Probing activation of the prokaryotic arginine transcriptional regulator using chimeric proteins.

The major transcription factors controlling arginine metabolism in Escherichia coli and Bacillus subtilis, ArgR and AhrC, respectively, are homologous multimeric proteins that form l -arginine-dependent DNA-binding complexes capable of repressing transcription of the biosynthetic genes (both), activating transcription of catabolic genes (AhrC only) or facilitating plasmid dimer resolution (both). Multimerisation and l -arginine binding are associated with the C-terminal 70-80 residues; the N-terminal regions contain a winged helix-turn-helix DNA-binding domain. We have constructed chimeric genes in which the sequences for the N and C-terminal domains have been swapped. The resultant chimeric proteins and their corresponding native proteins have been analysed for their ability to multimerise and bind DNA operator sites in an L-arginine-dependent fashion. Gel filtration and equilibrium sedimentation analysis are consistent with the formation of hexamers by all four proteins in the presence of L-arginine and at high protein concentrations (>100 nM monomer). The hexamer sedimentation coefficients suggest that there is a reduction in molecular volume upon binding L-arginine, consistent with a conformational change accompanying an allosteric activation of DNA-binding. In the absence of L-arginine or at lower protein concentrations, the hexamers are clearly in rapid equilibrium with smaller subunits, whose dominant species appear to be based on trimers, as expected from the crystal structure of the ArgR C-terminal fragment, with the exception of the ArgR-C chimera, which apparently dissociates into dimers, suggesting that in the intact protein the DNA-binding domains may have a significant dimeric interaction. The hexamer-trimer Kdis in the micromolar range, suggesting that trimers are the principal species at in vivo concentrations.DNA binding by all four proteins has been probed by gel retardation and DNase I footprinting analysis using all three types of naturally occurring operators: biosynthetic sites encompassing two 18 bp ARG boxes separated by 2 bp; biosynthetic sites containing two such boxes and a third 18 bp ARG box at a distance of 100 bp downstream, i.e. within the structural gene; and finally a catabolic operator which contains a single ARG box site. The data show that all four proteins bind to the operators at the expected regions in an L-arginine-dependent fashion. From the apparent affinities of the chimeras for each target site, there is no obvious sequence-specificity associated with the N-terminal domains; rather the data can be interpreted in terms of differential allosteric activation, including DNA binding in the absence of L-arginine.Remarkably, the proteins show apparent "anti-competition" in the presence of excess, specific DNA fragments in gel retardation. This appears to be due to assembly of an activated form of the protein, probably hexamers, on the operator DNA. The data are discussed in terms of the current models for the mode of action of both native proteins.

Crystallization of the arginine-dependent repressor/activator AhrC from Bacillus subtilis.

The arginine-dependent repressor/activator AhrC from Bacillus subtilis has been crystallized in space group C222(1), with unit cell dimensions a = 229.8 A, b = 72.8 A, c = 137.7 A and one aporepressor hexamer per asymmetric unit. Preliminary X-ray photographs show measurable intensities beyond 3.0 A.

Nucleotide sequence of a Bacillus subtilis arginine regulatory gene and homology of its product to the Escherichia coli arginine repressor.

In Bacillus subtilis, arginine represses its biosynthetic enzymes and activates its catabolic ones via a regulator gene ahrC. A 6.2-kb EcoRI fragment of B. subtilis chromosomal DNA that includes the ahrC gene has previously been cloned. Gene ahrC was localised in a 0.8-kb HindIII sub-fragment whose nucleotide sequence was determined. An open reading frame (ORF) was present whose translated amino acid sequence showed homology to that of the Escherichia coli arginine repressor encoded by that organism's argR gene. That this ORF corresponded to ahrC was confirmed by (i) the location of the transposon in an ahrC::Tn917 insertion mutant within the ORF; and (ii) by the appearance of an AhrC- phenotype when plasmids carrying restriction fragments lying wholly within this ORF were permitted to integrate by Campbell-type recombination into the B. subtilis chromosome. This represents the first description of a repressor in a 'housekeeping' biosynthetic system in a Bacillus, and indeed of homology between regulatory proteins for any 'housekeeping' system across such a wide taxonomic barrier among prokaryotes.

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.

Sequence analysis of the Bacillus subtilis argC promoter region.

A previously characterised promoter region upstream from the Bacillus subtilis argC gene was sequenced. The in vivo position of transcription start point (+1), was determined by mung-bean-nuclease mapping. The nucleotide (nt) sequences in the '-10' (TATAAT) and '-35' (TTGAAT) regions closely resemble consensus promoter sequences recognised by B. subtilis sigma 43 and Escherichia coli sigma 70 RNA polymerases. Between +9 and -64 are three imperfect inverted repeats with high homology to the E. coli arginine biosynthetic gene putative operator sequences (ARG boxes) [Cunin et al., Nucl. Acids Res. II (1985) 5007-5019] and which contain variable intra-repeat distances. Upstream from the '-35' region, extending as far as -71, is a 97% AT-rich sequence. The argC mRNA has a short leader region containing a B. subtilis ribosome-binding site 8 nt upstream from a TTG start codon for an open reading frame (ORF). The deduced amino acid sequence for this ORF contains regions of homology to that for the E. coli argC N-terminal region.

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.

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.

Sequences of the genes encoding argininosuccinate synthetase in Escherichia coli and Saccharomyces cerevisiae: comparison with methanogenic archaebacteria and mammals.

The nucleotide (nt) sequences of the genes encoding argininosuccinate synthetase from Escherichia coli K-12 (argG) and Saccharomyces cerevisiae (ARG1) were determined. The deduced amino-acid sequences were compared to each other and to their counterparts in two methanogens and in mammals. Three regions are highly conserved. Two of them appear to contain possible Walker-type nt-binding sites [Walker et al., EMBO J. 1 (1982) 945-951] and are therefore candidates for ATP-binding sites. The third region shows some similarity to a short portion of the N-proximal part of the PurA enzyme which catalyses an analogous reaction.

Physiological factors affecting streptomycin production by Streptomyces griseus ATCC 12475 in batch and continuous culture.

Conditions of growth are described for the production of streptomycin by Streptomyces griseus ATCC 12475 using chemically defined minimal medium and complex medium. It was found using batch cultures that early synthesis of the antibiotic occurred during growth in minimal medium but was delayed until the onset of stationary phase in complex medium. This effect was independent of whether spores or vegetative cells were used as inoculum. Stability of streptomycin biosynthesis in continuous culture was dependent on dilution rate and medium employed. Cultures were highly unstable when grown on complex medium but could be maintained in steady states in continuous culture using minimal medium when the dilution rate was increased in a stepwise manner, starting at a dilution rate of 0.02 h-1 (15% of mumax). The effect of changing dilution rate on growth, streptomycin production and the level of streptomycin phosphotransferase was examined using this technique.

Regulation of anaerobic arginine catabolism in Bacillus licheniformis by a protein of the Crp/Fnr family.

Arginine anaerobic catabolism occurs in Bacillus licheniformis through the arginine deiminase pathway, encoded by the gene cluster arcABDC. We report here the involvement of a new protein, ArcR, in the regulation of the pathway. ArcR is a protein of the Crp/Fnr family encoded by a gene located 109 bp downstream from arcC. It binds to a palindromic sequence, very similar to an Escherichia coli Crp binding site, located upstream from arcA. Residues in the C-terminal domain of Crp that form the DNA binding motif, in particular residues Arg-180 and Glu-181 that make specific bonds with DNA, are conserved in ArcR, suggesting that the complexes formed with DNA by Crp and ArcR are similar. Moreover, the pattern of DNase I hypersensitivity sites induced by the binding of ArcR suggests that ArcR bends the DNA in the same way as Crp. From the absence of anaerobic induction following inactivation of arcR and from the existence of a binding site upstream of the arcA transcription start point, it can be inferred that ArcR is an activator of the arginine deiminase pathway.

The use of PCR to isolate a putative ABC transporter from Saccharopolyspora erythraea.

A gene (ertX) encoding a putative ABC transporter was cloned from the erythromycin producer Saccharopolyspora erythraea, using PCR. The primers were based on regions of homology from ABC transporters which confer resistance to macrolide antibiotics. While ertX encodes a protein with a strong degree of similarity to other macrolide ABC transporters from streptomycetes and staphylococci, it did not confer resistance to erythromycin, tylosin, spiramycin, oleandomycin, josamcin, chalcomycin or midecamycin when subcloned into sensitive streptomycete hosts. Southern blot analysis suggested that ertX did not constitute part of the erythromycin gene cluster as identified to date.

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.

Active membrane transport and receptor proteins from bacteria.

A general strategy for the expression of bacterial membrane transport and receptor genes in Escherichia coli is described. Expression is amplified so that the encoded proteins comprise 5-35% of E. coli inner membrane protein. Depending upon their topology, proteins are produced with RGSH6 or a Strep tag at the C-terminus. These enable purification in mg quantities for crystallization and NMR studies. Examples of one nutrient uptake and one multidrug extrusion protein from Helicobacter pylori are described. This strategy is successful for membrane proteins from H. pylori, E. coli, Enterococcus faecalis, Bacillus subtilis, Staphylococcus aureus, Microbacterium liquefaciens, Brucella abortus, Brucella melitensis, Campylobacter jejuni, Neisseria meningitides, Streptomyces coelicolor and Rhodobacter sphaeroides.

Implication of a repression system, homologous to those of other bacteria, in the control of arginine biosynthesis genes in Streptomyces coelicolor.

As with most amino acid biosynthetic pathways in streptomycetes, enzymes of arginine biosynthesis in Streptomyces coelicolor show only slight derepression in minimal medium without, as opposed to with, exogenous arginine. However, when an arginine auxotroph was cultured in limiting arginine, ornithine carbamoyltransferase (OCT) activities rose by as much as 100-fold. The response was not due to a general starvation effect. To elucidate the repression-derepression mechanism, a DNA fragment containing the upstream region of the previously isolated S. coelicolor argCJB cluster was cloned into a multicopy vector and transformed into wild-type S. coelicolor; a slight transient derepression of OCT was observed in minimal medium without, though not with, added arginine, consistent with titration by the insert of a negatively acting macromolecule such as a repressor. A subfragment carrying the 5' end of argC and the region immediately upstream showed specific binding, in mobility shift assays, to purified AhrC, the repressor/activator of genes of arginine metabolism in Bacillus subtilis. It is therefore likely that in S. coelicolor, expression of arginine biosynthesis genes is controlled by a protein homologous to the well-characterised B. subtilis and Escherichia coli repressors.

Further studies on the inviability of Escherichia coli K12 rho mutant strains carrying IncFII plasmids.

Previous results (Baumberg and Lovett 1977) showed that many conjugative plasmids, most notably those of the IncFII group, were only transferable to Escherichia coli K12 rho mutants at very low frequencies, and that this appeared to be due to a lethal interaction between plasmid and rho- allele. Experiments reported here were designed to examine this phenomenon further, and in particular to test the possibility that uncontrolled plasmid replication in the presence of the rho mutant allele occurs. The rifampicin resistance allele rpo203 antagonizes the effect of rho201 on conjugal plasmid transfer; since the former is known to counteract the latter's effect on transcription termination, this result indicates that the plasmid-rho interaction stems directly from the lack of transcription termination caused by a rho mutant allele. Direct estimation of Rldrd19 replication after conjugal transfer to rho+ and rho201 recipients showed no difference according to rho status. Also, cotransduction of rho101 and 201 with ilv::Tn5 into recipients carrying miniplasmid derivatives of R1, which retain a restriction fragment containing all the DNA known to be involved in plasmid replication, was normal and the rho- transductants retained the miniplasmids. It therefore appears that the lethal interaction between the Inc FII plasmids and rho mutant alleles does not involve plasmid replication functions.

Map locations of some mutations conferring resistance to arginine hydroxamate in Bacillus subtilis 168.

Mutations conferring resistance to arginine hydroxamate in Bacillus subtilis 168 have been located on the genetic map by PBS1-mediated transduction. The majority of these mutations, belonging to classes 1, 2 and 4 of Harwood and Baumberg (1977) and affecting only expression of arginine catabolic enzymes, map at a locus designated ahr A cotransducible with cysA, purA and sacA. The order of markers in this region appears to be sacA-ahrA-purA-cysA. Certain anomalies were observed in the properties of Pur+ transductants from crosses with an Ahr donor and a purA recipient. A single ahr mutation (class 3), also affecting only arginine catabolism, maps between ctrA and sacA at a locus designated ahrB. Two others (class 6), affecting simultaneously enzymes of both arginine biosynthesis and catabolism, map between lys and aroD at a locus designated ahrC. Preliminary attempts to define the nature of functional products specified by these ahr loci suggest that a protein is encoded at ahrA.

Variation in expression of sex factor genes in the Proteus-Providencia group relative to Escherichia coli.

Several instances of anomalous expression of genes introduced from Escherichia coli K-12 into Proteus mirabilis have been described. It is shown here that control of sex pilus synthesis directed by the F-like R factor R1 and its depressed derivatives R1-16 (O-C) and R1-19 (i-minus) is also anomalous in P. mirabilis. Piliation in cells bearing the depressed plasmids is expressed at a lower level than in E. coli K-12, and repression is absent in R1-carrying cells. Preliminary results show a similar effect in Providencia. In Proteus morganii, a similarly reduced level of piliation in R1-16-+ or R1-19-+ cultures is observed, but an intermediate level of repression occurs in R1-+ cultures. Less extensive data suggest that expression of the sex factor genes of an R factor of the N incompatibility group differs far less between E. coli and P. mirabilis hosts. Possible bases for these effects are discussed.

Conjugational behaviour of N plasmids in Escherichia coli K12.

R factors of the N incompatibility group apparently transfer at low frequencies (often 10(-6)-10(-5) per donor cell) in 30 min liquid matings. However, the level of transfer is greatly increased when donor-recipient mixtures are held on solid medium selective for the recipient only, prior to transconjugant selection. Increase in transconjugant recovery is still donor-dependent at plating and therefore arises from mating on the plate. The process limiting N-mediated conjugation in liquid is probably mating pair formation, suggesting that the increase in yield of transconjugants may result from provision of a solid substrate for mating pair formation rather than from the delay in selection per se. The kinetics of N plasmid transfer on solid medium resemble those of traniments suggest that RP4, the prototype P incompatibility group plasmid, shows a stimulation of transfer on solid medium similar to the N plasmids studied. We suggest that N, and probably P, plasmids are (like F and the col V's) naturally derepressed for fertility, but this is masked by the imcompetence in liquid matings of donors carrying them.