bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid DNA Library Construction for Functional Genomic and Metagenomic Screening
Authors: Schmitz Jonathan E. , Daniel Anu , Collin Mattias , Schuch Raymond , Fischetti Vincent A.
Published: December 2007

A rapid protocol was developed for constructing plasmid libraries from small quantities of genomic/metagenomic DNA. The technique utilizes linker amplification with topoisomerase cloning and allows for inducible transcription in
Escherichia coli
. As proof of principle, several anti-
Bacillus
lysins were cloned from bacteriophage genomes and an aerolysin was cloned from a metagenomic sample.
Determination of Specific Growth Rate by Measurement of Specific Rate of Ribosome Synthesis in Growing and Nongrowing Cultures of
Acinetobacter calcoaceticus
Authors: Cutter Matthew R. , Stroot Peter G.
Published: December 2007

RT-RiboSyn measures the specific rate of ribosome synthesis in distinct microbial populations by measuring the generation rate of precursor 16S rRNA relative to that of mature 16S rRNA when precursor 16S rRNA processing is inhibited. Good agreement was demonstrated between specific rate of ribosome synthesis and specific growth rate of
Acinetobacter calcoaceticus
.
Comparison of Fungal Activities on Wood and Leaf Litter in Unaltered and Nutrient-Enriched Headwater Streams
Authors: Gulis Vladislav , Suberkropp Keller , Rosemond Amy D.
Published: December 2007

Fungi are the dominant organisms decomposing leaf litter in streams and mediating energy transfer to other trophic levels. However, less is known about their role in decomposing submerged wood. This study provides the first estimates of fungal production on wood and compares the importance of fungi in the decomposition of submerged wood versus that of leaves at the ecosystem scale. We determined fungal biomass (ergosterol) and activity associated with randomly collected small wood (<40 mm diameter) and leaves in two southern Appalachian streams (reference and nutrient enriched) over an annual cycle. Fungal production (from rates of radiolabeled acetate incorporation into ergosterol) and microbial respiration on wood (per gram of detrital C) were about an order of magnitude lower than those on leaves. Microbial activity (per gram of C) was significantly higher in the nutrient-enriched stream. Despite a standing crop of wood two to three times higher than that of leaves in both streams, fungal production on an areal basis was lower on wood than on leaves (4.3 and 15.8 g C m
−2
year
−1
in the reference stream; 5.5 and 33.1 g C m
−2
year
−1
in the enriched stream). However, since the annual input of wood was five times lower than that of leaves, the proportion of organic matter input directly assimilated by fungi was comparable for these substrates (15.4 [wood] and 11.3% [leaves] in the reference stream; 20.0 [wood] and 20.2% [leaves] in the enriched stream). Despite a significantly lower fungal activity on wood than on leaves (per gram of detrital C), fungi can be equally important in processing both leaves and wood in streams.
Phenotypic and Proteomic Characterization of Multiply Antibiotic-Resistant Variants ofSalmonella entericaSerovar Typhimurium Selected Following Exposure to Disinfectants
Authors: Karatzas Kimon A. G. , Randall Luke P. , Webber Mark , Piddock Laura J. V. , Humphrey Tom J. , Woodward Martin J. , Coldham Nick G.
Published: December 2007

In previous work,Salmonella entericaserovar Typhimurium strain SL1344 was exposed to sublethal concentrations of three widely used farm disinfectants in daily serial passages for 7 days in an attempt to investigate possible links between the use of disinfectants and antimicrobial resistance. Stable variants OXCR1, QACFGR2, and TOPR2 were obtained following treatment with an oxidizing compound blend, a quaternary ammonium disinfectant containing formaldehyde and glutaraldehyde, and a tar acid-based disinfectant, respectively. All variants exhibited ca. fourfold-reduced susceptibility to ciprofloxacin, chloramphenicol, tetracycline, and ampicillin. This coincided with reduced levels of outer membrane proteins for all strains and high levels of AcrAB-TolC for OXCR1 and QACFGR2, as demonstrated by two-dimensional high-performance liquid chromatography-mass spectrometry. The protein profiles of OXCR1 and QACFGR2 were similar, but they were different from that of TOPR2. An array of different proteins protecting against oxidants, nitroaromatics, disulfides, and peroxides were overexpressed in all strains. The growth and motility of variants were reduced compared to the growth and motility of the parent strain, the expression of several virulence proteins was altered, and the invasiveness in an enteric epithelial cell line was reduced. The colony morphology of OXCR1 and QACFGR2 was smooth, and both variants exhibited a loss of modal distribution of the lipopolysaccharide O-antigen chain length, favoring the production of short O-antigen chain molecules. Metabolic changes were also detected, suggesting that there was increased protein synthesis and a shift from oxidative phosphorylation to substrate level phosphorylation. In this study, we obtained evidence that farm disinfectants can select for strains with reduced susceptibility to antibiotics, and here we describe changes in protein expression in such strains.
The Type II Secretion System ofLegionella pneumophilaElaborates Two Aminopeptidases, as Well as a Metalloprotease That Contributes to Differential Infection among Protozoan Hosts
Authors: Rossier Ombeline , Dao Jenny , Cianciotto Nicholas P.
Published: December 2007

Legionella pneumophila, the agent of Legionnaires' disease, is an intracellular parasite of aquatic amoebae and human macrophages. A key factor forL. pneumophilain intracellular infection is its type II protein secretion system (Lsp). In order to more completely define Lsp output, we recently performed a proteomic analysis of culture supernatants. Based upon the predictions of that analysis, we found thatL. pneumophilasecretes two distinct aminopeptidase activities encoded by the geneslapAandlapB. WhereaslapAconferred activity against leucine, phenylalanine, and tyrosine aminopeptides,lapBwas linked to the cleavage of lysine- and arginine-containing substrates. To assess the role of secreted aminopeptidases in intracellular infection, we examined the relative abilities oflapAandlapBmutants to infect human U937 cell macrophages as well asHartmannella vermiformisandAcanthamoeba castellaniiamoebae. Although these experiments identified a dispensable role for LapA and LapB, they uncovered a previously unrecognized role for the type II-dependent ProA (MspA) metalloprotease. WhereasproAmutants were not defective for macrophage orA. castellaniiinfection, they (but not their complemented derivatives) were impaired for growth upon coculture withH. vermiformis. Thus, ProA represents the first type II effector implicated in an intracellular infection event. Furthermore,proArepresents anL. pneumophilagene that shows differential importance among protozoan infection models, suggesting that the legionellae might have evolved some of its factors to especially target certain of their protozoan hosts.
Depth-Resolved Quantification of Anaerobic Toluene Degraders and Aquifer Microbial Community Patterns in Distinct Redox Zones of a Tar Oil Contaminant Plume
Authors: Winderl Christian , Anneser Bettina , Griebler Christian , Meckenstock Rainer U. , Lueders Tillmann
Published: December 2007

Microbial degradation is the only sustainable component of natural attenuation in contaminated groundwater environments, yet its controls, especially in anaerobic aquifers, are still poorly understood. Hence, putative spatial correlations between specific populations of key microbial players and the occurrence of respective degradation processes remain to be unraveled. We therefore characterized microbial community distribution across a high-resolution depth profile of a tar oil-impacted aquifer where benzene, toluene, ethylbenzene, and xylene (BTEX) degradation depends mainly on sulfate reduction. We conducted depth-resolved terminal restriction fragment length polymorphism fingerprinting and quantitative PCR of bacterial 16S rRNA and benzylsuccinate synthase genes (bssA) to quantify the distribution of total microbiota and specific anaerobic toluene degraders. We show that a highly specialized degrader community of microbes related to known deltaproteobacterial iron and sulfate reducers (GeobacterandDesulfocapsaspp.), as well as clostridial fermenters (Sedimentibacterspp.), resides within the biogeochemical gradient zone underneath the highly contaminated plume core. This zone, where BTEX compounds and sulfate—an important electron acceptor—meet, also harbors a surprisingly high abundance of the yet-unidentified anaerobic toluene degraders carrying the previously detected F1-clusterbssAgenes (C. Winderl, S. Schaefer, and T. Lueders, Environ. Microbiol. 9:1035-1046, 2007). Our data suggest that this biogeochemical gradient zone is a hot spot of anaerobic toluene degradation. These findings show that the distribution of specific aquifer microbiota and degradation processes in contaminated aquifers are tightly coupled, which may be of value for the assessment and prediction of natural attenuation based on intrinsic aquifer microbiota.
Novel Isolate of
Bacillus thuringiensis
subsp.
thuringiensis
That Produces a Quasicuboidal Crystal of Cry1Ab21 Toxic to Larvae of
Trichoplusia ni
Authors: Swiecicka Izabela , Bideshi Dennis K. , Federici Brian A.
Published: December 2007

A new isolate (IS5056) of
Bacillus thuringiensis
subsp.
thuringiensis
that produces a novel variant of Cry1Ab, Cry1Ab21, was isolated from soil collected in northeastern Poland. Cry1Ab21 was composed of 1,155 amino acids and had a molecular mass of 130.5 kDa, and a single copy of the gene coding for this endotoxin was located on a ∼75-kbp plasmid. When synthesized by the wild-type strain, Cry1Ab21 produced a unique, irregular, bipyramidal crystal whose long and short axes were both approximately 1 μm long, which gave it a cuboidal appearance in wet mount preparations. In diet incorporation bioassays, the 50% lethal concentrations of the crystal-spore complex were 16.9 and 29.7 μg ml
−1
for second- and fourth-instar larvae of the cabbage looper,
Trichoplusia ni
, respectively, but the isolate was essentially nontoxic to larvae of the beet armyworm,
Spodoptera exigua
. A bioassay of autoclaved spore-crystal preparations showed no evidence of β-exotoxin activity, indicating that toxicity was due primarily to Cry1Ab21. Studies of the pathogenesis of isolate IS5056 in second-instar larvae of
T. ni
showed that after larval death the bacterium colonized and subsequently sporulated extensively throughout the cadaver, suggesting that other bacteria inhabiting the midgut lumen played little if any role in mortality. As
T. ni
is among the most destructive pests of vegetable crops in North America and has developed resistance to
B. thuringiensis
, this new isolate may have applied value.
Enzymic Approach to Eurythermalism of
Alvinella pompejana
and Its Episymbionts
Authors: Lee Charles K. , Cary S. Craig , Murray Alison E. , Daniel Roy M.
Published: December 2007

The equilibrium model, which describes the influence of temperature on enzyme activity, has been established as a valid and useful tool for characterizing enzyme eurythermalism and thermophily. By introducing
K
eq
, a temperature-dependent equilibrium constant for the interconversion between E
act
, the active form of enzyme, and E
inact
, a reversibly inactive form of enzyme, the equilibrium model currently provides the most complete description of the enzyme-temperature relationship; its derived parameters are intrinsic and apparently universal and, being derived under reaction conditions, potentially have physiological significance. One of these parameters,
T
eq
, correlates with host growth temperature better than enzyme stability does. The vent-dwelling annelid
Alvinella pompejana
has been reported as an extremely eurythermal organism, and the symbiotic complex microbial community associated with its dorsal surface is likely to experience similar environmental thermal conditions. The
A. pompejana
episymbiont community, predominantly composed of epsilonproteobacteria, has been analyzed metagenomically, enabling direct retrieval of genes coding for enzymes suitable for equilibrium model applications. Two such genes, coding for isopropylmalate dehydrogenase and glutamate dehydrogenase, have been isolated from the
A. pompejana
episymbionts, heterologously expressed, and shown by reverse transcription-quantitative PCR to be actively expressed. The equilibrium model parameters of characterized expression products suggested that enzyme eurythermalism constitutes part of the thermal adaptation strategy employed by the episymbionts. Moreover, the enzymes' thermal characteristics correspond to their predicted physiological roles and the abundance and expression of the corresponding genes. This paper demonstrates the use of the equilibrium model as part of a top-down metagenomic approach to studying temperature adaptation of uncultured organisms.
The Genome Sequence of the Metal-Mobilizing, Extremely Thermoacidophilic Archaeon
Metallosphaera sedula
Provides Insights into Bioleaching-Associated Metabolism
Authors: Auernik Kathryne S. , Maezato Yukari , Blum Paul H. , Kelly Robert M.
Published: December 2007

Despite their taxonomic description, not all members of the order
Sulfolobales
are capable of oxidizing reduced sulfur species, which, in addition to iron oxidation, is a desirable trait of biomining microorganisms. However, the complete genome sequence of the extremely thermoacidophilic archaeon
Metallosphaera sedula
DSM 5348 (2.2 Mb, ∼2,300 open reading frames [ORFs]) provides insights into biologically catalyzed metal sulfide oxidation. Comparative genomics was used to identify pathways and proteins involved (directly or indirectly) with bioleaching. As expected, the
M. sedula
genome contains genes related to autotrophic carbon fixation, metal tolerance, and adhesion. Also, terminal oxidase cluster organization indicates the presence of hybrid quinol-cytochrome oxidase complexes. Comparisons with the mesophilic biomining bacterium
Acidithiobacillus ferrooxidans
ATCC 23270 indicate that the
M. sedula
genome encodes at least one putative rusticyanin, involved in iron oxidation, and a putative tetrathionate hydrolase, implicated in sulfur oxidation. The
fox
gene cluster, involved in iron oxidation in the thermoacidophilic archaeon
Sulfolobus metallicus
, was also identified. These iron- and sulfur-oxidizing components are missing from genomes of nonleaching members of the
Sulfolobales
, such as
Sulfolobus solfataricus
P2 and
Sulfolobus acidocaldarius
DSM 639. Whole-genome transcriptional response analysis showed that 88 ORFs were up-regulated twofold or more in
M. sedula
upon addition of ferrous sulfate to yeast extract-based medium; these included genes for components of terminal oxidase clusters predicted to be involved with iron oxidation, as well as genes predicted to be involved with sulfur metabolism. Many hypothetical proteins were also differentially transcribed, indicating that aspects of the iron and sulfur metabolism of
M. sedula
remain to be identified and characterized.
Isolation and Characterization of
Vibrio tubiashii
Outer Membrane Proteins and Determination of a
toxR
Homolog
Authors: Beaubrun J. Jean-Gilles , Kothary M. H. , Curtis S. K. , Flores N. C. , Eribo B. E. , Tall B. D.
Published: December 2007

Outer membrane proteins (OMPs) expressed by
Vibrio tubiashii
under different environmental growth conditions were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and PCR analyses. Results showed the presence of a 38- to 40-kDa OmpU-like protein and
ompU
gene, a maltoporin-like protein, several novel OMPs, and a regulatory
toxR
homolog.
Rottlerin Inhibits Chlamydial Intracellular Growth and Blocks Chlamydial Acquisition of Sphingolipids from Host Cells
Authors: Shivshankar Pooja , Lei Lei , Wang Jie , Zhong Guangming
Published: December 2007

We report that rottlerin, a plant-derived compound known to inhibit various mammalian kinases, profoundly inhibited chlamydial growth in cell culture with a minimal inhibition concentration of 1 μM. The inhibition was effective even when rottlerin was added as late as the middle stage of chlamydial infection cycle, against multiple
Chlamydia
species, and in different host cell lines. Pretreatment of host cells with rottlerin prior to infection also blocked chlamydial growth, suggesting that rottlerin targets host factors. Moreover, rottlerin did not alter the chlamydial infection rate and did not directly target chlamydial protein synthesis and secretion. The rottlerin-mediated inhibition of chlamydial replication and inclusion expansion correlated well with the rottlerin-induced blockade of host cell sphingolipid trafficking from the Golgi apparatus into chlamydial inclusions. These studies not only allowed us to identify a novel antimicrobial activity for rottlerin but also allowed us to uncover a potential mechanism for rottlerin inhibition of chlamydial growth.
Relevant Double Mutations in Bioengineered
Streptomyces clavuligerus
Deacetoxycephalosporin C Synthase Result in Higher Binding Specificities Which Improve Penicillin Bioconversion
Authors: Goo Kian Sim , Chua Chun Song , Sim Tiow-Suan
Published: December 2007

Streptomyces clavuligerus
deacetoxycephalosporin C synthase (ScDAOCS) is an important industrial enzyme for the production of 7-aminodeacetoxycephalosporanic acid, which is a precursor for cephalosporin synthesis. Single mutations of six amino acid residues, V275, C281, N304, I305, R306, and R307, were previously shown to result in enhanced levels of ampicillin conversion, with activities ranging from 129 to 346% of the wild-type activity. In this study, these mutations were paired to investigate their effects on enzyme catalysis. The bioassay results showed that the C-terminal mutations (N304X [where X is alanine, leucine, methionine, lysine, or arginine], I305M, R306L, and R307L) in combination with C281Y substantially increased the conversion of ampicillin; the activity was up to 491% of the wild-type activity. Similar improvements were observed for converting carbenicillin (up to 1,347% of the wild-type activity) and phenethicillin (up to 1,109% of the wild-type activity). Interestingly, the N304X R306L double mutants exhibited lower activities for penicillin G conversion, and activities that were 40 to 114% of wild-type enzyme activity were detected. Based on kinetic studies using ampicillin, it was clear that the increases in the activities of the double mutants relative to those of the corresponding single mutants were due to enhanced substrate binding affinities. These results also validated the finding that the N304R and I305M mutations are ideal for increasing the substrate binding affinity and turnover rate of the enzyme, respectively. This study provided further insight into the structure-function interaction of ScDAOCS with different penicillin substrates, thus providing a useful platform for further rational modification of its enzymatic properties.
Sensitivity of an
Acinetobacter baylyi mpl
Mutant to DNA Damage
Authors: Chakravorty Adityarup , Klovstad Martha , Peterson Greg , Lindeman Robin E. , Gregg-Jolly Leslie A.
Published: December 2007

A mutant derived from
Acinetobacter baylyi
ADP1 was isolated from a screen for genes involved in the response to DNA damage. This derivative has an insertion in the
mpl
gene which encodes a peptidoglycan-recycling protein. We demonstrate that the insertion renders cells sensitive to mitomycin C and to UV.
CTA4
Transcription Factor Mediates Induction of Nitrosative Stress Response in
Candida albicans
Authors: Chiranand Wiriya , McLeod Ian , Zhou Huaijin , Lynn Jed J. , Vega Luis A. , Myers Hadley , Yates John R. , Lorenz Michael C. , Gustin Michael C.
Published: December 2007

This work has identified regulatory elements in the major fungal pathogen
Candida albicans
that enable response to nitrosative stress. Nitric oxide (NO) is generated by macrophages of the host immune system and commensal bacteria, and the ability to resist its toxicity is one adaptation that promotes survival of
C. albicans
inside the human body. Exposing
C. albicans
to NO induces upregulation of the flavohemoglobin Yhb1p. This protein confers protection by enzymatically converting NO to harmless nitrate, but it is unknown how
C. albicans
is able to detect NO in its environment and thus initiate this defense only as needed. We analyzed this problem by incrementally mutating the
YHB1
regulatory region to identify a nitric oxide-responsive element (NORE) that is required for NO sensitivity. Five transcription factor candidates of the Zn(II)2-Cys6 family were then isolated from crude whole-cell extracts by using magnetic beads coated with this DNA element. Of the five, only deletion of the
CTA4
gene prevented induction of
YHB1
transcription during nitrosative stress and caused growth sensitivity to the NO donor dipropylenetriamine NONOate; Cta4p associates in vivo with NORE DNA from the
YHB1
regulatory region. Deletion of
CTA4
caused a small but significant decrease in virulence. A
CTA4
-dependent putative sulfite transporter encoded by
SSU1
is also implicated in NO response, but
C. albicans ssu1
mutants were not sensitive to NO, in contrast to findings in
Saccharomyces cerevisiae
. Cta4p is the first protein found to be necessary for initiating NO response in
C. albicans
.
Disruption of the PfPK7Gene Impairs Schizogony and Sporogony in the Human Malaria ParasitePlasmodium falciparum
Authors: Dorin-Semblat Dominique , Sicard Audrey , Doerig Caroline , Ranford-Cartwright Lisa , Doerig Christian
Published: December 2007

PfPK7 is an orphan protein kinase ofPlasmodium falciparumwith maximal homology to MEK3/6 and to fungal protein kinase A proteins in its C-terminal and N-terminal regions, respectively. We showed previously that recombinant PfPK7 is active on various substrates but is unable to phosphorylate thePlasmodium falciparummitogen-activated protein kinase homologues, suggesting that it is not a MEK functional homologue. Using a reverse genetics approach to investigate the function of this enzyme in live parasites, we now show that PfPK7−parasite clones display phenotypes at two stages of their life cycle: first, a decrease in the rate of asexual growth in erythrocytes associated with a lower number of daughter merozoites generated per schizont, and second, a dramatic reduction in the ability to produce oocysts in the mosquito vector. A normal asexual growth rate and the ability to produce oocysts are restored if a functional copy of the PfPK7gene is reintroduced into the PfPK7−parasites. Hence, PfPK7 is involved in a pathway that regulates parasite proliferation and development.
The L-Type Calcium Ion Channel Cch1 Affects Ascospore Discharge and Mycelial Growth in the Filamentous FungusGibberella zeae(AnamorphFusarium graminearum)
Authors: Hallen Heather E. , Trail Frances
Published: December 2007

Cch1, a putative voltage-gated calcium ion channel, was investigated for its role in ascus development inGibberella zeae. Gene replacement mutants ofCCH1were generated and found to have asci which did not forcibly discharge spores, although morphologically ascus and ascospore development in the majority of asci appeared normal. Additionally, mycelial growth was significantly slower, and sexual development was slightly delayed in the mutant; mutant mycelia showed a distinctive fluffy morphology, and no cirrhi were produced. Wheat infected with Δcch1mutants developed symptoms comparable to wheat infected with the wild type; however, the mutants showed a reduced ability to protect the infected stalk from colonization by saprobic fungi. Transcriptional analysis of gene expression in mutants using the AffymetrixFusariummicroarray showed 2,449 genes with significant, twofold or greater, changes in transcript abundance across a developmental series. This work extends the role ofCCH1to forcible spore discharge inG. zeaeand suggests that this channel has subtle effects on growth and development.
Excystation of
Eimeria tenella
Sporozoites Impaired by Antibody Recognizing Gametocyte/Oocyst Antigens GAM22 and GAM56
Authors: Krücken Jürgen , Hosse Ralf J. , Mouafo Aimdip N. , Entzeroth Rolf , Bierbaum Stefan , Marinovski Predrag , Hain Karolina , Greif Gisela , Wunderlich Frank
Published: December 2007

Eimeria tenella
is the causative agent of coccidiosis in poultry. Infection of the chicken intestine begins with ingestion of sporulated oocysts releasing sporocysts, which in turn release invasive sporozoites. The monoclonal antibody E2E5 recognizes wall-forming body type II (WFBII) in gametocytes and the WFBII-derived inner wall of oocysts. Here we describe that this antibody also binds to the stieda body of sporocysts and significantly impairs in vitro excystation of sporozoites. Using affinity chromatography and protein sequence analysis, E2E5 is shown to recognize EtGAM56, the
E. tenella
ortholog of the
Eimeria maxima
gametocyte-specific GAM56 protein. In addition, this antibody was used to screen a genomic phage display library presenting
E. tenella
antigens as fusion proteins with the gene VIII product on the surfaces of phagemid particles and identified the novel 22-kDa histidine- and proline-rich protein EtGAM22. The Et
gam22
mRNA is expressed predominantly at the gametocyte stage, as detected by Northern blotting. Southern blot analysis in combination with data from the
E. tenella
genome project revealed that Et
gam22
is an intronless multicopy gene, with approximately 12 to 22 copies in head-to-tail arrangement. Conspicuously, Et
gam56
is also intronless and is localized adjacent to another
gam56
-like gene, Et
gam59
. Our data suggest that amplification is common for genes encoding oocyst wall proteins.
Candida albicans
Als Adhesins Have Conserved Amyloid-Forming Sequences
Authors: Otoo Henry N. , Lee Kyeng Gea , Qiu Weigang , Lipke Peter N.
Published: December 2007

The cell wall-bound Als adhesins of
Candida albicans
mediate both yeast-to-host tissue adherence and yeast aggregation. This aggregation is amyloid-like, with self-propagating secondary-structure changes, amyloid-characteristic dye binding, and induced birefringence (J. M. Rauceo, N. K. Gaur, K. G. Lee, J. E. Edwards, S. A. Klotz, and P. N. Lipke, Infect. Immun. 72:4948-4955, 2004). Therefore, we determined whether Als proteins could form amyloid fibers with properties like those in cellular aggregation. The β-aggregation predictor TANGO identified a heptapeptide sequence present in a highly conserved sequence with amyloid-forming potential in Als1p, Als3p, and Als5p. A tridecapeptide containing this sequence formed fibers that bound Congo red and thioflavin T and had characteristic amyloid morphology. Als5p
20-431
and Als5p
20-664
, large fragments of Als5p containing the amyloid sequence, also formed amyloid-like fibers and bound Congo red under native conditions.

K
a

/

K
s

analysis showed that the amyloid-forming sequences are highly conserved in Als proteins and evolve more slowly than other regions of the proteins. Therefore, amyloid-forming ability itself is conserved in these proteins.
Atf2 Transcription Factor Binds to the APP1 Promoter in
Cryptococcus neoformans
: Stimulatory Effect of Diacylglycerol
Authors: Tommasino Nicola , Villani Maristella , Qureshi Asfia , Henry Jennifer , Luberto Chiara , Del Poeta Maurizio
Published: December 2007

The fungus
Cryptococcus neoformans
is an environmental human pathogen which enters the lung via the respiratory tract and produces a unique protein, called antiphagocytic protein 1 (App1), that protects it from phagocytosis by macrophages. In previous studies, we proposed genetic evidences that transcription of
APP1
is controlled by the enzymatic reaction catalyzed by inositol phosphorylceramide synthase 1 (Ipc1) via the production of diacylglycerol through the activating transcription factor 2 (Atf2). We investigated here the mechanism by which Atf2 binds to the
APP1
promoter in vitro and in vivo. To this end, we produced Atf2 recombinant proteins (rAtf2) and found that rAtf2 binds to ATF
cis
-acting element present in the
APP1
promoter. Indeed, mutation of two key nucleotides in the ATF consensus sequence abolishes the binding of rAtf2 to the
APP1
promoter. Next, we produced
C. neoformans
strains with a hemagglutinin-tagged
ATF2
gene and showed that endogenous Atf2 binds to
APP1
promoter in vivo. Finally, by a novel DNA protein-binding precipitation assay, we showed that treatment with 1,2-dioctanoylglycerol positively increases binding of Atf2-
APP1
promoter in vivo. These studies provide new insights into the molecular mechanism by which Atf2 regulates
APP1
transcription in vivo with important implications for a better understanding of how
C. neoformans
escapes the phagocytic process.
The Cytoplasmic Region of α-1,6-Mannosyltransferase Mnn9p Is Crucial for Retrograde Transport from the Golgi Apparatus to the Endoplasmic Reticulum in
Saccharomyce
s
cerevisiae
Authors: Okamoto Michiyo , Yoko-o Takehiko , Miyakawa Tokichi , Jigami Yoshifumi
Published: December 2007

In
Saccharomyces cerevisiae
, Och1p and Mnn9p mannosyltransferases are localized in the
cis
-Golgi. Attempts to live image Och1p and Mnn9p tagged with green fluorescent protein or red fluorescent protein, respectively, using a high-performance confocal laser scanning microscope system resulted in simultaneous visualization of the native proteins in a living cell. Our observations revealed that Och1p and Mnn9p are not always colocalized to the same cisternae. The difference in the dynamics of these mannosyltransferases may reflect differences in the mechanisms for their retention in the
cis
-Golgi, since it has been reported that Mnn9p cycles between the endoplasmic reticulum and the
cis
-Golgi whereas Och1p does not (Z. Todorow, A. Spang, E. Carmack, J. Yates, and R. Schekman, Proc. Natl. Acad. Sci. USA
97:
13643-13648, 2000). We investigated the localization of chimeric proteins of Mnn9p and Och1p in
sec12
and
erd1
mutant cells. A chimeric protein, M16/O16, which consists of the N-terminal cytoplasmic region of Mnn9p and the transmembrane and luminal region of Och1p, behaved like Mnn9p, suggesting that the N-terminal cytoplasmic region is important for the intracellular dynamics of Mnn9p. This observation is supported by results from subcellular-fractionation experiments. Mutational analysis revealed that two arginine residues in the N-terminal region of Mnn9p are important for the chimeric protein to cycle between the endoplasmic reticulum and the Golgi apparatus.
Impact of Ammonium Permeases MepA, MepB, and MepC on Nitrogen-Regulated Secondary Metabolism in
Fusarium fujikuroi
Authors: Teichert Sabine , Rutherford Julian C. , Wottawa Marieke , Heitman Joseph , Tudzynski Bettina
Published: December 2007

In
Fusarium fujikuroi
, the production of gibberellins and bikaverin is repressed by nitrogen sources such as glutamine or ammonium. Sensing and uptake of ammonium by specific permeases play key roles in nitrogen metabolism. Here, we describe the cloning of three ammonium permease genes,
mepA, mepB
, and
mepC
, and their participation in ammonium uptake and signal transduction in
F. fujikuroi
. The expression of all three genes is strictly regulated by the nitrogen regulator AreA. Severe growth defects of Δ
mepB
mutants on low-ammonium medium and methylamine uptake studies suggest that MepB functions as the main ammonium permease in
F. fujikuroi
. In Δ
mepB
mutants, nitrogen-regulated genes such as the gibberellin and bikaverin biosynthetic genes are derepressed in spite of high extracellular ammonium concentrations.
mepA mepB
and
mepC mepB
double mutants show a similar phenotype as Δ
mepB
mutants. All three
F. fujikuroi mep
genes fully complemented the
Saccharomyces cerevisiae mep1 mep2 mep3
triple mutant to restore growth on low-ammonium medium, whereas only MepA and MepC restored pseudohyphal growth in the
mep2/mep2
mutant. Overexpression of
mepC
in the Δ
mepB
mutants partially suppressed the growth defect but did not prevent derepression of AreA-regulated genes. These studies provide evidence that MepB functions as a regulatory element in a nitrogen sensing system in
F. fujikuroi
yet does not provide the sensor activity of Mep2 in yeast, indicating differences in the mechanisms by which nitrogen is sensed in
S. cerevisiae
and
F. fujikuroi
.
Interaction between Two Putative Glycosyltransferases Is Required for Glycosylation of a Serine-Rich Streptococcal Adhesin
Authors: Bu Su , Li Yirong , Zhou Meixian , Azadin Parastoo , Zeng Meiqin , Fives-Taylor Paula , Wu Hui
Published: December 2007

Fap1, a serine-rich glycoprotein, is essential for fimbrial biogenesis and biofilm formation of
Streptococcus parasanguinis
(formerly
S. parasanguis
). Fap1-like proteins are conserved in many streptococci and staphylococci and have been implicated in bacterial virulence. Fap1 contains two serine-rich repeat regions that are modified by O-linked glycosylation. A seven-gene cluster has been identified, and this cluster is implicated in Fap1 biogenesis. In this study, we investigated the initial step of Fap1 glycosylation by using a recombinant Fap1 as a model. This recombinant molecule has the same monosaccharide composition profile as the native Fap1 protein. Glycosyl linkage analyses indicated that
N
-acetylglucosamine (GlcNAc) is among the first group of sugar residues transferred to the Fap1 peptide. Two putative glycosyltransferases, Gtf1 and Gtf2, were essential for the glycosylation of Fap1 with GlcNAc-containing oligosaccharide(s) in both
S. parasanguinis
as well as in the Fap1 glycosylation system in
Escherichia coli
. Yeast two-hybrid analysis as well as in vitro and in vivo glutathione
S
-transferase pull-down assays demonstrated the two putative glycosyltransferases interacted with each other. The interaction domain was mapped to an N-terminal region of Gtf1 that was required for the Fap1 glycosylation. The data in this study suggested that the formation of the Gtf1 and Gtf2 complex was required for the initiation of the Fap1 glycosylation and that the N-terminal region of Gtf1 was necessary.
Characterization of a New Rhamnogalacturonan Acetyl Esterase from
Bacillus halodurans
C-125 with a New Putative Carbohydrate Binding Domain
Authors: Navarro-Fernández José , Martínez-Martínez Irene , Montoro-García Silvia , García-Carmona Francisco , Takami Hideto , Sánchez-Ferrer Álvaro
Published: December 2007

BH1115 is a gene from
Bacillus halodurans
strain C-125 that hypothetically encodes a rhamnogalacturonan acetyl esterase (RGAE) of the CE-12 family. As confirmation, this gene was cloned, and the product was expressed in
Escherichia coli
strain Rosetta (DE3) cells and purified. The enzyme obtained was monomeric, with a molecular mass of 45 kDa, and exhibited alkaliphilic properties. A study of the inhibition of the activity by some modulators confirmed that the catalytic triad for the esterase activity was Ser-His-Asp. This enzyme also presents broad substrate specificity and is active toward 7-aminocephalosporanic acid, cephalosporin C,
p
-nitrophenyl acetate, β-naphthyl acetate, glucose pentaacetate, and acetylated xylan. Moreover, RGAE from
B. halodurans
achieves a synergistic effect with xylanase A toward acetylated xylan. As a member of the SGNH family, it does not adopt the common α/β hydrolase fold. The homology between the folds of RGAE from
Aspergillus aculeatus
and the hypothetical YxiM precursor from
Bacillus subtilis
, which both belong to the SGNH family, illustrates the divergence of such proteins from a common ancestor. Furthermore, the enzyme possesses a putative substrate binding region at the N terminus of the protein which has never been described to date for any RGAE.
Gene Acquisition at the Insertion Site for SCC
mec
, the Genomic Island Conferring Methicillin Resistance in
Staphylococcus aureus
Authors: Noto Michael J. , Kreiswirth Barry N. , Monk Alastair B. , Archer Gordon L.
Published: December 2007

Staphylococcus aureus
becomes resistant to methicillin by acquiring a genomic island, known as staphylococcal chromosome cassette
mec
(SCC
mec
), which contains the methicillin resistance determinant,
mecA
. SCC
mec
is site-specifically integrated into the staphylococcal chromosome at a locus known as the SCC
mec
attachment site (
attB
). In an effort to gain a better understanding of the potential that methicillin-sensitive
S. aureus
(MSSA) isolates have for acquiring SCC
mec
, the nucleotide sequences of
attB
and surrounding DNA regions were examined in a diverse collection of 42 MSSA isolates. The chromosomal region surrounding
attB
varied among the isolates studied and appears to be a common insertion point for acquired foreign DNA. Insertions of up to 15.1 kb were found containing open reading frames with homology to enterotoxin genes, restriction-modification systems, transposases, and several sequences that have not been previously described in staphylococci. Two groups, containing eight and four isolates, had sequences found in known SCC
mec
elements, suggesting SCC
mec
elements may have evolved through repeated DNA insertions at this locus. In addition, the
attB
sequences of the majority of MSSA isolates in this collection differ from the
attB
sequences of strains for which integrase-mediated SCC
mec
insertion or excision has been demonstrated, suggesting that some
S. aureus
isolates may lack the ability to site-specifically integrate SCC
me
c into their chromosomes.
Pseudomonas aeruginosaPqsA Is an Anthranilate-Coenzyme A Ligase
Authors: Coleman James P. , Hudson L. Lynn , McKnight Susan L. , Farrow John M. , Calfee M. Worth , Lindsey Claire A. , Pesci Everett C.
Published: December 2007

Pseudomonas aeruginosais an opportunistic human pathogen which relies on several intercellular signaling systems for optimum population density-dependent regulation of virulence genes. ThePseudomonasquinolone signal (PQS) is a 3-hydroxy-4-quinolone with a 2-alkyl substitution which is synthesized by the condensation of anthranilic acid with a 3-keto-fatty acid. ThepqsABCDEoperon has been identified as being necessary for PQS production, and thepqsAgene encodes a predicted protein with homology to acyl coenzyme A (acyl-CoA) ligases. In order to elucidate the first step of the 4-quinolone synthesis pathway inP. aeruginosa, we have characterized the function of thepqsAgene product. Extracts prepared fromEscherichia coliexpressing PqsA were shown to catalyze the formation of anthraniloyl-CoA from anthranilate, ATP, and CoA. The PqsA protein was purified as a recombinant His-tagged polypeptide, and this protein was shown to have anthranilate-CoA ligase activity. The enzyme was active on a variety of aromatic substrates, including benzoate and chloro and fluoro derivatives of anthranilate. Inhibition of PQS formation in vivo was observed for the chloro- and fluoroanthranilate derivatives, as well as for several analogs which were not PqsA enzymatic substrates. These results indicate that the PqsA protein is responsible for priming anthranilate for entry into the PQS biosynthetic pathway and that this enzyme may serve as a useful in vitro indicator for potential agents to disrupt quinolone signaling inP. aeruginosa.
Xylanase Attachment to the Cell Wall of the Hyperthermophilic Bacterium
Thermotoga maritima
Authors: Liebl Wolfgang , Winterhalter Christoph , Baumeister Wolfgang , Armbrecht Martin , Valdez Michael
Published: December 2007

The cellular localization and processing of the endo-xylanases (1,4-β-
d
-xylan-xylanohydrolase; EC 3.2.1.8) of the hyperthermophile
Thermotoga maritima
were investigated, in particular with respect to the unusual outer membrane (“toga”) of this gram-negative bacterium. XynB (40 kDa) was detected in the periplasmic fraction of
T. maritima
cells and in the culture supernatant. XynA (120 kDa) was partially released to the surrounding medium, but most XynA remained cell associated. Immunogold labeling of thin sections revealed that cell-bound XynA was localized mainly in the outer membranes of
T. maritima
cells. Amino-terminal sequencing of purified membrane-bound XynA revealed processing of the signal peptide after the eighth residue, thereby leaving the hydrophobic core of the signal peptide attached to the enzyme. This mode of processing is reminiscent of type IV prepilin signal peptide cleavage. Removal of the entire XynA signal peptide was necessary for release from the cell because enzyme purified from the culture supernatant lacked 44 residues at the N terminus, including the hydrophobic part of the signal peptide. We conclude that toga association of XynA is mediated by residues 9 to 44 of the signal peptide. The biochemical and electron microscopic localization studies together with the amino-terminal processing data indicate that XynA is held at the cell surface of
T. maritima
via a hydrophobic peptide anchor, which is highly unusual for an outer membrane protein.
Molecular Characterization Reveals Similar Virulence Gene Content in Unrelated Clonal Groups of
Escherichia coli
of Serogroup O174 (OX3)
Authors: Tarr Cheryl L. , Nelson Adam M. , Beutin Lothar , Olsen Katharina E. P. , Whittam Thomas S.
Published: December 2007

Most severe illnesses that are attributed to Shiga toxin-producing
Escherichia coli
are caused by isolates that also carry a pathogenicity island called the locus of enterocyte effacement (LEE). However, many cases of severe disease are associated with LEE-negative strains. We characterized the virulence gene content and the evolutionary relationships of
Escherichia coli
isolates of serogroup O174 (formerly OX3), strains of which have been implicated in cases of hemorrhagic colitis and hemolytic uremic syndrome. A total of 56 isolates from humans, farm animals, and food were subjected to multilocus virulence gene profiling (MVGP), and a subset of 16 isolates was subjected to multilocus sequence analysis (MLSA). The MLSA revealed that the O174 isolates fall into four separate evolutionary clusters within the
E. coli
phylogeny and are related to a diverse array of clonal groups, including enteropathogenic
E. coli
2 (EPEC 2), enterohemorrhagic
E. coli
2 (EHEC 2), and EHEC-O121. Of the 15 genes that we surveyed with MVGP, only 6 are common in the O174 strains. The different clonal groups within the O174 serogroup appear to have independently acquired and maintained similar sets of genes that include the Shiga toxins (
stx
1
and
stx
2
) and two adhesins (
saa
and
iha
). The absence of certain O island (OI) genes, such as those found on OI-122, is consistent with the notion that certain pathogenicity islands act cooperatively with the LEE island.
Characterization of the Sequence Specificity Determinants Required for Processing and Control of Sex Pheromone by the Intramembrane Protease Eep and the Plasmid-Encoded Protein PrgY
Authors: Chandler Josephine R. , Dunny Gary M.
Published: December 2007

Conjugative transfer of theEnterococcus faecalisplasmid pCF10 is induced by the peptide pheromone cCF10 when recipient-produced cCF10 is detected by donors. cCF10 is produced by proteolytic processing of the signal sequence of a chromosomally encoded lipoprotein (CcfA). In donors, endogenously produced cCF10 is carefully controlled to prevent constitutive expression of conjugation functions, an energetically wasteful process, except in vivo, where endogenous cCF10 induces a conjugation-linked virulence factor. Endogenous cCF10 is controlled by two plasmid-encoded products; a membrane protein PrgY reduces pheromone levels in donors, and a secreted inhibitor peptide iCF10 inhibits the residual endogenous pheromone that escapes PrgY control. In this study we genetically determined the amino acid specificity determinants within PrgY, cCF10, and the cCF10 precursor that are necessary for cCF10 processing and for PrgY-mediated control. We showed that amino acid residues 125 to 241 of PrgY are required for specific recognition of cCF10 and that PrgY recognizes determinants within the heptapeptide cCF10 sequence, supporting a direct interaction between PrgY and mature cCF10. In addition, we found that a regulated intramembrane proteolysis (RIP) family pheromone precursor-processing protein Eep recognizes amino acids N-terminal to cCF10 in the signal sequence of CcfA. These results support a model where Eep directly targets pheromone precursors for RIP and PrgY interacts directly with the mature cCF10 peptide during processing. Despite evidence that both PrgY and Eep associate with cCF10 in or near the membrane, results presented here indicate that these two proteins function independently.
Interaction of CheY with the C-Terminal Peptide of CheZ
Authors: Guhaniyogi Jayita , Wu Ti , Patel Smita S. , Stock Ann M.
Published: December 2007

Chemotaxis, a means for motile bacteria to sense the environment and achieve directed swimming, is controlled by flagellar rotation. The primary output of the chemotaxis machinery is the phosphorylated form of the response regulator CheY (P∼CheY). The steady-state level of P∼CheY dictates the direction of rotation of the flagellar motor. The chemotaxis signal in the form of P∼CheY is terminated by the phosphatase CheZ. Efficient dephosphorylation of CheY by CheZ requires two distinct protein-protein interfaces: one involving the strongly conserved C-terminal helix of CheZ (CheZ
C
) tethering the two proteins together and the other constituting an active site for catalytic dephosphorylation. In a previous work (J. Guhaniyogi, V. L. Robinson, and A. M. Stock, J. Mol. Biol.
359:
624-645, 2006), we presented high-resolution crystal structures of CheY in complex with the CheZ
C
peptide that revealed alternate binding modes subject to the conformational state of CheY. In this study, we report biochemical and structural data that support the alternate-binding-mode hypothesis and identify key recognition elements in the CheY-CheZ
C
interaction. In addition, we present kinetic studies of the CheZ
C
-associated effect on CheY phosphorylation with its physiologically relevant phosphodonor, the histidine kinase CheA. Our results indicate mechanistic differences in phosphotransfer from the kinase CheA versus that from small-molecule phosphodonors, explaining a modest twofold increase of CheY phosphorylation with the former, observed in this study, relative to a 10-fold increase previously documented with the latter.
Characterization of the SOS Regulon ofCaulobacter crescentus
Authors: da Rocha Raquel Paes , de Miranda Paquola Apuã César , do Valle Marques Marilis , Menck Carlos Frederico Martins , Galhardo Rodrigo S.
Published: December 2007

The SOS regulon is a paradigm of bacterial responses to DNA damage. A wide variety of bacterial species possess homologs oflexAandrecA, the central players in the regulation of the SOS circuit. Nevertheless, the genes actually regulated by the SOS have been determined only experimentally in a few bacterial species. In this work, we describe 37 genes regulated in a LexA-dependent manner in the alphaproteobacteriumCaulobacter crescentus. In agreement with previous results, we have found that the direct repeat GTTCN7GTTC is the SOS operator ofC. crescentus, which was confirmed by site-directed mutagenesis studies of theimuApromoter. Several potential promoter regions containing the SOS operator were identified in the genome, and the expression of the corresponding genes was analyzed for both the wild type and thelexAstrain, demonstrating that the vast majority of these genes are indeed SOS regulated. Interestingly, many of these genes encode proteins with unknown functions, revealing the potential of this approach for the discovery of novel genes involved in cellular responses to DNA damage in prokaryotes, and illustrating the diversity of SOS-regulated genes among different bacterial species.
Tn
5
Synaptic Complex Formation: Role of Transposase Residue W450
Authors: Gradman Richard J. , Reznikoff William S.
Published: December 2007

A series of Tn
5
transposases (Tnp's) with mutations at the conserved amino acid position W450, which was structurally predicted to be important for synapsis, have been generated and characterized. This study demonstrates that W450 is involved in hydrophobic (and possibly aromatic) contacts within the Tnp monomer that negatively regulate synaptic complex formation.