Mitochondrial DNA Diversity of Honey Bees (Apis mellifera) from Unmanaged Colonies and Swarms in the United States

To study the genetic diversity of honey bees (Apis mellifera L.) from unmanaged colonies in the United States, we sequenced a portion of the mitochondrial DNA COI–COII region. From the 530 to 1,230 bp amplicon, we observed 23 haplotypes from 247 samples collected from 12 states, representing three of the four A. mellifera lineages known to have been imported into the United States (C, M, and O). Six of the 13 C lineage haplotypes were not found in previous queen breeder studies in the United States. The O lineage accounted for 9% of unmanaged colonies which have not yet been reported in queen breeder studies. The M lineage accounted for a larger portion of unmanaged samples (7%) than queen breeder samples (3%). Based on our mitochondrial DNA data, the genetic diversity of unmanaged honey bees in the United States differs significantly from that of queen breeder populations (p < 0.00001). The detection of genetically distinct maternal lineages of unmanaged honey bees suggests that these haplotypes may have existed outside the managed honey bee population for a long period.     

Alternative Mechanisms for Coordinating Polymerase α and MCM Helicase

Functional coordination between DNA replication helicases and DNA polymerases at replication forks, achieved through physical linkages, has been demonstrated in prokaryotes but not in eukaryotes. In Saccharomyces cerevisiae, we showed that mutations that compromise the activity of the MCM helicase enhance the physical stability of DNA polymerase α in the absence of their presumed linker, Mcm10. Mcm10 is an essential DNA replication protein implicated in the stable assembly of the replisome by virtue of its interaction with the MCM2-7 helicase and Polα. Dominant mcm2 suppressors of mcm10 mutants restore viability by restoring the stability of Polα without restoring the stability of Mcm10, in a Mec1-dependent manner. In this process, the single-stranded DNA accumulation observed in the mcm10 mutant is suppressed. The activities of key checkpoint regulators known to be important for replication fork stabilization contribute to the efficiency of suppression. These results suggest that Mcm10 plays two important roles as a linker of the MCM helicase and Polα at the elongating replication fork—first, to coordinate the activities of these two molecular motors, and second, to ensure their physical stability and the integrity of the replication fork.The key players of the replication machinery are the DNA polymerases that synthesize the leading and lagging daughter strands and the replicative helicase that unwinds the parental strands ahead of the polymerases. Coordination between the helicase and the polymerases is critical during replication. Uncoupling of these two molecular machines, especially during lagging strand synthesis, may result in an unrestrained helicase and the exposure of extensive single-stranded DNA (ssDNA), as observed in checkpoint mutants treated with hydroxyurea (HU) (37). Although there is no direct evidence, the implication is that the replicative helicase would be moving at a faster pace than would the DNA polymerase if synchrony were destroyed. In Escherichia coli, the replicative helicase (DnaB) and the primase (DnaG) are coupled by direct contact to form a tight complex (3). In T7, processivity of the gp5 polymerase in lagging strand synthesis requires coupling to the gp4 helicase (16). Recent studies of the budding yeast Saccharomyces cerevisiae suggest that Mrc1 may couple DNA polymerase ɛ and the MCM helicase on the leading strand as well as activate the checkpoint response under replication stress (1, 22, 28). A candidate for coupling DNA polymerase α primase and the MCM helicase on the lagging strand is Mcm10, because Mcm10 interacts with subunits of the Mcm2-7 helicase (26, 29) as well as Polα (14, 33) and the stability of Polα requires Mcm10 in both budding yeast and human cells (8, 33). Mcm10 is an essential protein known to be involved in various aspects of the replication process. It is required during both initiation and elongation steps of DNA replication and interacts with a wide range of replication factors, such as ORC (17, 23, 29), MCM helicase, DNA polymerases ɛ and δ (23), Cdc45 (34), and Polα (33). Therefore, Mcm10 is important for the overall stability of the elongation complex, but its essential function remains unknown.Accumulating evidence suggests that the major function of many checkpoint proteins is the stabilization of the replication machinery at the fork (9, 22, 39), in addition to regulation of the temporal and spatial firing of origins and prevention of premature mitosis (31, 35, 39). The main signal that leads to checkpoint activation is believed to be the exposure of RPA-coated ssDNA (42). In Xenopus, ssDNA exposure has been shown to be mediated by a functional uncoupling between the polymerase and the helicase (7), and it has been shown that the level of checkpoint activation depended on the extent of ssDNA accumulation. This observation suggests that uncoupling of the polymerase and the helicase activity would result in ssDNA accumulation that in turn would activate the checkpoint pathway to stabilize the fork.In our study, we carried out a random and a gene-targeted mutagenesis screen to identify mutations that suppress the conditional lethality of mcm10 caused by the lability of Mcm10 in budding yeast (27). We found suppressor mutations in MCM2, which encodes one of the six distinct subunits of the MCM helicase. These mcm2 mutations correct the fork defects of mcm10, particularly that which leads to Polα instability. The altered helicase activity and activation of the checkpoint pathway of the mcm2 mutants appeared to be required for viability of mcm10 mcm2. We showed that uncoupling the MCM helicase and DNA polymerase α by destabilizing Mcm10 leads to accumulation of ssDNA, which is suppressed by reducing the MCM helicase activity. Our findings suggest that the physical coupling of Polα and the helicase by Mcm10 may be replaced by an alternative stabilization mechanism that involves slowing down the helicase and activating the checkpoint proteins.     

A nutritional profile of the social wasp Polistes metricus: Differences in nutrient levels between castes and changes within castes during the annual life cycle

In wasps, nutrition plays a vital role for colony cohesion and caste determination. However, there is no baseline data set for the nutritional levels of wasps during the different stages of the colony cycle. Here we examined the levels of carbohydrates, lipids, protein, Ca, Cu, Fe, K, Mg, Mn, Na, and Zn in the wasp Polistes metricus at different stages of the wasp's lifecycle. Individuals were collected at the following stages (1) spring gynes, (2) foundress colonies, (3) early worker colonies, (4) late worker colonies, (5) emerging reproductives (gynes and males), (6) early fall reproductives, and (7) late fall reproductives. All eggs, larvae, pupae and adults were analyzed for their nutritional content to determine if there were any differences between the nutrient levels in the different castes and how these nutrients changed within a caste during its lifetime. The results show there are differences in macro and micronutrient levels between the reproductive females and workers during development. Gynes showed changes in nutrient levels during their lifetime especially as they changed roles from a solitary individual to a nesting queen. Males also showed distinct nutritional changes during their lifetime. The implications for these nutritional differences are discussed.     

Production, characterization, and application of antibodies against heat-labile type-I toxin for detection of enterotoxigenic Escherichia coli

Strains of enterotoxigenic Escherichia coli (ETEC) are responsible for significant rates of morbidity and mortality among children, particularly in developing countries. The majority of clinical and public health laboratories are capable of isolating and identifying Salmonella, Shigella, Campylobacter, and Escherichia coli O157:H7 from stool samples, but ETEC cannot be identified by routine methods. The method most often used to identify ETEC is polymerase chain reaction for heat-stable and heat-labile enterotoxin genes, and subsequent serotyping, but most clinical and public health laboratories do not have the capacity or resources to perform these tests. In this study, polyclonal rabbit and monoclonal mouse IgG2b antibodies against ETEC heat-labile toxin-I (LT) were characterized and the potential applicability of a capture assay was analyzed. IgG-enriched fractions from rabbit polyclonal and the IgG2b monoclonal antibodies recognized LT in a conformational shape and they were excellent tools for detection of LT-producing strains. These findings indicate that the capture immunoassay could be used as a diagnostic assay of ETEC LT-producing strains in routine diagnosis and in epidemiological studies of diarrhea in developing countries as enzyme linked immunosorbent assay techniques remain as effective and economical choice for the detection of specific pathogen antigens in cultures.     

The ability of haemolysins expressed by atypical enteropathogenic Escherichia coli to bind to extracellular matrix components

Typical and atypical enteropathogenic Escherichia coli (EPEC) are considered important bacterial causes of diarrhoea. Considering the repertoire of virulence genes, atypical EPEC (aEPEC) is a heterogeneous group, harbouring genes that are found in other diarrheagenic E. coli pathotypes, such as those encoding haemolysins. Haemolysins are cytolytic toxins that lyse host cells disrupting the function of the plasma membrane. In addition, these cytolysins mediate a connection to vascular tissue and/or blood components, such as plasma and cellular fibronectin. Therefore, we investigated the haemolytic activity of 72 aEPEC isolates and determined the correlation of this phenotype with the presence of genes encoding enterohaemolysins (Ehly) and cytolysin A (ClyA). In addition, the correlation between the expression of haemolysins and the ability of these secreted proteins to adhere to extracellular matrix (ECM) components was also assessed in this study. Our findings demonstrate that a subset of aEPEC presents haemolytic activity due to the expression of Ehlys and/or ClyA and that this activity is closely related to the ability of these isolates to bind to ECM components.     

Nitrogen Forms Influence Microcystin Concentration and Composition via Changes in Cyanobacterial Community Structure

The eutrophication of freshwaters is a global health concern as lakes with excess nutrients are often subject to toxic cyanobacterial blooms. Although phosphorus is considered the main element regulating cyanobacterial biomass, nitrogen (N) concentration and more specifically the availability of different N forms may influence the overall toxicity of blooms. In this study of three eutrophic lakes prone to cyanobacterial blooms, we examined the effects of nitrogen species and concentrations and other environmental factors in influencing cyanobacterial community structure, microcystin (MC) concentrations and MC congener composition. The identification of specific MC congeners was of particular interest as they vary widely in toxicity. Different nitrogen forms appeared to influence cyanobacterial community structure leading to corresponding effects on MC concentrations and composition. Total MC concentrations across the lakes were largely explained by a combination of abiotic factors: dissolved organic nitrogen, water temperature and ammonium, but Microcystis spp. biomass was overall the best predictor of MC concentrations. Environmental factors did not appear to affect MC congener composition directly but there were significant associations between specific MC congeners and particular species. Based on redundancy analyses (RDA), the relative biomass of Microcystis aeruginosa was associated with MC-RR, M. wesenbergii with MC-LA and Aphanizomenon flos-aquae with MC-YR. The latter two species are not generally considered capable of MC production. Total nitrogen, water temperature, ammonium and dissolved organic nitrogen influenced the cyanobacterial community structure, which in turn resulted in differences in the dominant MC congener and the overall toxicity.     

Complete genome sequence of Riemerella anatipestifer type strain (ATCC 11845)

Riemerella anatipestifer (Hendrickson and Hilbert 1932) Segers et al. 1993 is the type species of the genus Riemerella, which belongs to the family Flavobacteriaceae. The species is of interest because of the position of the genus in the phylogenetic tree and because of its role as a pathogen of commercially important avian species worldwide. This is the first completed genome sequence of a member of the genus Riemerella. The 2,155,121 bp long genome with its 2,001 protein-coding and 51 RNA genes consists of one circular chromosome and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Complete genome sequence of Cellulophaga algicola type strain (IC166)

Cellulophaga algicola Bowman 2000 belongs to the family Flavobacteriaceae within the phylum 'Bacteroidetes' and was isolated from Melosira collected from the Eastern Antarctic coastal zone. The species is of interest because its members produce a wide range of extracellular enzymes capable of degrading proteins and polysaccharides with temperature optima of 20-30°C. This is the first completed genome sequence of a member of the genus Cellulophaga. The 4,888,353 bp long genome with its 4,285 protein-coding and 62 RNA genes consists of one circular chromosome and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Complete genome sequence of Desulfurococcus mucosus type strain (O7/1)

Desulfurococcus mucosus Zillig and Stetter 1983 is the type species of the genus Desulfurococcus, which belongs to the crenarchaeal family Desulfurococcaceae. The species is of interest because of its position in the tree of life, its ability for sulfur respiration, and several biotechnologically relevant thermostable and thermoactive extracellular enzymes. This is the third completed genome sequence of a member of the genus Desulfurococcus and already the 8(th) sequence from a member the family Desulfurococcaceae. The 1,314,639 bp long genome with its 1,371 protein-coding and 50 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.