Ambient Microbial Community Research Articles

Cysteine-Mediated Extracellular Electron Transfer of Lysinibacillus varians GY32.

Microbial extracellular electron transfer (EET) is essential in many natural and engineering processes. Compared with the versatile EET pathways of Gram-negative bacteria, the EET of Gram-positive bacteria has been studied much less and is mainly limited to the flavin-mediated pathway. Here, we investigate the EET pathway of a Gram-positive filamentous bacterium Lysinibacillus varians GY32. Strain GY32 has a wide electron donor spectrum (including lactate, acetate, formate, and some amino acids) in electrode respiration. Transcriptomic, proteomic, and electrochemical analyses show that the electrode respiration of GY32 mainly depends on electron mediators, and c-type cytochromes may be involved in its respiration. Fluorescent sensor and electrochemical analyses demonstrate that strain GY32 can secrete cysteine and flavins. Cysteine added shortly after inoculation into microbial fuel cells accelerated EET, showing cysteine is a new endogenous electron mediator of Gram-positive bacteria, which provides novel information to understand the EET networks in natural environments. IMPORTANCE Extracellular electron transport (EET) is a key driving force in biogeochemical element cycles and microbial chemical-electrical-optical energy conversion on the Earth. Gram-positive bacteria are ubiquitous and even dominant in EET-enriched environments. However, attention and knowledge of their EET pathways are largely lacking. Gram-positive bacterium Lysinibacillus varians GY32 has extremely long cells (>1 mm) and conductive nanowires, promising a unique and enormous role in the microenvironments where it lives. Its capability to secrete cysteine renders it not only an EET pathway to respire and survive, but also an electrochemical strategy to connect and shape the ambient microbial community at a millimeter scale. Moreover, its incapability of using flavins as an electron mediator suggests that the common electron mediator is species-dependent. Therefore, our results are important to understanding the EET networks in natural and engineering processes.

Abstract SY27-01: Colon commensals and cancer

Abstract A clear intersection between the infectious diseases and oncology fields has emerged from our understanding that many cancers arise in the setting of chronic infection. Roughly one-third of cancers worldwide, particularly epithelial cancers, are associated with identified single microbial infections, leading to the conceptual paradigm that chronic infection with specific microbes cause these cancers independent of other components of the ambient microbial community. Among epithelial cancers, colon cancer is the second leading cancer killer of adults in the United States but specific microbial contributors have not yet been identified. Rather, we now understand that the colon is home to one of the most dense and diverse communities of bacteria in the body and that we are each remarkably unique in our bacterial makeup, reinforcing the contrasting view that the microbial contribution to colon cancer pathogenesis may lie in the composition of the colon microbiome. This talk will explore the scientific data supporting differing paradigms for how the colonic flora may contribute to colon oncogenesis. Specific, usually commensal, members of the colonic microbiome and the mechanisms by which they may initiate and/or promote colon tumors with be discussed with a particular emphasis on enterotoxigenic Bacteroides fragilis (ETBF). B. fragilis not only colonizes most humans but is the leading anaerobe in human disease. ETBF is a molecular subset of B. fragilis distinguished by secretion of a zinc-dependent metalloprotease toxin. In murine models, ETBF is a potent inducer of colonic tumors. Tumor induction by ETBF is dependent, in part, on a specific host immune response involving IL-17-producing CD3+CD4+ T cells. A framework for utilizing this knowledge to better understand the pathogenesis of colon cancer and to develop new approaches to colon cancer prevention and/or therapy will be discussed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY27-01. doi:10.1158/1538-7445.AM2011-SY27-01

Perspective: Alpha-Bugs, Their Microbial Partners, and the Link to Colon Cancer

A dramatic intersection between the infectious diseases and oncology fields has emerged from the appreciation that many cancers arise in the setting of chronic infection. For certain viral-induced cancers, such as human papilloma virus (HPV)–associated cervical cancer, the integrated virus encodes oncogenes that directly drive the transformation of infected cells. However, the vast majority of infection-associated cancers arise from chronic immune responses that, although ineffective in eliminating the inciting microbe, collaborate with microbial products to drive carcinogenesis. As many as one-third of cancers worldwide, particularly epithelial cancers, are associated with identified single microbial infections, leading to the conceptual paradigm that chronic infection with specific microbes causes these cancers, independent of other components of the ambient microbial community. In addition to cervical cancer and HPV, other well-established examples include gastric cancer and Helicobacter pylori, liver cancer and hepatitis B and C viruses, urinary bladder cancer and Schistosoma hematobium, and biliary tree cancer, Clonorchis sinensis, and Opisthorchis viverrini. A contrasting view of microbial carcinogenesis has been applied to colon cancer, the second leading cause of death due to cancer among adults in the United States. On the basis of increasingly high through put methods, we now understand that the colon is home to one of the most dense and diverse communities of bacteria in the body and that, although clustering of the colon microbiome sequences may occur in families or under similar close-contact conditions, we are all remarkably unique in our bacterial make up [1–2]. Indeed, beginning in the 1960s, investigators postulated and pursued links between the complex colon commensal flora and colon cancer [3]. Our recent work demonstrating the capacity of a specific human colonic bacterium, enterotoxigenic Bacteroides fragilis (ETBF), to induce colon tumors in multiple intestinal neoplasia (Min) mice [4] leads us to propose the “Alpha-bug hypothesis,” which integrates the single microbe and microbiome community views of microbial carcinogenesis. While we focus on colon carcinogenesis as an example, we propose that the Alpha-bug hypothesis might apply to other cancer types as well as other chronic immune-based pathology such as inflammatory bowel disease (IBD).

Abstract ED06-03: Colonic bacteria, inflammation, and colon cancer

Abstract A clear intersection between the infectious diseases and oncology fields has emerged from our understanding that many cancers arise in the setting of chronic infection. Roughly one-third of cancers worldwide, particularly epithelial cancers, are associated with identified single microbial infections, leading to the conceptual paradigm that chronic infection with specific microbes cause these cancers independent of other components of the ambient microbial community. Among epithelial cancers, colon cancer is the second leading cancer killer of adults in the United States but specific microbial contributors have not yet been identified. Rather, we now understand that the colon is home to one of the most dense and diverse communities of bacteria in the body and that we are each remarkably unique in our bacterial makeup reinforcing the contrasting view that the microbial contribution to colon cancer pathogenesis may lie in the composition of the colon microbiome. This talk will explore the scientific data supporting differing paradigms for how the colonic flora may contribute to colon oncogenesis. Particular attention will be paid to specific, usually commensal, members of the colonic microbiome and the mechanisms by which they may initiate and/or promote colon tumors. A framework for utilizing this knowledge to better understand the pathogenesis of colon cancer and to develop new approaches to colon cancer prevention will be discussed. Citation Information: Cancer Prev Res 2010;3(12 Suppl):ED06-03.

Influence of microbial interactions on the susceptibility of Karenia spp. to algicidal bacteria

A bacterial strain (D38BY) belonging to the family Flavobacteriaceae and antagonistic towards an algicidal bacterium (strain S03; Flavobacteriaceae) was isolated from a culture of the red tide dinoflagellate Karenia brevis that had previously been characterized as resistant to attack by strain S03. This antagonistic bacterium increased the survival time of otherwise susceptible, bacteria- free K. brevis cultures in a concentration-dependent manner during exposure to the algicidal bac- terium. Experimental evidence indicated that direct contact was required in order for strain D38BY to inhibit the killing activity of algicidal strain S03. While further work is needed to determine its pre- cise mode of action, the antagonistic properties of strain D38BY provide further evidence that the resistance or susceptibility of certain algal taxa to algicidal attack can be more a function of inter- actions within the ambient microbial community than an intrinsic property of the alga.

Interactions between marine snow and heterotrophic bacteria: aggregate formation and microbial dynamics

Macroscopic aggregates (marine snow) contribute to new production and nutrient dynamics in the upper ocean and vertical fluxes of organic matter to the deep ocean. To test whether microorganisms play a significant role in phytoplankton aggregate formation we studied particle abundance and size as well as abundance, colonization behaviour, and community composition of bacteria during the growth of 2 marine diatoms (Thalassiosira weissflogii and Navicula sp.) under axenic and non-axenic conditions. Community composition of free-living and attached bacteria during phytoplankton growth and aggregation was studied by amplification of 16S rRNA gene frag- ments and denaturing gradient gel electrophoresis (DGGE). Our results show that the presence of bacteria was a prerequisite for aggregation of T. weissflogii but not of Navicula sp. Occurrences of distinct populations of free-living and attached bacteria depended on phytoplankton growth and aggregation dynamics. The community composition of especially attached bacteria significantly differed between the 2 algal cultures. Our study suggests that phytoplankton aggregation and verti- cal fluxes are closely linked to interactions between the marine phytoplankton and the ambient microbial community.

Microbial community interactions and population dynamics of an algicidal bacterium active against Karenia brevis (Dinophyceae)

The population dynamics of Cytophaga strain 41-DBG2, a bacterium algicidal to the harmful algal bloom (HAB) dinoflagellate Karenia brevis, were investigated in laboratory experiments using fluorescent in-situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE). Following its introduction into non-axenic K. brevis cultures at concentrations of 10 3 or 10 5 bacterial cells per milliliter, 41-DBG2 increased to 10 6 cells per milliliter before initiation of its algicidal activity. Such threshold concentrations were not achieved when starting algal cell numbers were relatively low (10 3 cells per milliliter), suggesting that the growth of this bacterium may require high levels of dissolved organic matter (DOM) excreted by the algae. It remains to be determined whether this threshold concentration is required to trigger an algicidal response by 41-DBG2 or, alternatively, is the point at which the bacterium accumulates to an effective killing concentration. The ambient microbial community associated with these algal cultures, as determined by DGGE profiles, did not change until after K. brevis cells were in the process of lysing, indicating a response to the rapid input of algal-derived organic matter. Resistance to algicidal attack exhibited by several K. brevis clones was found to result from the inhibition of 41-DBG2 growth in the presence of currently unculturable bacteria associated with those clones. These bacteria apparently prevented 41-DBG2 from reaching the threshold concentration required for initiation of algicidal activity. Remarkably, resistance and susceptibility to the algicidal activity of 41-DBG2 could be transferred between K. brevis clones with the exchange of their respective unattached bacterial communities, which included several dominant phylotypes belonging to the α-proteobacteria, γ-proteobacteria, and Cytophaga–Flavobacterium–Bacteroides (CFB) groups. We hypothesize that CFB bacteria may be successfully competing with 41-DBG2 (also a member of the CFB) for nutrients, thereby inhibiting growth of the latter and indirectly providing resistance against algicidal attack. We conclude that if algicidal bacteria play a significant role in regulating HAB dynamics, as some authors have inferred, bacterial community interactions are crucial factors that must be taken into consideration in future studies.

ALGICIDAL BACTERIA ACTIVE AGAINST GYMNODINIUM BREVE (DINOPHYCEAE). I. BACTERIAL ISOLATION AND CHARACTERIZATION OF KILLING ACTIVITY1,3

Interactions between bacteria and species of harmful and/or toxic algae are potentially important factors affecting both the population dynamics and the toxicity of these algae. Recent reports of bacteria lethal to certain harmful algal bloom (HAB) species, coupled with a rapidly evolving interest in attempting to minimize the adverse effects of HABs through various prevention, control, and mitigation strategies, have focused attention on defining the role of algicidal bacteria in bloom termination. The aim of the present study was to determine whether algicidal bacteria active against Gymnodinium breve Davis, a dinoflagellate responsible for frequent and protracted red tides in the Gulf of Mexico, are present in the waters of the west Florida shelf. To date, we have isolated two bacterial strains from this region lethal to G. breve and have begun to characterize the algicidal activity of one of these strains, 41‐DBG2. This bacterium, a yellow‐pigmented, gram‐negative rod, was isolated from waters containing no detectable G. breve cells, suggesting that such bacteria are part of the ambient microbial community and are not restricted to areas of high G. breve abundance. Strain 41‐DBG2 produced a dissolved algicidal compound(s) that was released into the growth medium, and the algicide was effective against the four Gulf of Mexico G. breve isolates tested as well as a closely related HAB species that also occurs in this region, Gymnodinium mikimotoi Miyake et Kominami ex Oda. Nonetheless, data showing that a nontoxic isolate of Gymnodinium sanguineum Hirasaka from Florida Bay was not affected indicate that the algicidal activity of this bacterium does exhibit a degree of taxonomic specificity. Our efforts are currently being directed at resolving several critical issues, including the identity of the algicide(s), the mechanisms regulating its production and ability to discriminate between target algal species, and how the growth rate of 41‐DBG2 is affected by the presence of G. breve cells. We have also proposed a conceptual model for interactions between algicidal bacteria and their target species to serve as a testable framework for ensuing field studies.

Gene Transfer in Marine Water Column and Sediment Microcosms by Natural Plasmid Transformation

We investigated the possibility for natural transformation in the marine environment by using broad-host-range plasmid multimers and a high-frequency-of-transformation (HFT) Vibrio strain as the recipient. Water and sediment samples were taken from Tampa Bay, the eastern Gulf of Mexico, the Florida Shelf near Miami, and the Bahamas Bank. In water column microcosms, transformation frequencies ranged from 1.7 x 10 to 2.7 x 10 transformants per recipient, with highest frequencies occurring when low levels of nutrients (peptone and yeast extract) were added. The presence of the ambient community either reduced transformation frequency by an order of magnitude or had no effect. In sterile sediments, nutrient additions had no consistent effect on transformation, with transfer frequencies similar to those observed in the water column. Transformation was not observed in any sediment experiment when the ambient microbial community was present. These findings are the first report of natural plasmid transformation in seawater and in the presence of the ambient microbial community. This process may be a mechanism for the acquisition of small, nonconjugative plasmids, which are commonly found in aquatic bacteria. Our data also suggest that natural transformation may be more likely to occur in the water column than in native marine sediments, contradicting prior conclusions based on studies with sterile sediments.

Production of extracellular nucleic acids by genetically altered bacteria in aquatic-environment microcosms

The factors which affect the production of extracellular DNA by genetically altered strains of Escherichia coli, Pseudomonas aeruginosa, Pseudomonas cepacia, and Bradyrhizobium japonicum in aquatic environments were investigated. Cellular nucleic acids were labeled in vivo by incubation with [3H]thymidine or [3H]adenine, and production of extracellular DNA in marine waters, artificial seawater, or minimal salts media was determined by detecting radiolabeled macromolecules in incubation filtrates. The presence or absence of the ambient microbial community had little effect on the production of extracellular DNA. Three of four organisms produced the greatest amounts of extracellular nucleic acids when incubated in low-salinity media (2% artificial seawater) rather than high-salinity media (10 to 50% artificial seawater). The greatest production of extracellular nucleic acids by P. cepacia occurred at pH 7 and 37 degrees C, suggesting that extracellular-DNA production may be a normal physiologic function of the cell. Incubation of labeled P. cepacia cells in water from Bimini Harbor, Bahamas, resulted in labeling of macromolecules of the ambient microbial population. Collectively these results indicate that (i) extracellular-DNA production by genetically altered bacteria released into aquatic environments is more strongly influenced by physiochemical factors than biotic factors, (ii) extracellular-DNA production rates are usually greater for organisms released in freshwater than marine environments, and (iii) ambient microbial populations can readily utilize materials released by these organisms.