bluuebunny/crossref_metadata_2025_split huggingface dataset:
Enhanced Inactivation of Salmonella and Pseudomonas Biofilms on Stainless Steel by Use of T-128, a Fresh-Produce Washing Aid, in Chlorinated Wash Solutions
Authors: Shen Cangliang , Luo Yaguang , Nou Xiangwu , Bauchan Gary , Zhou Bin , Wang Qin , Millner Patricia 
Published: June 2012

ABSTRACT

The effect of the washing aid T-128 (generally recognized as safe [GRAS] formulation, composed mainly of phosphoric acid and propylene glycol) on inactivation of
Salmonella
and
Pseudomonas
populations in biofilms on stainless steel was evaluated under conditions of increasing organic matter loads in chlorinated wash solutions dominated by hypochlorous acid. Biofilms were formed statically on stainless steel coupons suspended in 2% lettuce extract after inoculation with
Salmonella enterica
serovar Thompson or Newport or with
Pseudomonas fluorescens
. Coupons with biofilms were washed in chlorine solutions (0, 0.5, 1, 2, 5, 10, or 20 mg/liter at pH 6.5, 5.0 and 2.9), with or without T-128, and with increasing loads of organic matter (0, 0.25, 0.5, 0.75, or 1.0% lettuce extract). Cell populations on coupons were dispersed using intermittent, pulsed ultrasonication and vortexing and enumerated by colony counts on XLT-4 or
Pseudomonas
agars. Cell responses to fluorescent viability staining of biofilm treatment washing solutions were examined using confocal laser scanning microscopy. Results showed that 0.1% T-128 (without chlorine) reduced
P. fluorescens
biofilm populations by 2.5 log
10
units but did not reduce
Salmonella
populations. For both
Salmonella
and
Pseudomonas
, the sanitizing effect of free chlorine (1.0 to 5.0 mg/liter) was enhanced (
P
< 0.05) when it was combined with T-128. Application of T-128 decreased the free chlorine depletion rate caused by increasing organic matter in wash waters and significantly (
P
< 0.05) augmented inactivation of bacteria in biofilms compared to treatments without T-128. Image analysis of surfaces stained with SYTO and propidium iodide corroborate the cultural assay results showing that T-128 can aid in reducing pathogen viability in biofilms and thus can aid in sanitizing stainless steel contact surfaces during processing of fresh-cut produce.
Inactivation of Chloramphenicol and Florfenicol by a Novel Chloramphenicol Hydrolase
Authors: Tao Weixin , Lee Myung Hwan , Wu Jing , Kim Nam Hee , Kim Jin-Cheol , Chung Eunsook , Hwang Eul Chul , Lee Seon-Woo 
Published: June 2012

ABSTRACT

Chloramphenicol and florfenicol are broad-spectrum antibiotics. Although the bacterial resistance mechanisms to these antibiotics have been well documented, hydrolysis of these antibiotics has not been reported in detail. This study reports the hydrolysis of these two antibiotics by a specific hydrolase that is encoded by a gene identified from a soil metagenome. Hydrolysis of chloramphenicol has been recognized in cell extracts of
Escherichia coli
expressing a chloramphenicol acetate esterase gene,
estDL136
. A hydrolysate of chloramphenicol was identified as
p
-nitrophenylserinol by liquid chromatography-mass spectroscopy and proton nuclear magnetic resonance spectroscopy. The hydrolysis of these antibiotics suggested a promiscuous amidase activity of EstDL136. When
estDL136
was expressed in
E. coli
, EstDL136 conferred resistance to both chloramphenicol and florfenicol on
E. coli
, due to their inactivation. In addition,
E. coli
carrying
estDL136
deactivated florfenicol faster than it deactivated chloramphenicol, suggesting that EstDL136 hydrolyzes florfenicol more efficiently than it hydrolyzes chloramphenicol. The nucleotide sequences flanking
estDL136
encode proteins such as amidohydrolase, dehydrogenase/reductase, major facilitator transporter, esterase, and oxidase. The most closely related genes are found in the bacterial family
Sphingomonadaceae
, which contains many bioremediation-related strains. Whether the gene cluster with
estDL136
in
E. coli
is involved in further chloramphenicol degradation was not clear in this study. While acetyltransferases for chloramphenicol resistance and drug exporters for chloramphenicol or florfenicol resistance are often detected in numerous microbes, this is the first report of enzymatic hydrolysis of florfenicol resulting in inactivation of the antibiotic.
Protection of Bacillus pumilus Spores by Catalases
Authors: Checinska Aleksandra , Burbank Malcolm , Paszczynski Andrzej J. 
Published: June 2012

ABSTRACT

Bacillus pumilus
SAFR-032, isolated at spacecraft assembly facilities of the National Aeronautics and Space Administration Jet Propulsion Laboratory, is difficult to kill by the sterilization method of choice, which uses liquid or vapor hydrogen peroxide. We identified two manganese catalases, YjqC and BPUM_1305, in spore protein extracts of several
B. pumilus
strains by using PAGE and mass spectrometric analyses. While the BPUM_1305 catalase was present in six of the
B. pumilus
strains tested, YjqC was not detected in ATCC 7061 and BG-B79. Furthermore, both catalases were localized in the spore coat layer along with laccase and superoxide dismutase. Although the initial catalase activity in ATCC 7061 spores was higher, it was less stable over time than the SAFR-032 enzyme. We propose that synergistic activity of YjqC and BPUM_1305, along with other coat oxidoreductases, contributes to the enhanced resistance of
B. pumilus
spores to hydrogen peroxide. We observed that the product of the catalase reaction, gaseous oxygen, forms expanding vesicles on the spore surface, affecting the mechanical integrity of the coat layer, resulting in aggregation of the spores. The accumulation of oxygen gas and aggregations may play a crucial role in limiting further exposure of
Bacilli
spore surfaces to hydrogen peroxide or other toxic chemicals when water is present.
Internalization of Sapovirus, a Surrogate for Norovirus, in Romaine Lettuce and the Effect of Lettuce Latex on Virus Infectivity
Authors: Esseili Malak A. , Wang Qiuhong , Zhang Zhenwen , Saif Linda J. 
Published: June 2012

ABSTRACT

Noroviruses are the leading cause of food-borne outbreaks, including those that involve lettuce. The culturable porcine sapovirus (SaV) was used as a norovirus surrogate to study the persistence and the potential transfer of the virus from roots to leaves and from outer to inner leaves of lettuce plants. Treatment of lettuce with SaV was done through the roots of young plants, the soil, or the outer leaves of mature plants. Sampling of roots, xylem sap, and inner and outer leaves followed by RNA extraction and SaV-specific real-time reverse transcription (RT)-PCR was performed at 2 h and on postinoculation days (PID) 2, 5, 7, 14, and/or 28. When SaV was inoculated through the roots, viral RNA persisted on the roots and in the leaves until PID 28. When the virus was inoculated through the soil, viral RNA was detected on the roots and in the xylem sap until PID 14; viral RNA was detected in the leaves only until PID 2. No infectious virus was detected inside the leaves for either treatment. When SaV was inoculated through the outer leaves, viral RNA persisted on the leaves until PID 14; however, the virus did not transfer to inner leaves. Infectious viral particles on leaves were detected only at 2 h postinoculation. The milky sap (latex) of leaves, but not the roots' xylem sap, significantly decreased virus infectivity when tested
in vitro
. Collectively, our results showed the transfer of SaV from roots to leaves through the xylem system and the capacity of the sap of lettuce leaves to decrease virus infectivity in leaves.
Epimerase (Msed_0639) and Mutase (Msed_0638 and Msed_2055) Convert (
S
)-Methylmalonyl-Coenzyme A (CoA) to Succinyl-CoA in the Metallosphaera sedula 3-Hydroxypropionate/4-Hydroxybutyrate Cycle
Authors: Han Yejun , Hawkins Aaron S. , Adams Michael W. W. , Kelly Robert M. 
Published: June 2012

ABSTRACT

Crenarchaeotal genomes encode the 3-hydroxypropionate/4-hydroxybutyrate (3-HP/4-HB) cycle for carbon dioxide fixation. Of the 13 enzymes putatively comprising the cycle, several of them, including methylmalonyl-coenzyme A (CoA) epimerase (MCE) and methylmalonyl-CoA mutase (MCM), which convert (
S
)-methylmalonyl-CoA to succinyl-CoA, have not been confirmed and characterized biochemically. In the genome of
Metallosphaera sedula
(optimal temperature [
T
opt
], 73°C), the gene encoding MCE (Msed_0639) is adjacent to that encoding the catalytic subunit of MCM-α (Msed_0638), while the gene for the coenzyme B
12
-binding subunit of MCM (MCM-β) is located remotely (Msed_2055). The expression of all three genes was significantly upregulated under autotrophic compared to heterotrophic growth conditions, implying a role in CO
2
fixation. Recombinant forms of MCE and MCM were produced in
Escherichia coli
; soluble, active MCM was produced only if MCM-α and MCM-β were coexpressed. MCE is a homodimer and MCM is a heterotetramer (α
2
β
2
) with specific activities of 218 and 2.2 μmol/min/mg, respectively, at 75°C. The heterotetrameric MCM differs from the homo- or heterodimeric orthologs in other organisms. MCE was activated by divalent cations (Ni
2+
, Co
2+
, and Mg
2+
), and the predicted metal binding/active sites were identified through sequence alignments with less-thermophilic MCEs. The conserved coenzyme B
12
-binding motif (
DXHXXG
-SXL-GG) was identified in
M. sedula
MCM-β. The two enzymes together catalyzed the two-step conversion of (
S
)-methylmalonyl-CoA to succinyl-CoA, consistent with their proposed role in the 3-HP/4-HB cycle. Based on the highly conserved occurrence of single copies of MCE and MCM in
Sulfolobaceae
genomes, the
M. sedula
enzymes are likely to be representatives of these enzymes in the 3-HP/4-HB cycle in crenarchaeal thermoacidophiles.
Peculiar H
+
Homeostasis of Saccharomyces cerevisiae during the Late Stages of Wine Fermentation
Authors: Viana Tiago , Loureiro-Dias Maria C. , Loureiro Virgílio , Prista Catarina 
Published: June 2012

ABSTRACT

Intracellular pH (pH
in
) is a tightly regulated physiological parameter, which controls cell performance in all living systems. The purpose of this work was to evaluate if and how H
+
homeostasis is accomplished by an industrial wine strain of
Saccharomyces cerevisiae
while fermenting real must under the harsh winery conditions prevalent in the late stages of the fermentation process, in particular low pH and high ethanol concentrations and temperature. Cells grown at 15, 25, and 30°C were harvested in exponential and early and late stationary phases. Intracellular pH remained in the range of 6.0 to 6.4, decreasing significantly only by the end of glucose fermentation, in particular at lower temperatures (pH
in
5.2 at 15°C), although the cells remained viable and metabolically active. The cell capability of extruding H
+
via H
+
-ATPase and of keeping H
+
out by means of an impermeable membrane were evaluated as potential mechanisms of H
+
homeostasis. At 30°C, H
+
efflux was higher in all stages. The most striking observation was that cells in late stationary phase became almost impermeable to H
+
. Even when these cells were challenged with high ethanol concentrations (up to 20%) added in the assay, their permeability to H
+
remained very low, being almost undetectable at 15°C. Comparatively, ethanol significantly increased the H
+
permeability of cells in exponential phase. Understanding the molecular and physiological events underlying yeast H
+
homeostasis at late stages of fermentations may contribute to the development of more robust strains suitable to efficiently produce a high-quality wine.
Synthesis of Short-Chain Diols and Unsaturated Alcohols from Secondary Alcohol Substrates by the Rieske Nonheme Mononuclear Iron Oxygenase MdpJ
Authors: Schäfer Franziska , Schuster Judith , Würz Birgit , Härtig Claus , Harms Hauke , Müller Roland H. , Rohwerder Thore 
Published: June 2012

ABSTRACT

The Rieske nonheme mononuclear iron oxygenase MdpJ of the fuel oxygenate-degrading bacterial strain
Aquincola tertiaricarbonis
L108 has been described to attack short-chain tertiary alcohols via hydroxylation and desaturation reactions. Here, we demonstrate that also short-chain secondary alcohols can be transformed by MdpJ. Wild-type cells of strain L108 converted 2-propanol and 2-butanol to 1,2-propanediol and 3-buten-2-ol, respectively, whereas an
mdpJ
knockout mutant did not show such activity. In addition, wild-type cells converted 3-methyl-2-butanol and 3-pentanol to the corresponding desaturation products 3-methyl-3-buten-2-ol and 1-penten-3-ol, respectively. The enzymatic hydroxylation of 2-propanol resulted in an enantiomeric excess of about 70% for the (
R
)-enantiomer, indicating that this reaction was favored. Likewise, desaturation of (
R
)-2-butanol to 3-buten-2-ol was about 2.3-fold faster than conversion of the (
S
)-enantiomer. The biotechnological potential of MdpJ for the synthesis of enantiopure short-chain alcohols and diols as building block chemicals is discussed.
Molecular Survey of the Occurrence of Legionella spp., Mycobacterium spp., Pseudomonas aeruginosa, and Amoeba Hosts in Two Chloraminated Drinking Water Distribution Systems
Authors: Wang Hong , Edwards Marc , Falkinham Joseph O. , Pruden Amy 
Published: June 2012

ABSTRACT

The spread of opportunistic pathogens via public water systems is of growing concern. The purpose of this study was to identify patterns of occurrence among three opportunistic pathogens (
Legionella pneumophila
,
Mycobacterium avium
, and
Pseudomonas aeruginosa
) relative to biotic and abiotic factors in two representative chloraminated drinking water distribution systems using culture-independent methods. Generally, a high occurrence of
Legionella
(≥69.0%) and mycobacteria (100%), lower occurrence of
L. pneumophila
(≤20%) and
M. avium
(≤33.3%), and rare detection of
Pseudomonas aeruginosa
(≤13.3%) were observed in both systems according to quantitative PCR. Also,
Hartmanella vermiformis
was more prevalent than
Acanthamoeba
, both of which are known hosts for opportunistic pathogen amplification, the latter itself containing pathogenic members. Three-minute flushing served to distinguish distribution system water from plumbing in buildings (i.e., premise plumbing water) and resulted in reduced numbers of copies of
Legionella
, mycobacteria,
H. vermiformis
, and 16S rRNA genes (
P
< 0.05) while yielding distinct terminal restriction fragment polymorphism (T-RFLP) profiles of 16S rRNA genes. Within certain subgroups of samples, some positive correlations, including correlations of numbers of mycobacteria and total bacteria (16S rRNA genes),
H. vermiformis
and total bacteria, mycobacteria and
H. vermiformis
, and
Legionella
and
H. vermiformis
, were noted, emphasizing potential microbial ecological relationships. Overall, the results provide insight into factors that may aid in controlling opportunistic pathogen proliferation in real-world water systems.
Membrane Vesicle Formation as a Multiple-Stress Response Mechanism Enhances Pseudomonas putida DOT-T1E Cell Surface Hydrophobicity and Biofilm Formation
Authors: Baumgarten Thomas , Sperling Stefanie , Seifert Jana , von Bergen Martin , Steiniger Frank , Wick Lukas Y. , Heipieper Hermann J. 
Published: June 2012

ABSTRACT

Among the adaptive responses of bacteria to rapid changes in environmental conditions, those of the cell envelope are known to be the most crucial. Therefore, several mechanisms with which bacteria change their cell surface and membranes in the presence of different environmental stresses have been elucidated. Among these mechanisms, the release of outer membrane vesicles (MV) in Gram-negative bacteria has attracted particular research interest because of its involvement in pathogenic processes, such as that of
Pseudomonas aeruginosa
biofilm formation in cystic fibrosis lungs. In this study, we investigated the role of MV formation as an adaptive response of
Pseudomonas putida
DOT-T1E to several environmental stress factors and correlated it to the formation of biofilms. In the presence of toxic concentrations of long-chain alcohols, under osmotic stress caused by NaCl, in the presence of EDTA, and after heat shock, cells of this strain released MV within 10 min in the presence of a stressor. The MV formed showed similar size and charge properties, as well as comparable compositions of proteins and fatty acids. MV release caused a significant increase in cell surface hydrophobicity, and an enhanced tendency to form biofilms was demonstrated in this study. Therefore, the release of MV as a stress response could be put in a physiological context.
Structural Insights into the Regulatory Mechanism of the Response Regulator RocR from Pseudomonas aeruginosa in Cyclic Di-GMP Signaling
Authors: Chen Ming Wei , Kotaka Masayo , Vonrhein Clemens , Bricogne Gérard , Rao Feng , Chuah Mary Lay Cheng , Svergun Dmitri , Schneider Gunter , Liang Zhao-Xun , Lescar Julien 
Published: June 2012

ABSTRACTThe nucleotide messenger cyclic di-GMP (c-di-GMP) plays a central role in the regulation of motility, virulence, and biofilm formation in many pathogenic bacteria. EAL domain-containing phosphodiesterases are the major signaling proteins responsible for the degradation of c-di-GMP and maintenance of its cellular level. We determined the crystal structure of a single mutant (R286W) of the response regulator RocR fromPseudomonas aeruginosato show that RocR exhibits a highly unusual tetrameric structure arranged around a single dyad, with the four subunits adopting two distinctly different conformations. Subunits A and B adopt a conformation with the REC domain located above the c-di-GMP binding pocket, whereas subunits C and D adopt an open conformation with the REC domain swung to the side of the EAL domain. Remarkably, the access to the substrate-binding pockets of the EAL domains of the open subunits C and D are blocked intransby the REC domains of subunits A and B, indicating that only two of the four active sites are engaged in the degradation of c-di-GMP. In conjunction with biochemical and biophysical data, we propose that the structural changes within the REC domains triggered by the phosphorylation are transmitted to the EAL domain active sites through a pathway that traverses the dimerization interfaces composed of a conserved regulatory loop and the neighboring motifs. This exquisite mechanism reinforces the crucial role of the regulatory loop and suggests that similar regulatory mechanisms may be operational in many EAL domain proteins, considering the preservation of the dimerization interface and the spatial arrangement of the regulatory domains.
Pseudomonas aeruginosa Directly Shunts β-Oxidation Degradation Intermediates into
De Novo
Fatty Acid Biosynthesis
Authors: Yuan Yanqiu , Leeds Jennifer A. , Meredith Timothy C. 
Published: June 2012

ABSTRACT

We identified the fatty acid synthesis (FAS) initiation enzyme in
Pseudomonas aeruginosa
as FabY, a β-ketoacyl synthase KASI/II domain-containing enzyme that condenses acetyl coenzyme A (acetyl-CoA) with malonyl-acyl carrier protein (ACP) to make the FAS primer β-acetoacetyl-ACP in the
accompanying article
(Y. Yuan, M. Sachdeva, J. A. Leeds, and T. C. Meredith, J. Bacteriol. 194:5171-5184, 2012). Herein, we show that growth defects stemming from deletion of
fabY
can be suppressed by supplementation of the growth media with exogenous decanoate fatty acid, suggesting a compensatory mechanism. Fatty acids eight carbons or longer rescue growth by generating acyl coenzyme A (acyl-CoA) thioester β-oxidation degradation intermediates that are shunted into FAS downstream of FabY. Using a set of perdeuterated fatty acid feeding experiments, we show that the open reading frame PA3286 in
P. aeruginosa
PAO1 intercepts C
8
-CoA by condensation with malonyl-ACP to make the FAS intermediate β-keto decanoyl-ACP. This key intermediate can then be extended to supply all of the cellular fatty acid needs, including both unsaturated and saturated fatty acids, along with the 3-hydroxyl fatty acid acyl groups of lipopolysaccharide. Heterologous PA3286 expression in
Escherichia coli
likewise established the fatty acid shunt, and characterization of recombinant β-keto acyl synthase enzyme activity confirmed
in vitro
substrate specificity for medium-chain-length acyl CoA thioester acceptors. The potential for the PA3286 shunt in
P. aeruginosa
to curtail the efficacy of inhibitors targeting FabY, an enzyme required for FAS initiation in the absence of exogenous fatty acids, is discussed.
The Escherichia coli SMC Complex, MukBEF, Shapes Nucleoid Organization Independently of DNA Replication
Authors: Badrinarayanan Anjana , Lesterlin Christian , Reyes-Lamothe Rodrigo , Sherratt David 
Published: June 2012

ABSTRACTSMC (structuralmaintenance ofchromosomes) complexes function ubiquitously in organizing and maintaining chromosomes. Functional fluorescent derivatives of theEscherichia coliSMC complex, MukBEF, form foci that associate with the replication origin region (ori). MukBEF impairment results in mispositioning oforiand other loci in steady-state cells. These observations led to an earlier proposal that MukBEF positions new replicated sisteroris. We show here that MukBEF generates and maintains the cellular positioning of chromosome loci independently of DNA replication. Rapid impairment of MukBEF function by depleting a Muk component in the absence of DNA replication leads to loss of MukBEF foci as well as mispositioning oforiand other loci, while rapid Muk synthesis leads to rapid MukBEF focus formation but slow restoration of normal chromosomal locus positioning.
Genomic Variation among Contemporary Pseudomonas aeruginosa Isolates from Chronically Infected Cystic Fibrosis Patients
Authors: Chung Jade C. S. , Becq Jennifer , Fraser Louise , Schulz-Trieglaff Ole , Bond Nicholas J. , Foweraker Juliet , Bruce Kenneth D. , Smith Geoffrey P. , Welch Martin 
Published: June 2012

ABSTRACTThe airways of individuals with cystic fibrosis (CF) often become chronically infected with unique strains of the opportunistic pathogenPseudomonas aeruginosa. Several lines of evidence suggest that the infectingP. aeruginosalineage diversifies in the CF lung niche, yet so far this contemporary diversity has not been investigated at a genomic level. In this work, we sequenced the genomes of pairs of randomly selected contemporary isolates sampled from the expectorated sputum of three chronically infected adult CF patients. Each patient was infected by a distinct strain ofP. aeruginosa. Single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) were identified in the DNA common to the paired isolates from different patients. The paired isolates from one patient differed due to just 1 SNP and 8 indels. The paired isolates from a second patient differed due to 54 SNPs and 38 indels. The pair of isolates from the third patient both contained amutSmutation, which conferred a hypermutator phenotype; these isolates cumulatively differed due to 344 SNPs and 93 indels. In two of the pairs of isolates, a different accessory genome composition, specifically integrated prophage, was identified in one but not the other isolate of each pair. We conclude that contemporary isolates from a single sputum sample can differ at the SNP, indel, and accessory genome levels and that the cross-sectional genomic variation among coeval pairs ofP. aeruginosaCF isolates can be comparable to the variation previously reported to differentiate between paired longitudinally sampled isolates.