MICROBE-OPOLY: exploring the microbial world through associations.

This commentary describes the creation of a microbiology-themed Monopoly™ gameboard assignment for studying microbial diversity. Students worked in groups to redesign the standard Monopoly™ playing tokens, currency, action cards, properties, and special spaces for any microbial-themed topic using a provided template. A postassignment anonymous survey found that the students enjoyed this assignment and the opportunity to share their knowledge in a unique and creative format. While this assignment is especially amenable to microbiology due the large diversity seen in the microbial world, it could easily be adapted to other topics such as genetics, chemistry, or even math, where categorical associations or patterns are prevalent.

Expression of Scytonemin Biosynthesis Genes under Alternative Stress Conditions in the Cyanobacterium Nostoc punctiforme.

The indole-alkaloid scytonemin is a sunscreen pigment that is widely produced among cyanobacteria as an ultraviolet radiation (UVR) survival strategy. Scytonemin biosynthesis is encoded by two gene clusters that are known to be induced by long-wavelength radiation (UVA). Previous studies have characterized the transcriptome of cyanobacteria in response to a wide range of conditions, but the effect on the expression of scytonemin biosynthesis genes has not been specifically targeted. Therefore, the aim of this study is to determine the variable response of scytonemin biosynthesis genes to a variety of environmental conditions. Cells were acclimated to white light before supplementation with UVA, UVB, high light, or osmotic stress for 48 h. The presence of scytonemin was determined by absorbance spectroscopy and gene expression of representative scytonemin biosynthesis genes was measured using quantitative PCR. Scytonemin genes were up-regulated in UVA, UVB, and high light, although the scytonemin pigment was not detected under high light. There was no scytonemin or upregulation of these genes under osmotic stress. The lack of pigment production under high light, despite increased gene expression, suggests a time-dependent delay for pigment production or additional mechanisms or genes that may be involved in scytonemin production beyond those currently known.

The Independent and Shared Transcriptomic Response to UVA, UVB and Oxidative Stress in the Cyanobacterium Nostoc punctiforme ATCC 29133.

Research on the UVA, UVB and oxidative (as reactive oxygen species, ROS) stress response in cyanobacteria has typically focused on each individual stress condition, with limited studies addressing the intersection. Here, we evaluated the transcriptomic responses of the model cyanobacterium Nostoc punctiforme after exposure to each of these conditions. Overall, response to UVA was characterized by more gene down-regulation than the UVB or ROS response, although UVB affected over fourfold more genes than UVA or ROS. Regarding expression patterns, responses to UVA and ROS were more similar and differentiated from those to UVB. For example, genes involved in ROS metabolism were up-regulated under both UVA and ROS. However, when it came to RNA and protein metabolism, there were more up-regulated genes under UVB and ROS compared to UVA. This suggests that the response to UVB and ROS is more active than the response to UVA, which stimulated more genes in secondary metabolism. Histidine kinases and response regulators were often differentially expressed, demonstrating that regulatory systems were at the base of the patterns. This study provides background for future studies targeting different genes, proteins and systems sensitive to these conditions. It also highlights the significance of considering multiple stress conditions.

Timing the Evolutionary Advent of Cyanobacteria and the Later Great Oxidation Event Using Gene Phylogenies of a Sunscreen.

The biosynthesis of the unique cyanobacterial (oxyphotobacterial) indole-phenolic UVA sunscreen, scytonemin, is coded for in a conserved operon that contains both core metabolic genes and accessory, aromatic amino acid biosynthesis genes dedicated to supplying scytonemin's precursors. Comparative genomics shows conservation of this operon in many, but not all, cyanobacterial lineages. Phylogenetic analyses of the operon's aromatic amino acid genes indicate that five of them were recruited into the operon after duplication events of their respective housekeeping cyanobacterial cognates. We combined the fossil record of cyanobacteria and relaxed molecular clock models to obtain multiple estimates of these duplication events, setting a minimum age for the evolutionary advent of scytonemin at 2.1 ± 0.3 billion years. The same analyses were used to estimate the advent of cyanobacteria as a group (and thus the appearance of oxygenic photosynthesis), at 3.6 ± 0.2 billion years before present. Post hoc interpretation of 16S rRNA-based Bayesian analyses was consistent with these estimates. Because of physiological constraints on the use of UVA sunscreens in general, and the biochemical constraints of scytonemin in particular, scytonemin's age must postdate the time when Earth's atmosphere turned oxic, known as the Great Oxidation Event (GOE). Indeed, our biological estimate is in agreement with independent geochemical estimates for the GOE. The difference between the estimated ages of oxygenic photosynthesis and the GOE indicates the long span (on the order of a billion years) of the era of "oxygen oases," when oxygen was available locally but not globally.IMPORTANCE The advent of cyanobacteria, with their invention of oxygenic photosynthesis, and the Great Oxidation Event are arguably among the most important events in the evolutionary history of life on Earth. Oxygen is a significant toxicant to all life, but its accumulation in the atmosphere also enabled the successful development and proliferation of many aerobic organisms, especially metazoans. The currently favored dating of the Great Oxidation Event is based on the geochemical rock record. Similarly, the advent of cyanobacteria is also often drawn from the same estimates because in older rocks paleontological evidence is scarce or has been discredited. Efforts to obtain molecular evolutionary alternatives have offered widely divergent estimates. Our analyses provide a novel means to circumvent these limitations and allow us to estimate the large time gap between the two events.

Characterization of the juvenile green turtle (Chelonia mydas) microbiome throughout an ontogenetic shift from pelagic to neritic habitats.

The gut microbiome of herbivorous animals consists of organisms that efficiently digest the structural carbohydrates of ingested plant material. Green turtles (Chelonia mydas) provide an interesting model of change in these microbial communities because they undergo a pronounced shift from a surface-pelagic distribution and omnivorous diet to a neritic distribution and herbivorous diet. As an alternative to direct sampling of the gut, we investigated the cloacal microbiomes of juvenile green turtles before and after recruitment to neritic waters to observe any changes in their microbial community structure. Cloacal swabs were taken from individual turtles for analysis of the 16S rRNA gene sequences using Illumina sequencing. One fecal sample was also obtained, allowing for a preliminary comparison with the bacterial community of the cloaca. We found significant variation in the juvenile green turtle bacterial communities between pelagic and neritic habitats, suggesting that environmental and dietary factors support different bacterial communities in green turtles from these habitats. This is the first study to characterize the cloacal microbiome of green turtles in the context of their ontogenetic shifts, which could provide valuable insight into the origins of their gut bacteria and how the microbial community supports their shift to herbivory.

Extracellular Polysaccharide Production in a Scytonemin-Deficient Mutant of Nostoc punctiforme Under UVA and Oxidative Stress.

Some cyanobacteria can protect themselves from ultraviolet radiation by producing sunscreen pigments. In particular, the sheath pigment scytonemin protects cells against long-wavelength UVA radiation and is only found in cyanobacteria which are capable of extracellular polysaccharide (EPS) production. The presence of a putative glycosyltransferase encoded within the scytonemin gene cluster, along with the localization of scytonemin and EPS to the extracellular sheath, prompted us to investigate the relationship between scytonemin and EPS production under UVA stress. In this study, it was hypothesized that there would be a relationship between the biosynthesis of scytonemin and EPS under both UVA and oxidative stress, since the latter is a by-product of UVA radiation. EPS production was measured following exposure of wild-type Nostoc punctiforme and the non-scytonemin-producing strain SCY59 to UVA and oxidative stress. Under UVA, SCY59 produced significantly more EPS than the unstressed controls and the wild type, while both strains produced more EPS under oxidative stress compared to the controls. The results suggest that EPS secretion occurs in response to the oxidative stress by-product of UVA rather than as a direct response to UVA radiation.

Effects of solution chemistry on antimicrobial activities of silver nanoparticles against Gordonia sp.

Silver nanoparticles (NPs) are the largest and fastest growing category of nanotechnology-based medicines and consumer products. Silver can have great toxicity to some aquatic organisms and, as a biocidal agent, may also damage or alter the most abundant and vulnerable beneficial microorganisms in the environment, such as Gordonia sp. However, considering the complex chemical background of natural waters, silver NPs can have complicated interactions with background chemicals such as chloride, surfactants, and dissolved natural organic matters (NOM). The results of this study show that the average particle size and dispersivity of silver NPs and the surface characteristics play an important role in the toxicity of silver NPs. Aggregation was enhanced for silver NPs in 10mM NaNO3, but not much in 10mM NaCl due to reactions with chloride. However, the presence of 3mM sodium dodecyl sulfate (SDS) or 8mgC/L Suwannee River (SR) NOM appeared to reduce the aggregation of silver NPs. Regarding the bactericidal effect of silver NPs, solubility analysis suggests silver NPs inactivate Gordonia sp. differently from Ag(+) and/or a slow release of Ag(+) from silver NPs. When the silver NP concentration was raised from 7.3 to 29.2mg/L in DI water, the log inactivation rate of Gordonia sp. increased from 0.16±0.04 to 0.45±0.13. However, with 29.2mg/L silver NPs the log inactivation rate reached 1.40±0.26 in 3mM SDS. The presence of SRNOM mitigated the bactericidal efficacy of silver NPs due to surface coating/adsorption. On the other hand, 10mM NaCl reduced the log inactivation rate to 0.07±0.07 due to the formation of likely less toxic silver chloride species, such as AgCl, AgCl2(-), AgCl3(2-), and AgCl4(3-).

The response regulator Npun_F1278 is essential for scytonemin biosynthesis in the cyanobacterium Nostoc punctiforme ATCC 29133.

Following exposure to long-wavelength ultraviolet radiation (UVA), some cyanobacteria produce the indole-alkaloid sunscreen scytonemin. The genomic region associated with scytonemin biosynthesis in the cyanobacterium Nostoc punctiforme includes 18 cotranscribed genes. A two-component regulatory system (Npun_F1277/Npun_F1278) directly upstream from the biosynthetic genes was identified through comparative genomics and is likely involved in scytonemin regulation. In this study, the response regulator (RR), Npun_F1278, was evaluated for its ability to regulate scytonemin biosynthesis using a mutant strain of N. punctiforme deficient in this gene, hereafter strain Δ1278. Following UVA radiation, the typical stimulus to initiate scytonemin biosynthesis, Δ1278 was incapable of producing scytonemin. A phenotypic characterization of Δ1278 suggests that aside from the ability to produce scytonemin, the deletion of the Npun_F1278 gene does not affect the cellular morphology, cellular differentiation capability, or lipid-soluble pigment complement of Δ1278 compared to the wildtype. The mutant, however, had a slower specific growth rate under white light and produced ~2.5-fold more phycocyanin per cell under UVA than the wildtype. Since Δ1278 does not produce scytonemin, this study demonstrates that the RR gene, Npun_F1278, is essential for scytonemin biosynthesis in N. punctiforme. While most of the evaluated effects of this gene appear to be specific for scytonemin, this regulator may also influence the overall health of the cell and phycobiliprotein synthesis, directly or indirectly. This is the first study to identify a regulatory gene involved in the biosynthesis of the sunscreen scytonemin and posits a link between cell growth, pigment synthesis, and sunscreen production.

Gene expression of a two-component regulatory system associated with sunscreen biosynthesis in the cyanobacterium Nostoc punctiforme ATCC 29133.

Long-wavelength ultraviolet radiation (UVA) can damage cells through photooxidative stress, leading to harmful photosensitized proteins and pigments in cyanobacteria. To mitigate damage, some cyanobacteria secrete the UVA-absorbing pigment scytonemin into their extracellular sheath. Comparative genomic analyses suggest that scytonemin biosynthesis is regulated by the two-component regulatory system (TCRS) proteins encoded by Npun_F1277 and Npun_F1278 in the cyanobacterium Nostoc punctiforme ATCC 29133. To understand the dynamics of these genes, their expression was measured following exposure to UVA, UVB, high visible (VIS) irradiance and oxidative stress for 20, 40 and 60 min. Overall, both genes had statistically similar patterns of expression for all four conditions and were generally upregulated, except for those exposed to UVB by 60 min and for the cells under oxidative stress. The greatest UVA response was an upregulation by 20 min, while the response to UVB was the most dramatic and persisted through 40 min. High VIS irradiance resulted in a modest upregulation, while oxidative stress caused a slight downregulation. Both genes were also found to occur on the same transcript. These results demonstrate that these genes are positively responding to several light-associated conditions, which suggests that this TCRS may regulate more than just scytonemin biosynthesis under UVA stress.

A diverse assemblage of indole-3-acetic acid producing bacteria associate with unicellular green algae.

Microalgae have tremendous potential as a renewable feedstock for the production of liquid transportation fuels. In natural waters, the importance of physical associations and biochemical interactions between microalgae and bacteria is generally well appreciated, but the significance of these interactions to algal biofuels production have not been investigated. Here, we provide a preliminary report on the frequency of co-occurrence between indole-3-acetic acid (IAA)-producing bacteria and green algae in natural and engineered ecosystems. Growth experiments with unicellular algae, Chlorella and Scenedesmus, revealed IAA concentration-dependent responses in chlorophyll content and dry weight. Importantly, discrete concentrations of IAA resulted in cell culture synchronization, suggesting that biochemical priming of cellular metabolism could vastly improve the reliability of high density cultivation. Bacterial interactions may have an important influence on algal growth and development; thus, the preservation or engineered construction of the algal-bacterial assembly could serve as a control point for achieving low input, reliable production of algal biofuels.

The global response of Nostoc punctiforme ATCC 29133 to UVA stress, assessed in a temporal DNA microarray study.

Cyanobacteria in nature are exposed not only to the visible spectrum of sunlight but also to its harmful ultraviolet components (UVA and UVB). We used Nostoc punctiforme ATCC 29133 as a model to study the UVA response by analyzing global gene expression patterns using genomic microarrays. UVA exposure resulted in the statistically detectable differential expression of 573 genes of the 6903 that were probed, compared with that of the control cultures. Of those genes, 473 were up-regulated, while only 100 were down-regulated. Many of the down-regulated genes were involved in photosynthetic pigment biosynthesis, indicating a significant shift in this metabolism. As expected, we detected the up-regulation of genes encoding antioxidant enzymes and the sunscreen, scytonemin. However, a majority of the up-regulated genes, 47%, were unassignable bioinformatically to known functional categories, suggesting that the UVA stress response is not well understood. Interestingly, the most dramatic up-regulation involved several contiguous genes of unassigned metabolism on plasmid A. This is the first global UVA stress response analysis of any phototrophic microorganism and the differential expression of 8% of the genes of the Nostoc genome indicates that adaptation to UVA in Nostoc has been an evolutionary force of significance.

A comparative genomics approach to understanding the biosynthesis of the sunscreen scytonemin in cyanobacteria.

BACKGROUND: The extracellular sunscreen scytonemin is the most common and widespread indole-alkaloid among cyanobacteria. Previous research using the cyanobacterium Nostoc punctiforme ATCC 29133 revealed a unique 18-gene cluster (NpR1276 to NpR1259 in the N. punctiforme genome) involved in the biosynthesis of scytonemin. We provide further genomic characterization of these genes in N. punctiforme and extend it to homologous regions in other cyanobacteria. RESULTS: Six putative genes in the scytonemin gene cluster (NpR1276 to NpR1271 in the N. punctiforme genome), with no previously known protein function and annotated in this study as scyA to scyF, are likely involved in the assembly of scytonemin from central metabolites, based on genetic, biochemical, and sequence similarity evidence. Also in this cluster are redundant copies of genes encoding for aromatic amino acid biosynthetic enzymes. These can theoretically lead to tryptophan and the tyrosine precursor, p-hydroxyphenylpyruvate, (expected biosynthetic precursors of scytonemin) from end products of the shikimic acid pathway. Redundant copies of the genes coding for the key regulatory and rate-limiting enzymes of the shikimic acid pathway are found there as well. We identified four other cyanobacterial strains containing orthologues of all of these genes, three of them by database searches (Lyngbya PCC 8106, Anabaena PCC 7120, and Nodularia CCY 9414) and one by targeted sequencing (Chlorogloeopsis sp. strain Cgs-089; CCMEE 5094). Genomic comparisons revealed that most scytonemin-related genes were highly conserved among strains and that two additional conserved clusters, NpF5232 to NpF5236 and a putative two-component regulatory system (NpF1278 and NpF1277), are likely involved in scytonemin biosynthesis and regulation, respectively, on the basis of conservation and location. Since many of the protein product sequences for the newly described genes, including ScyD, ScyE, and ScyF, have export signal domains, while others have putative transmembrane domains, it can be inferred that scytonemin biosynthesis is compartmentalized within the cell. Basic structural monomer synthesis and initial condensation are most likely cytoplasmic, while later reactions are predicted to be periplasmic. CONCLUSION: We show that scytonemin biosynthetic genes are highly conserved among evolutionarily diverse strains, likely include more genes than previously determined, and are predicted to involve compartmentalization of the biosynthetic pathway in the cell, an unusual trait for prokaryotes.

Gene expression patterns associated with the biosynthesis of the sunscreen scytonemin in Nostoc punctiforme ATCC 29133 in response to UVA radiation.

Under exposure to UV radiation, some cyanobacteria synthesize sunscreen compounds. Scytonemin is a heterocyclic indole-alkaloid sunscreen, the synthesis of which is induced upon exposure to UVA (long-wavelength UV) radiation. We previously identified and characterized an 18-gene cluster associated with scytonemin biosynthesis in the cyanobacterium Nostoc punctiforme ATCC 29133; we now report on the expression response of these genes to a step-up shift in UVA exposure. Using quantitative PCR on cDNAs from the N. punctiforme transcriptome and primers targeting each of the 18 genes in the cluster, we followed their differential expression in parallel subcultures incubated with and without UVA. All 18 genes are induced by UVA irradiation, with relative transcription levels that generally peak after 48 h of continuous UVA exposure. A five-gene cluster implicated in the process of scytonemin biosynthesis solely on the basis of comparative genomics was also upregulated. Furthermore, we demonstrate that all of the genes in the 18-gene region are cotranscribed as part of a single transcriptional unit.

Molecular genetics and genomic analysis of scytonemin biosynthesis in Nostoc punctiforme ATCC 29133.

The indole-alkaloid scytonemin is the most common and widespread sunscreen among cyanobacteria. Previous research has focused on its nature, distribution, ecology, physiology, and biochemistry, but its molecular genetics have not been explored. In this study, a scytonemin-deficient mutant of the cyanobacterium Nostoc punctiforme ATCC 29133 was obtained by random transposon insertion into open reading frame NpR1273. The absence of scytonemin under conditions of induction by UV irradiation was the single phenotypic difference detected in a comparative analysis of the wild type and the mutant. A cause-effect relationship between the phenotype and the mutation in NpR1273 was demonstrated by constructing a second scytoneminless mutant through directed mutagenesis of that gene. The genomic region flanking the mutation revealed an 18-gene cluster (NpR1276 to NpR1259). Four putative genes in the cluster, NpR1274 to NpR1271, with no previously known functions, are likely to be involved in the assembly of scytonemin. Also in this cluster, there is a redundant set of genes coding for shikimic acid and aromatic amino acid biosynthesis enzymes, leading to the production of tryptophan and tyrosine, which are likely to be biosynthetic precursors of the sunscreen.