Spores of Bacillus anthracis, the causative agent of anthrax, are enclosed by a prominent loose-fitting, balloon-like layer called the exosporium. Although the exosporium serves as the source of surface antigens and a primary permeability barrier of the spore, its molecular structure and function are not well characterized. In this study, we identified five major proteins in purified B. anthracis (Sterne strain) exosporia. One protein was the recently identified collagen-like glycoprotein BclA, which appears to be a structural component of the exosporium hair-like nap. Using a large panel of unique antispore monoclonal antibodies, we demonstrated that BclA is the immunodominant antigen on the B. anthracis spore surface. We also showed that the BclA protein and not a carbohydrate constituent directs the dominant immune response. In addition, the length of the central (GXX)(n) repeat region of BclA appears to be strain specific. Two other unique proteins, BxpA and BxpB, were identified. BxpA is unusually rich in Gln and Pro residues and contains several different tandem repeats, which also exhibit strain-specific variation. In addition, BxpA was found to be cleaved approximately in half. BxpB appears to be glycosylated or associated with glycosylated material and is encoded by a gene that (along with bclA) may be part of an exosporium genomic island. The other two proteins identified were alanine racemase and superoxide dismutase, both of which were reported to be associated with the surface of other Bacillus spores. Possible functions of the newly identified proteins are discussed.

The three-dimensional structure of pentameric human serum amyloid P component at high resolution, the first reported for a pentraxin, reveals that the tertiary fold is remarkably similar to that of the legume lectins. Carboxylate and phosphate compounds bind directly to two calcium ions; interactions with a carboxyethylidene ring are mediated by Asn 59 and Gln 148 ligands of the calcium ions. These X-ray results indicate the probable modes of binding of the biologically important ligands, DNA and amyloid fibrils.

BACKGROUND

Increased levels of total serum IgE are a strong risk factor for the development of asthma. IgE is also involved in host defenses against parasites and fungi. Linkage of total serum IgE with markers located close to the 3 Mb cluster of cytokine genes in chromosome 5q31 has been reported. IL-4 or IL-13 are regarded as essential for IgE synthesis.

OBJECTIVE

We tested whether polymorphisms in the IL-13 gene might explain the linkage between chromosome 5q31 and total serum IgE levels.

METHODS

We used denaturing HPLC to detect polymorphisms in overlapping PCR fragments of the IL-13 gene including promoter and 3' untranslated regions. After sequencing was performed to identify the locations of the polymorphisms, PCR and primer-induced restriction site assays were used to genotype subjects in 3 unselected populations.

RESULTS

We report here 7 polymorphisms (6 novel) in IL-13. Four of these polymorphisms are tightly linked to a variant in the terminal portion of the coding region of the gene that results in a predicted amino acid change in residue 130 (Arg130Gln). The Gln form is strongly associated (P =.000002) with increased serum IgE levels in 3 different populations comprising a total of 1399 children. Two additional polymorphisms in the promoter region of IL-13 are more loosely linked to Arg130Gln and are also less significantly associated with total serum IgE levels.

CONCLUSIONS

These data suggest that the Arg130Gln polymorphism in IL-13, or others in close linkage with it, is associated with the development of the elevated serum IgE phenotype.

The enzyme Cu, Zn superoxide dismutase (SOD) protects against oxidative damage by dismuting the superoxide radical O2-. to molecular oxygen and hydrogen peroxide at the active-site Cu ion in a reaction that is rate-limited by diffusion and enhanced by electrostatic guidance. SOD has evolved to be one of the fastest enzymes known (V(max) approximately 2 x 10(9) M-1 s-1). The new crystal structures of human SOD show that amino-acid site chains that are implicated in electrostatic guidance (Glu 132, Glu 133 and Lys 136) form a hydrogen-bonding network. Here we show that site-specific mutants that increase local positive charge while maintaining this orienting network (Glu----Gln) have faster reaction rates and increased ionic-strength dependence, matching brownian dynamics simulations incorporating electrostatic terms. Increased positive charge alone is insufficient: one charge reversal (Glu----Lys) mutant is slower than the equivalent charge neutralization (Glu----Gln) mutant, showing that the newly introduced positive charge disrupts the orienting network. Thus, electrostatically facilitated diffusion rates can be increased by design, provided the detailed structural integrity of the active-site electrostatic network is maintained.

Eight mutants of barley (Hordeum vulgare cv Maris Mink) lacking the chloroplast isozyme of glutamine synthetase (EC 6.3.1.2.) were isolated by their inability to grow under photorespiratory conditions. The cytoplasmic isozyme of glutamine synthetase was present in the leaves of all the mutants, with activities comparable to the wild-type (10-12 nanokatals per gram fresh weight). The mutant plants developed normally and were fully fertile under conditions that minimize photorespiration. In 1% O(2) the rate of CO(2) fixation in leaves of one of the mutants, RPr 83/32, was the same as the wild-type, but in air this rate declined to 60% of the wild-type after 30 minutes. During this time the ammonia concentration in leaves of the mutant rose from 1 to 50 micromoles per gram fresh weight. Such ammonia accumulation in air was found in all the mutant lines. In back-crosses with the parent line, F(1) plants were viable in air. In the F(2) generation, nonviability in air and the lack of chloroplast glutamine synthetase co-segregated, in both the lines tested. These two lines and four others proved to be allelic; we designate them gln 2a-f. The characteristics of these mutants conclusively demonstrate the major role of chloroplast glutamine synthetase in photorespiration and its associated nitrogen recycling.

The epothilones are naturally occurring antimitotic drugs that share with the taxanes a similar mechanism of action without apparent structural similarity. Although photoaffinity labeling and electron crystallographic studies have identified the taxane-binding site on beta-tubulin, similar data are not available for epothilones. To identify tubulin residues important for epothilone binding, we have isolated two epothilone-resistant human ovarian carcinoma sublines derived in a single-step selection with epothilone A or B. These epothilone-resistant sublines exhibit impaired epothilone- and taxane-driven tubulin polymerization caused by acquired beta-tubulin mutations (beta274(Thr->>Ile) and beta282(Arg->>Gln)) located in the atomic model of alphabeta-tubulin near the taxane-binding site. Using molecular modeling, we investigated the conformational behavior of epothilone, which led to the identification of a common pharmacophore shared by taxanes and epothilones. Although two binding modes for the epothilones were predicted, one mode was identified as the preferred epothilone conformation as indicated by the activity of a potent pyridine-epothilone analogue. In addition, the structure-activity relationships of multiple taxanes and epothilones in the tubulin mutant cells can be fully explained by the model presented here, verifying its predictive value. Finally, these pharmacophore and activity data from mutant cells were used to model the tubulin binding of sarcodictyins, a distinct class of microtubule stabilizers, which in contrast to taxanes and the epothilones interact preferentially with the mutant tubulins. The unification of taxane, epothilone, and sarcodictyin chemistries in a single pharmacophore provides a framework to study drug-tubulin interactions that should assist in the rational design of agents targeting tubulin.

Mutations causing constitutive synthesis of glutamine synthetase (GlnC(-) phenotype) were transferred from Klebsiella aerogenes into Klebsiella pneumoniae by P1-mediated transduction. Such GlnC(-) strains of K. pneumoniae have constitutive levels of glutamine synthetase. Two of three GlnC(-) strains of K. pneumoniae studied, each containing independently isolated mutations that confer the GlnC(-) phenotype, continue to synthesize nitrogenase in the presence of NH(4) (+). One strain, KP5069, produces 30% as much nitrogenase when grown in the presence of 15 mM NH(4) (+) as in its absence. The GlnC(-) phenotype allows the synthesis of nitrogenase to continue under conditions that completely repress nitrogenase synthesis in the wild-type strain. Glutamine auxotrophs of K. pneumoniae, that do not produce catalytically active glutamine synthetase, are unable to synthesize nitrogenase during nitrogen limited growth. Complementation of K. pneumoniae Gln(-) strains by an Escherichia coli episome (F'133) simultaneously restores glutamine synthetase activity and the ability to synthesize nitrogenase. These results indicate a role for glutamine synthetase as a positive control element for nitrogen fixation in K. pneumoniae.

Endopeptidase-24.11 (EC 3.4.24.11), purified to homogeneity from pig kidney, was shown to hydrolyse a wide range of neuropeptides, including enkephalins, tachykinins, bradykinin, neurotensin, luliberin and cholecystokinin. The sites of hydrolysis of peptides were identified, indicating that the primary specificity is consistent with hydrolysis occurring at bonds involving the amino group of hydrophobic amino acid residues. Of the substrates tested, the amidated peptide substance P is hydrolysed the most efficiently (Km = 31.9 microM; kcat. = 5062 min-1). A free alpha-carboxy group at the C-terminus of a peptide substrate is therefore not essential for efficient hydrolysis by the endopeptidase. A large variation in kcat./Km values was observed among the peptide substrates studied, a finding that reflects a significant influence of amino acid residues, remote from the scissile bond, on the efficiency of hydrolysis. These subsite interactions between peptide substrate and enzyme thus confer some degree of functional specificity on the endopeptidase. The inhibition of endopeptidase-24.11 by several compounds was compared with that of pig kidney peptidyldipeptidase A (EC 3.4.15.1). Of the inhibitors examined, only N-[1(R,S)-carboxy-2-phenylethyl]-Phe-p-aminobenzoate inhibited endopeptidase-24.11 but not peptidyldipeptidase. Captopril (D-3-mercapto-2-methylpropanoyl-L-proline), Teprotide (pGlu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro) and MK422 [N-[(S)-1-carboxy-3-phenylpropyl]-L-Ala-L-Pro] were highly selective as inhibitors of peptidyldipeptidase. Although not wholly specific, phosphoramidon was a more potent inhibitor of endopeptidase-24.11 than were any of the synthetic compounds tested.

The 11-kD protease (PR) encoded by the human immunodeficiency virus 1 (HIV-1) is essential for the correct processing of viral polyproteins and the maturation of infectious virus, and is therefore a target for the design of selective acquired immunodeficiency syndrome (AIDS) therapeutics. To facilitate the identification of novel inhibitors of HIV-1 PR, as well as to permit detailed studies on the enzymology and inhibition of this enzyme, a continuous assay for its activity was developed that was based on intramolecular fluorescence resonance energy transfer (RET). The assay used the quenched fluorogenic substrate 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL)--Ser Gln Asn Tyr Pro Ile Val Gln--5-[(2-aminoethyl)amino]naphthalene-1 sulfonic acid (EDANS), whose peptide sequence is derived from a natural processing site for HIV-1 PR. Incubation of recombinant HIV-1 PR with the fluorogenic substrate resulted in specific cleavage at the Tyr-Pro bond and a time-dependent increase in fluorescence intensity that was linearly related to the extent of substrate hydrolysis. An internally quenched fluorogenic substrate was also designed that was selectively cleaved by the related PR from avian myeloblastosis virus (AMV). The fluorescence quantum yields of the HIV-1 PR and AMV PR substrates in the RET assay increased by 40.0- and 34.4-fold, respectively, per mole of substrate cleaved. Because of its simplicity, rapidity, and precision in the determination of reaction rates required for kinetic analysis, this method offers many advantages over the commonly used high-performance liquid chromatography- or electrophoresis-based assays for peptide substrate hydrolysis by retroviral PRs.

The human endonuclease III (hNTH1), a homolog of the Escherichia coli enzyme (Nth), is a DNA glycosylase with abasic (apurinic/apyrimidinic (AP)) lyase activity and specifically cleaves oxidatively damaged pyrimidines in DNA. Its cDNA was cloned, and the full-length enzyme (304 amino acid residues) was expressed as a glutathione S-transferase fusion polypeptide in E. coli. Purified wild-type protein with two additional amino acid residues and a truncated protein with deletion of 22 residues at the NH2 terminus were equally active and had absorbance maxima at 280 and 410 nm, the latter due to the presence of a [4Fe-4S]cluster, as in E. coli Nth. The enzyme cleaved thymine glycol-containing form I plasmid DNA and a dihydrouracil (DHU)-containing oligonucleotide duplex. The protein had a molar extinction coefficient of 5.0 x 10(4) and a pI of 10. With the DHU-containing oligonucleotide duplex as substrate, the Km was 47 nM, and kcat was approximately 0.6/min, independent of whether DHU paired with G or A. The enzyme carries out beta-elimination and forms a Schiff base between the active site residue and the deoxyribose generated after base removal. The prediction of Lys-212 being the active site was confirmed by sequence analysis of the peptide-oligonucleotide adduct. Furthermore, replacing Lys-212 with Gln inactivated the enzyme. However, replacement with Arg-212 yielded an active enzyme with about 85-fold lower catalytic specificity than the wild-type protein. DNase I footprinting with hNTH1 showed protection of 10 nucleotides centered around the base lesion in the damaged strand and a stretch of 15 nucleotides (with the G opposite the lesion at the 5'-boundary) in the complementary strand. Immunological studies showed that HeLa cells contain a single hNTH species of the predicted size, localized in both the nucleus and the cytoplasm.

An avian-origin human-infecting influenza (H7N9) virus was recently identified in China. We have evaluated the viral hemagglutinin (HA) receptor-binding properties of two human H7N9 isolates, A/Shanghai/1/2013 (SH-H7N9) (containing the avian-signature residue Gln(226)) and A/Anhui/1/2013 (AH-H7N9) (containing the mammalian-signature residue Leu(226)). We found that SH-H7N9 HA preferentially binds the avian receptor analog, whereas AH-H7N9 HA binds both avian and human receptor analogs. Furthermore, an AH-H7N9 mutant HA (Leu(226) → Gln) was found to exhibit dual receptor-binding property, indicating that other amino acid substitutions contribute to the receptor-binding switch. The structures of SH-H7N9 HA, AH-H7N9 HA, and its mutant in complex with either avian or human receptor analogs show how AH-H7N9 can bind human receptors while still retaining the avian receptor-binding property.

Short peptides that contain significant alpha-helical structure in aqueous solution allow the investigation of the role of amino acid side chains in stabilizing or destabilizing alpha-helix structure. A host-guest system of soluble synthetic peptides was designed that consisted of chains with the block sequence TyrSerGlu4Lys4X3Glu4Lys4, denoted EXK, in which X represents any "guest" amino acid residue. Circular dichroism spectroscopy indicates that the extent of helicity of these peptides follows the order Ala greater than Leu greater than Met greater than Gln greater than Ile greater than Val greater than Ser greater than Thr greater than Asn greater than Gly. This order differs from both host-guest copolymer values (Met greater than Ile greater than Leu greater than Ala greater than Gln greater than Val greater than Thr greater than Asn greater than Ser greater than Gly) and the tendencies of these amino acids to occur in helices in globular proteins (Ala greater than Met greater than Leu greater than Gln greater than Ile greater than Val greater than Asn, Thr greater than Ser greater than Gly), but matches the order found in a series of synthetic coiled-coil alpha helices, except for Ser. Proton nuclear magnetic resonance analysis of several EXK peptides indicates that these peptides are partially helical, with the helical residues favoring the amino terminus.

We examined the response of Streptococcus pneumoniae 7785 to clinafloxacin, a novel C-8-substituted fluoroquinolone which is being developed as an antipneumococcal agent. Clinafloxacin was highly active against S. pneumoniae 7785 (MIC, 0.125 microg/ml), and neither gyrA nor parC quinolone resistance mutations alone had much effect on this activity. A combination of both mutations was needed to register resistance, suggesting that both gyrase and topoisomerase IV are clinafloxacin targets in vivo. The sparfloxacin and ciprofloxacin MICs for the parC-gyrA mutants were 16 to 32 and 32 to 64 microg/ml, respectively, but the clinafloxacin MIC was 1 microg/ml, i.e., within clinafloxacin levels achievable in human serum. S. pneumoniae 7785 mutants could be selected stepwise with clinafloxacin at a low frequency, yielding first-, second-, third-, and fourth-step mutants for which clinafloxacin MICs were 0.25, 1, 6, and 32 to 64 microg/ml, respectively. Thus, high-level resistance to clinafloxacin required four steps. Characterization of the quinolone resistance-determining regions of the gyrA, parC, gyrB, and parE genes by PCR, HinfI restriction fragment length polymorphism, and DNA sequence analysis revealed an invariant resistance pathway involving sequential mutations in gyrA or gyrB, in parC, in gyrA, and finally in parC or parE. No evidence was found for other resistance mechanisms. The gyrA mutations in first- and third-step mutants altered GyrA hot spots Ser-83 to Phe or Tyr (Escherichia coli coordinates) and Glu-87 to Gln or Lys; second- and fourth-step parC mutations changed equivalent hot spots Ser-79 to Phe or Tyr and Asp-83 to Ala. gyrB and parE changes produced novel alterations of GyrB Glu-474 to Lys and of Pro-454 to Ser in the ParE PLRGK motif. Difficulty in selecting first-step gyrase mutants (isolated with 0.125 [but not 0.25] microg of clinafloxacin per ml at a frequency of 5.0 x 10(-10) to 8.5 x 10(-10)) accompanied by the small (twofold) MIC increase suggested only a modest drug preference for gyrase. Given the susceptibility of defined gyrA or parC mutants, the results suggested that clinafloxacin displays comparable if unequal targeting of gyrase and topoisomerase IV. Dual targeting and the intrinsic potency of clinafloxacin against S. pneumoniae and its first- and second-step mutants are desirable features in limiting the emergence of bacterial resistance.

Asthma and atopy show epidemiological association and are biologically linked by T-helper type 2 (T(h)2) cytokine-driven inflammatory mechanisms. IL-4 operates through the IL-4 receptor (IL-4R, a heterodimer of IL-4Ralpha and either gammac or IL-13Ralpha1) and IL-13 operates through IL-13R (a heterodimer of IL-4Ralpha and IL-13Ralpha1) to promote IgE synthesis and IgE-based mucosal inflammation which typify atopy. Recent animal model data suggest that IL-13 is a central cytokine in promoting asthma, through the stimulation of bronchial epithelial mucus secretion and smooth muscle hyper-reactivity. We investigated the role of common genetic variants of IL-13 and IL-13Ralpha1 in human asthma, considering IgE levels. A novel variant of human IL-13, GlnGln, is important in the internal constitution of the ligand and crucial in ligand-receptor interaction. A non-coding variant of IL-13Ralpha1, A1398G, on chromosome Xq13, associated primarily with high IgE levels (OR = 3. 38 in males, 1.10 in females) rather than asthma. Thus, certain variants of IL-13 signalling are likely to be important promoters of human asthma; detailed functional analysis of their actions is needed.

Members of the PI 3-kinase-related kinase (PIKK) family function in mitogenic and stress-induced signaling pathways in eukaryotic cells. Here, we characterize the newest PIKK family member, hSMG-1, as a genotoxic stress-activated protein kinase that displays some functional overlap with the related kinase, ATM, in human cells. Both ATM and hSMG-1 phosphorylate Ser/Thr-Gln-containing target sequences in the checkpoint protein p53 and the nonsense-mediated mRNA decay (NMD) protein hUpf1. Expression of hSMG-1 is required for optimal p53 activation after cellular exposure to genotoxic stress, and depletion of hSMG-1 leads to spontaneous DNA damage and increased sensitivity to ionizing radiation (IR). Moreover, IR exposure triggers hUpf1 phosphorylation at Ser/Thr-Gln motifs, and both ATM and hSMG-1 contribute to these phosphorylation events. Finally, NMD is suppressed in hSMG-1- but not ATM-deficient cells. These results indicate that hSMG-1 plays important roles in the maintenance of both genome and transcriptome integrity in human cells.

The major mammalian heat shock or "stress" proteins (molecular masses of 90,000, 72,000, and 73,000 daltons) have been purified from stressed HeLa cells. The 90,000-dalton protein co-purified with small amounts of a 100,000-dalton protein which was identified as one of the other stress proteins in these cells. The 72,000- and 73,000-dalton proteins co-purified throughout the fractionation scheme, apparently as a mixture of monomeric forms of the two proteins. From sedimentation velocity and gel filtration analysis, it was found that the 90,000/100,000-dalton protein mixture had a Stokes radius of 69A and a s20,w value of 5.8 while the 72,000/73,000-dalton protein mixture had a Stokes radius of 42.6A and a s20,w value of 4.3. The purified proteins migrated identically in two-dimensional gel electrophoretograms with their counterparts from total cell lysates of [35S]methionine-labeled stressed HeLa cells. Peptide mapping experiments indicated that the 72,000- and 73,000-dalton proteins contained common peptides while the 90,000- and 100,000-dalton proteins appeared to be distinct. Amino acid analysis of the 90,000- and a mixture of the 72,000/73,000-dalton proteins showed that both contained relatively high amounts of Asp/Asn and Glu/Gln.

tRNA(2Gln) made in vitro by transcription with T7 RNA polymerase does not contain the pseudouridines at positions 38, 39, and 55, the 4-thiouridine at position 8, or any of the methylated bases found in the tRNA(2Gln) made in vivo. Cocrystals of unmodified tRNA(2Gln) complexed with glutaminyl-tRNA synthetase from Escherichia coli are isomorphous with those of the complex with modified tRNA(2Gln). A difference electron density map between the complexes with modified and unmodified tRNAs calculated at 2.5-A resolution shows no differences in the protein or tRNA structures, except for some very small shifts in atoms contacting the thiol at the 4 position of uridine 8 that are required to accommodate the smaller oxygen in the unmodified tRNA. Perhaps the most functionally significant change in the unmodified tRNA is the absence of the specifically bound water molecules that are observed to cross-link the N5 of the pseudo-uridines to their 5' phosphate. This suggests a possible role for pseudouridinylation in stabilization of the tRNA through water-mediated linking of these modified bases to the backbone, which is consistent with the lower thermal stability of the unmodified tRNA. An identical water-bridging structure is possible at four of the five other psuedo-uridines in known tRNA structures.

The structure of phosphate-free bovine ribonuclease A has been refined at 1.26-A resolution by a restrained least-squares procedure to a final R factor of 0.15. X-ray diffraction data were collected with an electronic position-sensitive detector. The final model consists of all atoms in the polypeptide chain including hydrogens, 188 water sites with full or partial occupancy, and a single molecule of 2-methyl-2-propanol. Thirteen side chains were modeled with two alternate conformations. Major changes to the active site include the addition of two waters in the phosphate-binding pocket, disordering of Gln-11, and tilting of the imidazole ring of His-119. The structure of the protein and of the associated solvent was extensively compared with three other high-resolution, refined structures of this enzyme.

The product of the glnR gene is required for nitrogen regulation of the synthesis of glutamine synthesis (Gln synthetase) [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] and two periplasmic transport proteins that are subject to nitrogen control in Salmonella. Strains with mutations to loss of function of the glnR product [e.g., a strain with a Tn10 insertion or one with an ICR-induced (frameshift) mutation in glnR] have about 3% as much Gln synthetase as a fully derepressed wild-type strain and are unable to increase synthesis of this enzyme or periplasmic transport proteins in response to nitrogen limitation. The structural gene for Gln synthetase, glnA, and those for the periplasmic transport proteins are unlinked on the chromosome; thus, glnR appears to encode a diffusible positive regulatory element. Consistent with this, the mutant glnR allele is recessive to the wild-type allele with regard to expression of glnA (synthesis of Gln synthetase). Although glnR is closely linked to glnA, strains with mutations to complete loss of function of the glnR product can be distinguished from glnA strains by their ability to produce detectable Gln synthetase and to grow in the absence of glutamine. To demonstrate unequivocally that glnR is distinct from glnA, we have purified and characterized Gln synthetase from a strain with a Tn10 insertion in glnR. Because the properties of Gln synthetase from the insertion mutant, most importantly the carboxyl-terminal sequence of amino acids, are the same as those of synthetase from wild type, the Tn10 insertion cannot be in glnA (if it were, the carboxyl terminus of Gln synthetase would have to be altered); therefore we conclude that the Tn10 insertion is in a regulatory gene, glnR, which is distinct from glnA. A model for the function of the glnR product together with the previously defined glnF product in mediating nitrogen control is discussed.

Vibrio cholerae, the bacterium that causes cholera, has a pathogenic cycle consisting of a free-swimming phase outside its host, and a sessile virulent phase when colonizing the human small intestine. We have cloned the V. cholerae homologue of the rpoN gene (encoding sigma54) and determined its role in the cholera pathogenic cycle by constructing an rpoN null mutant. The V. cholerae rpoN mutant is non-motile; examination of this mutant by electron microscopy revealed that it lacks a flagellum. In addition to flagellar synthesis, sigma54 is involved in glutamine synthetase expression. Moreover, the rpoN mutant is defective for colonization in an infant mouse model of cholera. We present evidence that the colonization defect is distinct from the non-motile and Gln phenotypes of the rpoN mutant, implicating multiple and distinct roles of sigma54 during the V. cholerae pathogenic cycle. RNA polymerase containing sigma54 (sigma54-holoenzyme) has an absolute requirement for an activator protein to initiate transcription. We have identified three regulatory genes, flrABC (flagellar regulatory proteins ABC) that are additionally required for flagellar synthesis. The flrA and flrC gene products are sigma54-activators and form a flagellar transcription cascade. flrA and flrC mutants are also defective for colonization; this phenotype is probably independent of non-motility. An flrC constitutive mutation (M114->>I) was isolated that is independent of its cognate kinase FlrB. Expression of the constitutive FlrCM114->>I from the cholera toxin promoter resulted in a change in cell morphology, implicating involvement of FlrC in cell division. Thus, sigma54 holoenzyme, FlrA and FlrC transcribe genes for flagellar synthesis and possibly cell division during the free-swimming phase of the V. cholerae life cycle, and some as yet unidentified gene(s) that aid colonization within the host.

We have investigated the mechanism by which cultured endothelial cells generate L-arginine (L-Arg), the substrate for the biosynthesis of endothelium-derived relaxing factor. When Arg-depleted endothelial cells were incubated in Krebs' solution for 60 min, L-Arg levels were significantly (9.7-fold) elevated. The generation of L-Arg coincided with a substantial decrease (90%) in intracellular L-glutamine (L-Gln), whereas all other amino acids were virtually unaffected. Changes in calcium, pH, or oxygen tension had no effect on L-Arg generation, which was, however, prevented when the cells were incubated in culture medium containing L-Gln. L-Arg generated by endothelial cells labeled with L-[14C]Arg was derived from an unlabeled intracellular source, for the specific activity of the intracellular L-Arg pool decreased substantially (8.8-fold) over 60 min. Arg-depleted endothelial cells did not form urea or metabolize L-ornithine but converted L-citrulline (L-Cit) to L-Arg possibly via formation of L-argininosuccinic acid. Nondepleted cells stimulated with the calcium ionophore A23187 showed only a transient accumulation of L-Cit, indicating that L-Cit is recycled to L-Arg during the biosynthesis of endothelium-derived relaxing factor. The generation of L-Arg by Arg-depleted endothelial cells was partially (45%) blocked by protease inhibitors, and various Arg-containing dipeptides were rapidly cleaved to yield L-Arg. Thus, cultured endothelial cells recycle L-Cit to L-Arg and possibly liberate peptidyl L-Arg. The Arg-Cit cycle appears to be the equivalent in the endothelial cell to the formation of urea by the liver. The biosynthesis of endothelium-derived relaxing factor may, therefore, not only produce a powerful vasodilator but also relieve the endothelial cell of excess nitrogen.

A method is presented that positions polar hydrogen atoms in protein structures by optimizing the total hydrogen bond energy. For this goal, an empirical hydrogen bond force field was derived from small molecule crystal structures. Bifurcated hydrogen bonds are taken into account. The procedure also predicts ionization states of His, Asp, and Glu residues. During optimization, side-chain conformations of His, Gln, and Asn residues are allowed to change their last chi angle by 180 degrees to compensate for crystallographic misassignments. Crystal structure symmetry is taken into account where appropriate. The results can have significant implications for molecular dynamics simulations, protein engineering, and docking studies. The largest impact, however, is in protein structure verification: over 85% of protein structures tested can be improved by using our procedure.

Mutations in the glnA region of the Escherichia coli chromosome due to Mu prophage insertion result in two phenotypic classes. One class is Gln- and does not synthesize glutamine synthetase[L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] under any growth condition. The other class produces a low level of glutamine synthetase under all growth conditions and is uncoupled from the regulatory effects of mutations in the glnF and glnD genes. Complementation analysis demonstrates that these two classes of insertions are in different cistrons. From these data we suggest that a regulatory gene, glnG, tightly linked to glnA, mediates both activation and repression of glutamine synthetase synthesis. An analysis of the evidence accumulated to date makes it unlikely that glnG is the only gene in the glnA region involved in the complex system of nitrogen regulation.

Genetic elements coding for proteins that present amino acid identity with the conserved motifs of retroviral reverse transcriptases constitute the retroid family. With the exception of reverse transcriptases encoded by mitochondrial plasmids of Neurospora, all reverse transcriptases have an absolute requirement for a primer to initiate DNA synthesis. In retroviruses, plant pararetroviruses, and retrotransposons (transposons containing long terminal repeats), DNA synthesis is primed by specific tRNAs. All these retroelements contain a primer binding site presenting a Watson-Crick complementarity with the primer tRNA. The tRNAs most widely used as primers are tRNA(Trp), tRNA(Pro), tRNA(1,2Lys), tRNA(3Lys), tRNA(iMet). Other tRNAs such as tRNA(Gln), tRNA(Leu), tRNA(Ser), tRNA(Asn) and tRNA(Arg) are also occasionally used as primers. In the retroviruses and plant pararetroviruses, the primer binding site is complementary to the 3' end of the primer tRNA. In the case of retrotransposons, the primer binding site is either complementary to the 3' end or to an internal region of the primer tRNA. Additional interactions taking place between the primer tRNA and the retro-RNA outside of the primer binding site have been evidenced in the case of Rous sarcoma virus, human immunodeficiency virus type I, and yeast retrotransposon Ty1. A selective encapsidation of the primer tRNA, probably promoted by interactions with reverse transcriptase, occurs during the formation of virus or virus-like particles. Annealing of the primer tRNA to the primer binding site appears to be mediated by reverse transcriptase and/or the nucleocapsid protein. Modified nucleosides of the primer tRNA have been shown to be important for replication of the primer binding site, encapsidation of the primer (in the case of Rous sarcoma virus), and interaction with the genomic RNA (in the case of human immunodeficiency virus type I).