A Deg-protease family protein in marine Synechococcus is involved in outer membrane protein organization

Deg-family proteases are a periplasm-associated group of proteins that are known to be involved in envelope stress responses and are found in most microorganisms. Orthologous genes SYNW2176 (in strain WH8102) and sync_2523 (strain CC9311) are predicted members of the Deg-protease family and are among the few genes induced by copper stress in both open ocean and coastal marine Synechococcus strains. In contrast to the lack of a phenotype in a similar knockout in Synechocystis PCC6803, a SYNW2176 knockout mutant in strain WH8102 was much more resistant to copper than the wild-type. The mutant also exhibited a significantly altered outer membrane protein composition which may contribute to copper resistance, longer lag phase after transfer, low-level consistent alkaline phosphatase activity, and an inability to induce high alkaline phosphatase activity in response to phosphate stress. This phenotype suggests a protein-quality-control role for SYNW2176, the absence of which leads to a constitutively activated stress response. Deg-protease family proteins in this ecologically important cyanobacterial group thus help to determine outer membrane responses to both nutrients and toxins

Variability in Protist Grazing and Growth on Different Marine Synechococcus Isolates

Grazing mortality of the marine phytoplankton Synechococcus is dominated by planktonic protists, yet rates of consumption and factors regulating grazer-Synechococcus interactions are poorly understood. One aspect of predator-prey interactions for which little is known are the mechanisms by which Synechococcus avoids or resists predation and, in turn, how this relates to the ability of Synechococcus to support growth of protist grazer populations. Grazing experiments conducted with the raptorial dinoflagellate Oxyrrhis marina and phylogenetically diverse Synechococcus isolates (strains WH8102, CC9605, CC9311, and CC9902) revealed marked differences in grazing rates-specifically that WH8102 was grazed at significantly lower rates than all other isolates. Additional experiments using the heterotrophic nanoflagellate Goniomonas pacifica and the filter-feeding tintinnid ciliate Eutintinnis sp. revealed that this pattern in grazing susceptibility among the isolates transcended feeding guilds and grazer taxon. Synechococcus cell size, elemental ratios, and motility were not able to explain differences in grazing rates, indicating that other features play a primary role in grazing resistance. Growth of heterotrophic protists was poorly coupled to prey ingestion and was influenced by the strain of Synechococcus being consumed. Although Synechococcus was generally a poor-quality food source, it tended to support higher growth and survival of G. pacifica and O. marina relative to Eutintinnis sp., indicating that suitability of Synechococcus varies among grazer taxa and may be a more suitable food source for the smaller protist grazers. This work has developed tractable model systems for further studies of grazer-Synechococcus interactions in marine microbial food webs

Final Report: Transport and its regulation in Marine Microorganisms: A Genomic Based Approach

This grant funded the analysis and annotation of the genomes of Synechococcus and Ostreococcus, major marine primary producers. Particular attention was paid to the analysis of transporters using state of the art bioinformatics analyses. During the analysis of the Synechococcus genome, some of the components of the unique bacterial swimming apparatus of one species of Synechococcus (Clade III, strain WH8102) were determined and these included transporters, novel giant proteins and glycosyltransferases. This grant funded the analysis of gene expression in Synechococcus using whole genome microarrays. These analyses revealed the strategies by which marine cyanobacteria respond to environmental conditions such as the absence of phosphorus, a common limiting nutrient, and the interaction of Synechococcus with other microbes. These analyses will help develop models of gene regulation in cyanobacteria and thus help predict their responses to changes in environmental conditions

Diverse Microbial Hot Spring Mat Communities at Black Canyon of the Colorado River.

The thermophilic microbial mat communities at hot springs in the Black Canyon of the Colorado River, thought to harbor the protistan human pathogen Naegleria fowleri, were surveyed using both culture-independent and -dependent methods to further understand the ecology of these hot spring microbiomes. Originating from Lake Mead source water, seven spring sites were sampled, varying in temperature from 25 to 55 °C. Amplicon-based high-throughput sequencing of twelve samples using 16S rRNA primers (hypervariable V4 region) revealed that most mats are dominated by cyanobacterial taxa, some but not all similar to those dominating the mats at other studied hot spring systems. 18S rRNA amplicon sequencing (V9 region) demonstrated a diverse community of protists and other eukaryotes including a highly abundant amoebal sequence related to Echinamoeba thermarum. Additional taxonomic and diversity metric analyses using near full-length 16S and 18S rRNA gene sequencing allowed a higher sequence-based resolution of the community. The mat sequence data suggest a major diversification of the cyanobacterial orders Leptolyngbyales, as well as microdiversity among several cyanobacterial taxa. Cyanobacterial isolates included some representatives of ecologically abundant taxa. A Spearman correlation analysis of short-read amplicon sequencing data supported the co-occurrences of populations of cyanobacteria, chloroflexi, and bacteroidetes providing evidence of common microbial co-occurrences across the Black Canyon hot springs

An outer membrane receptor critical for heme utilization in a marine Roseobacter

<p><strong>Conference:</strong> 2014 Ocean Sciences Meeting - Honolulu, Hawaii<br><strong>Poster Number:</strong> 554<br><strong>Session:</strong> 151: Microbial growth factors in the sea: Characterizing their importance at the molecular to ecosystem level<br><strong>Presentation Day:</strong> Monday 2/24/2014<br><strong>Time:</strong> 16:00 - 17:00</p> <p> </p> <p><strong>TITLE:</strong> An outer membrane receptor critical for heme utilization in a marine Roseobacter</p> <p><strong>ABSTRACT:</strong> Heme compounds have been detected in particulate organic matter in the oceans, but very little is known regarding the transformations of these molecules in the marine environment. The Roseobacter clade is a biogeochemically important bacterial lineage in marine systems. Prior studies have shown that nearly 50% of Roseobacter isolate genomes contain putative heme uptake and utilization loci. Here we demonstrate that one isolate, Ruegeria sp. TM1040, can maintain growth using heme b as the Fe source. Further, we show that insertional inactivation of a putative heme uptake TonB dependent outer membrane receptor in Ruegeria sp. TM1040 results in complete inability to utilize heme b as an Fe source. This is the first functional characterization of an outer membrane heme receptor in a non-pathogenic marine organism. As heme is fundamental to diverse enzymes, it could be advantageous for microbes in the iron-poor oceans to have dedicated systems for assimilating heme directly. We propose that intact heme-containing molecules are a component of the bioavailable Fe pool for marine heterotrophic bacteria, and much like vitamins can be considered “growth factors” in the sea.</p

Heterotrophic bacterioplankton utilize heme-based molecules for growth: a case study in the biological controls on a model iron-binding ligand

<p>Presented at 2013 ASLO meeting New Orleans, Louisiana</p> <p><strong>TITLE:</strong> Heterotrophic bacterioplankton utilize heme-based molecules for growth: a case study in the biological controls on a model iron-binding ligand</p> <p><strong>ABSTRACT: </strong>Iron-ligand complexes in marine waters are structurally diverse, and much is yet to be learned of their structure. Heme, a form of porphyrin bound iron, has been detected at nanomolar concentrations in seawater, but the bioavailability and utilization of heme by marine organisms and its biogeochemical significance has rarely been examined. Here we investigate the molecular physiological basis of marine bacterial heme uptake and assimilation pathways that have been inferred bioinformatically. We show that putatively annotated heme uptake gene clusters in isolates of the marine Roseobacter clade are in fact viable systems for acquiring iron from heme and porphyrin-like ligands. A particular isolate, Silicibacter sp. TrichCH4B, can maintain growth solely on ferric-porphyrin complexes, and its putative heme uptake and utilization system is up-regulated under iron stress. We also describe our efforts in generating Roseobacter genetic mutants with inactivated heme uptake and utilization genes. This functional characterization is a step forward in understanding the specific biogeochemical origin, role, and fate of heme-based molecules in seawater.</p> <p> </p

Evolution, ecology, and mechanisms of heme uptake in the marine Roseobacter lineage

<p>Presented at 2015 ASLO meeting Granada, Spain</p> <p><strong>TITLE: </strong>Evolution, ecology, and mechanisms of heme uptake in the marine Roseobacter lineage</p> <p><strong>ABSTRACT: </strong>The sources, transformations, and ultimate fate of heme and hemoproteins in seawater are poorly understood. The marine Roseobacters, an abundant and cosmopolitan bacterial lineage, are frequently found in environmental niches where heme concentrations are likely to be greatest. Here we show that in the Roseobacter group, heme uptake genes represent the most common strategy for organic-iron acquisition and are highly conserved with respect to synteny, sequence similarity, and transcription factor binding. We also argue that Roseobacters specialized to use heme as an iron source after genome divergence by way of horizontal gene transfer. We show that heme uptake genes of a model Roseobacter, Ruegeria sp. TM1040, are co-transcribed as an operon and are upregulated under iron stress. The insertional inactivation of a predicted TonB dependent outer membrane receptor in the heme uptake operon results in the inability of TM1040 to grow on heme as an iron source. Our results identify and confirm genes responsible for heme uptake in a model Roseobacter strain and highlight heme uptake as the dominant organic-iron uptake pathway within the rest of the lineage.</p> <p> </p

Broad-host-range vector system for synthetic biology and biotechnology in cyanobacteria.

Inspired by the developments of synthetic biology and the need for improved genetic tools to exploit cyanobacteria for the production of renewable bioproducts, we developed a versatile platform for the construction of broad-host-range vector systems. This platform includes the following features: (i) an efficient assembly strategy in which modules released from 3 to 4 donor plasmids or produced by polymerase chain reaction are assembled by isothermal assembly guided by short GC-rich overlap sequences. (ii) A growing library of molecular devices categorized in three major groups: (a) replication and chromosomal integration; (b) antibiotic resistance; (c) functional modules. These modules can be assembled in different combinations to construct a variety of autonomously replicating plasmids and suicide plasmids for gene knockout and knockin. (iii) A web service, the CYANO-VECTOR assembly portal, which was built to organize the various modules, facilitate the in silico construction of plasmids, and encourage the use of this system. This work also resulted in the construction of an improved broad-host-range replicon derived from RSF1010, which replicates in several phylogenetically distinct strains including a new experimental model strain Synechocystis sp. WHSyn, and the characterization of nine antibiotic cassettes, four reporter genes, four promoters, and a ribozyme-based insulator in several diverse cyanobacterial strains