Wolbachia replication and host cell division in Aedes albopictus

Wolbachia pipientis is an obligate intracellular endosymbiont of a range of arthropod species. The microbe is best known for its manipulations of host reproduction that include inducing cytoplasmic incompatibility, parthenogenesis, feminization, and male-killing. Like other vertically transmitted intracellular symbionts, Wolbachiarsquos replication rate must not outpace that of its host cells if it is to remain benign. The mosquito Aedes albopictus is naturally infected both singly and doubly with different strains of Wolbachia pipientis. During diapause in mosquito eggs, no host cell division is believed to occur. Further development is triggered only by subsequent exposure of the egg to water. This study uses diapause in Wolbachia-infected Aedes albopictus eggs to determine whether symbiont replication slows or stops when host cell division ceases or whether it continues at a low but constant rate. We have shown that Wolbachia densities in eggs are greatest during embryonation and then decline throughout diapause, suggesting that Wolbachia replication is dependent on host cell replication.

Shared and unique environmental factors determine the ecology of methanogens in humans and rats

OBJECTIVE: This study ascertains the relative contributions of genetics and environment in determining methane emission in humans and rats. There is considerable interest in the factors determining the microbial species that inhabit the colon. Methanogens, which are archaebacteria, are an easily detected colonic luminal bacteria because they respire methane. They are present in some but not all human colons and lower animal hindguts. Opinion varies on the nature of the factors influencing this ecology with some studies proposing the existence of host genetic influences. METHODS: Methane emission was measured in human twin pairs by gas chromatography, and structural equation modeling was used to determine the proportion of genetic and environmental determinants. The importance of the timing of environmental effects and rat strain on the trait of methane emission were ascertained by experiments with cohabiting methanogenic and nonmethanogenic rats. RESULTS: Analysis of breath samples from 274 adolescent twin pairs and their families indicated that the major influences on the trait of methane emission are the result of shared (53%, 95% confidence interval 39-61) and unique environmental (47%, 95% confidence interval 38-56) effects. No significant autosomal genetic effects were detected, but as observed in other studies, men (37%) were less likely to excrete methane in their breath than women (63%). Investigation of methane emission in rats indicated that environmental effects in this animal are most potent during the weaning period, with stable gut microbial ecology thereafter for some but not all rat strains. CONCLUSIONS: These results are consistent with shared and unique environmental factors being the main determinants of the ecology of this colonic microbe. (Am J Gastroenterol 2000;95:2872-2879. (C) 2000 by Am. Coll. of Gastroenterology).

Activation of NK cell cytotoxicity

Natural killer (NK) cells are innate effector lymphocytes necessary for defence against stressed, microbe-infected, or malignant cells. NK cells kill target cells by either of two major mechanisms that require direct contact between NK cells and target cells. In the first pathway, cytoplasmic granule toxins, predominantly a membrane-disrupting protein known as perforin, and a family of structurally related serine C, proteases (granzymes) with various substrate specificities, are secreted by exocytosis and together induce apoptosis of the target cell. The granule-exocytosis pathway potently activates cell-death mechanisms that operate through the activation of apoptotic cysteine proteases (caspases), but can also cause cell death in the absence of activated caspases. The second pathway involves the engagement of death receptors (e.g. Fas/CD95) on target cells by their cognate ligands (e.g. FasL on NK cells, resulting in classical caspase-dependent apoptosis. The comparative role of these pathways in the pathophysiology of many diseases is being dissected by analyses of gene-targeted mice that lack these molecules, and humans who have genetic mutations affecting these pathways. We are also now learning that the effector function of NK cells is controlled by interactions involving specific NK cell receptors and their cognate ligands, either on target cells, or other cells of the immune system. This review will discuss the functional importance of NK cell cytotoxicity and the receptor/ligand interactions that control these processes. (C) 2004 Elsevier Ltd. All rights reserved.

Guadalupian algae-sponge reefs in siliciclastic environments - the reefs at Lengwu (South China) compared with the reef at Iwaizaki (Japan)

Guadalupian reefs occur locally in Guangxi, Guizhou, Yunnan and Western Zhejiang, South China. Two types of Guadalupian reefs can be recognized, one is developed in carbonate platforms, e.g. those in the juncture areas of Guangxi, Yunnan and Guizhou; the other occurs in a littoral clastic shelf. The Lengwu reef in Western Zhejiang is a representative of the latter type, which is a major topic of this paper. Lengwu algae-sponge reef, more than one hundred meters in thickness, are composed mainly of sponges, hydrozoans, algae, bryozoans, microbes and lime mud. Reef limestones sit on the mudstone interbedded with fine sandstone of the proximal prodelta facies and are overlain by coarse clasts of the delta front sediments. Lengwu reef displays a lens-shaped relief, dipping and thinning from the reef core, which is remarkably different from the surrounding sediments, showing a protruding relief. Sponges and microbe/algae form bafflestone, bindstone and framestone of the reef core facies. Fore-reef facies is characterized by lithoclastic rudstone and bioclastic packstone. Reef limestone sequence is composed of three cycles and controlled by sea level changes and sediment influx. Such reef is unique among the Guadalupian reefs in South China, but seems similar in some aspects to lwaizaki reef limestones of south Kitakami in Japan. Algae and microbes growing around sponges to form rigid structure in Lengwu reef are a typical feature, which is distinctly different to Guadalupian reefs in a stable platform facies of Guizhou, Yunnan and Guangxi, South China.

Characterization of Pantoea dispersa UQ68J: producer of a highly efficient sucrose isomerase for isomaltulose biosynthesis

Aims: Isolation, identification and characterization of a highly efficient isomaltulose producer. Methods and Results: After an enrichment procedure for bacteria likely to metabolize isomaltulose in sucrose-rich environments, 578 isolates were screened for efficient isomaltulose biosynthesis using an aniline/diphenylamine assay and capillary electrophoresis. An isolate designated UQ68J was exceptionally efficient in sucrose isomerase activity. Conversion of sucrose into isomaltulose by UQ68J (enzyme activity of 90-100 U mg(-1) DW) was much faster than the current industrial strain Protaminobacter rubrum CBS574.77 (41-66 U mg(-1) DW) or a reference strain of Erwinia rhapontici (0.3-0.9 U mg(-1) DW). Maximum yield of isomaltulose at 78-80% of supplied sucrose was achieved in less than half the reaction time needed by CBS574.77, and the amount of contaminating trehalulose (4%) was the lowest recorded from an isomaltulose-producing microbe. UQ68J is a Gram negative, facultatively anaerobic, motile, noncapsulate, straight rod-shaped bacterium producing acid but no gas from glucose. Based on 16S rDNA analysis UQ68J is closest to Klebsiella oxytoca, but it differs from Klebsiella in defining characteristics and most closely resembles Pantoea dispersa in phenotype. Significance and Impact of Study: This organism is likely to have substantial advantage over previously characterized sucrose isomerase producers for the industrial production of isomaltulose.

Rhizodeposits of Trifolium pratense and Lolium perenne: Their comparative effects on 2,4-D mineralization in two contrasting soils

Rhizosphere enhanced biodegradation of organic pollutants has been reported frequently and a stimulatory role for specific components of rhizodeposits postulated. As rhizodeposit composition is a function of plant species and soil type, we compared the effect of Lolium perenne and Trifolium pratense grown in two different soils (a sandy silt loam: pH 4, 2.8% OC, no previous 2,4-D exposure and a silt loam: pH 6.5, 4.3% OC, previous 2,4-D exposure) on the mineralization of the herbicide 2,4-D (2,4-dichlorophenoxyacetic acid). We investigated the relationship of mineralization kinetics to dehydrogenase activity, most probable number of 2,4-D degraders (MPN2,4-D) and 2,4-D degrader composition (using sequence analysis of the gene encoding alpha-ketoglutarate/2,4-D dioxygenase (tfdA)). There were significant (P < 0.01) plant-soil interaction effects on MPN2,4-D and 2,4-D mineralization kinetics (e.g. T pratense rhizodeposits enhanced the maximum mineralization rate by 30% in the acid sandy silt loam soil, but not in the neutral silt loam soil). Differences in mineralization kinetics could not be ascribed to 2,4-D degrader composition as both soils had tfdA sequences which clustered with tfdAs representative of two distinct classes of 2,4-D degrader: canonical R. eutropha JMP134-like and oligotrophic alpha-proteobacterial-like. Other explanations for the differential rhizodeposit effect between soils and plants (e.g. nutrient competition effects) are discussed. Our findings stress that complexity of soil-plant-microbe interactions in the rhizosphere make the occurrence and extent of rhizosphere-enhanced xenobiotic degradation difficult to predict.

Rhizodeposition and the enhanced mineralization of 2,4-dichlorophenoxyacetic acid in soil from the Trifolium pratense rhizosphere

Enhanced biodegradation of organic xenobiotic compounds in the rhizosphere is frequently recorded although the specific mechanisms are poorly understood. We have shown that the mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D) is enhanced in soil collected from the rhizosphere of Trifolium pratense[e.g. maximum mineralization rate = 7.9 days(-1) and time at maximum rate (t(1)) = 16.7 days for 12-day-old T. pratense soil in comparison with 4.7 days(-1) and 25.4 days, respectively, for non-planted controls). The purpose of this study was to gain a better understanding of the plant-microbe interactions involved in rhizosphere-enhanced biodegradation by narrowing down the identity of the T. pratense rhizodeposit responsible for stimulating the microbial mineralization of 2,4-D. Specifically, we investigated the distribution of the stimulatory component(s) among rhizodeposit fractions (exudates or root debris) and the influence of soil properties and plant species on its production. Production of the stimulatory rhizodeposit was dependent on soil pH (e.g. t(1) for roots grown at pH 6.5 was significantly lower than for those grown at pH 4.4) but independent of soil inorganic N concentration. Most strikingly, the stimulatory rhizodeposit was only produced by T. pratense grown in non-sterile soil and was present in both exudates and root debris. Comparison of the effect of root debris from plant species (three each) from the classes monocotyledon, dicotyledon (non-legume) and dicotyledon (legume) revealed that legumes had by far the greatest positive impact on 2,4-D mineralization kinetics. We discuss the significance of these findings with respect to legume-rhizobia interactions in the rhizosphere.

Detection and discrimination of herpes simplex viruses, Haemophilus ducreyi, Treponema pallidum, and Calymmatobacterium (Klebsiella) granulomatis from genital ulcers

Background. Genital ulcer disease (GUD) is commonly caused by pathogens for which suitable therapies exist, but clinical and laboratory diagnoses may be problematic. This collaborative project was undertaken to address the need for a rapid, economical, and sensitive approach to the detection and diagnosis of GUD using noninvasive techniques to sample genital ulcers. Methods. The genital ulcer disease multiplex polymerase chain reaction (GUMP) was developed as an inhouse nucleic acid amplification technique targeting serious causes of GUD, namely, herpes simplex viruses (HSVs), Haemophilus ducreyi, Treponema pallidum, and Klebsiella species. In addition, the GUMP assay included an endogenous internal control. Amplification products from GUMP were detected by enzyme linked amplicon hybridization assay (ELAHA). Results. GUMP-ELAHA was sensitive and specific in detecting a target microbe in 34.3% of specimens, including 1 detection of HSV-1, three detections of HSV-2, and 18 detections of T. pallidum. No H. ducreyi has been detected in Australia since 1998, and none was detected here. No Calymmatobacterium ( Klebsiella) granulomatis was detected in the study, but there were 3 detections during ongoing diagnostic use of GUMP-ELAHA in 2004 and 2005. The presence of C. granulomatis was confirmed by restriction enzyme digestion and nucleotide sequencing of the 16S rRNA gene for phylogenetic analysis. Conclusions. GUMP-ELAHA permitted comprehensive detection of common and rare causes of GUD and incorporated noninvasive sampling techniques. Data obtained by using GUMP-ELAHA will aid specific treatment of GUD and better define the prevalence of each microbe among at-risk populations with a view to the eradication of chancroid and donovanosis in Australia.

Frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections

Viruses are the major cause of pediatric acute respiratory tract infection (ARTI) and yet many suspected cases of infection remain uncharacterized. We employed 17 PCR assays and retrospectively screened 315 specimens selected by season from a predominantly pediatric hospital-based population. Before the Brisbane respiratory virus research study commenced, one or more predominantly viral pathogens had been detected in 15.2% (n = 48) of all specimens. The Brisbane study made an additional 206 viral detections, resulting in the identification of a microbe in 67.0% of specimens. After our study, the majority of microbes detected were RNA viruses (89.9%). Overall, human rhinoviruses (HRVs) were the most frequently identified target (n=140) followed by human adenoviruses (HAdVs; n = 25), human metapneumovirus (HMPV; n=18), human bocavirus (HBoV; n = 15), human respiratory syncytial virus (HRSV; n = 12), human coronaviruses (HCoVs; n = 11), and human herpesvirus-6 (n = 11). HRVs were the sole microbe detected in 37.8% (n = 31) of patients with suspected lower respiratory tract infection (LRTI). Genotyping of the HRV VP4/VP2 region resulted in a proposed subdivision of HRV type A into sublineages A1 and A2. Most of the genotyped HAdV strains were found to be type C. This study describes the high microbial burden imposed by HRVs, HMPV, HRSV, HCoVs, and the newly identified virus, HBoV on a predominantly paediatric hospital population with suspected acute respiratory tract infections and proposes a new formulation of viral targets for future diagnostic research studies.

Agrobacterium rhizogenes transformation of the Phaseolus spp.: A tool for functional genomics

A fast, reproducible, and efficient transformation procedure employing Agrobacterium rhizogenes was developed for Phaseolus vulgaris L. wild accessions, landraces, and cultivars and for three other species belonging to the genus Phaseolus: R coccineus, P lunatus, and P acutifolius. Induced hairy roots are robust and grow quickly. The transformation frequency is between 75 and 90% based on the 35-S promoter-driven green fluorescent protein and beta-glucuronidase expression reporter constructs. When inoculated with Rhizobium tropici, transgenic roots induce normal determinate nodules that fix nitrogen as efficiently as inoculated standard roots. The A. rhizogenes-induced hairy root transformation in the genus Phaseolus sets the foundation for functional genomics programs focused on root physiology, root metabolism, and root-microbe interactions.