Different mechanisms for thermal inactivation of Bacillus subtilis signal peptidase mutants.

The type I signal peptidase SipS of Bacillus subtilis is of major importance for the processing of secretory precursor proteins. In the present studies, we have investigated possible mechanisms of thermal inactivation of five temperature-sensitive SipS mutants. The results demonstrate that two of these mutants, L74A and Y81A, are structurally stable but strongly impaired in catalytic activity at 48 degrees C, showing the (unprecedented) involvement of the conserved leucine 74 and tyrosine 81 residues in the catalytic reaction of type I signal peptidases. This conclusion is supported by the crystal structure of the homologous signal peptidase of Escherichia coli (Paetzel, M., Dalbey, R. E., and Strynadka, N. C. J. (1998) Nature 396, 186-190). In contrast, the SipS mutant proteins R84A, R84H, and D146A were inactivated by proteolytic degradation, indicating that the conserved arginine 84 and aspartic acid 146 residues are required to obtain a protease-resistant conformation. The cell wall-bound protease WprA was shown to be involved in the degradation of SipS D146A, which is in accord with the fact that SipS has a large extracytoplasmic domain. As WprA was not involved in the degradation of the SipS mutant proteins R84A and R84H, we conclude that multiple proteases are responsible for the thermal inactivation of temperature-sensitive SipS mutants.     

Rapid diagnostic assays in the genomic biology era: detection and identification of infectious disease and biological weapon agents

In this special section of BioTechniques, we examine the role of rapid molecular technologies in the detection and identification of agents of infectious disease (ID) and biological weapons (BWs). Besides the threat posed by the global proliferation of BW technologies, there are numerous emerging and reemerging ID agents with significant public health consequences. Further compounding this already complicated situation are the estimated 600 million international tourists annually, many with the potential to the spread disease globally in a matter of hours. While clinical laboratories have key roles in the detection and identification of potential ID/BW agents, most staff are unfamiliar with these agents because of their rarity and the often laborious conventional methodologies needed to identify them. To meet this challenge, a vast array of rapid assay strategies has been developed for use in clinical diagnostics and environmental detection. Technologies have been developed or adapted to the challenges posed by these agents, permitting detection and identification in several minutes to hours. In particular, the development of improved reagents and detection systems has led to dramatic improvements in the sensitivity and specificity of immunological and nucleic acid-based systems, allowing an ever-increasing range of analytes to be identified and quantitated. In the accompanying articles, we have brought together experts from the many overlapping aspects of this arena in order to present a comprehensive and critical analysis of these technologies.     

The effects of submerged aquatic vegetation on the persistence of environmental populations of Enterococcus spp.

Enterococcus spp. are utilized worldwide as faecal indicator bacteria, but certain strains exhibit extended survival in environmental habitats and the factors influencing their persistence are poorly understood. We used flowing freshwater mesocosms to explore the effect of submerged aquatic vegetation (SAV) on the persistence of natural enterococci populations from a subtropical lake. The highest mean densities of culturable enterococci over 2 weeks occurred in SAV [8.6 × 10² colony‐forming units (cfu) per 100 g wet weight], followed by sediments (1.3 × 10² cfu per 100 g) and water (18 cfu per 100 ml). However, due to relative differences in the total mass of each substrate in the entire system (water > sediments > SAV), SAV‐associated enterococci represented only a minor proportion of the total population. Vegetated mesocosms harboured significantly higher mean cfu per mesocosm and cfu densities in sediments compared with their unvegetated counterparts, suggesting that SAV indirectly facilitates persistence in aquatic habitats. Populations were dominated (> 96%) by a single Enterococcus casseliflavus strain according to BOX‐PCR genotyping, which did not change over the 10‐month study and strongly suggests bacterial replication in the lake. The presence of such strains in the environment may represent highly competitive, naturalized and reproducing indicator bacteria populations that are not directly related to pollution events.     

Persistence thresholds for phocine distemper virus infection in harbour seal Phoca vitulina metapopulations

1. This paper explores the concept of the critical community size for persistence of infection in wildlife populations. We use as a case study the 1988 epidemic of phocine distemper virus in the North Sea population of harbour seals, Phoca vitulina .
2. We summarize the available data on this epidemic and use it to parameterize a stochastic compartmental model for an infection spreading through a spatial array of patches coupled by nearest-neighbour mixing, with replacement of susceptibles occurring as a discrete annual event.
3. A combination of analytical and simulation techniques is used to show that the high levels of transmission between different seal subpopulations, combined with the small annual birth cohort, act to make persistence of infection impossible in this harbour seal population at realistic population levels. The well known mechanisms by which metapopulation structures may act to promote persistence can be seen to have an effect only at weaker levels of spatial coupling, and higher levels of host recruitment, than those empirically observed.     

Pollen interception by linyphiid spiders in a corn agroecosystem: implications for dietary diversification and risk-assessment

Dietary diversification, including consumption of plant tissues such as pollen, can enhance the fecundity of generalist predators, resulting in improved control of pest prey. Supplemental pollen feeding has been observed in many natural enemies, including sheet-web spiders (Araneae: Linyphiidae), which represent a major component of food webs in agroecosystems. Their horizontal, ground-based webs have the potential to intercept pollen grains during anthesis of crop plants, providing the opportunity for consumption of pollen to occur. In laboratory feeding trials, Frontinella communis and Tennesseellum formicum (Araneae: Linyphiidae) readily fed on pollen grains dusted on their webs, with 82 and 92% of spiders consuming pollen within the 210 min trial. These results revealed a strong potential for dietary supplementation with pollen in ground-based sheet-web spiders, indicating that pollen feeding may be an important component of the feeding biology of linyphiids. To measure pollen and prey interception in simulated linyphiid webs, a 20 m × 20 m grid of miniature sticky traps was established within and downwind of a corn agroecosystem. Traps were exposed for 24 h, all intercepted material was transferred to the laboratory for subsequent identification, and replaced with additional traps for 28 consecutive days in July and August 2008, to encompass periods before, during and after anthesis. Over 150,000 corn pollen grains and 5,000 prey items (dominated by Collembola and Hemiptera) were intercepted at simulated web-sites. Dates of peak anthesis resulted in pollen counts as high as 4,000 grains per web-site in the interior of the cornfield. Spatial Analysis by Distance Indices (SADIE) indicated significant temporal and spatial variability in pollen interception within and outside the corn field, but interestingly there was no significant spatial association between pollen and prey. Furthermore, transgenic Bacillus thuringiensis corn expresses insecticidal proteins in pollen, posing an exposure risk to non-target arthropods. Consumption of corn pollen may be a route to transgenic protein exposure in this important taxon of generalist predators.