Simon Baumberg

Inducible erythromycin resistance in staphlyococci is encoded by a member of the ATP‐binding transport super‐gene family

A Staphylococcus epidermidis plasmid conferring inducible resistance to 14‐membered ring macrolides and type B streptogramins has been analysed and the DNA sequence of the gene responsible for resistance determined. A single open reading frame of 1.464kbp, preceded by a complex control region containing a promoter and two ribosomal binding sites, was identified. The deduced sequence of the 488‐amino‐acid protein (MsrA) revealed the presence of two ATP‐binding motifs homologous to those of a family of transport‐related proteins from Gram‐negative bacteria and eukaryotic cells, including the P‐glycoprotein responsible for multidrug resistance. In MsrA, but not these other proteins, the two potential ATP‐binding domains are separated by a Q‐linker of exceptional length. Q‐linkers comprise a class of flexible inter‐domain fusion junctions that are typically rich in glutamine and other hydrophilic amino acids and have a characteristic spacing of hydrophobic amino acids, as found in the MsrA sequence. Unlike the other transport‐related proteins, which act in concert with one or more hydrophobic membrane proteins, MsrA appears to function independently when cloned in a heterologous host (Staphylococcus aureus RN4220). MsrA might, therefore, interact with and confer antibiotic specificity upon other transmembrane efflux complexes of staphylococcal cells. The active efflux of [14C]‐erythromycin from cells of S. aureus RN4220 containing msrA has been demonstrated.

Transcriptional activation of the pathway-specific regulator of the actinorhodin biosynthetic genes in Streptomyces coelicolor

The Streptomyces produce a plethora of secondary metabolites including antibiotics and undergo a complex developmental cycle. As a means of establishing the pathways that regulate secondary metabolite production by this important bacterial genus, the model species Streptomyces coelicolor and its relatives have been the subject of several genetic screens. However, despite the identification and characterization of numerous genes that affect antibiotic production, there is still no overall understanding of the network that integrates the various environmental and growth signals to bring about changes in the expression of biosynthetic genes. To establish new links, we are taking a biochemical approach to identify transcription factors that regulate antibiotic production in S. coelicolor. Here we describe the identification and characterization of a transcription factor, designated AtrA, that regulates transcription of actII‐ORF4, the pathway‐specific activator of the actinorhodin biosynthetic gene cluster in S. coelicolor. Disruption of the corresponding atrA gene, which is not associated with any antibiotic gene cluster, reduced the production of actinorhodin, but had no detectable effect on the production of undecylprodigiosin or the calcium‐dependent antibiotic. These results indicate that atrA has specificity with regard to the biosynthetic genes it influences. An orthologue of atrA is present in the genome of Streptomyces avermitilis, the only other streptomycete for which there is a publicly available complete sequence. We also show that S. coelicolor AtrA can bind in vitro to the promoter of strR, a transcriptional activator unrelated to actII‐ORF4 that is the final regulator of streptomycin production in Streptomyces griseus. These findings provide further evidence that the path leading to the expression of pathway‐specific activators of antibiotic biosynthesis genes in disparate Streptomyces may share evolutionarily conserved components in at least some cases, even though the final activators are not related, and suggests that the regulation of streptomycin production, which serves an important paradigm, may be more complex than represented by current models.

Sequence and transcriptional analysis of the nourseothricin acetyltransferase-encoding gene nat1 from Streptomyces noursei

We have determined the nucleotide (nt) sequence of nat1, a gene encoding nourseothricin (Nc) acetyltransferase (AT) from Streptomyces noursei, and its transcriptional start point (tsp). The nt sequence upstream from the coding region is completely different from that of the stat gene (encoding streptothricin AT) from Streptomyces lavendulae [S. Horinouchi, K. Furuya, M. Nishiyama, H. Suzuki and T. Beppu, J. Bacteriol. 169 (1987) 1929-1937], even though the nt sequences of the two genes and the deduced amino acid (aa) sequences of the two enzymes show a high degree of similarity. Another stat gene, derived from a Gram-negative plasmid, showed only deduced aa similarity, but not nt sequence similarity, to the above two. A database search for related aa sequences did not reveal any clear-cut homologies to other types of protein. A multiple aa sequence alignment of several ATs is presented.

Identification of a chromosomally encoded ABC-transport system with which the staphylococcal erythromycin exporter MsrA may interact

The energy-dependent efflux of erythromycin (Er) in staphylococci is due to the presence of msr A, which encodes an ATP-binding protein. MsrA is related to the multi-component ATP-binding cassette (ABC) transporters which characteristically also contain membrane-spanning domains. Since MsrA functions in a heterologous host in the absence of other plasmid-encoded products, the requirement for a transmembrane (TM) complex might be fulfilled by hijacking a chromosomally encoded protein. Two genes, stpA and smpA, were identified upstream from msrA on the original Staphylococcus epidermidis plasmid, encoding an ATP-binding protein and a hydrophobic TM protein, respectively. Sequences highly similar to stpA and smpA (stpB and smpB) were also found adjacent to a chromosomal copy of msrA in S. hominis. In Southern blots, internal fragments of stpA or smpA hybridized to the chromosome of the Ers S. aureus RN4220. Cloning and sequence analysis of the region identified revealed the presence of two genes, stpC and smpC, related to stpA and smpA. The deduced amino-acid sequences of the gene products showed that StpA and StpC were 85% identical, whereas SmpA and SmpC were 65% identical. A gene similar to msrA was not present in the S. aureus chromosome. There was no further sequence similarity outside these conserved regions. These results indicate that the chromosomes of S. hominis and S. aureus contain sequences encoding a potential TM protein with which MsrA might interact.

Operator interactions by the Bacillus subtilis arginine repressor/activator, AhrC: novel positioning and DNA-mediated assembly of a transcriptional activator at catabolic sites.

We have previously reported the initial characterization of a catabolic operator site (OrocA) for the Bacillus subtilis arginine repressor/activator protein AhrC. Here, we present the characterization by gel retardation and DNase I footprinting of both OrocA and a second catabolic operator site, OrocD. Both operator sites encompass a single recognition site, an ARG box, located immediately upstream of the transcriptional start points, a unique positioning for a transcriptional activator protein. Although there is considerable sequence homology between the two catabolic operator sites, they vary significantly, around twofold, in their apparent affinities for the protein (Kd′  ≈ 90 nM for OrocA and ≈ 190 nM for OrocD). This difference may result from the lower match to the ARG box consensus of the OrocD site. Both catabolic operators show evidence for co‐operative binding with respect to protein concentration. Determination of the sequences of two AhrC catabolic operator sites, in combination with the three such biosynthetic sites, has allowed the derivation of an improved B. subtilis ARG box consensus sequence, CATGAATAAAAATg/tCAAg/t. This is not identical to the Escherichia coli consensus operator for the AhrC homologue, ArgR, which may explain the only partial cross‐functioning of these proteins in vivo. The OrocA site is adjacent to a sharp, stable bend located 5′ to the catabolic operator. Circular permutation analysis has been used to determine the relative angle of bend (≈ 50°), its location and the effect of adding magnesium ions and/or AhrC protein. Protein binding increases the relative bend angle to ≈ 85°. Bending is shown to be associated with a number of A‐tracts in the upstream sequence. However, altering the phasing of the A‐tracts has little effect on the affinity for AhrC. Truncation and competition experiments have been used to investigate the possible role of sequences flanking the operator on affinity. Very surprisingly, the affinity of the OrocA site appears to increase in the presence of excess, specific competitor fragment, i.e. the system shows anti‐competitive effects. Competition is restored at high molar excesses of specific fragment over the protein. We propose a novel model for the assembly of a higher affinity form of AhrC at operator sites that is consistent with both the apparent co‐operativity of binding and the anti‐competitive effects. These data suggest that the molecular interactions occurring between the prokaryotic arginine‐regulatory proteins and their operators may be more complex than is generally appreciated.

Purification and initial characterization of AhrC: the regulator of arginine metabolism genes in Bacillus subtilis

The arginine‐dependent repressor‐activator from Bacillus subtilis, AhrC, has been overexpressed in Escherichia coli and purified to homogeneity. AhrC, expressed in E. coli, is able to repress a Bacillus promoter (argCp), which lies upstream of the argC gene. The purified protein is a hexamer with a subunit molecular mass of 16.7 kDa. Its ability to recognize DNA has been examined in vitro using argCp in both DNase I and hydroxyl radical protection assays. AhrC binds at two distinct sites within the argCp fragment. One site, argCO1, with the highest affinity for protein, is located within the 5′ promoter sequences, whilst the other, argCO2, is within the coding region of argC. The data are consistent with the binding of a single hexamer of AhrC to argCO1 via four of its subunits, possibly allowing the remaining two subunits to bind at argCO2in vivo forming a repression loop similar to those observed for the E coli Lac repressor.

MINIMAL FUNCTIONAL SYSTEM REQUIRED FOR EXPRESSION OF ERYTHROMYCIN RESISTANCE BY MSRA IN STAPHYLOCOCCUS AUREUS RN4220

Previous studies have suggested that inducible erythromycin (Er) resistance in staphylococci mediated by the plasmid-borne ABC-transporter msrA is dependent on additional unidentified chromosomally encoded transmembrane (TM) domains. The requirement for two S. aureus candidate sequences, stpC and smpC, highly similar to sequences adjacent to msrA on the original S. epidermidis plasmid was investigated. Deletion of the sequences by allelic replacement was accomplished by electroporation of S. aureus RN4220 with a nonreplicating suicide vector. S. aureus strains carrying a delta(stpC-smpC) mutation showed an identical ErR phenotype to those arising from single crossover events and unmutated RN4220 containing msrA. This proves that neither stpC nor smpC is required for ErR. To further define the minimal functional unit required for MSR, the control region within the leader sequence of msrA was deleted. This resulted in constitutive resistance to Er and type B streptogramins (Sg), proving that SgR does not require the presence of Er. Deletion constructs containing the N- or C-terminal ABC regions of MsrA did not confer ErR in RN4220 singly or in combination.

Cloning of a Bacillus subtilis restriction fragment complementing auxotrophic mutants of eight Escherichia coli genes of arginine biosynthesis

SummaryFollowing shotgun cloning of EcoRI fragments of Bacillus subtilis 168 chromosomal DNA in pBR322 a hybrid plasmid, pUL720, was isolated which complements Escherichia coli K12 mutants defective for argA, B, C, D, E, F/I, carA and carB. Restriction analysis revealed that the insert of pUL720 comprises four EcoRI fragments, of sizes 12.0, 6.0, 5.0 and 0.8 kbp. Evidence was obtained from subcloning, Southern blot hybridisation, enzyme stability studies and transformation of B. subtilis arginine auxotrophs that the 12 kbp EcoRI fragment carries all the arg genes. It proved impossible to subclone the intact fragment in isolation in the multicopy vectors pBR322, pBR325 or pACYC184, and although it could be subcloned in the low copy vector pGV1106, propagation of the hybrid rapidly resulted in the selection of stable derivatives carrying, near one end, an insertion of 1 kbp of DNA originating from the E. coli chromosome. These and other stable derivatives resulting from subcloning the 12 kbp EcoRI fragment have lost only the ability to complement for E. coli argC, and it is suggested that sequences located close to the equivalent of argC are involved in destabilising plasmids bearing the 12 kbp fragment in E. coli in a copy number dependent manner.

A binding site for activation by theBacillus subtilis AhrC protein, a repressor/activator of arginine metabolism

InBacillus subtilis, the AhrC protein represses genes encoding enzymes of arginine biosynthesis and activates those mediating its catabolism. To determine how this repressor also functions as an activator, we attempted to clone catabolic genes by searching for insertions of the Tn917-lacZ transposon that express AhrC-dependent, arginine-inducibleβ-galactosidase activity. One such isolate was obtained. The region upstream oflacZ was subcloned inEscherichia coli in such a way that it could be replaced in theB. subtilis chromosome after appropriate manipulation. Analysis of exonuclease III-derived deletions located an AhrC-dependent, arginine-inducible promoter to within a ca. 1.9 kb fragment. The sequence revealed: the 3′ end of an ORF homologous togdh genes encoding glutamate dehydrogenase, with highest homology to the homologue fromClostridium difficile; the 5′ end of an ORF homologous to aSaccharomyces cerevisiae gene encoding Δ1-pyrroline 5-carboxylate dehydrogenase (P5CDH), an enzyme of arginine catabolism ; and just upstream of the latter, a sequence with homology to known AhrC binding sites in the upstream part of the biosyntheticargCJBD-cpa-F cluster. The same region has also been sequenced by others as part of theB. subtilis genome sequencing project, revealing that the P5CDH gene is the first in a cluster termedrocABC. Restriction fragments containing the putative AhrC-binding sequence, but not those lacking it, showed retarded electrophoretic mobility in the presence of purified AhrC. A 277 by AhrC-binding fragment also showed anomalous mobility in the absence of AhrC, consistent with its being intrinsically bent. DNAse I footprinting localized AhrC binding to by − 16/ − 22 to + 1 (the transcription startpoint). Such a location for an activator binding site, i.e. overlapping the transcription start, is unusual.

Dissecting the molecular details of prokaryotic transcriptional control by surface plasmon resonance: the methionine and arginine repressor proteins.

The commercial surface plasmon resonance (SPR) biosensors, BIACORE, have been used to investigate the molecular details of macromolecular interactions at prokaryotic promoter-operators. For the Escherichia coli methionine repressor, MetJ, we have quantitated the interaction of the protein with synthetic and natural operator sites and shown that the SPR response is directly related to the stoichiometry of the complexes being formed. The utility of a continuous flow system has also been exploited to investigate transcription from an immobilised promoter-operator fragment; with transcripts collected and subsequently characterised by RT-PCR. This technique has enabled us to investigate how repressor binding affects (i) the interaction of the RNA polymerase (RNAP) with the promoter and (ii) the ability of RNAP to initiate transcription. Remarkably, the repression complex appears to stabilise binding of RNAP, whilst having the expected effects on the levels of transcripts produced. This may well be a general mechanism allowing rapid transcription initiation to occur as soon as the repression complex dissociates. These techniques have also been used to examine protein-DNA interactions in the E. coli and Bacillus subtilis arginine repressor systems. The repressors are the products of the argR and ahrC genes, respectively. Both proteins form hexamers in rapid equilibrium with smaller subunits believed to be trimers. There are three types of operator in these systems, autoregulatory, biosynthetic and catabolic (B. subtilis only). Sensorgrams show that each protein recognises the three types of immobilised operator differently and that binding is stimulated over 100-fold by the presence of L-arginine.