Rebecca J. Case

Use of 16S rRNA and rpoB Genes as Molecular Markers for Microbial Ecology Studies

ABSTRACT Several characteristics of the 16S rRNA gene, such as its essential function, ubiquity, and evolutionary properties, have allowed it to become the most commonly used molecular marker in microbial ecology. However, one fact that has been overlooked is that multiple copies of this gene are often present in a given bacterium. These intragenomic copies can differ in sequence, leading to identification of multiple ribotypes for a single organism. To evaluate the impact of such intragenomic heterogeneity on the performance of the 16S rRNA gene as a molecular marker, we compared its phylogenetic and evolutionary characteristics to those of the single-copy gene rpoB. Full-length gene sequences and gene fragments commonly used for denaturing gradient gel electrophoresis were compared at various taxonomic levels. Heterogeneity found between intragenomic 16S rRNA gene copies was concentrated in specific regions of rRNA secondary structure. Such “heterogeneity hot spots” occurred within all gene fragments commonly used in molecular microbial ecology. This intragenomic heterogeneity influenced 16S rRNA gene tree topology, phylogenetic resolution, and operational taxonomic unit estimates at the species level or below. rpoB provided comparable phylogenetic resolution to that of the 16S rRNA gene at all taxonomic levels, except between closely related organisms (species and subspecies levels), for which it provided better resolution. This is particularly relevant in the context of a growing number of studies focusing on subspecies diversity, in which single-copy protein-encoding genes such as rpoB could complement the information provided by the 16S rRNA gene.

The Jekyll-and-Hyde chemistry of Phaeobacter gallaeciensis

Emiliania huxleyi, an environmentally important marine microalga, has a bloom-and-bust lifestyle in which massive algal blooms appear and fade. Phaeobacter gallaeciensis belongs to the roseobacter clade of α-Proteobacteria, the populations of which wax and wane with that of E. huxleyi. Roseobacter are thought to promote algal growth by biosynthesizing and secreting antibiotics and growth stimulants (auxins). Here we show that P. gallaeciensis switches its secreted small molecule metabolism to the production of potent and selective algaecides, the roseobacticides, in response to p-coumaric acid, an algal lignin breakdown product that is symptomatic of aging algae. This switch converts P. gallaeciensis into an opportunistic pathogen of its algal host.

AHL-driven quorum-sensing circuits: their frequency and function among the Proteobacteria

It is now apparent that bacteria utilize regulatory systems called quorum sensing (QS) to sense their population density. Such systems are dependant on the production of signaling molecules that activate specific genes when the signal reaches a critical threshold concentration. Such QS-regulated genes produce phenotypes that require coordinate behavior to convey competitive advantage to the population (such as biofilm formation and pathogenesis). The best-characterized QS system is that driven by acylated homoserine lactone (AHL) molecules. Quorum sensing-regulated phenotypes are diverse; however, their evolutionary selection is based on the competitive advantage conveyed by coordinating gene expression with the establishment of a quorum. Population density and coordinated gene expression are coupled for either (1) the multicellular characteristic of behaviors such as cell differentiation (for example swarming, biofilm formation), or (2) the fitness benefit of many individual cells simultaneously expressing the same phenotype (for example virulence factors, luminescence). QS enables a population to differentiate under favorable conditions where the population is dense enough to support the division and coordination of labor into subpopulations. In undifferentiated populations, QS coordinates gene expression so that it is simultaneous for cells within the population. In both scenarios, having QS regulation provides a competitive advantage for a population to both produce and respond to QS molecules. A selective pressure also exists for non-QS bacteria to sense and respond to QS molecules produced within the community. Examples of QS bacteria and bacteria able to detect and respond to exogenous signals are found in the literature; however, the frequency of QS and QS cheaters in the environment is poorly documented. With the growing number of bacterial genomes sequenced, especially genomes of nonclinically isolated bacteria, it may not be surprising that the number of genomes containing homologs of AHL-QS circuitry is ever growing. In this article, we use all current bacterial genomes to examine the frequency of AHL-QS among these bacteria, and the surprising number of bacteria with the genetic potential for eavesdropping on AHL signals from other bacteria.

Low Densities of Epiphytic Bacteria from the Marine Alga Ulva australis Inhibit Settlement of Fouling Organisms

ABSTRACT Bacteria that produce inhibitory compounds on the surface of marine algae are thought to contribute to the defense of the host plant against colonization of fouling organisms. However, the number of bacterial cells necessary to defend against fouling on the plant surface is not known. Pseudoalteromonas tunicata and Phaeobacter sp. strain 2.10 (formerly Roseobacter gallaeciensis) are marine bacteria often found in association with the alga Ulva australis and produce a range of extracellular inhibitory compounds against common fouling organisms. P. tunicata and Phaeobacter sp. strain 2.10 biofilms with cell densities ranging from 102 to 108 cells cm−2 were established on polystyrene petri dishes. Attachment and settlement assays were performed with marine fungi (uncharacterized isolates from U. australis), marine bacteria (Pseudoalteromonas gracilis, Alteromonas sp., and Cellulophaga fucicola), invertebrate larvae (Bugula neritina), and algal spores (Polysiphonia sp.) and gametes (U. australis). Remarkably low cell densities (102 to 103 cells cm−2) of P. tunicata were effective in preventing settlement of algal spores and marine fungi in petri dishes. P. tunicata also prevented settlement of invertebrate larvae at densities of 104 to 105 cells cm−2. Similarly, low cell densities (103 to 104cells cm−2) of Phaeobacter sp. strain 2.10 had antilarval and antibacterial activity. Previously, it has been shown that abundance of P. tunicata on marine eukaryotic hosts is low (<1 × 103 cells cm−2) (T. L. Skovhus et al., Appl. Environ. Microbiol. 70:2373-2382, 2004). Despite such low numbers of P. tunicata on U. australis in situ, our data suggest that P. tunicata and Phaeobacter sp. strain 2.10 are present in sufficient quantities on the plant to inhibit fouling organisms. This strongly supports the hypothesis that P. tunicata and Phaeobacter sp. strain 2.10 can play a role in defense against fouling on U. australis at cell densities that commonly occur in situ.

Temperature induced bacterial virulence and bleaching disease in a chemically defended marine macroalga

Host-pathogen interactions have been widely studied in humans and terrestrial plants, but are much less well explored in marine systems. Here we show that a marine macroalga, Delisea pulchra, utilizes a chemical defence - furanones - to inhibit colonization and infection by a novel bacterial pathogen, Ruegeria sp. R11, and that infection by R11 is temperature dependent. Ruegeria sp. R11 formed biofilms, invaded and bleached furanone-free, but not furanone-producing D. pulchra thalli, at high (24°C) but not low (19°C) temperatures. Bleaching is commonly observed in natural populations of D. pulchra near Sydney, Australia, during the austral summer when ocean temperatures are at their peak and the chemical defences of the alga are reduced. Furanones, produced by D. pulchra as a chemical defence, inhibit quorum sensing (QS) in bacteria, and this may play a role in furanone inhibition of R11 infection of furanone-free thalli as R11 produces QS signals. This interplay between temperature, an algal chemical defence mechanism and bacterial virulence demonstrates the complex impact environmental change can have on an ecosystem.

The integron/gene cassette system: an active player in bacterial adaptation.

The integron includes a site-specific recombination system capable of integrating and expressing genes contained in structures called mobile gene cassettes. Integrons were originally identified on mobile elements from pathogenic bacteria and were found to be a major reservoir of antibiotic-resistance genes. Integrons are now known to be ancient structures that are phylogenetically diverse and, to date, have been found in approximately 9% of sequenced bacterial genomes. Overall, gene diversity in cassettes is extraordinarily high, suggesting that the integron/gene cassette system has a broad role in adaptation rather than being confined to simply conferring resistance to antibiotics. In this chapter, we provide a review of the integron/gene cassette system highlighting characteristics associated with this system, diversity of elements contained within it, and their importance in driving bacterial evolution and consequently adaptation. Ideas on the evolution of gene cassettes and gene cassette arrays are discussed.

Genomes and Virulence Factors of Novel Bacterial Pathogens Causing Bleaching Disease in the Marine Red Alga Delisea pulchra

Nautella sp. R11, a member of the marine Roseobacter clade, causes a bleaching disease in the temperate-marine red macroalga, Delisea pulchra. To begin to elucidate the molecular mechanisms underpinning the ability of Nautella sp. R11 to colonize, invade and induce bleaching of D. pulchra, we sequenced and analyzed its genome. The genome encodes several factors such as adhesion mechanisms, systems for the transport of algal metabolites, enzymes that confer resistance to oxidative stress, cytolysins, and global regulatory mechanisms that may allow for the switch of Nautella sp. R11 to a pathogenic lifestyle. Many virulence effectors common in phytopathogenic bacteria are also found in the R11 genome, such as the plant hormone indole acetic acid, cellulose fibrils, succinoglycan and nodulation protein L. Comparative genomics with non-pathogenic Roseobacter strains and a newly identified pathogen, Phaeobacter sp. LSS9, revealed a patchy distribution of putative virulence factors in all genomes, but also led to the identification of a quorum sensing (QS) dependent transcriptional regulator that was unique to pathogenic Roseobacter strains. This observation supports the model that a combination of virulence factors and QS-dependent regulatory mechanisms enables indigenous members of the host algas epiphytic microbial community to switch to a pathogenic lifestyle, especially under environmental conditions when innate host defence mechanisms are compromised.

The biosynthesis of cyanobacterial sunscreen scytonemin in intertidal microbial mat communities

We have examined the biosynthesis and accumulation of cyanobacterial sunscreening pigment scytonemin within intertidal microbial mat communities using a combination of chemical, molecular, and phylogenetic approaches. Both laminated (layered) and nonlaminated mats contained scytonemin, with morphologically distinct mats having different cyanobacterial community compositions. Within laminated microbial mats, regions with and without scytonemin had different dominant oxygenic phototrophs, with scytonemin-producing areas consisting primarily of Lyngbya aestuarii and scytonemin-deficient areas dominated by a eukaryotic alga. The nonlaminated mat was populated by a diverse group of cyanobacteria and did not contain algae. The amplification and phylogenetic assignment of scytonemin biosynthetic gene scyC from laminated mat samples confirmed that the dominant cyanobacterium in these areas, L. aestuarii, is likely responsible for sunscreen production. This study is the first to utilize an understanding of the molecular basis of scytonemin assembly to explore its synthesis and function within natural microbial communities.

Ancient origin of the biosynthesis of lignin precursors.

BackgroundLignin plays an important role in plant structural support and water transport, and is considered one of the hallmarks of land plants. The recent discovery of lignin or its precursors in various algae has raised questions on the evolution of its biosynthetic pathway, which could be much more ancient than previously thought. To determine the taxonomic distribution of the lignin biosynthesis genes, we screened all publicly available genomes of algae and their closest non-photosynthetic relatives, as well as representative land plants. We also performed phylogenetic analysis of these genes to decipher the evolution and origin(s) of lignin biosynthesis.ResultsEnzymes involved in making p-coumaryl alcohol, the simplest lignin monomer, are found in a variety of photosynthetic eukaryotes, including diatoms, dinoflagellates, haptophytes, cryptophytes as well as green and red algae. Phylogenetic analysis of these enzymes suggests that they are ancient and spread to some secondarily photosynthetic lineages when they acquired red and/or green algal endosymbionts. In some cases, one or more of these enzymes was likely acquired through lateral gene transfer (LGT) from bacteria.ConclusionsGenes associated with p-coumaryl alcohol biosynthesis are likely to have evolved long before the transition of photosynthetic eukaryotes to land. The original function of this lignin precursor is therefore unlikely to have been related to water transport. We suggest that it participates in the biological defense of some unicellular and multicellular algae.ReviewersThis article was reviewed by Mark Ragan, Uri Gophna, Philippe Deschamps.

Indole-3-Acetic Acid Is Produced by Emiliania huxleyi Coccolith-Bearing Cells and Triggers a Physiological Response in Bald Cells

Indole-3-acetic acid (IAA) is an auxin produced by terrestrial plants which influences development through a variety of cellular mechanisms, such as altering cell orientation, organ development, fertility, and cell elongation. IAA is also produced by bacterial pathogens and symbionts of plants and algae, allowing them to manipulate growth and development of their host. They do so by either producing excess exogenous IAA or hijacking the IAA biosynthesis pathway of their host. The endogenous production of IAA by algae remains contentious. Using Emiliania huxleyi, a globally abundant marine haptophyte, we investigated the presence and potential role of IAA in algae. Homologs of genes involved in several tryptophan-dependent IAA biosynthesis pathways were identified in E. huxleyi. This suggests that this haptophyte can synthesize IAA using various precursors derived from tryptophan. Addition of L-tryptophan to E. huxleyi stimulated IAA production, which could be detected using Salkowskis reagent and GC × GC-TOFMS in the C cell type (coccolith bearing), but not in the N cell type (bald). Various concentrations of IAA were exogenously added to these two cell types to identify a physiological response in E. huxleyi. The N cell type, which did not produce IAA, was more sensitive to it, showing an increased variation in cell size, membrane permeability, and a corresponding increase in the photosynthetic potential quantum yield of Photosystem II (PSII). A roseobacter (bacteria commonly associated with E. huxleyi) Ruegeria sp. R11, previously shown to produce IAA, was co-cultured with E. huxleyi C and N cells. IAA could not be detected from these co-cultures, and even when stimulated by addition of L-tryptophan, they produced less IAA than axenic C type culture similarly induced. This suggests that IAA plays a novel role signaling between different E. huxleyi cell types, rather than between a bacteria and its algal host.