Genetic manipulation allows in vivo tracking of the life cycle of the son-killer symbiont,Arsenophonus nasoniae,and reveals patterns of host invasion,tropism and pathology

Maternally heritable symbionts are common in arthropods and represent important partners and antagonists. A major impediment to understanding the mechanistic basis of these symbioses has been lack of genetic manipulation tools, for instance, those enabling transgenic GFP expression systems for in vivo visualization. Here, we transform the ‘son-killer’ reproductive parasite Arsenophonus nasoniae that infects the parasitic wasp Nasonia vitripennis with the plasmid pOM1-gfp, re-introduce this strain to N. vitripennis and then used this system to track symbiont life history in vivo. These data revealed transfer of the symbiont into the fly pupa by N. vitripennis during oviposition and N. vitripennis larvae developing infection over time through feeding. A strong tropism of A. nasoniae to the N. vitripennis ovipositor developed during wasp pupation, which aids onward transmission. The symbiont was also visualized in diapause larvae. Occasional necrotic diapause larvae were observed which displayed intense systemic infection alongside widespread melanotic nodules indicative of an active but failed immune response. Our results provide the foundation for the study of this symbiosis through in vivo tracking of the fate of symbionts through host development, which is rarely achieved in heritable microbe/insect interactions.     

Characterization of a novel gene for strain typing reveals substructuring of Aspergillus fumigatus across North America

Fifty-five epidemiologically linked Aspergillus fumigatus isolates obtained from six nosocomial outbreaks of invasive aspergillosis were subtyped by sequencing the polymorphic region of the gene encoding a putative cell surface protein, Afu3g08990 (denoted as CSP). Comparative sequence analysis showed that genetic diversity was generated in the coding region of this gene by both tandem repeats and point mutations. Each unique sequence in an outbreak cluster was assigned an arbitrary number or CSP sequence type. The CSP typing method was able to identify "clonal" and genotypically distinct A. fumigatus isolates, and the results of this method were concordant with those of another discriminatory genotyping technique, the Afut1 restriction fragment length polymorphism typing method. The novel single-locus sequence typing (CSP typing) strategy appears to be a simple, rapid, discriminatory tool that can be readily shared across laboratories. In addition, we found that A. fumigatus isolates substructured into multiple clades; interestingly, one clade consisted of isolates predominantly representing invasive clinical isolates recovered from cardiac transplant patients from two different outbreak situations. We also found that the A. fumigatus isolate Af293, whose genome has been sequenced, possesses a CSP gene structure that is substantially different from those of the other A. fumigatus strains studied here, highlighting the need for further taxonomic study.     

Variation in Release Factor Abundance Is Not Needed to Explain Trends in Bacterial Stop Codon Usage

In bacteria stop codons are recognized by one of two class I release factors (RF1) recognizing TAG, RF2 recognizing TGA, and TAA being recognized by both. Variation across bacteria in the relative abundance of RF1 and RF2 is thus hypothesized to select for different TGA/TAG usage. This has been supported by correlations between TAG:TGA ratios and RF1:RF2 ratios across multiple bacterial species, potentially also explaining why TAG usage is approximately constant despite extensive variation in GC content. It is, however, possible that stop codon trends are determined by other forces and that RF ratios adapt to stop codon usage, rather than vice versa. Here, we determine which direction of the causal arrow is the more parsimonious. Our results support the notion that RF1/RF2 ratios become adapted to stop codon usage as the same trends, notably the anomalous TAG behavior, are seen in contexts where RF1:RF2 ratios cannot be, or are unlikely to be, causative, that is, at 3′untranslated sites never used for translation termination, in intragenomic analyses, and across archaeal species (that possess only one RF1). We conclude that specifics of RF biology are unlikely to fully explain TGA/TAG relative usage. We discuss why the causal relationships for the evolution of synonymous stop codon usage might be different from those affecting synonymous sense codon usage, noting that transitions between TGA and TAG require two-point mutations one of which is likely to be deleterious.     

The competing countermarks hypothesis: reliable assessment of competitive ability by potential mates

Scent marking on top of (overmarking), or in the vicinity of, a scent mark already present is commonly termed countermarking. Scent marks and countermarks provide a continuous record of competitive challenges between conspecifics, thus providing a reliable advertisement of an individual's ability to dominate or defend an area to other competitors and potential mates. To test the hypothesis that females should prefer males that countermark competing scent marks in their territory over those whose own marks are partially countermarked by a competing male, we manipulated scent marks in the territories of neighbouring male house mice (captive-bred Mus domesticus). As predicted, oestrous females were more strongly attracted to approach territory owners that countermarked the scent mark challenges of competitors than those that had been countermarked, and females themselves deposited more scent marks near the scents of these males. To investigate whether female mice use scent age, overlap or intrinsic qualitative or quantitative differences between scent marks and countermarks to make this discrimination, we redeposited male scent marks artificially as marks and partially overlapping countermarks, with or without a 24-h age difference between them. While the intrinsic quality or quantity of countermarks did not affect discrimination, an age difference between the original mark and subsequent countermark was important for consistent discrimination. The ultimate function of such competitive scent signalling thus may be to provide potential mates with a reliable indicator of the competitive ability of individuals advertising their high status. Since scent marks remain in the environment and are continuously available to challenge and investigation, they may provide a particularly effective and reliable means of dominance advertisement. Copyright 1999 The Association for the Study of Animal Behaviour.     

Expression of mdm-2 oncoprotein in the primary and metastatic sites of mammary tumor (GI-101) implanted athymic nude mice

The expression of mdm-2 oncoprotein (p90) was determined in a human breast tumor xenograft line (GI-101) that was derived from a 57 year old female cancer patient with recurrent, infiltrating ductal adenocarcinoma (Stage IIIa, T3N2MX). Immunoprecipitation coupled western blot analysis of the primary tumors that have been obtained from xenograft implanted athymic nude mice, using mdm-2 (Ab-1) mouse monoclonal antibody, primarily revealed high level expression of a 90 kD full length mdm-2 protein. In the GI-101 tumor the level of full length mdm-2 (p90) protein expression increased with the increase in the size of the tumor (100 to 2,000 mm(3)) and a maximum expression was detected in 2,000 mm(3) size tumors. In addition to the expression in the primary site, a significantly high level expression of mdm-2 protein (p90) was detected in the lung and liver tissues also, which are the known metastatic sites for GI-101 xenograft tumors. However, the level of mdm-2 protein expression was undetectable in the lung and liver tissues obtained from control mice. A cell line (GI-101A) derived from the GI-101 xenograft tumor also showed a high level expression of mdm-2 protein after several generations of cell passage. When the GI-101A cells were treated with DES (Diethylstilbestrol) the mdm-2 protein expression increased after 10 min treatment and reached a peak level at 40 min. Interestingly, DES (10 and 20 microM) treatment increased the total cell number also after 96 hr treatment compared to the non-treated cells. It appears that mdm-2 (p90) may have a significant role in supporting the tumor cell growth as well as the metastatic process of the GI-101A cells.     

How biologically relevant are interaction-based modules in protein networks?

By applying a graph-based algorithm to yeast protein-interaction networks we have extracted modular structures and show that they can be validated using information from the phylogenetic conservation of the network components. We show that the module cores, the parts with the highest intramodular connectivity, are biologically relevant components of the networks. These constituents correlate only weakly with other levels of organization. We also discuss how such structures could be used for finding targets for antimicrobial drugs.     

Characterization of the 70S Ribosome from Rhodopseudomonas palustris using an integrated "top-down" and "bottom-up" mass spectrometric approach

We present a comprehensive mass spectrometric approach that integrates intact protein molecular mass measurement ("top-down") and proteolytic fragment identification ("bottom-up") to characterize the 70S ribosome from Rhodopseudomonas palustris. Forty-two intact protein identifications were obtained by the top-down approach and 53 out of the 54 orthologs to Escherichia coli ribosomal proteins were identified from bottom-up analysis. This integrated approach simplified the assignment of post-translational modifications by increasing the confidence of identifications, distinguishing between isoforms, and identifying the amino acid positions at which particular post-translational modifications occurred. Our combined mass spectrometry data also allowed us to check and validate the gene annotations for three ribosomal proteins predicted to possess extended C-termini. In particular, we identified a highly repetitive C-terminal "alanine tail" on L25. This type of low complexity sequence, common to eukaryotic proteins, has previously not been reported in prokaryotic proteins. To our knowledge, this is the most comprehensive protein complex analysis to date that integrates two MS techniques.     

How many species are infected with Wolbachia? Cryptic sex ratio distorters revealed to be common by intensive sampling.

Inherited bacterial symbionts from the genus Wolbachia have attracted much attention by virtue of their ability to manipulate the reproduction of their arthropod hosts. The potential importance of these bacteria has been underlined by surveys, which have estimated that 17% of insect species are infected. We examined whether these surveys have systematically underestimated the proportion of infected species through failing to detect the low-prevalence infections that are expected when Wolbachia distorts the sex ratio. We estimated the proportion of species infected with Wolbachia within Acraea butterflies by testing large collections of each species for infection. Seven out of 24 species of Acraea were infected with Wolbachia. Four of these were infected with Wolbachia at high prevalence, a figure compatible with previous broad-scale surveys, whilst three carried low-prevalence infections that would have had a very low likelihood of being detected by previous sampling methods. This led us to conclude that sex-ratio-distorting Wolbachia may be common in insects that have an ecology and/or genetics that permit the invasion of these parasites and that previous surveys may have seriously underestimated the proportion of species that are infected.