Crystallization of the NAD-dependent malic enzyme from the parasitic nematode Ascaris suum.

The malic enzyme from muscle mitochondria of the parasitic nematode Ascaris suum is a tetramer of 65 kDa monomers that catalyzes the oxidative decarboxylation of malate to pyruvate and CO2 with NAD cofactor as oxidant. This malic enzyme is critical to the nematode for muscle function under anaerobic conditions. Unlike mammalian versions of the enzyme such as that found in rat liver, which require NADP as cofactor, the nematode version is an NAD-dependent enzyme. We report the crystallization of samples of the nematode enzyme at room temperature from pH 7.5 solutions of polyethylene glycol 4000 containing magnesium sulfate, NAD and sodium tartronate. Immediately upon mixing of protein and precipitant solutions, a marked precipitation of the protein occurs. Out of this precipitate, crystals appear almost immediately, most commonly in a truncated cube form that can grow to 0.5 to 0.7 mm on a cube edge in two to three days. The crystals are trigonal, space group P3(1)21 or its enantiomer, with a = b = 131.2(7) A, c = 152.6(9) A, and two monomers per asymmetric unit. Fresh crystals diffract X-radiation from a synchrotron source (lambda = 0.95 A) to about 3.0 A resolution. Rotational analysis of Patterson functions indicates that the malic enzyme tetramer has 222 symmetry.     

Urease-null and hydrogenase-null phenotypes of a phylloplane bacterium reveal altered nickel metabolism in two soybean mutants

Mutation at either of two genetic loci (Eu2 or Eu3) in soybean (Glycine max [L.] Merr.) results in a pleiotropic elimination of the activity of both major urease isozymes. Surprisingly, the phenotype of a phylloplane bacterium, Methylobacterium mesophilicum, living on the leaves of eu2/eu2 or eu3-e1/eu3-e1 mutants is also affected by these plant mutations. The bacteria isolated from leaves of these soybean mutants have transient urease- and hydrogenase-deficient phenotypes that can be corrected by the addition of nickel to free-living cultures. The same bacterium growing on wild-type soybeans or on urease mutants eu1-sun/eu1-sun or eu4/eu4, each deficient in only one urease isozyme, are urease-positive. These results suggest that the bacterium living on the eu2/eu2 or eu3-e1/eu3-e1 mutant is unable to produce an active urease or hydrogenase because it is effectively starved for nickel. We infer that mutations at Eu2 or Eu3 result in defects in nickel metabolism but not in Ni²⁺ uptake or transport, because eu2/eu2 and eu3-e1/eu3-e1 mutants exhibit normal uptake of ⁶³NiCl₂. Moreover, wild-type plants grafted on mutant rootstocks produce seeds with fully active urease, indicating unimpeded transport of nickel through mutant roots and stems.     

Calcium in bacteria: a solution to which problem?

Calcium and calcium-binding proteins including those resembling calmodulin are implicated in numerous diverse processes in bacteria. These processes include chemotaxis, sporulation, virulence, the transport of sugars and proteins, phosphorylation, heat shock, the initiation of DNAS replication, septation, nucleoid structure, nuclease activity and recombination, the stability of the envelope, and phospholipids synthesis and configuration. That such varied processes should have a common factor, calcium, suggests major underlying principles of calcium metabolism metabolism which have yet to be discovered.     

Electron Microscopic Study of Development in a Sea Cucumber,Stichopus tremulus (Holothuroidea), from Unfertilized Egg through Hatched Blastula

In this, the first fine structural study of sea cucumber embryology, eggs and embryos of Stichopus tremulus developing at 7.5°C are described from spawning through hatched blastulae. Spawned eggs are at about first meiotic metaphase and are surrounded by a jelly layer that remains around the embryos until hatching. No vitelline coat can be demonstrated, but whether it is truly absent or removed by electron microscopic processing is not known. Insemination initiates a rapid cortical reaction, completed within 2 min., which involves a wave of cortical granule exocytosis and fertilization envelope formation. The compactly fibrous fertilization envelope is about 50 nm thick and appears to consist entirely of ejected cortical granule material (if one assumes that there is no vitelline coat). As the fertilization envelope elevates, no hyaline layer appears in the perivitelline space. The first and second polar bodies are emitted, respectively, at about 9 and 15 min. after insemination. The first seven or so cleavages are equal, radial, and occur approximately every 4 hr. The blastocoel opens up at the four-cell stage and, during the earlier cleavages, remains connected with the perivitelline space via numerous gaps between the roughly spherical blastomeres. At the 64-cell stage, these gaps begin to close as the blastomeres start to become cuboidal; in addition, an embryonic cuticle is produced on the apical surface of each blastomere. In embryos of several hundred cells, the blastomeres become associated apicolaterally by junctional complexes, each consisting of a zonula adherens and a septate junction. Several hours before hatching, a single cilium is produced at the apical surface of most blastomeres. At hatching (about 50 hr after insemination), the ciliated blastula leaves behind the fertilization envelope and jelly layer. Swimming blastulae soon begin to elongate in the animal-vegetal axis, and a basal lamina develops on blastomere surfaces facing the blastocoel. The discussion includes a fine structural comparison of egg coats among the five classes of the phylum Echinodermata.     

Insertion of a MalE β-Galactosidase Fusion Protein into the Envelope of Escherichia coli Disrupts Biogenesis of Outer Membrane Proteins and Processing of Inner Membrane Proteins

The synthesis of a membrane-bound MalE β-galactosidase hybrid protein, when induced by growth of Escherichia coli on maltose, leads to inhibition of cell division and eventually a reduced rate of mass increase. In addition, the relative rate of synthesis of outer membrane proteins, but not that of inner membrane proteins, was reduced by about 50%. Kinetic experiments demonstrated that this reduction coincided with the period of maximum synthesis of the hybrid protein (and another maltose-inducible protein, LamB). The accumulation of this abnormal protein in the envelope therefore appeared specifically to inhibit the synthesis, the assembly of outer membrane proteins, or both, indicating that the hybrid protein blocks some export site or causes the sequestration of some limiting factor(s) involved in the export process. Since the MalE protein is normally located in the periplasm, the results also suggest that the synthesis of periplasmic and outer membrane proteins may involve some steps in common. The reduced rate of synthesis of outer membrane proteins was also accompanied by the accumulation in the envelope of at least one outer membrane protein and at least two inner membrane proteins as higher-molecular-weight forms, indicating that processing (removal of the N-terminal signal sequence) was also disrupted by the presence of the hybrid protein. These results may indicate that the assembly of these membrane proteins is blocked at a relatively late step rather than at the level of primary recognition of some site by the signal sequence. In addition, the results suggest that some step common to the biogenesis of quite different kinds of envelope protein is blocked by the presence of the hybrid protein.     

Envelope protein synthesis and inhibition of cell division in Escherichia coli during inactivation of the B subunit of DNA gyrase

The rates of synthesis of inner and outer membrane proteins of Escherichia coli K12 during inhibition of cell division have been studied. When cell division was inhibited, either by treatment of wild-type cells with the antibiotic clorobiocin (an inhibitor of the B subunit of DNA gyrase) or by a temperature shift of a gyrB-ts mutant, a 40% reduction in the rate of synthesis of total outer membrane protein relative to that of the inner membrane was observed. When a gyrB-ts mutant was shifted to high temperature under conditions which allowed continued cell division, this large reduction in the rate of synthesis of outer membrane protein relative to inner membrane protein was not observed. In contrast to the results obtained with clorobiocin, inhibition of cell division by the beta-lactam antibiotic cefuroxime did not cause any detectable disturbance in the rate of synthesis of either inner or outer membrane protein. This demonstrates that inhibition of septum formation per se does not perturb synthesis of envelope protein. The data obtained are consistent with a model in which the rate of synthesis and therefore expansion of outer membrane is one of many conditions which must be satisfied before septum formation can occur. The results are discussed in relation to such a model, and to previous findings which have shown that the rate of synthesis of outer membrane proteins displays a linear mode with an abrupt doubling in rate at a discrete point in the cell cycle.     

Viruses isolated from cells persistently infected with vesicular stomatitis virus show altered interactions with defective interfering particles.

Virus mutants isolated from persistent infections of vesicular stomatitis virus in BHK-21 cells were much less susceptible to interference mediated by the defective interfering particle used to establish the persistent infection. This mutational change occurred as early as 34 days in the persistent infection and continued for over 5 years. The earliest variants showed no oligonucleotide map changes and no difference in the temperature-sensitive phenotype from the original virus, but the later variants exhibited extensive map changes. These results suggest a possible role for defective interfering particles in the selection of the mutants.     

Characterization of Herpes Simplex Virus Type 1 RNA Present in the Absence of De Novo Protein Synthesis

We used herpes simplex virus type 1 (HSV-1) DNA and restriction fragments of HSV-1 DNA covalently coupled to cellulose as a reagent to isolate for further characterization the major and minor HSV-1 immediate-early mRNA species in HeLa cells infected and maintained in the absence of de novo protein synthesis. Five major and several minor immediate-early mRNA species were characterized. One major species was a 4.2-kilobase mRNA mapping in the TR(S)/IR(S) region with its 3' end distal to the U(S) region; this mRNA encoded a 170,000-dalton polypeptide in vitro. A 2.8-kilobase mRNA, encoding a 120,000-dalton polypeptide, was mapped in the TR(L)/IR(L) region with its 3' end directed toward the U(L) region. Three 1.8-kilobase mRNA species were mapped. One, mapping in the IR(S) region with its 3' end in the U(S), encoded a 68,000-dalton polypeptide. One mapped in the TR(S) region and had its 3' end in the U(S) region; the third one encoded a 64,000-dalton polypeptide and mapped in the U(L) region near the IR(L) region. One minor species 5.2 kilobases in size was clearly detectable mapping in the U(L) region. Furthermore, there were indications that one or more immediate-early mRNA species approximately 3 kilobases in size hybridized to regions near the TR(L) and in or near the TR(S)/IR(S) regions. Nuclear immediate-early RNA mapped only in those regions where polyribosomal immediate-early mRNA mapped, although minor differences were seen. Finally, we demonstrated that at least three major immediate-early mRNA's-4.2 kilobases, 2.8 kilobases, and the 1.8-kilobase one mapping in the IR(S)/U(S) region-continued to appear on polyribosomes as functional mRNA late after infection.