bluuebunny/crossref_metadata_2025 huggingface dataset:
slightEffect of damping on the propensity of squeal instability: An experimental investigation
Authors: Massi Francesco , Giannini Oliviero
Published: April 2008

Friction induced vibrations in automotive brakes is recognized as a major problem in industry. Squeal is a difficult subject because of its unpredictability caused by a not completely understood sensitivity to variation of the system parameters. In the literature several analytical and numerical studies deal with the relationship between damping and system propensity to have instability. These studies highlight the existence of a nonintuitive effect of damping distribution on modal coupling that gives rise to the unstable vibrations. The complexity of commercial brakes and the difficulties to identify the values of modal damping in brake assemblies lead to the necessity to rely on experimental analysis using simplified test rigs. This paper presents an experimental investigation of the relationship between the distribution of modal damping and the propensity to develop squeal in a beam-on-disk setup, which reliably reproduces squeal events with easy control and measurement of the damping of the disk and the beam, respectively. The experiments highlight the key role played by the modal damping distribution on squeal: A nonuniform repartition of the modal damping causes an increase of the squeal propensity.
Spectral weighting strategies for hearing-impaired listeners measured using a correlational method
Authors: Calandruccio Lauren , Doherty Karen A.
Published: April 2008

Spectral weighting strategies using a correlational method [R. A. Lutfi, J. Acoust. Soc. Am. 97, 1333–1334 (1995); V. M. Richards and S. Zhu, J. Acoust. Soc. Am. 95, 423–424 (1994)] were measured in ten listeners with sensorineural-hearing loss on a sentence recognition task. Sentences and a spectrally matched noise were filtered into five separate adjacent spectral bands and presented to listeners at various signal-to-noise ratios (SNRs). Five point-biserial correlations were computed between the listeners’ response (correct or incorrect) on the task and the SNR in each band. The stronger the correlation between performance and SNR, the greater that given band was weighted by the listener. Listeners were tested with and without hearing aids on. All listeners were experienced hearing aid users. Results indicated that the highest spectral band (∼2800–11000Hz) received the greatest weight in both listening conditions. However, the weight on the highest spectral band was less when listeners performed the task with their hearing aids on in comparison to when listening without hearing aids. No direct relationship was observed between the listeners’ weights and the sensation level within a given band.
Tolevamer, an Anionic Polymer, Neutralizes Toxins Produced by the BI/027 Strains of
Clostridium difficile
Authors: Hinkson Paul L. , Dinardo Carol , DeCiero Daniel , Klinger Jeffrey D. , Barker Robert H.
Published: April 2008

Clostridium difficile
-associated diarrhea (CDAD) is caused by the toxins the organism produces when it overgrows in the colon as a consequence of antibiotic depletion of normal flora. Conventional antibiotic treatment of CDAD increases the likelihood of recurrent disease by again suppressing normal bacterial flora. Tolevamer, a novel toxin-binding polymer, was developed to ameliorate the disease without adversely affecting normal flora. In the current study, tolevamer was tested for its ability to neutralize clostridial toxins produced by the epidemic BI/027 strains, thereby preventing toxin-mediated tissue culture cell rounding. The titers of toxin-containing
C. difficile
culture supernatants were determined using confluent cell monolayers, and then the supernatants were used in assays containing dilutions of tolevamer to determine the lowest concentration of tolevamer that prevented ≥90% cytotoxicity. Tolevamer neutralized toxins in the supernatants of all
C. difficile
strains tested. Specific antibodies against the large clostridial toxins TcdA and TcdB also neutralized the cytopathic effect, suggesting that tolevamer is specifically neutralizing these toxins and that the binary toxin (whose genes are carried by the BI/027 strains) is not a significant source of cytopathology against tissue culture cells in vitro.
Bactericidal and Sterilizing Activities of Several Orally Administered Combined Regimens against
Mycobacterium ulcerans
in Mice
Authors: Ji Baohong , Chauffour Aurélie , Robert Jérome , Jarlier Vincent
Published: April 2008

Treatment with rifampin-clarithromycin or moxifloxacin-clarithromycin for 8 weeks displayed promising bactericidal activity against
Mycobacterium ulcerans
in mice; none of the mice treated with rifampin-clarithromycin relapsed, whereas 59% of those treated with moxifloxacin-clarithromycin relapsed after treatment was stopped. The bactericidal and sterilizing activities of the five-times-weekly (5/7) administration of 5 mg of rifapentine/kg of body weight, either alone or in combination, were virtually identical to those of the corresponding regimens with 10 mg of rifampin/kg of body weight; however, because of the long half-life of rifapentine, accumulation of the drug after 5/7 administration is a concern. The bactericidal activity of 20 mg/kg rifapentine in monotherapy or 20 mg/kg rifapentine in combination with 150 mg/kg streptomycin or 200 mg/kg moxifloxacin administered twice weekly was as effective as the corresponding regimens containing 10 mg/kg rifampin administered 5/7, suggesting that Buruli ulcer might be treated with intermittently administered rifapentine-containing combinations.
In Vitro Activity of Anidulafungin against
Candida albicans
Biofilms
Authors: Jacobson Melissa J. , Piper Kerryl E. , Nguyen Gary , Steckelberg James M. , Patel Robin
Published: April 2008

We tested the activity of anidulafungin against 30
Candida albicans
isolates. The planktonic MICs for 50 and 90% of the isolates tested (MIC
50
and MIC
90
) were ≤0.03 and 0.125 μg/ml, respectively (MIC range, ≤0.03 to 2 μg/ml). The sessile MIC
50
and MIC
90
were ≤0.03 and ≤0.03 μg/ml, respectively (MIC range, ≤0.03 to >16 μg/ml).
First Report of
cfr
-Mediated Resistance to Linezolid in Human Staphylococcal Clinical Isolates Recovered in the United States
Authors: Mendes Rodrigo E. , Deshpande Lalitagauri M. , Castanheira Mariana , DiPersio Joseph , Saubolle Michael A. , Jones Ronald N.
Published: April 2008

Linezolid resistance has dominantly been mediated by mutations in 23S rRNA or ribosomal protein L4 genes. Recently,
cfr
has demonstrated the ability to produce a phenotype of resistance to not only oxazolidinones, but also other antimicrobial classes (phenicols, lincosamides, pleuromutilins, and streptogramin A). We describe the first detection of
cfr
-mediated linezolid resistance in
Staphylococcus aureus
and
Staphylococcus epidermidis
recovered from human infection cases monitored during the 2007 LEADER Program.
Apoptosis Induced by Aminoglycosides in LLC-PK1 Cells: Comparative Study of Neomycin, Gentamicin, Amikacin, and Isepamicin Using Electroporation
Authors: Denamur Sophie , Van Bambeke Françoise , Mingeot-Leclercq Marie-Paule , Tulkens Paul M.
Published: April 2008

Levels of apoptosis induction (4′,6′-diamidino-2-phenylindole staining, activation of caspase 3) for aminoglycosides were compared by using renal LLC-PK1 cells. Amikacin caused less apoptosis than gentamicin in incubated cells. In electroporated cells, neomycin B and gentamicin caused apoptosis in the 0.03 to 0.1 mM range, isepamicin required larger concentrations (0.2 mM), and amikacin was without effect.
Characterization of Replication and Conjugation of
Streptomyces
Circular Plasmids pFP1 and pFP11 and Their Ability To Propagate in Linear Mode with Artificially Attached Telomeres
Authors: Zhang Ran , Zeng Ana , Fang Ping , Qin Zhongjun
Published: April 2008

Many
Streptomyces
species harbor circular plasmids (8 to 31 kb) as well as linear plasmids (12 to 1,700 kb). We report the characterization of two newly detected circular plasmids, pFP11 (35,139 bp) and pFP1 (39,360 bp). As on linear plasmids, their replication loci comprise
repA
genes and adjacent iterons, to which RepA proteins bind specifically in vitro. Plasmids containing the minimal iterons plus the
repA
locus of pFP11 were inherited extremely unstably;
par
and additional loci were required for stable inheritance. Surprisingly, plasmids containing replication loci from pFP11 or
Streptomyces
circular plasmid SCP2 but not from pFP1, SLP1, or pIJ101 propagated in a stable linear mode when the telomeres of a linear plasmid were attached. These results indicate bidirectional replication for pFP11 and SCP2. Both pFP11 and pFP1 contain, for plasmid transfer, a major functional
traB
gene (encoding a DNA translocase typical for
Streptomyces
plasmids) as well as, surprisingly, a putative
traA
gene (encoding a DNA nickase, characteristic of single-stranded DNA transfer of gram-negative plasmids), but this did not appear to be functional, at least in isolation.
Transcriptional Analysis of
l
-Methionine Catabolism in the Cheese-Ripening Yeast
Yarrowia lipolytica
in Relation to Volatile Sulfur Compound Biosynthesis
Authors: Cholet Orianne , Hénaut Alain , Hébert Agnès , Bonnarme Pascal
Published: April 2008

Yarrowia lipolytica
is one of the yeasts most frequently isolated from the surface of ripened cheeses. In previous work, it has been shown that this yeast is able to convert
l
-methionine into various volatile sulfur compounds (VSCs) that may contribute to the typical flavors of several cheeses. In the present study, we show that
Y. lipolytica
does not assimilate lactate in the presence of
l
-methionine in a cheeselike medium. Nineteen presumptive genes associated with
l
-methionine catabolism or pyruvate metabolism in
Y. lipolytica
were transcriptionally studied in relation to
l
-methionine degradation. The expression levels of the Yl
ARO8
(YALI0E20977g), Yl
BAT1
(YALI0D01265g), and Yl
BAT2
(YALI0F19910g) genes (confirmed by real-time PCR experiments) were found to be strongly up-regulated by
l
-methionine, and a greater variety and larger amounts of VSCs, such as methanethiol and its autooxidation products (dimethyl disulfide and dimethyl trisulfide), were released in the medium when
Y. lipolytica
was grown in the presence of a high concentration of
l
-methionine. In contrast, other genes related to pyruvate metabolism were found to be down-regulated in the presence of
l
-methionine; two exceptions were the Yl
PDB1
(YALI0E27005g) and Yl
PDC6
(YALI0D06930g) genes, which encode a pyruvate dehydrogenase and a pyruvate decarboxylase, respectively. Both transcriptional and biochemical results corroborate the view that transamination is the first step of the enzymatic conversion of
l
-methionine to VSCs in
Y. lipolytica
and that the Yl
ARO8
, Yl
BAT1
, and Yl
BAT2
genes could play a key role in this process.
Inactivation of
Bacillus anthracis
Spores by a Combination of Biocides and Heating under High-Temperature Short-Time Pasteurization Conditions
Authors: Xu Sa , Labuza Theodore P. , Diez-Gonzalez Francisco
Published: April 2008

The milk supply is considered a primary route for a bioterrorism attack with
Bacillus anthracis
spores because typical high-temperature short-time (HTST) pasteurization conditions cannot inactivate spores. In the event of intentional contamination, an effective method to inactivate the spores in milk under HTST processing conditions is needed. This study was undertaken to identify combinations and concentrations of biocides that can inactivate
B. anthracis
spores at temperatures in the HTST range in less than 1 min. Hydrogen peroxide (HP), sodium hypochlorite (SH), and peroxyacetic acid (PA) were evaluated for their efficacy in inactivating spores of strains 7702, ANR-1, and 9131 in milk at 72, 80, and 85°C using a sealed capillary tube technique. Strains ANR-1 and 9131 were more resistant to all of the biocide treatments than strain 7702. Addition of 1,260 ppm SH to milk reduced the number of viable spores of each strain by 6 log CFU/ml in less than 90 and 60 s at 72 and 80°C, respectively. After neutralization, 1,260 ppm SH reduced the time necessary to inactivate 6 log CFU/ml (TTI6-log) at 80°C to less than 20 s. Treatment of milk with 7,000 ppm HP resulted in a similar level of inactivation in 60 s. Combined treatment with 1,260 ppm SH and 1,800 ppm HP inactivated spores of all strains in less than 20 s at 80°C. Mixing 15 ppm PA with milk containing 1,260 ppm SH resulted in TTI6-log of 25 and 12 s at 72 and 80°C, respectively. TTI6-log of less than 20 s were also achieved at 80°C by using two combinations of biocides: 250 ppm SH, 700 ppm HP, and 150 ppm PA; and 420 ppm SH (pH 7), 1,100 ppm HP, and 15 ppm PA. These results indicated that different combinations of biocides could consistently result in 6-log reductions in the number of
B. anthracis
spores in less than 1 min at temperatures in the HTST range. This information could be useful for developing more effective thermal treatment strategies which could be used in HTST milk plants to process contaminated milk for disposal and decontamination, as well as for potential protective measures.
Characterization and Genomic Analysis of Phage asccφ28, a Phage of the Family
Podoviridae
Infecting
Lactococcus lactis
Authors: Kotsonis Steven E. , Powell Ian B. , Pillidge Christopher J. , Limsowtin Gaëtan K. Y. , Hillier Alan J. , Davidson Barrie E.
Published: April 2008

Bacteriophage asccφ28 infects dairy fermentation strains of
Lactococcus lactis
. This report describes characterization of asccφ28 and its full genome sequence. Phage asccφ28 has a prolate head, whiskers, and a short tail (C2 morphotype). This morphology and DNA hybridization to
L. lactis
phage P369 DNA showed that asccφ28 belongs to the P034 phage species, a group rarely encountered in the dairy industry. The burst size of asccφ28 was found to be 121 ± 18 PFU per infected bacterial cell after a latent period of 44 min. The linear genome (18,762 bp) contains 28 possible open reading frames (ORFs) comprising 90% of the total genome. The ORFs are arranged bidirectionally in recognizable functional modules. The genome contains 577 bp inverted terminal repeats (ITRs) and putatively eight promoters and four terminators. The presence of ITRs, a phage-encoded DNA polymerase, and a terminal protein that binds to the DNA, along with BLAST and morphology data, show that asccφ28 more closely resembles streptococcal phage Cp-1 and the φ29-like phages that infect
Bacillus subtilis
than it resembles common lactococcal phages. The sequence of this phage is the first published sequence of a P034 species phage genome.
Factors Controlling the Distribution of Archaeal Tetraethers in Terrestrial Hot Springs
Authors: Pearson Ann , Pi Yundan , Zhao Weidong , Li WenJun , Li Yiliang , Inskeep William , Perevalova Anna , Romanek Christopher , Li Shuguang , Zhang Chuanlun L.
Published: April 2008

Glycerol dialkyl glycerol tetraethers (GDGTs) found in hot springs reflect the abundance and community structure of
Archaea
in these extreme environments. The relationships between GDGTs, archaeal communities, and physical or geochemical variables are underexamined to date and when reported often result in conflicting interpretations. Here, we examined profiles of GDGTs from pure cultures of
Crenarchaeota
and from terrestrial geothermal springs representing a wide distribution of locations, including Yellowstone National Park (United States), the Great Basin of Nevada and California (United States), Kamchatka (Russia), Tengchong thermal field (China), and Thailand. These samples had temperatures of 36.5 to 87°C and pH values of 3.0 to 9.2. GDGT abundances also were determined for three soil samples adjacent to some of the hot springs. Principal component analysis identified four factors that accounted for most of the variance among nine individual GDGTs, temperature, and pH. Significant correlations were observed between pH and the GDGTs crenarchaeol and GDGT-4 (four cyclopentane rings,
m
/
z
1,294); pH correlated positively with crenarchaeol and inversely with GDGT-4. Weaker correlations were observed between temperature and the four factors. Three of the four GDGTs used in the marine TEX
86
paleotemperature index (GDGT-1 to -3, but not crenarchaeol isomer) were associated with a single factor. No correlation was observed for GDGT-0 (acyclic caldarchaeol): it is effectively its own variable. The biosynthetic mechanisms and exact archaeal community structures leading to these relationships remain unknown. However, the data in general show promise for the continued development of GDGT lipid-based physiochemical proxies for archaeal evolution and for paleo-ecology or paleoclimate studies.
Comparison of
Campylobacter
Populations in Wild Geese with Those in Starlings and Free-Range Poultry on the Same Farm
Authors: Colles F. M. , Dingle K. E. , Cody A. J. , Maiden M. C. J.
Published: April 2008

Wild geese are a potential source of
Campylobacter
infection for humans and farm animals and have been implicated in at least two large waterborne disease outbreaks. There have been few investigations into the population biology of
Campylobacter
in geese, carriage rates are reported to vary (0 to 100%), and no genetic characterization of isolates has been performed. Fecal samples collected from wild geese in Oxfordshire, United Kingdom, were culture positive for
C. jejuni
(50.2%) and
C. coli
(0.3%). The
C. jejuni
(
n
= 166) isolates were characterized by using multilocus sequence typing and were compared with isolates collected from free-range broiler chickens and wild starlings sampled at the same location. A total of 38 STs, six clonal complexes, and 23
flaA
SVR nucleotide STs were identified. The ST-21 and ST-45 complexes (5.4% of isolates) were the only complexes to be identified among isolates from the geese and the other bird species sampled in the same location. These clonal complexes were also identified among human disease isolates collected in the same health care region. The results indicate that large numbers of wild geese carry
Campylobacter
; however, there was limited mixing of
Campylobacter
populations among the different sources examined, and the host source could be predicted with high probability from the allelic profile of a
C. jejuni
isolate. In conclusion, genotypes of
C. jejuni
isolated from geese are highly host specific, and a comparison with isolates from Oxfordshire cases of human disease revealed that while geese cannot be excluded as a source of infection for humans and farm animals, their contribution is likely to be minor.
Bayesian-Integrated Microbial Forensics
Authors: Jarman Kristin H. , Kreuzer-Martin Helen W. , Wunschel David S. , Valentine Nancy B. , Cliff John B. , Petersen Catherine E. , Colburn Heather A. , Wahl Karen L.
Published: April 2008

In the aftermath of the 2001 anthrax letters, researchers have been exploring ways to predict the production environment of unknown-source microorganisms. Culture medium, presence of agar, culturing temperature, and drying method are just some of the broad spectrum of characteristics an investigator might like to infer. The effects of many of these factors on microorganisms are not well understood, but the complex way in which microbes interact with their environments suggests that numerous analytical techniques measuring different properties will eventually be needed for complete characterization. In this work, we present a Bayesian statistical framework for integrating disparate analytical measurements. We illustrate its application to the problem of characterizing the culture medium of
Bacillus
spores using three different mass spectral techniques. The results of our study suggest that integrating data in this way significantly improves the accuracy and robustness of the analyses.
High-Yield Production of Secreted Active Proteins by the
Pseudomonas aeruginosa
Type III Secretion System
Authors: Derouazi M. , Toussaint B. , Quénée L. , Epaulard O. , Guillaume M. , Marlu R. , Polack B.
Published: April 2008

The
Escherichia coli
system is the system of choice for recombinant protein production because it is possible to obtain a high protein yield in inexpensive media. The accumulation of protein in an insoluble form in inclusion bodies remains a major disadvantage. Use of the
Pseudomonas aeruginosa
type III secretion system can avoid this problem, allowing the production of soluble secreted proteins.
Characterization of Endogenous Plasmids from
Lactobacillus salivarius
UCC118
Authors: Fang Fang , Flynn Sarah , Li Yin , Claesson Marcus J. , van Pijkeren Jan-Peter , Collins J. Kevin , van Sinderen Douwe , O'Toole Paul W.
Published: April 2008

The genome of
Lactobacillus salivarius
UCC118 comprises a 1.83-Mb chromosome, a 242-kb megaplasmid (pMP118), and two smaller plasmids of 20 kb (pSF118-20) and 44 kb (pSF118-44). Annotation and bioinformatic analyses suggest that both of the smaller plasmids replicate by a theta replication mechanism. Furthermore, it appears that they are transmissible, although neither possesses a complete set of conjugation genes. Plasmid pSF118-20 encodes a toxin-antitoxin system composed of
pemI
and
pemK
homologs, and this plasmid could be cured when PemI was produced in
trans
. The minimal replicon of pSF118-20 was determined by deletion analysis. Shuttle vector derivatives of pSF118-20 were generated that included the replication region (pLS203) and the replication region plus mobilization genes (pLS208). The plasmid pLS203 was stably maintained without selection in
Lactobacillus plantarum
,
Lactobacillus fermentum
, and the pSF118-20-cured derivative strain of
L. salivarius
UCC118 (strain LS201). Cloning in pLS203 of genes encoding luciferase and green fluorescent protein, and expression from a constitutive
L. salivarius
promoter, demonstrated the utility of this vector for the expression of heterologous genes in
Lactobacillus
. This study thus expands the knowledge base and vector repertoire of probiotic lactobacilli.
Improvement of the Glutaryl-7-Aminocephalosporanic Acid Acylase Activity of a Bacterial γ-Glutamyltranspeptidase
Authors: Yamada Chiaki , Kijima Kyoko , Ishihara Sayaka , Miwa Chinatsu , Wada Kei , Okada Toshihiro , Fukuyama Keiichi , Kumagai Hidehiko , Suzuki Hideyuki
Published: April 2008

7-Aminocephalosporanic acid (7-ACA) is an important material in the production of semisynthetic cephalosporins, which are the best-selling antibiotics worldwide. 7-ACA is produced from cephalosporin C via glutaryl-7-ACA (GL-7-ACA) by a bioconversion process usingd-amino acid oxidase and cephalosporin acylase (or GL-7-ACA acylase). Previous studies demonstrated that a single amino acid substitution, D433N, provided GL-7-ACA acylase activity for γ-glutamyltranspeptidase (GGT) ofEscherichia coliK-12. In this study, based on its three-dimensional structure, residues involved in substrate recognition ofE. coliGGT were rationally mutagenized, and effective mutations were then combined. A novel screening method, activity staining followed by a GL-7-ACA acylase assay with whole cells, was developed, and it enabled us to obtain mutant enzymes with enhanced GL-7-ACA acylase activity. The best mutant enzyme for catalytic efficiency, with akcat/Kmvalue for GL-7-ACA almost 50-fold higher than that of the D433N enzyme, has three amino acid substitutions: D433N, Y444A, and G484A. We also suggest that GGT fromBacillus subtilis168 can be another source of GL-7-ACA acylase for industrial applications.
Complete Genome Sequence of
Nitrosospira multiformis
, an Ammonia-Oxidizing Bacterium from the Soil Environment
Authors: Norton Jeanette M. , Klotz Martin G. , Stein Lisa Y. , Arp Daniel J. , Bottomley Peter J. , Chain Patrick S. G. , Hauser Loren J. , Land Miriam L. , Larimer Frank W. , Shin Maria W. , Starkenburg Shawn R.
Published: April 2008

The complete genome of the ammonia-oxidizing bacterium
Nitrosospira multiformis
(ATCC 25196
T
) consists of a circular chromosome and three small plasmids totaling 3,234,309 bp and encoding 2,827 putative proteins. Of the 2,827 putative proteins, 2,026 proteins have predicted functions and 801 are without conserved functional domains, yet 747 of these have similarity to other predicted proteins in databases. Gene homologs from
Nitrosomonas europaea
and
Nitrosomonas eutropha
were the best match for 42% of the predicted genes in
N. multiformis
. The
N. multiformis
genome contains three nearly identical copies of
amo
and
hao
gene clusters as large repeats. The features of
N. multiformis
that distinguish it from
N. europaea
include the presence of gene clusters encoding urease and hydrogenase, a ribulose-bisphosphate carboxylase/oxygenase-encoding operon of distinctive structure and phylogeny, and a relatively small complement of genes related to Fe acquisition. Systems for synthesis of a pyoverdine-like siderophore and for acyl-homoserine lactone were unique to
N. multiformis
among the sequenced genomes of ammonia-oxidizing bacteria. Gene clusters encoding proteins associated with outer membrane and cell envelope functions, including transporters, porins, exopolysaccharide synthesis, capsule formation, and protein sorting/export, were abundant. Numerous sensory transduction and response regulator gene systems directed toward sensing of the extracellular environment are described. Gene clusters for glycogen, polyphosphate, and cyanophycin storage and utilization were identified, providing mechanisms for meeting energy requirements under substrate-limited conditions. The genome of
N. multiformis
encodes the core pathways for chemolithoautotrophy along with adaptations for surface growth and survival in soil environments.
Dynamics of
Legionella
spp. and Bacterial Populations during the Proliferation of
L. pneumophila
in a Cooling Tower Facility
Authors: Wéry Nathalie , Bru-Adan Valérie , Minervini Céline , Delgénes Jean-Philippe , Garrelly Laurent , Godon Jean-Jacques
Published: April 2008

The dynamics of
Legionella
spp. and of dominant bacteria were investigated in water from a cooling tower plant over a 9-month period which included several weeks when
Legionella pneumophila
proliferated. The structural diversity of both the bacteria and the
Legionella
spp. was monitored by a fingerprint technique, single-strand conformation polymorphism, and
Legionella
spp. and
L. pneumophila
were quantified by real-time quantitative PCR. The structure of the bacterial community did not change over time, but it was perturbed periodically by chemical treatment or biofilm detachment. In contrast, the structure of the
Legionella
sp. population changed in different periods, its dynamics at times showing stability but also a rapid major shift during the proliferation of
L. pneumophila
in July. The dynamics of the
Legionella
spp. and of dominant bacteria were not correlated. In particular, no change in the bacterial community structure was observed during the proliferation of
L. pneumophila. Legionella
spp. present in the cooling tower system were identified by cloning and sequencing of 16S rRNA genes. A high diversity of
Legionella
spp. was observed before proliferation, including
L. lytica, L. fallonii
, and other
Legionella
-like amoebal pathogen types, along with as-yet-undescribed species. During the proliferation of
L. pneumophila, Legionella
sp. diversity decreased significantly,
L. fallonii
and
L. pneumophila
being the main species recovered.
Evolutionary Relationships of “
Candidatus
Endobugula” Bacterial Symbionts and Their
Bugula
Bryozoan Hosts
Authors: Lim-Fong Grace E. , Regali Lindsay A. , Haygood Margo G.
Published: April 2008

Ribosomal gene sequences were obtained from bryozoans in the genus
Bugula
and their bacterial symbionts; analyses of host and symbiont phylogenetic trees did not support a history of strict cospeciation. Symbiont-derived compounds known to defend host larvae from predation were only detected in two out of four symbiotic
Bugula
species.
Characterization of the RND-Type Multidrug Efflux Pump MexAB-OprM of the Plant Pathogen
Pseudomonas syringae
Authors: Stoitsova Savina O. , Braun Yvonne , Ullrich Matthias S. , Weingart Helge
Published: April 2008

In gram-negative bacteria, transporters belonging to the RND family are the transporters most relevant for resistance to antimicrobial compounds. In
Pseudomonas aeruginosa
, a clinically important pathogen, the RND-type pump MexAB-OprM has been recognized as one of the major multidrug efflux systems. Here, homologues of MexAB-OprM in the plant pathogens
Pseudomonas syringae
pv. phaseolicola 1448A,
P. syringae
pv. syringae B728a, and
P. syringae
pv. tomato DC3000 were identified, and
mexAB-oprM
-deficient mutants were generated. Determination of MICs revealed that mutation of MexAB-OprM dramatically reduced the tolerance to a broad range of antimicrobials. Moreover, the ability of the
mexAB
-
oprM
-deficient mutants to multiply in planta was reduced. RNA dot blot hybridization revealed growth-dependent regulation of the
mexAB-oprM
operon in
P. syringae
; the expression of this operon was maximal in early exponential phase and decreased gradually during further growth.
Genomewide Location Analysis of
Candida albicans
Upc2p, a Regulator of Sterol Metabolism and Azole Drug Resistance
Authors: Znaidi Sadri , Weber Sandra , Zin Al-Abdin Osman , Bomme Perrine , Saidane Saloua , Drouin Simon , Lemieux Sébastien , De Deken Xavier , Robert François , Raymond Martine
Published: April 2008

Upc2p, a transcription factor of the zinc cluster family, is an important regulator of sterol biosynthesis and azole drug resistance in
Candida albicans
. To better understand Upc2p function in
C. albicans
, we used genomewide location profiling to identify the transcriptional targets of Upc2p in vivo. A triple hemagglutinin epitope, introduced at the C terminus of Upc2p, conferred a gain-of-function effect on the fusion protein. Location profiling identified 202 bound promoters (
P
< 0.05). Overrepresented functional groups of genes whose promoters were bound by Upc2p included 12 genes involved in ergosterol biosynthesis (
NCP1
,
ERG11
,
ERG2
, and others), 18 genes encoding ribosomal subunits (
RPS30
,
RPL32
,
RPL12
, and others), 3 genes encoding drug transporters (
CDR1
,
MDR1
, and
YOR1
), 4 genes encoding transcription factors (
INO2
,
ACE2
,
SUT1
, and
UPC2
), and 6 genes involved in sulfur amino acid metabolism (
MET6
,
SAM2
,
SAH1
, and others). Bioinformatic analyses suggested that Upc2p binds to the DNA motif 5′-VNCGBDTR that includes the previously characterized Upc2p binding site 5′-TCGTATA. Northern blot analysis showed that increased binding correlates with increased expression for the analyzed Upc2p targets (
ERG11
,
MDR1
,
CDR1
,
YOR1
,
SUT1
,
SMF12
, and
CBP1
). The analysis of
ERG11
,
MDR1
, and
CDR1
transcripts in wild-type and
upc2
Δ/
upc2
Δ strains grown under Upc2p-activating conditions (lovastatin treatment and hypoxia) showed that Upc2p regulates its targets in a complex manner, acting as an activator or as a repressor depending upon the target and the activating condition. Taken together, our results indicate that Upc2p is a key regulator of ergosterol metabolism. They also suggest that Upc2p may contribute to azole resistance by regulating the expression of drug efflux pump-encoding genes in addition to ergosterol biosynthesis genes.
The Nuclear Migration Protein NUDF/LIS1 Forms a Complex with NUDC and BNFA at Spindle Pole Bodies
Authors: Helmstaedt Kerstin , Laubinger Karen , Voßkuhl Katja , Bayram Özgür , Busch Silke , Hoppert Michael , Valerius Oliver , Seiler Stephan , Braus Gerhard H.
Published: April 2008

Nuclear migration depends on microtubules, the dynein motor complex, and regulatory components like LIS1 and NUDC. We sought to identify new binding partners of the fungal LIS1 homolog NUDF to clarify its function in dynein regulation. We therefore analyzed the association between NUDF and NUDC inAspergillus nidulans. NUDF and NUDC directly interacted in yeast two-hybrid experiments via NUDF's WD40 domain. NUDC-green fluorescent protein (NUDC-GFP) was localized to immobile dots in the cytoplasm and at the hyphal cortex, some of which were spindle pole bodies (SPBs). We showed by bimolecular fluorescence complementation microscopy that NUDC directly interacted with NUDF at SPBs at different stages of the cell cycle. Applying tandem affinity purification, we isolated the NUDF-associated protein BNFA (forbinding toNUDF). BNFA was dispensable for growth and for nuclear migration. GFP-BNFA fusions localized to SPBs at different stages of the cell cycle. This localization depended on NUDF, since the loss of NUDF resulted in the cytoplasmic accumulation of BNFA. BNFA did not bind to NUDC in a yeast two-hybrid assay. These results show that the conserved NUDF and NUDC proteins play a concerted role at SPBs at different stages of the cell cycle and that NUDF recruits additional proteins specifically to the dynein complex at SPBs.
Molecular Signals in the Trafficking of
Toxoplasma gondii
Protein MIC3 to the Micronemes
Authors: El Hajj Hiba , Papoin Julien , Cérède Odile , Garcia-Réguet Nathalie , Soête Martine , Dubremetz Jean-François , Lebrun Maryse
Published: April 2008

The protozoan parasite
Toxoplasma gondii
is equipped with a sophisticated secretory apparatus, including three distinct exocytic organelles, named micronemes, rhoptries, and dense granules. We have dissected the requirements for targeting the microneme protein MIC3, a key component of
T. gondii
infection. We have shown that MIC3 is processed in a post-Golgi compartment and that the MIC3 propeptide and epidermal growth factor (EGF) modules contain microneme-targeting information. The minimal requirement for microneme delivery is defined by the propeptide plus any one of the three EGF domains. We have demonstrated that the cleavage of the propeptide, the dimerization of MIC3, and the chitin binding-like sequence, which are crucial for host cell binding and virulence, are dispensable for proper targeting. Finally, we have shown that part of MIC3 is withheld in the secretory pathway in a cell cycle-dependent manner.
Photoregulation of Cellular Morphology during Complementary Chromatic Adaptation Requires Sensor-Kinase-Class Protein RcaE in
Fremyella diplosiphon
Authors: Bordowitz Juliana R. , Montgomery Beronda L.
Published: April 2008

We used wild-type UTEX481; SF33, a shortened-filament mutant strain that shows normal complementary chromatic adaptation pigmentation responses; and FdBk14, an RcaE-deficient strain that lacks light-dependent pigmentation responses, to investigate the molecular basis of the photoregulation of cellular morphology in the cyanobacterium
Fremyella diplosiphon
. Detailed microscopic and biochemical analyses indicate that RcaE is required for the photoregulation of cell and filament morphologies of
F. diplosiphon
in response to red and green light.
Mobilization of the
incQ
Plasmid R300B with a Chromosomal Conjugation System in
Salmonella enterica
Serovar Typhi
Authors: Baker Stephen , Pickard Derek , Whitehead Sally , Farrar Jeremy , Dougan Gordon
Published: April 2008

Salmonella
pathogenicity island 7 (SPI-7) in
Salmonella enterica
serovar Typhi appears to be related to other genomic islands. Evidence suggests that SPI-7 is susceptible to spontaneous circularization, loss, and transposition. Here, we demonstrate that a region within SPI-7 has the ability to mobilize the small
incQ
plasmid R300B.
Norepinephrine Mediates Acquisition of Transferrin-Iron in
Bordetella bronchiseptica
Authors: Anderson Mark T. , Armstrong Sandra K.
Published: April 2008

Previous research demonstrated that the sympathoadrenal catecholamine norepinephrine could promote the growth of
Bordetella bronchiseptica
in iron-restricted medium containing serum. In this study, norepinephrine was demonstrated to stimulate growth of this organism in the presence of partially iron-saturated transferrin but not lactoferrin. Although norepinephrine is known to induce transcription of the
Bordetella bfeA
enterobactin catechol xenosiderophore receptor gene, neither a
bfeA
mutant nor a
bfeR
regulator mutant was defective in growth responsiveness to norepinephrine. However, growth of a
tonB
mutant strain was not enhanced by norepinephrine, indicating that the response to this catecholamine was the result of high-affinity outer membrane transport. The
B. bronchiseptica
genome encodes a total of 19 known and predicted iron transport receptor genes, none of which, when mutated individually, were found to confer a defect in norepinephrine-mediated growth stimulation in the presence of transferrin. Labeling experiments demonstrated a TonB-dependent increase in cell-associated iron levels when bacteria grown in the presence of
55
Fe-transferrin were exposed to norepinephrine. In addition, TonB was required for maximum levels of cell-associated norepinephrine. Together, these results demonstrate that norepinephrine facilitates
B. bronchiseptica
iron acquisition from the iron carrier protein transferrin and this process may represent a mechanism by which some bacterial pathogens obtain this essential nutrient in the host environment.
The
Xenorhabdus nematophila nilABC
Genes Confer the Ability of
Xenorhabdus
spp. To Colonize
Steinernema carpocapsae
Nematodes
Authors: Cowles Charles E. , Goodrich-Blair Heidi
Published: April 2008

Members of the
Steinernema
genus of nematodes are colonized mutualistically by members of the
Xenorhabdus
genus of bacteria. In nature,
Steinernema carpocapsae
nematodes are always found in association with
Xenorhabdus nematophila
bacteria. Thus, this interaction, like many microbe-host associations, appears to be species specific.
X. nematophila
requires the
nilA
,
nilB
, and
nilC
genes to colonize
S. carpocapsae
. In this work, we showed that of all the
Xenorhabdus
species examined, only
X. nematophila
has the
nilA
,
nilB
, and
nilC
genes. By exposing
S. carpocapsae
to other
Xenorhabdus
spp., we established that only
X. nematophila
is able to colonize
S. carpocapsae
; therefore, the
S. carpocapsae-X. nematophila
interaction is species specific. Further, we showed that introduction of the
nilA
,
nilB
, and
nilC
genes into other
Xenorhabdus
species enables them to colonize the same
S. carpocapsae
host tissue that is normally colonized by
X. nematophila
. Finally, sequence analysis supported the idea that the
nil
genes were horizontally acquired. Our findings indicate that a single genetic locus determines host specificity in this bacteria-animal mutualism and that host range expansion can occur through the acquisition of a small genetic element.
Pseudomonas aeruginosa
Twitching Motility-Mediated Chemotaxis towards Phospholipids and Fatty Acids: Specificity and Metabolic Requirements
Authors: Miller Rhea M. , Tomaras Andrew P. , Barker Adam P. , Voelker Dennis R. , Chan Edward D. , Vasil Adriana I. , Vasil Michael L.
Published: April 2008

Pseudomonas aeruginosa
demonstrates type IV pilus-mediated directional twitching motility up a gradient of phosphatidylethanolamine (PE). Only one of four extracellular phospholipases C of
P. aeruginosa
(i.e., PlcB), while not required for twitching motility per se, is required for twitching-mediated migration up a gradient of PE or phosphatidylcholine. Whether other lipid metabolism genes are associated with this behavior was assessed by analysis of transcription during twitching up a PE gradient in comparison to transcription during twitching in the absence of any externally applied phospholipid. Data support the hypothesis that PE is further degraded and that the long-chain fatty acid (LCFA) moieties of PE are completely metabolized via β-oxidation and the glyoxylate shunt. It was discovered that
P. aeruginosa
exhibits twitching-mediated chemotaxis toward unsaturated LCFAs (e.g., oleic acid), but not saturated LCFAs (e.g., stearic acid) of corresponding lengths. Analysis of mutants that are deficient in glyoxylate shunt enzymes, specifically isocitrate lyase (Δ
aceA
) and malate synthase (Δ
aceB
), suggested that the complete metabolism of LCFAs through this pathway was required for the migration of
P. aeruginosa
up a gradient of PE or unsaturated LCFAs. At this point, our data suggested that this process should be classified as energy taxis. However, further evaluation of the ability of the Δ
aceA
and Δ
aceB
mutants to migrate up a gradient of PE or unsaturated LCFAs in the presence of an alternative energy source clearly indicated that metabolism of LCFAs for energy is not required for chemotaxis toward these compounds.
Insight from TonB Hybrid Proteins into the Mechanism of Iron Transport through the Outer Membrane
Authors: Kaserer Wallace A. , Jiang Xiaoxu , Xiao Qiaobin , Scott Daniel C. , Bauler Matthew , Copeland Daniel , Newton Salete M. C. , Klebba Phillip E.
Published: April 2008

We created hybrid proteins to study the functions of TonB. We first fused the portion ofEscherichia coli tonBthat encodes the C-terminal 69 amino acids (amino acids 170 to 239) of TonB downstream fromE. coli malE(MalE-TonB69C). Production of MalE-TonB69C intonB+bacteria inhibited siderophore transport. After overexpression and purification of the fusion protein on an amylose column, we proteolytically released the TonB C terminus and characterized it. Fluorescence spectra positioned its sole tryptophan (W213) in a weakly polar site in the protein interior, shielded from quenchers. Affinity chromatography showed the binding of the TonB C-domain to other proteins: immobilized TonB-dependent (FepA and colicin B) and TonB-independent (FepAΔ3-17, OmpA, and lysozyme) proteins adsorbed MalE-TonB69C, revealing a general affinity of the C terminus for other proteins. Additional constructions fused full-length TonB upstream or downstream of green fluorescent protein (GFP). TonB-GFP constructs had partial functionality but no fluorescence; GFP-TonB fusion proteins were functional and fluorescent. The activity of the latter constructs, which localized GFP in the cytoplasm and TonB in the cell envelope, indicate that the TonB N terminus remains in the inner membrane during its biological function. Finally, sequence analyses revealed homology in the TonB C terminus toE. coliYcfS, a proline-rich protein that contains the lysin (LysM) peptidoglycan-binding motif. LysM structural mimicry occurs in two positions of the dimeric TonB C-domain, and experiments confirmed that it physically binds to the murein sacculus. Together, these findings infer that the TonB N terminus remains associated with the inner membrane, while the downstream region bridges the cell envelope from the affinity of the C terminus for peptidoglycan. This architecture suggests a membrane surveillance model of action, in which TonB finds occupied receptor proteins by surveying the underside of peptidoglycan-associated outer membrane proteins.
Cyclic AMP in Mycobacteria: Characterization and Functional Role of the Rv1647 Ortholog in
Mycobacterium smegmatis
Authors: Dass Bob Kennedy M. , Sharma Ritu , Shenoy Avinash R. , Mattoo Rohini , Visweswariah Sandhya S.
Published: April 2008

Mycobacterial genomes are endowed with many eukaryote-like nucleotide cyclase genes encoding proteins that can synthesize 3′,5′-cyclic AMP (cAMP). However, the roles of cAMP and the need for such redundancy in terms of adenylyl cyclase genes remain unknown. We measured cAMP levels in
Mycobacterium smegmatis
during growth and under various stress conditions and report the first biochemical and functional characterization of the MSMEG_3780 adenylyl cyclase, whose orthologs in
Mycobacterium tuberculosis
(Rv1647) and
Mycobacterium leprae
(ML1399) have been recently characterized in vitro. MSMEG_3780 was important for producing cAMP levels in the logarithmic phase of growth, since the ΔMSMEG_3780 strain showed lower intracellular cAMP levels at this stage of growth. cAMP levels decreased in wild-type
M. smegmatis
under conditions of acid stress but not in the ΔMSMEG_3780 strain. This was correlated with a reduction in MSMEG_3780 promoter activity, indicating that the effect of the reduction in cAMP levels on acid stress was caused by a decrease in the transcription of MSMEG_3780. Complementation of the ΔMSMEG_3780 strain with the genomic integration of MSMEG_3780 or the Rv1647 gene could restore cAMP levels during logarithmic growth. The Rv1647 promoter was also acid sensitive, emphasizing the biochemical and functional similarities in these two adenylyl cyclases. This study therefore represents the first detailed biochemical and functional analysis of an adenylyl cyclase that is important for maintaining cAMP levels in mycobacteria and underscores the subtle roles that these genes may play in the physiology of the organism.
Characterization of a Novel Methanol Dehydrogenase in Representatives of
Burkholderiales
: Implications for Environmental Detection of Methylotrophy and Evidence for Convergent Evolution
Authors: Kalyuzhnaya Marina G. , Hristova Krassimira R. , Lidstrom Mary E. , Chistoserdova Ludmila
Published: April 2008

Some members of
Burkholderiales
are able to grow on methanol but lack the genes (
mxaFI
) responsible for the well-characterized two-subunit pyrroloquinoline quinone-dependent quinoprotein methanol dehydrogenase that is widespread in methylotrophic
Proteobacteria
. Here, we characterized novel, mono-subunit enzymes responsible for methanol oxidation in four strains,
Methyloversatilis universalis
FAM5,
Methylibium petroleiphilum
PM1, and unclassified
Burkholderiales
strains RZ18-153 and FAM1. The enzyme from
M. universalis
FAM5 was partially purified and subjected to matrix-assisted laser desorption ionization-time of fight peptide mass fingerprinting. The resulting peptide spectrum was used to identify a gene candidate in the genome of
M. petroleiphilum
PM1 (
mdh2
) predicted to encode a type I alcohol dehydrogenase related to the characterized methanol dehydrogenase large subunits but at less than 35% amino acid identity. Homologs of
mdh2
were amplified from
M. universalis
FAM5 and strains RZ18-153 and FAM1, and mutants lacking
mdh2
were generated in three of the organisms. These mutants lost their ability to grow on methanol and ethanol, demonstrating that
mdh2
is responsible for oxidation of both substrates. Our findings have implications for environmental detection of methylotrophy and indicate that this ability is widespread beyond populations possessing
mxaF
, the gene traditionally used as a genetic marker for environmental detection of methanol-oxidizing capability. Our findings also have implications for understanding the evolution of methanol oxidation, suggesting a convergence toward the enzymatic function for methanol oxidation in MxaF and Mdh2-type proteins.