FKBP22, a protein expressed by Escherichia coli, possesses PPIase (peptidyl-prolyl cis-trans isomerase) activity, binds FK506 (an immunosuppressive drug), and shares homology with Legionella Mip (a virulence factor) and its related proteins. To understand the domain structure and the folding-unfolding mechanism of Mip-like proteins, we investigated a recombinant E. coli FKBP22 (His-FKBP22) as a model protein. Limited proteolysis indicated that His-FKBP22 harbors an N-terminal domain (NTD), a C-terminal domain (CTD), and a long flexible region linking the two domains. His-FKBP22, NTD(+) (NTD with the entire flexible region), and CTD(+) (CTD with a truncated flexible region) were unfolded by a two-state mechanism in the presence of urea. Urea induced the swelling of dimeric His-FKBP22 molecules at the pretransition state but dissociated it at the early transition state. In contrast, guanidine hydrochloride (GdnCl)-induced equilibrium unfolding of His-FKBP22 or NTD(+) and CTD(+) seemed to follow three-step and two-step mechanisms, respectively. Interestingly, the intermediate formed during the unfolding of His-FKBP22 with GdnCl was not a molten globule but was thought to be composed of the partially unfolded dimeric as well as various multimeric His-FKBP22 molecules. Dimeric His-FKBP22 did not dissociate gradually with increasing concentrations of GdnCl. Very low GdnCl concentrations also had little effect on the molecular dimensions of His-FKBP22. Unfolding with either denaturant was found to be reversible, as refolding of the unfolded His-FKBP22 completely, or nearly completely, restored the structure and function of the protein. Additionally, denaturation of His-FKBP22 appeared to begin at the CTD(+).

RsbW, an anti-sigma factor possessing kinase activity, is expressed by many Gram-positive bacteria including Staphylococcus aureus. To obtain clues about the domain structure and the folding-unfolding mechanism of RsbW, we have elaborately studied rRsbW, a recombinant S. aureus RsbW. Sequence analysis of the protein fragments, generated by the limited proteolysis of rRsbW, has proposed it to be a single-domain protein. The unfolding of rRsbW in the presence of GdnCl or urea was completely reversible in nature and occurred through the formation of at least two intermediates. The structure, shape, and the surface hydrophobicity of no intermediate completely matches with those of other intermediates or the native rRsbW. Interestingly, one of the intermediates, formed in the presence of less GdnCl concentrations, has a molten globule-like structure. Conversely, all of the intermediates, like native rRsbW, exist as dimers in aqueous solution. The putative molten globule and the urea-generated intermediates also have retained some kinase activity. Additionally, the putative ATP binding site/catalytic site of rRsbW shows higher denaturant sensitivity than the tentative dimerization region of this enzyme.

The basic regulatory elements that most temperate phages use for the establishment and maintenance of their lysogeny are the phage-encoded repressor and the cognate operator DNA [1][2][3][4][5][6][7][8][9][10][11][12]. A temperate phage generally enters into the lysogenic life cycle once its repressor inhibits the transcription of the phagespecific lytic genes from the early promoter by binding to the overlapped operator DNA. Repressors of the temperate phages, although varying greatly in size and in primary sequence level, mostly harbor a DNA binding domain and an oligomerization domain. The size and type of the operator DNAs also vary from phage to phage. Although some repressors bind to operators with dyad symmetry [1,[5][6][7][8][9] or operators with direct repeats [10], other repressors bind to asymmetric operators [2,3,[11][12][13] to establish lysogeny. Interestingly, the repressor of Vibrio cholerae phage CTX/ binds to extended operators, stopping lytic growth, as well as ensuring lysogeny of this phage [4]. Although these regulatory elements have enriched both basic and applied molecular biology enormously, they have not been cloned from most temperate phages or characterized in any depth.The temperate Staphylococcus aureus phage /11 [14] harbors the cI and cro genes in a divergent orientation to that in lambdoid phages [1,8]. The sequence of the immunity region of /11, however, differs significantly from those of the lambdoid phages and other temper- The repressor protein and cognate operator DNA of any temperate Staphylococcus aureus phage have not been investigated in depth, despite having the potential to enrich the molecular biology of the staphylococcal system. In the present study, using the extremely pure repressor of temperate Staphylococcus aureus phage /11 (CI), we demonstrate that CI is composed of a-helix and b-sheet to a substantial extent at room temperature, possesses two domains, unfolds at temperatures above 39°C and binds to two sites in the /11 cI-cro intergenic region with variable affinity. The above CI binding sites harbor two homologous 15 bp inverted repeats (O1 and O2), which are spaced 18 bp apart. Several guanine bases located in and around O1 and O2 demonstrate interaction with CI, indicating that these 15 bp sites are used as operators for repressor binding. CI interacted with O1 and O2 in a cooperative manner and was found to bind to operator DNA as a homodimer. Interestingly, CI did not show appreciable binding to another homologous 15 bp site (O3) that was located in the same primary immunity region as O1 and O2. Taken together, these results suggest that /11 CI and the /11 CI-operator complex resemble significantly those of the lambdoid phages at the structural level. The mode of action of /11 CI, however, may be distinct from that of the repressor proteins of k and related phages.Abbreviations CI, repressor of temperate Staphylococcus aureus phage /11; CTD, C-terminal domain; DMS, dimethyl sulfate; DTNB, 5,5¢-dithiobis-(2-nitrobenzoic acid); NTD, N-terminal domain.

Proteins expressed by the bacterial cold shock genes are highly conserved at sequence level and perform various biological functions in both the cold-stressed and normal cells. To study the effects of various agents on the cold shock genes of Staphylococcus aureus, we have cloned the upstream region of cspC from S. aureus Newman and found that the above region possesses appreciable promoter (P(c)) activity even at 37 degrees C. A reporter S. aureus strain CHANDA2, constructed by inserting the P(c)-lacZ transcriptional fusion into S. aureus RN4220 genome, was found to express very low level of beta-galactosidase after cold shock, indicating that low temperature induces P(c) very weakly. Interestingly, transcription from P(c ) was induced very strongly by several antibiotics, hydrogen peroxide and arsenate salt. Cold shock proteins expressed by S. aureus are highly identical at sequence level and bear single-strand nucleic acid binding motifs. A 16 nt downstream box and a 13 nt upstream box were identified at the downstream of initiation codon and at the upstream of ribosome binding site of csp transcripts. Their roles in S. aureus cold shock gene expression have been discussed elaborately.

Previously, the repressor protein of mycobacteriophage L1 bound to two operator DNAs with dissimilar affinity. Surprisingly, the putative operator consensus sequence, 5'GGTGGa/cTGTCAAG, lacks the dyad symmetry reported for the repressor binding operators of lambda and related phages. To gain insight into the structure of the L1 repressor-asymmetric operator DNA complex, we have performed various in vitro experiments. A dimethyl sulfate protection assay revealed that five guanine bases, mostly distributed in the two adjacent major grooves of the 13 bp operator DNA helix, participate in repressor binding. Hydroxyl radical footprinting demonstrated that interaction between the repressor and operator DNA is asymmetric in nature and occurs primarily through one face of the DNA helix. Genetic studies not only confirmed the results of the dimethyl sulfate protection assay but also indicated that other bases in the 13 bp operator DNA are critical for repressor binding. Interestingly, repressor that weakly induced bending in the asymmetric operator DNA interacted with this operator as a monomer. The tertiary structure of the L1 repressor-operator DNA complex therefore appears to be distinct from those of the lambdoid phages even though the number of repressor molecules per operator site closely matched that of the lambda phage system.