bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Novel Angular Dioxygenase Gene Cluster Encoding 3-Phenoxybenzoate 1′,2′-Dioxygenase in Sphingobium wenxiniae JZ-1
Authors: Wang Chenghong , Chen Qing , Wang Rui , Shi Chao , Yan Xin , He Jian , Hong Qing , Li Shunpeng 
Published: April 2014

ABSTRACT

Sphingobium wenxiniae
JZ-1 utilizes a wide range of pyrethroids and their metabolic product, 3-phenoxybenzoate, as sources of carbon and energy. A mutant JZ-1 strain, MJZ-1, defective in the degradation of 3-phenoxybenzoate was obtained by successive streaking on LB agar. Comparison of the draft genomes of strains JZ-1 and MJZ-1 revealed that a 29,366-bp DNA fragment containing a putative angular dioxygenase gene cluster (
pbaA1A2B
) is missing in strain MJZ-1. PbaA1, PbaA2, and PbaB share 65%, 52%, and 10% identity with the corresponding α and β subunits and the ferredoxin component of dioxin dioxygenase from
Sphingomonas wittichii
RW1, respectively. Complementation of
pbaA1A2B
in strain MJZ-1 resulted in the active 3-phenoxybenzoate 1′,2′-dioxygenase, but the enzyme activity in
Escherichia coli
was achieved only through the coexpression of
pbaA1A2B
and a glutathione reductase (GR)-type reductase gene,
pbaC
, indicating that the 3-phenoxybenzoate 1′,2′-dioxygenase belongs to a type IV Rieske non-heme iron aromatic ring-hydroxylating oxygenase system consisting of a hetero-oligomeric oxygenase, a [2Fe-2S]-type ferredoxin, and a GR-type reductase. The
pbaC
gene is not located in the immediate vicinity of
pbaA1A2B
. 3-Phenoxybenzoate 1′,2′-dioxygenase catalyzes the hydroxylation in the 1′ and 2′ positions of the benzene moiety of 3-phenoxybenzoate, yielding 3-hydroxybenzoate and catechol. Transcription of
pbaA1A2B
and
pbaC
was induced by 3-phenoxybenzoate, but the transcriptional level of
pbaC
was far less than that of
pbaA1A2B
, implying the possibility that PbaC may not be the only reductase that can physiologically transfer electrons to PbaA1A2B in strain JZ-1. Some GR-type reductases from other sphingomonad strains could also transfer electrons to PbaA1A2B, suggesting that PbaA1A2B has a low specificity for reductase.
Structure-Based Mutational Studies of Substrate Inhibition of Betaine Aldehyde Dehydrogenase BetB from Staphylococcus aureus
Authors: Chen Chao , Joo Jeong Chan , Brown Greg , Stolnikova Ekaterina , Halavaty Andrei S. , Savchenko Alexei , Anderson Wayne F. , Yakunin Alexander F. 
Published: April 2014

ABSTRACT

Inhibition of enzyme activity by high concentrations of substrate and/or cofactor is a general phenomenon demonstrated in many enzymes, including aldehyde dehydrogenases. Here we show that the uncharacterized protein BetB (SA2613) from
Staphylococcus aureus
is a highly specific betaine aldehyde dehydrogenase, which exhibits substrate inhibition at concentrations of betaine aldehyde as low as 0.15 mM. In contrast, the aldehyde dehydrogenase YdcW from
Escherichia coli
, which is also active against betaine aldehyde, shows no inhibition by this substrate. Using the crystal structures of BetB and YdcW, we performed a structure-based mutational analysis of BetB and introduced the YdcW residues into the BetB active site. From a total of 32 mutations, those in five residues located in the substrate binding pocket (Val288, Ser290, His448, Tyr450, and Trp456) greatly reduced the substrate inhibition of BetB, whereas the double mutant protein H448F/Y450L demonstrated a complete loss of substrate inhibition. Substrate inhibition was also reduced by mutations of the semiconserved Gly234 (to Ser, Thr, or Ala) located in the BetB NAD
+
binding site, suggesting some cooperativity between the cofactor and substrate binding sites. Substrate docking analysis of the BetB and YdcW active sites revealed that the wild-type BetB can bind betaine aldehyde in both productive and nonproductive conformations, whereas only the productive binding mode can be modeled in the active sites of YdcW and the BetB mutant proteins with reduced substrate inhibition. Thus, our results suggest that the molecular mechanism of substrate inhibition of BetB is associated with the nonproductive binding of betaine aldehyde.
Variables Influencing Extraction of Nucleic Acids from Microbial Plankton (Viruses, Bacteria, and Protists) Collected on Nanoporous Aluminum Oxide Filters
Authors: Mueller Jaclyn A. , Culley Alexander I. , Steward Grieg F. 
Published: April 2014

ABSTRACT
Anodic aluminum oxide (AAO) filters have high porosity and can be manufactured with a pore size that is small enough to quantitatively capture viruses. These properties make the filters potentially useful for harvesting total microbial communities from water samples for molecular analyses, but their performance for nucleic acid extraction has not been systematically or quantitatively evaluated. In this study, we characterized the flux of water through commercially produced nanoporous (0.02 μm) AAO filters (Anotop; Whatman) and used isolates (a virus, a bacterium, and a protist) and natural seawater samples to test variables that we expected would influence the efficiency with which nucleic acids are recovered from the filters. Extraction chemistry had a significant effect on DNA yield, and back flushing the filters during extraction was found to improve yields of high-molecular-weight DNA. Using the back-flush protocol, the mass of DNA recovered from microorganisms collected on AAO filters was ≥100% of that extracted from pellets of cells and viruses and 94% ± 9% of that obtained by direct extraction of a liquid bacterial culture. The latter is a minimum estimate of the relative recovery of microbial DNA, since liquid cultures include dissolved nucleic acids that are retained inefficiently by the filter. In conclusion, we demonstrate that nucleic acids can be extracted from microorganisms on AAO filters with an efficiency similar to that achievable by direct extraction of microbes in suspension or in pellets. These filters are therefore a convenient means by which to harvest total microbial communities from multiple aqueous samples in parallel for subsequent molecular analyses.
Deciphering the Effect of the Different N-Glycosylation Sites on the Secretion, Activity, and Stability of Cellobiohydrolase I from Trichoderma reesei
Authors: Qi Feifei , Zhang Weixin , Zhang Fengjie , Chen Guanjun , Liu Weifeng 
Published: April 2014

ABSTRACT

N-linked glycosylation modulates and diversifies the structures and functions of the eukaryotic proteome through both intrinsic and extrinsic effects on proteins. We investigated the significance of the three N-linked glycans on the catalytic domain of cellobiohydrolase I (CBH1) from the filamentous fungus
Trichoderma reesei
in its secretion and activity. While the removal of one or two N-glycosylation sites hardly affected the extracellular secretion of CBH1, eliminating all of the glycosylation sites did induce expression of the unfolded protein response (UPR) target genes, and secretion of this CBH1 variant was severely compromised in a calnexin gene deletion strain. Further characterization of the purified CBH1 variants showed that, compared to Asn270, the thermal reactivity of CBH1 was significantly decreased by removal of either Asn45 or Asn384 glycosylation site during the catalyzed hydrolysis of soluble substrate. Combinatorial loss of these two N-linked glycans further exacerbated the temperature-dependent inactivation. In contrast, this thermal labile property was less severe when hydrolyzing insoluble cellulose. Analysis of the structural integrity of CBH1 variants revealed that removal of N-glycosylation at Asn384 had a more pronounced effect on the integrity of regular secondary structure compared to the loss of Asn45 or Asn270. These data implicate differential roles of N-glycosylation modifications in contributing to the stability of specific functional regions of CBH1 and highlight the potential of improving the thermostability of CBH1 by tuning proper interactions between glycans and functional residues.
HrcT Is a Key Component of the Type III Secretion System in Xanthomonas spp. and Also Regulates the Expression of the Key
hrp
Transcriptional Activator HrpX
Authors: Liu Zhi-Yang , Zou Li-Fang , Xue Xiao-Bo , Cai Lu-Lu , Ma Wen-Xiu , Xiong Li , Ji Zhi-Yuan , Chen Gong-You 
Published: April 2014

ABSTRACT

The type III secretion system (T3SS), encoded by
hrp
(hypersensitive response and pathogenicity) genes in Gram-negative phytopathogenic bacteria, delivers repertoires of T3SS effectors (T3SEs) into plant cells to trigger the hypersensitive response (HR) in nonhost or resistant-host plants and promote pathogenicity in susceptible plants. The expression of
hrp
genes in
Xanthomonas
is regulated by two key regulatory proteins, HrpG and HrpX. However, the interactions between
hrp
gene products in directing T3SE secretion are largely unknown. Here we demonstrated that HrcT of
X. oryzae
pv. oryzicola functions as a T3SS component and positively regulates the expression of
hrpX
. Transcription of
hrcT
occurs via two distinct promoters; one (T1) is with the
hrpB
operon and the second (T3) within
hrpB7
Via either promoter T1 or T3, the defect in Hrp phenotype by
hrcT
deletion was corrected in the presence of
hrcT
only from
Xanthomonas
species but not from other phytopathogenic bacteria. An N-terminally truncated HrcT was able to bind the
hrpX
promoter and activate the expression of
hrpX
, supporting that HrcT is a positive regulator of
hrpX
. A revised model showing the regulatory interactions between HrcT, HrpX, and HrpG is proposed.
High-Efficiency Scarless Genetic Modification in Escherichia coli by Using Lambda Red Recombination and I-SceI Cleavage
Authors: Yang Junjie , Sun Bingbing , Huang He , Jiang Yu , Diao Liuyang , Chen Biao , Xu Chongmao , Wang Xin , Liu Jinle , Jiang Weihong , Yang Sheng 
Published: April 2014

ABSTRACT

Genetic modifications of bacterial chromosomes are important for both fundamental and applied research. In this study, we developed an efficient, easy-to-use system for genetic modification of the
Escherichia coli
chromosome, a two-plasmid method involving lambda Red (λ-Red) recombination and I-SceI cleavage. An intermediate strain is generated by integration of a resistance marker gene(s) and I-SceI recognition sites in or near the target gene locus, using λ-Red PCR targeting. The intermediate strain is transformed with a donor plasmid carrying the target gene fragment with the desired modification flanked by I-SceI recognition sites, together with a bifunctional helper plasmid for λ-Red recombination and I-SceI endonuclease. I-SceI cleavage of the chromosome and the donor plasmid allows λ-Red recombination between chromosomal breaks and linear double-stranded DNA from the donor plasmid. Genetic modifications are introduced into the chromosome, and the placement of the I-SceI sites determines the nature of the recombination and the modification. This method was successfully used for
cadA
knockout,
gdhA
knock-in, seamless deletion of
pepD
, site-directed mutagenesis of the essential
metK
gene, and replacement of
metK
with the
Rickettsia
S
-adenosylmethionine transporter gene. This effective method can be used with both essential and nonessential gene modifications and will benefit basic and applied genetic research.
Genomic Features of a Bumble Bee Symbiont Reflect Its Host Environment
Authors: Martinson Vincent G. , Magoc Tanja , Koch Hauke , Salzberg Steven L. , Moran Nancy A. 
Published: April 2014

ABSTRACT

Here, we report the genome of one gammaproteobacterial member of the gut microbiota, for which we propose the name “
Candidatus
Schmidhempelia bombi,” that was inadvertently sequenced alongside the genome of its host, the bumble bee,
Bombus impatiens
. This symbiont is a member of the recently described bacterial order
Orbales
, which has been collected from the guts of diverse insect species; however, “
Ca
. Schmidhempelia” has been identified exclusively with bumble bees. Metabolic reconstruction reveals that “
Ca
. Schmidhempelia” lacks many genes for a functioning NADH dehydrogenase I, all genes for the high-oxygen cytochrome
o
, and most genes in the tricarboxylic acid (TCA) cycle. “
Ca
. Schmidhempelia” has retained NADH dehydrogenase II, the low-oxygen specific cytochrome
bd
, anaerobic nitrate respiration, mixed-acid fermentation pathways, and citrate fermentation, which may be important for survival in low-oxygen or anaerobic environments found in the bee hindgut. Additionally, a type 6 secretion system, a Flp pilus, and many antibiotic/multidrug transporters suggest complex interactions with its host and other gut commensals or pathogens. This genome has signatures of reduction (2.0 megabase pairs) and rearrangement, as previously observed for genomes of host-associated bacteria. A survey of wild and laboratory
B. impatiens
revealed that “
Ca
. Schmidhempelia” is present in 90% of individuals and, therefore, may provide benefits to its host.
Distributions of Salmonella Subtypes Differ between Two U.S. Produce-Growing Regions
Authors: Strawn Laura K. , Danyluk Michelle D. , Worobo Randy W. , Wiedmann Martin 
Published: April 2014

ABSTRACT

Salmonella
accounts for approximately 50% of produce-associated outbreaks in the United States, several of which have been traced back to contamination in the produce production environment. To quantify
Salmonella
diversity and aid in identification of
Salmonella
contamination sources, we characterized
Salmonella
isolates from two geographically diverse produce-growing regions in the United States. Initially, we characterized the
Salmonella
serotype and subtype diversity associated with 1,677 samples collected from 33 produce farms in New York State (NYS). Among these 1,677 samples, 74 were
Salmonella
positive, yielding 80 unique isolates (from 147 total isolates), which represented 14 serovars and 23 different pulsed-field gel electrophoresis (PFGE) types. To explore regional
Salmonella
diversity associated with production environments, we collected a smaller set of samples (
n
= 65) from South Florida (SFL) production environments and compared the
Salmonella
diversity associated with these samples with the diversity found among NYS production environments. Among these 65 samples, 23 were
Salmonella
positive, yielding 32 unique isolates (from 81 total isolates), which represented 11 serovars and 17 different PFGE types. The most common serovars isolated in NYS were
Salmonella enterica
serovars Newport, Cerro, and Thompson, while common serovars isolated in SFL were
Salmonella
serovars Saphra and Newport and
S. enterica
subsp.
diarizonae
serovar 50:r:z. High PFGE type diversity (Simpson's diversity index, 0.90 ± 0.02) was observed among
Salmonella
isolates across both regions; only three PFGE types were shared between the two regions. The probability of three or fewer shared PFGE types was <0.000001; therefore,
Salmonella
isolates were considerably different between the two sampled regions. These findings suggest the potential for PFGE-based source tracking of
Salmonella
in production environments.
Stress Response and Adaptation of Listeria monocytogenes 08-5923 Exposed to a Sublethal Dose of Carnocyclin A
Authors: Liu Xiaoji , Basu Urmila , Miller Petr , McMullen Lynn M. 
Published: April 2014

ABSTRACT

Carnocyclin A (CCLA) is an antimicrobial peptide produced by
Carnobacterium maltaromaticum
ATCC PTA-5313, which can be used to control the growth of
Listeria monocytogenes
in ready-to-eat meat products. The aim of this research was to elucidate the cellular responses of
L. monocytogenes
08-5923 exposed to a sublethal dose of CCLA. Microarray, quantitative reverse transcription-PCR, tandem mass spectrometry, and electron microscopy were used to investigate the alteration in gene expression, protein production, and morphological changes in cells of
Listeria
following treatment with CCLA. The genes involved in metabolism (
baiE
,
trn
, and
pykA
), cell wall synthesis (
murZ
and
dacB2
), and cell division (
clpE
and
divIVA
) were upregulated following a 15-min exposure to CCLA as a result of stress responses. Genes involved in cell division, cell wall synthesis, flagellar synthesis, and metabolism were downregulated after 4 h as a result of adaptation. Analysis of total soluble proteins confirmed the downregulation of
pykA
and
gnd
after 4 h of exposure to CCLA. The absence of flagella was observed in
L. monocytogenes
following 30 h of exposure to CCLA. A sublethal dose of CCLA induced adaptation in
L. monocytogenes
08-5923 by inhibition of expression of genes and proteins critical for synthesis of cell wall structures and maintaining metabolic functions. Both the mannose- and cellobiose-specific phosphotransferase systems could be targets for CCLA.
Biotic Interactions and Sunlight Affect Persistence of Fecal Indicator Bacteria and Microbial Source Tracking Genetic Markers in the Upper Mississippi River
Authors: Korajkic Asja , McMinn Brian R. , Shanks Orin C. , Sivaganesan Mano , Fout G. Shay , Ashbolt Nicholas J. 
Published: April 2014

ABSTRACT

The sanitary quality of recreational waters that may be impacted by sewage is assessed by enumerating fecal indicator bacteria (FIB) (
Escherichia coli
and enterococci); these organisms are found in the gastrointestinal tracts of humans and many other animals, and hence their presence provides no information about the pollution source. Microbial source tracking (MST) methods can discriminate between different pollution sources, providing critical information to water quality managers, but relatively little is known about factors influencing the decay of FIB and MST genetic markers following release into aquatic environments. An
in situ
mesocosm was deployed at a temperate recreational beach in the Mississippi River to evaluate the effects of ambient sunlight and biotic interactions (predation, competition, and viral lysis) on the decay of culture-based FIB, as well as molecularly based FIB (Entero1a and GenBac3) and human-associated MST genetic markers (HF183 and HumM2) measured by quantitative real-time PCR (qPCR). In general, culturable FIB decayed the fastest, while molecularly based FIB and human-associated genetic markers decayed more slowly. There was a strong correlation between the decay of molecularly based FIB and that of human-associated genetic markers (
r
2
, 0.96 to 0.98;
P
< 0.0001) but not between culturable FIB and any qPCR measurement. Overall, exposure to ambient sunlight may be an important factor in the early-stage decay dynamics but generally was not after continued exposure (i.e., after 120 h), when biotic interactions tended to be the only/major influential determinant of persistence.
Promotion of Endodontic Lesions in Rats by a Novel Extraradicular Biofilm Model Using Obturation Materials
Authors: Kuremoto Katsutaka , Noiri Yuichiro , Ishimoto Takuya , Yoneda Naomichi , Yamamoto Reiko , Maezono Hazuki , Nakano Takayoshi , Hayashi Mikako , Ebisu Shigeyuki 
Published: April 2014

ABSTRACT

Although extraradicular biofilm formation is related to refractory periapical periodontitis, the mechanism of extraradicular biofilm development, as well as its effect on periapical lesions, is unknown. Therefore, we aimed to develop an
in vivo
extraradicular biofilm model in rats and to identify and quantify extraradicular biofilm-forming bacteria while investigating the effect of extraradicular biofilms on periapical lesions. Periapical lesions were induced by exposing the pulpal tissue of the mandibular first molars of male Wistar rats to their oral environment. Four weeks later, gutta-percha points were excessively inserted into the mesial root canals of the right first molars (experimental sites) but not the left first molars (control sites). After 6 and 8 weeks of pulp exposure, the presence of extraradicular biofilms was confirmed histomorphologically, and biofilm-forming bacteria were identified by using classical culture methods. The biofilms were observed in the extraradicular area of the experimental sites. Similar species were detected both inside and outside the root canals. The bacterial count, quantified by real-time PCR assays, in the extraradicular area gradually increased in the experimental sites until 20 weeks after pulp exposure. After 8 weeks of pulp exposure, the periapical lesion volume that was measured by micro-computed tomography was significantly larger in the experimental sites than in the control sites (
P
< 0.05 by Welch's
t
test). These results suggest that we developed an extraradicular biofilm model in rats and that extraradicular biofilms affect developing periapical lesions.
From Metagenomics to Pure Culture: Isolation and Characterization of the Moderately Halophilic Bacterium Spiribacter salinus gen. nov., sp. nov
Authors: León María José , Fernández Ana B. , Ghai Rohit , Sánchez-Porro Cristina , Rodriguez-Valera Francisco , Ventosa Antonio 
Published: April 2014

ABSTRACT

Recent metagenomic studies on saltern ponds with intermediate salinities have determined that their microbial communities are dominated by both
Euryarchaeota
and halophilic bacteria, with a gammaproteobacterium closely related to the genera
Alkalilimnicola
and
Arhodomonas
being one of the most predominant microorganisms, making up to 15% of the total prokaryotic population. Here we used several strategies and culture media in order to isolate this organism in pure culture. We report the isolation and taxonomic characterization of this new, never before cultured microorganism, designated M19-40
T
, isolated from a saltern located in Isla Cristina, Spain, using a medium with a mixture of 15% salts, yeast extract, and pyruvic acid as the carbon source. Morphologically small curved cells (young cultures) with a tendency to form long spiral cells in older cultures were observed in pure cultures. The organism is a Gram-negative, nonmotile bacterium that is strictly aerobic, non-endospore forming, heterotrophic, and moderately halophilic, and it is able to grow at 10 to 25% (wt/vol) NaCl, with optimal growth occurring at 15% (wt/vol) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence comparison showed that strain M19-40
T
has a low similarity with other previously described bacteria and shows the closest phylogenetic similarity with species of the genera
Alkalilimnicola
(94.9 to 94.5%),
Alkalispirillum
(94.3%), and
Arhodomonas
(93.9%) within the family
Ectothiorhodospiraceae
. The phenotypic, genotypic, and chemotaxonomic features of this new bacterium showed that it constitutes a new genus and species, for which the name
Spiribacter salinus
gen. nov., sp. nov., is proposed, with strain M19-40
T
(= CECT 8282
T
= IBRC-M 10768
T
= LMG 27464
T
) being the type strain.
Posttranscriptional Regulation of 2,4-Diacetylphloroglucinol Production by GidA and TrmE in Pseudomonas fluorescens 2P24
Authors: Zhang Wei , Zhao Zhao , Zhang Bo , Wu Xiao-Gang , Ren Zheng-Guang , Zhang Li-Qun 
Published: April 2014

ABSTRACT

Pseudomonas fluorescens
2P24 is a soilborne bacterium that synthesizes and excretes multiple antimicrobial metabolites. The polyketide compound 2,4-diacetylphloroglucinol (2,4-DAPG), synthesized by the
phlACBD
locus, is its major biocontrol determinant. This study investigated two mutants defective in antagonistic activity against
Rhizoctonia solani
. Deletion of the
gidA
(PM701) or
trmE
(PM702) gene from strain 2P24 completely inhibited the production of 2,4-DAPG and its precursors, monoacetylphloroglucinol (MAPG) and phloroglucinol (PG). The transcription of the
phlA
gene was not affected, but the translation of the
phlA
and
phlD
genes was reduced significantly. Two components of the Gac/Rsm pathway, RsmA and RsmE, were found to be regulated by
gidA
and
trmE
, whereas the other components, RsmX, RsmY, and RsmZ, were not. The regulation of 2,4-DAPG production by
gidA
and
trmE
, however, was independent of the Gac/Rsm pathway. Both the
gidA
and
trmE
mutants were unable to produce PG but could convert PG to MAPG and MAPG to 2,4-DAPG. Overexpression of PhlD in the
gidA
and
trmE
mutants could restore the production of PG and 2,4-DAPG. Taken together, these findings suggest that GidA and TrmE are positive regulatory elements that influence the biosynthesis of 2,4-DAPG posttranscriptionally.