bluuebunny/crossref_metadata_2025_split huggingface dataset:
Potential Regrowth and Recolonization of Salmonellae and Indicators in Biosolids and Biosolid-Amended Soil
Authors: Zaleski Kathleen J. , Josephson Karen L. , Gerba Charles P. , Pepper Ian L. 
Published: July 2005

ABSTRACT
This study evaluated the potential for conversion of Class B to Class A biosolids with respect to salmonellae and fecal coliforms during solar drying in concrete lined drying beds. Anaerobically (8% solids) and aerobically (2% solids) digested Class B biosolids were pumped into field-scale drying beds, and microbial populations and environmental conditions were monitored. Numbers of fecal coliforms and salmonellae decreased as temperature and rate of desiccation increased. After 3 to 4 weeks, Class A requirements were achieved in both biosolids for the pathogens and the indicators. However, following rainfall events, significant increase in numbers was observed for both fecal coliforms and salmonellae. In laboratory studies, regrowth of fecal coliforms was observed in both biosolids and biosolid-amended soil, but the regrowth of salmonellae observed in the concrete-lined drying beds did not occur. These laboratory studies demonstrated that pathogens decreased in numbers when soil was amended with biosolids. Based on serotyping, the increased numbers of salmonellae seen in the concrete lined drying beds following rainfall events was most likely due to recolonization due to contamination from fecal matter introduced by animals and not from regrowth of salmonellae indigenous to biosolids. Overall, we conclude that the use of concrete-lined beds created a situation in which moisture added as rainfall accumulated in the beds, promoting the growth of fecal coliforms and salmonellae added from external sources.
AmyA, an α-Amylase with β-Cyclodextrin-Forming Activity, and AmyB from the Thermoalkaliphilic OrganismAnaerobranca gottschalkii: Two α-Amylases Adapted to Their Different Cellular Localizations
Authors: Ballschmiter Meike , Armbrecht Martin , Ivanova Krasimira , Antranikian Garabed , Liebl Wolfgang 
Published: July 2005

ABSTRACTTwo α-amylase genes from the thermophilic alkaliphileAnaerobranca gottschalkiiwere cloned, and the corresponding enzymes, AmyA and AmyB, were investigated after purification of the recombinant proteins. Based on their amino acid sequences, AmyA is proposed to be a lipoprotein with extracellular localization and thus is exposed to the alkaline milieu, while AmyB apparently represents a cytoplasmic enzyme. The amino acid sequences of both enzymes bear high similarity to those of GHF13 proteins. The different cellular localizations of AmyA and AmyB are reflected in their physicochemical properties. The alkaline pH optimum (pH 8), as well as the broad pH range, of AmyA activity (more than 50% activity between pH 6 and pH 9.5) mirrors the conditions that are encountered by an extracellular enzyme exposed to the medium ofA. gottschalkii, which grows between pH 6 and pH 10.5. AmyB, on the other hand, has a narrow pH range with a slightly acidic pH optimum at 6 to 6.5, which is presumably close to the pH in the cytoplasm. Also, the intracellular AmyB is less tolerant of high temperatures than the extracellular AmyA. While AmyA has a half-life of 48 h at 70°C, AmyB has a half-life of only about 10 min at that temperature, perhaps due to the lack of stabilizing constituents of the cytoplasm. AmyA and AmyB were very similar with respect to their substrate specificity profiles, clearly preferring amylose over amylopectin, pullulan, and glycogen. Both enzymes also hydrolyzed α-, β-, and γ-cyclodextrin. Very interestingly, AmyA, but not AmyB, displayed high transglycosylation activity on maltooligosaccharides and also had significant β-cyclodextrin glycosyltransferase (CGTase) activity. CGTase activity has not been reported for typical α-amylases before. The mechanism of cyclodextrin formation by AmyA is unknown.
Suppression of Damping-Off Disease in Host Plants by the Rhizoplane Bacterium
Lysobacter
sp. Strain SB-K88 Is Linked to Plant Colonization and Antibiosis against Soilborne Peronosporomycetes
Authors: Islam Md. Tofazzal , Hashidoko Yasuyuki , Deora Abhinandan , Ito Toshiaki , Tahara Satoshi 
Published: July 2005

ABSTRACT

We previously demonstrated that xanthobaccin A from the rhizoplane bacterium
Lysobacter
sp. strain SB-K88 suppresses damping-off disease caused by
Pythium
sp. in sugar beet. In this study we focused on modes of
Lysobacter
sp. strain SB-K88 root colonization and antibiosis of the bacterium against
Aphanomyces cochlioides
, a pathogen of damping-off disease. Scanning electron microscopic analysis of 2-week-old sugar beet seedlings from seeds previously inoculated with SB-K88 revealed dense colonization on the root surfaces and a characteristic perpendicular pattern of
Lysobacter
colonization possibly generated via development of polar, brush-like fimbriae. In colonized regions a semitransparent film apparently enveloping the root and microcolonies were observed on the root surface. This
Lysobacter
strain also efficiently colonized the roots of several plants, including spinach, tomato,
Arabidopsis thaliana
, and
Amaranthus gangeticus
. Plants grown from both sugar beet and spinach seeds that were previously treated with
Lysobacter
sp. strain SB-K88 displayed significant resistance to the damping-off disease triggered by
A. cochlioides
. Interestingly, zoospores of
A. cochlioides
became immotile within 1 min after exposure to a SB-K88 cell suspension, a cell-free supernatant of SB-K88, or pure xanthobaccin A (MIC, 0.01 μg/ml). In all cases, lysis followed within 30 min in the presence of the inhibiting factor(s). Our data indicate that
Lysobacter
sp. strain SB-K88 has a direct inhibitory effect on
A. cochlioides
, suppressing damping-off disease. Furthermore, this inhibitory effect of
Lysobacter
sp. strain SB-K88 is likely due to a combination of antibiosis and characteristic biofilm formation at the rhizoplane of the host plant.
Isolation of Soil Bacteria Adapted To Degrade Humic Acid-Sorbed Phenanthrene
Authors: Vacca D. J. , Bleam W. F. , Hickey W. J. 
Published: July 2005

ABSTRACTThe goal of these studies was to determine how sorption by humic acids affected the bioavailability of polynuclear aromatic hydrocarbons (PAHs) to PAH-degrading microbes. Micellar solutions of humic acid were used as sorbents, and phenanthrene was used as a model PAH. Enrichments from PAH-contaminated soils established with nonsorbed phenanthrene yielded a total of 25 different isolates representing a diversity of bacterial phylotypes. In contrast, only three strains ofBurkholderiaspp. and one strain each ofDelftiasp. andSphingomonassp. were isolated from enrichments with humic acid-sorbed phenanthrene (HASP). Using [14C]phenanthrene as a radiotracer, we verified that only HASP isolates were capable of mineralizing HASP, a phenotype hence termed “competence.” Competence was an all-or-nothing phenotype: noncompetent strains showed no detectable phenanthrene mineralization in HASP cultures, but levels of phenanthrene mineralization effected by competent strains in HASP and NSP cultures were not significantly different. Levels and rates of phenanthrene mineralization exceeded those predicted to be supported solely by the metabolism of phenanthrene in the aqueous phase of HASP cultures. Thus, competent strains were able to directly access phenanthrene sorbed by the humic acids and did not rely on desorption for substrate uptake. To the best of our knowledge, this is the first report of (i) a selective interaction between aerobic bacteria and humic acid molecules and (ii) differential bioavailability to bacteria of PAHs sorbed to a natural biogeopolymer.
Purification, Characterization, and Crystallization of the Components of the Nitrobenzene and 2-Nitrotoluene Dioxygenase Enzyme Systems
Authors: Parales R. E. , Huang R. , Yu C.-L. , Parales J. V. , Lee F. K. N. , Lessner D. J. , Ivkovic-Jensen M. M. , Liu W. , Friemann R. , Ramaswamy S. , Gibson D. T. 
Published: July 2005

ABSTRACT

The protein components of the 2-nitrotoluene (2NT) and nitrobenzene dioxygenase enzyme systems from
Acidovorax
sp. strain JS42 and
Comamonas
sp. strain JS765, respectively, were purified and characterized. These enzymes catalyze the initial step in the degradation of 2-nitrotoluene and nitrobenzene. The identical shared reductase and ferredoxin components were monomers of 35 and 11.5 kDa, respectively. The reductase component contained 1.86 g-atoms iron, 2.01 g-atoms sulfur, and one molecule of flavin adenine dinucleotide per monomer. Spectral properties of the reductase indicated the presence of a plant-type [2Fe-2S] center and a flavin. The reductase catalyzed the reduction of cytochrome
c
, ferricyanide, and 2,6-dichlorophenol indophenol. The ferredoxin contained 2.20 g-atoms iron and 1.99 g-atoms sulfur per monomer and had spectral properties indicative of a Rieske [2Fe-2S] center. The ferredoxin component could be effectively replaced by the ferredoxin from the
Pseudomonas
sp. strain NCIB 9816-4 naphthalene dioxygenase system but not by that from the
Burkholderia
sp. strain LB400 biphenyl or
Pseudomonas putida
F1 toluene dioxygenase system. The oxygenases from the 2-nitrotoluene and nitrobenzene dioxygenase systems each had spectral properties indicating the presence of a Rieske [2Fe-2S] center, and the subunit composition of each oxygenase was an α
3
β
3
hexamer. The apparent

K
m

of 2-nitrotoluene dioxygenase for 2NT was 20 μM, and that for naphthalene was 121 μM. The specificity constants were 7.0 μM
−1
min
−1
for 2NT and 1.2 μM
−1
min
−1
for naphthalene, indicating that the enzyme is more efficient with 2NT as a substrate. Diffraction-quality crystals of the two oxygenases were obtained.
A Multigene Phylogenetic Study of Clonal Diversity and Divergence in North American Strains of the Plant Pathogen
Xylella fastidiosa
Authors: Schuenzel Erin L. , Scally Mark , Stouthamer Richard , Nunney Leonard 
Published: July 2005

ABSTRACT

Xylella fastidiosa
is a pathogen that causes leaf scorch and related diseases in over 100 plant species, including Pierce's disease in grapevines (PD), phony peach disease (PP), plum leaf scald (PLS), and leaf scorch in almond (ALS), oak (OAK), and oleander (OLS). We used a high-resolution DNA sequence approach to investigate the evolutionary relationships, geographic variation, and divergence times among the
X. fastidiosa
isolates causing these diseases in North America. Using a large data set of 10 coding loci and 26 isolates, the phylogeny of
X. fastidiosa
defined three major clades. Two of these clades correspond to the recently identified
X. fastidiosa
subspecies
piercei
(PD and some ALS isolates) and
X. fastidiosa
subsp.
multiplex
(OAK, PP, PLS, and some ALS isolates). The third clade grouped all of the OLS isolates into a genetically distinct group, named
X. fastidiosa
subsp.
sandyi
. These well-differentiated clades indicate that, historically,
X. fastidiosa
has been a clonal organism. Based on their synonymous-site divergence (∼3%), these three clades probably originated more than 15,000 years ago, long before the introduction of the nonnative plants that characterize most infections. The sister clades of
X. fastidiosa
subsp.
sandyi
and
X. fastidiosa
subsp.
piercei
have synonymous-site evolutionary rates 2.9 times faster than
X. fastidiosa
subsp.
multiplex
, possibly due to generation time differences. Within
X. fastidiosa
subsp.
multiplex
, a low level (∼0.1%) of genetic differentiation indicates the recent divergence of ALS isolates from the PP, PLS, and OAK isolates due to host plant adaptation and/or allopatry. The low level of variation within the
X. fastidiosa
subsp.
piercei
and
X. fastidiosa
subsp.
sandyi
clades, despite their antiquity, suggests strong selection, possibly driven by host plant adaptation.
Role for Glycine Betaine Transport in
Vibrio cholerae
Osmoadaptation and Biofilm Formation within Microbial Communities
Authors: Kapfhammer Dagmar , Karatan Ece , Pflughoeft Kathryn J. , Watnick Paula I. 
Published: July 2005

ABSTRACT

Vibrio cholerae
is a halophilic facultative human pathogen found in marine and estuarine environments. Accumulation of compatible solutes is important for growth of
V. cholerae
at NaCl concentrations greater than 250 mM. We have identified and characterized two compatible solute transporters, OpuD and PutP, that are involved in uptake of glycine betaine and proline by
V. cholerae. V. cholerae
does not, however, possess the
bet
genes, suggesting that it is unable to synthesize glycine betaine. In contrast, many
Vibrio
species are able to synthesize glycine betaine from choline. It has been shown that many bacteria not only synthesize but also secrete glycine betaine. We hypothesized that sharing of compatible solutes might be a mechanism for cooperativity in microbial communities. In fact, we have demonstrated that, in high-osmolarity medium,
V. cholerae
growth and biofilm development are enhanced by supplementation with either glycine betaine or spent media from other bacterial species. Thus, we propose that compatible solutes provided by other microorganisms may contribute to survival of
V. cholerae
in the marine environment through facilitation of osmoadaptation and biofilm development.
Improved and Versatile Transformation System Allowing Multiple Genetic Manipulations of the Hyperthermophilic Archaeon
Thermococcus kodakaraensis
Authors: Sato Takaaki , Fukui Toshiaki , Atomi Haruyuki , Imanaka Tadayuki 
Published: July 2005

ABSTRACT

We have recently developed a gene disruption system for the hyperthermophilic archaeon
Thermococcus kodakaraensis
by utilizing a
pyrF
-deficient mutant, KU25, as a host strain and the
pyrF
gene as a selectable marker. To achieve multiple genetic manipulations for more advanced functional analyses of genes in vivo, it is necessary to establish multiple host-marker systems or to develop a system in which repeated utilization of one marker gene is possible. In this study, we first constructed a new host strain, KU216 (Δ
pyrF
), by specific and almost complete deletion of endogenous
pyrF
through homologous recombination. In this refined host, there is no need to consider unknown mutations caused by random mutagenesis, and unlike in the previous host, KU25, there is little, if any, possibility that unintended recombination between the marker gene and the chromosomal allele occurs. Furthermore, a new host-marker combination of a
trpE
deletant, KW128 (Δ
pyrF
Δ
trpE
::
pyrF
), and the
trpE
gene was developed. This system made it possible to isolate transformants through a more simple selection procedure as well as to deduce the transformation efficiency, overcoming practical disadvantages of the first system. The effects of the transformation conditions were also investigated using this system. Finally, we have also established a system in which repeated utilization of the counterselectable
pyrF
marker is possible through its excision by pop-out recombination. Both endogenous and exogenous sequences could be applied as tandem repeats flanking the marker
pyrF
for pop-out recombination. A double deletion mutant, KUW1 (Δ
pyrF
Δ
trpE
), constructed with the pop-out strategy, was demonstrated to be a useful host for the dual markers
pyrF
and
trpE
. Likewise, a triple deletion mutant, KUWH1 (Δ
pyrF
Δ
trpE
Δ
hisD
), could also be constructed. The transformation systems developed here now provide the means for extensive genetic studies in this hyperthermophilic archaeon.
Development of a
recA
Gene-Based Identification Approach for the Entire
Burkholderia
Genus
Authors: Payne George W. , Vandamme Peter , Morgan Sara H. , LiPuma John J. , Coenye Tom , Weightman Andrew J. , Jones T. Hefin , Mahenthiralingam Eshwar 
Published: July 2005

ABSTRACT

Burkholderia
is an important bacterial genus containing species of ecological, biotechnological, and pathogenic interest. With their taxonomy undergoing constant revision and the phenotypic similarity of several species, correct identification of
Burkholderia
is difficult. A genetic scheme based on the
recA
gene has greatly enhanced the identification of
Burkholderia cepacia
complex species. However, the PCR developed for the latter approach was limited by its specificity for the complex. By alignment of existing and novel
Burkholderia recA
sequences, we designed new PCR primers and evaluated their specificity by testing a representative panel of
Burkholderia
strains. PCR followed by restriction fragment length polymorphism analysis of an 869-bp portion of the
Burkholderia recA
gene was not sufficiently discriminatory. Nucleotide sequencing followed by phylogenetic analysis of this
recA
fragment differentiated both putative and known
Burkholderia
species and all members of the
B. cepacia
complex. In addition, it enabled the design of a
Burkholderia
genus-specific
recA
PCR that produced a 385-bp amplicon, the sequence of which was also able to discriminate all species examined. Phylogenetic analysis of 188 novel
recA
genes enabled clarification of the taxonomic position of several important
Burkholderia
strains and revealed the presence of four novel
B. cepacia
complex
recA
lineages. Although the
recA
phylogeny could not be used as a means to differentiate
B. cepacia
complex strains recovered from clinical infection versus the natural environment, it did facilitate the identification of clonal strain types of
B. cepacia
,
B. stabilis
, and
B. ambifaria
capable of residing in both niches.
Location of theBombyx moriAminopeptidase N Type 1 Binding Site onBacillus thuringiensisCry1Aa Toxin
Authors: Atsumi Shogo , Mizuno Eri , Hara Hirotaka , Nakanishi Kazuko , Kitami Madoka , Miura Nami , Tabunoki Hiroko , Watanabe Ayako , Sato Ryoichi 
Published: July 2005

ABSTRACTWe analyzed the binding site on Cry1Aa toxin for the Cry1Aa receptor inBombyx mori, 115-kDa aminopeptidase N type 1 (BmAPN1) (K. Nakanishi, K. Yaoi, Y. Nagino, H. Hara, M. Kitami, S. Atsumi, N. Miura, and R. Sato, FEBS Lett.519:215-220, 2002), by using monoclonal antibodies (MAbs) that block binding between the binding site and the receptor. First, we produced a series of MAbs against Cry1Aa and obtained two MAbs, MAbs 2C2 and 1B10, that were capable of blocking the binding between Cry1Aa and BmAPN1 (blocking MAbs). The epitope of the Fab fragments of MAb 2C2 overlapped the BmAPN1 binding site, whereas the epitope of the Fab fragments of MAb 1B10 did not overlap but was located close to the binding site. Using three approaches for epitope mapping, we identified two candidate epitopes for the blocking MAbs on Cry1Aa. We constructed two Cry1Aa toxin mutants by substituting a cysteine on the toxin surface at each of the two candidate epitopes, and the small blocking moleculeN-(9-acridinyl)maleimide (NAM) was introduced at each cysteine substitution to determine the true epitope. The Cry1Aa mutant with NAM bound to Cys582 did not bind either of the two blocking MAbs, suggesting that the true epitope for each of the blocking MAbs was located at the site containing Val582, which also consisted of508STLRVN513and582VFTLSAHV589. These results indicated that the BmAPN1 binding site overlapped part of the region blocked by MAb 2C2 that was close to but excluded the actual epitope of MAb 2C2 on domain III of Cry1Aa toxin. We also discuss another area on Cry1Aa toxin as a new candidate site for BmAPN1 binding.
Diel Variations in Carbon Metabolism by Green Nonsulfur-Like Bacteria in Alkaline Siliceous Hot Spring Microbial Mats from Yellowstone National Park
Authors: van der Meer Marcel T. J. , Schouten Stefan , Bateson Mary M. , Nübel Ulrich , Wieland Andrea , Kühl Michael , de Leeuw Jan W. , Sinninghe Damsté Jaap S. , Ward David M. 
Published: July 2005

ABSTRACT

Green nonsulfur-like bacteria (GNSLB) in hot spring microbial mats are thought to be mainly photoheterotrophic, using cyanobacterial metabolites as carbon sources. However, the stable carbon isotopic composition of typical
Chloroflexus
and
Roseiflexus
lipids suggests photoautotrophic metabolism of GNSLB. One possible explanation for this apparent discrepancy might be that GNSLB fix inorganic carbon only during certain times of the day. In order to study temporal variability in carbon metabolism by GNSLB, labeling experiments with [
13
C]bicarbonate, [
14
C]bicarbonate, and [
13
C]acetate were performed during different times of the day. [
14
C]bicarbonate labeling indicated that during the morning, incorporation of label was light dependent and that both cyanobacteria and GNSLB were involved in bicarbonate uptake.
13
C-labeling experiments indicated that during the morning, GNSLB incorporated labeled bicarbonate at least to the same degree as cyanobacteria. The incorporation of [
13
C]bicarbonate into specific lipids could be stimulated by the addition of sulfide or hydrogen, which both were present in the morning photic zone. The results suggest that GNSLB have the potential for photoautotrophic metabolism during low-light periods. In high-light periods, inorganic carbon was incorporated primarily into
Cyanobacteria
-specific lipids. The results of a pulse-labeling experiment were consistent with overnight transfer of label to GNSLB, which could be interrupted by the addition of unlabeled acetate and glycolate. In addition, we observed direct incorporation of [
13
C]acetate into GNSLB lipids in the morning. This suggests that GNSLB also have a potential for photoheterotrophy in situ.
Identity and Ecophysiology of Uncultured Actinobacterial Polyphosphate-Accumulating Organisms in Full-Scale Enhanced Biological Phosphorus Removal Plants
Authors: Kong Yunhong , Nielsen Jeppe Lund , Nielsen Per Halkjær 
Published: July 2005

ABSTRACTMicroautoradiography combined with fluorescence in situ hybridization (MAR-FISH) was used to screen for potential polyphosphate-accumulating organisms (PAO) in a full-scale enhanced biological phosphorus removal (EBPR) plant. The results showed that, in addition to unculturedRhodocyclus-related PAO, two morphotypes hybridizing with gene probes for the gram-positiveActinobacteriawere also actively involved in uptake of orthophosphate (Pi). Clone library analysis and further investigations by MAR-FISH using two new oligonucleotide probes revealed that both morphotypes, cocci in clusters of tetrads and short rods in clumps, were relatively closely related to the genusTetrasphaerawithin the familyIntrasporangiaceaeof theActinobacteria(93 to 98% similarity in their 16S rRNA genes). FISH analysis of the community biomass in the treatment plant investigated showed that the short rods (targeted by probe Actino-658) were the most abundant (12% of allBacteriahybridizing with general bacterial probes), while the cocci in tetrads (targeted by probe Actino-221) made up 7%. Both morphotypes took up Piaerobically only if, in a previous anaerobic phase, they had taken up organic matter from wastewater or a mixture of amino acids. They could not take up short-chain fatty acids (e.g., acetate), glucose, or ethanol under anaerobic or aerobic conditions. The storage compound produced during the anaerobic period was not polyhydroxyalkanoates, as forRhodocyclus-related PAO, and its identity is still unknown. Growth and uptake of Pitook place in the presence of oxygen and nitrate but not nitrite, indicating a lack of denitrifying ability. A survey of the occurrence of these actinobacterial PAO in 10 full-scale EBPR plants revealed that both morphotypes were widely present, and in several plants more abundant than theRhodocyclus-related PAO, thus playing a very important role in the EBPR process.
Multiple-Mutation Reaction: a Method for Simultaneous Introduction of Multiple Mutations into theglpKGene ofMycoplasma pneumoniae
Authors: Hames Claudine , Halbedel Sven , Schilling Oliver , Stülke Jörg 
Published: July 2005

ABSTRACTInMycoplasma pneumoniae, the UGA opal codon specifies tryptophan rather than a translation stop site. This often makes it difficult to expressMycoplasmaproteins inE. coliisolates. In this work, we developed a strategy for the one-step introduction of several mutations. This method, the multiple-mutation reaction, is used to simultaneously replace nine opal codons in theM. pneumoniae glpKgene.