bluuebunny/crossref_metadata_2025 huggingface dataset:
Laser‐assisted microdissection for the study of the ecology of parasites in their hosts
Authors: POST R. J. , CRAINEY J. L. , BIVAND A. , RENZ A.
Published: January 2009

The population biology of internal parasites is difficult to study because the adult parasites are often inaccessible, deep within the host's body. Developing stages, such as eggs in the faeces or larvae in the skin are more easily obtained, but are difficult to handle because they are often very small and with a tough cuticle. This has limited their use in molecular ecology for estimating population biology parameters of the adults (their parents). We have usedOnchocerca ochengi(a filarial nematode parasite of cattle) to describe a novel and generally applicable method of easily and conveniently isolating individual larvae (microfilariae) from the host using laser‐assisted microdissection. Furthermore, we have been able to improve the isolation of DNA by using the laser to bisect the larva to release DNA from the tissues enclosed within the parasite cuticle, and in this way we have achieved amplification of fragments over 1400 bp, and routinely PCR‐amplified single‐copy sequences from 5% of the DNA from a single larva (the equivalent of approximately 15 nuclei), and regularly from 0.5%.
Characterization of 11 new microsatellite loci in taro (Colocasia esculenta)
Authors: HU KAN , HUANG XING FANG , KE WEI DONG , DING YI
Published: January 2009

Eleven new microsatellite markers were isolated from taro, Colocasia esculenta (L.) Schott, a root crop widely distributed all over the world. Forty‐eight primer pairs were designed from a microsatellite‐enriched genomic library, of which 11 primer pairs have polymorphisms in 30 individuals tested from a population in China, which revealed two to six alleles per locus with the observed and expected heterozygosity levels ranging from 0 to 0.733 and from 0.381 to 0.731, respectively. These new genetic markers will be useful for the study of taro germplasm management and population evolution in the future.
Isolation and characterization of microsatellite loci from Lochmaea suturalis, the heather beetle (Coleoptera: Chrysomelidae), a pest in Europe and a biocontrol agent in New Zealand
Authors: HARMAN HELEN M. , WEIR BEVAN S. , WELLS SARAH J. , HOWITT ROBYN L. J.
Published: January 2009

The heather beetle Lochmaea suturalis which is native to northwest Europe has been released as a biocontrol agent for heather in New Zealand. We have isolated and optimized eight microsatellite loci from New Zealand beetles. These loci provide markers with high polymorphism ranging from four to 20 alleles per locus. Observed heterozygosity averaged 0.631 per locus. These results suggest the markers are useful for population studies that will contribute to assessment of L. suturalis as a biocontrol agent.
Microsatellite loci for population and parentage analysis in the Amazon River dolphin (Inia geoffrensis de Blainville, 1817)
Authors: GRAVENA WALESKA , HRBEK TOMAS , DA SILVA VERA M. S. , ASTOLFI‐FILHO SPARTACO , FARIAS IZENI P.
Published: January 2009

We developed specific primers for microsatellite DNA regions for the Amazon River dolphin or boto Inia geoffrensis, for use in population and conservation genetic studies. We also tested their transferability for two other species, Pontoporia blainvillei (sister taxon of I. geoffrensis) and Sotalia guianensis. A total of 12 microsatellite loci were polymorphic for the boto. An additional 25 microsatellite loci previously isolated from other cetacean species were also tested in the boto. The 26 polymorphic microsatellite loci indicate they will be excellent markers for studies of population structure and kinship relations of the boto.
Polymorphic microsatellite loci in the widespread amphidromous goby Sicyopterus lagocephalus and cross‐genus amplification among Sicydiinae
Authors: HOAREAU T. B. , BARBISAN F. , DUBOIS S. , ZANE L. , BERREBI P.
Published: January 2009

Microsatellite DNA markers were isolated in an amphidromous goby (Sicyopterus lagocephalus) from a partial genomic library enriched for AC repeats. Eight microsatellites were highly polymorphic with six to 33 alleles per locus and expected heterozygosities ranging from 0.53 to 0.97. Cross‐species amplifications were performed within the sub‐family Sicydiinae by genotyping individuals from two species of the genus Cotylopus. Some of these loci were successfully amplified and showed polymorphism in the second genus.
Four cleaved amplified polymorphic sequence (CAPS) markers for the detection of the Juglans ailantifolia chloroplast in putatively native J. cinerea populations
Authors: MCCLEARY TIM S. , ROBICHAUD RODNEY L. , NUANES STEVE , ANAGNOSTAKIS SANDRA L. , SCHLARBAUM SCOTT E. , ROMERO‐SEVERSON JEANNE
Published: January 2009

Hybridization between butternut (Juglans cinerea), a forest tree native to eastern North America, and Japanese walnut (J. ailantifolia), a tree tolerant to the lethal fungal disease butternut canker, casts doubt on the genetic identity of the remaining butternuts. We report a diagnostic test to distinguish the J. cinerea chloroplast from the J. ailantifolia chloroplast using cleaved amplified polymorphic sequences resolvable in 1.5% agarose gels. J. ailantifolia maternal ancestry in naturally regenerated stands provides a site selection criterion for studies of introgression dynamics when the non‐native parent and the hybrids tolerate a disease to which the native species is susceptible.
Rapid polymerase chain reaction–restriction fragment length polymorphism method for discrimination of the two Atlantic cryptic deep‐sea species of scabbardfish
Authors: STEFANNI SERGIO , BETTENCOURT RAUL , KNUTSEN HALVOR , MENEZES GUI
Published: January 2009

The present investigation provides an efficient diagnostic method based on polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis to discriminate between two cryptic species of scabbardfish, Aphanopus carbo and A. intermedius, with commercial relevance in several European fish markets. Two DNA fragments from the mtDNA, including control region and partial cytochrome oxidase subunit I genes of about 1100 bp and 700 bp, respectively, were isolated by PCR amplification. Digestion of the amplicon including the control region with HaeII and the amplicon including the COI gene with Sau3AI restriction enzymes allowed an unequivocal discrimination between the two scabbardfish species. This PCR–RFLP method allowed a clear and rapid discrimination of the trichiurid species studied.
Eight microsatellite loci for the sexually transmitted, parasitic mite Coccipolipus hippodamiae
Authors: GORDON LAURA K. , HURST GREGORY D. D. , WATTS PHILLIP C.
Published: January 2009

Seven dinucleotide and one trinucleotide polymorphic microsatellite loci were isolated from the mite Coccipolipus hippodamiae. This mite is an ectoparasite of coccinellid beetles (ladybirds), principally the European two‐spot ladybird Adalia bipunctata, where it causes sterility in the female host. Levels of genetic diversity were assessed using 32 mites from Warsaw, Poland. We observed moderate variability, with the number of alleles per locus varying between 2 and 4, and observed and expected heterozygosities ranging from 0.031 to 0.267 and between 0.062 and 0.526, respectively. This is the first description of microsatellite loci from the genus Coccipolipus and these loci are currently being employed to answer fundamental questions about the epidemiology of C. hippodamiae infections on A. bipunctata.
Eight novel tetranucleotide and five cross‐species dinucleotide microsatellite loci for the ornate chorus frog (Pseudacris ornata)
Authors: DEGNER JACOB F. , HETHER TYLER D. , HOFFMAN ERIC A.
Published: January 2009

We describe the cloning and characterization of eight novel tetranucleotide microsatellite loci in the ornate chorus frog (Pseudacris ornata). We also screened 26 loci from GenBank that were isolated from other Pseudacris species and obtained consistent product from five of these dinucleotide loci. All loci are polymorphic. In our sample of 26 frogs from a natural population, polymorphism ranged from 1 to 22 alleles per locus with expected heterozygosities ranging from 0 to 0.958. These loci enable high‐resolution studies of P. ornata. Moreover, cross‐species amplification success suggests they will also be useful for other chorus frog species.
Isolation of 11 polymorphic tri‐ and tetranucleotide microsatellite loci in a North American sedge (Carex scoparia: Cyperaceae) and cross‐species amplification in three additional Carex species
Authors: HIPP ANDREW L. , KETTENRING KARIN M. , FELDHEIM KEVIN A. , WEBER JAIME A.
Published: January 2009

We report on the isolation and evaluation of 11 microsatellites from a widespread eastern North American wetland sedge, Carex scoparia. Loci exhibit 3–9 alleles over five populations and significant FIS (0.204–0.717) in most populations. All primers cross‐amplify in at least two other species, and 10 cross‐amplify in the more distantly related C. stipata. These markers will be used to examine population genetics and patterns of chromosomal diversification in this ecologically important sedge species and its relatives.
Microsatellites in the Karoo scrub‐robin, Cercotrichas coryphaeus (Passeriformes: Muscicapinae): isolation and characterization of 13 autosomal and two sex‐linked loci
Authors: RIBEIRO ÂNGELA , LLOYD PENN , FELDHEIM KEVIN A. , BOWIE RAURI C. K.
Published: January 2009

Fifteen polymorphic microsatellites were developed for the Karoo scrub‐robin, Cercotrichas coryphaeus. Here we describe and characterize microsatellite variation of 13 autosomal loci and two Z‐linked loci in 42 individuals from two distinct South African populations. The number of alleles per locus varied from three to 13 and values of observed heterozygosity ranged from 0.318 to 0.900. These loci will be used to test hypotheses relating to fine‐scale social structure and mating strategies in this cooperatively breeding species.
A novel manual pooling system for preparing three‐dimensional pools of a deep coverage soybean bacterial artificial chromosome library
Authors: XIA ZHENGJUN , WATANABE SATOSHI , CHEN QINGSHAN , SATO SHUSEI , HARADA KYUYA
Published: January 2009

Compared with hybridization‐based techniques, polymerase chain reaction‐based screening of large insert libraries has been used widely as it is fast, easy and sensitive. However, various pooling strategies are needed to ensure efficient screening. It is time‐consuming and labourious to prepare three‐dimensional pools for a deep coverage bacterial artificial chromosome (BAC) library of soybean (1.12 × 109 bp) in the absence of robotic facility. In the present study, we describe a novel manual pooling system for preparing three‐dimensional pools of a soybean BAC library. This simple technique enables a single researcher to construct three‐dimensional pools for a deep‐coverage (12 haploid genome equivalents) BAC library of soybean in less than 2 months without any robotic manipulation. When the prepared three‐dimensional pools were screened with 29 polymerase chain reaction‐based markers, an average of 9.2 clones per marker were identified. These identified clones will be useful either in quantitative trait loci gene isolation or in synteny study between soybean and other legumes including Lotus japonicus. This efficient pooling system could be applied to any other BAC libraries without the need for robotic manipulation.
Development and characterization of microsatellite markers in Zanthoxylum ailanthoides (Rutaceae)
Authors: NAGAI H , YOSHIDA T , KAMIYA K , YAHARA T , TACHIDA H
Published: January 2009

Nine microsatellite markers were developed for Zanthoxylum ailanthoides, a typical pioneer tree. Averaged over the nine loci, the number of alleles per locus was 5.1. The observed and expected heterozygosities ranged from 0.233 to 0.833 and from 0.314 to 0.823, with averages of 0.606 and 0.641, respectively. No loci showed significant departures from Hardy–Weinberg equilibrium or linkage equilibrium after Bonferroni correction (P > 0.05). These markers will be useful for parentage analyses and studies of population genetic structure in the species.
Foveal function in children treated for amblyopia
Authors: Agervi Pia , Nilsson Maria , Martin Lene
Published: January 2009

Purpose:  This study aimed to evaluate foveal function, using three different methods, in children treated for monocular amblyopia.

Methods:  A sample of 24 otherwise healthy children with treated amblyopia and an age‐matched control group of 25 healthy children were examined for best corrected visual acuity (BCVA) using a standard decimal (KM) chart and the computerized TriVA method at 50% and 10% contrasts. Foveal function was also measured with the rarebit fovea test (RFT), which is included in the rarebit perimetry program package. This test uses very small and bright dots against a dark background. The result is expressed as mean hit rate (MHR).

Results:  Amblyopic eyes showed significantly lower BCVA when evaluated with the KM chart and with the TriVA test at different contrast levels, compared with both fellow eyes and control eyes. No statistically significant difference between amblyopic and fellow eyes was found when foveal function was evaluated with the RFT (median MHRs 91.5% and 94.5%, respectively), although results for both amblyopic and fellow eyes were statistically lower than those of the control group (median MHR 97%) (p = 0.001 and p = 0.046, respectively). This might indicate that the RFT provides different information about foveal function than conventional VA tests.

Conclusions:  The findings in the current study accord with those of other studies reporting abnormalities in the fellow eyes of previously treated amblyopic patients. These findings may reflect a general disturbance in the visual system rather than a monocular adaptation to refractive error or ocular motor disturbance.
Indole-Diterpene Biosynthetic Capability ofEpichloëEndophytes as Predicted byltmGene Analysis
Authors: Young Carolyn A. , Tapper Brian A. , May Kimberley , Moon Christina D. , Schardl Christopher L. , Scott Barry
Published: January 2009

Bioprotective alkaloids produced byEpichloëand closely related asexualNeotyphodiumfungal endophytes protect their grass hosts from insect and mammalian herbivory. One class of these compounds, known for antimammalian toxicity, is the indole-diterpenes. TheLTMlocus ofNeotyphodium lolii(Lp19) andEpichloë festuce(Fl1), required for the biosynthesis of the indole-diterpene lolitrem, consists of 10ltmgenes. We have used PCR and Southern analysis to screen a broad taxonomic range of 44 endophyte isolates to determine why indole-diterpenes are present in so few endophyte-grass associations in comparison to that of the other bioprotective alkaloids, which are more widespread among the endophtyes. All 10ltmgenes were present in only three epichloë endophytes. A predominance of the asexualNeotyphodiumspp. examined contained 8 of the 10ltmgenes, with only oneN. loliicontaining the entireLTMlocus and the ability to produce lolitrems. Liquid chromatography-tandem mass spectrometry profiles of indole-diterpenes from a subset of endophyte-infected perennial ryegrass showed that endophytes that contained functional genes present inltmclusters 1 and 2 were capable of producing simple indole-diterpenes such as paspaline, 13-desoxypaxilline, and terpendoles, compounds predicted to be precursors of lolitrem B. Analysis of toxin biosynthesis genes by PCR now enables a diagnostic method to screen endophytes for both beneficial and detrimental alkaloids and can be used as a resource for screening isolates required for forage improvement.
Interaction of
Bacillus thuringiensis
Cry1 and Vip3A Proteins with
Spodoptera frugiperda
Midgut Binding Sites
Authors: Sena Janete A. D. , Hernández-Rodríguez Carmen Sara , Ferré Juan
Published: January 2009

Vip3Aa, Vip3Af, Cry1Ab, and Cry1Fa were tested for their toxicities and binding interactions. Vip3A proteins were more toxic than Cry1 proteins. Binding assays showed independent specific binding sites for Cry1 and Vip3A proteins. Cry1Ab and Cry1Fa competed for the same binding sites, whereas Vip3Aa competed for those of Vip3Af.
Selection of
Sphingomonadaceae
at the Base of
Laccaria proxima
and
Russula exalbicans
Fruiting Bodies
Authors: Boersma F. G. Hidde , Warmink Jan A. , Andreote Fernando A. , van Elsas Jan Dirk
Published: January 2009

The dense hyphal network directly underneath the fruiting bodies of ectomycorrhizal fungi might exert strong influences on the bacterial community of soil. Such fruiting bodies might serve as hot spots for bacterial activity, for instance by providing nutrients and colonization sites in soil. Here, we assessed the putative selection of specific members of the
Sphingomonadaceae
family at the bases of the fruiting bodies of the ectomycorrhizal fungi
Laccaria proxima
and
Russula exalbicans
in comparison to the adjacent bulk soil. To do so, we used a previously designed
Sphingomonadaceae
-specific PCR-denaturing gradient gel electrophoresis (DGGE) system and complemented this with analyses of sequences from a
Sphingomonadaceae
-specific clone library. The analyses showed clear selective effects of the fruiting bodies of both fungi on the
Sphingomonadaceae
community structures. The effect was especially prevalent with
R. exalbicans
. Strikingly, similar fungi sampled approximately 100 m apart showed similar DGGE patterns, while corresponding bulk soil-derived patterns differed from each other. However, the mycospheres of
L. proxima
and
R. exalbicans
still revealed divergent community structures, indicating that different fungi select for different members of the
Sphingomonadaceae
family. Excision of specific bands from the DGGE patterns, as well as analyses of the clone libraries generated from both habitats, revealed fruiting body-specific
Sphingomonadaceae
types. It further showed that major groups from the mycospheres of
R. exalbicans
and
L. proxima
did not cluster with known bacteria from the database, indicating new groups within the family of
Sphingomonadaceae
present in these environments.
Diverse and Novel
nifH
and
nifH
-Like Gene Sequences in the Deep-Sea Methane Seep Sediments of the Okhotsk Sea
Authors: Dang Hongyue , Luan Xiwu , Zhao Jingyi , Li Jing
Published: January 2009

Diverse
nifH
and
nifH
-like gene sequences were obtained from the deep-sea surface sediments of the methane hydrate-bearing Okhotsk Sea. Some sequences formed novel families of the NifH or NifH-like proteins, of currently unresolved bacterial or archaeal origin. Comparison with other marine environments indicates environmental specificity of some of the sequences, either unique to the methane seep sediments of the Okhotsk Sea or to the general deep-sea methane seep sedimentary environments.
Interactions between Coexisting Intracellular Genomes: Mitochondrial Density and
Wolbachia
Infection
Authors: Mouton L. , Henri H. , Fleury F.
Published: January 2009

Many arthropods are infected with maternally transmitted microorganisms, leading to the coexistence of several intracellular genomes within the host cells, including their own mitochondria. As these genomes are cotransmitted, their patterns of evolution have been intimately linked, with possible consequences for the diversity and evolution of the host mitochondrial DNA. The evolutionary aspects of the situation have been thoroughly investigated, especially the selective sweep on the mitochondria as a result of
Wolbachia
invasion, whereas direct interactions between mitochondria and intracellular symbionts within the host cells or body have received little attention. Since endosymbionts exploit host resources but mitochondria supply energy to meet the bioenergetic demands of organisms, an unanswered question concerns the correlation between their densities. Here, we investigated the influence of
Wolbachia
symbiosis on mitochondrial density in two parasitic wasps of
Drosophila
species, both of which are naturally infected by three
Wolbachia
strains, but they differ in their degree of dependency on these bacteria. In
Leptopilina heterotoma
, all
Wolbachia
strains are facultative, whereas
Asobara tabida
requires a strain of
Wolbachia
for oogenesis to occur. In both species,
Wolbachia
infections are stable and well regulated, since the density of each strain does not depend on the presence or absence of other strains. Using lines that harbor various
Wolbachia
infection statuses, we found that mitochondrial density was not affected by the infection regardless of the sex and age of the host, which is strongly reminiscent of the independent regulation of specific
Wolbachia
strains and suggest that the protagonists coexist independently of each other as the result of a long-term coevolutionary interaction.
HacA-Dependent Transcriptional Switch Releases
hacA
mRNA from a Translational Block upon Endoplasmic Reticulum Stress
Authors: Mulder Harm J. , Nikolaev Igor
Published: January 2009

Activation of the unfolded protein response (UPR) in eukaryotes involves the splicing of an unconventional intron from the mRNA encoding the transcriptional activator of the pathway. In
Saccharomyces cerevisiae
a 252-nucleotide (nt) unconventional intron is spliced out of the transcript of
HAC1
, changing the 3′ end of the
HAC1
open reading frame and relieving the transcript from a translational block in a single step. The translational block is caused by the base pairing of part of the unconventional intron with the 5′-untranslated region (5′UTR). In
Aspergillus niger
and other aspergilli, the unconventional intron in
hacA
mRNA is only 20 nt long. Since this intron is part of a stable stem-loop structure, base pairing with the 5′UTR, in contrast to the case with yeast
HAC1
, is not possible. However, analysis of the
hacA
mRNA revealed a GC-rich inverted repeat (18 base pairings). Upon the activation of the UPR, the 5′UTR of
hacA
mRNA is truncated by 230 nt, removing the left part of this inverted repeat. This implies a similar release of a translational block as in the case of
S. cerevisiae HAC1
but in two steps. The mechanism behind the 5′ truncation, which does not take place in either yeast
HAC1
or mammalian
xbp1
mRNA, has been hitherto unknown. Here we show that during secretion stress in
A. niger
,
hacA
transcription starts from a new start site closer to the ATG, relieving the transcript from translational attenuation. This transcriptional switch is mediated by HacA itself and the unfolded protein response element 2 (UPRE2) in the
hacA
promoter.
Analysis of All Protein Phosphatase Genes in
Aspergillus nidulans
Identifies a New Mitotic Regulator, Fcp1
Authors: Son Sunghun , Osmani Stephen A.
Published: January 2009

Reversible protein phosphorylation is an important regulatory mechanism of cell cycle control in which protein phosphatases counteract the activities of protein kinases. In
Aspergillus nidulans
, 28 protein phosphatase catalytic subunit genes were identified. Systematic deletion analysis identified four essential phosphatases and four required for normal growth. Conditional alleles of these were generated using the
alcA
promoter. The deleted phosphatase strain collection and regulatable versions of the essential and near-essential phosphatases provide an important resource for further analysis of the role of reversible protein phosphorylation to the biology of
A. nidulans
. We further demonstrate that
nimT
and
bimG
have essential functions required for mitotic progression since their deletions led to classical G
2
- and M-phase arrest. Although not as obvious, cells with An
pphA
and An
nem1
deleted also have mitotic abnormalities. One of the essential phosphatases, the RNA polymerase II C-terminal domain phosphatase An
fcp1
, was further examined for potential functions in mitosis because a temperature-sensitive An
fcp1
allele was isolated in a genetic screen showing synthetic interaction with the
cdk1F
mutation, a hyperactive mitotic kinase. The An
fcp1

ts

cdk1F
double mutant had severe mitotic defects, including inability of nuclei to complete mitosis in a normal fashion. The severity of the An
fcp1

ts

cdk1F
mitotic phenotypes were far greater than either single mutant, confirming the synthetic nature of their genetic interaction. The mitotic defects of the An
fcp1

ts

cdk1F
double mutant suggests a previously unrealized function for AnFCP1 in regulating mitotic progression, perhaps counteracting Cdk1-mediated phosphorylation.
Isolation and Characterization of
Cryptococcus neoformans
Spores Reveal a Critical Role for Capsule Biosynthesis Genes in Spore Biogenesis
Authors: Botts Michael R. , Giles Steven S. , Gates Marcellene A. , Kozel Thomas R. , Hull Christina M.
Published: January 2009

Spores are essential particles for the survival of many organisms, both prokaryotic and eukaryotic. Among the eukaryotes, fungi have developed spores with superior resistance and dispersal properties. For the human fungal pathogens, however, relatively little is known about the role that spores play in dispersal and infection. Here we present the purification and characterization of spores from the environmental fungus
Cryptococcus neoformans
. For the first time, we purified spores to homogeneity and assessed their morphological, stress resistance, and surface properties. We found that spores are morphologically distinct from yeast cells and are covered with a thick spore coat. Spores are also more resistant to environmental stresses than yeast cells and display a spore-specific configuration of polysaccharides on their surfaces. Surprisingly, we found that the surface of the spore reacts with antibodies to the polysaccharide glucuronoxylomannan, the most abundant component of the polysaccharide capsule required for
C. neoformans
virulence. We explored the role of capsule polysaccharide in spore development by assessing spore formation in a series of acapsular strains and determined that capsule biosynthesis genes are required for proper sexual development and normal spore formation. Our findings suggest that
C. neoformans
spores may have an adapted cell surface that facilitates persistence in harsh environments and ultimately allows them to infect mammalian hosts.
Identification and Specific Localization of Tyrosine-Phosphorylated Proteins in
Trypanosoma brucei
Authors: Nett Isabelle R. E. , Davidson Lindsay , Lamont Douglas , Ferguson Michael A. J.
Published: January 2009

Phosphorylation on tyrosine residues is a key signal transduction mechanism known to regulate intercellular and intracellular communication in multicellular organisms. Despite the lack of conventional tyrosine kinases in the genome of the single cell organism
Trypanosoma brucei
, phosphorylation on trypanosomal protein tyrosine residues has been reported for this parasite. However, the identities of most of the tyrosine-phosphorylated proteins and their precise site(s) of phosphorylation were unknown. Here, we have applied a phosphotyrosine-specific proteomics approach to identify 34 phosphotyrosine-containing proteins from whole-cell extracts of procyclic form
T. brucei
. A significant proportion of the phosphotyrosine-containing proteins identified in this study were protein kinases of the CMGC kinase group as well as some proteins of unknown function and proteins involved in energy metabolism, protein synthesis, and RNA metabolism. Interestingly, immunofluorescence microscopy using anti-phosphotyrosine antibodies suggests that there is a concentration of tyrosine-phosphorylated proteins associated with cytoskeletal structures (basal body and flagellum) and in the nucleolus of the parasite. This localization of tyrosine-phosphorylated proteins supports the idea that the function of signaling molecules is controlled by their precise location in
T. brucei
, a principle well known from higher eukaryotes.
Anthrax Protective Antigen Delivered bySalmonella entericaSerovar Typhi Ty21a Protects Mice from a Lethal Anthrax Spore Challenge
Authors: Osorio Manuel , Wu Yanping , Singh Sunil , Merkel Tod J. , Bhattacharyya Siba , Blake Milan S. , Kopecko Dennis J.
Published: January 2009

Bacillus anthracis, the etiological agent of anthrax disease, is a proven weapon of bioterrorism. Currently, the only licensed vaccine against anthrax in the United States is AVA Biothrax, which, although efficacious, suffers from several limitations. This vaccine requires six injectable doses over 18 months to stimulate protective immunity, requires a cold chain for storage, and in many cases has been associated with adverse effects. In this study, we modified theB. anthracisprotective antigen (PA) gene for optimal expression and stability, linked it to an inducible promoter for maximal expression in the host, and fused it to the secretion signal of theEscherichia colialpha-hemolysin protein (HlyA) on a low-copy-number plasmid. This plasmid was introduced into the licensed typhoid vaccine strain,Salmonella entericaserovar Typhi strain Ty21a, and was found to be genetically stable. Immunization of mice with three vaccine doses elicited a strong PA-specific serum immunoglobulin G response with a geometric mean titer of 30,000 (range, 5,800 to 157,000) and lethal-toxin-neutralizing titers greater than 16,000. Vaccinated mice demonstrated 100% protection against a lethal intranasal challenge with aerosolized spores ofB. anthracis7702. The ultimate goal is a temperature-stable, safe, oral human vaccine against anthrax infection that can be self-administered in a few doses over a short period of time.
Structural and Functional Importance of Transmembrane Domain 3 (TM3) in the Aspartate:Alanine Antiporter AspT: Topology and Function of the Residues of TM3 and Oligomerization of AspT
Authors: Nanatani Kei , Maloney Peter C. , Abe Keietsu
Published: January 2009

AspT, the aspartate:alanine antiporter ofTetragenococcus halophilus, a membrane protein of 543 amino acids with 10 putative transmembrane (TM) helices, is the prototype of the aspartate:alanine exchanger (AAE) family of transporters. Because TM3 (isoleucine 64 to methionine 85) has many amino acid residues that are conserved among members of the AAE family and because TM3 contains two charged residues and four polar residues, it is thought to be located near (or to form part of) the substrate translocation pathway that includes the binding site for the substrates. To elucidate the role of TM3 in the transport process, we carried out cysteine-scanning mutagenesis. The substitutions of tyrosine 75 and serine 84 had the strongest inhibitory effects on transport (initial rates ofl-aspartate transport were below 15% of the rate for cysteine-less AspT). Considerable but less-marked effects were observed upon the replacement of methionine 70, phenylalanine 71, glycine 74, arginine 76, serine 83, and methionine 85 (initial rates between 15% and 30% of the rate for cysteine-less AspT). Introduced cysteine residues at the cytoplasmic half of TM3 could be labeled with Oregon green maleimide (OGM), whereas cysteines close to the periplasmic half (residues 64 to 75) were not labeled. These results suggest that TM3 has a hydrophobic core on the periplasmic half and that hydrophilic residues on the cytoplasmic half of TM3 participate in the formation of an aqueous cavity in membranes. Furthermore, the presence ofl-aspartate protected the cysteine introduced at glycine 62 against a reaction with OGM. In contrast,l-aspartate stimulated the reactivity of the cysteine introduced at proline 79 with OGM. These results demonstrate that TM3 undergoesl-aspartate-induced conformational alterations. In addition, nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses and a glutaraldehyde cross-linking assay suggest that functional AspT forms homo-oligomers as a functional unit.
Identification of the Polyketide Synthase Involved in the Biosynthesis of the Surface-Exposed Lipooligosaccharides in Mycobacteria
Authors: Etienne Gilles , Malaga Wladimir , Laval Françoise , Lemassu Anne , Guilhot Christophe , Daffé Mamadou
Published: January 2009

Lipooligosaccharides (LOS) are highly antigenic glycolipids produced by a number of
Mycobacterium
species, which include “
M. canettii
,” a member of the
M. tuberculosis
complex, and the opportunistic pathogens
M. marinum
and
M. kansasii
. The various LOS share a core composed of trehalose esterified by at least 1 mole of polymethyl-branched fatty acid (PMB-FA) and differ from one another by their oligosaccharide extensions. In this study, we identified a cluster of genes, MSMEG_4727 through MSMEG_4741, likely involved in the synthesis of LOS in
M. smegmatis
. Disruption of MSMEG_4727 (the ortholog of
pks5
of
M. tuberculosis
), which encodes a putative polyketide synthase, resulted in the concomitant abrogation of the production of both PMB-FA and LOS in the mutant strain. Complementation of the mutant with the wild-type gene fully restored the phenotype. We also showed that, in contrast to the case for “
M. canettii
” and
M. marinum
, LOS are located in deeper compartments of the cell envelope of
M. smegmatis
. The availability of two mycobacterial strains differing only in LOS production should help in defining the biological role(s) of this important glycolipid.
The Rv1712 Locus from
Mycobacterium tuberculosis
H37Rv Codes for a Functional CMP Kinase That Preferentially Phosphorylates dCMP
Authors: Thum Caroline , Schneider Cristopher Z. , Palma Mario S. , Santos Diógenes S. , Basso Luiz A.
Published: January 2009

The
Mycobacterium tuberculosis cmk
gene, predicted to encode a CMP kinase (CMK), was cloned and expressed, and its product was purified to homogeneity. Steady-state kinetics confirmed that
M. tuberculosis
CMK is a monomer that preferentially phosphorylates CMP and dCMP by a sequential mechanism. A plausible role for CMK is discussed.
Properties of HflX, an Enigmatic Protein from
Escherichia coli
Authors: Dutta Dipak , Bandyopadhyay Kaustav , Datta Ajit Bikram , Sardesai Abhijit A. , Parrack Pradeep
Published: January 2009

The
Escherichia coli
gene
hflX
was first identified as part of the
hflA
operon, mutations in which led to an increased frequency of lysogenization upon infection of the bacterium by the temperate coliphage lambda. Independent mutational studies have also indicated that the HflX protein has a role in transposition. Based on the sequence of its gene, HflX is predicted to be a GTP-binding protein, very likely a GTPase. We report here purification and characterization of the HflX protein. We also specifically examined its suggested functional roles mentioned above. Our results show that HflX is a monomeric protein with a high (30% to 40%) content of helices. It exhibits GTPase as well as ATPase activities, but it has no role in lambda lysogeny or in transposition.
Expression and Mutational Analysis of the
glnB
Genomic Region in the Heterocyst-Forming Cyanobacterium
Anabaena
sp. Strain PCC 7120
Authors: Paz-Yepes Javier , Flores Enrique , Herrero Antonia
Published: January 2009

In the filamentous, heterocyst-forming cyanobacterium
Anabaena
sp. strain PCC 7120, the
glnB
gene is expressed at considerable levels both in the presence and in the absence of combined nitrogen, although induction, influenced by NtcA, takes place upon combined-nitrogen deprivation likely localized to vegetative cells. In spite of extensive efforts, a derivative of PCC 7120 lacking a functional
glnB
gene could be obtained only with constructs that lead to overexpression of a downstream open reading frames (ORF), particularly all2318. Strain CSP10 [
glnB
all2318(Con)] exhibited growth rates similar to those of the wild type when it was using nitrate or ammonium, but its diazotrophic growth was impaired. However, it differentiated heterocysts with a time course and distribution pattern similar to those of the wild type, although with no cyanophycin-containing polar granules, and exhibited impaired nitrogenase activity under oxic conditions, but not under microoxic conditions. In the mutant, NtcA-dependent inducion of the
hetC
and
nifH
genes was unaltered, but induction of the
urtA
gene and urea transport activity were increased. Active uptake of nitrite was also increased and insensitive to the ammonium-promoted inhibition observed for the wild type. Thus, regulation of the nitrite transport activity requires the
glnB
gene product. In the presence of a wild-type
glnB
gene, neither inactivation nor overexpression of all2318 produced an apparent phenotype. Thus, in an otherwise wild-type background, the
glnB
gene appears to be essential for growth of strain PCC 7120. For growth with combined nitrogen but not for diazotrophic growth, the requirement for
glnB
can be overridden by increasing the expression of all2318 (and/or ORFs downstream of it).
Constant
c
10
Ring Stoichiometry in the
Escherichia coli
ATP Synthase Analyzed by Cross-Linking
Authors: Ballhausen Britta , Altendorf Karlheinz , Deckers-Hebestreit Gabriele
Published: January 2009

The subunit
c
stoichiometry of
Escherichia coli
ATP synthase was studied by intermolecular cross-linking via oxidation of bi-cysteine-substituted subunit
c
(
c
A21C/
c
M65C). Independent of the carbon source used for growth and independent of the presence of other F
o
F
1
subunits, an equal pattern of cross-link formation stopping at the formation of decamers was obtained.
Conjugative Transfer of the Integrative Conjugative Elements ICE
St1
and ICE
St3
from
Streptococcus thermophilus
Authors: Bellanger Xavier , Roberts Adam P. , Morel Catherine , Choulet Frédéric , Pavlovic Guillaume , Mullany Peter , Decaris Bernard , Guédon Gérard
Published: January 2009

Integrative and conjugative elements (ICEs), also called conjugative transposons, are genomic islands that excise, self-transfer by conjugation, and integrate in the genome of the recipient bacterium. The current investigation shows the intraspecies conjugative transfer of the first described ICEs in
Streptococcus thermophilus
, ICE
St1
and ICE
St3
. Mitomycin C, a DNA-damaging agent, derepresses ICE
St3
conjugative transfer almost 25-fold. The ICE
St3
host range was determined using various members of the
Firmicutes
as recipients. Whereas numerous ICE
St3
transconjugants of
Streptococcus pyogenes
and
Enterococcus faecalis
were recovered, only one transconjugant of
Lactococcus lactis
was obtained. The newly incoming ICEs, except the one from
L. lactis
, are site-specifically integrated into the 3′ end of the
fda
gene and are still able to excise in these transconjugants. Furthermore, ICE
St3
was retransferred from
E. faecalis
to
S. thermophilus
. Recombinant plasmids carrying different parts of the ICE
St1
recombination module were used to show that the integrase gene is required for the site-specific integration and excision of the ICEs, whereas the excisionase gene is required for the site-specific excision only.
Different Biochemical Mechanisms Ensure Network-Wide Balancing of Reducing Equivalents in Microbial Metabolism
Authors: Fuhrer Tobias , Sauer Uwe
Published: January 2009

To sustain growth, the catabolic formation of the redox equivalent NADPH must be balanced with the anabolic demand. The mechanisms that ensure such network-wide balancing, however, are presently not understood. Based on13C-detected intracellular fluxes, metabolite concentrations, and cofactor specificities for all relevant central metabolic enzymes, we have quantified catabolic NADPH production inAgrobacterium tumefaciens, Bacillus subtilis, Escherichia coli, Paracoccus versutus, Pseudomonas fluorescens, Rhodobacter sphaeroides, Sinorhizobium meliloti, andZymomonas mobilis. For six species, the estimated NADPH production from glucose catabolism exceeded the requirements for biomass synthesis. Exceptions wereP. fluorescens, with balanced rates, andE. coli, with insufficient catabolic production, in which about one-third of the NADPH is supplied via the membrane-bound transhydrogenase PntAB.P. versutusandB. subtiliswere the only species that appear to rely on transhydrogenases for balancing NADPH overproduction during growth on glucose. In the other four species, the main but not exclusive redox-balancing mechanism appears to be the dual cofactor specificities of several catabolic enzymes and/or the existence of isoenzymes with distinct cofactor specificities, in particular glucose 6-phosphate dehydrogenase. An unexpected key finding for all species, exceptE. coliandB. subtilis, was the lack of cofactor specificity in the oxidative pentose phosphate pathway, which contrasts with the textbook view of the pentose phosphate pathway dehydrogenases as being NADP+dependent.
Role of Potassium Uptake Systems in
Sinorhizobium meliloti
Osmoadaptation and Symbiotic Performance
Authors: Domínguez-Ferreras Ana , Muñoz Socorro , Olivares José , Soto María J. , Sanjuán Juan
Published: January 2009

Stimulation of potassium uptake is the most rapid response to an osmotic upshock in bacteria. This cation accumulates by a number of different transport systems whose importance has not been previously addressed for rhizobia. In silico analyses reveal the presence of genes encoding four possible potassium uptake systems in the genome of
Sinorhizobium meliloti
1021: Kup1, Kup2, Trk, and Kdp. The study of the relevance of these systems under a number of different growth conditions and in symbiosis showed that the integrity of Kup1 or Trk is essential for growth under laboratory conditions even in osmotically balanced media and the absence of both systems leads to a reduced infectivity and competitiveness of the bacteria in alfalfa roots. Trk is the main system involved in the accumulation of potassium after an osmotic upshift and the most important system for growth of
S. meliloti
under hyperosmotic conditions. The other three systems, especially Kup1, are also relevant during the osmotic adaptation of the cell, and the relative importance of the Kdp system increases at low potassium concentrations.
In Vitro Characterization of the Enzyme Properties of the Phospholipid
N
-Methyltransferase PmtA from
Agrobacterium tumefaciens
Authors: Aktas Meriyem , Narberhaus Franz
Published: January 2009

Agrobacterium tumefaciens
requires phosphatidylcholine (PC) in its membranes for plant infection. The phospholipid
N
-methyltransferase PmtA catalyzes all three transmethylation reactions of phosphatidylethanolamine (PE) to PC via the intermediates monomethylphosphatidylethanolamine (MMPE) and dimethylphosphatidylethanolamine (DMPE). The enzyme uses
S
-adenosylmethionine (SAM) as the methyl donor, converting it to
S
-adenosylhomocysteine (SAH). Little is known about the activity of bacterial Pmt enzymes, since PC biosynthesis in prokaryotes is rare. In this article, we present the purification and in vitro characterization of
A. tumefaciens
PmtA, which is a monomeric protein. It binds to PE, the intermediates MMPE and DMPE, the end product PC, and phosphatidylglycerol (PG) and phosphatidylinositol. Binding of the phospholipid substrates precedes binding of SAM. We used a coupled in vitro assay system to demonstrate the enzymatic activity of PmtA and to show that PmtA is inhibited by the end products PC and SAH and the antibiotic sinefungin. The presence of PG stimulates PmtA activity. Our study provides insights into the catalysis and control of a bacterial phospholipid
N
-methyltransferase.
Roles of Germination-Specific Lytic Enzymes CwlJ and SleB in
Bacillus anthracis
Authors: Heffron Jared D. , Orsburn Benjamin , Popham David L.
Published: January 2009

The structural characteristics of a spore enable it to withstand stresses that typically kill a vegetative cell. Spores remain dormant until small molecule signals induce them to germinate into vegetative bacilli. Germination requires degradation of the thick cortical peptidoglycan by germination-specific lytic enzymes (GSLEs).
Bacillus anthracis
has four putative GSLEs, based upon sequence similarities with enzymes in other species: SleB, CwlJ1, CwlJ2, and SleL. In this study, the roles of SleB, CwlJ1, and CwlJ2 were examined. The expression levels of all three genes peak 3.5 h into sporulation. Genetic analysis revealed that, similar to other known GSLEs, none of these gene products are individually required for growth, sporulation, or triggering of germination. However, later germination events are affected in spores lacking CwlJ1 or SleB. Compared to the wild type, germinating spores without CwlJ1 suffer a delay in optical density loss and cortex peptidoglycan release. The absence of SleB also causes a delay in cortex fragment release. A double mutant lacking both SleB and CwlJ1 is completely blocked in cortex hydrolysis and progresses through outgrowth to produce colonies at a frequency 1,000-fold lower than that of the wild-type strain. A null mutation eliminating CwlJ2 has no effect on germination. High-performance liquid chromatography and mass spectroscopy analysis revealed that SleB is required for lytic transglycosylase activity. CwlJ1 also clearly participates in cortex hydrolysis, but its specific mode of action remains unclear. Understanding the lytic germination activities that naturally diminish spore resistance can lead to methods for prematurely inducing them, thus simplifying the process of treating contaminated sites.
Both Thiamine Uptake and Biosynthesis of Thiamine Precursors Are Required for Intracellular Replication ofListeria monocytogenes
Authors: Schauer Kristina , Stolz Jürgen , Scherer Siegfried , Fuchs Thilo M.
Published: January 2009

Thiamine pyrophosphate is an essential cofactor involved in central metabolism and amino acid biosynthesis and is derived from thiamine (vitamin B1). The extent to which this metabolite is available to bacterial pathogens replicating within host cells is still little understood. Growth studies using modified minimal Welshimer′s broth (mMWB) supplemented with thiamine or the thiamine precursor hydroxymethylpyrimidine (HMP) showed thatListeria monocytogenes, in agreement with bioinformatic prediction, is able to synthesize thiamine only in the presence of HMP. This appears to be due to a lack of ThiC, which is involved in HMP synthesis. The knockout ofthiD(lmo0317), which probably catalyzes the phosphorylation of HMP, inhibited growth in mMWB supplemented with HMP and reduced the replication rate ofL. monocytogenesin epithelial cells. Mutation of a predicted thiamine transporter gene, lmo1429, led to reduced proliferation ofL. monocytogenesin mMWB containing thiamine or thiamine phosphates and also within epithelial cells but had no influence on the expression of the virulence factors Hly and ActA. The toxic thiamine analogue pyrithiamine inhibited growth of wild-type strain EGD but not of the transporter mutant EGDΔthiT. We also demonstrated that ThiT binds thiamine, a finding compatible with ThiT acting as the substrate-binding component of a multimeric thiamine transporter complex. These data provide experimental evidence that Lmo1429 homologs includingBacillusYuaJ are necessary for thiamine transport in gram-positive bacteria and are therefore proposed to be annotated “ThiT.” Taken together, these data indicate that concurrent thiamine uptake and biosynthesis of thiamine precursors is a strategy ofL. monocytogenesand possibly other facultative intracellular pathogens to enable proliferation within the cytoplasm.