Interdomain hybrid Tet proteins confer tetracycline resistance only when they are derived from closely related members of the tet gene family.
Inner membrane Tet proteins encoded by tet genes in gram-negative bacteria mediate resistance to tetracycline (Tcr) by directing its export. Total sequences for class A, B, and C tet genes demonstrate that their products have a common ancestor, with Tet(A) and Tet(C) being more closely related (78% identical) than either is to Tet(B) (45% identical). The N- and C-terminal halves of Tet(B) and Tet(C) appear to comprise separate domains, and trans-complementation observed between tetracycline sensitive mutants in either domain of Tet(B) suggests separate but interactive functions for these domains. In this present study, interdomain hybrid genes were constructed to express hybrid tet products whose N- and C-terminal halves were derived from different family members [Tet(A/C), Tet(B/C), and Tet(C/B)]. Tet(A/C) specified a level of Tcr comparable to wild-type Tet(C) and 60% that of Tet(A), indicating that domains from these closely related tet products can function in cis. Although neither Tet(B/C) nor Tet(C/B) hybrids conferred significant Tcr, cells producing both of these types of hybrid proteins expressed substantial Tcr, indicating that productive interactions can occur in trans between Tet(B/C) and Tet(C/B). Taken together, these results suggest that highly specific interactions between the N- and C-terminal domains are necessary for Tcr and do not occur in individual hybrids derived from the more distant relatives, Tet(B) and Tet(C). This requirement for specific interactions suggests that N- and C-terminal domains have coevolved in each member of the Tet family.
Rubin
R A
RA
Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
Levy
S B
SB
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D005838:Genotype; D008969:Molecular Sequence Data; D005810:Multigene Family; D010957:Plasmids; D011815:R Factors; D012097:Repressor Proteins / Q000235:genetics* / Q000302:isolation & purification; D015183:Restriction Mapping; D013753:Tetracycline Resistance / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*
D001426:Bacterial Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C034811:tetracycline resistance-encoding transposon repressor protein