The Composition of Saccharogenic Amylase from Rhizopus javanicus and the Isolation of Glycopeptides

Saccharogenic amylase from Rhizopus javanicus sp. 3–46 was known to be a glycoprotein which contained 27 residues of mannose and 4 residues of N-acetylglucosamine per mole of the saccharogenic amylase. Attempts have been made to obtain glycopeptides from the saccharogenic amylase. Three glycopeptides, GP-I-a, GP-I-b and GP-II, were separated from a Pronase digest of heat-denatured saccharogenic amylase by gel filtration on Sephadex G-50 and chromatography on DEAE-Sephadex A-25. GP-I-a contained asparagine, glycine, mannose and N-acetylglucosamine in a molar ratio of 1: 1: 6: 2. GP-I-b contained asparagine, threonine, mannose and N-acetylglucosamine in a molar ratio of 1: 1: 9:2. GP-II consisted of threonine, serine, proline, alanine and mannose in a molar ratio of 6: 2: 2: 2: 12.     

Physiological and genetical studies on a mutant of Salmonella typhimurium which is temperature-sensitive for DNA synthesis

Summary The following evidence supports the view that a temperature-sensitive mutant of Salmonella typhimurium (11 G) is defective in DNA synthesis initiation: a) the increment in DNA synthesis at 38° is abolished by prior completion of rounds of replication at 25°. b) The extent of the increment at 38° is greatly increased by prior growth in the presence of a DNA synthesis inhibitor. c) Density gradient centrifugation demonstrates that the terminal region of the chromosomes is preferentially replicated at 38°. d) Preferential replication of the chromosome origins occurs at 25° after a period at 38°. The parental strain in the presence of a DNA synthesis inhibitor or the mutant at 38° (without inhibitor) show increased sensitivity to the detergent sodium deoxycholate, possibly due to a secondary effect of DNA synthesis inhibition on membrane composition. Strains of 11 G carrying episomes transfer the episomes very poorly at 38° suggesting a rôle for the chromosomal initiation apparatus in episome transfer. Continued cell division of 11 G with the production of DNA-less cells at 38° is not due to the presence of a rec mutation and no secondary mutation responsible for such division has been found. The lesion maps close to leu on the Salmonella chromosome.     

Cell division and the production of cells lacking nuclear bodies in a mutant of Salmonella typhimurium

Summary There are two distinct division phases when the temperature-sensitive DNA synthesis initiation mutant Salmonella typhimurium strain 11G is shifted from 25° to 38°. The first phase appears to represent segregation of the nuclear bodies formed at 38°. Division in this phase takes place at the normal size and produces mainly organisms with one nuclear body. It is dependent on the termination of the rounds of replication in operation at the time of the temperature shift and sensitive to low doses of penicillin. This division phase continues for 60–75 min and then after a short lag division restarts. At first the cells undergoing the second division phase are only slightly larger than normal but they soon grow into short filaments which bud off cells at both ends even if only one nuclear body is present. The cells budded off in this division phase are about 3 long on both broth and M M. They lack nuclear bodies but have a small amount of DNA which may be exclusively in the form of a large plasmid. This second division phase is also dependent on rounds of replication being allowed to terminate at 38° and is sensitive to low levels of penicillin. It is 80–90 min after the temperature shift before the second division phase starts and this lag is maintained even if rounds of replication have been completed prior to the temperature shift by amino acid starvation at 25°. The occurrence of this lag and the demonstration (using penicillin) of potential division sites at regular intervals along 11G filaments suggest that division is initiated some time before the actual division event.     

The attenuated phenotype of a Salmonella typhimurium flgM mutant is related to expression of FliC flagellin.

The flgM gene of Salmonella typhimurium encodes a negative regulator of flagellin synthesis that acts by inhibiting the flagellum-specific sigma factor FliA (sigma 28), but only when a mutation in a flagellar basal body, hook, or switch gene is present. We previously showed that FlgM is also necessary for the virulence of S. typhimurium in the mouse model of typhoid fever and proposed that FlgM is required to modulate the activity of the FliA sigma factor, which, in turn, regulates a gene involved in virulence. In this investigation, we observed that (i) the in vitro generation times of flgM mutant and wild-type strains of S. typhimurium were indistinguishable, as were the amounts of flagellin produced by the strains; (ii) the 50% lethal doses of fliA mutant and wild-type strains of S. typhimurium were similar in orally infected mice; and (iii) inactivation of the FliA-regulated flagellin gene fliC in an flgM S. typhimurium mutant resulted in a virulent phenotype. Therefore, we now conclude that expression of the FliC flagellin subunit in an flgM strain is responsible for the attenuated phenotype of an flgM mutant and that FliA does not appear to positively regulate virulence genes in S. typhimurium. Our results suggest that the normal regulation of flagellum synthesis appears to be necessary for virulence and that there may be an advantage conferred in vivo by expression of a particular flagellar phenotype of S. typhimurium.     

prsB is an allele of the Salmonella typhimurium prsA gene: characterization of a mutant phosphoribosylpyrophosphate synthetase.

The Salmonella typhimurium prsB mutation was previously mapped at 45 min on the chromosome, and a prsB strain was reported to produce undetectable levels of phosphoribosylpyrophosphate (PRPP) synthetase activity and very low levels of immunologically cross-reactive protein in vitro (N.K. Pandey and R.L. Switzer, J. Gen. Microbiol, 128:1863-1871, 1982). We have shown by P22-mediated transduction that the prsB gene is actually an allele of prsA, the structural gene for PRPP synthetase, which maps at 35 min. The prsB (renamed prs-100) mutant produces about 20% of the activity and 100% of the cross-reactive material of wild-type strains. prs-100 mutant strains are temperature sensitive, as is the mutant PRPP synthetase in vitro. The prs-100 mutation is a C-to-T transition which results in replacement of Arg-78 in the mature wild-type enzyme by Cys. The mutant PRPP synthetase was purified to greater than 98% purity. It possessed elevated Michaelis constants for both ATP and ribose-5-phosphate, a reduced maximal velocity, and reduced sensitivity to the allosteric inhibitor ADP. The mutant enzyme had altered physical properties and was susceptible to specific cleavage at the Arg-101-to-Ser-102 bond in vivo. It appears that the mutation alters the enzyme's kinetic properties through substantial structural alterations rather than by specific perturbation of substrate binding or catalysis.     

Crystallization of lipopolysaccharide from a Salmonella typhimurium semi-rough (SR) mutant.

Salmonella typhimurium SR-form lipopolysaccharide (LPS), consisting of a single repeating unit of the O-antigenic polysaccharide, linked to the R-core consisting of oligosaccharide that is, in turn, linked to lipid A, formed crystals whose shapes were hexagonal plates, discoids, and solid columns when precipitated by the addition of 2 volumes of 95% ethanol containing 375 mM MgCl2 and kept in 70% ethanol containing 250 mm MgCl2 at 4 C for 10 days. Among these crystals, the basic form is considered to be the hexagonal plates. Analyses of hexagonal plate crystals showed that they consist of hexagonal lattices with a lattice constant (a axis) of 4.62 A and longitudinal axis (c axis) of approximately 100 A. In X-ray diffraction patterns in the low-angle region, crystals of S. typhimurium SR-form LPS exhibited much less distinct reflections when compared with crystals of synthetic Escherichia coli-type lipid A. In contrast to the previous finding that S. minnesota S-form LPS possessing the O-antigenic polysaccharide does not crystallize under the same experimental conditions as used in the present study, the presence of a single repeating unit of the O-antigenic polysaccharide does not inhibit crystallization.     

Energetics of glucose uptake in a Salmonella typhimurium mutant containing uncoupled enzyme IIGlc

Uncoupled enzyme II^Glc of the phosphoenolpyruvate (PEP): glucose phosphotransferase system (PTS) in Salmonella typhimurium is able to catalyze glucose transport in the absence of PEP-dependent phosphorylation. We have studied the energetics of glucose uptake catalyzed by this uncoupled enzyme II^Glc. The molar growth yields on glucose of two strains cultured anaerobically in glucose-limited chemostat-and batch cultures were compared. Strain PP 799 transported and phosphorylated glucose via an intact PTS, while strain PP 952 took up glucose exclusively via uncoupled enzyme II^Glc, followed by ATP-dependent phosphorylation by glucokinase. Thus the strains were isogenic except for the mode of uptake and phosphorylation of the growth substrate. PP 799 and PP 952 exhibited similar Y _Glc values. Assuming equal Y _ATP values for both strains this result indicated that there were no energetic demands for glucose uptake via uncoupled enzyme II^Glc.Abbreviations PTS phosphoenolpyruvate: carbohydrate phosphotransferase system - HPr histidine-containing phosphocarrier protein - GalP galactose permease     

Galactose epimeraseless (GalE) mutant G30 of Salmonella typhimurium is a good potential live oral vaccine carrier for fimbrial antigens

Abstract Extracts of the transformable cyanobacterial strain Anacystis nidulans R2 were analyzed for the presence of restriction endonuclease. One enzyme, Ani I, was found and determined to be sequence-specific on the basis of its ability to cleave several Bacillus plasmids at a limited number of sites. The activity of this enzyme is significantly reduced in extracts prepared from cell cultures grown at 38°C.     

A single point mutation in 3-deoxy-D-manno-octulosonate-8-phosphate synthase is responsible for temperature sensitivity in a mutant strain of Salmonella typhimurium

Salmonella typhimurium mutants conditionally deficient in 3-deoxy-d-manno-octulosonate-8-phosphate (KDO8P) synthase activity play a central role in our understanding of lipopolysaccharide function in enteric bacteria. The detailed characterization of KDO8P synthase from such a mutant, however, has not been previously reported. To address this issue KDO8P synthase from S. typhimurium AG701 and from a related temperature-sensitive strain (S. typhimurium AG701i50) have been overexpressed in Escherichia coli and purified to homogeneity. The enzyme from the temperature-sensitive strain has a single proline to serine substitution at position 145, leading to an increase in K(m) for both substrates, d-arabinose 5-phosphate and phosphoenolpyruvate. Analytical gel filtration and native polyacrylamide gel electrophoresis indicate that this enzyme also has an altered oligomeric state. These observations are rationalized through an examination of the structure of E. coli KDO8P synthase, which has 93% sequence identity to the enzyme from S. typhimurium.     

Biochemical and genetic characterization of a mutant of Salmonella typhimurium defective in a locus for glutamate dehydrogenase activity

Summary By two consecutive treatments with N-methyl-N-nitro-N-nitrosoguanidine we obtained mutant SM151 of Salmonella typhimurium which differs from the parental LT2 strain in: a) is able to use l-glutamate as carbon source (first mutation), and b) requires that amino acid for growth (second mutation). It was found that the requirement of mutant SM151 for glutamate was due to a very low activity of glutamate dehydrogenase. Both glutamate-oxaloacetate transaminase and aspartase activities were present at normal levels. Glutamate dehydrogenase activity was strongly repressed by glutamate; aspartase activity was under severe catabolite (glucose) repression, while glutamate-oxaloacetate transaminase was partially repressed by glutamate. By conjugation and transduction the locus gdh, responsible for the low activity of the glutamate dehydrogenase of mutant SM151, was located at about minute 128 of the bacterial chromosome and found to be linked to the argC, argF, and metB loci.     

The isolation and characterisation of a mutant of Salmonella typhimurium defective in mutation frequency decline

A mutant of Salmonella typhimurium strain trpC3 has been isolated which is defective in mutation frequency decline (MFD) for UV-induced suppressor revertants to tryptophan independence. Several characteristics of this mutant, PW₄, suggest that it is altered in the timing or rate of the general excision repair mechanism. Survival is greater in strain PW₄ when the first post-irradiation cell division is delayed by the inhibition of immediate protein synthesis. Similarly, stationary phase cells, which show an extended lag after irradiation, are more UV-resistant than lag-phase cells, which recover more rapidly. These data are consistent with the hypothesis that, in contrast with the parent strain trpC₃, the time available in the mutant strain for the action of excision repair is critical in the determination of survival after UV treatment. Contransductional analysis of the mutant locus indicates close linkage to metE, a region in which excision repair genes have been located.     

An unstable mutant gene of the am locus of Neurospora results from a small duplication

We have cloned the unstable am mutant gene, am126, as well as the am gene from an am126 revertant. The mutation is a result of a 33-bp duplication that repeats a sequence starting 13 bp upstream of the 3' splice junction between intron 1 and exon 2 and extends 20 bp into exon 2. In addition, there is a G----A transition 2 bp upstream of the first copy of the duplicated sequence. In the revertant gene the wild-type sequence is precisely recovered, involving both the loss of the duplication and a reversion (A----G) of the associated transition. Our data suggest that only the more 5' of the two 3' splice junctions present in the duplicated version of the gene is used. This favors a 5'----3' scanning mechanism for exon splicing.     

Isolation and characterization of a temperature-sensitive lethal mutant of Salmonella typhimurium that is conditionally defective in 3-deoxy-D-manno-octulosonate-8-phosphate synthesis.

A new mutant of Salmonella typhimurium was isolated which possesses a temperature-sensitive defect in the synthesis of 3-deoxy-D-manno-octulosonic acid. The defect in 3-deoxy-D-manno-octulosonic acid synthesis is due to a temperature-sensitive 3-deoxy-D-manno-octulosonate-8-phosphate synthetase, and the mutant accumulates an incomplete lipid A under nonpermissive conditions. Evidence is presented which indicates that the incomplete lipid A molecule is most likely identical in structure to the lipid A precursor synthesized by previously characterized mutants conditionally defective in 3-deoxy-D-manno-octulosonic acid synthesis. However, unlike related mutants which undergo growth stasis under nonpermissive conditions, the accumulation of lipid A precursor in the new mutant results in cell death at elevated temperatures.     

Methanol oxidation in a spontaneous mutant of Thiosphaera pantotropha with a methanol-positive phenotype is catalysed by a dye-linked ethanol dehydrogenase

Abstract A spontaneous Thiosphaera pantotropha mutant (Tp9002) that is able to grow on methanol has been isolated. With hybridization experiments it has been demonstrated that mxaF , the gene encoding the large subunit of methanol dehydrogenase, is absent from T. pantotropha . In Tp9002, a dye-linked enzyme activity was found with a substrate specificity similar to that of the dye-linked ethanol dehydrogenase from Pseudomonas aeruginosa . The N-terminus of a 26-kDa cytochrome c , exclusively synthesized in Tp9002, is homologous to the N-terminus of the electron acceptor of ethanol dehydrogenase. These results suggest that in Tp9002 a dye-linked ethanol dehydrogenase is responsible for methanol oxidation, using a 26-kDa cytochrome c as electron acceptor.     

Biochemical analysis of SopE from Salmonella typhimurium, a highly efficient guanosine nucleotide exchange factor for RhoGTPases.

RhoGTPases are key regulators of eukaryotic cell physiology. The bacterial enteropathogen Salmonella typhimurium modulates host cell physiology by translocating specific toxins into the cytoplasm of host cells that induce responses such as apoptotic cell death in macrophages, the production of proinflammatory cytokines, the rearrangement of the host cell actin cytoskeleton (membrane ruffling), and bacterial entry into host cells. One of the translocated toxins is SopE, which has been shown to bind to RhoGTPases of the host cell and to activate RhoGTPase signaling. SopE is sufficient to induce profuse membrane ruffling in Cos cells and to facilitate efficient bacterial internalization. We show here that SopE belongs to a novel class of bacterial toxins that modulate RhoGTPase function by transient interaction. Surface plasmon resonance measurements revealed that the kinetics of formation and dissociation of the SopE.CDC42 complex are in the same order of magnitude as those described for complex formation of GTPases of the Ras superfamily with their cognate guanine nucleotide exchange factors (GEFs). In the presence of excess GDP, dissociation of the SopE.CDC42 complex was accelerated more than 1000-fold. SopE-mediated guanine nucleotide exchange was very efficient (e.g. exchange rates almost 10(5)-fold above the level of the uncatalyzed reaction; substrate affinity), and the kinetic constants were similar to those described for guanine nucleotide exchange mediated by CDC25 or RCC1. Far-UV CD spectroscopy revealed that SopE has a high content of alpha-helical structure, a feature also found in Dbl homology domains, Sec7-like domains, and the Ras-GEF domain of Sos. Despite the lack of any obvious sequence similarity, our data suggest that SopE may closely mimic eukaryotic GEFs.     

The Salmonella typhimurium flagellar basal body protein FliE is required for flagellin production and to induce a proinflammatory response in epithelial cells

During apical colonization by Salmonella typhimurium, intestinal epithelial cells orchestrate a proinflammatory response that involves secretion of chemoattractants, predominantly interleukin-8, which coordinate neutrophil trans-epithelial migration at the site of infection. This host-pathogen interaction requires several S. typhimurium genes. To identify novel genes that participate in this pathogen-induced proinflammatory response, we created S. typhimurium Tn-10 transposon mutants and identified a single mutant with Tn-10 insertional inactivation within the fliE flagellar locus that was able to adhere to and invade intestinal epithelial cells normally but was unable to induce interleukin-8 secretion in host cells. The fliE-deficient mutant failed to secrete flagellin and lacked any surface assembly of flagellae. Unlike wild-type S. typhimurium, the fliE-deficient mutant did not activate the IkappaBalpha/NF-kappaB signaling pathway or induce the coordinated trans-epithelial migration of isolated human neutrophils. Transcomplementation of the fliE-deficient mutant with a wild-type fliE-harboring plasmid restored all defects and produced a wild-type S. typhimurium phenotype. Furthermore, functional down-regulation of basolateral TLR5 completely inhibited the monolayers' ability to respond to both wild-type S. typhimurium and purified flagellin but had no affect on tumor necrosis factor alpha-induced responses. We therefore conclude that S. typhimurium fliE is essential for flagellin secretion, flagellar assembly, and S. typhimurium-induced proinflammatory responses through basolateral TLR5 and is consistent with the emerging model of S. typhimurium flagellin-induced inflammation.     

Synthesis of (2S,4R,5S)-2,4,6-Trimethyl-5-heptanolide,a Sex Pheromone Component for Macrocentvus grandii

(2S,4R,5S)-2,4,6-Trimethyl-5-heptanolide (1), a sex pheromone component for Macrocentvus grandii, was synthesized by starting from methyl (R)-citronellate (2) and employing bromolactonization (10→11) as the key reaction.     

The reduced stability of a plant alcohol dehydrogenase is due to the substitution of serine for a highly conserved phenylalanine residue

The zinc-binding long-chain alcohol dehydrogenases from plants and animals exhibit a considerable level of amino acid sequence conservation. While the functional importance of many of the conserved residues is known, the role of others has not yet been determined. We have identified a naturally occurring Adh-1 allele in the legume Phaseolus acutifolius with several unusual characteristics. Individuals homozygous for this allele, Adh-1CN, possess a single isozyme starch gel electrophoretic pattern suggestive of a null allele, and exhibit ADH enzyme activity levels ca. 60% lower than the standard wild-type Adh-1F line. Interestingly, analysis of Adh-1CN homozygotes on an alternative gel system indicates that Adh-1CN does encode a polypeptide capable of forming functional homo- and heterodimers. However, the levels of ADH activity displayed by these isozymes are far lower than those observed for the corresponding wild type ADH-1F isozymes. Dialysis experiments indicate that isozymes containing the ADH-1CN polypeptide are inactivated by slightly acidic conditions, which may explain the apparent null phenotype on starch gels. Elevated temperatures cause a similar loss of enzyme activity. The deduced amino acid sequences of ADH-1CN and ADH-1F were obtained from their corresponding cDNA clones, and the only significant difference detected between the two is a single amino acid replacement substitution. Residue 144 is occupied by phenylalanine in the ADH-1F polypeptide, whereas serine occupies this position in the ADH-1CN polypeptide. The proximity of residue 144 to the catalytic zinc in the substrate-binding pocket, coupled with the fact that it is integral to a defined hydrophobic core of the ADH polypeptide, may explain the observed disruptive effect that the serine substitution has on both the activity and stability of the ADH-1CN polypeptide. It also provides an explanation for the maintenance of phenylalanine or the structurally similar tyrosine at this residue in Zn-binding long-chain ADHs.