bluuebunny/crossref_metadata_2025_split huggingface dataset:
High Levels of the Xenorhabdus nematophila Transcription Factor Lrp Promote Mutualism with the Steinernema carpocapsae Nematode Host
Authors: Cao Mengyi , Patel Tilak , Rickman Tara , Goodrich-Blair Heidi , Hussa Elizabeth A. 
Published: April 2017

ABSTRACT

Xenorhabdus nematophila
bacteria are mutualistic symbionts of
Steinernema carpocapsae
nematodes and pathogens of insects. The
X. nematophila
global regulator Lrp controls the expression of many genes involved in both mutualism and pathogenic activities, suggesting a role in the transition between the two host organisms. We previously reported that natural populations of
X. nematophila
exhibit various levels of Lrp expression and that cells expressing relatively low levels of Lrp are optimized for virulence in the insect
Manduca sexta
. The adaptive advantage of the high-Lrp-expressing state was not established. Here we used strains engineered to express constitutively high or low levels of Lrp to test the model in which high-Lrp-expressing cells are adapted for mutualistic activities with the nematode host. We demonstrate that high-Lrp cells form more robust biofilms in laboratory media than do low-Lrp cells, which may reflect adherence to host tissues. Also, our data showed that nematodes cultivated with high-Lrp strains are more frequently colonized than are those associated with low-Lrp strains. Taken together, these data support the idea that high-Lrp cells have an advantage in tissue adherence and colonization initiation. Furthermore, our data show that high-Lrp-expressing strains better support nematode reproduction than do their low-Lrp counterparts under both
in vitro
and
in vivo
conditions. Our data indicate that heterogeneity of Lrp expression in
X. nematophila
populations provides diverse cell populations adapted to both pathogenic (low-Lrp) and mutualistic (high-Lrp) states.

IMPORTANCE
Host-associated bacteria experience fluctuating conditions during both residence within an individual host and transmission between hosts. For bacteria that engage in evolutionarily stable, long-term relationships with particular hosts, these fluctuations provide selective pressure for the emergence of adaptive regulatory mechanisms. Here we present evidence that the bacterium
Xenorhabdus nematophila
uses various levels of the transcription factor Lrp to optimize its association with its two animal hosts, nematodes and insects, with which it behaves as a mutualist and a pathogen, respectively. Building on our previous finding that relatively low cellular levels of Lrp are optimal for pathogenesis, we demonstrate that, conversely, high levels of Lrp promote mutualistic activities with the
Steinernema carpocapsae
nematode host. These data suggest that
X. nematophila
has evolved to utilize phenotypic variation between high- and low-Lrp-expression states to optimize its alternating behaviors as a mutualist and a pathogen.
Genetic Stability and Evolution of thesigBAllele, Used for Listeria Sensu Stricto Subtyping and Phylogenetic Inference
Authors: Liao Jingqiu , Wiedmann Martin , Kovac Jasna 
Published: April 2017

ABSTRACTSequencing of single genes remains an important tool that allows the rapid classification of bacteria. Sequencing of a portion ofsigB, which encodes a stress-responsive alternative sigma factor, has emerged as a commonly used molecular tool for the initial characterization of diverseListeriaisolates. In this study, evolutionary approaches were used to assess the validity ofsigBallelic typing forListeria. For a data set of 4,280 isolates,sigBallelic typing showed a Simpson's index of diversity of 0.96. Analyses of 164sigBallelic types (ATs) found among the 6Listeriasensu strictospecies, representing these 4,280 isolates, indicate that neither frequent homologous recombination nor positive selection significantly contributed to the evolution ofsigB, confirming its genetic stability. The molecular clock test provided evidence for unequal evolution rates across clades;Listeria welshimeridisplayed the lowestsigBdiversity and was the only species in whichsigBevolved in a clocklike manner, implying a unique natural history. Among the fourL. monocytogeneslineages,sigBevolution followed a molecular clock only in lineage IV. Moreover,sigBdisplayed a significant negative TajimaDvalue in lineage II, suggesting a recent population bottleneck followed by lineage expansion. The absence of positive selection along with the violation of the molecular clock suggested a nearly neutral mechanism ofListeriasensu strictosigBevolution. While comparison with a whole-genome sequence-based phylogeny revealed that thesigBphylogeny did not correctly reflect the ancestry ofL. monocytogeneslineage IV, the availability of a largesigBAT database allowed accurate species classification.IMPORTANCEsigBallelic typing has been widely used for species delineation and subtyping ofListeria. However, an informative evaluation of this method from an evolutionary perspective was missing. Our data indicate that the genetic stability ofsigBis affected by neither frequent homologous recombination nor positive selection, which supports thatsigBallelic typing provides reliable subtyping and classification ofListeria sensu strictostrains. However, multigene data are required for accurate phylogeny reconstruction ofListeria. This study thus contributes to a better understanding of the evolution ofsigBand confirms the robustness of thesigBsubtyping system forListeria.
Engineering of Bacteriophages Y2::
dpoL1-C
and Y2::
luxAB
for Efficient Control and Rapid Detection of the Fire Blight Pathogen, Erwinia amylovora
Authors: Born Yannick , Fieseler Lars , Thöny Valentin , Leimer Nadja , Duffy Brion , Loessner Martin J. 
Published: April 2017

ABSTRACT

Erwinia amylovora
is the causative agent of fire blight, a devastating plant disease affecting members of the
Rosaceae
. Alternatives to antibiotics for control of fire blight symptoms and outbreaks are highly desirable, due to increasing drug resistance and tight regulatory restrictions. Moreover, the available diagnostic methods either lack sensitivity, lack speed, or are unable to discriminate between live and dead bacteria. Owing to their extreme biological specificity, bacteriophages are promising alternatives for both aims. In this study, the virulent broad-host-range
E. amylovora
virus Y2 was engineered to enhance its killing activity and for use as a luciferase reporter phage, respectively. Toward these aims, a depolymerase gene of
E. amylovora
virus L1 (
dpoL1-C
) or a bacterial
luxAB
fusion was introduced into the genome of Y2 by homologous recombination. The genes were placed downstream of the major capsid protein
orf68
, under the control of the native promoter. The modifications did not affect viability of infectivity of the recombinant viruses. Phage Y2::
dpoL1-C
demonstrated synergistic activity between the depolymerase degrading the exopolysaccharide capsule and phage infection, which greatly enhanced bacterial killing. It also significantly reduced the ability of
E. amylovora
to colonize the surface of detached flowers. The reporter phage Y2::
luxAB
transduced bacterial luciferase into host cells and induced synthesis of large amounts of a LuxAB luciferase fusion. After the addition of aldehyde substrate, bioluminescence could be readily monitored, and this enabled rapid and specific detection of low numbers of viable bacteria, without enrichment, both
in vitro
and in plant material.

IMPORTANCE
Fire blight, caused by
Erwinia amylovora
, is the major threat to global pome fruit production, with high economic losses every year. Bacteriophages represent promising alternatives to not only control the disease, but also for rapid diagnostics. To enhance biocontrol efficacy, we combined the desired properties of two phages, Y2 (broad host range) and L1 (depolymerase for capsule degradation) in a single recombinant phage. This phage showed enhanced biocontrol and could reduce
E. amylovora
on flowers. Phage Y2 was also genetically engineered into a luciferase reporter phage, which transduces bacterial bioluminescence into infected cells and allows detection of low numbers of viable target bacteria. The combination of speed, sensitivity, and specificity is superior to previously used diagnostic methods. In conclusion, genetic engineering could improve the properties of phage Y2 toward better killing efficacy and sensitive detection of
E. amylovora
cells.
Acid Evolution of Escherichia coli K-12 Eliminates Amino Acid Decarboxylases and Reregulates Catabolism
Authors: He Amanda , Penix Stephanie R. , Basting Preston J. , Griffith Jessie M. , Creamer Kaitlin E. , Camperchioli Dominic , Clark Michelle W. , Gonzales Alexandra S. , Chávez Erazo Jorge Sebastian , George Nadja S. , Bhagwat Arvind A. , Slonczewski Joan L. 
Published: April 2017

ABSTRACT

Acid-adapted strains of
Escherichia coli
K-12 W3110 were obtained by serial culture in medium buffered at pH 4.6 (M. M. Harden, A. He, K. Creamer, M. W. Clark, I. Hamdallah, K. A. Martinez, R. L. Kresslein, S. P. Bush, and J. L. Slonczewski, Appl Environ Microbiol 81:1932–1941, 2015,
https://doi.org/10.1128/AEM.03494-14
). Revised genomic analysis of these strains revealed insertion sequence (IS)-driven insertions and deletions that knocked out regulators CadC (acid induction of lysine decarboxylase), GadX (acid induction of glutamate decarboxylase), and FNR (anaerobic regulator). Each acid-evolved strain showed loss of one or more amino acid decarboxylase systems, which normally help neutralize external acid (pH 5 to 6) and increase survival in extreme acid (pH 2). Strains from populations B11, H9, and F11 had an IS
5
insertion or IS-mediated deletion in
cadC
, while population B11 had a point mutation affecting the arginine activator
adiY
. The
cadC
and
adiY
mutants failed to neutralize acid in the presence of exogenous lysine or arginine. In strain B11-1, reversion of an
rpoC
(RNA polymerase) mutation partly restored arginine-dependent neutralization. All eight strains showed deletion or downregulation of the Gad acid fitness island. Strains with the Gad deletion lost the ability to produce GABA (gamma-aminobutyric acid) and failed to survive extreme acid. Transcriptome sequencing (RNA-seq) of strain B11-1 showed upregulated genes for catabolism of diverse substrates but downregulated acid stress genes (the biofilm regulator
ariR
,
yhiM
, and Gad). Other strains showed downregulation of H
2
consumption mediated by hydrogenases (
hya
and
hyb
) which release acid. Strains F9-2 and F9-3 had a deletion of
fnr
and showed downregulation of FNR-dependent genes (
dmsABC
,
frdABCD
,
hybABO
,
nikABCDE
, and
nrfAC
). Overall, strains that had evolved in buffered acid showed loss or downregulation of systems that neutralize unbuffered acid and showed altered regulation of catabolism.

IMPORTANCE
Experimental evolution of an enteric bacterium under a narrow buffered range of acid pH leads to loss of genes that enhance fitness above or below the buffered pH range, including loss of enzymes that may raise external pH in the absence of buffer. Prominent modes of evolutionary change involve IS-mediated insertions and deletions that knock out key regulators. Over generations of acid stress, catabolism undergoes reregulation in ways that differ for each evolving strain.
Pumilacidin-Like Lipopeptides Derived from Marine Bacterium Bacillus sp. Strain 176 Suppress the Motility of Vibrio alginolyticus
Authors: Xiu Pengyuan , Liu Rui , Zhang Dechao , Sun Chaomin 
Published: April 2017

ABSTRACT

Bacterial motility is a crucial factor during the invasion and colonization processes of pathogens, which makes it an attractive therapeutic drug target. Here, we isolated a marine bacterium (
Vibrio alginolyticus
strain 178) from a seamount in the tropical West Pacific that exhibits vigorous motility on agar plates and severe pathogenicity to zebrafish. We found that
V. alginolyticus
178 motility was significantly suppressed by another marine bacterium,
Bacillus
sp. strain 176, isolated from the same niche. We isolated, purified, and characterized two different cyclic lipopeptides (CLPs) from
Bacillus
sp. 176 using high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy. The two related CLPs have a pumilacidin-like structure and were both effective inhibitors of
V. alginolyticus
178 motility. The CLPs differ by only one methylene group in their fatty acid chains. In addition to motility suppression, the CLPs also induced cell aggregation in the medium and reduced adherence of
V. alginolyticus
178 to glass substrates. Notably, upon CLP treatment, the expression levels of two
V. alginolyticus
flagellar assembly genes (
flgA
and
flgP
) dropped dramatically. Moreover, the CLPs inhibited biofilm formation in several other strains of pathogenic bacteria without inducing cell death. This study indicates that CLPs from
Bacillus
sp. 176 show promise as antimicrobial lead compounds targeting bacterial motility and biofilm formation with a low potential for eliciting antibiotic resistance.

IMPORTANCE
Pathogenic bacteria often require motility to establish infections and subsequently spread within host organisms. Thus, motility is an attractive therapeutic target for the development of novel antibiotics. We found that cyclic lipopeptides (CLPs) produced by marine bacterium
Bacillus
sp. strain 176 dramatically suppress the motility of the pathogenic bacterium
Vibrio alginolyticus
strain 178, reduce biofilm formation, and promote cellular aggregation without inducing cell death. These findings suggest that CLPs hold great promise as potential drug candidates targeting bacterial motility and biofilm formation with a low overall potential for triggering antibiotic resistance.
Nitrous Oxide Reduction by an Obligate Aerobic Bacterium, Gemmatimonas aurantiaca Strain T-27
Authors: Park Doyoung , Kim Hayeon , Yoon Sukhwan 
Published: April 2017

ABSTRACT

N
2
O-reducing organisms with nitrous oxide reductases (NosZ) are known as the only biological sink of N
2
O in the environment. Among the most abundant
nosZ
genes found in the environment are
nosZ
genes affiliated with the understudied
Gemmatimonadetes
phylum. In this study, a unique regulatory mechanism of N
2
O reduction in
Gemmatimonas aurantiaca
strain T-27, an isolate affiliated with the
Gemmatimonadetes
phylum, was examined. Strain T-27 was incubated with N
2
O and/or O
2
as the electron acceptor. Significant N
2
O reduction was observed only when O
2
was initially present. When batch cultures of strain T-27 were amended with O
2
and N
2
O, N
2
O reduction commenced after O
2
was depleted. In a long-term incubation with the addition of N
2
O upon depletion, the N
2
O reduction rate decreased over time and came to an eventual stop. Spiking of the culture with O
2
resulted in the resuscitation of N
2
O reduction activity, supporting the hypothesis that N
2
O reduction by strain T-27 required the transient presence of O
2
. The highest level of
nosZ
transcription (8.97
nosZ
transcripts/
recA
transcript) was observed immediately after O
2
depletion, and transcription decreased ∼25-fold within 85 h, supporting the observed phenotype. The observed difference between responses of strain T-27 cultures amended with and without N
2
O to O
2
starvation suggested that N
2
O helped sustain the viability of strain T-27 during temporary anoxia, although N
2
O reduction was not coupled to growth. The findings in this study suggest that obligate aerobic microorganisms with
nosZ
genes may utilize N
2
O as a temporary surrogate for O
2
to survive periodic anoxia.

IMPORTANCE
Emission of N
2
O, a potent greenhouse gas and ozone depletion agent, from the soil environment is largely determined by microbial sources and sinks. N
2
O reduction by organisms with N
2
O reductases (NosZ) is the only known biological sink of N
2
O at environmentally relevant concentrations (up to ∼1,000 parts per million by volume [ppmv]). Although a large fraction of
nosZ
genes recovered from soil is affiliated with
nosZ
found in the genomes of the obligate aerobic phylum
Gemmatimonadetes
, N
2
O reduction has not yet been confirmed in any of these organisms. This study demonstrates that N
2
O is reduced by an obligate aerobic bacterium,
Gemmatimonas aurantiaca
strain T-27, and suggests a novel regulation mechanism for N
2
O reduction in this organism, which may also be applicable to other obligate aerobic organisms possessing
nosZ
genes. We expect that these findings will significantly advance the understanding of N
2
O dynamics in environments with frequent transitions between oxic and anoxic conditions.
Microbiota in Exhaled Breath Condensate and the Lung
Authors: Glendinning Laura , Wright Steven , Tennant Peter , Gill Andrew C. , Collie David , McLachlan Gerry 
Published: April 2017

ABSTRACT
The lung microbiota is commonly sampled using relatively invasive bronchoscopic procedures. Exhaled breath condensate (EBC) collection potentially offers a less invasive alternative for lung microbiota sampling. We compared lung microbiota samples retrieved by protected specimen brushings (PSB) and exhaled breath condensate collection. We also sought to assess whether aerosolized antibiotic treatment would influence the lung microbiota and whether this change could be detected in EBC. EBC was collected from 6 conscious sheep and then from the same anesthetized sheep during mechanical ventilation. Following the latter EBC collection, PSB samples were collected from separate sites within each sheep lung. On the subsequent day, each sheep was then treated with nebulized colistimethate sodium. Two days after nebulization, EBC and PSB samples were again collected. Bacterial DNA was quantified using 16S rRNA gene quantitative PCR. The V2-V3 region of the 16S rRNA gene was amplified by PCR and sequenced using Illumina MiSeq. Quality control and operational taxonomic unit (OTU) clustering were performed with mothur. The EBC samples contained significantly less bacterial DNA than the PSB samples. The EBC samples from anesthetized animals clustered separately by their bacterial community compositions in comparison to the PSB samples, and 37 bacterial OTUs were identified as differentially abundant between the two sample types. Despite only low concentrations of colistin being detected in bronchoalveolar lavage fluid, PSB samples were found to differ by their bacterial compositions before and after colistimethate sodium treatment. Our findings indicate that microbiota in EBC samples and PSB samples are not equivalent.

IMPORTANCE
Sampling of the lung microbiota usually necessitates performing bronchoscopic procedures that involve a hospital visit for human participants and the use of trained staff. The inconvenience and perceived discomfort of participating in this kind of research may deter healthy volunteers and may not be a safe option for patients with advanced lung disease. This study set out to evaluate a less invasive method for collecting lung microbiota samples by comparing samples taken via protected specimen brushings (PSB) to those taken via exhaled breath condensate (EBC) collection. We found that there was less bacterial DNA in EBC samples compared with that in PSB samples and that there were differences between the bacterial communities in the two sample types. We conclude that while EBC and PSB samples do not produce equivalent microbiota samples, the study of the EBC microbiota may still be of interest.
Holotrichia oblita Midgut Proteins That Bind to Bacillus thuringiensis Cry8-Like Toxin and Assembly of the H. oblita Midgut Tissue Transcriptome
Authors: Jiang Jian , Huang Ying , Shu Changlong , Soberón Mario , Bravo Alejandra , Liu Chunqing , Song Fuping , Lai Jinsheng , Zhang Jie 
Published: April 2017

ABSTRACT

The
Bacillus thuringiensis
strain HBF-18 (CGMCC 2070), containing two
cry
genes (
cry8
-like and
cry8Ga
), is toxic to
Holotrichia oblita
larvae. Both Cry8-like and Cry8Ga proteins are active against this insect pest, and Cry8-like is more toxic. To analyze the characteristics of the binding of Cry8-like and Cry8Ga proteins to brush border membrane vesicles (BBMVs) in
H. oblita
larvae, binding assays were conducted with a fluorescent DyLight488-labeled Cry8-like toxin. The results of saturation binding assays demonstrated that Cry8-like bound specifically to binding sites on BBMVs from
H. oblita
, and heterologous competition assays revealed that Cry8Ga shared binding sites with Cry8-like. Furthermore, Cry8-like-binding proteins in the midgut from
H. oblita
larvae were identified by pulldown assays and liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, the
H. oblita
midgut transcriptome was assembled by high-throughput RNA sequencing and used for identification of Cry8-like-binding proteins. Eight Cry8-like-binding proteins were obtained from pulldown assays conducted with BBMVs. The LC-MS/MS data for these proteins were successfully matched with the
H. oblita
transcriptome, and BLASTX results identified five proteins as serine protease, transferrin-like, uncharacterized protein LOC658236 of
Tribolium castaneum
, ATPase catalytic subunit, and actin. These identified Cry8-like-binding proteins were different from those confirmed previously as receptors for Cry1A proteins in lepidopteran insect species, such as aminopeptidase, alkaline phosphatase, and cadherin.

IMPORTANCE
Holotrichia oblita
is one of the main soil-dwelling pests in China. The larvae damage the roots of crops, resulting in significant yield reductions and economic losses.
H. oblita
is difficult to control, principally due to its soil-dwelling habits. In recent years, some Cry8 toxins from
Bacillus thuringiensis
were shown to be active against this pest. Study of the mechanism of action of these Cry8 toxins is needed for their effective use in the control of
H. oblita
and for their future utilization in transgenic plants. Our work provides important basic data and promotes understanding of the insecticidal mechanism of Cry8 proteins against
H. oblita
larvae.