Search for: All records

Polycyclic aromatic hydrocarbons (PAHs) are common toxic and carcinogenic pollutants in marine ecosystems. Despite their prevalence in these habitats, relatively little is known about the natural microflora and biochemical pathways that contribute to their degradation. Approaches to investigate marine microbial PAH degraders often heavily rely on genetic biomarkers, which requires prior knowledge of specific degradative enzymes and genes encoding them. As such, these biomarker-reliant approaches cannot efficiently identify novel degradation pathways or degraders. Here, we screen 18 marine bacterial strains representing the Pseudomonadota, Bacillota, and Bacteroidota phyla for degradation of two model PAHs, pyrene (high molecular weight) and phenanthrene (low molecular weight). Using a qualitative PAH plate screening assay, we determined that 16 of 18 strains show some ability to degrade either or both compounds. Degradative ability was subsequently confirmed with a quantitative high-performance liquid chromatography approach, where an additional strain showed some degradation in liquid culture. Several members of the prominent marineRoseobacteraceaefamily degraded pyrene and phenanthrene with varying efficiency (1.2%–29.6% and 5.2%–52.2%, respectively) over 26 days. Described PAH genetic biomarkers were absent in all PAH degrading strains for which genome sequences are available, suggesting that these strains harbor novel transformation pathways. These results demonstrate the utility of culture-based approaches in expanding the knowledge landscape concerning PAH degradation in marine systems.

Polycyclic aromatic hydrocarbon (PAH) pollution is widespread throughout marine environments and significantly affects native flora and fauna. Investigating microbes responsible for degrading PAHs in these environments provides a greater understanding of natural attenuation in these systems. In addition, the use of culture-based approaches to inform bioinformatic and omics-based approaches is useful in identifying novel mechanisms of PAH degradation that elude genetic biomarker-based investigations. Furthermore, culture-based approaches allow for the study of PAH co-metabolism, which increasingly appears to be a prominent mechanism for PAH degradation in marine microbes.

 

Interactions amongst marine microalgae and heterotrophic bacteria drive processes underlying major biogeochemical cycles and are important for many artificial systems. These dynamic and complex interactions span the range from cooperative to competitive, and it is the diverse and intricate networks of metabolites and chemical mediators that are predicted to principally dictate the nature of the relationship at any point in time. Recent advances in technologies to identify, analyze, and quantify metabolites have allowed for a comprehensive view of the molecules available for exchange and/or reflective of organismal interactions, setting the stage for development of mechanistic understanding of these systems. Here, we (i) review the current knowledge landscape of microalgal–bacterial interactions by focusing on metabolomic studies of selected, simplified model systems; (ii) describe the state of the field of metabolomics, with specific focus on techniques and approaches developed for microalga–bacterial interaction studies; and (iii) outline the main approaches for development of mathematical models of these interacting systems, which collectively have the power to enhance interpretation of experimental data and generate novel testable hypotheses. We share the viewpoint that a comprehensive and integrated series of -omics approaches that include theoretical formulations are necessary to develop predictive and mechanistic understanding of these biological entities.

 

In the oceans, viruses that infect bacteria (phages) influence a variety of microbially mediated processes that drive global biogeochemical cycles. The nature of their influence is dependent upon infection mode, be it lytic or lysogenic. Temperate phages are predicted to be prevalent in marine systems where they are expected to execute both types of infection modes. Understanding the range and outcomes of temperate phage–host interactions is fundamental for evaluating their ecological impact. Here, we (i) review phage‐mediated rewiring of host metabolism, with a focus on marine systems, (ii) consider the range and nature of temperate phage–host interactions, and (iii) draw on studies of cultivated model systems to examine the consequences of lysogeny among several dominant marine bacterial lineages. We also readdress the prevalence of lysogeny among marine bacteria by probing a collection of 1239 publicly available bacterial genomes, representing cultured and uncultivated strains, for evidence of complete prophages. Our conservative analysis, anticipated to underestimate true prevalence, predicts 18% of the genomes examined contain at least one prophage, the majority (97%) were found within genomes of cultured isolates. These results highlight the need for cultivation of additional model systems to better capture the diversity of temperate phage–host interactions in the oceans.

 

ABSTRACT Bacterial growth substrates influence a variety of biological functions, including the biosynthesis and regulation of lipid intermediates. The extent of this rewiring is not well understood nor has it been considered in the context of virally infected cells. Here, we used a one-host-two-temperate phage model system to probe the combined influence of growth substrate and phage infection on host carbon and lipid metabolism. Using untargeted metabolomics and lipidomics, we reported the detection of a suite of metabolites and lipid classes for two Sulfitobacter lysogens provided with three growth substrates of differing complexity and nutrient composition (yeast extract/tryptone [complex], glutamate and acetate). The growth medium led to dramatic differences in the detectable intracellular metabolites, with only 15% of 175 measured metabolites showing overlap across the three growth substrates. Between-strain differences were most evident in the cultures grown on acetate, followed by glutamate then complex medium. Lipid distribution profiles were also distinct between cultures grown on different substrates as well as between the two lysogens grown in the same medium. Five phospholipids, three aminolipid, and one class of unknown lipid-like features were identified. Most (≥94%) of these 75 lipids were quantifiable in all samples. Metabolite and lipid profiles were strongly determined by growth medium composition and modestly by strain type. Because fluctuations in availability and form of carbon substrates and nutrients, as well as virus pressure, are common features of natural systems, the influence of these intersecting factors will undoubtedly be imprinted in the metabolome and lipidome of resident bacteria. IMPORTANCE Community-level metabolomics approaches are increasingly used to characterize natural microbial populations. These approaches typically depend upon temporal snapshots from which the status and function of communities are often inferred. Such inferences are typically drawn from lab-based studies of select model organisms raised under limited growth conditions. To better interpret community-level data, the extent to which ecologically relevant bacteria demonstrate metabolic flexibility requires elucidation. Herein, we used an environmentally relevant model heterotrophic marine bacterium to assess the relationship between growth determinants and metabolome. We also aimed to assess the contribution of phage activity to the host metabolome. Striking differences in primary metabolite and lipid profiles appeared to be driven primarily by growth regime and, secondarily, by phage type. These findings demonstrated the malleable nature of metabolomes and lipidomes and lay the foundation for future studies that relate cellular composition with function in complex environmental microbial communities. 

ABSTRACT Microbial interactions are often mediated by diffusible small molecules, including secondary metabolites, that play roles in cell-to-cell signaling and inhibition of competitors. Biofilms are often “hot spots” for high concentrations of bacteria and their secondary metabolites, which make them ideal systems for the study of small-molecule contributions to microbial interactions. Here, we use a five-member synthetic community consisting of Roseobacteraceae representatives to investigate the role of secondary metabolites on microbial biofilm dynamics. One synthetic community member, Rhodobacterales strain Y4I, possesses two acylated homoserine lactone (AHL)-based cell-to-cell signaling systems ( pgaRI and phaRI ) as well as a nonribosomal peptide synthase gene ( igi ) cluster that encodes the antimicrobial indigoidine. Through serial substitution of Y4I with mutants deficient in single signaling molecule pathways, the contribution of these small-molecule systems could be assessed. As secondary metabolite production is dependent upon central metabolites, the influence of growth substrate (i.e., complex medium versus defined medium with a single carbon substrate) on these dynamics was also considered. Depending on the Y4I mutant genotype included, community dynamics ranged from competitive to cooperative. The observed interactions were mostly competitive in nature. However, the community harboring a Y4I variant that was both impaired in quorum sensing (QS) pathways and unable to produce indigoidine ( pgaR variant) shifted toward more cooperative interactions over time. These cooperative interactions were enhanced in the defined growth medium. The results presented provide a framework for deciphering complex, small-molecule-mediated interactions that have broad application to microbial biology. IMPORTANCE Microbial biofilms play critical roles in marine ecosystems and are hot spots for microbial interactions that play a role in the development and function of these communities. Roseobacteraceae are an abundant and active family of marine heterotrophic bacteria forming close associations with phytoplankton and carrying out key transformations in biogeochemical cycles. Group members are aggressive primary colonizers of surfaces, where they set the stage for the development of multispecies biofilm communities. Few studies have examined the impact of secondary metabolites, such as cell-to-cell signaling and antimicrobial production, on marine microbial biofilm community structure. Here, we assessed the impact of secondary metabolites on microbial interactions using a synthetic, five-member Roseobacteraceae community by measuring species composition and biomass production during biofilm growth. We present evidence that secondary metabolites influence social behaviors within these multispecies microbial biofilms, thereby improving understanding of bacterial secondary metabolite production influence on social behaviors within marine microbial biofilm communities. 

ABSTRACT Widespread usage of high-throughput sequencing (HTS) in the LIFE SCIENCES has produced a demand for undergraduate and graduate institutions to offer classes exposing students to all aspects of HTS (sample acquisition, laboratory work, sequencing technologies, bioinformatics, and statistical analyses). Despite the increase in demand, many challenges exist for these types of classes. We advocate for the usage of the sourdough starter microbiome for implementing meta-amplicon sequencing. The relatively small community, dominated by a few taxa, enables potential contaminants to be easily identified, while between-sample differences can be quickly statistically assessed. Finally, bioinformatic pipelines and statistical analyses can be carried out on personal student laptops or in a teaching computer lab. In two semesters adopting this system, 12 of 14 students were able to effectively capture the sourdough starter microbiome, using the instructor’s paired sample as reference. 

ABSTRACT Mobile genetic elements (MGEs) drive bacterial evolution, alter gene availability within microbial communities, and facilitate adaptation to ecological niches. In natural systems, bacteria simultaneously possess or encounter multiple MGEs, yet their combined influences on microbial communities are poorly understood. Here, we investigate interactions among MGEs in the marine bacterium Sulfitobacter pontiacus . Two related strains, CB-D and CB-A, each harbor a single prophage. These prophages share high sequence identity with one another and an integration site within the host genome, yet these strains exhibit differences in “spontaneous” prophage induction (SPI) and consequent fitness. To better understand mechanisms underlying variation in SPI between these lysogens, we closed their genomes, which revealed that in addition to harboring different prophage genotypes, CB-A lacks two of the four large, low-copy-number plasmids possessed by CB-D. To assess the relative roles of plasmid content versus prophage genotype on host physiology, a panel of derivative strains varying in MGE content were generated. Characterization of these derivatives revealed a robust link between plasmid content and SPI, regardless of prophage genotype. Strains possessing all four plasmids had undetectable phage in cell-free lysates, while strains lacking either one plasmid (pSpoCB-1) or a combination of two plasmids (pSpoCB-2 and pSpoCB-4) produced high (>10 5 PFU/mL) phage titers. Homologous plasmid sequences were identified in related bacteria, and plasmid and phage genes were found to be widespread in Tara Oceans metagenomic data sets. This suggests that plasmid-dependent stabilization of prophages may be commonplace throughout the oceans. IMPORTANCE The consequences of prophage induction on the physiology of microbial populations are varied and include enhanced biofilm formation, conferral of virulence, and increased opportunity for horizontal gene transfer. These traits lead to competitive advantages for lysogenized bacteria and influence bacterial lifestyles in a variety of niches. However, biological controls of “spontaneous” prophage induction, the initiation of phage replication and phage-mediated cell lysis without an overt stressor, are not well understood. In this study, we observed a novel interaction between plasmids and prophages in the marine bacterium Sulfitobacter pontiacus . We found that loss of one or more distinct plasmids—which we show carry genes ubiquitous in the world’s oceans—resulted in a marked increase in prophage induction within lysogenized strains. These results demonstrate cross talk between different mobile genetic elements and have implications for our understanding of the lysogenic-lytic switches of prophages found not only in marine environments, but throughout all ecosystems. 

Abstract Microbial sulfur metabolism contributes to biogeochemical cycling on global scales. Sulfur metabolizing microbes are infected by phages that can encode auxiliary metabolic genes (AMGs) to alter sulfur metabolism within host cells but remain poorly characterized. Here we identified 191 phages derived from twelve environments that encoded 227 AMGs for oxidation of sulfur and thiosulfate ( dsrA , dsrC/tusE , soxC , soxD and soxYZ ). Evidence for retention of AMGs during niche-differentiation of diverse phage populations provided evidence that auxiliary metabolism imparts measurable fitness benefits to phages with ramifications for ecosystem biogeochemistry. Gene abundance and expression profiles of AMGs suggested significant contributions by phages to sulfur and thiosulfate oxidation in freshwater lakes and oceans, and a sensitive response to changing sulfur concentrations in hydrothermal environments. Overall, our study provides fundamental insights on the distribution, diversity, and ecology of phage auxiliary metabolism associated with sulfur and reinforces the necessity of incorporating viral contributions into biogeochemical configurations. 

Microbial biofilms associated with marine particulate organic matter carry out transformations that influence local and regional biogeochemical cycles. Early microbial colonizers are often hypothesized to “set the stage” for biofilm structure, dynamics, and function via N -acyl homoserine lactone (AHL)-mediated quorum sensing (QS). Production of AHLs, as well as antimicrobials, contributes to the colonization success of members of the Roseobacter clade. One member of this group of abundant marine bacteria, Rhodobacterales sp. Y4I, possesses two QS systems, phaRI (QS1) and pgaRI (QS2). Here, we characterize mutants in both QS systems to provide genetic evidence that the two systems work in hierarchical fashion to coordinate production of the antimicrobial indigoidine as well as biofilm formation. A mutation in pgaR (QS2) results in decreased expression of genes encoding both QS systems as well as those governing the biosynthesis of indigoidine. In contrast, mutations in QS1 did not significantly influence gene expression of QS2. Addition of exogenous AHLs to QS1 and QS2 mutants led to partial restoration of indigoidine production (45–60% of WT) for QS1 but not QS2. Mutational disruptions of QS1 had a more pronounced effect on biofilm development than those in QS2. Finally, we demonstrate that c-di-GMP levels are altered in QS and indigoidine biosynthesis Y4I mutants. Together, these results indicate that pgaRI (QS2) is at the top of a regulatory hierarchy governing indigoidine biosynthesis and that the global regulatory metabolite, c-di-GMP, is likely integrated into the QS circuitry of this strain. These findings provide mechanistic understanding of physiological processes that are important in elucidating factors driving competitiveness of Roseobacters in nature.