bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development of a Direct Isolation Procedure for Free-Living Diazotrophs under Controlled Hypoxic Conditions
Authors: Mirza Babur S. , Rodrigues Jorge L. M. 
Published: June 2012

ABSTRACT

Free-living diazotrophs are diverse and ubiquitous in soil, contributing the nitrogen pool in natural ecosystems. The isolation of nitrogen-fixing microorganisms has relied on semisolid nitrogen-free medium enrichment, followed by multiple subculturing steps. These procedures limit the diversity of recovered isolates. In the current study, we investigated three different isolation strategies for free-living diazotrophs using a soil sample from the Amazon forest. The methods were (i) direct plating on solid nitrogen-free medium under a 2% O
2
concentration, (ii) enrichment in semisolid nitrogen-free medium before plating on solid nitrogen-free medium under 2% O
2
, and (iii) enrichment followed by subculturing in the semisolid nitrogen-free medium before plating on nitrogen containing medium under a 21% O
2
concentration. A total of 794 isolates were differentiated by their genomic fingerprinting patterns, and strains with unique profiles were identified on the basis of sequencing of their 16S rRNA gene. Isolates belonged to four bacterial phyla:
Proteobacteria
,
Firmicutes
,
Actinobacteria
, and
Bacteriodetes
. The novel strategy of combining a solid N-free medium and hypoxic conditions showed an increase of 62.6% in the diversity of diazotrophs in comparison to that obtained by the conventional semisolid medium-based methods. All isolates grew on the nitrogen-free medium under a 2% O
2
concentration, 78% of them showed the presence of the
nifH
gene, and 39% tested positive for acetylene reduction activity. Our results suggest that direct plating of soil dilutions on nitrogen-free solid medium under a 2% O
2
concentration is a useful strategy for the isolation of the diverse diazotrophic communities.
Effects of Sequential and Simultaneous Applications of Bacteriophages on Populations of Pseudomonas aeruginosa
In Vitro
and in Wax Moth Larvae
Authors: Hall Alex R. , De Vos Daniel , Friman Ville-Petri , Pirnay Jean-Paul , Buckling Angus 
Published: June 2012

ABSTRACT

Interest in using bacteriophages to treat bacterial infections (phage therapy) is growing, but there have been few experiments comparing the effects of different treatment strategies on both bacterial densities and resistance evolution. While it is established that multiphage therapy is typically more effective than the application of a single phage type, it is not clear if it is best to apply phages simultaneously or sequentially. We tried single- and multiphage therapy against
Pseudomonas aeruginosa
PAO1
in vitro
, using different combinations of phages either simultaneously or sequentially. Across different phage combinations, simultaneous application was consistently equal or superior to sequential application in terms of reducing bacterial population density, and there was no difference (on average) in terms of minimizing resistance. Phage-resistant bacteria emerged in all experimental treatments and incurred significant fitness costs, expressed as reduced growth rate in the absence of phages. Finally, phage therapy increased the life span of wax moth larvae infected with
P. aeruginosa
, and a phage cocktail was the most effective short-term treatment. When the ratio of phages to bacteria was very high, phage cocktails cured otherwise lethal infections. These results suggest that while adding all available phages simultaneously tends to be the most successful short-term strategy, there are sequential strategies that are equally effective and potentially better over longer time scales.
Exploiting a Natural Auxotrophy for Genetic Selection
Authors: Ramage Elizabeth , Gallagher Larry , Manoil Colin 
Published: June 2012

ABSTRACT

We exploited the natural histidine auxotrophy of
Francisella
species to develop
hisD
(encodes histidinol dehydrogenase) as a positive selection marker. A shuttle plasmid (pBR103) carrying
Escherichia coli hisD
and designed for cloning of PCR fragments replicated in both attenuated and highly virulent
Francisella
strains. During this work, we formulated a simplified defined growth medium for
Francisella novicida
.
Actinobacterial Nitrate Reducers and Proteobacterial Denitrifiers Are Abundant in N
2
O-Metabolizing Palsa Peat
Authors: Palmer Katharina , Horn Marcus A. 
Published: June 2012

ABSTRACT

Palsa peats are characterized by elevated, circular frost heaves (peat soil on top of a permanently frozen ice lens) and are strong to moderate sources or even temporary sinks for the greenhouse gas nitrous oxide (N
2
O). Palsa peats are predicted to react sensitively to global warming. The acidic palsa peat Skalluvaara (approximate pH 4.4) is located in the discontinuous permafrost zone in northwestern Finnish Lapland.
In situ
N
2
O fluxes were spatially variable, ranging from 0.01 to −0.02 μmol of N
2
O m
−2
h
−1
. Fertilization with nitrate stimulated
in situ
N
2
O emissions and N
2
O production in anoxic microcosms without apparent delay. N
2
O was subsequently consumed in microcosms. Maximal reaction velocities (
v
max
) of nitrate-dependent denitrification approximated 3 and 1 nmol of N
2
O per h per gram (dry weight [g
DW
]) in soil from 0 to 20 cm and below 20 cm of depth, respectively.
v
max
values of nitrite-dependent denitrification were 2- to 5-fold higher than the
v
max
nitrate-dependent denitrification, and
v
max
of N
2
O consumption was 1- to 6-fold higher than that of nitrite-dependent denitrification, highlighting a high N
2
O consumption potential. Up to 12 species-level operational taxonomic units (OTUs) of
narG
,
nirK
and
nirS
, and
nosZ
were retrieved. Detected OTUs suggested the presence of diverse uncultured soil denitrifiers and dissimilatory nitrate reducers, hitherto undetected species, as well as
Actino
-,
Alpha
-, and
Betaproteobacteria
. Copy numbers of
nirS
always outnumbered those of
nirK
by 2 orders of magnitude. Copy numbers of
nirS
tended to be higher, while copy numbers of
narG
and
nosZ
tended to be lower in 0- to 20-cm soil than in soil below 20 cm. The collective data suggest that (i) the source and sink functions of palsa peat soils for N
2
O are associated with denitrification, (ii) actinobacterial nitrate reducers and
nirS
-type and
nosZ
-harboring proteobacterial denitrifiers are important players, and (iii) acidic soils like palsa peats represent reservoirs of diverse acid-tolerant denitrifiers associated with N
2
O fluxes.
A Synthetic Arabinose-Inducible Promoter Confers High Levels of Recombinant Protein Expression in Hyperthermophilic Archaeon Sulfolobus islandicus
Authors: Peng Nan , Deng Ling , Mei Yuxia , Jiang Dongqing , Hu Yongmei , Awayez Mariana , Liang Yunxiang , She Qunxin 
Published: June 2012

ABSTRACT

Despite major progresses in genetic studies of hyperthermophilic archaea, recombinant protein production in these organisms always suffers from low yields and a robust expression system is still in great demand. Here we report a versatile vector that confers high levels of protein expression in
Sulfolobus islandicus
, a hyperthermophilic crenarchaeon. Two expression vectors, pSeSD and pEXA, harboring 11 unique restriction sites were constructed. They contain coding sequences of two hexahistidine (6×His) peptide tags and those coding for two protease sites, the latter of which make it possible to remove the peptide tags from expressed recombinant proteins. While pEXA employed an
araS
promoter for protein expression, pSeSD utilized P

araS
-SD

, an
araS
derivative promoter carrying an engineered ribosome-binding site (RBS; a Shine-Dalgarno [SD] sequence). We found that P

araS
-SD

directed high levels of target gene expression. More strikingly, N-terminal amino acid sequencing of recombinant proteins unraveled that the protein synthesized from pEXA-N-
lacS
lacked the designed 6×His tag and that translation initiation did not start at the ATG codon of the fusion gene. Instead, it started at multiple sites downstream of the 6×His codons. Intriguingly, inserting an RBS site upstream of the ATG codon regained the expression of the 6×His tag, as shown with pSeSD-N-
lacS
. These results have yielded novel insight into the archaeal translation mechanism. The crenarchaeon
Sulfolobus
can utilize N-terminal coding sequences of proteins to specify translation initiation in the absence of an RBS site.
Association of Campylobacter jejuni Metabolic Traits with Multilocus Sequence Types
Authors: de Haan Caroline P. A. , Llarena Ann-Katrin , Revez Joana , Hänninen Marja-Liisa 
Published: June 2012

ABSTRACT

In this study, we describe the association of three
Campylobacter jejuni
metabolism-related traits, γ-glutamyl-transpeptidase (GGT), fucose permease (
fucP
), and secreted
l
-asparaginase [
ansB
(s)], with multilocus sequence types (STs). A total of 710
C. jejuni
isolates with known STs were selected and originated from humans, poultry, bovines, and the environment. Among these isolates, we found 31.1% to produce GGT and 49.3% and 30.3% to be positive for
ansB
(s) and
fucP
, respectively. The combination of GGT production, the presence of
ansB
(s), and the absence of
fucP
was associated with ST-22, ST-586, and the ST-45 and ST-283 clonal complexes (CCs), which were the main STs and CCs found among the human and chicken isolates. The ST-21 CC was associated with the presence of
fucP
and was the major CC among the bovine isolates. Although the ST-61 CC was the second major CC among the bovine isolates, these isolates did not have any of the markers studied, making the role of
fucP
in bovine gut colonization questionable. The ST-45 CC was subdivided into three groups that were attributed solely to ST-45. One group showed a marker combination described previously, another group was found to be positive for
ansB
(s) only, and the third group did not have any of the markers studied. These results suggest that the host association of these markers seems to be indirect and may arise as a consequence of host-ST and -CC associations. Thus, a representative collection of STs should be tested to draw sensible conclusions in similar studies.
The Novel Kasugamycin 2′-
N
-Acetyltransferase Gene
aac(2′)-IIa
, Carried by the IncP Island, Confers Kasugamycin Resistance to Rice-Pathogenic Bacteria
Authors: Yoshii Atsushi , Moriyama Hiromitsu , Fukuhara Toshiyuki 
Published: June 2012

ABSTRACT

Kasugamycin (KSM), a unique aminoglycoside antibiotic, has been used in agriculture for many years to control not only rice blast caused by the fungus
Magnaporthe grisea
but also rice bacterial grain and seedling rot or rice bacterial brown stripe caused by
Burkholderia glumae
or
Acidovorax avenae
subsp.
avenae
, respectively. Since both bacterial pathogens are seed-borne and cause serious injury to rice seedlings, the emergence of KSM-resistant
B. glumae
and
A. avenae
isolates highlights the urgent need to understand the mechanism of resistance to KSM. Here, we identified a novel gene,
aac(2′)-IIa
, encoding a KSM 2′-
N
-acetyltransferase from both KSM-resistant pathogens but not from KSM-sensitive bacteria. AAC(2′)-IIa inactivates KSM, although it reveals no cross-resistance to other aminoglycosides. The
aac(2′)-IIa
gene from
B. glumae
strain 5091 was identified within the IncP genomic island inserted into the bacterial chromosome, indicating the acquisition of this gene by horizontal gene transfer. Although excision activity of the IncP island and conjugational gene transfer was not detected under the conditions tested, circular intermediates containing the
aac(2′)-IIa
gene were detected. These results indicate that the
aac(2′)-IIa
gene had been integrated into the IncP island of a donor bacterial species. Molecular detection of the
aac(2′)-IIa
gene could distinguish whether isolates are resistant or susceptible to KSM. This may contribute to the production of uninfected rice seeds and lead to the effective control of these pathogens by KSM.
Transovarial Transmission of Rickettsia spp. and Organ-Specific Infection of the Whitefly Bemisia tabaci
Authors: Brumin Marina , Levy Maggie , Ghanim Murad 
Published: June 2012

ABSTRACTThe whiteflyBemisia tabaciis a cosmopolitan insect pest that harborsPortiera aleyrodidarum, the primary obligatory symbiotic bacterium, and several facultative secondary symbionts. Secondary symbionts inB. tabaciare generally associated with the bacteriome, ensuring their vertical transmission; however,Rickettsiais an exception and occupies most of the body cavity, except the bacteriome. The mode ofRickettsiatransfer between generations and its subcellular localization in insect organs have not been investigated. Using electron and fluorescence microscopy, we show thatRickettsiainfects the digestive, salivary, and reproductive organs of the insect; however, it was not observed in the bacteriome.Rickettsiainvades the oocytes during early developmental stages and resides in follicular cells and cytoplasm; it is mostly excluded when the egg matures; however, some bacterial cells remain in the egg, ensuring their transfer to subsequent generations.Rickettsiawas localized to testicles and the spermatheca, suggesting a horizontal transfer between males and females during mating. The bacterium was further observed at large amounts in midgut cells, concentrating in vacuole-like structures, and was located in the hemolymph, specifically at exceptionally large amounts around bacteriocytes and in fat bodies. Organs further infected byRickettsiaincluded the primary salivary glands and stylets, sites of possible secretion of the bacterium outside the whitefly body. The close association betweenRickettsiaand theB. tabacidigestive system might be important for digestive purposes. The vertical transmission ofRickettsiato subsequent generations occurs via the oocyte and not, like other secondary symbionts, the bacteriome.
Two Gene Clusters Coordinate Galactose and Lactose Metabolism in Streptococcus gordonii
Authors: Zeng Lin , Martino Nicole C. , Burne Robert A. 
Published: June 2012

ABSTRACT

Streptococcus gordonii
is an early colonizer of the human oral cavity and an abundant constituent of oral biofilms. Two tandemly arranged gene clusters, designated
lac
and
gal
, were identified in the
S. gordonii
DL1 genome, which encode genes of the tagatose pathway (
lacABCD
) and sugar phosphotransferase system (PTS) enzyme II permeases. Genes encoding a predicted phospho-β-galactosidase (LacG), a DeoR family transcriptional regulator (LacR), and a transcriptional antiterminator (LacT) were also present in the clusters. Growth and PTS assays supported that the permease designated EII
Lac
transports lactose and galactose, whereas EII
Gal
transports galactose. The expression of the gene for EII
Gal
was markedly upregulated in cells growing on galactose. Using promoter-
cat
fusions, a role for LacR in the regulation of the expressions of both gene clusters was demonstrated, and the
gal
cluster was also shown to be sensitive to repression by CcpA. The deletion of
lacT
caused an inability to grow on lactose, apparently because of its role in the regulation of the expression of the genes for EII
Lac
, but had little effect on galactose utilization.
S. gordonii
maintained a selective advantage over
Streptococcus mutans
in a mixed-species competition assay, associated with its possession of a high-affinity galactose PTS, although
S. mutans
could persist better at low pHs. Collectively, these results support the concept that the galactose and lactose systems of
S. gordonii
are subject to complex regulation and that a high-affinity galactose PTS may be advantageous when
S. gordonii
is competing against the caries pathogen
S. mutans
in oral biofilms.
Arcobacter in Lake Erie Beach Waters: an Emerging Gastrointestinal Pathogen Linked with Human-Associated Fecal Contamination
Authors: Lee Cheonghoon , Agidi Senyo , Marion Jason W. , Lee Jiyoung 
Published: June 2012

ABSTRACT

The genus
Arcobacter
has been associated with human illness and fecal contamination by humans and animals. To better characterize the health risk posed by this emerging waterborne pathogen, we investigated the occurrence of
Arcobacter
spp. in Lake Erie beach waters. During the summer of 2010, water samples were collected 35 times from the Euclid, Villa Angela, and Headlands (East and West) beaches, located along Ohio's Lake Erie coast. After sample concentration,
Arcobacter
was quantified by real-time PCR targeting the
Arcobacter
23S rRNA gene. Other fecal genetic markers (
Bacteroides
16S rRNA gene [HuBac],
Escherichia coli uidA
gene,
Enterococcus
23S rRNA gene, and tetracycline resistance genes) were also assessed.
Arcobacter
was detected frequently at all beaches, and both the occurrence and densities of
Arcobacter
spp. were higher at the Euclid and Villa Angela beaches (with higher levels of fecal contamination) than at the East and West Headlands beaches. The
Arcobacter
density in Lake Erie beach water was significantly correlated with the human-specific fecal marker HuBac according to Spearman's correlation analysis (
r
= 0.592;
P
< 0.001). Phylogenetic analysis demonstrated that most of the identified
Arcobacter
sequences were closely related to
Arcobacter cryaerophilus
, which is known to cause gastrointestinal diseases in humans. Since human-pathogenic
Arcobacter
spp. are linked to human-associated fecal sources, it is important to identify and manage the human-associated contamination sources for the prevention of
Arcobacter
-associated public health risks at Lake Erie beaches.
Solubility-Promoting Function of Hsp90 Contributes to Client Maturation and Robust Cell Growth
Authors: Pursell Natalie W. , Mishra Parul , Bolon Daniel N. A. 
Published: June 2012

ABSTRACT
The Hsp90 chaperone is required for the maturation of signal transduction clients, including many kinases and nuclear steroid hormone receptors. The binding and hydrolysis of ATP by Hsp90 drive conformational rearrangements in three structure domains. Two intrinsically disordered regions of Hsp90 located between these domains and at the C terminus have traditionally been considered to impart flexibility. We discovered that the charged nature of these acid-rich disordered regions imparts a solubility-promoting function to Hsp90 that is important for its cellular activity in yeast. Both the solubility-promoting function and ATPase activity must occur in the same Hsp90 molecule in order to support robust growth, suggesting that the solubility-promoting function is required during the ATP-driven client maturation process. Expression of model clients together with Hsp90 variants indicated interdependent solubilities mediated by the aggregation propensities of both the client and Hsp90. We propose a model whereby the charge-rich disordered regions of Hsp90 serve a solubility-promoting function important for complexes with aggregation-prone clients. These findings demonstrate a novel biological function of the intrinsically disordered regions in Hsp90 and provide a compelling rationale for why their charged properties are conserved throughout eukaryotic evolution.
Acetate Regulation of Spore Formation Is under the Control of the Ras/Cyclic AMP/Protein Kinase A Pathway and Carbon Dioxide in Saccharomyces cerevisiae
Authors: Jungbluth Marc , Mösch Hans-Ulrich , Taxis Christof 
Published: June 2012

ABSTRACT

In
Saccharomyces cerevisiae
, the Ras/cyclic AMP (cAMP)/protein kinase A (PKA) pathway is a nutrient-sensitive signaling cascade that regulates vegetative growth, carbohydrate metabolism, and entry into meiosis. How this pathway controls later steps of meiotic development is largely unknown. Here, we have analyzed the role of the Ras/cAMP/PKA pathway in spore formation by the meiosis-specific manipulation of Ras and PKA or by the disturbance of cAMP production. We found that the regulation of spore formation by acetate takes place after commitment to meiosis and depends on PKA and appropriate A kinase activation by Ras/Cyr1 adenylyl cyclase but not by activation through the Gpa2/Gpr1 branch. We further discovered that spore formation is regulated by carbon dioxide/bicarbonate, and an analysis of mutants defective in acetate transport (
ady2
Δ) or carbonic anhydrase (
nce103
Δ) provided evidence that these metabolites are involved in connecting the nutritional state of the meiotic cell to spore number control. Finally, we observed that the potential PKA target Ady1 is required for the proper localization of the meiotic plaque proteins Mpc70 and Spo74 at spindle pole bodies and for the ability of these proteins to initiate spore formation. Overall, our investigation suggests that the Ras/cAMP/PKA pathway plays a crucial role in the regulation of spore formation by acetate and indicates that the control of meiotic development by this signaling cascade takes places at several steps and is more complex than previously anticipated.
SrfJ, a Salmonella Type III Secretion System Effector Regulated by PhoP, RcsB, and IolR
Authors: Cordero-Alba Mar , Bernal-Bayard Joaquín , Ramos-Morales Francisco 
Published: June 2012

ABSTRACT

Virulence-related type III secretion systems are present in many Gram-negative bacterial pathogens. These complex devices translocate proteins, called effectors, from the bacterium into the eukaryotic host cell. Here, we identify the product of
srfJ
, a
Salmonella enterica
serovar Typhimurium gene regulated by SsrB, as a new substrate of the type III secretion system encoded by
Salmonella
pathogenicity island 2. The N-terminal 20-amino-acid segment of SrfJ was recognized as a functional secretion and translocation signal specific for this system. Transcription of
srfJ
was positively regulated by the PhoP/PhoQ system in an SsrB-dependent manner and was negatively regulated by the Rcs system in an SsrB-independent manner. A screen for regulators of an
srfJ-lacZ
transcriptional fusion using the T-POP transposon identified IolR, the regulator of genes involved in
myo
-inositol utilization, as an
srfJ
repressor. Our results suggest that SrfJ is synthesized both inside the host, in response to intracellular conditions, and outside the host, in
myo
-inositol-rich environments.
Growing Unculturable Bacteria
Authors: Stewart Eric J. 
Published: June 2012

ABSTRACT
The bacteria that can be grown in the laboratory are only a small fraction of the total diversity that exists in nature. At all levels of bacterial phylogeny, uncultured clades that do not grow on standard media are playing critical roles in cycling carbon, nitrogen, and other elements, synthesizing novel natural products, and impacting the surrounding organisms and environment. While molecular techniques, such as metagenomic sequencing, can provide some information independent of our ability to culture these organisms, it is essentially impossible to learn new gene and pathway functions from pure sequence data. A true understanding of the physiology of these bacteria and their roles in ecology, host health, and natural product production requires their cultivation in the laboratory. Recent advances in growing these species include coculture with other bacteria, recreating the environment in the laboratory, and combining these approaches with microcultivation technology to increase throughput and access rare species. These studies are unraveling the molecular mechanisms of unculturability and are identifying growth factors that promote the growth of previously unculturable organisms. This minireview summarizes the recent discoveries in this area and discusses the potential future of the field.