The global distribution and climate resilience of marine heterotrophic prokaryotes.

Heterotrophic Bacteria and Archaea (prokaryotes) are a major component of marine food webs and global biogeochemical cycles. Yet, there is limited understanding about how prokaryotes vary across global environmental gradients, and how their global abundance and metabolic activity (production and respiration) may be affected by climate change. Using global datasets of prokaryotic abundance, cell carbon and metabolic activity we reveal that mean prokaryotic biomass varies by just under 3-fold across the global surface ocean, while total prokaryotic metabolic activity increases by more than one order of magnitude from polar to tropical coastal and upwelling regions. Under climate change, global prokaryotic biomass in surface waters is projected to decline ~1.5% per °C of warming, while prokaryotic respiration will increase ~3.5% ( ~ 0.85 Pg C yr-1). The rate of prokaryotic biomass decline is one-third that of zooplankton and fish, while the rate of increase in prokaryotic respiration is double. This suggests that future, warmer oceans could be increasingly dominated by prokaryotes, diverting a growing proportion of primary production into microbial food webs and away from higher trophic levels as well as reducing the capacity of the deep ocean to sequester carbon, all else being equal.

Quantifying functional redundancy in polysaccharide-degrading prokaryotic communities.

BACKGROUND: Functional redundancy (FR) is widely present, but there is no consensus on its formation process and influencing factors. Taxonomically distinct microorganisms possessing genes for the same function in a community lead to within-community FR, and distinct assemblies of microorganisms in different communities playing the same functional roles are termed between-community FR. We proposed two formulas to respectively quantify the degree of functional redundancy within and between communities and analyzed the FR degrees of carbohydrate degradation functions in global environment samples using the genetic information of glycoside hydrolases (GHs) encoded by prokaryotes. RESULTS: Our results revealed that GHs are each encoded by multiple taxonomically distinct prokaryotes within a community, and the enzyme-encoding prokaryotes are further distinct between almost any community pairs. The within- and between-FR degrees are primarily affected by the alpha and beta community diversities, respectively, and are also affected by environmental factors (e.g., pH, temperature, and salinity). The FR degree of the prokaryotic community is determined by deterministic factors. CONCLUSIONS: We conclude that the functional redundancy of GHs is a stabilized community characteristic. This study helps to determine the FR formation process and influencing factors and provides new insights into the relationships between prokaryotic community biodiversity and ecosystem functions. Video Abstract.

Atomic Force Microscopy Imaging and Analysis of Prokaryotic Genome Organization.

This protocol describes the application of atomic force microscopy for structural analysis of prokaryotic and organellar nucleoids. It is based on a simple cell manipulation procedure that enables stepwise dissection of the nucleoid. The procedure includes (i) on-substrate lysis of cells and (ii) enzyme treatment, followed by atomic force microscopy. This type of dissection analysis permits analysis of nucleoid structure ranging from the fundamental units assembled on DNA to higher-order levels of organization. The combination with molecular-genetic and biochemical techniques further permits analysis of the functions of key nucleoid factors relevant to signal-induced structural reorganization or building up of basic structures, as seen for Dps in Escherichia coli and TrmBL2 in Thermococcus kodakarensis. These systems are described here as examples of the successful application of AFM for this purpose. Moreover, we describe the procedures needed for quantitative analysis of the data.

Adaptation of a eukaryote-like ProRS to a prokaryote-like tRNAPro.

Prolyl-tRNA synthetases (ProRSs) are unique among aminoacyl-tRNA synthetases (aaRSs) in having two distinct structural architectures across different organisms: prokaryote-like (P-type) and eukaryote/archaeon-like (E-type). Interestingly, Bacillus thuringiensis harbors both types, with P-type (BtProRS1) and E-type ProRS (BtProRS2) coexisting. Despite their differences, both enzymes are constitutively expressed and functional in vivo. Similar to BtProRS1, BtProRS2 selectively charges the P-type tRNAPro and displays higher halofuginone tolerance than canonical E-type ProRS. However, these two isozymes recognize the primary identity elements of the P-type tRNAPro-G72 and A73 in the acceptor stem-through distinct mechanisms. Moreover, BtProRS2 exhibits significantly higher tolerance to stresses (such as heat, hydrogen peroxide, and dithiothreitol) than BtProRS1 does. This study underscores how an E-type ProRS adapts to a P-type tRNAPro and how it may contribute to the bacterium's survival under stress conditions.

Internal carbon recycling by heterotrophic prokaryotes compensates for mismatches between phytoplankton production and heterotrophic consumption.

Molecular observational tools are useful for characterizing the composition and genetic endowment of microbial communities but cannot measure fluxes, which are critical for the understanding of ecosystems. To overcome these limitations, we used a mechanistic inference approach to estimate dissolved organic carbon (DOC) production and consumption by phytoplankton operational taxonomic units and heterotrophic prokaryotic amplicon sequence variants and inferred carbon fluxes between members of this microbial community from Western English Channel time-series data. Our analyses focused on phytoplankton spring and summer blooms, as well as bacteria summer blooms. In spring blooms, phytoplankton DOC production exceeds heterotrophic prokaryotic consumption, but in bacterial summer blooms heterotrophic prokaryotes consume three times more DOC than produced by the phytoplankton. This mismatch is compensated by heterotrophic prokaryotic DOC release by death, presumably from viral lysis. In both types of summer blooms, large amounts of the DOC liberated by heterotrophic prokaryotes are reused through internal recycling, with fluxes between different heterotrophic prokaryotes being at the same level as those between phytoplankton and heterotrophic prokaryotes. In context, internal recycling accounts for approximately 75% and 30% of the estimated net primary production (0.16 vs 0.22 and 0.08 vs 0.29 µmol l-1 d-1) in bacteria and phytoplankton summer blooms, respectively, and thus represents a major component of the Western English Channel carbon cycle. We have concluded that internal recycling compensates for mismatches between phytoplankton DOC production and heterotrophic prokaryotic consumption, and we encourage future analyses on aquatic carbon cycles to investigate fluxes between heterotrophic prokaryotes, specifically internal recycling.

Enhancing strategies of photosynthetic hydrogen production from microalgae: Differences in hydrogen production between prokaryotic and eukaryotic algae.

Hydrogen production through the metabolic bypass of microalgae photosynthesis is an environmentally friendly method. This review examines the genetic differences in hydrogen production between prokaryotic and eukaryotic microalgae. Additionally, the pathways for enhancing microalgae-based photosynthetic hydrogen generation are summarized. The main strategies for enhancing microalgal hydrogen production involve inhibiting the oxygen-generating process of photosynthesis and promoting the oxygen tolerance of hydrogenase. Future research is needed to explore the regulation of physiological metabolism through quorum sensing in microalgae to enhance photosynthetic hydrogen production. Moreover, effective evaluation of carbon emissions and sequestration across the entire photosynthetic hydrogen production process is crucial for determining the sustainability of microalgae-based production approaches through comprehensive lifecycle assessment. This review elucidates the prospects and challenges associated with photosynthetic hydrogen production by microalgae.

Nitrate reduction to ammonium: a phylogenetic, physiological, and genetic aspects in Prokaryotes and eukaryotes.

The microbe-mediated conversion of nitrate (NO3-) to ammonium (NH4+) in the nitrogen cycle has strong implications for soil health and crop productivity. The role of prokaryotes, eukaryotes and their phylogeny, physiology, and genetic regulations are essential for understanding the ecological significance of this empirical process. Several prokaryotes (bacteria and archaea), and a few eukaryotes (fungi and algae) are reported as NO3- reducers under certain conditions. This process involves enzymatic reactions which has been catalysed by nitrate reductases, nitrite reductases, and NH4+-assimilating enzymes. Earlier reports emphasised that single-cell prokaryotic or eukaryotic organisms are responsible for this process, which portrayed a prominent gap. Therefore, this study revisits the similarities and uniqueness of mechanism behind NO3- -reduction to NH4+ in both prokaryotes and eukaryotes. Moreover, phylogenetic, physiological, and genetic regulation also shed light on the evolutionary connections between two systems which could help us to better explain the NO3--reduction mechanisms over time. Reports also revealed that certain transcription factors like NtrC/NtrB and Nit2 have shown a major role in coordinating the expression of NO3- assimilation genes in response to NO3- availability. Overall, this review provides a comprehensive information about the complex fermentative and respiratory dissimilatory nitrate reduction to ammonium (DNRA) processes. Uncovering the complexity of this process across various organisms may further give insight into sustainable nitrogen management practices and might contribute to addressing global environmental challenges.

Investigating the nature of prokaryotic genomic island locations within a genome.

Horizontal gene transfer (HGT) is a powerful evolutionary force that considerably shapes the structure of prokaryotic genomes and is associated with genomic islands (GIs). A GI is a DNA segment composed of transferred genes that can be found within a prokaryotic genome, obtained through HGT. Much research has focused on detecting GIs in genomes, but here we pursue a new course, which is identifying possible preferred locations of GIs in the prokaryotic genome. Here, we identify the locations of the GIs within prokaryotic genomes to examine patterns in those locations. Prokaryotic GIs were analyzed according to the genome structure that they are located in, whether it be a circular or a linear genome. The analytical investigations employed are: (1) studying the GI locations in relation to the origin of replication (oriC); (2) exploring the distances between GIs; and (3) determining the distribution of GIs across the genomes. For each of the investigations, the analysis was performed on all of the GIs in the data set. Moreover, to void bias caused by the distribution of the genomes represented, the GIs in one genome from each species and the GIs of the most frequent species are also analyzed. Overall, the results showed that there are preferred sites for the GIs in the genome. In the linear genomes, these sites are usually located in the oriC region and terminus region, while in the circular genomes, they are located solely in the terminus region. These results also showed that the distance distribution between the GIs is almost exponential, which proves that GIs have preferred sites within genomes. The oriC and termniuns are preferred sites for the GIs and a possible natural explanation for this could be connected to the content of the oriC region. Moreover, the content of the GIs in terms of its protein families was studied and the results demonstrated that the majority of frequent protein families are close to identical in each section.

Macroalgal virosphere assists with host-microbiome equilibrium regulation and affects prokaryotes in surrounding marine environments.

The microbiomes in macroalgal holobionts play vital roles in regulating macroalgal growth and ocean carbon cycling. However, the virospheres in macroalgal holobionts remain largely underexplored, representing a critical knowledge gap. Here we unveil that the holobiont of kelp (Saccharina japonica) harbors highly specific and unique epiphytic/endophytic viral species, with novelty (99.7% unknown) surpassing even extreme marine habitats (e.g. deep-sea and hadal zones), indicating that macroalgal virospheres, despite being closest to us, are among the least understood. These viruses potentially maintain microbiome equilibrium critical for kelp health via lytic-lysogenic infections and the expression of folate biosynthesis genes. In-situ kelp mesocosm cultivation and metagenomic mining revealed that kelp holobiont profoundly reshaped surrounding seawater and sediment virus-prokaryote pairings through changing surrounding environmental conditions and virus-host migrations. Some kelp epiphytic viruses could even infect sediment autochthonous bacteria after deposition. Moreover, the presence of ample viral auxiliary metabolic genes for kelp polysaccharide (e.g. laminarin) degradation underscores the underappreciated viral metabolic influence on macroalgal carbon cycling. This study provides key insights into understanding the previously overlooked ecological significance of viruses within macroalgal holobionts and the macroalgae-prokaryotes-virus tripartite relationship.

Substrate uptake patterns shape niche separation in marine prokaryotic microbiome.

Marine heterotrophic prokaryotes primarily take up ambient substrates using transporters. The patterns of transporters targeting particular substrates shape the ecological role of heterotrophic prokaryotes in marine organic matter cycles. Here, we report a size-fractionated pattern in the expression of prokaryotic transporters throughout the oceanic water column due to taxonomic variations, revealed by a multi-"omics" approach targeting ATP-binding cassette (ABC) transporters and TonB-dependent transporters (TBDTs). Substrate specificity analyses showed that marine SAR11, Rhodobacterales, and Oceanospirillales use ABC transporters to take up organic nitrogenous compounds in the free-living fraction, while Alteromonadales, Bacteroidetes, and Sphingomonadales use TBDTs for carbon-rich organic matter and metal chelates on particles. The expression of transporter proteins also supports distinct lifestyles of deep-sea prokaryotes. Our results suggest that transporter divergency in organic matter assimilation reflects a pronounced niche separation in the prokaryote-mediated organic matter cycles.

The radical impact of oxygen on prokaryotic evolution-enzyme inhibition first, uninhibited essential biosyntheses second, aerobic respiration third.

Molecular oxygen is a stable diradical. All O2-dependent enzymes employ a radical mechanism. Generated by cyanobacteria, O2 started accumulating on Earth 2.4 billion years ago. Its evolutionary impact is traditionally sought in respiration and energy yield. We mapped 365 O2-dependent enzymatic reactions of prokaryotes to phylogenies for the corresponding 792 protein families. The main physiological adaptations imparted by O2-dependent enzymes were not energy conservation, but novel organic substrate oxidations and O2-dependent, hence O2-tolerant, alternative pathways for O2-inhibited reactions. Oxygen-dependent enzymes evolved in ancestrally anaerobic pathways for essential cofactor biosynthesis including NAD+, pyridoxal, thiamine, ubiquinone, cobalamin, heme, and chlorophyll. These innovations allowed prokaryotes to synthesize essential cofactors in O2-containing environments, a prerequisite for the later emergence of aerobic respiratory chains.

Protein-Coding Gene Families in Prokaryote Genome Comparisons.

The identification of orthologous genes is relevant for comparative genomics, phylogenetic analysis, and functional annotation. There are many computational tools for the prediction of orthologous groups as well as web-based resources that offer orthology datasets for download and online analysis. This chapter presents a simple and practical guide to the process of orthologous group prediction, using a dataset of 10 prokaryotic proteomes as example. The orthology methods covered are OrthoMCL, COGtriangles, OrthoFinder2, and OMA. The authors compare the number of orthologous groups predicted by these various methods, and present a brief workflow for the functional annotation and reconstruction of phylogenies from inferred single-copy orthologous genes. The chapter also demonstrates how to explore two orthology databases: eggNOG6 and OrthoDB.

Annotation and Comparative Genomics of Prokaryotic Transposable Elements.

The data generated in nearly 30 years of bacterial genome sequencing has revealed the abundance of transposable elements (TE) and their importance in genome and transcript remodeling through the mediation of DNA insertions and deletions, structural rearrangements, and regulation of gene expression. Furthermore, what we have learned from studying transposition mechanisms and their regulation in bacterial TE is fundamental to our current understanding of TE in other organisms because much of what has been observed in bacteria is conserved in all domains of life. However, unlike eukaryotic TE, prokaryotic TE sequester and transmit important classes of genes that impact host fitness, such as resistance to antibiotics and heavy metals and virulence factors affecting animals and plants, among other acquired traits. This provides dynamism and plasticity to bacteria, which would otherwise be propagated clonally. The insertion sequences (IS), the simplest form of prokaryotic TE, are autonomous and compact mobile genetic elements. These can be organized into compound transposons, in which two similar IS can flank any DNA segment and render it transposable. Other more complex structures, called unit transposons, can be grouped into four major families (Tn3, Tn7, Tn402, Tn554) with specific genetic characteristics. This chapter will revisit the prominent structural features of these elements, focusing on a genomic annotation framework and comparative analysis. Relevant aspects of TE will also be presented, stressing their key position in genome impact and evolution, especially in the emergence of antimicrobial resistance and other adaptive traits.

An evolutionary optimum amid moderate heritability in prokaryotic cell size.

We investigate the distribution and evolution of prokaryotic cell size based on a compilation of 5,380 species. Size spans four orders of magnitude, from 100 nm (Mycoplasma) to more than 1 cm (Thiomargarita); however, most species congregate heavily around the mean. The distribution approximates but is distinct from log normality. Comparative phylogenetics suggests that size is heritable, yet the phylogenetic signal is moderate, and the degree of heritability is independent of taxonomic scale (i.e., fractal). Evolutionary modeling indicates the presence of an optimal cell size to which most species gravitate. The size is equivalent to a coccus of 0.70 µm in diameter. Analyses of 1,361 species with sequenced genomes show that genomic traits contribute to size evolution moderately and synergistically. Given our results, scaling theory, and empirical evidence, we discuss potential drivers that may expand or shrink cells around the optimum and propose a stability landscape model for prokaryotic cell size.

DeepReg: a deep learning hybrid model for predicting transcription factors in eukaryotic and prokaryotic genomes.

Deep learning models (DLMs) have gained importance in predicting, detecting, translating, and classifying a diversity of inputs. In bioinformatics, DLMs have been used to predict protein structures, transcription factor-binding sites, and promoters. In this work, we propose a hybrid model to identify transcription factors (TFs) among prokaryotic and eukaryotic protein sequences, named Deep Regulation (DeepReg) model. Two architectures were used in the DL model: a convolutional neural network (CNN), and a bidirectional long-short-term memory (BiLSTM). DeepReg reached a precision of 0.99, a recall of 0.97, and an F1-score of 0.98. The quality of our predictions, the bias-variance trade-off approach, and the characterization of new TF predictions were evaluated and compared against those produced by DeepTFactor, as well as against experimental data from three model organisms. Predictions based on our DLM tended to exhibit less variance and bias than those from DeepTFactor, thus increasing reliability and decreasing overfitting.

Can Sb(III)-oxidizing prokaryote also oxidize As(III) under aerobic and anaerobic conditions, and vice versa?

Sb(III) and As(III) share similar chemical features and coexist in the environment. However, their oxidase enzymes have completely different sequences and structures. This raises an intriguing question: Could Sb(III)-oxidizing prokaryotes (SOPs) also oxidize As(III), and vice versa? Regarding this issue, previous investigations have yielded unclear, incorrect and even conflicting data. This work aims to address this matter. First, we prepared an enriched population of SOPs that comprises 55 different AnoA genes, lacking AioAB and ArxAB genes. We found that these SOPs can oxidize both Sb(III) and As(III) with comparable capabilities. To further confirm this finding, we isolated three cultivable SOP strains that have AnoA gene, but lack AioAB and ArxAB genes. We observed that they also oxidize both Sb(III) and As(III) under both anaerobic and aerobic conditions. Secondly, we obtained an enriched population of As(III)-oxidizing prokaryotes (AOPs) from As-contaminated soils, which comprises 69 different AioA genes, lacking AnoA gene. We observed that the AOP population has significant As(III)-oxidizing activities, but lack detectable Sb(III)-oxidizing activities under both aerobic and anaerobic conditions. Therefore, we convincingly show that SOPs can oxidize As(III), but AOPs cannot oxidize Sb(III). These findings clarify the previous ambiguities, confusion, errors or contradictions regarding how SOPs and AOPs oxidize each other's substrate.

CRISPR Tools for Engineering Prokaryotic Systems: Recent Advances and New Applications.

In the past decades, the broad selection of CRISPR-Cas systems has revolutionized biotechnology by enabling multimodal genetic manipulation in diverse organisms. Rooted in a molecular engineering perspective, we recapitulate the different CRISPR components and how they can be designed for specific genetic engineering applications. We first introduce the repertoire of Cas proteins and tethered effectors used to program new biological functions through gene editing and gene regulation. We review current guide RNA (gRNA) design strategies and computational tools and how CRISPR-based genetic circuits can be constructed through regulated gRNA expression. Then, we present recent advances in CRISPR-based biosensing, bioproduction, and biotherapeutics across in vitro and in vivo prokaryotic systems. Finally, we discuss forthcoming applications in prokaryotic CRISPR technology that will transform synthetic biology principles in the near future.

Co-transcriptional gene regulation in eukaryotes and prokaryotes.

Many steps of RNA processing occur during transcription by RNA polymerases. Co-transcriptional activities are deemed commonplace in prokaryotes, in which the lack of membrane barriers allows mixing of all gene expression steps, from transcription to translation. In the past decade, an extraordinary level of coordination between transcription and RNA processing has emerged in eukaryotes. In this Review, we discuss recent developments in our understanding of co-transcriptional gene regulation in both eukaryotes and prokaryotes, comparing methodologies and mechanisms, and highlight striking parallels in how RNA polymerases interact with the machineries that act on nascent RNA. The development of RNA sequencing and imaging techniques that detect transient transcription and RNA processing intermediates has facilitated discoveries of transcription coordination with splicing, 3'-end cleavage and dynamic RNA folding and revealed physical contacts between processing machineries and RNA polymerases. Such studies indicate that intron retention in a given nascent transcript can prevent 3'-end cleavage and cause transcriptional readthrough, which is a hallmark of eukaryotic cellular stress responses. We also discuss how coordination between nascent RNA biogenesis and transcription drives fundamental aspects of gene expression in both prokaryotes and eukaryotes.

A widespread bacterial protein compartment sequesters and stores elemental sulfur.

Subcellular compartments often serve to store nutrients or sequester labile or toxic compounds. As bacteria mostly do not possess membrane-bound organelles, they often have to rely on protein-based compartments. Encapsulins are one of the most prevalent protein-based compartmentalization strategies found in prokaryotes. Here, we show that desulfurase encapsulins can sequester and store large amounts of crystalline elemental sulfur. We determine the 1.78-angstrom cryo-EM structure of a 24-nanometer desulfurase-loaded encapsulin. Elemental sulfur crystals can be formed inside the encapsulin shell in a desulfurase-dependent manner with l-cysteine as the sulfur donor. Sulfur accumulation can be influenced by the concentration and type of sulfur source in growth medium. The selectively permeable protein shell allows the storage of redox-labile elemental sulfur by excluding cellular reducing agents, while encapsulation substantially improves desulfurase activity and stability. These findings represent an example of a protein compartment able to accumulate and store elemental sulfur.

Intracellular signaling in proto-eukaryotes evolves to alleviate regulatory conflicts of endosymbiosis.

The complex eukaryotic cell resulted from a merger between simpler prokaryotic cells, yet the role of the mitochondrial endosymbiosis with respect to other eukaryotic innovations has remained under dispute. To investigate how the regulatory challenges associated with the endosymbiotic state impacted genome and network evolution during eukaryogenesis, we study a constructive computational model where two simple cells are forced into an obligate endosymbiosis. Across multiple in silico evolutionary replicates, we observe the emergence of different mechanisms for the coordination of host and symbiont cell cycles, stabilizing the endosymbiotic relationship. In most cases, coordination is implicit, without signaling between host and symbiont. Signaling only evolves when there is leakage of regulatory products between host and symbiont. In the fittest evolutionary replicate, the host has taken full control of the symbiont cell cycle through signaling, mimicking the regulatory dominance of the nucleus over the mitochondrion that evolved during eukaryogenesis.