Analysis of Genes for Succinoyl Trehalose Lipid Production and Increasing Production in Rhodococcus sp. Strain SD-74

Succinoyl trehalose lipids (STLs) are promising glycolipid biosurfactants produced from n-alkanes that are secreted by Rhodococcus species bacteria. These compounds not only exhibit unique interfacial properties but also demonstrate versatile biochemical actions. In this study, three novel types of genes involved in the biosynthesis of STLs, including a putative acyl coenzyme A (acyl-CoA) transferase (tlsA), fructose-bisphosphate aldolase (fda), and alkane monooxygenase (alkB), were identified. The predicted functions of these genes indicate that alkane metabolism, sugar synthesis, and the addition of acyl groups are important for the biosynthesis of STLs. Based on these results, we propose a biosynthesis pathway for STLs from alkanes in Rhodococcus sp. strain SD-74. By overexpressing tlsA, we achieved a 2-fold increase in the production of STLs. This study advances our understanding of bacterial glycolipid production in Rhodococcus species.     

Proteome analysis of rat hippocampal neurons by multiple large gel two-dimensional electrophoresis

The goal of the present study was to detect as many protein spots as possible in mammalian cells using two-dimensional gel electrophoresis (2-DE). For proteome analysis, it is of importance to reveal as many proteins as possible. A single standard 2-DE gel (pH 3-10, 18 cm x 20 cm, 13.5% gel) could detect 853 spots from proteins of cultured rat hippocampal neurons when visualized by silver staining. To increase the resolution of the separation and the number of detectable proteins by 2-DE, we utilized seven different narrow pH range immobilized pH gradients in the first dimension. In the second dimension, fourteen long SDS polyacrylamide gels were used: seven 7.5% gels for the separation of high molecular mass proteins (> or = 40 kDa) and seven 13.5% gels for the separation of low molecular mass proteins (< or = 40 kDa). Three hundred and sixty microg of proteins from cultured hippocampal neurons were loaded on to individual gels and visualized by silver staining. All 14 gel images were assembled into a 70 cm x 67 cm cybergel that contained 6677 protein spots, thereby indicating that the utilization of the present strategy led to a 783% increase in the number of detected spots in comparison to the standard procedure. Loading double the amount (720 microg) of proteins on to a 13.5% gel led to a 184% increase in the number of detected spots, thereby indicating that the present strategy has a potential to display more protein spots in the cybergels.     

Natural variation in population densities and vertical transmission rates of a Spiroplasma endosymbiont in Drosophila hydei

A bacterium belonging to the genus Spiroplasma, an endosymbiont of the fly Drosophila hydei, is vertically transmitted through host egg cytoplasm. To infer vertical transmission rates of Spiroplasma in natural populations of D. hydei, the infection status of Spiroplasma was examined for offspring produced by Spiroplasma-positive females that were collected in two geographical populations. In both populations, nearly half of the broods consisted of only infected offspring. Infection frequencies of the rest of the broods ranged from 0.364 to 0.975. Quantitative PCR demonstrated that the Spiroplasma titers in the whole body of wild-caught females were highly variable (1.81?×?10⁶–5.60?×?10⁸ cells per insect). Contrary to our expectations, however, the Spiroplasma titers did not account for the variation in infection frequencies among offspring (i.e., vertical transmission rates). These results suggest that the spatial distribution of Spiroplasma, particularly in somatic tissues and germ tissues, is highly variable among host individuals, which may be caused by environmental stochasticity or some unknown effects.     

Application of a powdered artificial diet to promote the establishment of the predatory bug Geocoris varius (Hemiptera: Geocoridae) on strawberry plants

We examined whether the application of a powdered artificial diet on target plants would promote the establishment of Geocoris varius (Uhler) (Hemiptera: Geocoridae). Laboratory trials showed that, after 72 h, 93 % of the G. varius individuals remained on plants to which the artificial diet was applied as opposed to only 30 % of G. varius remaining on untreated strawberry plants. Moreover, application of the powdered diet promoted the development of G. varius from the third to the fifth nymphal stages and extended the average longevity by 12 days. The results suggested that the application of powdered artificial diet promotes the establishment of G. varius released in a greenhouse, even when pest densities are low, increasing the effectiveness of biological control. Furthermore, the increase in predation associated with greater numbers of more developed G. varius would improved the effectiveness of this biocontrol method.     

The ABC-primosome. A novel priming system employing dnaA, dnaB, dnaC, and primase on a hairpin containing a dnaA box sequence

A priming mechanism requiring dnaA, dnaB, and dnaC proteins operates on a single-stranded DNA coated with single-stranded DNA-binding protein. This novel priming, referred to as "ABC-priming," requires a specific hairpin structure whose stem carries a dnaA protein recognition sequence (dnaA box). In conjunction with primase and DNA polymerase III holoenzyme, ABC-priming can efficiently convert single-stranded DNA into the duplex replicative form. dnaA protein specifically recognizes and binds the single-stranded hairpin and permits the loading of dnaB protein to form a prepriming protein complex containing dnaA and dnaB proteins which can be physically isolated. ABC-priming can replace phi X174 type priming on the lagging strand template of pBR322 in vitro, suggesting a possible function of ABC-priming for the lagging strand synthesis and duplex unwinding. Similar to the phi X174 type priming, a mobile nature of ABC-priming was indicated by helicase activity in the presence of ATP of a prepriming protein complex formed at the hairpin. The implications of this novel priming in initiation of replication at the chromosomal origin, oriC, and in its contribution to the replication fork are discussed.     

Two regions in human DNA polymerase beta mRNA suppress translation in Escherichia coli.

Although human DNA polymerase beta (DNA pol beta) shows 96% identity with rat DNA pol beta at the amino acid level, it is weakly expressed in Escherichia (E.) coli relative to the rat enzyme. The mechanism of this suppression was investigated. Pulse-chase protein labeling and steady state mRNA analysis showed that mature human DNA pol beta protein is relatively stable in E. coli and the levels of human and rat DNA pol beta mRNA were comparable indicating that the human DNA pol beta expression is suppressed at the translational level. By systematic expression analysis of a number of chimeric genes composed of human and rat cDNAs, two strong translational suppression regions were mapped in the human DNA pol beta mRNA; one was named TSR-1, corresponding to CGG encoding arginine (arg) at position 4 and the other, termed TSR-2, is located between codons 153 and 199. Since substitution of the rat Arg-4 codon with synonymous codons showed strong effects upon the expression level, we propose that the arg codon at the N-terminal coding region plays a role in modulating expression.     

Comparative characterization of spirosomes isolated from Lactobacillus brevis, Lactobacillus fermentum, and Lactobacillus buchneri

Spirosomes, cytoplasmic fine spirals, were isolated and purified from Lactobacillus brevis ATCC 8287, L. fermentum F-1, and L. buchneri ATCC 4005, and their morphological, biochemical, and immunological properties were investigated. The spirosomes of these lactobacilli were morphologically indistinguishable from one another, and they had the same buoyant density of 1.320 g/cm3 in CsCl. All of the spirosomes were composed of a single protein, spirosin, with an apparent molecular weight of about 95,000 for L. brevis and L. fermentum and of about 96,000 for L. buchneri as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The spirosins from the three lactobacilli were compared by peptide mapping on SDS-PAGE after cleavage with N-chlorosuccinimide and limited proteolysis with Staphylococcus aureus V8 protease. The peptide map of the L. brevis spirosin was identical with that of the L. fermentum spirosin, whereas it was markedly different from the L. buchneri spirosin. The amino acid composition of the L. brevis spirosin was almost similar to that of the L. fermentum spirosin, while it differed appreciably from the L. buchneri spirosin. Using antiserum against the L. brevis spirosin, immunodiffusion test revealed that the antigenicity of the spirosomes from L. brevis was identical with that from L. fermentum, whereas it was partially different from that from L. buchneri.     

Pulmonary surfactant protein A modulates the cellular response to smooth and rough lipopolysaccharides by interaction with CD14.

Pulmonary surfactant protein A (SP-A) plays an important part in Ab-independent host defense mechanisms of the lung. In this study we investigated how SP-A interacts with distinct serotypes of bacterial LPS and modulates LPS-elicited cellular responses. SP-A bound to rough forms but not to smooth forms of LPS. In the macrophage-like cell line U937, SP-A inhibited mRNA expression and secretion of TNF-alpha induced by smooth LPS, but rough LPS-induced TNF-alpha expression was unaffected by SP-A. When U937 cells and rat alveolar macrophages were preincubated with SP-A, smooth LPS failed to induce TNF-alpha secretion, whereas rough LPS-induced TNF-alpha secretion was modestly increased. To clarify the mechanism by which SP-A modulates LPS-elicited cellular responses, we further examined the interaction of SP-A with CD14, which is known as a major LPS receptor. Western blot analysis revealed that CD14 was one of the SP-A binding proteins isolated from solubilized U937 cells. In addition, SP-A directly bound to recombinant soluble CD14 (rsCD14). When rsCD14 was preincubated with SP-A, the binding of rsCD14 to smooth LPS was significantly reduced but the association of rsCD14 with rough LPS was augmented. These results demonstrate the different actions of SP-A upon distinct serotypes of LPS and indicate that the direct interaction of SP-A with CD14 constitutes a likely mechanism by which SP-A modulates LPS-elicited cellular responses.     

Reduced resistance to experimental viral and bacterial infections of mice treated with polychlorinated biphenyl

When mice given diet containing 100, 200 or 400 micrograms of polychlorinated biphenyl (PCB) per g, or PCB-free diet for 21 days were inoculated intranasally with influenza virus, the mortality was higher in some groups given PCB than in the control group. When Staphylococcus aureus was inoculated intraperitoneally into mice given a diets with or without PCB, a significant difference was observed in the mortalities in the groups. Subcutaneous injection of S. aureus also caused a larger subcutaneous abscess in the mice given diets containing PCB than in those given control diet. Thus, it is suggested that PCB ingestion reduces host resistance to systemic or local infection with viruses or bacteria.