Treatment of whole blood (WB) and red blood cells (RBC) with S-303 inactivates pathogens and retains in vitro quality of stored RBC.

A pathogen inactivation (PI) process has been developed using the frangible anchor linker effector (FRALE) compound S-303. A series of experiments were performed in whole blood (WB) to measure the level of viral and bacterial inactivation. The results showed that 0.2mM S-303 and 2mM glutathione (GSH) inactivated >6.5 logs of HIV, >5.7 logs of Bluetongue virus, >7.0 logs of Yersinia enterocolitica, 4.2 logs of Serratia marcescens, and 7.5 logs of Staphylococcus epidermidis. Recent development for S-303 is focused on optimization of the PI process for red blood cell concentrates (RBC). A series of studies in RBC showed that 0.2mM S-303 and 20mM GSH inactivated approximately 5 logs or greater of Y. enterocolitica, E. coli, S. marcescens, S. aureus, HIV, bovine viral diarrhoea virus, bluetongue virus and human adenovirus 5. In both applications of the S-303 process, in vitro parameters of RBC function and physiology were retained compared to conventional RBC. Results from these studies indicate that S-303 can be applicable for PI of RBC and WB.

Mutant forms of the enhancer-binding protein NtrC can activate transcription from solution.

Activators of the sigma54-holoenzyme catalyze the isomerization of closed complexes between this polymerase and a promotor to open complexes in a reaction that depends upon hydrolysis of a nucleoside triphosphate. The activators normally bind to DNA sites with the properties of transcriptional enhancers and contact the polymerase by means of DNA loop formation. Here, we demonstrate that mutant forms of the activator nitrogen regulatory protein C (NtrC) that lack one helix of the helix-turn-helix (HTH) DNA-binding motif or the entire motif retain residual capacity to activate transcription from solution, despite the fact that they are largely unable to dimerize and have greatly decreased ability to hydrolyze ATP. We show that substitution of alanine for three hydrophilic residues in the second helix of the HTH yields a stable, dimeric form of NtrC defective in DNA-binding. Like mutant forms with deletions of one or both helices, the NtrC3ala protein failed to bind DNA in a sensitive affinity co-electrophoresis assay, indicating that its affinity for a strong enhancer was reduced by at least 5000-fold. (The assay detected enhancer-binding by two mutant forms of NtrC with single amino acid substitutions in the HTH and non-specific DNA-binding by the wild-type protein.) The phosphorylated NtrC3ala protein had normal ATPase activity in solution but, unlike the activity of the phosphorylated wild-type protein, which could be stimulated at least tenfold by an oligonucleotide carrying a strong enhancer, the ATPase activity of the phosphorylated NtrC3ala protein was not stimulated. At concentrations of 100 nM or greater, the phosphorylated NtrC3ala protein activated transcription from the major glnA promoter. In agreement with the fact that it did not show detectable DNA-binding in other assays, its ability to activate transcription was no greater on templates carrying the glnA enhancer than on templates lacking an enhancer. The results indicate that both roles of the glnA enhancer, tethering and facilitation of the formation of an active oligomer of NtrC, can be bypassed if the protein is present at high concentrations in solution.

Unusual oligomerization required for activity of NtrC, a bacterial enhancer-binding protein.

Nitrogen regulatory protein C (NtrC) contacts a bacterial RNA polymerase from distant enhancers by means of DNA loops and activates transcription by allowing polymerase to gain access to the template DNA strand. It was shown that NtrC from Salmonella typhimurium must build large oligomers to activate transcription. In contrast to eukaryotic enhancer-binding proteins, most of which must bind directly to DNA, some NtrC dimers were bound solely by protein-protein interactions. NtrC oligomers were visualized with scanning force microscopy. Evidence of their functional importance was provided by showing that some inactive non-DNA-binding and DNA-binding mutant forms of NtrC can cooperate to activate transcription.

Repressor forms of the enhancer-binding protein NrtC: some fail in coupling ATP hydrolysis to open complex formation by sigma 54-holoenzyme.

NtrC (nitrogen regulatory protein C) is a bacterial enhancer-binding protein that activates transcription by catalyzing isomerization of closed complexes between sigma54-holoenzyme and a promoter to open complexes. To catalyze this reaction, NtrC must be phosphorylated and form an appropriate oligomer so that it can hydrolyze ATP. NtrC can also repress transcription by sigma70-holoenzyme. In this paper we characterize "repressor" mutant forms of NtrC from Salmonella typhimurium, forms that have lost the ability to activate transcription by sigma54-holoenzyme (in vitro activity at least 1000-fold lower than wild-type) but retain the ability to repress transcription by sigma70-holoenzyme. The amino acid substitutions in NtrCrepressor proteins that were obtained by classical genetic techniques alter residues in the central domain of the protein, the domain directly responsible for transcriptional activation. Commensurate with this, phosphorylation and the autophosphatase activities of NtrCrepressor proteins, which are functions of the amino-terminal regulatory domain of NtrC, are normal. In addition, these proteins have essentially normal DNA-binding, which is a function of the C-terminal region of NtrC and bind cooperatively to enhancers. (The NtrC(G219K) protein has "improved" DNA-binding, which is discussed.) We previously presented evidence that several NtrCrepressor proteins have impaired ATPase activity. We now show that two other repressor proteins, NtrC(A216V) and NtrC(A220T), have as much ATPase activity as wild-type NtrC when they are phosphorylated and bound to an enhancer and that they have considerably more activity than an unphosphorylated NtrC(constitutive) protein, which is capable of activating transcription. These results demonstrate that NtrC(A216V) and NtrC(A220T) fail in a function of the central domain other than ATPase activity. Although they may fail in contact with sigma54-holoenzyme per se, the fact that alanine is the amino acid normally found at these positions leads us to speculate that these proteins fail in coupling energy to a change in conformation of the polymerase.

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

In Bacillus 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-inducible beta-galactosidase activity. One such isolate was obtained. The region upstream of lacZ was subcloned in Escherichia coli in such a way that it could be replaced in the B. 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 to gdh genes encoding glutamate dehydrogenase, with highest homology to the homologue from Clostridium difficile; the 5' end of an ORF homologous to a Saccharomyces cerevisiae gene encoding delta 1-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 biosynthetic argCJBD-cpa-F cluster. The same region has also been sequenced by others as part of the B. subtilis genome sequencing project, revealing that the P5CDH gene is the first in a cluster termed rocABC. 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 bp 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 bp -16/-22 to +1 (the transcription startpoint). Such a location for an activator binding site, i.e. overlapping the transcription start, is unusual.

The major dimerization determinants of the nitrogen regulatory protein NTRC from enteric bacteria lie in its carboxy-terminal domain.

The NTRC protein (nitrogen regulatory protein C) of enteric bacteria is an enhancer-binding protein that activates transcription by the sigma54-holoenzyme form of RNA polymerase. NTRC is a homodimeric protein that binds to a dyad-symmetrical site in DNA. To activate transcription NTRC must be phosphorylated and must form an appropriate oligomeric species at an enhancer. In order to study subunit exchange between NTRC dimers, we constructed a fusion of the maltose-binding protein (MBP) to the amino-terminal end of NTRC (MBP-NTRC) and visualized the formation of heterodimers between MBP-NTRC and wild-type NTRC by a gel-mobility shift assay for DNA-binding. When MBP-NTRC is mixed with wild-type NTRC at 37 degrees C, subunit exchange occurs rapidly. The apparent half-life for dissociation of homodimers of NTRC is two to three minutes at 37 degrees C and is not changed by phosphorylation. The isolated carboxy-terminal domain of NTRC (91 amino acid residues) forms heterodimers with both wild-type NTRC and MBP-NTRC, indicating that the C-terminal domain is sufficient for dimerization. The apparent rate of dissociation of homodimers of the C-terminal domain is essentially the same as that of full-length NTRC, indicating that the major dimerization determinants of the protein lie in its C-terminal domain. Congruent with this, a truncated form of NTRC from which the last 58 amino acid residues were removed is a monomer in solution. Moreover, truncated forms of NTRC from which the last 16 or 26 amino acid residues were removed are predominantly monomeric in solution, as is a mutant form with the amino acid substitution A410E in its C-terminal domain. Monomerization of the above mutant forms of NTRC can be rationalized on the basis of homology between the C-terminal region of NTRC and a 50 amino acid residue region of the factor for inversion stimulation (FIS) protein.

Oligomerization of NTRC at the glnA enhancer is required for transcriptional activation.

To activate transcription of the glnA gene, the dimeric NTRC protein (nitrogen regulatory protein C) of enteric bacteria binds to an enhancer located approximately 100 bp upstream of the promoter. The enhancer is composed of two binding sites for NTRC that are three turns of the DNA helix apart. One role of the enhancer is to tether NTRC in high local concentration near the promoter to allow for its frequent interaction with sigma 54 holoenzyme by DNA looping. We have found that a second role of the enhancer is to ensure oligomerization of NTRC into a complex of at least two dimers that is required for transcriptional activation. Formation of this complex is greatly facilitated by a protein-protein interaction between NTRC dimers that is increased when the protein is phosphorylated.

Role of integration host factor in stimulating transcription from the sigma 54-dependent nifH promoter.

In a wide variety of nitrogen-fixing organisms among the Purple Bacteria (large division of Gram-negative bacteria) the nitrogen fixation (nif) operons are transcribed by an alternative holoenzyme form of RNA polymerase, sigma 54-holoenzyme. Transcription depends on the activator protein NIFA (nitrogen fixation protein A), which catalyzes isomerization of closed complexes between this polymerase and a promoter to transcriptionally productive open complexes. NIFA-mediated activation of transcription from the nifH promoter of Klebsiella pneumoniae is greatly stimulated by the integration host factor IHF, which binds to a site between the upstream binding site for NIFA and the promoter, and bends the DNA. IHF fails to stimulate activation of transcription from this promoter by another activator of sigma 54-holoenzyme, NTRC (nitrogen regulatory protein C), which lacks a specific binding site in the nifH promoter region. As predicted, if the IHF-induced bend facilitates interaction between NIFA and sigma 54-holoenzyme, substitution of an NTRC-binding site for the NIFA-binding site allowed IHF to stimulate NTRC-mediated activation of transcription from the nifH promoter. The stimulation was of the same order of magnitude as that for NIFA in the native configuration of the promoter-regulatory region (up to 20-fold). With purified NTRC and the substitution construct we could demonstrate that stimulation by IHF in a purified transcription system was comparable to that in a crude coupled transcription-translation system, indicating that the stimulation in the crude system could be accounted for by IHF. The IHF stimulation was observed on linear as well as supercoiled templates, indicating that the geometric requirements are relatively simple. We have attempted to visualize the arrangement of proteins on DNA fragments carrying the nifH promoter-regulatory region of K. pneumoniae by electron microscopy. IHF stimulated NIFA-mediated activation of transcription from the nifH and nifD promoters of Bradyrhizobium japonicum and less so from the nifH promoters of Rhizobium meliloti and Thiobacillus ferrooxidans, consistent with previous observations that stimulation is greatest at promoters that are weak binding sites for sigma 54-holoenzyme in closed complexes.

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 argCo2 in vivo forming a repression loop similar to those observed for the E. coli Lac repressor.

Prokaryotic transcriptional enhancers and enhancer-binding proteins.

A number of prokaryotic enhancer-binding proteins activate transcription by specialized forms of RNA polymerase. The enhancer-binding proteins catalyse isomerization of the initial complex formed between RNA polymerase and a promoter from the closed to the open state. To do so, one class of enhancer-binding proteins contacts its cognate polymerase by DNA loop formation but the other, which is represented by a single member, does not. Despite this difference, both classes of enhancer-binding proteins must hydrolyse ATP to catalyse open complex formation.

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.

Sequences required for regulation of arginine biosynthesis promoters are conserved between Bacillus subtilis and Escherichia coli.

The region required for regulation of a previously characterized arginine-regulatable promoter upstream from the argC gene in the argCAEBD-cpa-argF cluster of Bacillus subtilis was defined by integration of argC-lacZ translational fusions into the chromosome at a site distant from the arginine loci. Some sequence similarity was detected between the argC regulatory region and the well-characterized Escherichia coli arginine operators (ARG boxes). This similarity was shown to be functional in vivo in that the B. subtilis repressor regulated the E. coli arginine genes, but the E. coli repressor, even when encoded by a multicopy plasmid, could not repress the B. subtilis argC promoter. In vitro binding studies using purified repressors on DNA fragments encoding operators from both E. coli and B. subtilis demonstrated interactions by both proteins.