bluuebunny/crossref_metadata_2025_split huggingface dataset:
Anaerobic Methyl
tert
-Butyl Ether-Degrading Microorganisms Identified in Wastewater Treatment Plant Samples by Stable Isotope Probing
Authors: Sun Weimin , Sun Xiaoxu , Cupples Alison M. 
Published: February 2012

ABSTRACT

Anaerobic methyl
tert
-butyl ether (MTBE) degradation potential was investigated in samples from a range of sources. From these 22 experimental variations, only one source (from wastewater treatment plant samples) exhibited MTBE degradation. These microcosms were methanogenic and were subjected to DNA-based stable isotope probing (SIP) targeted to both bacteria and archaea to identify the putative MTBE degraders. For this purpose, DNA was extracted at two time points, subjected to ultracentrifugation, fractioning, and terminal restriction fragment length polymorphism (TRFLP). In addition, bacterial and archaeal 16S rRNA gene clone libraries were constructed. The SIP experiments indicated bacteria in the phyla
Firmicutes
(family
Ruminococcaceae
) and
Alphaproteobacteria
(genus
Sphingopyxis
) were the dominant MTBE degraders. Previous studies have suggested a role for
Firmicutes
in anaerobic MTBE degradation; however, the putative MTBE-degrading microorganism in the current study is a novel MTBE-degrading phylotype within this phylum. Two archaeal phylotypes (genera
Methanosarcina
and
Methanocorpusculum
) were also enriched in the heavy fractions, and these organisms may be responsible for minor amounts of MTBE degradation or for the uptake of metabolites released from the primary MTBE degraders. Currently, limited information exists on the microorganisms able to degrade MTBE under anaerobic conditions. This work represents the first application of DNA-based SIP to identify anaerobic MTBE-degrading microorganisms in laboratory microcosms and therefore provides a valuable set of data to definitively link identity with anaerobic MTBE degradation.
Assessing the Contributions of the LiaS Histidine Kinase to the Innate Resistance of Listeria monocytogenes to Nisin, Cephalosporins, and Disinfectants
Authors: Collins Barry , Guinane Caitriona M. , Cotter Paul D. , Hill Colin , Ross R. Paul 
Published: February 2012

ABSTRACT

The
Listeria monocytogenes
LiaSR two-component system (2CS) encoded by
lmo1021
and
lmo1022
plays an important role in resistance to the food preservative nisin. A nonpolar deletion in the histidine kinase-encoding component (Δ
liaS
) resulted in a 4-fold increase in nisin resistance. In contrast, the Δ
liaS
strain exhibited increased sensitivity to a number of cephalosporin antibiotics (and was also altered with respect to its response to a variety of other antimicrobials, including the active agents of a number of disinfectants). This pattern of increased nisin resistance and reduced cephalosporin resistance in
L. monocytogenes
has previously been associated with mutation of a second histidine kinase, LisK, which is a predicted regulator of
liaS
and a penicillin binding protein encoded by
lmo2229
. We noted that
lmo2229
transcription is increased in the Δ
liaS
mutant and in a Δ
liaS
Δ
lisK
double mutant and that disruption of
lmo2229
in the Δ
liaS
Δ
lisK
mutant resulted in a dramatic sensitization to nisin but had a relatively minor impact on cephalosporin resistance. We anticipate that further efforts to unravel the complex mechanisms by which LiaSR impacts on the antimicrobial resistance of
L. monocytogenes
could facilitate the development of strategies to increase the susceptibility of the pathogen to these agents.
Microbiota Dynamics and Diversity at Different Stages of Industrial Processing of Cocoa Beans into Cocoa Powder
Authors: Lima Lídia J. R. , van der Velpen Vera , Wolkers-Rooijackers Judith , Kamphuis Henri J. , Zwietering Marcel H. , Nout M. J. Rob 
Published: February 2012

ABSTRACT

We sampled a cocoa powder production line to investigate the impact of processing on the microbial community size and diversity at different stages. Classical microbiological methods were combined with 16S rRNA gene PCR-denaturing gradient gel electrophoresis, coupled with clone library construction, to analyze the samples. Aerobic thermoresistant spores (ThrS) (100°C; 10 min) were also isolated and characterized (identity, genetic diversity, and spore heat resistance), in view of their relevance to the quality of downstream heat-treated cocoa-flavored drinks. In the nibs (broken, shelled cocoa beans), average levels of total aerobic microorganisms (TAM) (4.4 to 5.6 log CFU/g) and aerobic total spores (TS) (80°C; 10 min; 4.3 to 5.5 log CFU/g) were significantly reduced (
P
< 0.05) as a result of alkalizing, while fungi (4.2 to 4.4 log CFU/g) and
Enterobacteriaceae
(1.7 to 2.8 log CFU/g) were inactivated to levels below the detection limit, remaining undetectable throughout processing. Roasting further decreased the levels of TAM and TS, but they increased slightly during subsequent processing. Molecular characterization of bacterial communities based on enriched cocoa samples revealed a predominance of members of the
Bacillaceae
,
Pseudomonadaceae
, and
Enterococcaceae
. Eleven species of ThrS were found, but
Bacillus licheniformis
and the
Bacillus subtilis
complex were prominent and revealed great genetic heterogeneity. We concluded that the microbiota of cocoa powder resulted from microorganisms that could have been initially present in the nibs, as well as microorganisms that originated during processing.
B. subtilis
complex members, particularly
B. subtilis
subsp.
subtilis
, formed the most heat-resistant spores. Their occurrence in cocoa powder needs to be considered to ensure the stability of derived products, such as ultrahigh-temperature-treated chocolate drinks.
Ribosome Display of Combinatorial Antibody Libraries Derived from Mice Immunized with Heat-Killed Xylella fastidiosa and the Selection of MopB-Specific Single-Chain Antibodies
Authors: Azizi Armaghan , Arora Arinder , Markiv Anatoliy , Lampe David J. , Miller Thomas A. , Kang Angray S. 
Published: February 2012

ABSTRACT

Pierce's disease is a devastating lethal disease of
Vitus vinifera
grapevines caused by the bacterium
Xylella fastidiosa
. There is no cure for Pierce's disease, and control is achieved predominantly by suppressing transmission of the glassy-winged sharpshooter insect vector. We present a simple robust approach for the generation of panels of recombinant single-chain antibodies against the surface-exposed elements of
X. fastidiosa
that may have potential use in diagnosis and/or disease transmission blocking studies.
In vitro
combinatorial antibody ribosome display libraries were assembled from immunoglobulin transcripts rescued from the spleens of mice immunized with heat-killed
X. fastidiosa
. The libraries were used in a single round of selection against an outer membrane protein, MopB, resulting in the isolation of a panel of recombinant antibodies. The potential use of selected anti-MopB antibodies was demonstrated by the successful application of the 4XfMopB3 antibody in an enzyme-linked immunosorbent assay (ELISA), a Western blot assay, and an immunofluorescence assay (IFA). These immortalized
in vitro
recombinant single-chain antibody libraries generated against heat-killed
X. fastidiosa
are a resource for the Pierce's disease research community that may be readily accessed for the isolation of antibodies against a plethora of
X. fastidiosa
surface-exposed antigenic molecules.
BrpA Is Involved in Regulation of Cell Envelope Stress Responses in Streptococcus mutans
Authors: Bitoun J. P. , Liao S. , Yao X. , Ahn S.-J. , Isoda R. , Nguyen A. H. , Brady L. J. , Burne R. A. , Abranches J. , Wen Z. T. 
Published: February 2012

ABSTRACT

Previous studies have shown that BrpA plays a major role in acid and oxidative stress tolerance and biofilm formation by
Streptococcus mutans
. Mutant strains lacking BrpA also display increased autolysis and decreased viability, suggesting a role for BrpA in cell envelope integrity. In this study, we examined the impact of BrpA deficiency on cell envelope stresses induced by envelope-active antimicrobials. Compared to the wild-type strain UA159, the BrpA-deficient mutant (TW14D) was significantly more susceptible to antimicrobial agents, especially lipid II inhibitors. Several genes involved in peptidoglycan synthesis were identified by DNA microarray analysis as downregulated in TW14D. Luciferase reporter gene fusion assays also revealed that expression of
brpA
is regulated in response to environmental conditions and stresses induced by exposure to subinhibitory concentrations of cell envelope antimicrobials. In a
Galleria mellonella
(wax worm) model, BrpA deficiency was shown to diminish the virulence of
S. mutans
OMZ175, which, unlike
S. mutans
UA159, efficiently kills the worms. Collectively, these results suggest that BrpA plays a role in the regulation of cell envelope integrity and that deficiency of BrpA adversely affects the fitness and diminishes the virulence of OMZ175, a highly invasive strain of
S. mutans
.
Prevalence of Yersinia enterocolitica in Pigs Slaughtered in Chinese Abattoirs
Authors: Liang Junrong , Wang Xin , Xiao Yuchun , Cui Zhigang , Xia Shengli , Hao Qiong , Yang Jinchuan , Luo Longze , Wang Shukun , Li Kewei , Yang Haoshu , Gu Wenpeng , Xu Jianguo , Kan Biao , Jing Huaiqi 
Published: February 2012

ABSTRACT

The distribution of
Yersinia enterocolitica
in slaughtered pigs in China was studied. A total of 8,773 samples were collected and examined from different pig abattoirs in 11 provinces from 2009 to 2011. Of these, 4,495 were oral-pharyngeal swab (tonsils) samples from pigs, 1,239 were from intestinal contents, and 3,039 were feces samples from abattoirs or local pigpens. The data showed that 1,132 strains were obtained, from which the isolation rate for
Yersinia enterocolitica
was 19.53% (878/4,495) from the tonsil samples, 7.51% (93/1,239) from intestinal contents, and 5.30% (161/3,039) from feces. Of the 850 pathogenic
Yersinia
strains, except for three of bioserotype 2/O:9 and three of bioserotype 4/O:3, most (844/850) were of bioserotype 3/O:3. Interestingly, pathogenic
Y. enterocolitica
accounted for the majority of the isolated strains from most provinces (85.17% to 100%), whereas from Heilongjiang, 96.52% (111/115) were classified as nonpathogenic biotype 1A with various serotypes, and only 3.48% of the strains (4/115) were pathogenic 3/O:3. All of the pathogenic strains were analyzed using pulsed-field gel electrophoresis (PFGE), and 49 patterns were obtained for the O:3 pathogenic strains; most of them were K6GN11C30021 (53.13%: 450/847) and K6GN11C30012 (21.37%: 181/847). Several strains from diarrhea patient samples revealed PFGE patterns identical to that from samples of local pigs, suggesting a possible link between porcine isolates and human infection. The results above suggested that
Yersinia enterocolitica
in slaughtered pigs from Chinese abattoirs was characterized by region-specific PFGE patterns and confirmed that strains isolated from pigs are closely related to those from human infections.
Rapid and Specific Detection of Escherichia coli Serogroups O26, O45, O103, O111, O121, O145, and O157 in Ground Beef, Beef Trim, and Produce by Loop-Mediated Isothermal Amplification
Authors: Wang Fei , Jiang Lin , Yang Qianru , Prinyawiwatkul Witoon , Ge Beilei 
Published: February 2012

ABSTRACT

Escherichia coli
O157 and six additional serogroups of Shiga toxin-producing
E. coli
(STEC) (O26, O45, O103, O111, O121, and O145) account for the majority of STEC infections in the United States. In this study, O serogroup-specific genes (
wzx
or
wzy
) were used to design loop-mediated isothermal amplification (LAMP) assays for the rapid and specific detection of these leading STEC serogroups. The assays were evaluated in pure culture and spiked food samples (ground beef, beef trim, lettuce, and spinach) and compared with real-time quantitative PCR (qPCR). No false-positive or false-negative results were observed among 120 bacterial strains used to evaluate assay specificity. The limits of detection of various STEC strains belonging to these target serogroups were approximately 1 to 20 CFU/reaction mixture in pure culture and 10
3
to 10
4
CFU/g in spiked food samples, which were comparable to those of qPCR. Standard curves generated suggested good linear relationships between STEC cell numbers and LAMP turbidity signals. In various beef and produce samples spiked with two low levels (1 to 2 and 10 to 20 CFU/25 g) of respective STEC strains, the LAMP assays consistently achieved accurate detection after 6 to 8 h of enrichment. In conclusion, these newly developed LAMP assays may facilitate rapid and reliable detection of the seven major STEC serogroups in ground beef, beef trim, and produce during routine sample testing.
Regulation of Vacuolar H
+
-ATPase Activity by the Cdc42 Effector Ste20 in Saccharomyces cerevisiae
Authors: Lin Meng , Li Sheena Claire , Kane Patricia M. , Höfken Thomas 
Published: February 2012

ABSTRACT

In the budding yeast
Saccharomyces cerevisiae
, the Cdc42 effector Ste20 plays a crucial role in the regulation of filamentous growth, a response to nutrient limitation. Using the split-ubiquitin technique, we found that Ste20 forms a complex with Vma13, an important regulatory subunit of vacuolar H
+
-ATPase (V-ATPase). This protein-protein interaction was confirmed by a pulldown assay and coimmunoprecipitation. We also demonstrate that Ste20 associates with vacuolar membranes and that Ste20 stimulates V-ATPase activity in isolated vacuolar membranes. This activation requires Ste20 kinase activity and does not depend on increased assembly of the V
1
and V
0
sectors of the V-ATPase, which is a major regulatory mechanism. Furthermore, loss of V-ATPase activity leads to a strong increase in invasive growth, possibly because these cells fail to store and mobilize nutrients efficiently in the vacuole in the absence of the vacuolar proton gradient. In contrast to the wild type, which grows in rather small, isolated colonies on solid medium during filamentation, hyperinvasive
vma
mutants form much bigger aggregates in which a large number of cells are tightly clustered together. Genetic data suggest that Ste20 and the protein kinase A catalytic subunit Tpk2 are both activated in the
vma13
Δ strain. We propose that during filamentous growth, Ste20 stimulates V-ATPase activity. This would sustain nutrient mobilization from vacuolar stores, which is beneficial for filamentous growth.
Genomic Rearrangements Leading to Overexpression of Aldo-Keto Reductase YafB of Escherichia coli Confer Resistance to Glyoxal
Authors: Kwon Minsuk , Lee Junghoon , Lee Changhan , Park Chankyu 
Published: February 2012

ABSTRACT

Glyoxal is toxic and mutagenic
α
-oxoaldehyde generated
in vivo
as an oxidation by-product of sugar metabolism. We selected glyoxal-resistant mutants from an
Escherichia coli
strain lacking major glyoxal-detoxifying genes,
gloA
and
yqhD
, by growing cells in medium containing a lethal concentration of glyoxal. The mutants carried diverse genomic rearrangements, such as multibase deletions and recombination, in the upstream region of the
yafB
gene, encoding an aldo-keto reductase. Since these genomic lesions create transcriptional fusions of the
yafB
gene to the upstream
rrn
regulon or eliminate a negative regulatory site, the mutants generally enhanced an expression of the
yafB
gene. Glyoxal resistances of the mutants are correlated with the levels of
yafB
transcripts as well as the activities of aldo-keto reductase. An overproduction of YafB in the glyoxal-resistant mutant lacking the putative NsrR-binding site provides evidence that the
yafB
gene is negatively regulated by this protein. We also observed that the expression of
yafB
is enhanced with an increased concentration of glyoxal as well as a mutation in the
fnr
gene, encoding a putative regulator. The bindings of NsrR and Fnr to the
yafB
promoter were also demonstrated by gel mobility shift assays.
ExpR Is Not Required for Swarming but Promotes Sliding in Sinorhizobium meliloti
Authors: Nogales Joaquina , Bernabéu-Roda Lydia , Cuéllar Virginia , Soto María J. 
Published: February 2012

ABSTRACT

Swarming is a mode of translocation dependent on flagellar activity that allows bacteria to move rapidly across surfaces. In several bacteria, swarming is a phenotype regulated by quorum sensing. It has been reported that the swarming ability of the soil bacterium
Sinorhizobium meliloti
Rm2011 requires a functional ExpR/Sin quorum-sensing system. However, our previous published results demonstrate that strains Rm1021 and Rm2011, both known to have a disrupted copy of
expR
, are able to swarm on semisolid minimal medium. In order to clarify these contradictory results, the role played by the LuxR-type regulator ExpR has been reexamined. Results obtained in this work revealed that
S. meliloti
can move over semisolid surfaces using at least two different types of motility. One type is flagellum-independent surface spreading or sliding, which is positively influenced by a functional
expR
gene mainly through the production of exopolysaccharide II (EPS II). To a lesser extent, EPS II-deficient strains can also slide on surfaces by a mechanism that is at least dependent on the siderophore rhizobactin 1021. The second type of surface translocation shown by
S. meliloti
is swarming, which is greatly dependent on flagella and rhizobactin 1021 but does not require ExpR. We have extended our study to demonstrate that the production of normal amounts of succinoglycan (EPS I) does not play a relevant role in surface translocation but that its overproduction facilitates both swarming and sliding motilities.
Novel, Highly Specific
N
-Demethylases Enable Bacteria To Live on Caffeine and Related Purine Alkaloids
Authors: Summers Ryan M. , Louie Tai Man , Yu Chi-Li , Gakhar Lokesh , Louie Kailin C. , Subramanian Mani 
Published: February 2012

ABSTRACT

The molecular basis for the ability of bacteria to live on caffeine as a sole carbon and nitrogen source is unknown.
Pseudomonas putida
CBB5, which grows on several purine alkaloids, metabolizes caffeine and related methylxanthines via sequential
N
-demethylation to xanthine. Metabolism of caffeine by CBB5 was previously attributed to one broad-specificity methylxanthine
N
-demethylase composed of two subunits, NdmA and NdmB. Here, we report that NdmA and NdmB are actually two independent Rieske nonheme iron monooxygenases with
N
1
- and
N
3
-specific
N
-demethylation activity, respectively. Activity for both enzymes is dependent on electron transfer from NADH via a redox-center-dense Rieske reductase, NdmD. NdmD itself is a novel protein with one Rieske [2Fe-2S] cluster, one plant-type [2Fe-2S] cluster, and one flavin mononucleotide (FMN) per enzyme. All
ndm
genes are located in a 13.2-kb genomic DNA fragment which also contained a formaldehyde dehydrogenase.
ndmA
,
ndmB
, and
ndmD
were cloned as His
6
fusion genes, expressed in
Escherichia coli
, and purified using a Ni-NTA column. NdmA-His
6
plus His
6
-NdmD catalyzed
N
1
-demethylation of caffeine, theophylline, paraxanthine, and 1-methylxanthine to theobromine, 3-methylxanthine, 7-methylxanthine, and xanthine, respectively. NdmB-His
6
plus His
6
-NdmD catalyzed
N
3
-demethylation of theobromine, 3-methylxanthine, caffeine, and theophylline to 7-methylxanthine, xanthine, paraxanthine, and 1-methylxanthine, respectively. One formaldehyde was produced from each methyl group removed. Activity of an
N
7
-specific
N
-demethylase, NdmC, has been confirmed biochemically. This is the first report of bacterial
N
-demethylase genes that enable bacteria to live on caffeine. These genes represent a new class of Rieske oxygenases and have the potential to produce biofuels, animal feed, and pharmaceuticals from coffee and tea waste.
The Early Divisome Protein FtsA Interacts Directly through Its 1c Subdomain with the Cytoplasmic Domain of the Late Divisome Protein FtsN
Authors: Busiek Kimberly K. , Eraso Jesus M. , Wang Yipeng , Margolin William 
Published: February 2012

ABSTRACTInEscherichia coli, FtsN localizes late to the cell division machinery, only after a number of additional essential proteins are recruited to the early FtsZ-FtsA-ZipA complex. FtsN has a short, positively charged cytoplasmic domain (FtsNCyto), a single transmembrane domain (FtsNTM), and a periplasmic domain that is essential for FtsN function. Here we show that FtsA and FtsN interact directlyin vitro. FtsNCytois sufficient to bind to FtsA, but only when it is tethered to FtsNTMor to a leucine zipper. Mutation of a conserved patch of positive charges in FtsNCytoto negative charges abolishes the interaction with FtsA. We also show that subdomain 1c of FtsA is sufficient to mediate this interaction with FtsN. Finally, although FtsNCyto-TMis not essential for FtsN function, its overproduction causes a modest dominant-negative effect on cell division. These results suggest that basic residues within a dimerized FtsNCytoprotein interact directly with residues in subdomain 1c of FtsA. Since FtsA binds directly to FtsZ and FtsN interacts with enzymes involved in septum synthesis and splitting, this interaction between early and late divisome proteins may be one of several feedback controls for Z ring constriction.
The Branched-Chain Amino Acid Aminotransferase Encoded by
ilvE
Is Involved in Acid Tolerance in Streptococcus mutans
Authors: Santiago Brendaliz , MacGilvray Matthew , Faustoferri Roberta C. , Quivey Robert G. 
Published: February 2012

ABSTRACT

The ability of
Streptococcus mutans
to produce and tolerate organic acids from carbohydrate metabolism represents a major virulence factor responsible for the formation of carious lesions. Pyruvate is a key metabolic intermediate that, when rerouted to other metabolic pathways such as amino acid biosynthesis, results in the alleviation of acid stress by reducing acid end products and aiding in maintenance of intracellular pH. Amino acid biosynthetic genes such as
ilvC
and
ilvE
were identified as being upregulated in a proteome analysis of
Streptococcus mutans
under acid stress conditions (A. C. Len, D. W. Harty, and N. A. Jacques, Microbiology
150:
1353–1366, 2004). In
Lactococcus lactis
and
Staphylococcus carnosus
, the
ilvE
gene product is involved with biosynthesis and degradation of branched-chain amino acids, as well as in the production of branched-chain fatty acids (B. Ganesan and B. C. Weimer, Appl. Environ. Microbiol.
70:
638–641, 2004; S. M. Madsen et al., Appl. Environ. Microbiol.
68:
4007–4014, 2002; and M. Yvon, S. Thirouin, L. Rijnen, D. Fromentier, and J. C. Gripon, Appl. Environ. Microbiol.
63:
414–419, 1997). Here we constructed and characterized an
ilvE
deletion mutant of
S. mutans
UA159. Growth experiments revealed that the
ilvE
mutant strain has a lag in growth when nutritionally limited for branched-chain amino acids. We further demonstrated that the loss of
ilvE
causes a decrease in acid tolerance. The
ilvE
strain exhibits a defect in F
1
-F
o
ATPase activity and has reduced catabolic activity for isoleucine and valine. Results from transcriptional studies showed that the
ilvE
promoter is upregulated during growth at low pH. Collectively, the results of this investigation show that amino acid metabolism is a component of the acid-adaptive repertoire of
S. mutans
.