bluuebunny/crossref_metadata_2025_split huggingface dataset:
Increased Thermal and Osmotic Stress Resistance in Listeria monocytogenes 568 Grown in the Presence of Trehalose Due to Inactivation of the Phosphotrehalase-Encoding Gene
treA
Authors: Ells Timothy C. , Truelstrup Hansen Lisbeth 
Published: August 2011

ABSTRACT

The food-borne pathogen
Listeria monocytogenes
is a problem for food processors and consumers alike, as the organism is resistant to harsh environmental conditions and inimical barriers implemented to prevent the survival and/or growth of harmful bacteria. One mechanism by which listeriae mediate survival is through the accumulation of compatible solutes, such as proline, betaine and carnitine. In other bacteria, including
Escherichia coli
, the synthesis and accumulation of another compatible solute, trehalose, are known to aid in the survival of stressed cells. The objective of this research was to investigate trehalose metabolism in
L. monocytogenes
, where the sugar is thought to be transferred across the cytoplasmic membrane via a specific phosphoenolpyruvate phosphotransferase system and phosphorylation to trehalose-6-phosphate (T6P). The latter is subsequently broken down into glucose and glucose-6-phosphate by α,α-(1,1) phosphotrehalase, the putative product of the
treA
gene. Here we report on an isogenic
treA
mutant of
L. monocytogenes
568 (568:ΔTreA) which, relative to the wild-type strain, displays increased tolerances to multiple stressors, including heat, high osmolarity, and desiccation. This is the first study to examine the putative trehalose operon in
L. monocytogenes
, and we demonstrate that
lmo1254
(
tre
A
) in
L. monocytogenes
568 indeed encodes a phosphotrehalase required for the hydrolysis of T6P. Disruption of the
treA
gene results in the accumulation of T6P which is subsequently dephosphorylated to trehalose in the cytosol, thereby contributing to the stress hardiness observed in the
treA
mutant. This study highlights the importance of compatible solutes for microbial survival in adverse environments.
Maturation of Released Spores Is Necessary for Acquisition of Full Spore Heat Resistance during Bacillus subtilis Sporulation
Authors: Sanchez-Salas Jose-Luis , Setlow Barbara , Zhang Pengfei , Li Yong-qing , Setlow Peter 
Published: August 2011

ABSTRACT

The first ∼10% of spores released from sporangia (early spores) during
Bacillus subtilis
sporulation were isolated, and their properties were compared to those of the total spores produced from the same culture. The early spores had significantly lower resistance to wet heat and hypochlorite than the total spores but identical resistance to dry heat and UV radiation. Early and total spores also had the same levels of core water, dipicolinic acid, and Ca and germinated similarly with several nutrient germinants. The wet heat resistance of the early spores could be increased to that of total spores if early spores were incubated in conditioned sporulation medium for ∼24 h at 37°C (maturation), and some hypochlorite resistance was also restored. The maturation of early spores took place in pH 8 buffer with Ca
2+
but was blocked by EDTA; maturation was also seen with early spores of strains lacking the CotE protein or the coat-associated transglutaminase, both of which are needed for normal coat structure. Nonetheless, it appears to be most likely that it is changes in coat structure that are responsible for the increased resistance to wet heat and hypochlorite upon early spore maturation.
Acquired Antibiotic Resistance: Are We Born with It?
Authors: Zhang Lu , Kinkelaar Daniel , Huang Ying , Li Yingli , Li Xiaojing , Wang Hua H. 
Published: August 2011

ABSTRACT

The rapid emergence of antibiotic resistance (AR) is a major public health concern. Recent findings on the prevalence of food-borne antibiotic-resistant (ART) commensal bacteria in ready-to-consume food products suggested that daily food consumption likely serves as a major avenue for dissemination of ART bacteria from the food chain to human hosts. To properly assess the impact of various factors, including the food chain, on AR development in hosts, it is important to determine the baseline of ART bacteria in the human gastrointestinal (GI) tract. We thus examined the gut microbiota of 16 infant subjects, from the newborn stage to 1 year of age, who fed on breast milk and/or infant formula during the early stages of development and had no prior exposure to antibiotics. Predominant bacterial populations resistant to several antibiotics and multiple resistance genes were found in the infant GI tracts within the first week of age. Several ART population transitions were also observed in the absence of antibiotic exposure and dietary changes. Representative AR gene pools including
tet
(M),
ermB
,
sul2
, and
bla
TEM
were detected in infant subjects.
Enterococcus
spp.,
Staphylococcus
spp.,
Klebsiella
spp.,
Streptococcus
spp., and
Escherichia coli
/
Shigella
spp. were among the identified AR gene carriers. ART bacteria were not detected in the infant formula and infant foods examined, but small numbers of skin-associated ART bacteria were found in certain breast milk samples. The data suggest that the early development of AR in the human gut microbiota is independent of infants' exposure to antibiotics but is likely impacted by exposure to maternal and environmental microbes during and after delivery and that the ART population is significantly amplified within the host even in the absence of antibiotic selective pressure.
Lytic Myophage Abp53 Encodes Several Proteins Similar to Those Encoded by Host Acinetobacter baumannii and Phage phiKO2
Authors: Lee Chia-Ni , Tseng Tsai-Tien , Lin Juey-Wen , Fu Yung-Chieh , Weng Shu-Fen , Tseng Yi-Hsiung 
Published: August 2011

ABSTRACT

Acinetobacter baumannii
is an important Gram-negative opportunistic pathogen causing nosocomial infections. The emergence of multiple-drug-resistant
A. baumannii
isolates has increased in recent years. Directed toward phage therapy, a lytic phage of
A. baumannii
, designated Abp53, was isolated from a sputum sample in this study. Abp53 has an isometric head and a contractile tail with tail fibers (belonging to
Myoviridae
), a latent period of about 10 min, and a burst size of approximately 150 PFU per infected cell. Abp53 could completely lyse 27% of the
A. baumannii
isolates tested, which were all multiple drug resistant, but not other bacteria. Mg
2+
enhanced the adsorption and productivity of, and host lysis by, Abp53. Twenty Abp53 virion proteins were visualized in SDS-polyacrylamide gel electrophoresis, with a 47-kDa protein being the predicted major capsid protein. Abp53 has a double-stranded DNA genome of 95 kb. Sequence analyses of a 10-kb region revealed 8 open reading frames. Five of the encoded proteins, including 3 tail components and 2 hypothetical proteins, were similar to proteins encoded by
A. baumannii
strain ACICU. ORF1176 (one of the tail components, 1,176 amino acids [aa]), which is also similar to tail protein gp21 of
Klebsiella
phage phiKO2, contained repeated domains similar to those within the ACICU_02717 protein of
A. baumannii
ACICU and gp21. These findings suggest a common ancestry and horizontal gene transfer during evolution. As phages can expand the host range by domain duplication in tail fiber proteins, repeated domains in ORF1176 might have a similar significance in Abp53.
Cell Surface Attachment Structures Contribute to Biofilm Formation and Xylem Colonization by Erwinia amylovora
Authors: Koczan Jessica M. , Lenneman Bryan R. , McGrath Molly J. , Sundin George W. 
Published: August 2011

ABSTRACT

Biofilm formation plays a critical role in the pathogenesis of
Erwinia amylovora
and the systemic invasion of plant hosts. The functional role of the exopolysaccharides amylovoran and levan in pathogenesis and biofilm formation has been evaluated. However, the role of biofilm formation, independent of exopolysaccharide production, in pathogenesis and movement within plants has not been studied previously. Evaluation of the role of attachment in
E. amylovora
biofilm formation and virulence was examined through the analysis of deletion mutants lacking genes encoding structures postulated to function in attachment to surfaces or in cellular aggregation. The genes and gene clusters studied were selected based on
in silico
analyses. Microscopic analyses and quantitative assays demonstrated that attachment structures such as fimbriae and pili are involved in the attachment of
E. amylovora
to surfaces and are necessary for the production of mature biofilms. A time course assay indicated that type I fimbriae function earlier in attachment, while type IV pilus structures appear to function later in attachment. Our results indicate that multiple attachment structures are needed for mature biofilm formation and full virulence and that biofilm formation facilitates entry and is necessary for the buildup of large populations of
E. amylovora
cells in xylem tissue.
Modification of the Technical Properties of Lactobacillus johnsonii NCC 533 by Supplementing the Growth Medium with Unsaturated Fatty Acids
Authors: Muller J. A. , Ross R. P. , Sybesma W. F. H. , Fitzgerald G. F. , Stanton C. 
Published: August 2011

ABSTRACT

The aim of this study was to investigate the influence of supplementing growth medium with unsaturated fatty acids on the technical properties of the probiotic strain
Lactobacillus johnsonii
NCC 533, such as heat and acid tolerance, and inhibition of
Salmonella enterica
serovar Typhimurium infection. Our results showed that the membrane composition and morphology of
L. johnsonii
NCC 533 were significantly changed by supplementing a minimal
Lactobacillus
medium with oleic, linoleic, and linolenic acids. The ratio of saturated to unsaturated plus cyclic fatty acids in the bacterial membrane decreased by almost 2-fold when minimal medium was supplemented with unsaturated fatty acids (10 μg/ml). The subsequent acid and heat tolerance of
L. johnsonii
decreased by 6- and 20-fold when the strain was grown in the presence of linoleic and linolenic acids, respectively, compared with growth in oleic acid (all at 10 μg/ml). Following acid exposure, significantly higher (
P
< 0.05) oleic acid content was detected in the membrane when growth medium was supplemented with linoleic or linolenic acid, indicating that saturation of the membrane fatty acids occurred during acid stress. Cell integrity was determined in real time during stressed conditions using a fluorescent viability kit in combination with flow cytometric analysis. Following heat shock (at 62.5°C for 5 min),
L. johnsonii
was unable to form colonies; however, 60% of the bacteria showed no cell integrity loss, which could indicate that the elevated heat inactivated vital processes within the cell, rendering it incapable of replication. Furthermore,
L. johnsonii
grown in fatty acid-enriched minimal medium had different adhesion properties and caused a 2-fold decrease in
S. enterica
serovar Typhimurium UK1-lux invasion of HT-29 epithelial cells compared with bacteria grown in minimal medium alone. This could be related to changes in the hydrophobicity and fluidity of the membrane. Our study shows that technical properties underlying probiotic survivability can be affected by nutrient composition of the growth medium.
Overcoming Fluctuation and Leakage Problems in the Quantification of Intracellular 2-Oxoglutarate Levels in Escherichia coli
Authors: Yan Dalai , Lenz Peter , Hwa Terence 
Published: August 2011

ABSTRACT

2-Oxoglutarate is located at the junction between central carbon and nitrogen metabolism, serving as an intermediate for both. In nitrogen metabolism, 2-oxoglutarate acts as both a carbon skeletal carrier and an effector molecule. There have been only sporadic reports of its internal concentrations. Here we describe a sensitive and accurate method for determination of the 2-oxoglutarate pool concentration in
Escherichia coli
. The detection was based on fluorescence derivatization followed by reversed-phase high-pressure liquid chromatography separation. Two alternative cell sampling strategies, both of which were based on a fast filtration protocol, were sequentially developed to overcome both its fast metabolism and contamination from 2-oxoglutarate that leaks into the medium. We observed rapid changes in the 2-oxoglutarate pool concentration upon sudden depletion of nutrients: decreasing upon carbon depletion and increasing upon nitrogen depletion. The latter was studied in mutants lacking either of the two enzymes using 2-oxoglutarate as the carbon substrate for glutamate biosynthesis. The results suggest that flux restriction on either reaction greatly influences the internal 2-oxoglutarate level. Additional study indicates that KgtP, a 2-oxoglutarate proton symporter, functions to recover the leakage loss of 2-oxoglutarate. This recovery mechanism benefits the measurement of cellular 2-oxoglutarate level in practice by limiting contamination from 2-oxoglutarate leakage.
Whole-Genome Sequencing and Phenotypic Analysis of Bacillus subtilis Mutants following Evolution under Conditions of Relaxed Selection for Sporulation
Authors: Brown Christopher T. , Fishwick Laura K. , Chokshi Binna M. , Cuff Marissa A. , Jackson Jay M. , Oglesby Travis , Rioux Alison T. , Rodriguez Enrique , Stupp Gregory S. , Trupp Austin H. , Woollcombe-Clarke James S. , Wright Tracy N. , Zaragoza William J. , Drew Jennifer C. , Triplett Eric W. , Nicholson Wayne L. 
Published: August 2011

ABSTRACT

Little is known about how genetic variation at the nucleotide level contributes to competitive fitness within species. During a 6,000-generation study of
Bacillus subtilis
evolved under relaxed selection for sporulation, a new strain, designated WN716, emerged with significantly different colony and cell morphologies; loss of sporulation, competence, acetoin production, and motility; multiple auxotrophies; and increased competitive fitness (H. Maughan and W. L. Nicholson, Appl. Environ. Microbiol.
77:
4105–4118, 2011). The genome of WN716 was analyzed by OpGen optical mapping, whole-genome 454 pyrosequencing, and the CLC Genomics Workbench. No large chromosomal rearrangements were found; however, 34 single-nucleotide polymorphisms (SNPs) and +1 frameshifts were identified in WN716 that resulted in amino acid changes in coding sequences of annotated genes, and 11 SNPs were located in intergenic regions. Several classes of genes were affected, including biosynthetic pathways, sporulation, competence, and DNA repair. In several cases, attempts were made to link observed phenotypes of WN716 with the discovered mutations, with various degrees of success. For example, a +1 frameshift was identified at codon 13 of
sigW
, the product of which (SigW) controls a regulon of genes involved in resistance to bacteriocins and membrane-damaging antibiotics. Consistent with this finding, WN716 exhibited sensitivity to fosfomycin and to a bacteriocin produced by
B. subtilis
subsp.
spizizenii
and exhibited downregulation of SigW-dependent genes on a transcriptional microarray, consistent with WN716 carrying a knockout of
sigW
. The results suggest that propagation of
B. subtilis
for less than 2,000 generations in a nutrient-rich environment where sporulation is suppressed led to rapid initiation of genomic erosion.
Matrix-Assisted Laser Desorption Ionization (MALDI)-Time of Flight Mass Spectrometry- and MALDI Biotyper-Based Identification of Cultured Biphenyl-Metabolizing Bacteria from Contaminated Horseradish Rhizosphere Soil
Authors: Uhlik Ondrej , Strejcek Michal , Junkova Petra , Sanda Miloslav , Hroudova Miluse , Vlcek Cestmir , Mackova Martina , Macek Tomas 
Published: August 2011

ABSTRACT

Bacteria that are able to utilize biphenyl as a sole source of carbon were extracted and isolated from polychlorinated biphenyl (PCB)-contaminated soil vegetated by horseradish. Isolates were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The usage of MALDI Biotyper for the classification of isolates was evaluated and compared to 16S rRNA gene sequence analysis. A wide spectrum of bacteria was isolated, with
Arthrobacter
,
Serratia
,
Rhodococcus
, and
Rhizobium
being predominant.
Arthrobacter
isolates also represented the most diverse group. The use of MALDI Biotyper in many cases permitted the identification at the level of species, which was not achieved by 16S rRNA gene sequence analyses. However, some isolates had to be identified by 16S rRNA gene analyses if MALDI Biotyper-based identification was at the level of probable or not reliable identification, usually due to a lack of reference spectra included in the database. Overall, this study shows the possibility of using MALDI-TOF MS and MALDI Biotyper for the fast and relatively nonlaborious identification/classification of soil isolates. At the same time, it demonstrates the dominant role of employing 16S rRNA gene analyses for the identification of recently isolated strains that can later fill the gaps in the protein-based identification databases.
Identification of a Transcriptional Repressor Involved in Benzoate Metabolism in Geobacter bemidjiensis
Authors: Ueki Toshiyuki 
Published: August 2011

ABSTRACT

Subsurface environments contaminated with aromatic compounds can be remediated
in situ
by
Geobacter
species. A transcription factor that represses expression of
bamA
, a benzoate-inducible gene, in
Geobacter bemidjiensis
during growth with acetate was identified. It is likely that this repressor also regulates other genes involved in aromatic compound metabolism.
Bacterial Display and Screening of Posttranslationally Thioether-Stabilized Peptides
Authors: Bosma Tjibbe , Kuipers Anneke , Bulten Erna , de Vries Louwe , Rink Rick , Moll Gert N. 
Published: August 2011

ABSTRACT

A major hurdle in the application of therapeutic peptides is their rapid degradation by peptidases. Thioether bridges effectively protect therapeutic peptides against breakdown, thereby strongly increasing bioavailability, enabling oral and pulmonary delivery and potentially significantly optimizing the receptor interaction of selected variants. To efficiently select optimal variants, a library of DNA-coupled thioether-bridged peptides is highly desirable. Here, we present a unique cell surface display system of thioether-bridged peptides and successfully demonstrate highly selective screening. Peptides are posttranslationally modified by thioether bridge-installing enzymes in
Lactococcus lactis
, followed by export and sortase-mediated covalent coupling to the lactococcal cell wall. This allows the combinatorial optimization and selection of medically and economically highly important therapeutic peptides with strongly enhanced therapeutic potential.
High-Throughput Typing Method To Identify a Non-Outbreak-Involved Legionella pneumophila Strain Colonizing the Entire Water Supply System in the Town of Rennes, France
Authors: Sobral D. , Le Cann P. , Gerard A. , Jarraud S. , Lebeau B. , Loisy-Hamon F. , Vergnaud G. , Pourcel C. 
Published: August 2011

ABSTRACT

Two legionellosis outbreaks occurred in the city of Rennes, France, during the past decade, requiring in-depth monitoring of
Legionella pneumophila
in the water network and the cooling towers in the city. In order to characterize the resulting large collection of isolates, an automated low-cost typing method was developed. The multiplex capillary-based variable-number tandem repeat (VNTR) (multiple-locus VNTR analysis [MLVA]) assay requiring only one PCR amplification per isolate ensures a high level of discrimination and reduces hands-on and time requirements. In less than 2 days and using one 4-capillary apparatus, 217 environmental isolates collected between 2000 and 2009 and 5 clinical isolates obtained during outbreaks in 2000 and 2006 in Rennes were analyzed, and 15 different genotypes were identified. A large cluster of isolates with closely related genotypes and representing 77% of the population was composed exclusively of environmental isolates extracted from hot water supply systems. It was not responsible for the known Rennes epidemic cases, although strains showing a similar MLVA profile have regularly been involved in European outbreaks. The clinical isolates in Rennes had the same genotype as isolates contaminating a mall's cooling tower. This study further demonstrates that unknown environmental or genetic factors contribute to the pathogenicity of some strains. This work illustrates the potential of the high-throughput MLVA typing method to investigate the origin of legionellosis cases by allowing the systematic typing of any new isolate and inclusion of data in shared databases.
Chemoenzymatic Synthesis of
N
-Acetyl-
d
-Neuraminic Acid from
N
-Acetyl-
d
-Glucosamine by Using the Spore Surface-Displayed
N
-Acetyl-
d
-Neuraminic Acid Aldolase
Authors: Gao Chao , Xu Xiaoman , Zhang Xifeng , Che Bin , Ma Cuiqing , Qiu Jianhua , Tao Fei , Xu Ping 
Published: August 2011

ABSTRACT

Chemoenzymatic synthesis of
N
-acetyl-
d
-neuraminic acid from
N
-acetyl-
d
-glucosamine using the spore surface-displayed
N
-acetyl-
d
-neuraminic acid aldolase at a high concentration (53.9 g liter
−1
) was achieved in this study. Thus, displaying a target enzyme on the surface of spores might be an alternative for integration of biocatalytic conversion into chemical synthesis.
Initiation of Polyene Macrolide Biosynthesis: Interplay between Polyketide Synthase Domains and Modules as Revealed via Domain Swapping, Mutagenesis, and Heterologous Complementation
Authors: Heia Sondre , Borgos Sven E. F. , Sletta Håvard , Escudero Leticia , Seco Elena M. , Malpartida Francisco , Ellingsen Trond E. , Zotchev Sergey B. 
Published: August 2011

ABSTRACT

Polyene macrolides are important antibiotics used to treat fungal infections in humans. In this work, acyltransferase (AT) domain swaps, mutagenesis, and cross-complementation with heterologous polyketide synthase domain (PKS) loading modules were performed in order to facilitate production of new analogues of the polyene macrolide nystatin. Replacement of AT
0
in the nystatin PKS loading module NysA with the propionate-specific AT
1
from the nystatin PKS NysB, construction of hybrids between NysA and the loading module of rimocidin PKS RimA, and stepwise exchange of specific amino acids in the AT
0
domain by site-directed mutagenesis were accomplished. However, none of the NysA mutants constructed was able to initiate production of new nystatin analogues. Nevertheless, many NysA mutants and hybrids were functional, providing for different levels of nystatin biosynthesis. An interplay between certain residues in AT
0
and an active site residue in the ketosynthase (KS)-like domain of NysA in initiation of nystatin biosynthesis was revealed. Some hybrids between the NysA and RimA loading modules carrying the NysA AT
0
domain were able to prime rimocidin PKS with both acetate and butyrate units upon complementation of a
rimA
-deficient mutant of the rimocidin/CE-108 producer
Streptomyces diastaticus
. Expression of the PimS0 loading module from the pimaricin producer in the same host, however, resulted in production of CE-108 only. Taken together, these data indicate relaxed substrate specificity of NysA AT
0
domain, which is counteracted by a strict specificity of the first extender module KS domain in the nystatin PKS of
Streptomyces noursei
.