New Shuttle Vector-Based Expression System To Generate Polyhistidine-Tagged Fusion Proteins in Staphylococcus aureus and Escherichia coli

Abstract

Four Staphylococcus aureus-Escherichia coli shuttle vectors were constructed for gene expression and production of tagged fusion proteins. Vectors pBUS1-HC and pTSSCm have no promoter upstream of the multiple cloning site (MCS), and this allows study of genes under the control of their native promoters, and pBUS1-P cap -HC and pTSSCm-P cap contain the strong constitutive promoter of S. aureus type 1 capsule gene 1A (P cap ) upstream of a novel MCS harboring codons for the peptide tag Arg-GlySer-hexa-His (rgs-his 6 ). All plasmids contained the backbone derived from pBUS1, including the E. coli origin ColE1, five copies of terminator rrnB T 1 , and tetracycline resistance marker tet(L) for S. aureus and E. coli. The minimum pAM␣1 replicon from pBUS1 was improved through either complementation with the single-strand origin oriL from pUB110 (pBUS1-HC and pBUS1-P cap -HC) or substitution with a pT181-family replicon (pTSSCm and pTSSCm-P cap ). The new constructs displayed increased plasmid yield and segregational stability in S. aureus. Furthermore, pBUS1-P cap -HC and pTSSCm-P cap offer the potential to generate C-terminal RGS-His 6 translational fusions of cloned genes using simple molecular manipulation. BcgI-induced DNA excision followed by religation converts the TGA stop codon of the MCS into a TGC codon and links the rgs-his 6 codons to the 3= end of the target gene. The generation of the rgs-his 6 codon-fusion, gene expression, and protein purification were demonstrated in both S. aureus and E. coli using the macrolide-lincosamide-streptogramin B resistance gene erm(44) inserted downstream of P cap . The new His tag expression system represents a helpful tool for the direct analysis of target gene function in staphylococcal cells. Staphylococci are commensals of human and animal skin. In some species, particularly in Staphylococcus aureus, community-and hospital-associated clones have acquired a multitude of virulence and antibiotic resistance mechanisms, representing a serious public health risk (1, 2). An increased understanding of the resistance and pathogenicity mechanisms of staphylococci is needed. Therefore, molecular tools for genetic analysis, including gene disruption and ectopic expression, have been developed (3). S. aureus-Escherichia coli shuttle vectors have been constructed using restriction fragments derived from small, natural highcopy-number S. aureus plasmids, such as pUB110, pT181, pE194, and pC194 (4-7). These plasmids propagate through rolling-circle replication (RCR) in staphylococci, requiring the replication initiator protein (Rep) and the double-strand origin (dso) to initiate leading-strand synthesis as well as the single-strand origin (sso) for subsequent efficient lagging-strand synthesis (8). The absence of a functional sso sequence in RCR plasmids has been associated with single-stranded DNA (ssDNA) accumulation, low copy numbers, and plasmid instability (9-11). While some sso sequences support replication only in the native host, other sequences are functional in a number of ...

Characterization of four β-glucosidases acting on isoflavone-glycosides from Bifidobacterium pseudocatenulatum IPLA 36007

“…In this work, the promoter of the erythromycin resistance gene of pE194 from S. aureus and the ρ-independent terminator of the rrnB operon from E. coli were used as signals for heterologous expression of the glucosidase genes. These signals have already been shown to be recognized by Gram-positive and Gram-negative bacteria (Schwendener and Perreten, 2015;Horinouchi and Weisblum, 1982).…”

Staphylococcus sciuriC2865 from a distinct subspecies cluster as reservoir of the novel transferable trimethoprim resistance gene,dfrE,and adaptation driving mobile elements

“…To address functionality of the candidate TMP resistance gene and for potential synergistic activity by its immediate 3'-end thymidylate synthase gene (thy), three different dfrE containing regions were amplified and cloned into pBUS-Pcap-HC (constitutive promoter) or pBUS-HC vectors using the Gibson assembly workflow: (i) dfrE gene alone, (ii) dfrE gene plus flanking regions, and (iii) dfrE gene, thy gene and flanking regions (Table S2; S1 Data) [50]. Constructs of interest were transformed into Escherichia coli DH5α and subsequently into S. aureus RN4220 (S1 Data).…”

Comparison of the stability of eGFP displayed on the Bacillus subtilis spore surface using CotB and C-terminally truncated CotB proteins as an anchoring motif under extreme conditions

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