bluuebunny/crossref_metadata_2025 huggingface dataset:
Reduced Drug Uptake in Phenotypically Resistant Nutrient-Starved Nonreplicating Mycobacterium tuberculosis
Authors: Sarathy Jansy , Dartois Véronique , Dick Thomas , Gengenbacher Martin
Published: January 2013

During active tuberculosis a spectrum of physiologically different
Mycobacterium tuberculosis
bacilli reside in human tissues. Subpopulations of the pathogen survive antibiotic treatment for a prolonged time in a dormant state of phenotypic drug resistance, a phenomenon independent of genetic mutations. Here, we used an established culture model of nutrient deprivation to shift down
M. tuberculosis
from growth to nonreplicating survival, which is characterized by a drastic loss of drug susceptibility. Liquid chromatography coupled with mass spectrometry techniques were employed to quantify drug penetration in replicating and nutrient-starved nonreplicating bacilli. We found that intracellular concentrations of fluoroquinolones, rifamycins, and linezolid were lower in nonreplicating
M. tuberculosis
. Studies with pump inhibitors suggest that the observed differences were independent of efflux processes. We conclude that decreased drug permeability contributes to phenotypic drug resistance of dormant
M. tuberculosis
.
rRNA (
rrn
) Operon-Engineered Bacillus subtilis as a Feasible Test Organism for Antibiotic Discovery
Authors: Tanaka Yukinori , Nanamiya Hideaki , Yano Koichi , Kakugawa Koji , Kawamura Fujio , Ochi Kozo
Published: January 2013

Bacillus subtilis
contains 10 rRNA (
rrn
) operons. We found that rRNA operon-engineered
B. subtilis
strain RIK543, with only the
rrnO
operon, is specifically hypersensitive to RNA polymerase inhibitors such as rifamycin SV and rifampin (80-fold and 20-fold, respectively). In pilot screening experiments, we found actinomycete isolates successfully at an incidence of 1.9% (18/945) that produced antibacterials that were detectable only with RIK543 as the test organism. Strain RIK543 may be a feasible test organism for the discovery of novel RNA polymerase inhibitors.
wksl3, a New Biocontrol Agent for Salmonella enterica Serovars Enteritidis and Typhimurium in Foods: Characterization, Application, Sequence Analysis, and Oral Acute Toxicity Study
Authors: Kang Hyun-Wol , Kim Jae-Won , Jung Tae-Sung , Woo Gun-Jo
Published: January 2013

Of theSalmonella entericaserovars,S. Enteritidis andS. Typhimurium are responsible for most of theSalmonellaoutbreaks implicated in the consumption of contaminated foods in the Republic of Korea. Because of the widespread occurrence of antimicrobial-resistantSalmonellain foods and food processing environments, bacteriophages have recently surfaced as an alternative biocontrol tool. In this study, we isolated a virulent bacteriophage (wksl3) that could specifically infectS. Enteritidis,S. Typhimurium, and several additional serovars. Transmission electron microscopy revealed that phage wksl3 belongs to the familySiphoviridae. Complete genome sequence analysis and bioinformatic analysis revealed that the DNA of phage wksl3 is composed of 42,766 bp with 64 open reading frames. Since it does not encode any phage lysogeny factors, toxins, pathogen-related genes, or food-borne allergens, phage wksl3 may be considered a virulent phage with no side effects. Analysis of genetic similarities between phage wksl3 and four of its relatives (SS3e, vB_SenS-Ent1, SE2, and SETP3) allowed wksl3 to be categorized as a SETP3-like phage. A single-dose test of oral toxicity with BALB/c mice resulted in no abnormal clinical observations. Moreover, phage application to chicken skin at 8ï¿½C resulted in an about 2.5-log reduction in the number ofSalmonellabacteria during the test period. The strong, stable lytic activity, the significant reduction of the number ofS. Enteritidis bacteria after application to food, and the lack of clinical symptoms of this phage suggest that wksl3 may be a useful agent for the protection of foods againstS. Enteritidis andS. Typhimurium contamination.
Efficient Production of Active Polyhydroxyalkanoate Synthase in Escherichia coli by Coexpression of Molecular Chaperones
Authors: Thomson Nicholas M. , Saika Azusa , Ushimaru Kazunori , Sangiambut Smith , Tsuge Takeharu , Summers David K. , Sivaniah Easan
Published: January 2013

The type I polyhydroxyalkanoate synthase from
Cupriavidus necator
was heterologously expressed in
Escherichia coli
with simultaneous overexpression of chaperone proteins. Compared to expression of synthase alone (14.55 mg liter
−1
), coexpression with chaperones resulted in the production of larger total quantities of enzyme, including a larger proportion in the soluble fraction. The largest increase was seen when the GroEL/GroES system was coexpressed, resulting in approximately 6-fold-greater enzyme yields (82.37 mg liter
−1
) than in the absence of coexpressed chaperones. The specific activity of the purified enzyme was unaffected by coexpression with chaperones. Therefore, the increase in yield was attributed to an enhanced soluble fraction of synthase. Chaperones were also coexpressed with a polyhydroxyalkanoate production operon, resulting in the production of polymers with generally reduced molecular weights. This suggests a potential use for chaperones to control the physical properties of the polymer.
Directed Construction and Analysis of a Sinorhizobium meliloti pSymA Deletion Mutant Library
Authors: Yurgel Svetlana N. , Mortimer Michael W. , Rice Jennifer T. , Humann Jodi L. , Kahn Michael L.
Published: January 2013

Resources from the
Sinorhizobium meliloti
Rm1021 open reading frame (ORF) plasmid libraries were used in a medium-throughput method to construct a set of 50 overlapping deletion mutants covering all of the Rm1021 pSymA megaplasmid except the replicon region. Each resulting pSymA derivative carried a defined deletion of approximately 25 ORFs. Various phenotypes, including cytochrome
c
respiration activity, the ability of the mutants to grow on various carbon and nitrogen sources, and the symbiotic effectiveness of the mutants with alfalfa, were analyzed. This approach allowed us to systematically evaluate the potential impact of regions of Rm1021 pSymA for their free-living and symbiotic phenotypes.
Defining the Bacteroides Ribosomal Binding Site
Authors: Wegmann Udo , Horn Nikki , Carding Simon R.
Published: January 2013

The human gastrointestinal tract, in particular the colon, hosts a vast number of commensal microorganisms. Representatives of the genus
Bacteroides
are among the most abundant bacterial species in the human colon.
Bacteroidetes
diverged from the common line of eubacterial descent before other eubacterial groups. As a result, they employ unique transcription initiation signals and, because of this uniqueness, they require specific genetic tools. Although some tools exist, they are not optimal for studying the roles and functions of these bacteria in the human gastrointestinal tract. Focusing on translation initiation signals in
Bacteroides
, we created a series of expression vectors allowing for different levels of protein expression in this genus, and we describe the use of
pepI
from
Lactobacillus delbrueckii
subsp.
lactis
as a novel reporter gene for
Bacteroides
. Furthermore, we report the identification of the 3′ end of the 16S rRNA of
Bacteroides ovatus
and analyze in detail its ribosomal binding site, thus defining a core region necessary for efficient translation, which we have incorporated into the design of our expression vectors. Based on the sequence logo information from the 5′ untranslated region of other
Bacteroidales
ribosomal protein genes, we conclude that our findings are relevant to all members of this order.
Involvement of Intermediate Sulfur Species in Biological Reduction of Elemental Sulfur under Acidic, Hydrothermal Conditions
Authors: Boyd Eric S. , Druschel Gregory K.
Published: January 2013

The thermoacidophile and obligate elemental sulfur (S
8
0
)-reducing anaerobe
Acidilobus sulfurireducens
18D70 does not associate with bulk solid-phase sulfur during S
8
0
-dependent batch culture growth. Cyclic voltammetry indicated the production of hydrogen sulfide (H
2
S) as well as polysulfides after 1 day of batch growth of the organism at pH 3.0 and 81ï¿½C. The production of polysulfide is likely due to the abiotic reaction between S
8
0
and the biologically produced H
2
S, as evinced by a rapid cessation of polysulfide formation when the growth temperature was decreased, inhibiting the biological production of sulfide. After an additional 5 days of growth, nanoparticulate S
8
0
was detected in the cultivation medium, a result of the hydrolysis of polysulfides in acidic medium. To examine whether soluble polysulfides and/or nanoparticulate S
8
0
can serve as terminal electron acceptors (TEA) supporting the growth of
A. sulfurireducens
, total sulfide concentration and cell density were monitored in batch cultures with S
8
0
provided as a solid phase in the medium or with S
8
0
sequestered in dialysis tubing. The rates of sulfide production in 7-day-old cultures with S
8
0
sequestered in dialysis tubing with pore sizes of 12 to 14 kDa and 6 to 8 kDa were 55% and 22%, respectively, of that of cultures with S
8
0
provided as a solid phase in the medium. These results indicate that the TEA existed in a range of particle sizes that affected its ability to diffuse through dialysis tubing of different pore sizes. Dynamic light scattering revealed that S
8
0
particles generated through polysulfide rapidly grew in size, a rate which was influenced by the pH of the medium and the presence of organic carbon. Thus, S
8
0
particles formed through abiological hydrolysis of polysulfide under acidic conditions appeared to serve as a growth-promoting TEA for
A. sulfurireducens
.
Thaumarchaeotal Signature Gene Distribution in Sediments of the Northern South China Sea: an Indicator of the Metabolic Intersection of the Marine Carbon, Nitrogen, and Phosphorus Cycles?
Authors: Dang Hongyue , Zhou Haixia , Yang Jinying , Ge Huangmin , Jiao Nianzhi , Luan Xiwu , Zhang Chuanlun , Klotz Martin G.
Published: January 2013

Thaumarchaeota are abundant and active in marine waters, where they contribute to aerobic ammonia oxidation and light-independent carbon fixation. The ecological function of thaumarchaeota in marine sediments, however, has rarely been investigated, even though marine sediments constitute the majority of the Earth's surface. Thaumarchaeota in the upper layer of sediments may contribute significantly to the reservoir of nitrogen oxides in ocean waters and thus to productivity, including the assimilation of carbon. We tested this hypothesis in the northern South China Sea (nSCS), a section of a large oligotrophic marginal sea with limited influx of nutrients, including nitrogen, by investigating the diversity, abundance, community structure, and spatial distribution of thaumarchaeotal signatures in surface sediments. Quantitative real-time PCR using primers designed to detect 16S rRNA and
amoA
genes in sediment community DNA revealed a significantly higher abundance of pertinent thaumarchaeotal than betaproteobacterial genes. This finding correlates with high levels of
hcd
genes, a signature of thaumarchaeotal autotrophic carbon fixation. Thaumarchaeol, a signature lipid biomarker for thaumarchaeota, constituted the majority of archaeal lipids in marine sediments. Sediment temperature and organic P and silt contents were identified as key environmental factors shaping the community structure and distribution of the monitored thaumarchaeotal
amoA
genes. When the pore water PO
4
3−
concentration was controlled for via partial-correlation analysis, thaumarchaeotal
amoA
gene abundance significantly correlated with the sediment pore water NO
2
−
concentration, suggesting that the
amoA
-bearing thaumarchaeota contribute to nitrite production. Statistical analyses also suggest that thaumarchaeotal metabolism could serve as a pivotal intersection of the carbon, nitrogen, and phosphorus cycles in marine sediments.
Distinct Actions by Paenibacillus sp. Strain E18 α-
l
-Arabinofuranosidases and Xylanase in Xylan Degradation
Authors: Shi Pengjun , Chen Xiaoyan , Meng Kun , Huang Huoqing , Bai Yingguo , Luo Huiying , Yang Peilong , Yao Bin
Published: January 2013

We cloned a
Paenibacillus
sp. strain E18 5.3-kb xylanolytic gene cluster that contains three open reading frames encoding two family 43 α-
l
-arabinofuranosidases (Abf43A and Abf43B) and one family 10 xylanase (XynBE18). The deduced amino acid sequences of Abf43A and Abf43B were at most 68% and 63% identical to those of two putative family 43 proteins from
Clostridium
sp. strain DL-VIII (EHI98634.1 and EHI98635.1), respectively, but were only 11% identical to each other. Recombinant Abf43A and Abf43B had similar activities at 45ï¿½C and pH 6.0 but varied in thermostabilities and substrate specificities. Abf43B was active against only 4-nitrophenyl α-
l
-arabinofuranoside, whereas Abf43A acted on 4-nitrophenyl α-
l
-arabinofuranoside, wheat arabinoxylan, 4-nitrophenyl α-
d
-xylopyranoside, and sugar beet arabinan. The sequential and combined effects on xylan degradation by XynBE18, Abf43A, and Abf43B were characterized. For beechwood, birchwood, and oat spelt xylans as the substrates, synergistic effects were found when XynBE18 and Abf43A or Abf43B were incubated together and when the substrates were first incubated with Abf43A or Abf43B and then with XynBE18. Further high-performance liquid chromatography (HPLC) analysis showed that the amounts of xylobiose and xylose increased sharply in the aforementioned reactions. For water-soluble wheat arabinoxylan as the substrate, Abf43A not only released arabinose but also had a synergistic effect with XynBE18. Synergy may arise as the result of removal of arabinose residues from xylans by α-
l
-arabinofuranosidases, which eliminates steric hindrance caused by the arabinose side chains and which allows xylanases to then degrade the xylan backbone, producing short xylooligosaccharides.
Evolutionary Acquisition and Loss of Saxitoxin Biosynthesis in Dinoflagellates: the Second “Core” Gene,
sxtG
Authors: Orr Russell J. S. , Stï¿½ken Anke , Murray Shauna A. , Jakobsen Kjetill S.
Published: January 2013

Saxitoxin and its derivatives are potent neurotoxins produced by several cyanobacteria and dinoflagellate species. SxtA is the initial enzyme in the biosynthesis of saxitoxin. The dinoflagellate full mRNA and partial genomic sequences have previously been characterized, and it appears that
sxtA
originated in dinoflagellates through a horizontal gene transfer from a bacterium. So far, little is known about the remaining genes involved in this pathway in dinoflagellates. Here we characterize
sxtG
, an amidinotransferase enzyme gene that putatively encodes the second step in saxitoxin biosynthesis. In this study, the entire
sxtG
transcripts from
Alexandrium fundyense
CCMP1719 and
Alexandrium minutum
CCMP113 were amplified and sequenced. The transcripts contained typical dinoflagellate spliced leader sequences and eukaryotic poly(A) tails. In addition, partial
sxtG
transcript fragments were amplified from four additional
Alexandrium
species and
Gymnodinium catenatum
. The phylogenetic inference of dinoflagellate
sxtG
, congruent with
sxtA
, revealed a bacterial origin. However, it is not known if
sxtG
was acquired independently of
sxtA
. Amplification and sequencing of the corresponding genomic
sxtG
region revealed noncanonical introns. These introns show a high interspecies and low intraspecies variance, suggesting multiple independent acquisitions and losses. Unlike
sxtA
,
sxtG
was also amplified from
Alexandrium
species not known to synthesize saxitoxin. However, amplification was not observed for 22 non-saxitoxin-producing dinoflagellate species other than those of the genus
Alexandrium
or
G. catenatum
. This result strengthens our hypothesis that saxitoxin synthesis has been secondarily lost in conjunction with
sxtA
for some descendant species.
CO-Dependent H
2
Production by Genetically Engineered Thermococcus onnurineus NA1
Authors: Kim Min-Sik , Bae Seung Seob , Kim Yun Jae , Kim Tae Wan , Lim Jae Kyu , Lee Seong Hyuk , Choi Ae Ran , Jeon Jeong Ho , Lee Jung-Hyun , Lee Hyun Sook , Kang Sung Gyun
Published: January 2013

Hydrogenogenic CO oxidation (CO + H
2
O → CO
2
+ H
2
) has the potential for H
2
production as a clean renewable fuel.
Thermococcus onnurineus
NA1, which grows on CO and produces H
2
, has a unique gene cluster encoding the carbon monoxide dehydrogenase (CODH) and the hydrogenase. The gene cluster was identified as essential for carboxydotrophic hydrogenogenic metabolism by gene disruption and transcriptional analysis. To develop a strain producing high levels of H
2
, the gene cluster was placed under the control of a strong promoter. The resulting mutant, MC01, showed 30-fold-higher transcription of the mRNA encoding CODH, hydrogenase, and Na
+
/H
+
antiporter and a 1.8-fold-higher specific activity for CO-dependent H
2
production than did the wild-type strain. The H
2
production potential of the MC01 mutant in a bioreactor culture was 3.8-fold higher than that of the wild-type strain. The H
2
production rate of the engineered strain was severalfold higher than those of any other CO-dependent H
2
-producing prokaryotes studied to date. The engineered strain also possessed high activity for the bioconversion of industrial waste gases created as a by-product during steel production. This work represents the first demonstration of H
2
production from steel mill waste gas using a carboxydotrophic hydrogenogenic microbe.
Catabolism of
N
-Acetylneuraminic Acid, a Fitness Function of the Food-Borne Lactic Acid Bacterium Lactobacillus sakei, Involves Two Newly Characterized Proteins
Authors: Anba-Mondoloni Jamila , Chaillou Stï¿½phane , Zagorec Monique , Champomier-Vergï¿½s Marie-Christine
Published: January 2013

In silico
analysis of the genome sequence of the meat-borne lactic acid bacterium (LAB)
Lactobacillus sakei
23K has revealed a repertoire of potential functions related to the adaptation of this bacterium to the meat environment. Among these functions, the ability to use
N
-acetyl-neuraminic acid (NANA) as a carbon source could provide a competitive advantage for growth on meat in which this amino sugar is present. In this work, we proposed to analyze the functionality of a gene cluster encompassing
nanTEAR
and
nanK
(
nanTEAR-nanK
). We established that this cluster encoded a pathway allowing transport and early steps of the catabolism of NANA in this genome. We also demonstrated that this cluster was absent from the genome of other
L. sakei
strains that were shown to be unable to grow on NANA. Moreover,
L. sakei
23K
nanA
,
nanT
,
nanK
, and
nanE
genes were able to complement
Escherichia coli
mutants. Construction of different mutants in
L. sakei
23K Δ
nanR
, Δ
nanT
, and Δ
nanK
and the double mutant
L. sakei
23K Δ(
nanA-nanE
) made it possible to show that all were impaired for growth on NANA. In addition, two genes located downstream from
nanK
,
lsa1644
and
lsa1645
, are involved in the catabolism of sialic acid in
L. sakei
23K, as a
L. sakei
23K Δ
lsa1645
mutant was no longer able to grow on NANA. All these results demonstrate that the gene cluster
nanTEAR-nanK-lsa1644-lsa1645
is indeed involved in the use of NANA as an energy source by
L. sakei
.
Selection for Loss of RpoS in Cronobacter sakazakii by Growth in the Presence of Acetate as a Carbon Source
Authors: ï¿½lvarez-Ordï¿½ï¿½ez Avelino , Begley Mï¿½ire , Hill Colin
Published: January 2013

We demonstrate that growth of
Cronobacter sakazakii
in the presence of acetate as a carbon source promotes loss of RpoS, with a consequent reduction in stress tolerance. This suggests that
C. sakazakii
is capable of regulating cell fitness through mutation of the
rpoS
gene.
Characterization of the Biosynthetic Genes for 10,11-Dehydrocurvularin, a Heat Shock Response-Modulating Anticancer Fungal Polyketide from Aspergillus terreus
Authors: Xu Yuquan , Espinosa-Artiles Patricia , Schubert Vivien , Xu Ya-ming , Zhang Wei , Lin Min , Gunatilaka A. A. Leslie , Sï¿½ssmuth Roderich , Molnï¿½r Istvï¿½n
Published: January 2013

10,11-Dehydrocurvularin is a prevalent fungal phytotoxin with heat shock response and immune-modulatory activities. It features a dihydroxyphenylacetic acid lactone polyketide framework with structural similarities to resorcylic acid lactones like radicicol or zearalenone. A genomic locus was identified from the dehydrocurvularin producer strain
Aspergillus terreus
AH-02-30-F7 to reveal genes encoding a pair of iterative polyketide synthases (
A. terreus
CURS1 [AtCURS1] and AtCURS2) that are predicted to collaborate in the biosynthesis of 10,11-dehydrocurvularin. Additional genes in this locus encode putative proteins that may be involved in the export of the compound from the cell and in the transcriptional regulation of the cluster. 10,11-Dehydrocurvularin biosynthesis was reconstituted in
Saccharomyces cerevisiae
by heterologous expression of the polyketide synthases. Bioinformatic analysis of the highly reducing polyketide synthase AtCURS1 and the nonreducing polyketide synthase AtCURS2 highlights crucial biosynthetic programming differences compared to similar synthases involved in resorcylic acid lactone biosynthesis. These differences lead to the synthesis of a predicted tetraketide starter unit that forms part of the 12-membered lactone ring of dehydrocurvularin, as opposed to the penta- or hexaketide starters in the 14-membered rings of resorcylic acid lactones. Tetraketide
N
-acetylcysteamine thioester analogues of the starter unit were shown to support the biosynthesis of dehydrocurvularin and its analogues, with yeast expressing AtCURS2 alone. Differential programming of the product template domain of the nonreducing polyketide synthase AtCURS2 results in an aldol condensation with a different regiospecificity than that of resorcylic acid lactones, yielding the dihydroxyphenylacetic acid scaffold characterized by an S-type cyclization pattern atypical for fungal polyketides.
RubisCO Gene Clusters Found in a Metagenome Microarray from Acid Mine Drainage
Authors: Guo Xue , Yin Huaqun , Cong Jing , Dai Zhimin , Liang Yili , Liu Xueduan
Published: January 2013

The enzyme responsible for carbon dioxide fixation in the Calvin cycle, ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), is always detected as a phylogenetic marker to analyze the distribution and activity of autotrophic bacteria. However, such an approach provides no indication as to the significance of genomic content and organization. Horizontal transfers of RubisCO genes occurring in eubacteria and plastids may seriously affect the credibility of this approach. Here, we presented a new method to analyze the diversity and genomic content of RubisCO genes in acid mine drainage (AMD). A metagenome microarray containing 7,776 large-insertion fosmids was constructed to quickly screen genome fragments containing RubisCO form I large-subunit genes (
cbbL
). Forty-six
cbbL
-containing fosmids were detected, and six fosmids were fully sequenced. To evaluate the reliability of the metagenome microarray and understand the microbial community in AMD, the diversities of
cbbL
and the 16S rRNA gene were analyzed. Fosmid sequences revealed that the form I RubisCO gene cluster could be subdivided into form IA and IB RubisCO gene clusters in AMD, because of significant divergences in molecular phylogenetics and conservative genomic organization. Interestingly, the form I RubisCO gene cluster coexisted with the form II RubisCO gene cluster in one fosmid genomic fragment. Phylogenetic analyses revealed that horizontal transfers of RubisCO genes may occur widely in AMD, which makes the evolutionary history of RubisCO difficult to reconcile with organismal phylogeny.
Interactions of Nitrifying Bacteria and Heterotrophs: Identification of a Micavibrio-Like Putative Predator of Nitrospira spp
Authors: Dolinšek Jan , Lagkouvardos Ilias , Wanek Wolfgang , Wagner Michael , Daims Holger
Published: January 2013

Chemolithoautotrophic nitrifying bacteria release soluble organic compounds, which can be substrates for heterotrophic microorganisms. The identities of these heterotrophs and the specificities of their interactions with nitrifiers are largely unknown. In this study, we incubated nitrifying activated sludge with
13
C-labeled bicarbonate and used stable isotope probing of 16S rRNA to monitor the flow of carbon from uncultured nitrifiers to heterotrophs. To facilitate the identification of heterotrophs, the abundant 16S rRNA molecules from nitrifiers were depleted by catalytic oligonucleotides containing locked nucleic acids (LNAzymes), which specifically cut the 16S rRNA of defined target organisms. Among the
13
C-labeled heterotrophs were organisms remotely related to
Micavibrio
, a microbial predator of Gram-negative bacteria. Fluorescence
in situ
hybridization revealed a close spatial association of these organisms with microcolonies of nitrite-oxidizing sublineage I
Nitrospira
in sludge flocs. The high specificity of this interaction was confirmed by confocal microscopy and a novel image analysis method to quantify the localization patterns of biofilm microorganisms in three-dimensional (3-D) space. Other isotope-labeled bacteria, which were affiliated with
Thermomonas
, colocalized less frequently with nitrifiers and thus were commensals or saprophytes rather than specific symbionts or predators. These results suggest that
Nitrospira
spp. are subject to bacterial predation, which may influence the abundance and diversity of these nitrite oxidizers and the stability of nitrification in engineered and natural ecosystems.
In silico
screening of published next-generation sequencing data sets revealed a broad environmental distribution of the uncultured
Micavibrio
-like lineage.
Characterization of the Denitrification-Associated Phosphorus Uptake Properties of “Candidatus Accumulibacter phosphatis” Clades in Sludge Subjected to Enhanced Biological Phosphorus Removal
Authors: Kim Jeong Myeong , Lee Hyo Jung , Lee Dae Sung , Jeon Che Ok
Published: January 2013

To characterize the denitrifying phosphorus (P) uptake properties of “
Candidatus
Accumulibacter phosphatis,” a sequencing batch reactor (SBR) was operated with acetate. The SBR operation was gradually acclimated from anaerobic-oxic (AO) to anaerobic-anoxic-oxic (A2O) conditions by stepwise increases of nitrate concentration and the anoxic time. The communities of “
Ca
. Accumulibacter” and associated bacteria at the initial (AO) and final (A2O) stages were compared using 16S rRNA and polyphosphate kinase genes and using fluorescence
in situ
hybridization (FISH). The acclimation process led to a clear shift in the relative abundances of recognized “
Ca
. Accumulibacter” subpopulations from clades IIA > IA > IIF to clades IIC > IA > IIF, as well as to increases in the abundance of other associated bacteria (
Dechloromonas
[from 1.2% to 19.2%] and “
Candidatus
Competibacter phosphatis” [from 16.4% to 20.0%]), while the overall “
Ca
. Accumulibacter” abundance decreased (from 55.1% to 29.2%). A series of batch experiments combined with FISH/microautoradiography (MAR) analyses was performed to characterize the denitrifying P uptake properties of the “
Ca
. Accumulibacter” clades. In FISH/MAR experiments using slightly diluted sludge (∼0.5 g/liter), all “
Ca
. Accumulibacter” clades successfully took up phosphorus in the presence of nitrate. However, the “
Ca
. Accumulibacter” clades showed no P uptake in the presence of nitrate when the sludge was highly diluted (∼0.005 g/liter); under these conditions, reduction of nitrate to nitrite did not occur, whereas P uptake by “
Ca
. Accumulibacter” clades occurred when nitrite was added. These results suggest that the “
Ca
. Accumulibacter” cells lack nitrate reduction capabilities and that P uptake by “
Ca
. Accumulibacter” is dependent upon nitrite generated by associated nitrate-reducing bacteria such as
Dechloromonas
and “
Ca
. Competibacter.”
Arabinose Induces Pellicle Formation by Vibrio fischeri
Authors: Visick Karen L. , Quirke Kevin P. , McEwen Sheila M.
Published: January 2013

Biofilms are multicellular communities of bacteria attached to a surface and embedded in a protective matrix. In many cases, the signals that induce biofilm formation are unknown. Here, we report that biofilm formation by the marine bacterium
Vibrio fischeri
can be induced by the addition of arabinose to LBS (Luria-Bertani-salt), a tryptone-based medium. Growth of cells in the presence of 0.2% arabinose, but not other sugars, induced the production of a pellicle at the air/liquid interfaces of static cultures.
V. fischeri
failed to grow on arabinose as the sole carbon source, suggesting that pellicle production did not occur as a result of increased growth, but experiments using the acid/base indicator phenol red suggested that
V. fischeri
may partially metabolize arabinose. Pellicle production was independent of the
syp
polysaccharide locus but was altered upon disruption of the
bcs
cellulose locus. Through a screen for mutants defective for pellicle production, we found that loss of motility disrupted the formation of the arabinose-induced pellicle. Among the ∼20 mutants that retained motility were strains with insertions in a putative
msh
pilus locus and a strain with a defect in
yidK
, which is involved in galactose catabolism. Mutants with the
msh
gene disrupted grew poorly in the presence of arabinose, while the
yidK
mutant appeared to be “blind” to the presence of arabinose. Finally, arabinose impaired symbiotic colonization by
V. fischeri
. This work thus identifies a novel signal and new pathways involved in control of biofilm formation by
V. fischeri
.
Characterization of a Novel Metagenome-Derived 6-Phospho-β-Glucosidase from Black Liquor Sediment
Authors: Yang Chunyu , Niu Yu , Li Chunfang , Zhu Deyu , Wang Wei , Liu Xinqiang , Cheng Bin , Ma Cuiqing , Xu Ping
Published: January 2013

The enzyme 6-phospho-β-glucosidase is an important member of the glycoside hydrolase family 1 (GH1). However, its catalytic mechanisms, especially the key residues determining substrate specificity and affinity, are poorly understood. A metagenome-derived gene sequence, encoding a novel 6-phospho-β-glucosidase designated Pbgl25-217, was isolated and characterized. The optimal conditions for enzymatic activity were 37ï¿½C and pH 7; Ca
2+
, Mg
2+
, and Mn
2+
stabilized the activity of Pbgl25-217, whereas Ni
2+
, Fe
2+
, Zn
2+
, Cu
2+
, and Fe
3+
inhibited its activity. The

K
m

and
V
max
of Pbgl25-217 were 4.8 mM and 1,987.0 U mg
−1
, respectively. Seven conserved residues were recognized by multiple alignments and were tested by site-directed mutagenesis for their functions in substrate recognition and catalytic reaction. The results suggest that residues S427, Lys435, and Tyr437 act as “gatekeepers” in a phosphate-binding loop and play important roles in phosphate recognition. This functional identification may provide insights into the specificity of 6-phospho-β-glycosidases in GH1 and be useful for designing further directed evolution.
Effects of Temperature and pH on Reduction of Bacteria in a Point-of-Use Drinking Water Treatment Product for Emergency Relief
Authors: Marois-Fiset Jean-Thomas , Carabin Anne , Lavoie Audrey , Dorea Caetano C.
Published: January 2013

The effects of temperature and pH on the water treatment performance of a point-of-use (POU) coagulant/disinfectant product were evaluated. Cold temperatures (∼5ï¿½C) reduced the bactericidal efficiency of the product with regard to
Escherichia coli
and total coliform log
10
reductions.
Development of a Continuous Bioconversion System Using a Thermophilic Whole-Cell Biocatalyst
Authors: Ninh Pham Huynh , Honda Kohsuke , Yokohigashi Yukako , Okano Kenji , Omasa Takeshi , Ohtake Hisao
Published: January 2013

The heat treatment of recombinant mesophilic cells having heterologous thermophilic enzymes results in the denaturation of indigenous mesophilic enzymes and the elimination of undesired side reactions; therefore, highly selective whole-cell catalysts comparable to purified enzymes can be readily prepared. However, the thermolysis of host cells leads to the heat-induced leakage of thermophilic enzymes, which are produced as soluble proteins, limiting the exploitation of their excellent stability in repeated and continuous reactions. In this study,
Escherichia coli
cells having the thermophilic fumarase from
Thermus thermophilus
(
Tt
FTA) were treated with glutaraldehyde to prevent the heat-induced leakage of the enzyme, and the resulting cells were used as a whole-cell catalyst in repeated and continuous reactions. Interestingly, although electron microscopic observations revealed that the cellular structure of glutaraldehyde-treated
E. coli
was not apparently changed by the heat treatment, the membrane permeability of the heated cells to relatively small molecules (up to at least 3 kDa) was significantly improved. By applying the glutaraldehyde-treated
E. coli
having
Tt
FTA to a continuous reactor equipped with a cell-separation membrane filter, the enzymatic hydration of fumarate to malate could be operated for more than 600 min with a molar conversion yield of 60% or higher.
In Situ
Determination of Clostridium Endospore Membrane Fluidity during Pressure-Assisted Thermal Processing in Combination with Nisin or Reutericyclin
Authors: Hofstetter S. , Winter R. , McMullen L. M. , Gï¿½nzle M. G.
Published: January 2013

This study determined the membrane fluidity of clostridial endospores during treatment with heat and pressure with nisin or reutericyclin. Heating (90ï¿½C) reduced laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) general polarization, corresponding to membrane fluidization. Pressure (200 MPa) stabilized membrane order. Reutericyclin and nisin exhibit divergent effects on heat- and pressure-induced spore inactivation and membrane fluidity.
Bifunctional Gene Cluster
lnqBCDEF
Mediates Bacteriocin Production and Immunity with Differential Genetic Requirements
Authors: Iwatani Shun , Horikiri Yuko , Zendo Takeshi , Nakayama Jiro , Sonomoto Kenji
Published: January 2013

A comprehensive gene disruption of lacticin Q biosynthetic cluster
lnqQBCDEF
was carried out. The results demonstrated the necessity of the complete set of
lnqQBCDEF
for lacticin Q production, whereas immunity was flexible, with LnqEF (ABC transporter) being essential for and LnqBCD partially contributing to immunity.
Rates of Species Accumulation and Taxonomic Diversification during Phototrophic Biofilm Development Are Controlled by both Nutrient Supply and Current Velocity
Authors: Larson Chad A. , Passy Sophia I.
Published: January 2013

The accumulation of new and taxonomically diverse species is a marked feature of community development, but the role of the environment in this process is not well understood. To address this problem, we subjected periphyton in laboratory streams to low (10-cm ï¿½ s−1), high (30-cm ï¿½ s−1), and variable (9- to 32-cm ï¿½ s−1) current velocity and low- versus high-nutrient inputs. We examined how current velocity and resource supply constrained (i) the rates of species accumulation, a measure of temporal beta-diversity, and (ii) the rates of diversification of higher taxonomic categories, defined here as the rate of higher taxon richness increase with the increase of species richness. Temporal biofilm dynamics were controlled by a strong nutrient-current interaction. Nutrients accelerated the rates of accumulation of new species, when flow velocity was not too stressful. Species were more taxonomically diverse under variable than under low-flow conditions, indicating that flow heterogeneity increased the niche diversity in the high-nutrient treatments. Conversely, the lower diversification rates under high- than under low-nutrient conditions at low velocity are explained with finer resource partitioning among species, belonging to a limited number of related genera. The overall low rates of diversification in high-current treatments suggest that the ability to withstand current stress was conserved within closely related species. Temporal heterogeneity of disturbance has been shown to promote species richness, but here we further demonstrate that it also affects two other components of biodiversity, i.e., temporal beta-diversity and diversification rate. Therefore, management efforts for preserving the inherent temporal heterogeneity of natural ecosystems will have detectable positive effects on biodiversity.
Cloning of a Novel Nicotine Oxidase Gene from Pseudomonas sp. Strain HZN6 Whose Product Nonenantioselectively Degrades Nicotine to Pseudooxynicotine
Authors: Qiu Jiguo , Ma Yun , Zhang Jing , Wen Yuezhong , Liu Weiping
Published: January 2013

Pseudomonas
sp. strain HZN6 utilizes nicotine as its sole source of carbon, nitrogen, and energy. However, its catabolic mechanism has not been elucidated. In this study, self-formed adaptor PCR was performed to amplify the upstream sequence of the pseudooxynicotine amine oxidase gene. A 1,437-bp open reading frame (designated
nox
) was found to encode a nicotine oxidase (NOX) that shows 30% amino acid sequence identity with 6-hydroxy-l-nicotine oxidase from
Arthrobacter nicotinovorans
. The
nox
gene was cloned into a broad-host-range cloning vector and transferred into the non-nicotine-degrading bacteria
Escherichia coli
DH5α (DH-nox) and
Pseudomonas putida
KT2440 (KT-nox). The transconjugant KT-nox obtained nicotine degradation ability and yielded an equimolar amount of pseudooxynicotine, while DH-nox did not. Reverse transcription-PCR showed that the
nox
gene is expressed in both DH5α and KT2440, suggesting that additional factors required for nicotine degradation are present in a
Pseudomonas
strain(s), but not in
E. coli
. The mutant of strain HZN6 with
nox
disrupted lost the ability to degrade nicotine, but not pseudooxynicotine. These results suggested that the
nox
gene is responsible for the first step of nicotine degradation. The (
RS
)-nicotine degradation results showed that the two enantiomers were degraded at approximately the same rate, indicating that NOX does not show chiral selectivity. Site-directed mutagenesis revealed that both the conserved flavin adenine dinucleotide (FAD)-binding GXGXXG motif and His456 are essential for nicotine degradation activity.
Persistence of the 2009 Pandemic Influenza A (H1N1) Virus on N95 Respirators
Authors: Coulliette A. D. , Perry K. A. , Edwards J. R. , Noble-Wang J. A.
Published: January 2013

In the United States, the 2009 pandemic influenza A (H1N1) virus (pH1N1) infected almost 20% of the population and caused >200,000 hospitalizations and >10,000 deaths from April 2009 to April 2010. On 24 April 2009, the CDC posted interim guidance on infection control measures in health care settings explicitly for pH1N1 and recommended using filtering face respirators (FFRs) when in close contact with a suspected- or confirmed-to-be-infected individual, particularly when performing aerosol-generating procedures. The persistence and infectivity of pH1N1 were evaluated on FFRs, specifically N95 respirators, under various conditions of absolute humidity (AH) (4.1 ï¿½ 10
5
mPa, 6.5 ï¿½ 10
5
mPa, and 14.6 ï¿½ 10
5
mPa), sample matrices (2% fetal bovine serum [FBS], 5 mg/ml mucin, and viral medium), and times (4, 12, 24, 48, 72, and 144 h). pH1N1 was distributed onto N95 coupons (3.8 to 4.2 cm
2
) and extracted by a vortex-centrifugation-filtration process, and the ability of the remaining virus to replicate was quantified using an enzyme-linked immunosorbent assay (ELISA) to determine the log
10
concentration of the infectious virus per coupon. Overall, pH1N1 remained infectious for 6 days, with an approximately 1-log
10
loss of virus concentrations over this time period. Time and AH both affected virus survival. We found significantly higher (
P
≤ 0.01) reductions in virus concentrations at time points beyond 24 to 72 h (−0.52-log
10
reduction) and 144 h (−0.74) at AHs of 6.5 ï¿½ 10
5
mPa (−0.53) and 14.6 ï¿½ 10
5
mPa (−0.47). This research supports discarding respirators after close contact with a person with suspected or confirmed influenza infection due to the virus's demonstrated ability to persist and remain infectious.
Inactivation of Pathogens in Feces by Desiccation and Urea Treatment for Application in Urine-Diverting Dry Toilets
Authors: Magri Maria Elisa , Philippi Luiz Sï¿½rgio , Vinnerï¿½s Bjï¿½rn
Published: January 2013

Ecological sanitation technologies can be effective in providing health and environmental pollution control if they can efficiently reduce the pathogenicity of microorganisms carried in fecal material to safe levels. This study evaluated the sanitizing effects of different additives for dry treatment of feces from urine-diverting dry toilets, based on inactivation ofEnterococcus faecalis,Salmonella entericaserovar Typhimurium, bacteriophages MS2 and ΦX, andAscaris suum. The additives, ash (A) and oyster shell (O) in different amounts and urea (U) to optimize the process, were compared with no additive, solely urea, and sawdust as controls (C) and were covered ([x%O:A]) or uncovered (x%O:A). The main inactivation factors found were desiccation, ammonia content, and pH.S.Typhimurium andE. faecaliswere more affected by the ammonia content. A combination of neutral to high pH and desiccation was most effective for inactivation of MS2, and desiccation was most effective for inactivation of ΦX andA. suum. The inactivation rate was modeled for all combinations studied. The most promising treatments were [150%O:A+U], 150%O:A+U, and 150%O:A. According to the models, these could inactivate, for example, 7 log10units of all bacteria and bacteriophages within 83, 125, and 183 days, respectively. The inactivation ofA. suumwas modeled, albeit the measured decay in egg viability was low.
Dual Roles of Pseudomonas aeruginosa AlgE in Secretion of the Virulence Factor Alginate and Formation of the Secretion Complex
Authors: Rehman Zahid U. , Rehm Bernd H. A.
Published: January 2013

AlgE is a monomeric 18-stranded β-barrel protein required for secretion of the extracellular polysaccharide alginate in
Pseudomonas aeruginosa
. To assess the molecular mechanism of alginate secretion, AlgE was subjected to site-specific and FLAG epitope insertion mutagenesis. Except for β-strands 6 and 10, epitope insertions into the transmembrane β-strands abolished localization of AlgE to the outer membrane. Interestingly, an epitope insertion into β-strand 10 produced alginate and was only detectable in outer membranes isolated from cells grown on solid media. The deletion of nine C-terminal amino acid residues destabilized AlgE. Replacement of amino acids that constitute the highly electropositive pore constriction showed that individual amino acid residues have a specific function in alginate secretion. Two of the triple mutants (K47E+R353A+R459E and R74E+R362A+R459E) severely reduced alginate production. Mutual stability analysis using the
algE
deletion mutant PDO300Δ
algE
(miniCTX) showed the periplasmic alginate biosynthesis proteins AlgK and AlgX were completely destabilized, while the copy number of the inner membrane c-di-GMP receptor Alg44 was reduced. Chromosomal integration of
algE
restored AlgK, AlgX, and Alg44, providing evidence for a multiprotein complex that spans the cell envelope. Periplasmic turn 4 of AlgE was identified as an important region for maintaining the stability of the putative multiprotein complex.
Uniformity of Glycyl Bridge Lengths in the Mature Cell Walls of Fem Mutants of Methicillin-Resistant Staphylococcus aureus
Authors: Sharif Shasad , Kim Sung Joon , Labischinski Harald , Chen Jiawei , Schaefer Jacob
Published: January 2013

Peptidoglycan (PG) composition in intact cells of methicillin-resistant
Staphylococcus aureus
(MRSA) and its isogenic Fem mutants has been characterized by measuring the glycine content of PG bridge structures by solid-state nuclear magnetic resonance (NMR). The glycine content estimated from integrated intensities (rather than peak heights) in the cell walls of whole cells was increased by approximately 30% for the FemA mutant and was reduced by 25% for the FemB mutant relative to expected values for homogeneous structures. In contrast, the expected compositions were observed in isolated cell walls of the same mutants. For FemA mutant whole cells, the increase was due to the presence of triglycyl bridge PG units (confirmed directly by mass spectrometric analysis), which constituted 10% of the total PG. These species were coalesced in some sort of a lattice or aggregate with spatial proximity to other PG bridges. This result suggests that the triglycyl-bridged PG units form a PG-like structure that is not incorporated into the mature cell wall.
Localization of FtsZ in Helicobacter pylori and Consequences for Cell Division
Authors: Specht Mara , Dempwolff Felix , Schätzle Sarah , Thomann Ralf , Waidner Barbara
Published: January 2013

Of the various kinds of cell division, the most common mode is binary fission, the division of a cell into two morphologically identical daughter cells. However, in the case of asymmetric cell division,
Caulobacter crescentus
produces two morphologically and functionally distinct cell types. Here, we have studied cell cycle progression of the human pathogen
Helicobacter pylori
using a functional green fluorescent protein (GFP) fusion of FtsZ protein and membrane staining. In small cells, representing newly divided cells, FtsZ localizes to a single cell pole. During the cell cycle, spiral intermediates are formed until an FtsZ ring is positioned with very little precision, such that central as well as acentral rings can be observed. Daughter cells showed considerably different sizes, suggesting that
H. pylori
divides asymmetrically. Fluorescence recovery after photobleaching (FRAP) analyses demonstrate that the
H. pylori
FtsZ ring is about as dynamic as that of
Escherichia coli
but that polar assemblies show less turnover. Strikingly, our results demonstrate that
H. pylori
cell division follows a different route from that in
E. coli
and
Bacillus subtilis.
It is also different from that in
C. crescentus
, where cytokinesis regulation proteins like MipZ play a role. Therefore, this report provides the first cell-biological analysis of FtsZ dynamics in the human pathogen
H. pylori
and even in epsilonproteobacteria to our knowledge. In addition, analysis of the filament architecture of
H. pylori
and
E. coli
FtsZ filaments in the heterologous system of
Drosophila melanogaster
S2 Schneider cells revealed that both have different filamentation properties
in vivo
, suggesting a unique intrinsic characteristic of each protein.
Xenocin Export by the Flagellar Type III Pathway in Xenorhabdus nematophila
Authors: Singh Preeti , Park Dongjin , Forst Steven , Banerjee Nirupama
Published: January 2013

The xenocin operon of
Xenorhabdus nematophila
consists of
xciA
and
ximB
genes encoding a 64-kDa xenocin and 42-kDa immunity protein to kill competing microbes in the insect larva. The catalytic domain of xenocin has RNase activity and is responsible for its cytotoxicity. Under SOS conditions, xenocin is produced with immunity protein as a complex. Here, we show that xenocin and immunity protein complex are exported through the flagellar type III system of
X. nematophila
. Secretion of xenocin complex was abolished in an
flhA
strain but not in an
fliC
strain. The xenocin operon is not linked to the flagellar operon transcriptionally. The immunity protein is produced alone from a second, constitutive promoter and is targeted to the periplasm in a flagellum-independent manner. For stable expression of xenocin, coexpression of immunity protein was necessary. To examine the role of immunity protein in xenocin export, an enzymatically inactive protein was produced by site-directed mutagenesis in the active site of the catalytic domain. Toxicity was abolished in D535A and H538A variants of xenocin, which were expressed alone without an immunity domain and secreted in the culture supernatant through flagellar export. Secretion of xenocin through the flagellar pathway has important implications in the evolutionary success of
X. nematophila
.