Campylobacter fetus Uses Multiple Loci for DNA Inversion within the 5′ Conserved Regions of sap Homologs

Campylobacter fetus cells possess multiple promoterless sap homologs, each capable of expressing a surface layer protein (SLP) by utilizing a unique promoter present on a 6.2-kb invertible element. Each sap homolog includes a 626-bp 5′ conserved region (FCR) with 74 bp upstream and 552 bp within the open reading frame. After DNA inversion, the splice is seamless because the FCRs are identical. In mutant strain 23D:ACA2K101, in which sapA and sapA2 flanking the invertible element in opposite orientations were disrupted by promoterless chloramphenicol resistance (Cm(r)) and kanamycin resistance (Km(r)) cassettes, respectively, the frequency of DNA inversion is 100-fold lower than that of wild-type strain 23D. To define the roles of a 15-bp inverted repeat (IR) and a Chi-like site (CLS) in the FCR, we mutagenized each upstream of sapA2 in 23D:ACA2K101 by introducing NotI and KpnI sites to create strains 23D:ACA2K101N and 23D:ACA2K101K, respectively. Alternatively selecting colonies for Cm(r) or Km(r) showed that mutagenizing the IR or CLS had no apparent effect on the frequency of the DNA inversion. However, mapping the unique NotI or KpnI site in relation to the Cm(r) or Km(r) cassette in the cells that changed phenotype showed that splices occurred both upstream and downstream of the mutated sites. PCR and sequence analyses also showed that the splice could occur in the 425-bp portion of the FCR downstream of the cassettes. In total, these data indicate that C. fetus can use multiple sites within the FCR for its sap-related DNA inversion.

胎儿弯曲杆菌（Campylobacter fetus）细胞携带多个不带启动子的sap同源基因，每个同源基因均可通过利用6.2kb可逆元件上的独特启动子，表达表面层蛋白（surface layer protein, SLP）。每个sap同源基因均包含一段626bp的5'端保守区域（5' conserved region, FCR），该区域包含74bp的上游序列以及开放阅读框内的552bp序列。DNA反转完成后，由于各FCR序列完全一致，因此剪接过程无痕迹残留。
在突变菌株23D:ACA2K101中，位于可逆元件两侧且方向相反的sapA与sapA2，分别被不带启动子的氯霉素抗性（chloramphenicol resistance, Cm^r）基因盒与卡那霉素抗性（kanamycin resistance, Km^r）基因盒破坏，其DNA反转频率较野生型菌株23D低100倍。为明确15bp反向重复序列（inverted repeat, IR）及类Chi位点（Chi-like site, CLS）在FCR中的功能，我们分别在23D:ACA2K101的sapA2上游引入NotI与KpnI酶切位点，以此构建得到菌株23D:ACA2K101N与23D:ACA2K101K。
分别以Cm^r或Km^r抗性筛选菌落的实验结果显示，对IR或CLS进行诱变并未对DNA反转频率产生显著影响。然而，通过对表型发生改变的菌株中，与Cm^r或Km^r基因盒对应的独特NotI或KpnI酶切位点进行定位，可发现剪接既发生在突变位点的上游，也发生在其下游。PCR与序列分析结果还表明，剪接可发生在抗性基因盒下游的FCR 425bp片段区域内。综上，上述实验数据表明，胎儿弯曲杆菌可利用FCR内的多个位点完成其sap相关的DNA反转过程。