Abstract
ABSTRACTThe cell envelope of Gram-negative bacteria consists of two membranes surrounding the periplasm and peptidoglycan layer. β-Lactam antibiotics target the periplasmic penicillin-binding proteins that synthesize peptidoglycan, resulting in cell death. The primary means by which bacterial species resist the effects of β-lactam drugs is to populate the periplasmic space with β-lactamases. Resistance to β-lactam drugs is spread by lateral transfer of genes encoding β-lactamases from one species of bacteria to another. However, the resistance phenotype depends in turn on these “alien” protein sequences being recognized and exported across the cytoplasmic membrane by either the Sec or Tat protein translocation machinery of the new bacterial host. Here, we examine BKC-1, a carbapenemase from an unknown bacterial source that has been identified in a single clinical isolate of Klebsiella pneumoniae. BKC-1 was shown to be located in the periplasm, and functional in both K. pneumoniae and Escherichia coli. Sequence analysis revealed the presence of an unusual signal peptide with a twin arginine motif and a duplicated hydrophobic region. Biochemical assays showed this signal peptide directs BKC-1 for translocation by both Sec and Tat translocons. This is one of the few descriptions of a periplasmic protein that is functionally translocated by both export pathways in the same organism, and we suggest it represents a snapshot of evolution for a β-lactamase adapting to functionality in a new host.
Highlights
The cell envelope of Gram-negative bacteria consists of two membranes surrounding the periplasm and peptidoglycan layer. b-Lactam antibiotics target the periplasmic penicillin-binding proteins that synthesize peptidoglycan, resulting in cell death
There is a global distribution of the CTX-M family of b-lactamases in E. coli [24, 25], K. pneumoniae [26, 27], other Enterobacteriaceae [28, 29], and Pseudomonas aeruginosa [30, 31], implying that all of the species acquiring these genes are capable of efficiently translating the polypeptide through what might be nonoptimal codon bias, transporting it across the inner membrane via protein translocases that must recognize its signal peptide, and folding the CTX-M protein so that the functional form populates the periplasm
Through the production of BKC-1 in a model strain of E. coli with or without a functional twin arginine translocase (Tat) system, we show that while a subpopulation of protein molecules are translocated by the Sec pathway, the rest are translocated via Tat translocon
Summary
The cell envelope of Gram-negative bacteria consists of two membranes surrounding the periplasm and peptidoglycan layer. b-Lactam antibiotics target the periplasmic penicillin-binding proteins that synthesize peptidoglycan, resulting in cell death. Sequence analysis suggested the signal peptide of BKC-1 has features consistent with the protein being a substrate of the Tat pathway (Table 2).
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