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The dimeric repressor MecI regulates the mecA gene that encodes the penicillin-binding protein PBP-2a in methicillin-resistant Staphylococcus aureus (MRSA). MecI is similar to BlaI, the repressor for the blaZ gene of beta-lactamase. MecI and BlaI can bind to both operator DNA sequences. The crystal structure of MecI in complex with the 32 base-pair cognate DNA of mec was determined to 3.8 A resolution. MecI is a homodimer and each monomer consists of a compact N-terminal winged-helix domain, which binds to DNA, and a loosely packed C-terminal helical domain, which intertwines with its counter-monomer. The crystal contains horizontal layers of virtual DNA double helices extending in three directions, which are separated by perpendicular DNA segments. Each DNA segment is bound to two MecI dimers. Similar to the BlaI-mec complex, but unlike the MecI-bla complex, the MecI repressors bind to both sides of the mec DNA dyad that contains four conserved sequences of TACA/TGTA. The results confirm the up-and-down binding to the mec operator, which may account for cooperative effect of the repressor.

β-Lactam antibiotics are becoming less effective therapy for treating staphylococcal infections as resistance to them increases. Resistance is mediated by a β-lactamase (encoded by blaZ) that hydrolyzes penicillins (4) and an alternate penicillin binding protein target (PBP2a, encoded by mecA) to which β-lactam antibiotics bind poorly (17). The transcription of blaZ and mecA is corepressed by related regulators (encoded by blaI and mecI). Both BlaI and MecI bind to palindromic promoter-operator sequences, presumably as dimers, and can interchangeably repress the transcription of either target gene (8, 26, 36). Signal transduction through either of two transmembrane inducers, BlaR1 or MecR1, leads to proteolytic autocleavage of the cytoplasmic domains of these molecules. Autocleavage of the signal transducer is followed by cleavage of the cognate repressor (BlaI or MecI, respectively) and induction of the transcription of blaZ or mecA (42). The exact mechanism of cleavage is unclear at present. The current hypothesis (1, 42) is that β-lactam binding to the extramembrane sensor of BlaR1 or MecR1 triggers autocleavage of its cytoplasmic domain at a single site, leaving the putative cytoplasmic protease tethered to the membrane. Sequence-specific repressor cleavage then ensues, either through direct interaction with the activated BlaR1 or MecR1 cytoplasmic protease domain or, more likely, through interaction with an intermediary molecule (BlaR2 or MecR2). Candidate R2 molecules have not yet been identified. Genes for the signal transducers (blaR1 and mecR1) are part of two-gene operons that also contain genes for each repressor (blaI or mecI). Each regulon is divergently transcribed from its regulated gene, and promoter-operators for each resistance gene and its regulator overlap on opposite DNA strands. Thus, not only does binding of BlaI or MecI repress transcription of blaZ or mecA, but the regulatory operon is autorepressed as well. BlaI or MecI dimers are believed to bind specifically to two regions of dyad symmetry in the bla operator, the Z and R1 dyads located in the blaZ-blaR1 intergenic region (8). Each dyad contains an 18-bp palindrome, and they are separated from each other by a 13-bp linker (Fig. ​(Fig.1A).1A). These two repressors also bind the mec operator, which consists of a single 30-bp palindrome with two 15-bp half-sites (36). Within each 15-bp half-site of the mec operator is 12 bp of dyad symmetry (Fig. ​(Fig.1B).1B). Fingerprint studies show that each repressor binds to both the bla and mec operators and that each repressor protects the same sequences (8, 26, 36). The molecular masses of the monomers are about 14 kDa for both BlaI and MecI, with 61% of the residues identical, as shown in Fig. ​Fig.2A.2A. The high degree of sequence homology suggests that the two structures should have the same fold. Extensive characterization of the activities of the repressors also suggests that the structures have two domains, one for dimerization and another for DNA binding. However, it is not clear how the structures of BlaI and MecI change to allow binding to two different targets that vary markedly in the number and spacing of contacted nucleotides. FIG. 1. bla and mec DNA promoter-operator sequences (7, 36). (A) The bla operator consists of the R1 dyad and Z dyad (shaded). Each dyad contains an 18-bp palindrome, and they are separated from each other by a 13-bp linker. (B) The mec operator (shaded) consists ... FIG. 2. Sequence alignment, structure, and electron density maps of the BlaI-DNA complex. (A) Sequence alignment of BlaI and MecI repressor proteins. The proteins used in the alignment are as follows: BlaI, S. aureus N395 ({"type":"entrez-protein","attrs":{"text":"Q9AC78","term_id":"81536386","term_text":"Q9AC78"}} ... The three-dimensional structure of the N-terminal DNA binding domain of BlaI from Bacillus licheniformis was determined from heteronuclear nuclear magnetic resonance (NMR) spectroscopy (27). It is composed of three α-helices and three β-strands typical of the winged helix protein family, and helix α3 makes specific contacts with the TACA motif of the target DNA. The crystal structure of MecI (Protein Data Bank [PDB] code 1OKR) determined recently by Garcia-Castellanos et al. (14) shows a dimer of two independent N-terminal winged helix domains and two intertwined C-terminal dimerization domains of spiral helical architecture. Further elucidation of interactions between the MecI dimer and the blaZ operator DNA (PDB code 1SAX) confirmed the involvement of helix α3 of the protein and the GTAXT motif of the DNA for specific recognition (13) (Fig. ​(Fig.2B).2B). However, Garcia-Castellanos et al. (14) did not investigate the binding of MecI to its homologous target, the mec promoter-operator, and did not address the question of how related repressors could bind to two different targets. In this paper we report the crystal structures of BlaI from Staphylococcus aureus, both in free form and in complex with 32 bp of DNA of the mec operator, determined to 2.0- and 2.7-A resolutions, respectively. Comparison of the BlaI and MecI structures suggests that the proteins may assume different quaternary conformations, a closed form and an open form. Based on the crystal structures, as well as data from DNase protection experiments, we propose a model for binding of the repressors to both bla and mec operators.

Among 23 patients carrying methicillin-resistant Staphylococcus aureus (MRSA) in their anterior nares, 6 (26%) also carried methicillin-susceptible S. aureus (MSSA) as less prevalent flora. In 4 of the 6 patients, the MSSA was unrelated to prevalent MRSA, as determined by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and staphylococcal protein A (spa) typing. However, in two patients, the strains were identical except for the absence of spontaneous staphylococcal cassette chromosome mec (SCC mec ). We consider this evidence of spontaneous SCC mec excision in vivo .

The exbBD genes of Pseudomonas aeruginosa PAO were cloned by complementation of the growth defect of an Escherichia coli exbB tolQ double mutant on iron-restricted medium. Nucleotide sequence analysis confirmed that these genes are contiguous and preceded by a second tonB gene in this organism, which we have designated tonB2. lacZ promoter fusions confirmed that expression of the tonB2-exbB-exbD genes is increased under conditions of iron limitation. Deletions within any of these genes, in contrast to deletions in the first tonB gene, tonB1, did not adversely affect growth on iron-restricted medium. On the other hand, tonB1 tonB2 double mutants were more compromised as regards growth in an iron-restricted medium than a tonB1 deletion, indicating that TonB2 could partially replace TonB1 in its role in iron acquisition. TonB1 but not TonB2 deletion strains were also compromised as regards the utilization of hemin or hemoglobin as sole iron sources, indicating that heme transport requires TonB1.

The region upstream of the multiple antibiotic resistance efflux operon mexA-mexB-oprM in Pseudomonas aeruginosa was sequenced, and a gene, mexR, was identified. The predicted MexR product contains 147 amino acids with a molecular mass of 16,964 Da, which is consistent with the observed size of the overexpressed mexR gene product. MexR was homologous to MarR, the repressor of MarA-dependent multidrug resistance in Escherichia coli, and other repressors of the MarR family. A mexR knockout mutant showed a twofold increase in expression of both plasmid-borne and chromosomal mexA-reporter gene fusions compared with the MexR+ parent strain, indicating that the mexR gene product negatively regulates expression of the mexA-mexB-oprM operon. Furthermore, the cloned mexR gene product reduced expression of a plasmid-borne mexA-lacZ fusion in E. coli, indicating that MexR represses mexA-mexB-oprM expression directly. Consistent with the increased expression of the efflux operon in the mexR mutant, the mutant showed an increase (relative to its MexR+ parent) in resistance to several antimicrobial agents. Expression of a mexR-lacZ fusion increased threefold in a mexR knockout mutant, indicating that mexR is negatively autoregulated. OCR1, a nalB multidrug-resistant mutant which overproduces OprM, exhibited a greater than sevenfold increase in expression of a chromosomal mexA-phoA fusion compared with its parent. Introduction of a mexR knockout mutation in strain OCR1 eliminated this increase in efflux gene expression and, as expected, increased the susceptibility of the strain to a variety of antibiotics. The nucleotide sequences of the mexR genes of OCR1 and its parental strain revealed a single base substitution in the former which would cause a predicted substitution of Trp for Arg at position 69 of its mexR product. These data suggest that MexR possesses both repressor and activator function in vivo, the activator form being favored in nalB multidrug-resistant strains.

OprJ, overproduced in nfxB multidrug-resistant strains of Pseudomonas aeruginosa, and OprK, overproduced in the multidrug-resistant strain K385, were demonstrated to be immunologically cross-reactive using an OprJ-specific monoclonal antibody. Treatment of the purified proteins with trypsin or chymotrypsin yielded virtually indistinguishable digestion patterns, and the N-terminal sequence of two trypsin fragments was identical for both proteins, indicating that OprJ and OprK share identity. The N-terminal amino acid sequences were used to facilitate cloning of the oprJ gene on a 5kbp Kpnl fragment and a 10 kbp BamHl fragment. Nucleotide sequencing of portions of these fragments revealed that oprJ was the terminal gene in a putative three-gene operon, mexC-mexD-oprJ. The predicted mexC-mexD-oprJ gene products exhibit homology to the MexA-MexB-OprM components of the multidrug-resistance efflux pump of P. aeruginosa (43-46% identity). Consistent with an implied role for mexC-mexD-oprJ in drug efflux, the mexC-mexD-oprJ-hyperexpressing strain K385 showed reduced accumulation of a variety of antibiotics as compared with its parent strain, and this drug 'exclusion' was abrogated by energy inhibitors. The mexC and oprJ products are putative lipoproteins of a molecular mass of 40,707 and 51,742 Da, respectively, while mexD was predicted to encode a protein of 111 936 Da. Sequencing upstream of mexC revealed the presence of the nfxB gene transcribed divergently from the efflux genes. Overproduction of OprJ and the attendant multiple-antibiotic resistance of strain K385 was shown to result from a point mutation in nfxB, resulting in a H87-->R change in the predicted NfxB polypeptide. OprJ overproduction and multidrug resistance in K385 was reversed by the cloned nfxB gene, suggesting that nfxB encodes a repressor of mexC-mexD-oprJ expression. Consistent with this, the cloned nfxB gene repressed synthesis of a mexC-lacZ fusion in Escherichia coli. nfxB also repressed expression of a nfxB-lacZ fusion, indicating that NfxB negatively regulates its own expression. These data indicate that the multidrug resistance of nfxB strains is due to overexpression of an efflux operon, mexC-mexD-oprJ, encoding components of a second efflux pump in P. aeruginosa.

Loss of tonB1 adversely affects iron acquisition and intrinsic multidrug resistance in Pseudomonas aeruginosa . Several mutations in tonB1 compromised the protein's contribution to both processes, although TonB1 derivatives altered in residues C35, Q268, R287, Q292, R300, and R304 were compromised vis-à-vis their contribution to drug resistance only.

Energy-dependent receptor-mediated ligand uptake across the outer membrane of gram-negative bacteria is dependent upon the function of the product of the tonB gene (5). This apparently dimeric (14) proline-rich protein is, in Escherichia coli, anchored to the cytoplasmic membrane via its N terminus and extends into the periplasm (23, 61, 62), where it interacts with receptor proteins in the outer membrane (6, 22, 26, 66, 68). This disposition of the protein reflects its apparent role in coupling the energized state of the cytoplasmic membrane to the outer membrane receptors (52). TonB interaction with receptors is promoted by ligand binding to these receptors (53, 54), suggesting that TonB is specifically recruited by ligand-loaded receptors. The ligand-loaded status is apparently reflected by changes in receptor conformation upon ligand binding (7, 19, 32, 46, 47, 48, 55), including a change in the N-terminal TonB box region (see below) implicated in TonB association (51). Previous models of TonB-dependent receptors described them as gated porins whose activities were modulated by the action of the TonB protein (38, 63), although the recently determined crystal structures of two of these, FhuA (19, 47) and FepA (12), indicate that the channels are not gated. Rather, a cork domain formed by the N terminus plugs the channel from the periplasmic side (12, 19, 47). These receptors, which include receptors for ferric siderophores (10), vitamin B12 (33), heme and hemin (73), and transferrin (10, 21), show conserved regions of homology (50), one of which, the so-called TonB box (67), is proposed to interact directly with TonB (6, 26). Such interaction appears to be mediated by the C-terminal region of the TonB protein (31, 40) at or near residue Glu160 (6, 26). Indeed, a recent cross-linking study confirmed the interaction of TonB and the TonB box region of the TonB-dependent vitamin B12 receptor, BtuB, mediated by residues in TonB surrounding GlnQ160 (13). Still, derivatives of another TonB-dependent receptor, FhuA, lacking the TonB box and, indeed, most of the N-terminal cork domain, was still TonB dependent (8, 39) and could interact with TonB (29), indicating that TonB also interacts with its receptors via regions other than the TonB box. Full TonB function is, in E. coli, dependent upon the products of the exbB and exbD genes (18, 24), which are localized in the cytoplasmic membrane (34, 35). The former is involved in the stabilization of TonB (1, 20, 37, 69), apparently via an interaction with the TonB N terminus (31, 36, 43, 72). ExbD also interacts with TonB (1) and ExbB (9, 20), apparently as part of a heterohexameric complex of ExbB and ExbD homotrimers (27). A recent model suggests that TonB shuttles between the outer and inner membranes, its association with the former dependent upon ExbBD and mediated by the TonB N terminus and its association with the latter mediated by its C terminus (45). This cycling between outer and inner membrane states is likely facilitated by conformational changes in TonB that occur in response to ExbB, the proton motive force, and the ligand-loaded receptor (42). The Pseudomonas aeruginosa siderophore receptors show conserved regions of homology shared by members of the family of so-called TonB-dependent receptors (2, 15, 59), and a tonB gene has been identified in this organism (60). Although it displays significant homology, e.g., to E. coli TonB, this TonB protein is distinguished by the presence of additional sequences at its N terminus, making it larger than all other examples of TonB (60). Still, disruption of this gene abrogates siderophore-mediated iron uptake (60) and heme uptake (76), a finding consistent with an involvement in iron acquisition, as for other TonB proteins. Homologues of exbB and exbD have recently been described in P. aeruginosa, linked to a second tonB gene, tonB2, in an apparent operon structure (76). Despite the obvious iron regulation of this putative operon, however, inactivation of tonB2, exbB, or exbD had no adverse effect on iron or heme acquisition in this organism (76). In the present report we assess the functional importance of the novel N-terminal extension of the P. aeruginosa TonB protein (now called TonB1 [76]), as well as the conserved C terminus, and we show that the various mutations examined differentially affect TonB1 function in E. coli versus that in P. aeruginosa.

The major outer membrane protein gene (omp1) was sequenced for each of six Chlamydia psittaci (guinea pig inclusion conjunctivitis [GPIC]) strains isolated from guinea pigs. Five of the isolates were obtained in the United States during the 1960s and 1970s, including the prototype strain isolated by Murray in 1962. The other isolate was obtained from a guinea pig in England. The nucleotide sequence of the omp1 gene for each strain was identical. The lack of omp1 allelic polymorphism among GPIC isolates suggests that, unlike C. trachomatis, the GPIC agent lacks antigenic variation in the major outer membrane protein.

Analysis of the 1,042,519–base pair Chlamydia trachomatis genome revealed unexpected features related to the complex biology of chlamydiae. Although chlamydiae lack many biosynthetic capabilities, they retain functions for performing key steps and interconversions of metabolites obtained from their mammalian host cells. Numerous potential virulence-associated proteins also were characterized. Several eukaryotic chromatin-associated domain proteins were identified, suggesting a eukaryotic-like mechanism for chlamydial nucleoid condensation and decondensation. The phylogenetic mosaic of chlamydial genes, including a large number of genes with phylogenetic origins from eukaryotes, implies a complex evolution for adaptation to obligate intracellular parasitism.

TonB couples the energized state of the cytoplasmic membrane to the operation of outer membrane receptors responsible for Fe(III) siderophore uptake across the outer membrane of gram-negative bacteria. A tonB mutant of Pseudomonas aeruginosa deficient in iron siderophore uptake was shown in the present study to be hypersusceptible to a wide variety of antibiotics, reminiscent of the phenotype of mutants defective in the mexAB-oprM antibiotic efflux operon. This was not related to influences of a tonB mutation on the iron status of the cell, and indeed, intrinsic antibiotic susceptibility and mexAB-oprM expression were unaffected by iron levels in the growth medium. The presence of tonB on a multicopy plasmid increased the level of resistance of a MexAB-OprM + strain but not that of a MexAB-OprM − strain to a variety of antimicrobial agents. mexAB-oprM expression was not, however, altered in a tonB deletion mutant, indicating that any influence of TonB on MexAB-OprM-mediated multidrug resistance was at the level of pump activity. Consistent with this, drug accumulation assays revealed that the tonB deletion mutant exhibited decreased levels of drug efflux. Still, the multidrug resistance of a nalB strain was not wholly abrogated by a tonB mutation, indicating that it is likely not an essential component of the efflux apparatus. Similarly, elimination of tonB from an nfxB strain only partially compromised MexCD-OprJ-mediated multidrug resistance. Intriguingly, the drug susceptibility of a mexAB-oprM deletion strain was increased following deletion of tonB , suggesting that TonB may also influence antibiotic resistance mediated by determinants other than MexAB-OprM (and MexCD-OprJ). Thus, TonB plays an important role in both intrinsic and acquired antibiotic resistance in P. aeruginosa .

A Pseudomonas aeruginosa strain carrying an insertion of an ΩHg interposon in the mexB gene ( mexB ::ΩHg; strain K879) produced markedly reduced but still detectable levels of OprM, the product of the third gene of the mexAB-oprM multidrug efflux operon. By using a lacZ transcriptional fusion vector, promoter activity likely responsible for OprM expression in the mexB ::ΩHg mutant was identified upstream of oprM . Introduction of the oprM gene, but not the mexAB genes, into a P. aeruginosa multidrug-susceptible Δ mexAB-oprM mutant increased resistance to quinolones, cephalosporins, erythromycin, and tetracycline. A Δ mexAB-oprM strain carrying the oprM gene accumulated markedly less antibiotic than the deletion strain without oprM . Antibiotic accumulation by the MexAB − OprM + strain was markedly enhanced upon treatment of cells with the uncoupler carbonyl cyanide m -chlorophenylhydrazone (CCCP), indicating that MexAB-independent OprM function likely involves an efflux process. Moreover, pretreatment of cells with CCCP prior to the accumulation assay abrogated any differences in accumulation levels between the MexAB − OprM + and MexAB − OprM − strains, indicating that reduced drug accumulation by the OprM + strain (in the absence of CCCP) cannot be due to OprM-mediated reduction in outer membrane permeability. It appears, therefore, that OprM can be expressed and function in a drug efflux capacity independent of MexAB.

The Pseudomonas aeruginosa tonB gene was cloned by complementation of the tonB mutation of Pseudomonas putida strain TE516 (W. Bitter, J. Tommassen & P. J. Weisbeek, 1993, Mol Microbiol 7, 117-130). The gene was 1025 bp in length, capable of encoding a protein of 36860 Da. As with previously described TonB proteins, the P. aeruginosa TonB (TonBp.a.) was rich in Pro residues (18.1 %) and contained Glu-Pro/Lys-Pro repeats. Unlike previously described TonB proteins, however, TonBp.a. lacked an N-terminal membrane anchor (signal) sequence and contained, instead, a predicted internal signal/anchor sequence, expected to yield an atypical N-terminal cytoplasmic domain in this protein. TonB proteins are essential components in iron-siderophore uptake in bacteria, apparently functioning as energy transducers in coupling the energized state of the cytoplasmic membrane to outer-membrane receptor function. As expected, tonB derivatives of P. aeruginosa were defective in siderophore-mediated iron acquisition. tonB gene expression was inducible by iron-limitation, consistent with the identification of a Fur consensus binding sequence upstream of the gene. TonBp.a. showed substantially greater similarity to the Escherichia coli TonB protein than the Pseudomonas putida protein (31 % identity vs. 20 % identity) and tonBP.a. was able to complement deficiencies in the acquisition of ferric enterobactin and vitamin B12# and sensitivity to phage ø80 of an E. coli tonB strain. The larger size of TonBP.a. and its ability to function in both E. coli and P. putida make it a unique TonB protein whose characterization should enhance our understanding of TonB function in bacteria.

Chlamydia trachomatis serovar-specific major outer membrane protein (MOMP) antigens are important targets of immune neutralization in vitro, and natural immunity to infection is associated with serovar specificity. Reinfection, often by different serovars, plays an essential role in chlamydial disease pathogenesis. By use of a murine model, the anamnestic serologic response was characterized following priming and challenge inoculations using 6 different serovars. The serologic response was evaluated using synthetic peptides representing MOMP variant segments (VS) 1, 2, 3, and 4 antigenic regions for each of the 6 serovars. One year after a single priming inoculation, challenge with the same serovar resulted in serologic responses to homologous VS1 peptides. After challenge with a different serovar, anti-VS1 serologic responses were often elicited with specificity to both the priming serovar and the challenge serovar. The recall of serologic response to the original serovar was typically dependent upon the antigenic relationship of the 2 serovars.