The repressor of phage 434 binds to six operator sites on the phage chromosome. A comparison of the sequences of these 14-base-pair (bp) operator sites reveals a striking pattern: at five of the six sites, the symmetrically arrayed outer eight base pairs (four in each half-site) are identical and the remaining site differs at only one position (Fig. 1b). In contrast, the sequences of the inner four base pairs are highly variable. Crystallographic analysis of the repressor-operator complex shows that at each half-site, the 'recognition alpha-helix' of the repressor is positioned in the major groove such that it could contact the outermost five base pairs, but not the innermost two (Fig. 1a). We show in this paper that the sequence of the central base pairs of the operator (two in each half-site) have a significant role in determining operator affinity for repressor, despite the evidence presented here and in the accompanying paper that these base pairs are not contacted by repressor. We also show that these central base pairs influence operator affinity for Cro, a second gene regulatory protein encoded by phage 434. We discuss the likely structural basis for this evidently indirect, but sequence-dependent, effect of the central base pairs of the operator on its affinity for the two regulatory proteins.

Bacterially derived exotoxins kill eukaryotic cells by inactivating factors and/or pathways that are universally conserved among eukaryotic organisms. The genes that encode these exotoxins are commonly found in bacterial viruses (bacteriophages). In the context of mammals, these toxins cause diseases ranging from cholera to diphtheria to enterohemorrhagic diarrhea. Phage-carried exotoxin genes are widespread in the environment and are found with unexpectedly high frequency in regions lacking the presumed mammalian "targets," suggesting that mammals are not the primary targets of these exotoxins. We suggest that such exotoxins may have evolved for the purpose of bacterial antipredator defense. We show here that Tetrahymena thermophila, a bacterivorous predator, is killed when cocultured with bacteria bearing a Shiga toxin (Stx)-encoding temperate bacteriophage. In cocultures with Tetrahymena, the Stx-encoding bacteria display a growth advantage over those that do not produce Stx. Tetrahymena is also killed by purified Stx. Disruption of the gene encoding the StxB subunit or addition of an excess of the nontoxic StxB subunit substantially reduced Stx holotoxin toxicity, suggesting that this subunit mediates intake and/or trafficking of Stx by Tetrahymena. Bacterially mediated Tetrahymena killing was blocked by mutations that prevented the bacterial SOS response (recA mutations) or by enzymes that breakdown H 2 O 2 (catalase), suggesting that the production of H 2 O 2 by Tetrahymena signals its presence to the bacteria, leading to bacteriophage induction and production of Stx.

The P22 c2 repressor protein (P22R) binds to DNA sequence-specifically and helps to direct the temperate lambdoid bacteriophage P22 to the lysogenic developmental pathway. We describe the 1.6 A X-ray structure of the N-terminal domain (NTD) of P22R in a complex with a DNA fragment containing the synthetic operator sequence [d(ATTTAAGATATCTTAAAT)]2. This operator has an A-T base pair at position 9L and a T-A base pair at position 9R and is termed DNA9T. Direct readout: nondirectional van der Waals interactions between protein and DNA appear to confer sequence-specificity. The structure of the P22R NTD-DNA9T complex suggests that sequence-specificity arises substantially from lock-and-key interaction of a valine with a complementary binding cleft on the major groove surface of DNA9T. The cleft is formed by four methyl groups on sequential base pairs of 5'-TTAA-3'. The valine cleft is intrinsic to the DNA sequence and does not arise from protein-induced DNA conformational changes. Protein-DNA hydrogen bonding plays a secondary role in specificity. Indirect readout: it is known that the noncontacted bases in the center of the complex are important determinants of affinity. The protein induces a transition of the noncontacted region from B-DNA to B'-DNA. The B' state is characterized by a narrow minor groove and a zigzag spine of hydration. The free energy of the transition from B- to B'-DNA is known to depend on the sequence. Thus, the observed DNA conformation and hydration allows for the formulation of a predictive model of the indirect readout phenomenon.

The affinity of the Eseherichia coli phage 434 operator for phage 434 repressor is affected by changes in the sequence of the noncontacted base pairs near the operator's center. The results presented here show that base composition near the center ofthe operator affects the operator's affinity for repressor by altering the ease with which the operator can be overtwisted into the proper configuration for complex formation. We show that both DNA flexibility and repressor flexibility influence the strength of the repressor-operator interaction: an operator with a single-strand nick at its center has a higher affinity for repressor than does the intact operator: and a repressor bearing a mutation that results in a relaxed dimer interaction is less sensitive than is wild type to changes in the flexibility of the operator. We show that the effect of noncontacted base pairs on operator affinity is independent of the slight overall bend of the operator seen in the repressoroperator complex. Central sequence effects on affinity for repressor are independent ofthe identity ofadjacent base pairs, suggesting that the structure of the individual base pairs, not interactions between them, are responsible for the different torsional rigidities of different operators.Our current picture of how the Escherichia coli phage 434 repressor recognizes its DNA operator is summarized in Fig. 1. In the repressor-operator complex, one a-helix of the repressor dimer lies in each half-site of the operator. Each of these helices is positioned in the major groove so that its side chains can contact the outermost 5 base pairs in one operator half-site (1). Biochemical and x-ray crystallographic studies suggest that neither this "recognition helix" nor any other part of the repressor contacts the innermost 2 base pairs of each half-site (1, 2). Nevertheless, the operator's affinity for repressor is determined, in part, by the composition of these base pairs: operators bearing APT or T'A base pairs at operator positions 6-9 bind the repressor more strongly than do operators bearing G-C or C-G base pairs at these positions (2). We have proposed that the base composition near the center of the operator affects its affinity for repressor by altering the ease with which operator DNA can be deformed into the optimal configuration for complex formation (2). Crystallographic analysis of the 434 repressor-operator complex shows that when 434 operator binds to 434 repressor, the DNA near the center of the operator is slightly overtwisted, so that the minor groove in this region is compressed (Fig. 1) (1). Evidently this DNA deformation is required to align the two operator half-sites so that each monomer of the bound repressor dimer can make optimal contacts with each half-site.In this paper we explore more directly the role of both DNA flexibility and repressor flexibility in determining the affinity of operator for repressor. We reasoned that introducing a single-strand break at the central phosphodiester bond of the operator would increase th...

The genes encoding Shiga toxin (stx), the major virulence factor of Shiga toxin-encoding Escherichia coli (STEC) strains, are carried on lambdoid prophages resident in all known STEC strains. The stx genes are expressed only during lytic growth of these temperate bacteriophages. We cloned the gene encoding the repressor of the Shiga toxin-encoding bacteriophage 933W and examined the DNA binding and transcriptional regulatory activities of the overexpressed, purified protein. Typical of nearly all lambdoid phage repressors, 933W repressor binds to three sites in 933W right operator (O R ). Also typical, when bound at O R , 933W repressor functions as an activator at the P RM promoter and a repressor at the P R promoter. In contrast to other lambdoid bacteriophages, 933W left operator (O L ) contains only two repressor binding sites, but the O L -bound repressor still efficiently represses P L transcription. Lambdoid prophage induction requires inactivation of the repressor's DNA binding activity. In all phages examined thus far, this inactivation requires a RecA-stimulated repressor autoproteolysis event, with cleavage occurring precisely in an Ala-Gly dipeptide sequence that is found within a "linker " region that joins the two domains of these proteins. However, 933W repressor protein contains neither an Ala-Gly nor an alternative Cys-Gly dipeptide cleavage site anywhere in its linker sequence. We show here that the autocleavage occurs at a Leu-Gly dipeptide. Thus, the specificity of the repressor autocleavage site is more variable than thought previously.Shiga toxins (stx) are the major virulence factors in enterohemorrhagic Escherichia coli infections, causing such diseases as hemorrhagic colitis, infantile diarrhea, and hemolytic uremic syndrome. In virtually all known Shiga toxin-encoding E. coli strains, the genes encoding Shiga toxins are carried on lambdoid prophages (8,19,31,34,35,45) as part of an operon whose activity is ultimately regulated by the bacteriophage repressor (32,33,46,47).Lambdoid phage genomes contain two operator regions, O L and O R , each of which includes promoters whose expression is controlled by the binding of the bacteriophage repressor to multiple binding sites found in each operator region. Efficient functioning of the genetic switch between lysis and lysogeny depends on the ability of the repressor to bind with different affinities to each of the individual sites within O R and O L (40). The repressor directs the establishment and maintenance of the lysogenic state by simultaneously repressing transcription of the genes needed for lytic phage growth and activating transcription of its own gene, the only gene needed for maintenance of the lysogenic state (40).The E. coli O157:H7 strain EDL933 is considered to be the reference strain for disease-causing O157:H7 isolates. Incubating this strain with agents that trigger induction of resident prophages causes this strain to express the disease-causing stx2 gene product and to produce a lambdoid bacteriophage (38). Sequence analysi...

In this review, we highlight recent work that has increased our understanding of the production and distribution of Shiga toxin in the environment. Specifically, we review studies that offer an expanded view of environmental reservoirs for Shiga toxin producing microbes in terrestrial and aquatic ecosystems. We then relate the abundance of Shiga toxin in the environment to work that demonstrates that the genetic mechanisms underlying the production of Shiga toxin genes are modified and embellished beyond the classical microbial gene regulatory paradigms in a manner that apparently “fine tunes” the trigger to modulate the amount of toxin produced. Last, we highlight several recent studies examining microbe/protist interactions that postulate an answer to the outstanding question of why microbes might harbor and express Shiga toxin genes in the environment.

The bacteriophage 434 repressor regulates gene expression by binding with differing affinities to the six operator sites on the phage chromosome. The symmetrically arrayed outer eight base pairs (four in each half-site) of these 14-base-pair operators are highly conserved but the middle four bases are divergent. Although these four base pairs are not in contact with repressor, operators with A.T or T.A base pairs at these positions bind repressor more strongly than those bearing C.G or G.C, suggesting that these bases are important for the repressor's ability to discriminate between operators. There is evidence that the central base pairs influence operator function by constraining the twisting and/or bending of DNA. Here we show that there is a relationship between the intrinsic twist of an operator, as determined by the sequence of its central bases, and its affinity for repressor; an operator with a lower affinity is undertwisted relative to an operator with higher affinity. In complex with repressor, the twist of both high- and low-affinity operators is the same. These results indicate that the intrinsic twist of DNA and its twisting flexibility both affect the affinity of 434 operator for repressor.

Phage-encoded Shiga toxin (Stx) acts as a bacterial defence against the eukaryotic predator Tetrahymena. To function as an effective bacterial anti-predator defence, Stx must kill a broad spectrum of predators. Consistent with that assertion, we show here that bacterially encoded Stx efficiently kills the bacteriovore Acanthamoeba castellanii in co-culture. We also show that, in addition to Stx, the phage-encoded exotoxin, diphtheria toxin (Dtx) expressed by Corynebacterium diphtheriae also can function as part of an anti-predator strategy; it kills Acanthamoeba in co-culture. Interestingly, only exotoxins produced by bacteria internalized by the Acanthamoeba predator are cytolethal; the presence of purified Dtx or Stx in culture medium has no effect on predator viability. This finding is consistent with our results indicating that intoxication of Acanthamoeba by these exotoxins does not require a receptor. Thus bacteria, in the disguise of a food source, function as a 'Trojan Horse', carrying genes encoding an exotoxin into target organisms. This 'Trojan Horse' mechanism of exotoxin delivery into predator cells allows intoxication of predators that lack a cell surface receptor for the particular toxin, allowing bacteria-bearing exotoxins to kill a broader spectrum of predators, increasing the fitness of the otherwise 'defenceless' prey bacteria.