Beta-lactamases are the most common agents of beta-lactam resistance in bacteria (Bush, 2010). Depending on their signal sequence at the N terminal (Palmer et al., 2012), most well-known beta-lactamases are known to translocate via the Sec pathway, which transports unfolded proteins across the inner membrane while a few translocate via the Tat pathway which transports folded proteins. Though several beta-lactamases have been characterized in terms of their activity and phenotype, little is known about the details of their biogenesis. Furthermore, newer beta-lactamases also receive limited attention. In this study, we aimed to investigate this differential beta-lactamase translocation by studying the BKC-1, a new plasmid-borne beta-lactamase recently reported in Brazilian clinical strains of Klebsiella pneumoniae (Nicoletti et al., 2015).
BKC-1 was analysed using TatP and SignalP and sequence alignment with similar predicted proteins from BLAST analysis was carried out using Clustal Omega. Antibiotic sensitivity assays were carried out using broth dilution and protein expression profiles were determined using Western blots.
BKC-1 was predicted to be a Tat peptide and found to possess a repeating h region in the signal peptide. Strains lacking TatC and expressing BKC-1 continued to depict beta-lactam resistant phenotypes similar to those of the wild type, with the exception of Ceftazidime. However, varied retardation in growth was observed for strains devoid of TatC in the presence of sub-inhibitory concentrations of different beta-lactams. The tatC mutants expressing BKC-1 with the original signal peptide as well its shorter variant did not grow in the presence of Ceftazidime and the wild type strain expressing the two variants exhibited different growth rates. This was also consistent with variable processed protein levels of BKC-1 expressed in the wild type and tatC mutants.
We thus suggest that BKC-1 might be a dual targeting enzyme and its duplicated h region could play a role in adaptive resistance given that BKC-1 was isolated from a clinical isolate. In the future we aim to dig deeper into the molecular mechanisms that facilitate the successful biogenesis of BKC-1 in the context of other plasmid associated beta-lactamases and understand the phenotypic evolution of the enzyme with sequence variation.