bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification by Quantitative Carrier Test of Surrogate Spore-Forming Bacteria To Assess Sporicidal Chemicals for Use against
Bacillus anthracis
Authors: Majcher Miles R. , Bernard Kathryn A. , Sattar Syed A. 
Published: December 2007

ABSTRACT

The spores of six strains of
Bacillus anthracis
(four virulent and two avirulent) were compared with those of four other types of spore-forming bacteria for their resistance to four liquid chemical sporicides (sodium hypochlorite at 5,000 ppm available chlorine, 70,000 ppm accelerated H
2
O
2
, 1,000 ppm chlorine dioxide, and 3,000 ppm peracetic acid). All test bacteria were grown in a 1:10 dilution of Columbia broth (with manganese) incubated at 37°C for 72 h. The spore suspensions, heat treated at 80°C for 10 min to rid them of any viable vegetative cells, contained 1 × 10
8
to 3 × 10
8
CFU/ml. The second tier of the quantitative carrier test (QCT-2), a standard of ASTM International, was used to assess for sporicidal activity, with disks (1 cm in diameter) of brushed and magnetized stainless steel as spore carriers. Each carrier, with 10 μl (≥10
6
CFU) of the test spore suspension in a soil load, was dried and then overlaid with 50 μl of the sporicide being evaluated. The contact time at room temperature ranged from 5 to 20 min, and the arbitrarily set criterion for acceptable sporicidal activity was a reduction of ≥10
6
in viable spore count. Each test was repeated at least three times. In the final analysis, the spores of
Bacillus licheniformis
(ATCC 14580
T
) and
Bacillus subtilis
(ATCC 6051
T
) proved to be generally more resistant than the spores of the strains of
B. anthracis
tested. The use of one or both of the safe and easy-to-handle surrogates identified here should help in developing safer and more-effective sporicides and also in evaluating the field effectiveness of existing and newer formulations in the decontamination of objects and surfaces suspected of
B. anthracis
contamination.
Development of Multiple-Locus Variable-Number Tandem-Repeat Analysis for the Molecular Subtyping of
Enterobacter sakazakii
Authors: Mullane N. R. , Ryan M. , Iversen C. , Murphy M. , O'Gaora P. , Quinn T. , Whyte P. , Wall P. G. , Fanning S. 
Published: December 2007

ABSTRACT

The genomic content of
Enterobacter sakazakii
strain ATCC BAA-894 was analyzed for variable-number tandem repeats (VNTRs). In this study we report the development of a multiple-locus VNTR analysis (MLVA) strategy for the subtyping of
E. sakazakii
. The method is based on a GeneScan analysis of four VNTR loci labeled with multiple fluorescent dyes. This approach was applied to a collection of 112 isolates representing all 16 of the currently defined
E. sakazakii
biogroups. MLVA successfully discriminated among these isolates and compared favorably with pulsed-field gel electrophoresis. The method was relatively fast and easy to perform. The potential value of MLVA as an epidemiological tool is discussed.
Suppressing Posttranslational Gluconoylation of Heterologous Proteins by Metabolic Engineering of
Escherichia coli
Authors: Aon Juan C. , Caimi Richard J. , Taylor Alexander H. , Lu Quinn , Oluboyede Femi , Dally Jennifer , Kessler Michelle D. , Kerrigan John J. , Lewis Tia S. , Wysocki Lisa A. , Patel Pramatesh S. 
Published: December 2007

ABSTRACT

Minimization of chemical modifications during the production of proteins for pharmaceutical and medical applications is of fundamental and practical importance. The gluconoylation of heterologously expressed protein which is observed in
Escherichia coli
BL21(DE3) constitutes one such undesired posttranslational modification. We postulated that formation of gluconoylated/phosphogluconoylated products of heterologous proteins is caused by the accumulation of 6-phosphogluconolactone due to the absence of phosphogluconolactonase (PGL) in the pentose phosphate pathway. The results obtained demonstrate that overexpression of a heterologous PGL in BL21(DE3) suppresses the formation of the gluconoylated adducts in the therapeutic proteins studied. When this
E. coli
strain was grown in high-cell-density fed-batch cultures with an extra copy of the
pgl
gene, we found that the biomass yield and specific productivity of a heterologous 18-kDa protein increased simultaneously by 50 and 60%, respectively. The higher level of PGL expression allowed
E. coli
strain BL21(DE3) to satisfy the extra demand for precursors, as well as the energy requirements, in order to replicate plasmid DNA and express heterologous genes, as metabolic flux analysis showed by the higher precursor and NADPH fluxes through the oxidative branch of the pentose phosphate shunt. This work shows that
E. coli
strain BL21(DE3) can be used as a host to produce three different proteins, a heterodimer of liver X receptors, elongin C, and an 18-kDa protein. This is the first report describing a novel and general strategy for suppressing this nonenzymatic modification by metabolic pathway engineering.
“Endomicrobia” and Other Bacteria Associated with the Hindgut ofDermolepida albohirtumLarvae
Authors: Pittman Geoffrey W. , Brumbley Stevens M. , Allsopp Peter G. , O'Neill Scott L. 
Published: December 2007

ABSTRACTSymbiotic bacteria residing in the hindgut chambers of scarab beetle larvae may be useful in paratransgenic approaches to reduce larval root-feeding activities on agricultural crops. We compared the bacterial community profiles associated with the hindgut walls of individualDermolepida albohirtumthird-instar larvae over 2 years and those associated with their plant root food source among different geographic regions. Denaturing gradient gel electrophoresis analysis was used with universal andActinobacteria-specific 16S rRNA primers to reveal a number of taxa that were found consistently in allD. albohirtumlarvae but not in samples from their food source, sugarcane roots. These taxa included representatives from the “Endomicrobia,”Firmicutes,Proteobacteria, andActinobacteriaand were related to previously described bacteria from the intestines of other scarab larvae and termites. These universally distributed taxa have the potential to form vertically transmitted symbiotic associations with these insects.
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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACTIn 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

ABSTRACTLegionella 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

ABSTRACTMicrobial 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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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.