Progeny production in the periplasm of Thermosipho globiformans

Thermotogales are rod-shaped, Gram-negative, anaerobic, (hyper) thermophiles distinguished by an outer sheath-like toga, which comprises an outer membrane (OM) and an amorphous layer (AL). Thermosipho globiformans bacteria can transform into spheroids with multiple cells concurrently with AL disintegration during early growth; the cell is defined as the cytoplasmic membrane (CM) plus the entity surrounded by the CM. Spheroids eventually produce rapidly moving periplasmic ‘progenies’ through an unknown mechanism. Here, we used high-temperature microscopy (HTM) to directly observe spheroid generation and growth. Rod OMs abruptly inflated to form ~2 μm-diameter balloons. Concurrently, multiple globular cells emerged in the balloons, suggesting their translocation and transformation from the rod state. During spheroid growth, the cells elongated and acquired a large dish shape by possible fusion. Spheroids with dish-shaped cells further enlarged to ~12 μm in diameter. HTM and epifluorescence-microscopy results collectively indicated that the nucleoids of dish-shaped cells transformed to form a ring shape, which then distorted to form a lip shape as the spheroid enlarged. HTM showed that ‘progenies’ were produced in the spheroid periplasm. Transmission electron microscopy results suggested that the ‘progenies’ represented immature progenies lacking togas, which were acquired subsequently.

     

Mutagenesis studies of protein farnesyltransferase implicate aspartate beta 352 as a magnesium ligand

Protein farnesyltransferase (FTase) catalyzes the addition of a farnesyl chain onto the sulfur of a C-terminal cysteine of a protein substrate. Magnesium ions enhance farnesylation catalyzed by FTase by several hundred-fold, with a KMg value of 4 mM. The magnesium ion is proposed to coordinate the diphosphate leaving group of farnesyldiphosphate (FPP) to stabilize the developing charge in the farnesylation transition state. Here we further investigate the magnesium binding site using mutagenesis and biochemical studies. Free FPP binds Mg2+ with a Kd of 120 microM. The 10-fold weaker affinity for Mg2+ observed for the FTase.FPP.peptide ternary complex is probably caused by the positive charges in the diphosphate binding pocket of FTase. Furthermore, mutation of aspartate beta 352 to alanine (D beta 352A) or lysine (D beta 352K) in FTase drastically alters the Mg2+ dependence of FTase catalysis without dramatically affecting the rate constant of farnesylation minus magnesium or the binding affinity of either substrate. In D beta 352A FTase, the KMg increases 28-fold to 110 +/- 30 mM, and the farnesylation rate constant at saturating Mg2+ decreases 27-fold to 0.30 +/- 0.05 s-1. Substitution of a lysine for Asp-beta 352 removes the magnesium activation of farnesylation catalyzed by FTase but does not significantly enhance the rate constant for farnesylation in the absence of Mg2+. In wild type FTase, Mg2+ can be replaced by Mn2+ with a 2-fold lower KMn (2 mM). These results suggest both that Mg2+ coordinates the side chain carboxylate of Asp-beta 352 and that the role of magnesium in the reaction includes positioning the FPP prior to catalysis.     

The aconitase function of iron regulatory protein 1. Genetic studies in yeast implicate its role in iron-mediated redox regulation

Iron regulatory proteins (IRP) are sequence-specific RNA-binding proteins that mediate iron-responsive gene regulation in animals. IRP1 is also the cytosolic isoform of aconitase (c-aconitase). This latter activity could complement a mitochondrial aconitase mutation (aco1) in Saccharomyces cerevisiae to restore glutamate prototrophy. In yeast, the c-aconitase activity of IRP1 was responsive to iron availability in the growth medium. Although IRP1 expression rescued aco1 yeast from glutamate auxotrophy, cells remained growth-limited by glutamate, displaying a slow-growth phenotype on glutamate-free media. Second site mutations conferring enhanced cytosolic aconitase-dependent (ECA) growth were recovered. Relative c-aconitase activity was increased in extracts of strains harboring these mutations. One of the ECA mutations was found to be in the gene encoding cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDP2). This mutation, an insertion of a Ty delta element into the 5' region of IDP2, markedly elevates expression of Idp2p in glucose media. Our results demonstrate the physiological significance of the aconitase activity of IRP1 and provide insight into the role of c-aconitase with respect to iron and cytoplasmic redox regulation.     

Microscopic studies of fatty acid vesicles

Microscopic vesicles enclosed by membranes formed entirely of oleic or linoleic acids (ufasomes) were studied by the freeze-etching and birefringence techniques. The results suggest the presence of one or more membranes around the particles, in which the fatty acid chains lie perpendicular to the surface. Comparison with results obtained with phospholipid liposomes shows that both types of particles are basically similar, although ufasomes have a less regular structure.     

Keratin Degradation by Fervidobacterium pennavorans, a Novel Thermophilic Anaerobic Species of the Order Thermotogales

From a hot spring of the Azores islands a novel thermophilic bacterium belonging to the Thermotogales order was isolated. This strain, which grows optimally at 70(deg)C and pH 6.5, is the first known extreme thermophile that is able to degrade native feathers at high temperatures. The enzyme system converts feather meal to amino acids and peptides. On the basis of physiological, morphological, and 16S rDNA studies the new isolate was found to be a member of the Thermotogales order and was identified as Fervidobacterium pennavorans. The strain was highly related to Fervidobacterium islandicum and Fervidobacterium pullulanolyticum. The cell-bound keratinolytic enzyme system was purified 32-fold by detergent treatment with CHAPS (3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was characterized as a serine protease with a molecular mass of 130 kDa and an isoelectric point of 3.8. Optimal activity was measured at 80(deg)C and pH 10.0. Furthermore, 19 anaerobic thermophilic archaea and bacteria belonging to the orders Thermococcales, Thermoproteales, Thermotogales, and Clostridiales (growth temperatures between 60 and 105(deg)C) were tested for their abilities to grow on feathers and produce heat-stable keratinolytic enzymes. None of the tested extremophilic microorganisms was able to attack the substrate in a native form.     

Genome‐wide tests for introgression between cactophilic Drosophila implicate a role of inversions during speciation

Models of speciation‐with‐gene‐flow have shown that the reduction in recombination between alternative chromosome arrangements can facilitate the fixation of locally adaptive genes in the face of gene flow and contribute to speciation. However, it has proven frustratingly difficult to show empirically that inversions have reduced gene flow and arose during or shortly after the onset of species divergence rather than represent ancestral polymorphisms. Here, we present an analysis of whole genome data from a pair of cactophilic fruit flies, Drosophila mojavensis and D. arizonae, which are reproductively isolated in the wild and differ by several large inversions on three chromosomes. We found an increase in divergence at rearranged compared to colinear chromosomes. Using the density of divergent sites in short sequence blocks we fit a series of explicit models of species divergence in which gene flow is restricted to an initial period after divergence and may differ between colinear and rearranged parts of the genome. These analyses show that D. mojavensis and D. arizonae have experienced postdivergence gene flow that ceased around 270 KY ago and was significantly reduced in chromosomes with fixed inversions. Moreover, we show that these inversions most likely originated around the time of species divergence which is compatible with theoretical models that posit a role of inversions in speciation with gene flow.     

Lipoteichoic acid is a major component of the Bacillus subtilis periplasm

Cryo-electron microscopy (cryo-EM) of frozen-hydrated specimens allows high-resolution observation of structures in optimally preserved samples. In gram-positive bacteria, this method reveals the presence of a periplasmic space between the plasma membrane and an often differentiated cell wall matrix. Since virtually nothing is known about the composition of its constituent matter (i.e., the periplasm), it is still unclear what structures (or mechanism) sustain a gram-positive periplasmic space. Here we have used cryo-EM of frozen-hydrated sections in combination with various labels to probe the model gram-positive organism Bacillus subtilis for major periplasmic components. Incubation of cells with positively charged gold nanoparticles showed almost similar levels of gold binding to the periplasm and the cell wall. On cells whose cell walls were enzymatically hydrolyzed (i.e., on protoplasts), a surface diffuse layer extending ~30 nm from the membrane was revealed. The thickness and density of this layer were not significantly altered after treatment with a nonspecific protease, whereas it was labeled with anti-lipoteichoic acid (LTA) antibodies conjugated to nanogold. Further, the LTA layer spans most of the thickness of the periplasmic space, which strongly suggests that LTA is a major component of the B. subtilis periplasm.     

Paternal effects in a wild‐type zebrafish implicate a role of sperm‐derived small RNAs

While the importance of maternal effects has long been appreciated, a growing body of evidence now points to the paternal environment having an important influence on offspring phenotype. Indeed, research on rodent models suggests that paternal stress leaves an imprint on the behaviour and physiology of offspring via nongenetic information carried in the spermatozoa; however, fish have been understudied with regard to these sperm‐mediated effects. Here, we investigated whether the zebrafish was subjected to heritable influences of paternal stress by exposing males to stressors (conspecific‐derived alarm cue, chasing and bright light) before mating and assessing the behavioural and endocrine responses of their offspring, including their behavioural response to conspecific‐derived alarm cue. We found that after males are exposed to stress, their larval offspring show weakened responses to stressors. Small RNA sequencing subsequently revealed that the levels of several small noncoding RNAs, including microRNAs, PIWI‐interacting RNAs and tRNA‐derived small RNAs, were altered in the spermatozoa of stressed fathers, suggesting that stress‐induced alterations to the spermatozoal RNA landscape may contribute to shaping offspring phenotype. The work demonstrates that paternal stress should not be overlooked as a source of phenotypic variation and that spermatozoal small RNAs may be important intergenerational messengers in fish.     

Isolation of the periplasm of Neisseria gonorrhoeae

The periplasm of Neisseria gonorrhoeae should be similar to other Gram-negative bacteria, but no published reports confirm this assumption. We used a periplasmic isolation procedure developed in Escherichia coli to release the periplasmic contents of N. gonorrhoeae. The resultant periplasmic extract lacked lipopolysaccharide, protein markers of inner or outer membranes, surface-radiolabelled protein components, or ribosomal proteins. The periplasmic extract contained a single haem protein believed to be a c-type cytochrome known to exist in the periplasm of other Gram-negative species, and retained significant alkaline phosphatase activity. The dominant protein species released in the periplasmic extract was the gonococcal homologue of elongation factor Tu, a major component released in similar periplasmic extracts of E. coli. These data showed that the extraction procedure selectively released periplasmic components and that the gonococcal periplasm was comparable to that of E. coli. Further analysis of the gonococcal periplasm may provide important insights into the physiology of this pathogen of humans.     

Polyamines of the thermophilic eubacteria belonging to the genera Thermosipho, Thermaerobacter and Caldicellulosiruptor

Cellular polyamines of four new thermophiles located in three early branched eubacterial clades, were investigated for the chemotaxonomic significance of polyamine distribution profiles. The thermophilic anaerobic Thermosipho japonicus, belonging to the order Thermotogales, contained norspermidine, norspermine and thermospermine in addition to spermidine and spermine. The polyamine profile was identical to the polyamine composition of Thermotoga, Fervidobacterium and Petrotoga species of the order. Spermidine, norspermidine, spermine, N4-bis(aminopropyl)spermidine and agmatine were found in thermophilic aerobic Thermaerobacter marianensis. Some differences were observed in the polyamine compositions of the phylogenetically related thermophilic anaerobes, Moorella, Dictyoglomus, Thermoanaerobacterium and Thermoanaerobacter species. Thermophilic anaerobic Caldicellulosiruptor kristianssonii and Caldicellulosiruptor owensensis contained a linear penta-amine, thermopentamine, and two quaternary branched penta-amines, N4-bis(aminopropyl)spermidine and N4-bis(aminopropyl)norspermidine, as the major polyamines. A novel tertiary branched penta-amine, N4-aminopropylspermine, was found in the two Caldicellulosiruptor species.     

Microscopic and chemical studies of a gelling polysaccharide from Lactobacillus hilgardii

The bacterium Lactobacillus hilgardii (previously named L. brevis), isolated from sugary kefir grains, has been found to produce a gelling extracellular polysaccharide. Methylation analysis and ¹H- and ¹³C-NMR analysis revealed a dextran-like structure for the polysaccharide. A high proportion of -(1,6)-Glc and small proportions of -(1,3,6)-Glc, -(1,4,6)-Glc and terminal -Glc were detected. The L. hilgardii polysaccharide differed from normal dextrans in containing a small proportion of -(1,3)-Glc. The structure of the polysaccharide was found to be independent of harvesting time and unaffected by changes in the growth medium. The development of the gel structure has been followed by transmission electron microscopy.