among microorganisms for living space and nutrients. Some microorganisms compete with others by producing antibiotics that are sensitive to antibiotics. For example, *streptomyces* can produce 80% of the antibiotics currently known [3]. At the same time, various microorganisms naturally have certain resistance to different antibiotics, which brings challenges to the genetic engineering of strains [4].

At present, four classes of antibiotics are mainly classified according to their inhibition pathway: (i) inhibition of cell wall synthesis, such as β−lactam antibiotics and glycopeptides; (ii) inhibition of protein synthesis including macrolides, oxazolidinones, amphenicols, lincosamides, tetracyclines, and aminoglycosides [5]; (iii) inhibition of DNA synthesis by targeting gyrase or DNA, such as fluoroquinolones and nitroimidazoles [6,7]; and (iv) inhibition of membrane integrity, such as lipopeptides (e.g., daptomycin) [8] and

**Citation:** Geng, B.; Huang, X.; Wu, Y.; He, Q.; Yang, S. Identification and Characterization of Genes Related to Ampicillin Antibiotic Resistance in *Zymomonas mobilis*. *Antibiotics* **2022**, *11*, 1476. https://doi.org/10.3390/ antibiotics11111476

Academic Editor: John E. Gustafson

Received: 10 September 2022 Accepted: 24 October 2022 Published: 25 October 2022

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polymyxins (e.g., colistin) [9]. β−Lactams are the most widely used antibiotics with the potential to interrupt bacterial cell wall formation as a result of covalent binding to essential penicillin−binding protein (PBP) enzymes that are involved in the terminal steps of peptidoglycan cross−linking in both Gram−negative and Gram−positive bacteria [10]. Ampicillin is the representative β−lactam antibiotic and commonly used in genetic engineering to screen colonies with ampicillin resistance.

To survive in an environment containing antibiotics, microorganisms have developed a variety of antibiotic resistance (AR) mechanisms during evolution. Based on the biochemical route involved in resistance, the mechanisms of antibiotic resistance currently are classified into four types. The first one is to modify the antimicrobial molecule. Some enzymes are capable of chemical alterations to inactivate the antibiotics with acetylation, phosphorylation, and adenylation of the antibiotics, such as aminoglycosides, chloramphenicol, and streptogramins [11]. Some enzymes can destroy the antibiotic molecule, such as β−lactamases, rendering the antibiotic unable to interact with the target sites [11]. The second is to prevent antibiotics from reaching their target by decreasing penetration or actively extruding the antimicrobial compound. A reduced number or differential expression of porins, such as the OprD porin protein in some microorganisms, prevented the entry of carbapenems [12]. Extruding the toxic compound out of the cell through an efflux pump [13,14] and the formation of biofilm [15] are also effective to prevent the entrance of antibiotics. The third one is to change or bypass target sites by avoiding the antibiotic to reach its binding site or to modify the target sites that results in decreased affinity for the antibiotic molecule. The last type is a global cell adaptive response to the antibacterial attack.

*Z. mobilis* is a facultative anaerobic Gram−negative bacterium with a unique Entner– Doudoroff (ED) pathway and many excellent physiological characteristics for industrial bioethanol production, such as the highly efficient utilization of sugar and high ethanol yield and ethanol tolerance [16,17]. Currently, the available antibiotics used for genetic engineering in *Z. mobilis* include ampicillin, kanamycin, spectinomycin, chloramphenicol, tetracycline, streptomycin, and gentamicin [18,19]. Among them, some antibiotics are naturally resisted by *Z. mobilis* at low concentrations, such as ampicillin < 300 µg/mL, kanamycin < 350 µg/mL, streptomycin < 300 µg/mL, gentamicin < 100 µg/mL, and tetracycline and chloramphenicol < 25 µg/mL [18,19]. In addition, different subspecies of *Z. mobilis* have variable susceptibility to different antibiotics. For example, the working concentrations of ampicillin, chloramphenicol, tetracycline, and kanamycin that are used for genetics studies were 300 vs. 500, 100 vs. 100, 25 vs. 25, and 350 vs. 250 µg/mL in *Z. mobilis* ZM4 and CP4, respectively [19].

Although *Z. mobilis* is tolerant to ampicillin, only one work reported that *ZMO0103* probably is an ampicillin−resistant gene, which reported the results of a heterologous protein expression and an enzymatic kinetic analysis [20]. The genome sequence of *Z. mobilis* ZM4 was published, and the genome annotation was further improved [21–23]. Moreover, the genome−editing tools including the native type I−F CRISPR−Cas system and the CRISPR−Cas12 system as well as the platform to identify and characterize biological parts have been established in *Z. mobilis* [24–26]. Therefore, we attempted to explore the ampicillin tolerance mechanism of *Z. mobilis* by identifying potential resistance genes using bioinformatics approaches and constructing ampicillin−sensitive mutant strains by genome engineering to verify its function.

### **2. Results**

#### *2.1. In Silico Analysis of the AR Genes of Z. mobilis ZM4*

A total of 100 candidate AR genes in *Z. mobilis* ZM4 were predicted using the databases of CARD and MEGARes. The results demonstrate that *Z. mobilis* ZM4 contains 9 putative lactamase−related genes (*ZMO0103*, *ZMO0108*, *ZMO0598*, *ZMO0675*, *ZMO0781*, *ZMO1336*, *ZMO1574*, *ZMO1914*, and *ZMO1967*), 7 putative transferase−related genes (*ZMO0111*, *ZMO0183*, *ZMO1143*, *ZMO1306*, *ZMO1355*, *ZMO1452*, and *ZMO1577*), 8

putative porin−related genes (*ZMO0079*, *ZMO0257*, *ZMO0478*, *ZMO1124*, *ZMO1164*, *ZMO1177*, *ZMO1322*, and *ZMO1387*), and 76 putative efflux pump−related genes (See Supplementary Materials Table S1). In addition, 68 β−lactamase genes were predicted in ZM4 by BLASTP with the gene sequences of the β−lactam class in the UniProt database (See Supplementary Materials Table S2). Among them, six genes were annotated as β−lactamase−encoding genes (*ZMO0103*, *ZMO0893*, *ZMO1094*, *ZMO1650*, *ZMO1866*, *ZMO1967*), two candidate genes in list 1 (*ZMO0103*, *ZMO1967*), and five genes in list 2 (*ZMO0103*, *ZMO0893*, *ZMO1650*, *ZMO1866*, and *ZMO1967*).

Five putative ampicillin−resistant (AR) candidate genes of *ZMO0103*, *ZMO0893*, *ZMO1650*, *ZMO1866*, and *ZMO1967* were predicted as the β−lactamase genes, and *ZMO1094* was annotated as metallo−beta−lactamase−like protein−encoding gene in ZM4. Multiple sequence alignment revealed that ZMO0103, ZMO0893, and ZMO1650 belong to the AmpC superfamily (Supplementary Materials Figure S1); ZMO1967 and ZMO1094 belong to the PenP superfamily (β−lactamase class A), while ZMO1866 belongs to the RnjA superfamily (Supplementary Materials Figure S1) according to the conserved domain search [27]. Moreover, ZMO0103, ZMO0893, and ZMO1650 have similar conserved structures based on the multisequence alignment results (Supplementary Materials Figure S2).
