Expression of the SNF1 gene from Candida tropicalis is required for growth on various carbon sources, including glucose

SNF1 of Saccharomyces cerevisiae is an essential gene for the derepression of glucose repression. A homolog of SNF1 (CtSNF1) was isolated from an n-alkane-assimilating diploid yeast, Candida tropicalis. CtSNF1 could complement the snf1 mutant of S. cerevisiae. The previously published method for introducing the exogenous DNA into C. tropicalis was employed to construct SNF1/ snf1 heterozygote and snf1/snf1 homozygote strains. The successfully constructed SNF1/snf1 heterozygote was named KO-1. Disruption of the second CtSNF1 allele was unsuccessful, suggesting that CtSNF1 might be essential for cell viability. Therefore, in order to control the expression of CtSNF1, a strain (named KO-1G) in which the promoter region of CtSNF1 was replaced with the GAL10 promoter of C. tropicalis was constructed, and the growth of strains KO-1 and KO-1G was compared with that of the parental strain. The growth of strain KO-1 on glucose, sucrose, or acetate did not differ from the growth of the parental strain, but strain KO-1 showed a slight growth retardation on n-alkane. The growth of strain KO-1G on galactose was normal, but the cells stopped growing when transferred to glucose-, acetate-, or n-alkane-containing medium. Northern blot analysis against mRNA from the n-alkane-grown KO-1G strain demonstrated a close relationship between the presence of CtSNF1 mRNA and the growth of the cells, indicating that CtSNF1 is essential for cell viability. Moreover, mRNA levels of isocitrate lyase, which is localized in peroxisomes of C. tropicalis, were significantly affected by the level of CtSNF1 mRNA. Received: 3 May 1999 / Accepted: 14 July 1999     

Non‐housekeeping,non‐essential GroEL (chaperonin) has acquired novel structure and function beneficial under stress in cyanobacteria

GroELs which are prokaryotic members of the chaperonin (Cpn)/Hsp60 family are molecular chaperones of which Escherichia coli GroEL is a model for subsequent research. The majority of bacterial species including E. coli and Bacillus subtilis have only one essential groEL gene that forms an operon with the co‐chaperone groES gene. In contrast to these model bacteria, two or three groEL genes exist in cyanobacterial genomes. One of them, groEL2, does not form an operon with the groES gene, whereas the other(s) does. In the case of cyanobacteria containing two GroEL homologs, one of the GroELs, GroEL1, substitutes for the native GroEL in an E. coli cell, but GroEL2 does not. Unlike the E. coli GroEL, GroEL2 is not essential, but it plays an important role which is not substitutable by GroEL1 under stress. Regulation of expression and biochemical properties of GroEL2 are different/diversified from GroEL1 and E. coli GroEL in many aspects. We postulate that the groEL2 gene has acquired a novel, beneficial function especially under stresses and become preserved by natural selection, with the groEL1 gene retaining the original, house‐keeping function. In this review, we will focus on difference between the two GroELs in cyanobacteria, and divergence of GroEL2 from the E. coli GroEL. We will also compare cyanobacterial GroELs with the chloroplast Cpns (60α and 60β) which are thought to be evolved from the cyanobacterial GroEL1. Chloroplast Cpns appear to follow the different path from cyanobacterial GroELs in the evolution after gene duplication of the corresponding ancestral groEL gene.     

<Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Cry11Ba works synergistically with Cry4Aa but not with Cry11Aa for toxicity against mosquito <Emphasis Type="Italic">Culex pipiens</Emphasis> (Diptera: Culicidae) larvae

A 2,175-bp modified gene (cry11Ba-S1) encoding Cry11Ba from Bacillus thuringiensis subsp. jegathesan was designed and the recombinant protein was expressed as a fusion protein with glutathione S-transferase in Escherichia coli. The recombinant Cry11Ba was highly toxic against Culex pipiens mosquito larvae, being nine and 17 times more toxic than mosquitocidal Cry4Aa and Cry11Aa from Bacillus thuringiensis subsp. israelensis, respectively. Interestingly, a further increase in the toxicity of the recombinant Cry11Ba was achieved by mixing with Cry4Aa, but not with Cry11Aa. These findings suggested that Cry11Ba worked synergistically with Cry4Aa, but not with Cry11Aa, in exhibiting toxicity against C. pipiens larvae. On the other hand, the amount of Cry toxin bound to brush border membrane vesicles (BBMVs) did not significantly change between individual toxins and the toxin mixtures, suggesting that the increase in toxins binding to BBMVs was not a reason for the observed synergistic effect. It is generally accepted that synergism of toxins is a potentially powerful tool for enhancing insecticidal activity and managing Cry toxin resistance in mosquitoes. The mixture of Cry4Aa and Cry11Ba in order to increase toxicity would be very valuable in terms of mosquito control.     

Histone-like DNA binding protein of Streptococcus intermedius induces the expression of pro-inflammatory cytokines in human monocytes via activation of ERK1/2 and JNK pathways

Streptococcus intermedius is a commensal associated with serious, deep-seated purulent infections in major organs, such as the brain and liver. Histone-like DNA binding protein (HLP) is an accessory architectural protein in a variety of bacterial cellular processes. In this study, we investigated the mechanisms of pro-inflammatory cytokine inductions in THP-1 cells by stimulation with recombinant HLP of S. intermedius (r Si -HLP). r Si -HLP stimulation-induced production of pro-inflammatory cytokines (IL-8, IL-1β and TNF-α) occurred in a time- and dose-dependent manner. In contrast with the heat-stable activity of DNA binding, the induction activity of r Si -HLP was heat-unstable. In subsequent studies, r Si -HLP acted cooperatively with lipoteichoic acid, the synthetic Toll-like receptor 2 agonist, Pam3CSK4, and the cytosolic nucleotide binding oligomerization domain 2 receptor agonist, muramyldipeptide. Furthermore, Western blot and blocking assays with specific inhibitors showed that r Si -HLP stimulation induced the activation of cell signal transduction pathways, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK). In addition to its physiological role in bacterial growth through DNA binding, these results indicate that Si -HLP can trigger a cascade of events that induce pro-inflammatory responses via ERK1/2 and JNK signal pathways, and suggest that bacterial HLP may contribute to the activation of host innate immunity during bacterial infection.     

The aux1 gene of the Ri plasmid is sufficient to confer auxin autotrophy in tobacco BY-2 cells

Tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells are rapidly proliferating meristematic cells that require auxin for culture in vitro. We have established several transgenic BY-2 cell lines that carry the T-DNA of Agrobacterium rhizogenes 15834, which harbors an agropine-type root-inducing (Ri) plasmid. Two of these lines, BYHR-3 and BYHR-7, were used to test the role of auxin in the proliferation of plant cells. The lines grew rapidly in Linsmaier-Skoog (LS) medium lacking auxin and other phytohormones. The TR-DNA, containing the aux1 (tryptophan monooxygenase) and aux2 (indoleacetamide hydrolase) genes, was present in the genomes of both transgenic lines, whereas the TL-DNA, containing the rolA, B, C and D genes, was present in the genome of BYHR-7 but not BYHR-3. Since the introduction of the rolABCD genes alone did not affect the auxin requirement of BY-2 cells, the aux1 and aux2 genes, but not the rolABCD genes, appear to be relevant to the auxin autotrophy of these transgenic lines. Furthermore, the overexpression of aux1 allowed BY-2 cells to grow rapidly in the absence of auxin, suggesting the existence in plant cells of an unidentified gene whose product is functionally equivalent or similar to that of aux2 of the Ri plasmid.     

The role of Arg-96 in Danio rerio creatine kinase in substrate recognition and active center configuration

In creatine kinases (CKs), the amino acid residue-96 is a strictly conserved arginine. This residue is not directly associated with substrate binding, but it is located close to the binding site of the substrate creatine. On the other hand, the residue-96 is known to be involved in expression in the substrate specificity of various other phosphagen (guanidino) kinases, since each enzyme has a specific residue at this position: arginine kinase (Tyr), glycocyamine kinase (Ile), taurocyamine kinase (His) and lombricine kinase (Lys). To gain a greater understanding of the role of residue-96 in CKs, we replaced this residue in zebra fish Danio rerio cytoplasmic CK with other 19 amino acids, and expressed these constructs in Escherichia coli. All the twenty recombinant enzymes, including the wild-type, were obtained as soluble form, and their activities were determined in the forward direction. Compared with the activity of wild-type, the R96K mutant showed significant activity (8.3% to the wild-type), but 10 mutants (R96Y, A, S, E, H, T, F, C, V and N) showed a weak activity (0.056–1.0%). In the remaining mutants (R96Q, G, M, P, L, W, D and I), the activity was less than 0.05%. Our mutagenesis studies indicated that Arg-96 in Danio CK can be substituted for partially by Lys, but other replacements caused remarkable loss of activity. From careful inspection of the crystal structures (transition state analog complex (TSAC) and open state) of Torpedo cytoplasmic CK, we found that the side chain of R96 forms hydrogen bonds with A339 and D340 only in the TSAC structure. Based on the assumption that CKs consist of four dynamic domains (domains 1–3, and fixed domain), the above hydrogen bonds act to link putative domains 1 and 3 in TSAC structure. We suggest that residue-96 in CK and equivalent residues in other phosphagen kinases, which are structurally similar, have dual roles: (1) one involves in distinguishing guanidino substrates, and (2) the other plays a key role in organizing the hydrogen-bond network around residue-96 which offers an appropriate active center for the high catalytic turnover. The mode of development of the network appears to be unique each phosphagen kinase, reflecting evolution of each enzyme.     

Early embryonic development and diapause stage in the band-legged ground cricket Dianemobius nigrofasciatus

The band-legged ground cricket Dianemobius nigrofasciatus enters diapause at an early embryonic stage when adults are reared under short-day conditions or the eggs are exposed to a low temperature. We examined the morphological features of the embryo during early development and determined the exact stage of entry into diapause. In non-diapause eggs, no periplasmic space was observed in the surface region and a small number of nuclei surrounded by cytoplasm (energids) were found among the yolk granules and lipid droplets 12 h after egg laying (AEL) at 25°C. The energids sparsely but evenly populated the surface region at 40 h AEL, but there were some gaps between these energids. A continuous thin layer of nuclei with cytoplasm had completely covered the egg surface at 56 h AEL, suggesting that the blastoderm is formed between 40 and 56 h AEL. At 72 h AEL, we found a germ band at the posterior pole. Electron microscopy revealed clear cell membranes at 40 h AEL. Staining with rhodamine-dextran dye demonstrated that the cell membrane is formed when the nuclei appear on the egg surface at 12–24 h AEL. These results indicate that cellularization occurs before blastoderm formation. In diapause eggs, neither the embryonic rudiment nor germ band was formed, but a continuous layer of cells covered the egg surface. It is concluded that D. nigrofasciatus enters diapause at the cellular blastoderm.     

Relationships among tributary length,census population size,and genetic variability of white-spotted charr, <Emphasis Type="Italic">Salvelinus leucomaenis</Emphasis>, in the Lake Biwa water system

The relationships between census population size and tributary length and between haplotype diversity of the mitochondrial DNA and census population size in ten white-spotted charr populations in the Lake Biwa water system and its adjacent basins were investigated. The census population size (number of fish with ≥100 mm in standard length) significantly increased with the tributary length. In the eastern part of the Lake Biwa water system, haplotype diversity increased with the census population size. On other hand, in the western part of the water system and adjacent basins, haplotype diversity was zero irrespective of the census population size. These results suggest that white-spotted charr populations in the eastern and western part of the Lake Biwa water system have undergone different levels of bottlenecks related to the habitat size in the postglacial warming.