Membrane transport proteins: implications of sequence comparisons.

Analyses of the sequences and structures of many transport proteins that differ in substrate specificity, direction of transport and mechanism of transport suggest that they form a family of related proteins. Their sequence similarities imply a common mechanism of action. This hypothesis provides an objective basis for examining their mechanisms of action and relationships to other transporters.

Structural mechanisms of QacR induction and multidrug recognition.

The Staphylococcus aureus multidrug binding protein QacR represses transcription of the qacA multidrug transporter gene and is induced by structurally diverse cationic lipophilic drugs. Here, we report the crystal structures of six QacR-drug complexes. Compared to the DNA bound structure, drug binding elicits a coil-to-helix transition that causes induction and creates an expansive multidrug-binding pocket, containing four glutamates and multiple aromatic and polar residues. These structures indicate the presence of separate but linked drug-binding sites within a single protein. This multisite drug-binding mechanism is consonant with studies on multidrug resistance transporters.

Surface exclusion genes traS and traT of the F sex factor of Escherichia coli K-12. Determination of the nucleotide sequence and promoter and terminator activities.

The DNA encoding the surface exclusion genes traS and traT of the F sex factor of Escherichia coli K-12 has been sequenced and the biological activity of the various terminators and promoters determined. The data show that traS encodes a 16,861 Mr protein with no apparent signal sequence, as expected for its cytoplasmic membrane location. The protein is extremely hydrophobic. traS has its own promoter and a weak terminator region follows the gene. After the traS termination loop there is a small intergenic region before the traT promoter. The traT gene encodes a 25,932 Mr precursor for the 23,709 Mr mature protein. The amino-terminal signal peptide is 21 amino acid residues, consistent with it being an outer membrane lipoprotein. A very strong termination loop follows the gene and adjacent to this a further loop can be predicted from the sequence. These secondary structures would be expected to enhance the stability of the mRNA in the presence of 3' specific ribonucleases accounting for the apparent long half-life of the messenger. The amino acid sequence of the mature product of traT of F differs from that of R100 by only a single amino acid substitution (Gly for Ala at position 119), whereas that of pED208 (Folac) differs at 40 positions. traT lies in a region of heteroduplex homology between F and R100, and the nucleotide sequence confirms this and demonstrates that this homology breaks down immediately preceding and following the coding region. Sequence analysis shows that this is also so for pED208. Thus the entire traS of F, R100 and pED208 are very different at the DNA level. An open reading frame, preceded by a typical promoter sequence and a weak and poorly located Shine-Dalgarno sequence, follows traT and corresponds to the start of traD. Alone, this promoter appears to be inactive.

IS257 from Staphylococcus aureus: member of an insertion sequence superfamily prevalent among gram-positive and gram-negative bacteria.

The nucleotide sequences for the IS257 family of insertion sequences from Staphylococcus aureus were compared with those of the ISS1 family from Streptococcus lactis and the IS15 family which is widespread amongst Gram-negative bacteria. These elements have a striking degree of similarity in both their putative transposase polypeptide sequences and their nucleotide sequences (40 to 64% between pairs), including 12 out of 14 bp conservation in their terminal inverted repeats. The evolutionary distance between the IS15 family and the IS257 and ISS1 families of Gram-positive origin is approximately twice that between the IS257 and ISS1 families. Analysis of base substitutions in the three sequences has provided insights into the effect of selection for the G + C content of immigrant genes to conform to that of their hosts, and into the evolution of biases in overall amino acid composition of cellular proteins in prokaryotes and eukaryotes. The IS257, ISS1, IS15 families form a superfamily of insertion sequences that has been involved in the spread of a number of antimicrobial resistance determinants in Gram-positive and Gram-negative pathogens.

Topology, structure and evolution of two families of proteins involved in antibiotic and antiseptic resistance in eukaryotes and prokaryotes--an analysis.

Analysis of deduced amino acid sequences has demonstrated that the sequences of eukaryotic and prokaryotic proteins mediating resistance to antibiotics and antiseptics are highly related. Hydropathy analysis and alignment of conserved motifs revealed that these proteins can be divided into two separate families with either 12 or 14 transmembrane segments (TMS). Conserved motifs have been identified which are either characteristic for each family or conserved in both families. The conservation of these motifs suggested that they may be essential for the function of these proteins. Phylogenetic and structural analysis revealed that the two families may have evolved from a common ancestor with six TMS.

Nucleotide sequence analysis of IS256 from the Staphylococcus aureus gentamicin-tobramycin-kanamycin-resistance transposon Tn4001.

Resistance to the aminoglycosides gentamicin, tobramycin and kanamycin (GmTmKmR) in Australian clinical strains of Staphylococcus aureus is commonly carried on the composite transposon Tn4001. The resistance gene aacA-aphD of Tn4001, which encodes a bifunctional AAC(6')-APH(2") modifying enzyme, is flanked by two 1324-bp inverted repeats, IS256L and IS256R, that are identical in sequence. Analysis of the IS256 sequence revealed structural features characteristic of IS elements including 26-bp imperfect terminal inverted repeats and a single open reading frame with coding capacity for a 45.6 kDa protein. The nucleotide sequence of IS256 described here, together with the sequence of the aacA-aphD gene reported previously [Rouch et al., J. Gen. Microbiol. 133 (1987) 3039-3052], completes the entire sequence of Tn4001, which totals 4566 bp.

Nucleotide sequence and transcriptional analysis of a third function (Flm) involved in F-plasmid maintenance.

The leading region of the conjugative F plasmid encodes for a function, Flm, capable of extending the maintenance of normally unstable plasmids. Nucleotide sequencing and functional studies of flm locus have shown that it consists of at least two genes, flmA and flmB, which are physically and functionally homologous to hok and sok of parB in plasmid R1. The 52-amino acid flmA-coded polypeptide is almost identical to the hok product which has been shown to be a membrane-associated lethal protein [Gerdes et al., EMBO J. 5 (1986) 2023-2029]. Gene flmB codes for a 100 nucleotide, non-translated, complementary RNA which overlaps the 5' leader sequence of the flmA RNA. The flmA RNA also encodes an open reading frame (ORF70) which overlaps the flmA-coding sequence and may be a third gene involved in the Flm function. S1 analysis and functional studies suggest that the antisense flmB RNA binds to the flmA RNA and suppresses the expression of the lethal product, presumably by blocking coupled translation of ORF70 and flmA. Secondary structure analysis predicts that the flmA RNA is extremely stable compared to the regulatory flmB RNA. We suggest that when these RNA species are retained by cells which have lost the F plasmid, the more stable flmA RNA will eventually be translated thus leading to cell death. This phenomenon provides a third mechanism, additional to ParFIA and Ccd functions, to ensure maintenance of the F plasmid in a growing bacterial population.

Structure and evolution of a family of genes encoding antiseptic and disinfectant resistance in Staphylococcus aureus.

Resistance to antiseptics and disinfectants in Staphylococcus aureus, encoded by the qacC/qacD gene family, is associated with genetically dissimilar small, nontransmissible (pSK89) and large conjugative (pSK41) plasmids. The qacC and qacD genes were analysed in detail through deletion mapping and nucleotide sequence analysis, and shown to encode the same polypeptide, predicted to be 107 aa in size. Direct repeat elements flank the qacD gene, elements which also flank the qacC gene in truncated forms. These elements contain palA sequences, regions of DNA required for replication of some plasmids in S. aureus. The qacC gene is predicted to have evolved from the qacD gene, and in the process to have become reliant on new promoter sequences for its expression. The entire sequence of the 2.4-kb plasmid pSK89 (which contains qacC) was determined, and is compared with other plasmids from Gram + bacteria.

Sequence and conservation of genes at the distal end of the transfer region on plasmids F and R6-5.

The nucleotide sequence of the region downstream of transfer gene traI, including fertility inhibition gene finO, on the conjugative plasmids F and R6-5, has been determined. Analysis of the F sequence revealed two open reading frames (ORF's), ORF248 and ORF186; ORF186 (finO) is interrupted by the insertion of IS3. The R6-5 sequence also contained ORF248 and an intact ORF186, although an additional ORF (ORF286) was located between the two genes. ORF248, which we have designated traX, and ORF186 (finO) are highly conserved on both plasmids. The organisation of these genes indicates that traI and traX on F, and traI, traX and ORF286 on R6-5 are co-transcribed from their respective promoters upstream of traI. Sequences homologous to traX were detected on a range of conjugative F-like plasmids, whereas sequences homologous to ORF286 were only found on plasmids R6-5, R100 and R1. The conservation of traX sequences suggests a functional importance for that gene and/or its product.

Characterisation of sin, a potential recombinase-encoding gene from Staphylococcus aureus.

The staphylococcal beta-lactamase (Bla) transposon Tn4002 has previously been reported to have a high level of insertional specificity for a 1.8-kb region on the plasmid pSK1. Nucleotide sequences of this region and of a related region on plasmid pI9789 were determined. Sequence analysis revealed that these two plasmids contain sin, a gene whose deduced product shows similarity to a family of DNA recombinases. Southern hybridisation analysis indicated that sin is located on alpha-, beta- and gamma-families of Bla plasmids and on pSK1 family plasmids. A region of dyad symmetry located upstream from sin on pSK1 and pI9789 was identified as the site of insertions of Tn552 and Tn4002 in separate isolates.

Analysis of a transfer region from the staphylococcal conjugative plasmid pSK41.

The nucleotide sequence of a 14.4-kb region (tra) associated with DNA transfer of the staphylococcal conjugative plasmid, pSK41, has been determined. Analysis of the sequence revealed the presence of 15 genes potentially involved in the conjugative process. Polypeptide products likely to correspond to ten of these genes have been identified, of which one was found to be a lipoprotein. Comparison of the deduced tra products to the protein databases revealed several interesting similarities, one of which suggests an evolutionary link between this Gram+ bacterial conjugation system and DNA transfer systems of Gram- bacteria, such as Escherichia coli and Agrobacterium tumefaciens. The nt sequence also provided an insight into the transcriptional organisation and regulation of the region.

Substrate specificity and energetics of antiseptic and disinfectant resistance in Staphylococcus aureus.

Clinical isolates of Staphylococcus aureus carry various antiseptic and disinfectant resistance determinants (qac genes) on a variety of plasmids. The biochemistry and specificity of these resistance genes in S. aureus is the subject of this report. The qac genes were separated into two families on the basis of resistance profiles and DNA homology. Isotopic and fluorimetric assays demonstrated that the qac genes encode efflux systems that rely on proton motive force.

Characterisation of chloramphenicol resistance plasmids of Staphylococcus aureus and S. epidermidis by restriction enzyme mapping techniques.

Chloramphenicol resistance (Cmr) plasmids pSK2 and pSK5 from Staphylococcus aureus and pSK102 and pSK103 from S. epidermidis have been characterised and detailed restriction endonuclease cleavage maps constructed. TaqI digestion profiles illustrated the identity of pSK5 and pSK102 and also revealed a high degree of similarity between these four Cmr plasmids from Australian staphylococci and three Cmr plasmids from S. aureus strains of geographically unrelated origin. DNA-DNA hybridisation indicated that the chloramphenicol acetyltransferase determinant carried by pSK5/pSK102 could be found on other structurally-distinct Cmr plasmids. The role of S. epidermidis as a reservoir for Cmr plasmids found in S. aureus is discussed.

Antibiotic resistance in Staphylococcus aureus isolated at an Australian hospital between 1946 and 1981.

A total of 517 strains of Staphylococcus aureus isolated at a hospital in Melbourne, Australia between 1946 and 1981 was examined for resistance to a range of antimicrobial agents and for the presence of plasmid DNA. The use of mixed-culture transfer and restriction endonuclease analysis showed that the determinants for resistance to penicillin and to the heavy metals were carried by several related plasmids of (15-23) X 10(6) mol. wt, and that tetracycline resistance was encoded on a plasmid of 2.8 X 10(6) mol. wt in strains isolated before 1970. These phenotypes were chromosomally encoded in the majority of strains isolated thereafter. Resistance to chloramphenicol throughout the study period was plasmid-mediated. Of five aminoglycoside-resistance phenotypes, one was plasmid-mediated and three were chromosomally encoded. The remaining phenotype, specifying low-level gentamicin resistance, was found to be located on the chromosome of early isolates, but in later strains was borne by an 18 X 10(6) mol. wt plasmid which also encoded resistance to quaternary ammonium compounds.

Antibiotic susceptibilities and plasmid profiles of nosocomial methicillin-resistant Staphylococcus aureus: a retrospective study.

Methicillin-resistant Staphylococcus aureus strains isolated at a single Melbourne Hospital between 1969 and 1981 were examined for susceptibility to a range of antimicrobial agents and for the presence of plasmid DNA. Isolates obtained during 1969 possessed a plasmid of mol. wt 20 X 10(6), encoding heavy metal resistance and penicillinase production, and a plasmid of mol. wt 2.8 X 10(6), mediating tetracycline resistance. In the majority of isolates obtained after 1973, these functions were chromosomally encoded. Before 1980, both high- and low-level chromosomally-encoded gentamicin resistances were encountered, whereas isolates from 1980 and 1981 displayed low-level gentamicin resistance only; the latter phenotype was most commonly mediated by a plasmid of mol. wt 18 X 10(6) that also encoded resistance to tobramycin and kanamycin. Chloramphenicol resistance in strains isolated throughout the period was mediated by one of three plasmids, each of mol. wt c. 3 X 10(6).

A retrospective molecular analysis of gentamicin resistance in Staphylococcus aureus strains from UK hospitals.

The composite transposon Tn4001 and a related chromosomal Tn4001-like element, encode resistance to the aminoglycosides gentamicin, tobramycin and kanamycin (GmTmKmr) in Australian strains of Staphylococcus aureus. Southern hybridisation analysis of GmTmKmr S. aureus strains isolated from various hospitals in the UK between 1975 and 1985 indicated that they predominantly encoded chromosomal copies of Tn4001 or a Tn4001-like element. However, a strain isolated in 1985 was found to carry Tn4001 on a plasmid related to pSK1, the prototypical multiresistance plasmid commonly detected in S. aureus strains from Australian hospitals.

Typing of methicillin-resistant Staphylococcus aureus by antibiotic resistance phenotypes.

The identification of new epidemic strains of methicillin-resistant Staphylococcus aureus is essential for rapid, effective infection control. We have developed a typing method which uses antibiotic sensitivity patterns to differentiate methicillin-resistant S. aureus and which is faster and more cost-effective than biochemical analysis or bacteriophage typing. Characterisation of phenotypes which are chromosomally-encoded, plasmid- or chromosomally-encoded or exclusively plasmid-mediated has enabled us to separate Australian strains of methicillin-resistant S. aureus into 11 classes, representatives of which were indistinguishable by bacteriophage type, or plasmid profile alone. The value of this procedure is thus clearly shown.

Trimethoprim resistance determinants encoding a dihydrofolate reductase in clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci.

The molecular and biochemical basis of resistance to high concentrations (MIC greater than or equal to 1000 mg/L) of trimethoprim (Tpr(H] was examined in Australian isolates of Staphylococcus aureus and coagulase-negative staphylococci. The Tpr(H) determinant (dfr A) was located within a 2.75-Kb Bg/II fragment on the S. aureus aminoglycoside-resistance plasmids pSK1 and pSK16 as judged by comparative restriction mapping with two naturally-occurring variants, pSK9 and pSK14, which did not encode trimethoprim resistance. This was confirmed in DNA-DNA hybridisation experiments in which a 0.9-Kb sequence of pSK1 DNA was used as a specific probe for the Tpr(H) gene. Plasmid DNA from three strains of coagulase-negative staphylococci, and the chromosomal material of one other isolate, were found to share homology with the probe DNA. Dihydrofolate reductases produced by these strains were virtually identical to the type S1 enzyme encoded by the S. aureus plasmid pSK1. Interspecies transfer may have been responsible for the conservation of Tpr(H) gene sequences among staphylococci.

Molecular epidemiology of multiresistant Staphylococcus aureus in Australian hospitals.

Antibiotic multiresistant isolates of Staphylococcus aureus from outbreaks of nosocomial infection throughout Australia were found to possess essentially similar patterns of antibiotic resistance. Plasmid DNA profiles from these isolates exhibited a common pattern of large plasmids, of (15-22) X 10(6) mol. wt, associated with resistance to gentamicin, kanamycin and tobramycin, plasmids of 3 X 10(6) mol. wt, mediating resistance to chloramphenicol, and cryptic plasmids of 1 X 10(6) mol. wt. Restriction endonuclease digestion confirmed the presence of related plasmids in isolates from all the hospitals that were surveyed. The homogeneity of these organisms suggests the dissemination of a multiresistant, plasmid-bearing strain of S. aureus, or its derivatives, among geographically-separated hospitals in Australia.

Multidrug resistance to antiseptics and disinfectants in coagulase-negative staphylococci.

The occurrence of resistance to antiseptics and disinfectants in clinical isolates of coagulase-negative staphylococci (CNS) was examined. Of 164 clinical strains of CNS isolated in the early 1980s, 65 were resistant to cationic antimicrobial compounds such as cetyltrimethylammonium bromide. Further characterisation of 40 resistant isolates by DNA-DNA hybridisation analysis and phenotypic resistance studies revealed that this resistance was mediated by the multidrug export genes qacA and qacC, characterised previously in Staphylococcus aureus. Of the resistant CNS isolates, 50% contained only qacA, 10% contained only qacC, and the remaining 40% contained both qacA and qacC. Both qacA and qacC genes resided on plasmids in all cases, with qacA located on plasmids of > 10 kb, whereas qacC was located primarily on plasmids of 2-3 kb. Representative qacA and qacC plasmids were characterised by restriction endonuclease mapping, and were found to be similar in some cases, but different in others, to those plasmids on which these genes are found in S. aureus.