Metabolically active bacteria detected with click chemistry in low organic matter rainwater.

Rain contains encapsulated bacteria that can be transported over vast distances during relatively short periods of time. However, the ecological significance of bacteria in "precontact" rainwater-rainwater prior to contact with non-atmospheric surfaces-remains relatively undefined given the methodological challenges of studying low-abundance microbes in a natural assemblage. Here, we implement single-cell "click" chemistry in a novel application to detect the protein synthesis of bacteria in precontact rainwater samples as a measure of metabolic activity. Using epifluorescence microscopy, we find approximately 103-104 bacteria cells mL-1 with up to 7.2% of the observed cells actively synthesizing protein. Additionally, our measurement of less than 30 µM total organic carbon in the samples show that some rainwater bacteria can metabolize substrates in very low organic matter conditions, comparable to extremophiles in the deep ocean. Overall, our results raise new questions for the field of rainwater microbiology and may help inform efforts to develop quantitative microbial risk assessments for the appropriate use of harvested rainwater.

Bacterial surface interactions with organic colloidal particles: Nanoscale hotspots of organic matter in the ocean.

Colloidal particles constitute a substantial fraction of organic matter in the global ocean and an abundant component of the organic matter interacting with bacterial surfaces. Using E. coli ribosomes as model colloidal particles, we applied high-resolution atomic force microscopy to probe bacterial surface interactions with organic colloids to investigate particle attachment and relevant surface features. We observed the formation of ribosome films associating with marine bacteria isolates and natural seawater assemblages, and that bacteria readily utilized the added ribosomes as growth substrate. In exposure experiments ribosomes directly attached onto bacterial surfaces as 40-200 nm clusters and patches of individual particles. We found that certain bacterial cells expressed surface corrugations that range from 50-100 nm in size, and 20 nm deep. Furthermore, our AFM studies revealed surface pits in select bacteria that range between 50-300 nm in width, and 10-50 nm in depth. Our findings suggest novel adaptive strategies of pelagic marine bacteria for colloid capture and utilization as nutrients, as well as storage as nanoscale hotspots of DOM.

Insight into the resilience and susceptibility of marine bacteria to T6SS attack by Vibrio cholerae and Vibrio coralliilyticus.

The type VI secretion system (T6SS) is a nanomachine capable of killing adjacent microbial cells in a contact-dependent manner. Due to limited studies, relatively little is known about the range of marine bacteria that are susceptible to T6SS attack. Here, 15 diverse marine bacterial isolates from the phyla Bacteroidetes and Ɣ-Proteobacteria were challenged against the marine bacterium and human pathogen, Vibrio cholerae, which has a well described T6SS. V. cholerae killed several of the tested Ɣ-Proteobacteria, including members of the orders Vibrionales, Alteromonadales, Oceanospirillales, and Pseudomonadales. In contrast, V. cholerae co-existed with multiple Bacteroidetes and Ɣ-Proteobacteria isolates, but was killed by Vibrio coralliilyticus. Follow-up experiments revealed that five V. coralliilyticus strains, including known coral and shellfish pathogens survived the T6SS challenge and killed V. cholerae. By using predicted protein comparisons and mutagenesis, we conclude that V. coralliilyticus protected itself in the challenge by using its own T6SS to kill V. cholerae. This study provides valuable insight into the resilience and susceptibility of marine bacteria to the V. cholerae T6SS, and provides the first evidence for a functional T6SS in V. coralliilyticus, both of which have implications for human and ocean health.

Viral Attachment to Biotic and Abiotic Surfaces in Seawater.

Viruses influence microbial community structure and biogeochemical cycles in marine environments. Viral attachment to nonhost surfaces could influence host viral infection rates; however, the prevalence of such viral attachment is not investigated quantitatively. We used coastal seawater viral assemblages and, as models, marine vibriophage (SIO-2) and enterobacteriophages (T2 and T4) to investigate their attachment to probable nonhost marine bacteria. We also studied viral attachment to colloids and other abiotic surfaces in seawater. Centrifugation experiments with bacterium-virus mixtures showed substantial viral loss in the supernatant presumably due to the viral attachment to bacteria. This attachment (0.04 to 24 viruses µm-2 [bacterial surface area]) varied with bacterium-virus combinations. Surprisingly, filtering seawater on 0.2-µm Anodisc or polycarbonate filters retained ∼12 to 84% of viruses presumably attached to ≥0.2-µm-sized particles and/or the filter surface. Enzymatic digestion followed by epifluorescence and atomic force microscopy suggested that 7 to 25% of the total viruses were attached via ß-glycosidic linkages. Furthermore, a substantial proportion (7 to 48%) of viruses became attached to model abiotic surfaces (polycarbonate, polypropylene, and glass), and this has significance for laboratory protocols as well as studies of virus ecology in particle-rich marine environments. Substantial attachment of viruses to nonhost surfaces could influence virus-driven biogeochemical cycles and microbial community structure.IMPORTANCE Viruses play important roles in altering microbial community structure and biogeochemical cycles in marine environments. Viral attachment to nonhost surfaces can influence host viral infection rates; however, the prevalence of viral attachment to nonhost surfaces and the ratio of attached viruses to total viruses are little known. We used coastal seawater viral assemblages and used marine vibriophage (SIO-2) and enterobacteriophages (T2 and T4) as models to investigate their attachment to abiotic and biotic surfaces in seawater. Viral attachment was observed on several surfaces, such as nonhost bacteria, polymers, filters, cover glasses, and tube surfaces. This study cautions against commonly used protocols that require viral incubation and seawater fractionation. More importantly, these results could influence virus-driven biogeochemical cycles and microbial community structure in the ocean.

Bacterioplankton drawdown of coral mass-spawned organic matter.

Coral reef ecosystems are highly sensitive to microbial activities that result from dissolved organic matter (DOM) enrichment of their surrounding seawater. However, the response to particulate organic matter (POM) enrichment is less studied. In a microcosm experiment, we tested the response of bacterioplankton to a pulse of POM from the mass-spawning of Orbicella franksi coral off the Caribbean coast of Panama. Particulate organic carbon (POC), a proxy measurement for POM, increased by 40-fold in seawater samples collected during spawning; 68% degraded within 66 h. The elevation of multiple hydrolases presumably solubilized the spawn-derived POM into DOM. A carbon budget constructed for the 275 µM of degraded POC showed negligible change to the concentration of dissolved organic carbon (DOC), indicating that the DOM was readily utilized. Fourier transform ion cyclotron resonance mass spectrometry shows that the DOM pool became enriched with heteroatom-containing molecules, a trend that suggests microbial alteration of organic matter. Our sensitivity analysis demonstrates that bacterial carbon demand could have accounted for a large proportion of the POC degradation. Further, using bromodeoxyuridine immunocapture in combination with 454 pyrosequencing of the 16S ribosomal RNA gene, we surmise that actively growing bacterial groups were the primary degraders. We conclude that coral gametes are highly labile to bacteria and that such large capacity for bacterial degradation and alteration of organic matter has implications for coral reef health and coastal marine biogeochemistry.

Response of bacterial communities from California coastal waters to alginate particles and an alginolytic Alteromonas macleodii strain.

Alginate is a major cell wall polysaccharide from marine macroalgae and nutrient source for heterotrophic bacteria. Alginate can form gel particles in contact with divalent cations as found in seawater. Here, we tested the hypothesis that alginate gel particles serve as carbon source and microhabitat for marine bacteria by adding sterile alginate particles to microcosms with seawater from coastal California, a habitat rich in alginate-containing macroalgae. Alginate particles were rapidly colonized and degraded, with three- to eightfold higher bacterial abundances and production among alginate particle-associated (PA) bacteria. 16S rRNA gene amplicon sequencing showed that alginate PA bacteria were enriched in OTUs related to Cryomorphaceae, Saprospiraceae (Bacteroidetes) and Phaeobacter (Alphaproteobacteria) towards the end of the experiment. In microcosms amended with alginate particles and the proficient alginolytic bacterium Alteromonas macleodii strain 83-1, this strain dominated the community and outcompeted Cryomorphaceae, Saprospiraceae and Phaeobacter, and PA hydrolytic activities were over 50% higher. Thus, alginolytic activity by strain 83-1 did not benefit non-alginolytic strains by cross-feeding on alginate hydrolysis or other metabolic products. Considering the global distribution and extensive biomass of alginate-containing macroalgae, the observed bacterial dynamics associated with the utilization and remineralization of alginate microhabitats promote the understanding of carbon cycling in macroalgae-rich waters worldwide.

miR-335 inhibits small cell lung cancer bone metastases via IGF-IR and RANKL pathways.

UNLABELLED: Small cell lung cancer (SCLC) is a rapidly progressing, incurable cancer that frequently spreads to bone. New insights are needed to identify therapeutic targets to prevent or retard SCLC metastatic progression. Human SCLC SBC-5 cells in mouse xenograft models home to skeletal and nonskeletal sites, whereas human SCLC SBC-3 cells only pervade nonskeletal sites. Because microRNAs (miRNA) often act as tumor regulators, we investigated their role in preclinical models of SCLC. miRNA expression profiling revealed selective and reduced expression of miRNA (miR)-335 and miR-29a in SBC-5 cells, compared with SBC-3 cells. In SBC-5 cells, miR-335 expression correlated with bone osteolytic lesions, whereas miR-29a expression did not. Overexpression of miR-335 in SBC-5 cells significantly reduced cell migration, invasion, proliferation, colony formation, and osteoclast induction in vitro. Importantly, in miR-335 overexpressing SBC-5 cell xenografts (n = 10), there were minimal osteolytic lesions in the majority of mice and none in three mice. Expression of RANK ligand (RANKL) and insulin-like growth factor-I receptor (IGF-IR), key mediators of bone metastases, were elevated in SBC-5 as compared with SBC-3 cells. Mechanistically, overexpression of miR-335 in SBC-5 cells reduced RANKL and IGF-IR expression. In conclusion, loss of miR-335 promoted SCLC metastatic skeletal lesions via deregulation of IGF-IR and RANKL pathways and was associated with metastatic osteolytic skeletal lesions. IMPLICATIONS: These preclinical findings establish a need to pursue the role of miR-335 in human SCLC with metastatic skeletal disease.

Stimulatory and suppressive effects of novaluron on the Colorado potato beetle reproduction.

The Colorado potato beetle, Leptinotarsa decemlineata (Say), is one of the most damaging insect pests of potato, Solanum tuberosum L. Novaluron is a relatively new benzoylphenyl urea insect growth regulator with good activity against this pest. Earlier studies revealed that feeding on potato foliage treated with novaluron induces reversible egg hatch inhibition in adult Colorado potato beetles. We investigated whether novaluron effects depend on physiological state of the beetles at the time of exposure. The following four treatments were created: young beetles unmated at the beginning of the experiment and feeding on potato foliage treated with novaluron, young beetles unmated at the beginning of the experiment and feeding on untreated foliage, older beetles mated at the beginning of the experiment and feeding on foliage treated with novaluron, and older beetles mated at the beginning of the experiment and feeding on untreated foliage. The beetles were exposed to the respective treatments for 5 d. After that, both young and older beetles feeding on novaluron-treated leaves were switched onto untreated leaves and monitored for another 5 d to test their ability to recover. Young beetles unmated at the beginning of the experiment produced more eggs after feeding on the treated foliage, possibly indicating the presence of a pesticide-induced homeostatic modulation. No such effect was observed in the older beetles. Regardless of beetle physiological state at the beginning of the experiment, eggs produced on treated foliage did not hatch. The beetles eventually resumed laying viable eggs after being switched onto untreated foliage, with the recovery being delayed by approximately 24 h in young beetles compared with older beetles. Our results corroborate that novaluron reduces fertility of treated adults.