Optimized infrared spectrum of ( H 2 O ) m : ( H C N ) n $$ {\left({H}_2O\right)}_m:{(HCN)}_n $$ mixtures.

Using density functional theory at D3-B3LYP/aug-cc-pVDZ level combined with the conductor-like polarizable continuum model (CPCM) solvent model, a study of the IR spectrum of H 2 O $$ {\mathrm{H}}_2\mathrm{O} $$ :HCN mixtures is reported. The CPCM solvent effect notably enhances the accuracy of the IR spectra compared to gas-phase calculations, while the dielectric constant value has minimum impact on the final spectrum. An optimized methodology is suggested that effectively minimizes the root mean square deviation between theoretical and experimental data. This novel approach not only enhances the quality of the final IR spectra but also captures relevant spectral features, highlighting its potential to decipher molecular interactions in such intricate mixtures.

H-NS involved in positive regulation of glycerol dehydratase gene expression in Klebsiella pneumoniae 2e.

Glycerol dehydratase is the key and rate-limiting enzyme in the 1,3-propanediol synthesis pathway of Klebsiella pneumoniae, which determined the producing rate and yield of 1,3-propanediol. However, the expression regulation mechanism of glycerol dehydratase gene dhaB remains poorly unknown. In this study, a histone-like nucleoid-structuring (H-NS) protein was identified and characterized as the positive transcription regulator for dhaB expression in K. pneumoniae 2e, which exhibited high tolerance against crude glycerol in our previous study. Deletion of hns gene significantly decreased the transcription level of dhaB in K. pneumoniae 2e, which led to a remarkable defect on strain growth, glycerol dehydratase activity, and 3-hydroxypropanal production during glycerol fermentation. The transcription level of dhaB was significantly up-regulated in crude glycerol relative to pure glycerol, while the inactivation of H-NS resulted in more negative effect for transcription level of dhaB in the former. Though the H-NS expression level was almost comparable in both substrates, its multimer state was reduced in crude glycerol relative to pure glycerol, suggesting that the oligomerization state of H-NS might have contributed for positive regulation of dhaB expression. Furthermore, electrophoretic mobility shift and DNase I footprinting assays showed that H-NS could directly bind to the upstream promoter region of dhaB by recognizing the AT-rich region. These findings provided new insight into the transcriptional regulation mechanism of H-NS for glycerol dehydratase expression in K. pneumoniae, which might offer new target for engineering bacteria to industrially produce 1,3-propanediol.IMPORTANCEThe biological production of 1,3-propanediol from glycerol by microbial fermentation shows great promising prospect on industrial application. Glycerol dehydratase catalyzes the penultimate step in glycerol metabolism and is regarded as one of the key and rate-limiting enzymes for 1,3-propanediol production. H-NS was reported as a pleiotropic modulator with negative effects on gene expression in most studies. Here, we reported for the first time that the expression of glycerol dehydratase gene is positively regulated by the H-NS. The results provide insight into a novel molecular mechanism of H-NS for positive regulation of glycerol dehydratase gene expression in K. pneumoniae, which holds promising potential for facilitating construction of engineering highly efficient 1,3-propanediol-producing strains.

In Vitro Transcription Assay to Quantify Effects of H-NS Filaments on RNA Chain Elongation by RNA Polymerase.

Bacterial chromosomal DNA is structured and compacted by proteins known as bacterial chromatin proteins (i.e., nucleoid-associated proteins or NAPs). DNA-dependent RNA polymerase (RNAP) must frequently interact with bacterial chromatin proteins because they often bind DNA genome-wide. In some cases, RNAP must overcome barriers bacterial chromatin proteins impose on transcription. One key bacterial chromatin protein in Escherichia coli that influences transcription is the histone-like nucleoid structuring protein, H-NS. H-NS binds to DNA and forms nucleoprotein filaments. To investigate the effect of H-NS filaments on RNAP elongation, we developed an in vitro transcription assay to monitor RNAP progression on a DNA template bound by H-NS. In this method, initiation and elongation by RNAP are uncoupled by first initiating transcription in the presence of only three ribonucleoside triphosphates (rNTPs) to halt elongation just downstream of the promoter. Before elongation is restarted by addition of the fourth NTP, an H-NS filament is formed on the DNA so that transcript elongation occurs on an H-NS nucleoprotein filament template. Here, we provide detailed protocols for performing in vitro transcription through H-NS filaments, analysis of the transcription products, and visualization of H-NS filament formation on DNA by electrophoretic mobility shift assay (EMSA). These methods enable insight into how H-NS affects RNAP transcript elongation and provide a starting point to determine effects of other bacterial chromatin proteins on RNAP elongation.

The histone-like nucleoid-structuring protein encoded by the plasmid pMBL6842 regulates both plasmid stability and host physiology of Pseudoalteromonas rubra SCSIO 6842.

Plasmids orchestrate bacterial adaptation across diverse environments and facilitate lateral gene transfer within bacterial communities. Their presence can perturb host metabolism, creating a competitive advantage for plasmid-free cells. Plasmid stability hinges on efficient replication and partition mechanisms. While plasmids commonly encode histone-like nucleoid-structuring (H-NS) family proteins, the precise influence of plasmid-encoded H-NS proteins on stability remains elusive. In this study, we examined the conjugative plasmid pMBL6842, harboring the hns gene, and observed its positive regulation of parAB transcription, critical for plasmid segregation. Deletion of hns led to rapid plasmid loss, which was remedied by hns complementation. Further investigations unveiled adverse effects of hns overexpression on the bacterial host. Transcriptome analysis revealed hns's role in regulating numerous bacterial genes, impacting both host growth and swimming motility in the presence of the hns gene. Therefore, our study unveils the multifaceted roles of H-NS in both plasmid stability and host physiology, underscoring its biological significance and paving the way for future inquiries into the involvement of H-NS in horizontal gene transfer events.

Bacterial chromatin proteins, transcription, and DNA topology: Inseparable partners in the control of gene expression.

DNA in bacterial chromosomes is organized into higher-order structures by DNA-binding proteins called nucleoid-associated proteins (NAPs) or bacterial chromatin proteins (BCPs). BCPs often bind to or near DNA loci transcribed by RNA polymerase (RNAP) and can either increase or decrease gene expression. To understand the mechanisms by which BCPs alter transcription, one must consider both steric effects and the topological forces that arise when DNA deviates from its fully relaxed double-helical structure. Transcribing RNAP creates DNA negative (-) supercoils upstream and positive (+) supercoils downstream whenever RNAP and DNA are unable to rotate freely. This (-) and (+) supercoiling generates topological forces that resist forward translocation of DNA through RNAP unless the supercoiling is constrained by BCPs or relieved by topoisomerases. BCPs also may enhance topological stress and overall can either inhibit or aid transcription. Here, we review current understanding of how RNAP, BCPs, and DNA topology interplay to control gene expression.

Nucleoid-associated proteins shape the global protein occupancy and transcriptional landscape of a clinical isolate of Vibrio cholerae.

Vibrio cholerae, the causative agent of the diarrheal disease cholera, poses an ongoing health threat due to its wide repertoire of horizontally acquired elements (HAEs) and virulence factors. New clinical isolates of the bacterium with improved fitness abilities, often associated with HAEs, frequently emerge. The appropriate control and expression of such genetic elements is critical for the bacteria to thrive in the different environmental niches they occupy. H-NS, the histone-like nucleoid structuring protein, is the best-studied xenogeneic silencer of HAEs in gamma-proteobacteria. Although H-NS and other highly abundant nucleoid-associated proteins (NAPs) have been shown to play important roles in regulating HAEs and virulence in model bacteria, we still lack a comprehensive understanding of how different NAPs modulate transcription in V. cholerae. By obtaining genome-wide measurements of protein occupancy and active transcription in a clinical isolate of V. cholerae, harboring recently discovered HAEs encoding for phage defense systems, we show that a lack of H-NS causes a robust increase in the expression of genes found in many HAEs. We further found that TsrA, a protein with partial homology to H-NS, regulates virulence genes primarily through modulation of H-NS activity. We also identified few sites that are affected by TsrA independently of H-NS, suggesting TsrA may act with diverse regulatory mechanisms. Our results demonstrate how the combinatorial activity of NAPs is employed by a clinical isolate of an important pathogen to regulate recently discovered HAEs. IMPORTANCE: New strains of the bacterial pathogen Vibrio cholerae, bearing novel horizontally acquired elements (HAEs), frequently emerge. HAEs provide beneficial traits to the bacterium, such as antibiotic resistance and defense against invading bacteriophages. Xenogeneic silencers are proteins that help bacteria harness new HAEs and silence those HAEs until they are needed. H-NS is the best-studied xenogeneic silencer; it is one of the nucleoid-associated proteins (NAPs) in gamma-proteobacteria and is responsible for the proper regulation of HAEs within the bacterial transcriptional network. We studied the effects of H-NS and other NAPs on the HAEs of a clinical isolate of V. cholerae. Importantly, we found that H-NS partners with a small and poorly characterized protein, TsrA, to help domesticate new HAEs involved in bacterial survival and in causing disease. A proper understanding of the regulatory state in emerging isolates of V. cholerae will provide improved therapies against new isolates of the pathogen.

Otolaryngology-head and neck surgery resident and medical school characteristics: Current paradigm amidst changing application criteria.

PURPOSE: To determine the characteristics of current US Otolaryngology-Head and Neck Surgery (Oto-HNS) residents and their medical school. METHODS: Data were manually collected between Dec 2022 and Jan 2023 for 1649 residents attending 163 US-based ACGME accredited Oto-HNS residency programs, reflecting the 2018-2022 cohort. All data were collected from publicly available sources including residency and medical school program websites, web of science, and professional networking sites (ex: LinkedIn, Doximity). Data were analyzed to determine the "feeder" schools which contributed the greatest number and percent of residents. Using univariable linear regression models, we characterized factors which were associated with feeder school status. RESULTS: Of 1649 residents analyzed, 364 (22 %) matched to their home program and 918 (56 %) stayed in the region of their medical school. The median [IQR] number of published papers and abstracts was 5 [3, 9] with an h-index of 2 [1,4]. Factors associated with producing a greater percent of Oto-HNS residents include presence of an interest group, presence of a home program, USNWR research rank of the medical school, Doximity reputation rank of the home residency program, average pre-residency h-index of the school's graduates, and total NIH research funding (each p < 0.001). CONCLUSIONS: In the changing landscape of residency applications after the USMLE Step 1 exam's transition in January 2022 to pass/fail scoring, it is important to objectively characterize current Oto-HNS residents. Findings from this study will inform prospective residents and residency programs seeking to improve access to Oto-HNS. Future small-scale studies may help further identify driving factors within medical school curricula.

MucR protein: Three decades of studies have led to the identification of a new H-NS-like protein.

MucR belongs to a large protein family whose members regulate the expression of virulence and symbiosis genes in α-proteobacteria species. This protein and its homologs were initially studied as classical transcriptional regulators mostly involved in repression of target genes by binding their promoters. Very recent studies have led to the classification of MucR as a new type of Histone-like Nucleoid Structuring (H-NS) protein. Thus this review is an effort to put together a complete and unifying story demonstrating how genetic and biochemical findings on MucR suggested that this protein is not a classical transcriptional regulator, but functions as a novel type of H-NS-like protein, which binds AT-rich regions of genomic DNA and regulates gene expression.

Improved levels of checkpoint molecule PD-L1 on peripheral blood monocyte subsets in obstructive sleep apnea syndrome patients upon hypoglossal nerve stimulation.

Oxidative stress in patients suffering from obstructive sleep apnea syndrome (OSAS) is associated with a low-grade systemic inflammation, immune disturbance, and increased invasion of monocytes into the endothelium. Besides continuous positive airway pressure (PAP), hypoglossal nerve stimulation (HNS) has become a promising treatment option for patients with OSAS. We aimed to analyse the influence of HNS therapy on the cellular characteristics relevant for adhesion and immune regulation of circulating CD14/CD16 monocyte subsets. Whole blood flow cytometric measurements were performed to analyse the expression levels of different adhesion molecules and checkpoint molecule PD-L1 (programmed death-ligand 1) in connection with pro-inflammatory plasma cytokine IL-8 and the clinical values of BMI (body mass index), AHI (apnea-hypopnea index), ODI (oxygen desaturation index), and ESS (Epworth sleepiness scale) upon HNS treatment. Hypoglossal nerve stimulation treatment significantly improved the expression of adhesion molecule CD162 (P-selectin receptor) on non-classical monocytes and significantly downregulated the expression of PD-L1 on all three monocyte subsets. We conclude that the holistic improvement of different parameters such as the oxygenation of the peripheral blood, a reduced systemic inflammation, and the individual sleeping situation upon HNS respiratory support, leads to an improved immunologic situation.

Fast-Tracking of Publication Times of Otolaryngology Papers During the COVID-19 Pandemic.

Introduction The outbreak of COVID-19 has produced an unprecedented number of trials and articles. Objective To study the impact of the COVID-19 pandemic on otolaryngology-head and neck surgery (ORL-HNS) journal processing times. Methods Original papers search of published in selected ORL-HNS journals in terms of times from submission-to-acceptance (S-A), acceptance-to-first online publication (A-P), and submission-to-online publication (S-P). Papers were divided into those published in the pre-COVID-19 era and those during the COVID-19 era. The latter were further divided into unrelated to COVID-19 and related to COVID-19. Results A total of 487 articles from 5 selected ORL-HNS journals were included, of which 236 (48.5%) were published during the pre-COVID-19 era and 251 (51.5%) were published during the COVID-19 era. Among them, 180 (37%) papers were not related to COVID-19, and 71 (14.5%) were related to COVID-19. The S-A duration of COVID-19-related articles was significantly shorter compared with that of papers submitted in the pre-COVID-19 era and to papers submitted in the COVID-19 era but unrelated to COVID-19 (median 6 to 34 days compared to 65 to 125 and 46 to 127, respectively) in all 5 journals. The most prominent reductions in S-A and S-P times were documented in the laryngology and otology/neurotology disciplines, respectively. Conclusions Processing times of the included papers were significantly shorter in most of the selected ORL-HNS journals during the COVID-19 era compared with the pre-COVID-19 era. COVID-19-related papers were processed more rapidly than non-COVID-19-related papers. These findings testify to the possibility of markedly expediting S-P times and hopefully set a precedent for postpandemic publishing schedules. Level Of Evidence: 5.

Palate shape is associated with Unilateral Hypoglossal Nerve Stimulation Outcomes.

OBJECTIVE: The aim was to determine the potential association between palate shape and unilateral hypoglossal nerve stimulation (HNS) outcomes. METHODS: Preoperative drug-induced sleep endoscopy (DISE) videos were reviewed and scored by 3 blinded reviewers to determine airway narrowing at the hard-soft palate junction (HP), soft palate genu, and inferior velum, as described by Woodson (2014). Scoring was as follows: 1-open airway, 2-narrow, 3-severe narrowing. Overall palate shape (oblique, intermediate, or vertical) was determined based on prior criteria. Successful surgical treatment was defined by the HNS titration polysomnogram as a reduction of ≥50% in the apnea-hypopnea index (AHI) to <15 events/h. RESULTS: Of 332 adults, the majority was male (77%) with an average BMI of 29.2 ± 3.6 kg/m2 . Overall success rate was 73%. Success rate was lower in patients with vertical palate shape compared with the other shapes (56% vs. 75%, p = 0.029). HP score 3 compared with scores 2 and 1 was associated with lower success rates (60% vs. 76%, p = 0.028), but genu and velum scores were not associated with outcomes. Patients with both HP score 3 and complete oropharyngeal lateral wall-related obstruction had notably worse outcomes (22% vs. 74%, p = 0.026). HP score 3 (OR 0.45, 95%CI 0.22-0.92) and vertical palate shape (OR 0.33, 95%CI 0.15-0.78) were independently associated with lower odds of surgical response after adjustment for DISE findings, age, gender, and BMI. CONCLUSION: Vertical palate shape and narrowing at the hard-soft palate junction are independently associated with lower HNS surgical success rates. LEVEL OF EVIDENCE: 3 Laryngoscope, 134:981-986, 2024.

Hierarchic regulation of a metabolic pathway: H-NS, CRP, and SsrB control myo-inositol utilization by Salmonella enterica.

IMPORTANCE: The capacity to utilize myo-inositol (MI) as sole carbon and energy source is widespread among bacteria, among them the intestinal pathogen S. Typhimurium. This study elucidates the complex and hierarchical regulation that underlies the utilization of MI by S. Typhimurium under substrate limitation. A total of seven regulatory factors have been identified so far, allowing the pathogen an environment-dependent, efficient, and fine-tuned regulation of a metabolic property that provides growth advantages in different environments.

Fast-Tracking of Publication Times of Otolaryngology Papers During the COVID-19 Pandemic

Abstract Introduction The outbreak of COVID-19 has produced an unprecedented number of trials and articles. Objective To study the impact of the COVID-19 pandemic on otolaryngology-head and neck surgery (ORL-HNS) journal processing times. Methods Original papers search of published in selected ORL-HNS journals in terms of times from submission-to-acceptance (S-A), acceptance-to-first online publication (A-P), and submission-to-online publication (S-P). Papers were divided into those published in the pre-COVID-19 era and those during the COVID-19 era. The latter were further divided into unrelated to COVID-19 and related to COVID-19. Results A total of 487 articles from 5 selected ORL-HNS journals were included, of which 236 (48.5%) were published during the pre-COVID-19 era and 251 (51.5%) were publishedduring theCOVID-19era.Amongthem, 180 (37%) papers werenot related to COVID-19, and 71 (14.5%) were related to COVID-19. The S-A duration of COVID-19-related articles was significantly shorter compared with that of papers submitted in the pre-COVID-19 era and to papers submitted in the COVID-19 era but unrelated to COVID-19 (median 6 to 34 days compared to 65 to 125 and 46 to 127, respectively) in all 5 journals. The most prominent reductions in S-A and S-P times were documented in the laryngology and otology/neurotology disciplines, respectively. Conclusions Processing times of the included papers were significantly shorter in most of the selected ORL-HNS journals during the COVID-19 era compared with the pre-COVID-19 era. COVID-19-related papers were processed more rapidly than non-COVID-19-related papers. These findings testify to the possibility of markedly expediting S-P times and hopefully set a precedent for postpandemic publishing schedules. Level Of Evidence: 5

Brucella MucR acts as an H-NS-like protein to silence virulence genes and structure the nucleoid.

Histone-like nucleoid structuring (H-NS) and H-NS-like proteins serve as global gene silencers and work with antagonistic transcriptional activators (counter-silencers) to properly coordinate the expression of virulence genes in pathogenic bacteria. In Brucella, MucR has been proposed as a novel H-NS-like gene silencer, but direct experimental evidence is lacking. Here, we show that MucR serves as an H-NS-like silencer of the Brucella abortus genes encoding the polar autotransporter adhesins BtaE and BmaC, the c-di-GMP-specific phosphodiesterase BpdB, and the quorum-sensing regulator BabR. We also demonstrate that the MarR-type transcriptional activator MdrA can displace MucR from the btaE promoter, supporting the existence of MucR counter-silencers in Brucella. Moreover, our chromatin immunoprecipitation (ChIP)-seq analysis identified 546 MucR enrichment peaks along the genome, including in the promoters of the genes encoding the Type IV secretion machinery and effectors and the quorum-sensing regulator VjbR. Importantly, MucR ChIP-seq peaks overlap with the previously described binding sites for the transcriptional activators VjbR, BvrR, and CtrA suggesting that these regulators serve as MucR counter-silencers and work in concert with MucR to coordinate virulence gene expression in Brucella. In addition, using chromosome conformation capture (Hi-C), we show that like H-NS in Escherichia coli, MucR alters the global structure of the Brucella nucleoid. Finally, a copy of the E. coli hns rescues the distinctive growth defect and elevated btaE expression of a B. abortus mucR mutant. Together, these findings solidify the role of MucR as a novel type of H-NS-like protein and suggest that MucR's gene-silencing properties play a key role in virulence in Brucella. IMPORTANCE Histone-like nucleoid structuring (H-NS) and H-NS-like proteins coordinate host-associated behaviors in many pathogenic bacteria, often through forming silencer/counter-silencer pairs with signal-responsive transcriptional activators to tightly control gene expression. Brucella and related bacteria do not encode H-NS or homologs of known H-NS-like proteins, and it is unclear if they have other proteins that perform analogous functions during pathogenesis. In this work, we provide compelling evidence for the role of MucR as a novel H-NS-like protein in Brucella. We show that MucR possesses many of the known functions attributed to H-NS and H-NS-like proteins, including the formation of silencer/counter-silencer pairs to control virulence gene expression and global structuring of the nucleoid. These results uncover a new role for MucR as a nucleoid structuring protein and support the importance of temporal control of gene expression in Brucella and related bacteria.

MucR from Sinorhizobium meliloti: New Insights into Its DNA Targets and Its Ability to Oligomerize.

Proteins of the MucR/Ros family play a crucial role in bacterial infection or symbiosis with eukaryotic hosts. MucR from Sinorhizobium meliloti plays a regulatory role in establishing symbiosis with the host plant, both dependent and independent of Quorum Sensing. Here, we report the first characterization of MucR isolated from Sinorhizobium meliloti by mass spectrometry and demonstrate that this protein forms higher-order oligomers in its native condition of expression by SEC-MALS. We show that MucR purified from Sinorhizobium meliloti can bind DNA and recognize the region upstream of the ndvA gene in EMSA, revealing that this gene is a direct target of MucR. Although MucR DNA binding activity was already described, a detailed characterization of Sinorhizobium meliloti DNA targets has never been reported. We, thus, analyze sequences recognized by MucR in the rem gene promoter, showing that this protein recognizes AT-rich sequences and does not require a consensus sequence to bind DNA. Furthermore, we investigate the dependence of MucR DNA binding on the length of DNA targets. Taken together, our studies establish MucR from Sinorhizobium meliloti as a member of a new family of Histone-like Nucleoid Structuring (H-NS) proteins, thus explaining the multifaceted role of this protein in many species of alpha-proteobacteria.

Absence of proline-peptide transporter YjiY in Salmonella Typhimurium leads to secretion of factors which inhibits intra-species biofilm formation.

Salmonella is a genus of widely spread Gram negative, facultative anaerobic bacteria, which is known to cause »th of diarrheal morbidity and mortality globally. It causes typhoid fever and gastroenteritis by gaining access to the host gut through contaminated food and water. Salmonella utilizes its biofilm lifestyle to strongly resist antibiotics and persist in the host. Although biofilm removal or dispersal has been studied widely, the inhibition of the initiation of Salmonella Typhimurium (STM WT) biofilm remains elusive. This study demonstrates the anti-biofilm property of the cell-free supernatant obtained from a carbon-starvation induced proline peptide transporter mutant (STM ΔyjiY) strain. The STM ΔyjiY culture supernatant primarily inhibits biofilm initiation by regulating biofilm-associated transcriptional network that is reversed upon complementation (STM ΔyjiY:yjiY). We demonstrate that abundance of FlgM correlates with the absence of flagella in the STM ΔyjiY supernatant treated WT cells. NusG works synergistically with the global transcriptional regulator H-NS. Relatively low abundances of flavoredoxin, glutaredoxin, and thiol peroxidase might lead to accumulation of ROS within the biofilm, and subsequent toxicity in STM ΔyjiY supernatant. This work further suggests that targeting these oxidative stress relieving proteins might be a good choice to reduce Salmonella biofilm.

NhaR, LeuO, and H-NS Are Part of an Expanded Regulatory Network for Ectoine Biosynthesis Expression.

Bacteria accumulate compatible solutes to maintain cellular turgor pressure when exposed to high salinity. In the marine halophile Vibrio parahaemolyticus, the compatible solute ectoine is biosynthesized de novo, which is energetically more costly than uptake; therefore, tight regulation is required. To uncover novel regulators of the ectoine biosynthesis ectABC-asp_ect operon, a DNA affinity pulldown of proteins interacting with the ectABC-asp_ect regulatory region was performed. Mass spectrometry analysis identified, among others, 3 regulators: LeuO, NhaR, and the nucleoid associated protein H-NS. In-frame non-polar deletions were made for each gene and PectA-gfp promoter reporter assays were performed in exponential and stationary phase cells. PectA-gfp expression was significantly repressed in the ΔleuO mutant and significantly induced in the ΔnhaR mutant compared to wild type, suggesting positive and negative regulation, respectively. In the Δhns mutant, PectA-gfp showed increased expression in exponential phase cells, but no change compared to wild type in stationary phase cells. To examine whether H-NS interacts with LeuO or NhaR at the ectoine regulatory region, double deletion mutants were created. In a ΔleuO/Δhns mutant, PectA-gfp showed reduced expression, but significantly more than ΔleuO, suggesting H-NS and LeuO interact to regulate ectoine expression. However, ΔnhaR/Δhns had no additional effect compared to ΔnhaR, suggesting NhaR regulation is independent of H-NS. To examine leuO regulation further, a PleuO-gfp reporter analysis was examined that showed significantly increased expression in the ΔleuO, Δhns, and ΔleuO/Δhns mutants compared to wild type, indicating both are repressors. Growth pattern analysis of the mutants in M9G 6%NaCl showed growth defects compared to wild type, indicating that these regulators play an important physiological role in salinity stress tolerance outside of regulating ectoine biosynthesis gene expression. IMPORTANCE Ectoine is a commercially used compatible solute that acts as a biomolecule stabilizer because of its additional role as a chemical chaperone. A better understanding of how the ectoine biosynthetic pathway is regulated in natural bacterial producers can be used to increase efficient industrial production. The de novo biosynthesis of ectoine is essential for bacteria to survive osmotic stress when exogenous compatible solutes are absent. This study identified LeuO as a positive regulator and NhaR as a negative regulator of ectoine biosynthesis and showed that, similar to enteric species, LeuO is an anti-silencer of H-NS. In addition, defects in growth in high salinity among all the mutants suggest that these regulators play a broader role in the osmotic stress response beyond ectoine biosynthesis regulation.

Determination of the Chromatin Openness in Bacterial Genomes.

The hyperactive Tn5 transposase in the ATAC-seq method has been widely used to determine the open DNA regions and understand the overall epigenomic regulation in the chromatins of eukaryotic cells. Here, we describe POP-seq (Prokaryotic chromatin Openness Profiling sequencing), an adaptation of the ATAC-seq method, to interrogate changes in the openness of prokaryotic nucleoids.

Serotype conversion gene rfbT is directly regulated by histone-like nucleoid structuring protein (H-NS) in V. cholerae O1.

Vibrio cholerae serogroup O1 (V. cholerae O1) is closely associated with cholera epidemics and has two main immunologically distinguishable serotypes, Ogawa and Inaba. Isolates serotype as Ogawa if the O-antigen polysaccharide (O-PS) is methylated or as Inaba if the O-PS is not methylated. This methylation is mediated by a methyltransferase encoded by the rfbT gene, and the mutation and low expression of rfbT results in serotype switch from Ogawa to Inaba. Previously, we have shown that cAMP receptor protein (CRP) activates rfbT. In this study, we demonstrated that histone-like nucleoid structuring protein (H-NS) is directly involved in the transcriptional repression of rfbT. This finding is supported by the analyses of rfbT mRNA level, rfbT-lux reporter fusions, electrophoretic mobility shift assay (EMSA), and DNase I footprinting assay. The rfbT mRNA abundances were significantly increased by deleting hns rather than fis which also preferentially associates with AT-rich sequences. A single-copy chromosomal complement of hns partly restored the down-regulation of rfbT. Analysis of rfbT-lux reporter fusions validated the transcriptional repression of hns. Subsequent EMSA and DNase I footprinting assay confirmed the direct binding of H-NS to rfbT promoter and mapped the exact binding site which was further verified by site-directed mutagenesis and promoter functional analysis. Furthermore, we found that in hns deletion mutant, CRP is no longer required for transcriptionally activating rfbT, suggesting that CRP functions as a dedicated transcription factor to relieve H-NS repression at rfbT. Together, this study expanded our understanding of the genetic regulatory mechanism of serotype conversion by global regulators in V. cholerae O1.

Field-portable seawater toxicity monitoring platform using lens-free shadow imaging technology.

The accidental spill of hazardous and noxious substances (HNSs) in the ocean has serious environmental and human health consequences. Assessing the ecotoxicity of seawater exposed to various HNS is challenging due to the constant development of new HNS or mixtures, and assessment methods are also limited. Microalgae viability tests are often used among the various biological indicators for ecotoxicity testing, as they are the primary producers in aquatic ecosystems. However, since the conventional cell growth rate test measures cell viability over three to four days using manual inspection under a conventional optical microscope, it is labor- and time-intensive and prone to subjective errors. In this study, we propose a rapid and automated method to evaluate seawater ecotoxicity by quantification of the morphological changes of microalgae exposed to more than 30 HNSs. This method was further validated using conventional growth rate test results. Dunaliella tertiolecta, a microalgae species without rigid cell walls, was selected as the test organism. Its morphological changes in response to HNS exposure were measured at the single cell level using a custom-developed device that uses lens-free shadow imaging technology. The ecotoxicity evaluation induced by the morphological change could be available in as little as 5 min using the proposed method and device, and it could be effective for 20 HNSs out of 30 HNSs tested. Moreover, the test results of six selected HNSs with high marine transport volume and toxicity revealed that the sensitivity of the proposed method extends to half the maximum effective concentration (EC50) and even to the lowest observed effective concentration (LOEC). Furthermore, the average correlation index between the growth inhibition test (three to four days) and the proposed morphology changes test (5 min) for the six selected HNSs was 0.84, indicating great promise in the field of various point-of-care water quality monitoring. Thus, the proposed equipment and technology may provide a viable alternative to traditional on-site toxicity testing, and the potential of rapid morphological analysis may replace traditional growth inhibition testing.