bluuebunny/crossref_metadata_2025_split huggingface dataset:
Improved Enumeration of Lactic Acid Bacteria in Mesophilic Dairy Starter Cultures by Using Multiplex Quantitative Real-Time PCR and Flow Cytometry-Fluorescence In Situ Hybridization
Authors: Friedrich Udo , Lenke Jan 
Published: June 2006

ABSTRACT

Nucleic acid-based assays were developed to enumerate members of the three taxa
Lactococcus lactis
subsp.
cremoris
,
L. lactis
subsp.
lactis
, and
Leuconostoc
spp. in mesophilic starter cultures. To our knowledge the present is the first study to present a multiplex quantitative PCR (qPCR) strategy for the relative enumeration of bacteria. The multiplex qPCR strategy was designed to quantify the target DNA simultaneously relative to total bacterial DNA. The assay has a high discriminatory power and resolves concentration changes as low as 1.3-fold. The methodology was compared with flow cytometric fluorescence in situ hybridization (FLOW-FISH) and 5-bromo-4-chloro-3-indolyl-β-
d
-galactopyranoside (X-Gal)-calcium citrate agar-based plate counting. For enumeration by FLOW-FISH, three new probes having the same specificity as the qPCR assay were designed and established. A combination with flow cytometry greatly reduced the time consumed compared to manual enumeration. Both qPCR and FLOW-FISH yielded similar community compositions for 10 complex starter cultures, with all detected subpopulations being highly significantly correlated (
P
< 0.001). Correlations between X-Gal-calcium citrate agar-based CFU and qPCR-derived counts were highly significant (
P
< 0.01 and
P
< 0.001, respectively) for the number of acidifiers versus
L. lactis
subsp.
cremoris
and for
Leuconostoc
spp. as quantified by the two techniques, respectively. This confirmed that most acidifiers in the studied PROBAT cultures are members of
L. lactis
subsp.
cremoris
. Quantitative real-time PCR and FLOW-FISH were found to be effective and accurate tools for the bacterial community analysis of complex starter cultures.
Tomato QM-Like Protein Protects
Saccharomyces cerevisiae
Cells against Oxidative Stress by Regulating Intracellular Proline Levels
Authors: Chen Changbin , Wanduragala Srimevan , Becker Donald F. , Dickman Martin B. 
Published: June 2006

ABSTRACT

Exogenous proline can protect cells of
Saccharomyces cerevisiae
from oxidative stress. We altered intracellular proline levels by overexpressing the proline dehydrogenase gene (
PUT1
) of
S. cerevisiae
. Put1p performs the first enzymatic step of proline degradation in
S. cerevisiae
. Overexpression of Put1p results in low proline levels and hypersensitivity to oxidants, such as hydrogen peroxide and paraquat. A
put1
-disrupted yeast mutant deficient in Put1p activity has increased protection from oxidative stress and increased proline levels. Following a conditional life/death screen in yeast, we identified a tomato (
Lycopersicon esculentum
) gene encoding a QM-like protein (tQM) and found that stable expression of
tQM
in the Put1p-overexpressing strain conferred protection against oxidative damage from H
2
O
2
, paraquat, and heat. This protection was correlated with reactive oxygen species (ROS) reduction and increased proline accumulation. A yeast two-hybrid system assay was used to show that tQM physically interacts with Put1p in yeast, suggesting that tQM is directly involved in modulating proline levels. tQM also can rescue yeast from the lethality mediated by the mammalian proapoptotic protein Bax, through the inhibition of ROS generation. Our results suggest that tQM is a component of various stress response pathways and may function in proline-mediated stress tolerance in plants.
Overproduction of Wild-Type and Bioengineered Derivatives of the Lantibiotic Lacticin 3147
Authors: Cotter Paul D. , Draper Lorraine A. , Lawton Elaine M. , McAuliffe Olivia , Hill Colin , Ross R. Paul 
Published: June 2006

ABSTRACT

Lacticin 3147 is a broad-spectrum two-peptide lantibiotic whose genetic determinants are located on two divergent operons on the lactococcal plasmid pMRC01. Here we introduce each of 14 subclones, containing different combinations of lacticin 3147 genes, into MG1363 (pMRC01) and determine that a number of them can facilitate overproduction of the lantibiotic. Based on these studies it is apparent that while the provision of additional copies of genes encoding the biosynthetic/production machinery and the regulator LtnR is a requirement for high-level overproduction, the presence of additional copies of the structural genes (i.e.,
ltnA1A2
) is not.
Potential of a 16S rRNA-Based Taxonomic Microarray for Analyzing the Rhizosphere Effects of Maize on
Agrobacterium
spp. and Bacterial Communities
Authors: Sanguin Hervé , Remenant Benoît , Dechesne Arnaud , Thioulouse Jean , Vogel Timothy M. , Nesme Xavier , Moënne-Loccoz Yvan , Grundmann Geneviève L. 
Published: June 2006

ABSTRACT

Bacterial diversity is central to ecosystem sustainability and soil biological function, for which the role of roots is important. The high-throughput analysis potential of taxonomic microarray should match the breadth of bacterial diversity. Here, the power of this technology was evidenced through methodological verifications and analysis of maize rhizosphere effect based on a 16S rRNA-based microarray developed from the prototype of H. Sanguin et al. (Environ. Microbiol. 8:289-307, 2006). The current probe set was composed of 170 probes (41 new probes in this work) that targeted essentially the
Proteobacteria
. Cloning and sequencing of 16S rRNA amplicons were carried out on maize rhizosphere and bulk soil DNA. All tested clones that had a perfect match with corresponding probes were positive in the hybridization experiment. The hierarchically nested probes were reliable, but the level of taxonomic identification was variable, depending on the probe set specificity. The comparison of experimental and theoretical hybridizations revealed 0.91% false positives and 0.81% false negatives. The microarray detection threshold was estimated at 0.03% of a given DNA type based on DNA spiking experiments. A comparison of the maize rhizosphere and bulk soil hybridization results showed a significant rhizosphere effect, with a higher predominance of
Agrobacterium
spp. in the rhizosphere, as well as a lower prevalence of
Acidobacteria
,
Bacteroidetes
,
Verrucomicrobia
, and
Planctomycetes
, a new taxon of interest in soil. In addition, well-known taxonomic groups such as
Sphingomonas
spp.,
Rhizobiaceae
, and
Actinobacteria
were identified in both microbial habitats with strong hybridization signals. The taxonomic microarray developed in the present study was able to discriminate and characterize bacterial community composition in related biological samples, offering extensive possibilities for systematic exploration of bacterial diversity in ecosystems.
Microbial Diversity Associated with Odor Modification for Production of Fertilizers from Chicken Litter
Authors: Enticknap Julie J. , Nonogaki Hirofumi , Place Allen R. , Hill Russell T. 
Published: June 2006

ABSTRACTLitter from the chicken industry can present several environmental challenges, including offensive odors and runoff into waterways leading to eutrophication. An economically viable solution to the disposal of waste from chicken houses is treatment to produce a natural, granulated fertilizer that can be commercially marketed for garden and commercial use. Odor of the final product is important in consumer acceptance, and an earthy odor is desirable. By understanding and manipulating the microbial processes occurring during this process, it may be possible to modify the odors produced. Geosmin and related volatiles produced by soil actinomycetes are responsible for earthy odors, and actinomycetes are likely to be present in the composting manure. Bacterial communities at each stage of the process were analyzed by culturing studies and denaturing gradient gel electrophoresis (DGGE). The processing steps changed the culturable bacterial community, but the total community was shown by DGGE to be stable throughout the process. A local agricultural soil was analyzed in parallel as a potential source of geosmin-producing actinomycetes. This agricultural soil had higher microbial diversity than the compost at both the culturable and the molecular levels. Actinomycete bacteria were isolated and analyzed by AromaTrax, a gas chromatography-olfactometry system. This system enables the odor production of individual isolates to be monitored, allowing for rational selection of strains for augmentation experiments to improve the odor of the final fertilizer product.
Production of Xylitol from
d
-Xylose by a Xylitol Dehydrogenase Gene-Disrupted Mutant of
Candida tropicalis
Authors: Ko Byoung Sam , Kim Jinmi , Kim Jung Hoe 
Published: June 2006

ABSTRACT

Xylitol dehydrogenase (XDH) is one of the key enzymes in
d
-xylose metabolism, catalyzing the oxidation of xylitol to
d
-xylulose. Two copies of the
XYL2
gene encoding XDH in the diploid yeast
Candida tropicalis
were sequentially disrupted using the Ura-blasting method. The
XYL2
-disrupted mutant, BSXDH-3, did not grow on a minimal medium containing
d
-xylose as a sole carbon source. An enzyme assay experiment indicated that BSXDH-3 lost apparently all XDH activity. Xylitol production by BSXDH-3 was evaluated using a xylitol fermentation medium with glucose as a cosubstrate. As glucose was found to be an insufficient cosubstrate, various carbon sources were screened for efficient cofactor regeneration, and glycerol was found to be the best cosubstrate. BSXDH-3 produced xylitol with a volumetric productivity of 3.23 g liter
−1
h
−1
, a specific productivity of 0.76 g g
−1
h
−1
, and a xylitol yield of 98%. This is the first report of gene disruption of
C. tropicalis
for enhancing the efficiency of xylitol production.
Molecular Epidemiology of Ceftiofur-Resistant
Escherichia coli
Isolates from Dairy Calves
Authors: Donaldson Sarah C. , Straley Beth A. , Hegde Narasimha V. , Sawant Ashish A. , DebRoy Chitrita , Jayarao Bhushan M. 
Published: June 2006

ABSTRACT

Healthy calves (
n
= 96, 1 to 9 weeks old) from a dairy herd in central Pennsylvania were examined each month over a five-month period for fecal shedding of ceftiofur-resistant gram-negative bacteria. Ceftiofur-resistant
Escherichia coli
isolates (
n
= 122) were characterized by antimicrobial resistance (disk diffusion and MIC), serotype, pulsed-field gel electrophoresis subtypes, beta-lactamase genes, and virulence genes. Antibiotic disk diffusion assays showed that the isolates were resistant to ampicillin (100%), ceftiofur (100%), chloramphenicol (94%), florfenicol (93%), gentamicin (89%), spectinomycin (72%), tetracycline (98%), ticarcillin (99%), and ticarcillin-clavulanic acid (99%). All isolates were multidrug resistant and displayed elevated MICs. The
E. coli
isolates belonged to 42 serotypes, of which O8:H25 was the predominant serotype (49.2%). Pulsed-field gel electrophoresis classified the
E. coli
isolates into 27 profiles. Cluster analysis showed that 77 isolates (63.1%) belonged to one unique group. The prevalence of pathogenic
E. coli
was low (8%). A total of 117 ceftiofur-resistant
E. coli
isolates (96%) possessed the
bla
CMY2
gene. Based on phenotypic and genotypic characterization, the ceftiofur-resistant
E. coli
isolates belonged to 59 clonal types. There was no significant relationship between calf age and clonal type. The findings of this study revealed that healthy dairy calves were rapidly colonized by antibiotic-resistant strains of
E. coli
shortly after birth. The high prevalence of multidrug-resistant nonpathogenic
E. coli
in calves could be a significant source of resistance genes to other bacteria that share the same environment.
Succession of Bacterial Communities during Early Plant Development: Transition from Seed to Root and Effect of Compost Amendment
Authors: Green Stefan J. , Inbar Ehud , Michel Frederick C. , Hadar Yitzhak , Minz Dror 
Published: June 2006

ABSTRACT

Compost amendments to soils and potting mixes are routinely applied to improve soil fertility and plant growth and health. These amendments, which contain high levels of organic matter and microbial cells, can influence microbial communities associated with plants grown in such soils. The purpose of this study was to follow the bacterial community compositions of seed and subsequent root surfaces in the presence and absence of compost in the potting mix. The bacterial community compositions of potting mixes, seed, and root surfaces sampled at three stages of plant growth were analyzed via general and newly developed
Bacteroidetes
-specific, PCR-denaturing gradient gel electrophoresis methodologies. These analyses revealed that seed surfaces were colonized primarily by populations detected in the initial potting mixes, many of which were not detected in subsequent root analyses. The most persistent bacterial populations detected in this study belonged to the genus
Chryseobacterium
(
Bacteroidetes
) and the family
Oxalobacteraceae
(
Betaproteobacteria
). The patterns of colonization by populations within these taxa differed significantly and may reflect differences in the physiology of these organisms. Overall, analyses of bacterial community composition revealed a surprising prevalence and diversity of
Bacteroidetes
in all treatments.
Diversity of Heteropolysaccharide-Producing Lactic Acid Bacterium Strains and Their Biopolymers
Authors: Mozzi Fernanda , Vaningelgem Frederik , Hébert Elvira María , Van der Meulen Roel , Foulquié Moreno María Remedios , Font de Valdez Graciela , De Vuyst Luc 
Published: June 2006

ABSTRACT

Thirty-one lactic acid bacterial strains from different species were evaluated for exopolysaccharide (EPS) production in milk. Thermophilic strains produced more EPS than mesophilic ones, but EPS yields were generally low. Ropiness or capsular polysaccharide formation was strain dependent. Six strains produced high-molecular-mass EPS. Polymers were classified into nine groups on the basis of their monomer composition. EPS from
Enterococcus
strains were isolated and characterized.
Genotypic Diversity of
Haemophilus parasuis
Field Strains
Authors: Olvera A. , Calsamiglia M. , Aragon V. 
Published: June 2006

ABSTRACT

Haemophilus
parasuis
is the cause of Glässer's disease and other
clinical disorders in pigs. It can also be isolated from the upper
respiratory tracts of healthy pigs, and isolates can have significant
differences in virulence. In this work, a partial sequence from the
60-kDa heat shock protein (Hsp60) gene was assessed as an
epidemiological marker. We analyzed partial sequences of
hsp60
and 16S rRNA genes from 103 strains of
H. parasuis
and other
related species to obtain a better classification of the strains and
examine the correlation with virulence. The results were compared with
those obtained by enterobacterial repetitive intergenic consensus PCR.
Our results showed that
hsp60
is a reliable marker for
epidemiological studies of
H. parasuis
and that the analysis
of its sequence is a better approach than fingerprinting methods.
Furthermore, the analysis of the
hsp60
and 16S rRNA gene
sequences revealed the presence of a separate lineage of virulent
strains and indicated the occurrence of lateral gene transfer among
H. parasuis
and
Actinobacillus
strains.
Dynamics and Characterization of Refractory Dissolved Organic Matter Produced by a Pure Bacterial Culture in an Experimental Predator-Prey System
Authors: Gruber David F. , Simjouw Jean-Paul , Seitzinger Sybil P. , Taghon Gary L. 
Published: June 2006

ABSTRACT

We studied the effects of a bacterium (
Pseudomonas chlororaphis
) and a bactivorous protozoan (
Uronema
sp.) on transformations of labile dissolved organic carbon (DOC). In 36-day time series experiments, bacteria were grown on glucose both with and without protozoa. We measured bulk organic carbon pools and used electrospray ionization mass spectrometry to characterize dissolved organic matter on a molecular level. Bacteria rapidly utilized glucose, depleting it to nondetectable levels and producing new DOC compounds of higher molecular weight within 2 days. Some of these new compounds, representing 3 to 5% of the initial glucose-C, were refractory and persisted for over a month. Other new compounds were produced and subsequently used by bacteria during the lag and exponential growth phases, pointing to a dynamic cycling of organic compounds. Grazers caused a temporary spike in the DOC concentration consisting of labile compounds subsequently utilized by the bacteria. Grazing did not increase the complexity of the DOC pool already established by the bacteria but did continually decrease the particulate organic carbon pool and expedited the conversion of glucose-C to CO
2
. After 36 days, 29% of initial glucose-C remained in pure bacteria cultures, while only 6% remained in cultures where a grazer was present. In this study the bacteria were the primary shapers of the complex DOC continuum, suggesting higher trophic levels possibly have less of an impact on the qualitative composition of DOC than previously assumed.
Characterization of Antimicrobial Substances Produced by
Enterococcus faecalis
MRR 10-3, Isolated from the Uropygial Gland of the Hoopoe (
Upupa epops
)
Authors: Martín-Platero Antonio M. , Valdivia Eva , Ruíz-Rodríguez Magdalena , Soler Juan J. , Martín-Vivaldi Manuel , Maqueda Mercedes , Martínez-Bueno Manuel 
Published: June 2006

ABSTRACT

The uropygial gland (preen gland) is a holocrine secretory gland situated at the base of the tail in birds which produces a hydrophobic fatty secretion. In certain birds, such as the hoopoe,
Upupa epops
, the composition of this secretion is influenced by both seasonal and sexual factors, becoming darker and more malodorous in females and in their nestlings during the nesting phase. The secretion is spread throughout the plumage when the bird preens itself, leaving its feathers flexible and waterproof. It is also thought to play a role in defending the bird against predators and parasites. We have isolated from the uropygial secretion of a nestling a bacterium that grows in monospecific culture which we have identified unambiguously by phenotypic and genotypic means as
Enterococcus faecalis
. The strain in question produces antibacterial substances that are active against all gram-positive bacteria assayed and also against some gram-negative strains. Its peptide nature identifies it as a bacteriocin within the group known as enterocins. Two peptides were purified to homogeneity (MR10A and MR10B), and matrix-assisted laser desorption ionization-time of flight (mass spectrometry) analysis showed masses of 5201.58 and 5207.7 Da, respectively. Amino acid sequencing of both peptides revealed high similarity with enterocin L50A and L50B (L. M. Cintas, P. Casaus, H. Holo, P. E. Hernández, I. F. Nes, and L. S. Håvarstein, J. Bacteriol.
180:
1988-1994, 1998). PCR amplification of total DNA from strain MRR10-3 with primers for the L50A/B structural genes and sequencing of the amplified fragment revealed almost identical sequences, except for a single conservative change in residue 38 (Glu→Asp) in MR10A and two changes in residues 9 (Thr→Ala) and 15 (Leu→Phe) in MR10B. This is the first time that the production of bacteriocins by a bacterium isolated from the uropygial gland has been described. The production of these broad-spectrum antibacterial substances by an enterococcal strain living in the uropygial gland may be important to the hygiene of the nest and thus to the health of the eggs and chicks.
Transcriptome of a
Nitrosomonas europaea
Mutant with a Disrupted Nitrite Reductase Gene (
nirK
)
Authors: Cho Catherine Mee-Hie , Yan Tingfen , Liu Xueduan , Wu Liyou , Zhou Jizhong , Stein Lisa Y. 
Published: June 2006

ABSTRACT

Global gene expression was compared between the
Nitrosomonas europaea
wild type and a nitrite reductase-deficient mutant using a genomic microarray. Forty-one genes were differentially regulated between the wild type and the
nirK
mutant, including the
nirK
operon, genes for cytochrome
c
oxidase, and seven iron uptake genes. Relationships of differentially regulated genes to the
nirK
mutant phenotype are discussed.
Protein Engineering of the 4-Methyl-5-Nitrocatechol Monooxygenase from
Burkholderia
sp. Strain DNT for Enhanced Degradation of Nitroaromatics
Authors: Leungsakul Thammajun , Johnson Glenn R. , Wood Thomas K. 
Published: June 2006

ABSTRACT

4-Methyl-5-nitrocatechol (4M5NC) monooxygenase (DntB) from
Burkholderia
sp. strain DNT catalyzes the second step of 2,4-dinitrotoluene degradation by converting 4M5NC to 2-hydroxy-5-methylquinone with the concomitant removal of the nitro group. DntB is a flavoprotein that has a very narrow substrate range. Here, error-prone PCR was used to create variant DntB M22L/L380I, which accepts the two new substrates 4-nitrophenol (4NP) and 3-methyl-4-nitrophenol (3M4NP). At 300 μM of 4NP, the initial rate of the variant expressing M22L/L380I enzyme (39 ± 6 nmol/min/mg protein) was 10-fold higher than that of the wild-type enzyme (4 ± 2 nmol/min/mg protein). The values of
k
cat
/

K
m

of the purified wild-type DntB enzyme and purified variant M22L/L380I were 40 and 450 (s
−1
M
−1
), respectively, which corroborates that the variant M22L/L380I enzyme has 11-fold-higher efficiency than the wild-type enzyme for 4NP degradation. In addition, the variant M22L/L380I enzyme has fourfold-higher activity toward 3M4NP; at 300 μM, the initial nitrite release rate of M22L/L380I enzyme was 17 ± 4 nmol/min/mg protein, while that of the wild-type enzyme was 4.4 ± 0.7 nmol/min/mg protein. Saturation mutagenesis was also used to further investigate the role of the individual amino acid residues at positions M22, L380, and M22/L380 simultaneously. Mutagenesis at the individual positions M22L and L380I did not show appreciable enhancement in 4NP activity, which suggested that these two sites should be mutated together; simultaneous saturation mutagenesis led to the identification of the variant M22S/L380V, with 20% enhanced degradation of 4NP compared to the variant M22L/L380I. This is the first report of protein engineering for nitrite removal by a flavoprotein.
Comparative and Functional Analysis of Sortase-Dependent Proteins in the Predicted Secretome of
Lactobacillus salivarius
UCC118
Authors: van Pijkeren Jan-Peter , Canchaya Carlos , Ryan Kieran A. , Li Yin , Claesson Marcus J. , Sheil Barbara , Steidler Lothar , O'Mahony Liam , Fitzgerald Gerald F. , van Sinderen Douwe , O'Toole Paul W. 
Published: June 2006

ABSTRACT

Surface proteins are important factors in the interaction of probiotic and pathogenic bacteria with their environment or host. We performed a comparative bioinformatic analysis of four publicly available
Lactobacillus
genomes and the genome of
Lactobacillus salivarius
subsp.
salivarius
strain UCC118 to identify secreted proteins and those linked to the cell wall. Proteins were identified which were predicted to be anchored by WXL-binding domains, N- or C-terminal anchors, GW repeats, lipoprotein anchors, or LysM-binding domains. We identified 10 sortase-dependent surface proteins in
L. salivarius
UCC118, including three which are homologous to mucus-binding proteins (LSL_0152, LSL_0311, and LSL_1335), a collagen-binding protein homologue (LSL_2020b), two hypothetical proteins (LSL_1838 and LSL_1902b), an enterococcal surface protein homologue (LSL_1085), a salivary agglutinin-binding homologue (LSL_1832b), an epithelial binding protein homologue (LSL_1319), and a proteinase homologue (LSL_1774b). However, two of the genes are gene fragments and four are pseudogenes, suggesting a lack of selection for their function. Two of the 10 genes were not transcribed in vitro, and 1 gene showed a 10-fold increase in transcript level in stationary phase compared to logarithmic phase. The sortase gene was deleted, and three genes encoding sortase-dependent proteins were disrupted. The sortase mutant and one sortase-dependent protein (mucus-binding homologue) mutant showed a significant reduction in adherence to human epithelial cell lines. The genome-wide investigation of surface proteins can thus help our understanding of their roles in host interaction.