Surface signaling in transcriptional regulation of the ferric citrate transport system of Escherichia coli: mutational analysis of the alternative sigma factor FecI supports its essential role in fec transport gene transcription.
Ferric citrate induces transcription of the ferric citrate transport genes (fec) in escherichia coli by binding to the outer membrane receptor protein FecA without entering the cell. The signal elicited by ferric citrate crosses the outer membrane via TonB, ExbB, and ExbD. FecR transmits the signal across the cytoplasmic membrane and activates FecI located in the cytoplasm. FecI belongs to a subgroup of sigma factors that respond to extracytoplasmic stimuli. Chromosomal insertion and deletion mutations were generated in fecI; the resulting mutants were totally devoid of FecA production and fecB-lacZ expression. Iron starvation did not derepress fec transport gene transcription in fecI mutants. Missense point mutations were generated in the predicted helix-turn-helix motif of FecI to examine its role in transcription initiation. Replacement of glutamate by alanine (E141A) at the third position in the first helix reduced the residual activity of FecI in the absence of ferric citrate to 30% of the wild-type level, but induced fec transcription almost normally n the presence of ferric citrate. Mutant FecI(K145E) displayed 156% of the activity of wild-type FecI in the absence of ferric citrate and conferred full induction by ferric citrate. Mutant FecI(K155E), which has a mutation in the second helix, showed 9% of the wild-type activity in the presence of ferric citrate and 78% in the absence of ferric citrate. The reduced activity of FecI(K155E) was also shown in vitro by DNA binding assays with cell lysates; in gel retardation experiments FecI(K155E) reduced the electrophoretic mobility of fecA promoter-containing DNA less than did wild-type FecI. fecI is not autoregulated, as demonstrated by the lack of FecI-induced fecI-lacZ expression in vivo and by the lack of specific fecI transcription in vitro. Instead, formation of fecI mRNA requires sigma 70. We conclude that transcription of the fec transport genes is regulated by FecI, which responds to ferric citrate via FecR. fecI and fecR co-transcription is inhibited by the iron-loaded Fur repressor, which then results in a low level of transcription of the fec transport genes.
Ochs
M
M
Mikrobiologie II, Universität Tübingen, Germany.
Angerer
A
A
Enz
S
S
Braun
V
V
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000502:physiology; D001426:Bacterial Proteins / Q000502:physiology; D001483:Base Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000096:biosynthesis; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005290:Ferric Compounds / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D019077:Helix-Turn-Helix Motifs / Q000235:genetics; D006706:Homeostasis; D007501:Iron / Q000502:physiology; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D011485:Protein Binding; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012097:Repressor Proteins / Q000502:physiology; D012808:Sigma Factor / Q000235:genetics* / Q000502:physiology; D015398:Signal Transduction / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics*
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C028589:FecA protein, E coli; C490539:FecI protein, E coli; C093541:FecR protein, E coli; D005290:Ferric Compounds; D026901:Membrane Transport Proteins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D012808:Sigma Factor; C068477:ferric uptake regulating proteins, bacterial; C025314:ferric citrate; D007501:Iron; D012321:DNA-Directed RNA Polymerases