Global Regulatory Mechanisms Research Articles

Implications of carbon catabolite repression for Aspergillus-based cell factories: A review.

Carbon catabolite repression (CCR) is a global regulatory mechanism that allows organisms to preferentially utilize a preferred carbon source (usually glucose) by suppressing the expression of genes associated with the utilization of nonpreferred carbon sources. Aspergillus is a large genus of filamentous fungi, some species of which have been used as microbial cell factories for the production of organic acids, industrial enzymes, pharmaceuticals, and other fermented products due to their safety, substrate convenience, and well-established post-translational modifications. Many recent studies have verified that CCR-related genetic alterations can boost the yield of various carbohydrate-active enzymes (CAZymes), even under CCR conditions. Based on these findings, we emphasize that appropriate regulation of the CCR pathway, especially the expression of the key transcription factor CreA gene, has great potential for further expanding the application of Aspergillus cell factories to develop strains for industrial CAZymes production. Further, the genetically modified CCR strains (chassis hosts) can also be used for the production of other useful natural products and recombinant proteins, among others. We here review the regulatory mechanisms of CCR in Aspergillus and its direct application in enzyme production, as well as its potential application in organic acid and pharmaceutical production to illustrate the effects of CCR on Aspergillus cell factories.

Navigating Governance Networks for Urban Issues: Global Trends and Prospects

This study examines the critical role of governance networks in addressing urban issues from a global perspective. We further elucidate the inception of these networks and how they can contribute to resolving the multifaceted challenges in urban issues. We employed a qualitative research method underpinned by an integrated literature review (ILR) approach. ILR, as a research methodology, allows for the critical examination, integration, and synthesis of extant literature, thereby enabling the generation of fresh frameworks and perspectives on the topic at hand. Our findings underscore the need for diverse actors, including government officials, societal figures, and entrepreneurs, to govern these networks and address urban issues. We also discovered that governance networks, by structuring societal dynamics, are systematically influenced by global socioeconomic and political regulatory mechanisms. Their applications span various public sector issues, such as mass media, healthcare, and water governance. The primary limitation of our study lies in the lack of field studies. Despite this, the outcomes offer significant insights into the theoretical concepts of governance networks from different regions, a distinctive contribution to the existing body of knowledge.
 Keywords: urban issues, governance networks, theoretical concept

Optimizing Port Multi-AGV Trajectory Planning through Priority Coordination: Enhancing Efficiency and Safety

Efficient logistics and transport at the port heavily relies on efficient AGV scheduling and planning for container transshipment. This paper presents a comprehensive approach to address the challenges in AGV path planning and coordination within the domain of intelligent transportation systems. We propose an enhanced graph search method for constructing the global path of a single AGV that mitigates the issues associated with paths closely aligned with obstacle corner points. Moreover, a centralized global planning module is developed to facilitate planning and scheduling. Each individual AGV establishes real-time communication with the upper layers to accurately determine its position at complex intersections. By computing its priority sequence within a coordination circle, the AGV effectively treats the high-priority trajectories of other vehicles as dynamic obstacles for its local trajectory planning. The feasibility of trajectory information is ensured by solving the online real-time Optimal Control Problem (OCP). In the trajectory planning process for a single AGV, we incorporate a linear programming-based obstacle avoidance strategy. This strategy transforms the obstacle avoidance optimization problem into trajectory planning constraints using Karush-Kuhn-Tucker (KKT) conditions. Consequently, seamless and secure AGV movement within the port environment is guaranteed. The global planning module encompasses a global regulatory mechanism that provides each AGV with an initial feasible path. This approach not only facilitates complexity decomposition for large-scale problems, but also maintains path feasibility through continuous real-time communication with the upper layers during AGV travel. A key advantage of our progressive solution lies in its flexibility and scalability. This approach readily accommodates extensions based on the original problem and allows adjustments in the overall problem size in response to varying port cargo throughput, all without requiring a complete system overhaul.

The regulation of bacterial two-partner secretion systems.

Two-partner secretion (TPS) systems, also known as Type Vb secretion systems, allow the translocation of effector proteins across the outer membrane of Gram-negative bacteria. By secreting different classes of effectors, including cytolysins and adhesins, TPS systems play important roles in bacterial pathogenesis and host interactions. Here, we review the current knowledge on TPS systems regulation and highlight specific and common regulatory mechanisms across TPS functional classes. We discuss in detail the specific regulatory networks identified in various bacterial species and emphasize the importance of understanding the context-dependent regulation of TPS systems. Several regulatory cues reflecting host environment during infection, such as temperature and iron availability, are common determinants of expression for TPS systems, even across relatively distant species. These common regulatory pathways often affect TPS systems across subfamilies with different effector functions, representing conserved global infection-related regulatory mechanisms.

Crossing bacterial boundaries: The carbon catabolite repression system Crc-Hfq of Pseudomonas putida KT2440 as a tool to control translation in E. coli

As a global regulatory mechanism, carbon catabolite repression allows bacteria and eukaryal microbes to preferentially utilize certain substrates from a mixture of carbon sources. The mechanism varies among different species. In Pseudomonas spp., it is mainly mediated by the Crc-Hfq complex which binds to the 5′ region of the target mRNAs, thereby inhibiting their translation. This molecular mechanism enables P. putida to rapidly adjust and fine-tune gene expression in changing environments. Hfq is an RNA-binding protein that is ubiquitous and highly conserved in bacterial species. Considering the characteristics of Hfq, and the widespread use and rapid response of Crc-Hfq in P. putida, this complex has the potential to become a general toolbox for post-transcriptional multiplex regulation. In this study, we demonstrate for the first time that transplanting the pseudomonal catabolite repression protein, Crc, into E. coli causes multiplex gene repression. Under the control of Crc, the production of a diester and its precursors was significantly reduced. The effects of Crc introduction on cell growth in both minimal and rich media were evaluated. Two potential factors - off-target effects and Hfq-sequestration - could explain negative effects on cell growth. Simultaneous reduction of off-targeting and increased sequestration of Hfq by the introduction of the small RNA CrcZ, indicated that Hfq sequestration plays a more prominent role in the negative side-effects. This suggests that the negative growth effect can be mitigated by well-controlled expression of Hfq. This study reveals the feasibility of controlling gene expression using heterologous regulation systems.

Chromosome-level genome assembly of Hydractinia symbiolongicarpus.

Hydractinia symbiolongicarpus is a pioneering model organism for stem cell biology, being one of only a few animals with adult pluripotent stem cells (known as i-cells). However, the unavailability of a chromosome-level genome assembly has hindered a comprehensive understanding of global gene regulatory mechanisms underlying the function and evolution of i-cells. Here, we report the first chromosome-level genome assembly of H. symbiolongicarpus (HSymV2.0) using PacBio HiFi long-read sequencing and Hi-C scaffolding. The final assembly is 483 Mb in total length with 15 chromosomes representing 99.8% of the assembly. Repetitive sequences were found to account for 296 Mb (61%) of the total genome; we provide evidence for at least two periods of repeat expansion in the past. A total of 25,825 protein-coding genes were predicted in this assembly, which include 93.1% of the metazoan Benchmarking Universal Single-Copy Orthologs (BUSCO) gene set. 92.8% (23,971 genes) of the predicted proteins were functionally annotated. The H. symbiolongicarpus genome showed a high degree of macrosynteny conservation with the Hydra vulgaris genome. This chromosome-level genome assembly of H. symbiolongicarpus will be an invaluable resource for the research community that enhances broad biological studies on this unique model organism.

Міокіни – один із ключових елементів взаємодії між скелетними м'язами та іншими системами організму людини, необхідних для адаптації до фізичних навантажень

Muscle tissue makes up a significant percentage of body weight, and its metabolism affects almost all body systems. Despite a sufficiently large number of physiological observations that confirmed the need for regular physical activity to maintain health, the molecular mechanisms of such an effect remained unestablished for a long time. Results of recent research confirmed that skeletal muscles are an endocrine organ that produces a wide range of bioregulators, which synthesis and excretion are stimulated during exercise. At present, many of these factors that mediate metabolic and physiological responses in muscles and other organs have been identified and named myokines. To date the most studied myokines are: interleukins (IL-6, LIF, IL-4, IL-7, IL-8, and IL-15), myostatin, myonectin (CTRP15), irisin, fibroblast growth factor 21 (FGF21), brain-derived neurotrophic factor (BDNF), insulin-like growth factor-1 (IGF-1), follistatin-like protein-1 (FSTL-1), decorin and SPARC (osteonectin). Most of the myokines exert their effects through paracrine and/or autocrine pathways within muscles, and many of them also act as endocrine agents – via lymph and blood. The biological activity of myokines is realized via modulation of the activity of general global regulatory mechanisms, such as the SMAD signaling cascade, p38/MAPK, Erk1/2 MAPK, PI3K/Akt/GSK-3β, cAMP/Akt, AMPK-dependent regulation, and JNK signaling pathway. At the same time, myokines are involved in the regulation of the activity of the myogenic transcription factors MyoD, myf5, myogenin and a number of proteins involved in the sensing and transport of glucose and fatty acids. Myokines play one of the main roles in the interaction between skeletal muscles, liver, bone and adipose tissues. They increase tissues sensitivity to insulin and are involved in the regulation of important metabolic processes such as carbohydrate, protein and lipid metabolism. Myokines play a significant role in the regulation of myogenesis, osteogenesis, thermogenesis, lipolysis, growth and division of muscle and nerve tissue cells, vascularization, etc. Given the fact that the expression of myokines is induced by muscle contraction, their study allows us to reveal the molecular mechanisms realizing the positive effects of physical exertion. Further studies of myokines and their mechanisms of action are necessary for the development of personalized recommendations for the physical activity of people with metabolic diseases in rehabilitation, physical therapy, medical supervision of physical education and sports.

Plant HEM1 specifies a condensation domain to control immune gene translation.

Translational reprogramming is a fundamental layer of immune regulation, but how such a global regulatory mechanism operates remains largely unknown. Here we perform a genetic screen and identify Arabidopsis HEM1 as a global translational regulator of plant immunity. The loss of HEM1 causes exaggerated cell death to restrict bacterial growth during effector-triggered immunity (ETI). By improving ribosome footprinting, we reveal that the hem1 mutant increases the translation efficiency of pro-death immune genes. We show that HEM1 contains a plant-specific low-complexity domain (LCD) absent from animal homologues. This LCD endows HEM1 with the capability of phase separation in vitro and in vivo. During ETI, HEM1 interacts and condensates with the translation machinery; this activity is promoted by the LCD. CRISPR removal of this LCD causes more ETI cell death. Our results suggest that HEM1 condensation constitutes a brake mechanism of immune activation by controlling the tissue health and disease resistance trade-off during ETI.

Diversity in Genetic Regulation of Bacterial Fimbriae Assembled by the Chaperone Usher Pathway.

Bacteria express different types of hair-like proteinaceous appendages on their cell surface known as pili or fimbriae. These filamentous structures are primarily involved in the adherence of bacteria to both abiotic and biotic surfaces for biofilm formation and/or virulence of non-pathogenic and pathogenic bacteria. In pathogenic bacteria, especially Gram-negative bacteria, fimbriae play a key role in bacteria-host interactions which are critical for bacterial invasion and infection. Fimbriae assembled by the Chaperone Usher pathway (CUP) are widespread within the Enterobacteriaceae, and their expression is tightly regulated by specific environmental stimuli. Genes essential for expression of CUP fimbriae are organised in small blocks/clusters, which are often located in proximity to other virulence genes on a pathogenicity island. Since these surface appendages play a crucial role in bacterial virulence, they have potential to be harnessed in vaccine development. This review covers the regulation of expression of CUP-assembled fimbriae in Gram-negative bacteria and uses selected examples to demonstrate both dedicated and global regulatory mechanisms.

Global transcriptome analyses and regulatory mechanisms in Halothece sp. PCC 7418 exposed to abiotic stresses.

Halotolerant species are of interest since they occur naturally in environments with excess toxic ions. The cyanobacterium Halothece sp. PCC 7418 (hereafter referred to as Halothece) exhibits remarkable halotolerance and was used to examine stress-responsive regulatory mechanisms. The effects of five different stimuli on Halothece transcriptomes were examined using RNA sequencing. In response to diverse stresses, there were both common and stress-specific transcriptional responses. A common upregulated gene set under all stresses consisted of nine differentially expressed genes (DEGs). We also found that osmotic stress elicited the largest set of DEGs. Salt- and osmotic-responsive regulatory mechanisms shared common pathways. DEGs that were upregulated under salt stress encoded proteins involved in photosynthesis and related machineries. Furthermore, DEGs encoding two-component system (TCS) factors, transcriptional factors, scaffolds for protein-protein interactions, transporters, protein turnover factors, and lipid biosynthesis enzymes were also identified under salt stress. Notably, one-carbon (1C) metabolism factors, glycine betaine (GB) synthesis enzymes, and GB transporters were upregulated under salt stress. Metabolic analyses revealed that GB accumulated under salt stress, while mycosporine-2-glycine (M2G) accumulated under salt or osmotic stress. None of the nutrient starvations induced GB nor M2G accumulation. These results suggested that GB and M2G are two osmoprotectants that contribute to halotolerance. Based on our results, we proposed regulatory mechanisms that are crucial for halotolerance, which are coordinated with the GB, M2G, 1C, amino acid, and central carbon interlinking metabolic pathways. 1C metabolism directly fulfills the high metabolite requirements for halotolerance together with the ancillary role of several metabolic pathways.Key Points• Global transcriptome surveys together with molecular and metabolite analyses provide insights into regulatory networks that are crucial for halotolerance• Regulatory networks that are crucial for halotolerance coordinated with the two key osmoprotectants, one carbon, amino acid, and central carbon interlinking metabolic pathways• The findings have translational relevance in genomic and transcriptomic mechanisms of halotolerance.

Contrasting regulatory effects of organic acids on aerobic vinyl chloride biodegradation in etheneotrophs.

Vinyl chloride (VC) is a common groundwater pollutant generated during anaerobic biodegradation of chlorinated solvents (e.g., trichloroethene (TCE) or tetrachloroethene (PCE)). Aerobic VC biodegradation by etheneotrophs can support anaerobic PCE and TCE bioremediation to achieve complete removal in situ. However, anaerobic bioremediation strategies necessitate biostimulation with electron donors that are fermented in situ, generating organic acids that could influence aerobic VC biodegradation processes. We examined the effect of organic acids (lactate, acetate, propionate, and butyrate) on aerobic VC biodegradation by VC-assimilating etheneotrophs Mycobacterium strain JS60 and Nocardioides strain JS614. Strain JS60 grew on all organic acids tested, while strain JS614 did not respond to lactate. VC-grown strain JS60 fed VC and one or more organic acids showed carbon catabolite repression (CCR) behavior where VC biodegradation occurred only after organic acids were depleted. In contrast, CCR was not evident in VC-grown strain JS614, which degraded VC and organic acids simultaneously. Acetate-grown JS60 showed similar CCR behavior when fed VC and a single organic acid, except that extended lag periods (5-12days) occurred before VC oxidation ensued. Acetate-grown JS614 fed VC and either acetate or butyrate displayed 5-8day lag periods before simultaneous VC and organic acid biodegradation. In contrast, acetate-grown JS614 degraded VC and propionate without a significant lag, suggesting a regulatory link between propionate and VC oxidation in JS614. Different global regulatory mechanisms controlling VC biodegradation in the presence of organic acids in etheneotrophs have implications for developing combined anaerobic-aerobic bioremediation strategies at chlorinated ethene-contaminated sites. KEY POINTS: • With organic acids present, VC utilization was repressed in JS60, but not in JS614 • Strain JS60 grew readily on lactate, while strain JS614 did not • Propionate alleviated lag periods for VC utilization in acetate-grown JS614.

Opportunities to Foster Conservation and Sustainable Use of Biodiversity Beyond National Jurisdiction: A Role for Scientists

AbstractCountries are currently negotiating a comprehensive global binding treaty at the United Nations to better protect biodiversity in the high seas (referred to from here as “the Treaty”). Developing a governance framework for the sharing of benefits from marine genetic resources (MGRs) of the high seas (or areas beyond national jurisdiction, referred to from here as “ABNJ”) is a crucial element of the proposed treaty. Negotiating countries' positions on MGRs vary widely, ranging from no regulation to the establishment of a new global regulatory mechanism. For the scientists who may be involved with collecting MGRs in ABNJ, the Treaty could impact their research activities, including mandatory precollection notification. There could also be new requirements for daily research activities associated with utilizing physical materials collected in ABNJ or data generated from such materials. For indigenous peoples and local communities (IPLC) that may hold relevant traditional knowledge, the Treaty could ensure their rights to prior, free, and informed consent and the use of mutually agreed terms. The scientific community and IPLC have an opportunity to engage with key stakeholders to increase the chances that any new requirements will be practical, remain relevant in decades to come, and provide a platform that will foster research, collaboration, and marine biological diversity conservation.

Quantitative contribution of the spacer length in the supercoiling-sensitivity of bacterial promoters.

DNA supercoiling acts as a global transcriptional regulator in bacteria, but the promoter sequence or structural determinants controlling its effect remain unclear. It was previously proposed to modulate the torsional angle between the −10 and −35 hexamers, and thereby regulate the formation of the closed-complex depending on the length of the ‘spacer’ between them. Here, we develop a thermodynamic model of this notion based on DNA elasticity, providing quantitative and parameter-free predictions of the relative activation of promoters containing a short versus long spacer when the DNA supercoiling level is varied. The model is tested through an analysis of in vitro and in vivo expression assays of mutant promoters with variable spacer lengths, confirming its accuracy for spacers ranging from 15 to 19 nucleotides, except those of 16 nucleotides where other regulatory mechanisms likely overcome the effect of this specific step. An analysis at the whole-genome scale in Escherichia coli then demonstrates a significant effect of the spacer length on the genomic expression after transient or inheritable superhelical variations, validating the model’s predictions. Altogether, this study shows an example of mechanical constraints associated to promoter binding by RNA Polymerase underpinning a basal and global regulatory mechanism.

Systems-Level Analysis of the Global Regulatory Mechanism of CodY in Lactococcus lactis Metabolism and Nisin Immunity Modulation.

Bacteria adapt to the constantly changing environment by regulating their metabolism. The global transcriptional regulator CodY is known to regulate metabolism in low-G+C Gram-positive bacteria. Systems-level identification of its direct targets by proteome and chromatin immunoprecipitation followed by sequencing (ChIP-seq) assays have rarely been reported. Here, we identified that CodY serves as an activator or a repressor of hundreds of genes involved in nitrogen metabolism, carbohydrate metabolism, and transcription through iTRAQ proteome and ChIP-seq. Combined with the electrophoretic mobility shift assay (EMSA), apart from the genes associated with amino acid biosynthesis (ilvD, leuA, optS, ybbD, dtpT, and pepN), genes involved in cell wall synthesis (murD and ftsW) and nisin immunity (nisI) were identified as being regulated by CodY. Moreover, it was demonstrated by nisin resistance assay that CodY activated the transcription of nisI and contributed to nisin immunity. Intriguingly, CodY showed a self-regulation through binding to the motif AAAGGTGTGACAACT in the coding sequence (CDS) region of codY, as verified by DNase I footprinting assay and MEME analysis. In addition, a novel conserved AT-rich motif, AATWTTCTGACAATT, was obtained in L. lactis F44. This study provides new insights into the comprehensive CodY regulation in L. lactis by controlling metabolism, nisin immunity, and self-expression. IMPORTANCE Lactococcus lactis, a species of lactic acid bacteria (LAB) widely used in food fermentation, has been the model strain in genetic engineering, and its application has extended from food to microbial cell factories. CodY is a global regulator in low-G+C Gram-positive bacteria. Its function and direct target genes at the genome-level are little known in L. lactis. In this study, we describe the comprehensive regulation mechanism of CodY. It widely modulated the metabolism of nitrogen and carbohydrate, cell wall synthesis, and nisin immunity in L. lactis F44, and its expression level was regulated by feedback control.

Is South Africa ready for the future of human germline genome editing? Comparing South African law and recent proposals for global governance

Over the past few years, developments in the science of precise editing of human genomes using CRISPR-Cas9 have led many countries that lack specific laws in this area, such as South Africa (SA), to contemplate legal reform. Thaldar et al. recently published five principles to guide legal reform in SA on heritable genome editing. In a similar vein, concerns about the global impact of human germline genome editing have led to calls for a global regulatory mechanism. This is what the World Health Organization has tried to achieve with the recently published ‘Draft governance framework for human genome editing’. In this article, we compare the policies proposed by the draft framework to the current SA legal position, as well as the five guiding principles. The article concludes that SA law is in need of reform in order to meet the global standards that the draft framework seems to be moving towards.

Theory of branching morphogenesis by local interactions and global guidance

Branching morphogenesis governs the formation of many organs such as lung, kidney, and the neurovascular system. Many studies have explored system-specific molecular and cellular regulatory mechanisms, as well as self-organizing rules underlying branching morphogenesis. However, in addition to local cues, branched tissue growth can also be influenced by global guidance. Here, we develop a theoretical framework for a stochastic self-organized branching process in the presence of external cues. Combining analytical theory with numerical simulations, we predict differential signatures of global vs. local regulatory mechanisms on the branching pattern, such as angle distributions, domain size, and space-filling efficiency. We find that branch alignment follows a generic scaling law determined by the strength of global guidance, while local interactions influence the tissue density but not its overall territory. Finally, using zebrafish innervation as a model system, we test these key features of the model experimentally. Our work thus provides quantitative predictions to disentangle the role of different types of cues in shaping branched structures across scales.

Insights Into Mechanism of the Naphthalene-Enhanced Biodegradation of Phenanthrene by Pseudomonas sp. SL-6 Based on Omics Analysis.

The existence of polycyclic aromatic hydrocarbons (PAHs) in contaminated environment is multifarious. At present, studies of metabolic regulation focus on the degradation process of single PAH. The global metabolic regulatory mechanisms of microorganisms facing coexisting PAHs are poorly understood, which is the major bottleneck for efficient bioremediation of PAHs pollution. Naphthalene (NAP) significantly enhanced the biodegradation of phenanthrene (PHE) by Pseudomonas sp. SL-6. To explore the underlying mechanism, isobaric tags for relative and absolute quantification (iTRAQ) labeled quantitative proteomics was used to characterize the differentially expressed proteins of SL-6 cultured with PHE or NAP + PHE as carbon source. Through joint analysis of proteome and genome, unique proteins were identified and quantified. The up-regulated proteins mainly concentrated in PAH catabolism, Transporters and Electron transfer carriers. In the process, the regulator NahR, activated by salicylate (intermediate of NAP-biodegradation), up-regulates degradation enzymes (NahABCDE and SalABCDEFGH), which enhances the biodegradation of PHE and accumulation of toxic intermediate–1-hydroxy-2-naphthoic acid (1H2Na); 1H2Na stimulates the expression of ABC transporter, which maintains intracellular physiological activity by excreting 1H2Na; the up-regulation of cytochrome C promotes the above process running smoothly. Salicylate works as a trigger that stimulates cell to respond globally. The conjecture was verified at transcriptional and metabolic levels. These new insights contribute to improving the overall understanding of PAHs-biodegradation processes under complex natural conditions, and promoting the application of microbial remediation technology for PAHs pollution.

Fis Connects Two Sensory Pathways, Quorum Sensing and Surface Sensing, to Control Motility in Vibrio parahaemolyticus.

Factor for inversion stimulation (Fis) is a global regulator that is highly expressed during exponential phase growth and undetectable in stationary phase growth. Quorum sensing (QS) is a global regulatory mechanism that controls gene expression in response to changes in cell density and growth phase. In Vibrio parahaemolyticus, a marine species and a significant human pathogen, the QS regulatory sRNAs, Qrr1 to Qrr5, are expressed during exponential growth and negatively regulate the high cell density QS master regulator OpaR. OpaR is a positive regulator of capsule polysaccharide (CPS) formation, which is required for biofilm formation, and is a repressor of lateral flagella required for swarming motility. In V. parahaemolyticus, we show that Fis is a positive regulator of the qrr sRNAs expression. In an in-frame fis deletion mutant, qrr expression was repressed and opaR expression was induced. The Δfis mutant produced CPS and biofilm, but swarming motility was abolished. Also, the fis deletion mutant was more sensitive to polymyxin B. Swarming motility requires expression of both the surface sensing scrABC operon and lateral flagella laf operon. Our data showed that in the Δfis mutant both laf and scrABC genes were repressed. Fis controlled swarming motility indirectly through the QS pathway and directly through the surface sensing pathway. To determine the effects of Fis on cellular metabolism, we performed in vitro growth competition assays, and found that Δfis was outcompeted by wild type in minimal media supplemented with intestinal mucus as a sole nutrient source. The data showed that Fis positively modulated mucus components L-arabinose, D-gluconate and N-acetyl-D-glucosamine catabolism gene expression. In an in vivo colonization competition assay, Δfis was outcompeted by wild type, indicating Fis is required for fitness. Overall, these data demonstrate a global regulatory role for Fis in V. parahaemolyticus that includes QS, motility, and metabolism.

Identification of Essential Genes Associated With Prodigiosin Production in Serratia marcescens FZSF02.

Prodigiosin is a promising secondary metabolite produced mainly by Serratia strains. To study the global regulatory mechanism of prodigiosin biosynthesis, a mutagenesis library containing 23,000 mutant clones was constructed with the EZ-Tn5 transposon, and 114 clones in the library showed altered prodigiosin production ability. For 37 of the 114 clones, transposon insertion occurred on the prodigiosin biosynthetic cluster genes; transposon inserted genes of the 77 clones belonged to 33 different outside prodigiosin biosynthetic cluster genes. These 33 genes can be divided into transcription-regulating genes, membrane protein-encoding genes, and metabolism enzyme-encoding genes. Most of the genes were newly reported to be involved in prodigiosin production. Transcriptional levels of the pigA gene were significantly downregulated in 22 mutants with different inserted genes, which was in accordance with the phenotype of decreased prodigiosin production. Functional confirmation of the mutant genes involved in the pyrimidine nucleotide biosynthesis pathway was carried out by adding orotate and uridylate (UMP) into the medium. Gene complementation confirmed the regulatory function of the EnvZ/OmpR two-component regulatory system genes envZ and ompR in prodigiosin production.

A Novel Family of RNA-Binding Proteins Regulate Polysaccharide Metabolism in Bacteroides thetaiotaomicron.

Human gut microbiome composition is constantly changing, and diet is a major driver of these changes. Gut microbial species that persist in mammalian hosts for long periods of time must possess mechanisms for sensing and adapting to nutrient shifts to avoid being outcompeted. Global regulatory mechanisms mediated by RNA-binding proteins (RBPs) that govern responses to nutrient shifts have been characterized in Proteobacteria and Firmicutes but remain undiscovered in the Bacteroidetes. Here, we report the identification of RBPs that are broadly distributed across the Bacteroidetes, with many genomes encoding multiple copies. Genes encoding these RBPs are highly expressed in many Bacteroides species. A purified RBP, RbpB, from Bacteroides thetaiotaomicron binds to single-stranded RNA in vitro with an affinity similar to other characterized regulatory RBPs. B. thetaiotaomicron mutants lacking RBPs show dramatic shifts in expression of polysaccharide utilization and capsular polysaccharide loci, suggesting that these RBPs may act as global regulators of polysaccharide metabolism. A B. thetaiotaomicron ΔrbpB mutant shows a growth defect on dietary sugars belonging to the raffinose family of oligosaccharides (RFOs). The ΔrbpB mutant had reduced expression of BT1871, encoding a predicted RFO-degrading melibiase, compared to the wild-type strain. Mutation of BT1871 confirmed that the enzyme it encodes is essential for growth on melibiose and promotes growth on the RFOs raffinose and stachyose. Our data reveal that RbpB is required for optimal expression of BT1871 and other polysaccharide-related genes, suggesting that we have identified an important new family of global regulatory proteins in the Bacteroidetes. IMPORTANCE The human colon houses hundreds of bacterial species, including many belonging to the genus Bacteroides, that aid in breaking down our food to keep us healthy. Bacteroides have many genes responsible for breaking down different dietary carbohydrates, and complex regulatory mechanisms ensure that specific genes are only expressed when the right carbohydrates are available. In this study, we discovered that Bacteroides use a family of RNA-binding proteins as global regulators to coordinate expression of carbohydrate utilization genes. The ability to turn different carbohydrate utilization genes on and off in response to changing nutrient conditions is critical for Bacteroides to live successfully in the gut, and thus the new regulators we have identified may be important for life in the host.