Reducing the Bacterial Lag Phase Through Methylated Compounds: Insights from Algal-Bacterial Interactions.

The bacterial lag phase is a key period for resuming growth. Despite its significance, the lag phase remains underexplored, particularly in environmental bacteria. Here, we explore the lag phase of the model marine bacterium Phaeobacter inhibens when it transitions from starvation to growth with a microalgal partner. Utilizing transcriptomics and 13 C-labeled metabolomics, our study reveals that methylated compounds, which are abundantly produced by microalgae, shorten the bacterial lag phase. Our findings underscore the significance of methyl groups as a limiting factor during the lag phase and demonstrate that methyl groups can be harvested from algal compounds and assimilated through the methionine cycle. Furthermore, we show that methylated compounds, characteristic of photosynthetic organisms, induce variable reductions in lag times among bacteria associated with algae and plants. These findings highlight the adjustability of the bacterial lag phase and emphasize the importance of studying bacteria in an environmental context. One-Sentence Summary: Bacteria use algal compounds as a metabolic shortcut to transition from starvation to growth.

Bacteria contribute exopolysaccharides to an algal-bacterial joint extracellular matrix.

Marine ecosystems are influenced by phytoplankton aggregation, which affects processes like marine snow formation and harmful events such as marine mucilage outbreaks. Phytoplankton secrete exopolymers, creating an extracellular matrix (ECM) that promotes particle aggregation. This ECM attracts heterotrophic bacteria, providing a nutrient-rich and protective environment. In terrestrial environments, bacterial colonization near primary producers relies on attachment and the formation of multidimensional structures like biofilms. Bacteria were observed attaching and aggregating within algal-derived exopolymers, but it is unclear if bacteria produce an ECM that contributes to this colonization. This study, using Emiliania huxleyi algae and Phaeobacter inhibens bacteria in an environmentally relevant model system, reveals a shared algal-bacterial ECM scaffold that promotes algal-bacterial aggregation. Algal exudates play a pivotal role in promoting bacterial colonization, stimulating bacterial exopolysaccharide (EPS) production, and facilitating a joint ECM formation. A bacterial biosynthetic pathway responsible for producing a specific EPS contributing to bacterial ECM formation is identified. Genes from this pathway show increased expression in algal-rich environments. These findings highlight the underestimated role of bacteria in aggregate-mediated processes in marine environments, offering insights into algal-bacterial interactions and ECM formation, with implications for understanding and managing natural and perturbed aggregation events.

Abundant Sulfitobacter marine bacteria protect Emiliania huxleyi algae from pathogenic bacteria.

Emiliania huxleyi is a unicellular micro-alga that forms massive oceanic blooms and plays key roles in global biogeochemical cycles. Mounting studies demonstrate various stimulatory and inhibitory influences that bacteria have on the E. huxleyi physiology. To investigate these algal-bacterial interactions, laboratory co-cultures have been established by us and by others. Owing to these co-cultures, various mechanisms of algal-bacterial interactions have been revealed, many involving bacterial pathogenicity towards algae. However, co-cultures represent a significantly simplified system, lacking the complexity of bacterial communities. In order to investigate bacterial pathogenicity within an ecologically relevant context, it becomes imperative to enhance the microbial complexity of co-culture setups. Phaeobacter inhibens bacteria are known pathogens that cause the death of E. huxleyi algae in laboratory co-culture systems. The bacteria depend on algal exudates for growth, but when algae senesce, bacteria switch to a pathogenic state and induce algal death. Here we investigate whether P. inhibens bacteria can induce algal death in the presence of a complex bacterial community. We show that an E. huxleyi-associated bacterial community protects the alga from the pathogen, although the pathogen occurs within the community. To study how the bacterial community regulates pathogenicity, we reduced the complex bacterial community to a five-member synthetic community (syncom). The syncom is comprised of a single algal host and five isolated bacterial species, which represent major bacterial groups that are naturally associated with E. huxleyi. We discovered that a single bacterial species in the reduced community, Sulfitobacter pontiacus, protects the alga from the pathogen. We further found that algal protection from P. inhibens pathogenicity is a shared trait among several Sulfitobacter species. Algal protection by bacteria might be a common phenomenon with ecological significance, which is overlooked in reduced co-culture systems.

Aerobic bacteria produce nitric oxide via denitrification and promote algal population collapse.

Microbial interactions govern marine biogeochemistry. These interactions are generally considered to rely on exchange of organic molecules. Here we report on a novel inorganic route of microbial communication, showing that algal-bacterial interactions between Phaeobacter inhibens bacteria and Gephyrocapsa huxleyi algae are mediated through inorganic nitrogen exchange. Under oxygen-rich conditions, aerobic bacteria reduce algal-secreted nitrite to nitric oxide (NO) through denitrification, a well-studied anaerobic respiratory mechanism. The bacterial NO is involved in triggering a cascade in algae akin to programmed cell death. During death, algae further generate NO, thereby propagating the signal in the algal population. Eventually, the algal population collapses, similar to the sudden demise of oceanic algal blooms. Our study suggests that the exchange of inorganic nitrogen species in oxygenated environments is a potentially significant route of microbial communication within and across kingdoms.

Utilization of diverse organophosphorus pollutants by marine bacteria.

Anthropogenic organophosphorus compounds (AOPCs), such as phosphotriesters, are used extensively as plasticizers, flame retardants, nerve agents, and pesticides. To date, only a handful of soil bacteria bearing a phosphotriesterase (PTE), the key enzyme in the AOPC degradation pathway, have been identified. Therefore, the extent to which bacteria are capable of utilizing AOPCs as a phosphorus source, and how widespread this adaptation may be, remains unclear. Marine environments with phosphorus limitation and increasing levels of pollution by AOPCs may drive the emergence of PTE activity. Here, we report the utilization of diverse AOPCs by four model marine bacteria and 17 bacterial isolates from the Mediterranean Sea and the Red Sea. To unravel the details of AOPC utilization, two PTEs from marine bacteria were isolated and characterized, with one of the enzymes belonging to a protein family that, to our knowledge, has never before been associated with PTE activity. When expressed in Escherichia coli with a phosphodiesterase, a PTE isolated from a marine bacterium enabled growth on a pesticide analog as the sole phosphorus source. Utilization of AOPCs may provide bacteria a source of phosphorus in depleted environments and offers a prospect for the bioremediation of a pervasive class of anthropogenic pollutants.

Coccolith Sr/Ca is a robust temperature and growth rate indicator that withstands dynamic microbial interactions.

Coccolithophores are a diverse group of calcifying microalgae that have left a prominent fossil record on Earth. Various coccolithophore relics, both organic and inorganic, serve as proxies for reconstruction of past oceanic conditions. Emiliania huxleyi is the most widely distributed representative of the coccolithophores in modern oceans and is known to engage in dynamic interactions with bacteria. Algal-bacterial interactions influence various aspects of algal physiology and alter algal alkenone unsaturation (UK'37 ), a frequently used organic coccolithophore-derived paleo-temperature proxy. Whether algal-bacterial interactions influence inorganic coccolithophore-derived paleo-proxies is yet unknown. A commonly used inorganic proxy for past productivity and sea surface temperature is the Sr/Ca ratio of the coccolith calcite. Interestingly, during interactions between bacteria and a population of calcifying algae, bacteria were shown to physically attach only to non-calcified algal cells, suggesting an influence on algal calcification. In this study, we explore the effects of algal-bacterial interactions on calcification and coccolith Sr/Ca ratios. We find that while bacteria attach only to non-calcified algal cells, coccolith cell coverage and overall calcite production in algal populations with and without bacteria is similar. Furthermore, we find that Sr/Ca values are impacted only by water temperature and algal growth rate, regardless of bacterial influences on algal physiology. Our observations reinforce the robustness of coccolith Sr/Ca ratios as a paleo-proxy independent of microbial interactions and highlight a fundamental difference between organic and inorganic paleo-proxies.

Resolving the Microalgal Gene Landscape at the Strain Level: a Novel Hybrid Transcriptome of Emiliania huxleyi CCMP3266.

Microalgae are key ecological players with a complex evolutionary history. Genomic diversity, in addition to limited availability of high-quality genomes, challenge studies that aim to elucidate molecular mechanisms underlying microalgal ecophysiology. Here, we present a novel and comprehensive transcriptomic hybrid approach to generate a reference for genetic analyses and resolve the microalgal gene landscape at the strain level. The approach is demonstrated for a strain of the coccolithophore microalga Emiliania huxleyi, which is a species complex with considerable genome variability. The investigated strain is commonly studied as a model for algal-bacterial interactions and was therefore sequenced in the presence of bacteria to elicit the expression of interaction-relevant genes. We applied complementary PacBio Iso-Seq full-length cDNA and poly(A)-independent Illumina total RNA sequencing, which resulted in a de novo-assembled, near-complete hybrid transcriptome. In particular, hybrid sequencing improved the reconstruction of long transcripts and increased the recovery of full-length transcript isoforms. To use the resulting hybrid transcriptome as a reference for genetic analyses, we demonstrate a method that collapses the transcriptome into a genome-like data set, termed "synthetic genome" (sGenome). We used the sGenome as a reference to visually confirm the robustness of the CCMP3266 gene assembly, to conduct differential gene expression analysis, and to characterize novel E. huxleyi genes. The newly identified genes contribute to our understanding of E. huxleyi genome diversification and are predicted to play a role in microbial interactions. Our transcriptomic toolkit can be implemented in various microalgae to facilitate mechanistic studies on microalgal diversity and ecology. IMPORTANCE Microalgae are key players in the ecology and biogeochemistry of our oceans. Efforts to implement genomic and transcriptomic tools in laboratory studies involving microalgae suffer from the lack of published genomes. In the case of coccolithophore microalgae, the problem has long been recognized; the model species Emiliania huxleyi is a species complex with genomes composed of a core and a large variable portion. To study the role of the variable portion in niche adaptation, and specifically in microbial interactions, strain-specific genetic information is required. Here, we present a novel transcriptomic hybrid approach, and generated strain-specific genome-like information. We demonstrate our approach on an E. huxleyi strain that is cocultivated with bacteria. By constructing a "synthetic genome," we generated comprehensive gene annotations that enabled accurate analyses of gene expression patterns. Importantly, we unveiled novel genes in the variable portion of E. huxleyi that play putative roles in microbial interactions.

Dynamic metabolic exchange governs a marine algal-bacterial interaction.

Emiliania huxleyi is a model coccolithophore micro-alga that generates vast blooms in the ocean. Bacteria are not considered among the major factors influencing coccolithophore physiology. Here we show through a laboratory model system that the bacterium Phaeobacter inhibens, a well-studied member of the Roseobacter group, intimately interacts with E. huxleyi. While attached to the algal cell, bacteria initially promote algal growth but ultimately kill their algal host. Both algal growth enhancement and algal death are driven by the bacterially-produced phytohormone indole-3-acetic acid. Bacterial production of indole-3-acetic acid and attachment to algae are significantly increased by tryptophan, which is exuded from the algal cell. Algal death triggered by bacteria involves activation of pathways unique to oxidative stress response and programmed cell death. Our observations suggest that bacteria greatly influence the physiology and metabolism of E. huxleyi. Coccolithophore-bacteria interactions should be further studied in the environment to determine whether they impact micro-algal population dynamics on a global scale.

Bacterial influence on alkenones in live microalgae.

The microalga Emiliania huxleyi produces alkenone lipids that are important proxies for estimating past sea surface temperatures. Field calibrations of this proxy are robust but highly variable results are obtained in culture. Here, we present results suggesting that algal-bacterial interactions may be responsible for some of this variability. Co-cultures of E. huxleyi and the bacterium Phaeobacter inhibens resulted in a 2.5-fold decrease in algal alkenone-containing lipid bodies. In addition levels of unsaturated alkenones increase in co-cultures. These changes result in an increase in the reconstructed growth temperature of up to 2°C relative to axenic algal cultures.

Morphological Heterogeneity and Attachment of Phaeobacter inhibens.

The Roseobacter clade is a key group of bacteria in the ocean exhibiting diverse metabolic repertoires and a wide range of symbiotic life-styles. Many Roseobacters possess remarkable capabilities of attachment to both biotic and abiotic surfaces. When attached to each other, these bacteria form multi-cellular structures called rosettes. Phaeobacter inhibens, a well-studied Roseobacter, exhibits various cell sizes and morphologies that are either associated with rosettes or occur as single cells. Here we describe the distribution of P. inhibens morphologies and rosettes within a population. We detect an N-acetylglucosamine-containing polysaccharide on the poles of some cells and at the center of all rosettes. We demonstrate that rosettes are formed by the attachment of individual cells at the polysaccharide-containing pole rather than by cell division. Finally, we show that P. inhibens attachment to abiotic surfaces is hindered by the presence of DNA from itself, but not from other bacteria. Taken together, our findings demonstrate that cell adhesiveness is likely to play a significant role in the life cycle of P. inhibens as well as other Roseobacters.

The molecular timeline of a reviving bacterial spore.

The bacterial spore can rapidly convert from a dormant to a fully active cell. Here we study this remarkable cellular transition in Bacillus subtilis and reveal the identity of the newly synthesized proteins throughout spore revival. Our analysis uncovers a highly ordered developmental program that correlates with the spore morphological changes and reveals the spatial and temporal molecular events fundamental to reconstruct a cell. As opposed to current knowledge, we found that translation takes place during the earliest revival event, termed germination, a process hitherto considered to occur without the need for any macromolecule synthesis. Furthermore, we demonstrate that translation is required for execution of germination and relies on the bona fide translational factors RpmE and Tig. Our study sheds light on the spore revival process and on the vital building blocks underlying cellular awakening, thereby paving the way for designing new antimicrobial agents to eradicate spore-forming pathogens.

Molecular kinetics of reviving bacterial spores.

Bacterial spores can remain dormant for years, yet they possess a remarkable potential to rapidly resume a vegetative life form. Here, we identified a distinct phase at the onset of spore outgrowth, designated the ripening period. This transition phase is exploited by the germinating spore for molecular reorganization toward elongation and subsequent cell division. We have previously shown that spores of different ages, kept under various temperatures, harbor dissimilar molecular reservoirs (E. Segev, Y. Smith, and S. Ben-Yehuda, Cell 148:139-149, 2012). Utilizing this phenomenon, we observed that the length of the ripening period can vary according to the spore molecular content. Importantly, the duration of the ripening period was found to correlate with the initial spore rRNA content and the kinetics of rRNA accumulation upon exiting dormancy. Further, the synthesis of the ribosomal protein RplA and the degradation of the spore-specific protein SspA also correlated with the duration of the ripening period. Our data suggest that the spore molecular cargo determines the extent of the ripening period, a potentially crucial phase for a germinating spore in obtaining limited resources during revival.

RNA dynamics in aging bacterial spores.

Upon starvation, the bacterium Bacillus subtilis enters the process of sporulation, lasting several hours and culminating in formation of a spore, the most resilient cell type known. We show that a few days following sporulation, the RNA profile of spores is highly dynamic. In aging spores incubated at high temperatures, RNA content is globally decreased by degradation over several days. This degradation might be a strategy utilized by the spore to facilitate its dormancy. However, spores kept at low temperature exhibit a different RNA profile with evidence supporting transcription. Further, we demonstrate that germination is affected by spore age, incubation temperature, and RNA state, implying that spores can acquire dissimilar characteristics at a time they are considered dormant. We propose that, in contrast to current thinking, entering dormancy lasts a few days, during which spores are affected by the environment and undergo corresponding molecular changes influencing their emergence from quiescence.