bluuebunny/crossref_metadata_2025_split huggingface dataset:
A complex regulatory cascade involving quorum sensing regulates siderophore-mediated iron homeostasis inChromobacterium violaceum
Authors: Batista Bianca B. , de Lima Vinicius M. , Picinato Beatriz A. , Koide Tie , da Silva Neto José F. 
Published: December 2023

AbstractIron is a transition metal used as a cofactor in many biochemical reactions. In bacteria, iron homeostasis involves Fur-mediated de-repression of iron uptake systems, such as the iron-chelating compounds siderophores. In this work, we identified and characterized novel regulatory systems that control siderophores in the environmental opportunistic pathogenChromobacterium violaceum. Screening of a 10,000-transposon mutant library for siderophore halos on PSA-CAS plates identified seven possible regulatory systems involved in siderophore-mediated iron homeostasis inC. violaceum. Further characterization revealed a regulatory cascade that controls siderophores involving the transcription factor VitR acting upstream of the quorum sensing (QS) system CviIR. Mutation of the regulator VitR led to an increase in siderophore halos, and a decrease in biofilm, violacein, and protease production. We determined that these effects occurred due to VitR-dependent de-repression ofvioS. Increased VioS leads to direct inhibition of the CviR regulator by protein-protein interaction. Indeed, insertion mutations incviRand null mutation ofcviIandcviRled to an increase of siderophore halos. RNA-seq of thecviIandcviRmutants revealed that CviR regulates CviI-dependent and CviI-independent regulons. Classical QS-dependent processes (violacein, proteases, and antibiotics) were activated at high cell density by both CviI and CviR. However, genes related to iron homeostasis and many other processes were regulated by CviR but not CviI, suggesting that CviR acts without its canonical CviI autoinducer. Our data revealed a complex regulatory cascade involving QS that controls siderophore-mediated iron homeostasis inC. violaceum.ImportanceThe iron-chelating compounds siderophores play a major role in bacterial iron acquisition. Here, we employed a genetic screen to identify novel siderophore regulatory systems inChromobacterium violaceum, an opportunistic human pathogen. Many mutants with increased siderophore halos had transposon insertions in genes encoding transcription factors, including a novel regulator called VitR, and CviR, the regulator of the quorum sensing (QS) system CviIR. We found that VitR is upstream in the pathway and acts as a dedicated repressor ofvioS, which encodes a direct CviR-inhibitory protein. Indeed, all QS-related phenotypes of avitRmutant were rescued in avitRvioSmutant. At high cell density, CviIR activated classical QS-dependent processes (violacein, proteases, and antibiotics production). However, genes related to iron homeostasis and type-III and type-VI secretion systems were regulated by CviR in a CviI- or cell density-independent manner. Our data unveil a complex regulatory cascade integrating QS and siderophores inC. violaceum.
Microbiome depletion and recovery in the sea anemone,Exaiptasia diaphana, following antibiotic exposure
Authors: MacVittie Sophie , Doroodian Saam , Alberto Aaron , Sogin Maggie 
Published: December 2023

AbstractMicrobial species that comprise host-associated microbiomes play an essential role in maintaining and mediating the health of plants and animals. While defining the role of individual or even complex communities is important towards quantifying the effect of the microbiome on host health, it is often challenging to develop causal studies that link microbial populations to changes in host fitness. Here, we investigated the impacts of reduced microbial load following antibiotic exposure on the fitness of the anemone,Exaiptasia diaphanaand subsequent recovery of the host’s microbiome. Anemones were exposed to two different types of antibiotic solutions for three weeks and subsequently held in sterilized seawater for a subsequent three-week recovery period. Our results revealed that both antibiotic treatments reduced the overall microbial load during and up to one week post treatment. The observed reduction in microbial load was coupled to reduced anemone biomass, halted asexual reproduction rates, and for one of the antibiotic treatments, the partial removal of the anemone’s algal symbiont. Finally, our amplicon sequencing results of the 16S rRNA gene revealed that anemone bacterial composition only shifted in treated individuals during the recovery phase of the experiment, where we also observed a significant reduction in the overall diversity of the microbial community. Our work implies that theE. diaphana’smicrobiome contributes to host fitness and that the recovery of the of the host’s microbiome following disturbance with antibiotics leads to a reduced, but stable microbial state.ImportanceExaiptasia diaphanais an emerging model used to define the cellular and molecular mechanisms of coral-algal symbioses.E. diaphanaalso houses a diverse microbiome, consisting of hundreds of microbial partners with undefined function. Here, we applied antibiotics to quantify the impact of microbiome removal on host fitness as well as define trajectories in microbiome recovery following disturbance. We showed that reduction of the microbiome leads to negative impacts on host fitness, and that the microbiome does not recover to its original composition while held under aseptic conditions. Rather the microbiome becomes less diverse, but more consistent across individuals. Our work is important because it suggests that anemone microbiomes play a role in maintaining host fitness, that they are susceptible to disturbance events, and it is possible to generate gnotobiotic individuals that can be leveraged in microbiome manipulation studies to investigate the role of individual species on host health.
Physiological and transcriptomic response to methyl-coenzyme M reductase limitation inMethanosarcina acetivorans
Authors: Chadwick Grayson L. , Dury Gavin A. , Nayak Dipti D. 
Published: December 2023

AbstractMethyl-coenzyme M reductase (MCR) catalyzes the final step of methanogenesis, the microbial metabolism responsible for nearly all biological methane emissions to the atmosphere. Decades of biochemical and structural studies have generated detailed insights into MCR functionin vitro, yet very little is known about the interplay between MCR and methanogen physiology. For instance, while it is routinely stated that MCR catalyzes the rate-limiting step of methanogenesis, this statement has not been categorically tested. Here, to gain a more direct understanding of MCR’s control on the growth ofMethanosarcina acetivorans,we generate a strain with an induciblemcroperon on the chromosome, allowing for careful control of MCR expression. We show that MCR is not growth rate limiting in substrate-replete batch cultures. However, through careful titration of MCR expression, growth-limiting state(s) can be obtained. Transcriptomic analysis ofM. acetivoransexperiencing MCR-limitation reveals a global response with hundreds of differentially expressed genes across diverse functional categories. Notably, MCR limitation leads to a strong induction of methylsulfide methyltransferases, likely due to insufficient recycling of metabolic intermediates. In addition, themcroperon does not seem to be transcriptionally regulated, i.e., it is constitutively expressed, suggesting that the overabundance of MCR might be beneficial when cells experience nutrient limitation or stressful conditions. Altogether, we show that there is wide range of cellular MCR concentrations that can sustain optimal growth, suggesting that other factors like anabolic reactions might be rate-limiting for methanogenic growth.ImportanceMethane is a potent greenhouse gas that has contributed toca.25% of global warming in the post-industrial era. Atmospheric methane is primarily of biogenic origin, mostly produced by microorganisms called methanogens. In methanogens, methyl-coenzyme M reductase (MCR) catalyzes methane formation. Even though MCR comprisesca.10% of the cellular proteome, it is hypothesized to be growth-limiting during methanogenesis. Here, we show thatMethanosarcina acetivoransgrown under standard laboratory conditions produces more MCR than its cellular demand for optimal growth. The tools outlined in this study can be used to refine metabolic models of methanogenesis and assay lesions in MCR in a higher throughput manner than isolation and biochemical characterization of pure protein.
Molecular definition of the BAK:VDAC2 interaction as a target to manipulate apoptosis
Authors: Yuan Zheng , van Delft Mark , Li Mark , Sumardy Fransisca , Smith Brian J , Huang David C. S. , Lessene Guillaume , Khakam Yelena , Jin Ruitao , He Sitong , Smith Nicholas A , Birkinshaw Richard W , Czabotar Peter E , Dewson Grant 
Published: December 2023

AbstractBAK and BAX execute intrinsic apoptosis by permeabilising the mitochondrial outer membrane. Their activity is regulated through interactions with pro-survival BCL-2 family proteins and with non-BCL-2 proteins including the mitochondrial porin VDAC2. VDAC2 is important for bringing both BAK and BAX to mitochondria where they execute their apoptotic function. Despite this important function in apoptosis, whilst interactions with pro-survival family members are well characterised and have culminated in the development of drugs that target these interfaces to induce cancer cell apoptosis, the interaction between BAK and VDAC2 remains largely undefined. Deep scanning mutagenesis coupled with cysteine linkage identified key residues in the interaction between BAK and VDAC2. Obstructive labelling of specific residues in the BH3 domain or hydrophobic groove of BAK disrupted this interaction. Conversely, mutating specific residues in a cytosol-exposed region of VDAC2 stabilised the interaction with BAK, and inhibited BAK apoptotic activity. Thus, this VDAC2–BAK interaction site can potentially be targeted to either inhibit BAK-mediated apoptosis in scenarios where excessive apoptosis contributes to disease, or to promote BAK-mediated apoptosis for cancer therapy.
Spatial Transcriptomic Characterization of Novel Pathologic Niches in IPF
Authors: Mayr Christoph H. , Santacruz Diana , Jarosch Sebastian , Lempp Charlotte , Neubert Lavinia , Rath Berenice , Kamp Jan C. , Jonigk Danny , Kühnel Mark , Schlueter Holger , Doerr Jonas , Dick Alec , Ramirez Fidel , Thomas Matthew J. 
Published: December 2023

ABSTRACTAn unmet medical need persists in Idiopathic Pulmonary fibrosis (IPF), for which treatments additional to anti-fibrotic therapy are needed. Single cell RNA sequencing (scRNA-seq) has advanced our understanding of IPF with cell type-specific insights but lacks cellular tissue context. Spatial transcriptomics addresses this by providing spatially resolved gene expression, enabling gene and cell type localization within the tissue environment. We profiled IPF and control patient lung tissue sections using spatial transcriptomics and combined the data with an atlas of integrated IPF scRNA-seq datasets. Through computational analysis, we identified three disease-associated pathologic niches with unique cellular composition / localization and analyzed their cell-cell communication. We identified the Fibrotic niche, comprising Myofibroblasts and Aberrant Basaloid cells, preferentially located around airways and close to the Airway Macrophage niche in the lumen, containing SPP1+ Macrophages. We also identified the Immune niche, distinct foci of lymphoid cells in fibrotic tissue, surrounded by remodeled endothelial vessels.TEASERSpatial transcriptomics localizes genes and cell types in the tissue and identifies pathological cellular niches in IPF and control lungs.
A translation-independent directed evolution strategy to engineer aminoacyl-tRNA synthetases
Authors: Soni Chintan , Prywes Noam , Hall Matthew , Savage David F. , Schepartz Alanna , Chatterjee Abhishek 
Published: December 2023

AbstractUsing directed evolution, engineered aminoacyl-tRNA synthetases (aaRS) have been developed that enable co-translational incorporation of numerous noncanonical amino acids (ncAAs) into proteins in living cells. Until now, the selection of such novel aaRS mutants has relied on coupling their activity to the expression of a reporter protein with a selectable phenotype. However, such translation-dependent selection schemes are incompatible with exotic monomers that diverge structurally from canonical α-amino acids and are suboptimal substrates for the ribosome. To enable the ribosomal incorporation of such exotic monomers, a two-step solution is needed: A) Engineering an aaRS to acylate its cognate tRNA with the exotic monomer, without relying on ribosomal translation as a readout, and B) Subsequent engineering of the ribosome to accept the resulting acylated tRNA for translation. Here, we report a platform for aaRS engineering that directly selects for tRNA-acylation without ribosomal translation (START). In START, each distinct aaRS mutant is correlated to a cognate tRNA containing a unique sequence barcode. Acylation by an active aaRS mutant protects the associated barcode-containing tRNAs from an oxidative treatment designed to damage the 3′-terminus of the uncharged tRNAs. Sequencing of these surviving barcode-containing tRNAs is then used to reveal the identity of aaRS mutants that acylated the correlated tRNA sequences. The efficacy of START was demonstrated by identifying novel mutants of theM. alvuspyrrolysyl-tRNA synthetase from a naïve library that charge noncanonical amino acids.
Exploring the impact of microbial manipulation on the early development of kelp (Saccharina latissima) using an ecological core microbiome framework
Authors: Park Jungsoo , Schenk Siobhan , Davis Katherine , Clark Jennifer , Parfrey Laura Wegener 
Published: December 2023

ABSTRACTKelp cultivation is a rapidly expanding economic sector, as kelp are valued for a wide range of commercial products and for restoration of kelp forest ecosystems. Microbes associated with kelp and other macroalgae play a critical role in processes such as nutrient exchange, chemical signaling, and defense against pathogens. Thus, manipulating the microbiome to enhance macroalgal growth and resilience is a promising, but largely untested, tool in sustainable kelp cultivation. The core microbiome hypothesis suggests that bacteria that are consistently found on a host (the core microbes) are likely to have a disproportionate impact on host biology, making them an attractive target for microbiome manipulation. Here, we surveyed wildSaccharina latissimaand their surrounding environment to identify core bacterial taxa, compared to cultivated kelp, and experimentally tested the effect of cultured bacterial isolates on kelp development. We find that core bacteria are nearly absent in cultivated juvenile sporophytes in nurseries but eventually colonized after outplanting kelp to ocean farm sites. We find that bacterial inoculants can have both positive and negative effects on kelp development. In line with predictions from the core microbiome hypothesis, we find a positive correlation between the frequency of the bacterial genus in the wild and the bacterial effect on the number of sporophytes in kelp co-culture experiments.IMPORTANCEThe core microbiome hypothesis suggests that symbiotic microorganisms consistently associated with hosts have functional effects on host biology and health. However, there is a lack of evidence to either support or refute this idea. This study surveys the distribution of bacteria on wild and cultivated kelp to identify the core microbiome and tests the ability of bacterial isolates cultured from the surface of wild kelp to influence kelp growth and development in laboratory microbial manipulation experiments. The frequency of bacterial genera on wild kelp was positively correlated with influence on kelp development in laboratory experiments, providing support for the core microbiome hypothesis.
Activity of Antibiotics againstBurkholderia cepaciacomplex in Artificial Sputum Medium
Authors: Shukla Anusha , Rodriguez Shade , Brennan-Krohn Thea 
Published: December 2023

ABSTRACTBurkholderia cepaciacomplex (Bcc) is a collection of intrinsically drug-resistant gram-negative bacteria that cause life-threatening pulmonary disease in people with cystic fibrosis (CF). Standard antimicrobial susceptibility testing methods have poor predictive value for clinical outcomes in people with Bcc infections, likely due in part to the significant differences betweenin vitrotesting conditions and the environment in which Bcc grow in the lungs of people with CF. We tested the activity of six commonly used antibiotics against two clinical Bcc strains grown to high density in an artificial sputum medium in order to assess their activity in conditions mimicking those foundin vivo. There were major discrepancies between standard susceptibility results and activity in our model, with some antibiotics, including ceftazidime, showing minimal activity despite low MICs, while others, notably tobramycin, were more active in high-density growth conditions than in standard assays. This work underscores the urgent need to develop more clinically relevant susceptibility testing approaches for Bcc.
The HmrABCX pathway regulates the transition between motile and sessile lifestyles inCaulobacter crescentusby a HfiA-independent mechanism
Authors: Zappa Sébastien , Berne Cecile , Morton Robert I. , De Stercke Jonathan , Brun Yves V. 
Published: December 2023

ABSTRACTThrough its cell cycle, the bacteriumCaulobacter crescentusswitches from a motile, free-living state, to a sessile surface-attached cell. During this coordinated process, cells undergo irreversible morphological changes, such as shedding of their polar flagellum and synthesis of an adhesive holdfast at the same pole. In this work, we used genetic screens to identify genes involved in the regulation of the motile to sessile lifestyle transition. We identified a predicted hybrid histidine kinase that inhibits biofilm formation and activates the motile lifestyle: HmrA (Holdfast andmotility regulator A). Genetic screens and genomic localization led to the identification of additional genes that regulate the proportion of cells harboring an active flagellum or a holdfast and that form a putative phosphorelay pathway with HmrA. Further genetic analysis indicates that the Hmr pathway is independent of the holdfast synthesis regulator HfiA and may impact c-di-GMP synthesis through the diguanylate cyclase DgcB pathway. Finally, we provide evidence that the Hmr pathway is involved in the regulation of sessile-to-motile lifestyle as a function of environmental stresses, namely excess copper and non-optimal temperatures.IMPORTANCEComplex communities attached to a surface, or biofilms, represent the major lifestyle of bacteria in the environment. Such a sessile state enables its inhabitants to be more resistant to adverse environmental conditions. Thus, having a deeper understanding of the underlying mechanisms that regulate the transition between the motile and the sessile states could help design strategies to improve biofilms when they are beneficial or impede them when they are detrimental. ForC. crescentusmotile cells, the transition to the sessile lifestyle is irreversible, and this decision is regulated at several levels. In this work, we describe a putative phosphorelay that promotes the motile lifestyle and inhibits biofilm formation, providing new insights into the control of adhesin production that leads to the formation of biofilms.
Bacterial diversity dominates variable macrophage responses of tuberculosis patients in Tanzania
Authors: Hiza Hellen , Zwyer Michaela , Hella Jerry , Arbués Ainhoa , Sasamalo Mohamed , Borrell Sonia , Xu Zhi Ming , Ross Amanda , Brites Daniela , Fellay Jacques , Reither Klaus , Gagneux Sébastien , Portevin Damien 
Published: December 2023

AbstractTheMycobacterium tuberculosiscomplex (MTBC) comprises nine human-adapted lineages that differ in their geographical distribution. Local adaptation of specific MTBC genotypes to the respective human host population has been invoked in this context. Here, we generated macrophages from cryopreserved blood mononuclear cells of Tanzanian tuberculosis patients, from which the infecting MTBC strains had previously been phylogenetically characterized. We infected these macrophages ex vivo with a phylogenetically similar MTBC strain (“matched infection”) or with strains representative of other MTBC lineages (“mismatched infection”). We found that L1 infections resulted in a significantly lower bacterial burden and that the intra-cellular replication rate of L2 strains was significantly higher compared the other MTBC lineages, irrespective of the MTBC lineage originally infecting the patients. Moreover, L4-infected macrophages released significantly greater amounts of TNF-α, IL-6, IL-10, MIP-1β, and IL-1β compared to macrophages infected by all other strains. Taken together, while our results revealed no measurable effect of local adaptation, they further highlight the strong impact of MTBC phylogenetic diversity on the variable outcome of the host-pathogen interaction in human tuberculosis.
Allantoic fluid-based qPCR for early-onsetin ovosexing
Authors: Santos Simão , Corion Matthias , De Ketelaere Bart , Lammertyn Jeroen , Spasic Dragana 
Published: December 2023

AbstractThe culling of day-old male chickens remains an important welfare issue in the poultry industry. Several governments (e.g., Germany, France or Italy) have prohibited this practice, pushing the hatcheries to look for alternatives. Although different solutions exist for solving this problem, sex determination during embryo’s incubation (so calledin ovosexing) is considered the most suitable both for consumers and the industry since it aligns with the increasing demand for ethical and sustainable agriculture. However, to be applied in the market,in ovosexing technologies have to meet a number of requirements, such as being compatible with all egg colors and early developmental stages, while maintaining high hatchability rate and accuracy at low cost and high throughput. To meet these requirements, we studied the use of the sexual genesHINTW(female-specific) andDMRT-1(present in both males and females) between incubation days 6 and 9. By utilizing quantitative polymerase chain reaction (qPCR) as analysis method and allantoic fluid (AF) as sample, our study confirmed specific detection of these genes in AF, remarkably already at day 6 of development. In a blind study performed with 80 eggs, we achieved a 95% accuracy rate in sorting embryos when using theHINTWgene alone and an outstanding 100% accuracy rate when using Δλ values (difference between theHINTWandDMRT-1qPCR cycle threshold (Ct)). Importantly, the AF sampling procedure did not reveal any significant detrimental effects on hatchability or embryo development, revealing high potential for this understudied type of sample to be used. In conclusion, the developed assay can provide more in-depth information about AF as a sample for genomicin ovosexing and open new industrial possibilities for developing faster and cheaper assays.Research highlightsA highly reliable and accurate method forin ovosexing in early incubation stages is established.Allantoic fluid can be easily extracted with minimal invasiveness while providing the necessary genomic material for sexual sorting.HINTWwas found to be present in both female and male samples due to possible maternal contamination.HINTWalone or combined withDMRT-1gene enables early-onset sexing with 100% accuracy.