Functional Determinants of the Escherichia coli fis Promoter: Roles of −35, −10, and Transcription Initiation Regions in the Response to Stringent Control and Growth Phase-Dependent Regulation

Fis is a small nucleoid-associated protein found in several enteric bacteria, including Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, Serratia marcescens, Erwinia carotovora, Yersinia pestis, Proteus vulgaris, and three nonenteric bacteria (6, 22, 27, 37). Its gene, located at 73.4 min on the E. coli chromosome, is part of a two-gene operon transcribed from a single promoter (4, 36). Between the promoter and fis lies a well-conserved open reading frame (ORF1), whose function has not been described. Fis, however, has been shown to be involved in a variety of cellular processes. For example, it stimulates certain site-specific DNA recombination reactions (2, 3, 16, 23, 26, 47), stimulates stable RNA transcription (34, 42), regulates initiation of DNA replication at oriC (11, 14, 51), and modulates DNA topology (44). Fis exhibits a distinct expression pattern (4, 35, 36, 47). When stationary-phase cells are batch cultured in rich medium such as Luria-Bertani (LB) medium, Fis protein levels rapidly increase from less than 250 to over 25,000 dimers per cell in the early logarithmic growth phase. Protein levels then decrease continually to less than 1% peak levels by early stationary phase (4). Transcription control is most likely the primary determinant of this peculiar expression pattern. mRNA and protein expression patterns are similar, and mRNA decay rates do not contribute to this pattern (4, 40). However, little is known about the regions of the fis promoter (fis P) that mediate this unique expression pattern. Several observations demonstrated that fis P is controlled by negative autoregulation. Maximal fis mRNA levels were found to be sixfold higher in fis mutant cells than in otherwise isogenic fis+ cells (4). Likewise, β-galactosidase activities from fis P fused to lacZ are over fourfold higher in fis mutant cells than in fis+ cells (36). Six Fis binding sites have been identified in the promoter region, and binding of RNA polymerase to fis P in vitro was prevented if Fis was also present (4). So far, Fis sites I (centered at +24) and II (centered at −44) have been shown to be critical for autoregulation, with site II playing the more important role (36, 40). On the other hand, fis P transcription is stimulated in vivo three- to fourfold by integration host factor (IHF) when bound to a site centered at −116 (40). However, while Fis and IHF have opposing effects on the magnitude of fis P expression, neither is responsible for the growth phase-dependent regulation (4, 40). In fact, this expression pattern can be generated by the fis P sequences from −38 to +5, which lack the IHF and Fis binding sites (36, 40). Thus, this 43-bp region must contain the minimal elements required for basal transcription activity, as well as growth phase-dependent regulation. Stringent control was also shown to negatively regulate expression from minimal fis P (36). When cells were starved for isoleucine, mRNA synthesized from fis P quickly subsided but was restored upon addition of chloramphenicol. A sequence of seven consecutive G · C base pairs starting 2 bp downstream from the putative −10 region is a likely candidate for a discriminator, which has been implicated in the stringent control of many promoters (9). Replacement of these sequences with the corresponding region from the β-lactamase (bla) promoter resulted in loss of both stringent and growth phase-dependent control (36). This was taken as evidence that the GC-rich motif was required for both regulatory processes and implied that growth phase-dependent regulation could be explained in terms of a sensitivity to variations in intracellular ppGpp levels. However, fis P maintains its unusual expression pattern in a relA spoT strain, indicating that growth phase-dependent regulation is not dependent on ppGpp levels (4). Much uncertainty remains regarding the function and regulation of fis P, and this warrants a more detailed characterization of the nucleotide sequences comprising this promoter. Thus, we generated a comprehensive collection of mutations within the fis P region and tested their effects on transcription in vivo, stringent control, and growth phase-dependent regulation. We show that three DNA elements that affect growth phase-dependent regulation include the suboptimal −10 and −35 regions and the nucleotide at or near the transcriptional start site. We also found that single-point mutations in the GC-rich sequence in fis P do not affect the stringent control, but changing four sequential G · C base pairs to A · T base pairs significantly reduces susceptibility to this regulation. Moreover, we found that promoter mutants altered in growth phase-dependent regulation are still subject to stringent control and vice versa. Finally, mutations obtained in this work provided a functional basis for a reliable identification of the fis P recognition sequences.