Phenotypic responses to interspecies competition and commensalism in a naturally derived microbial co-culture

The fundamental question of whether different microbial species will co-exist or compete in a given environment depends on context, composition and environmental constraints. Model microbial systems can yield some general principles related to this question. In this study we employed a naturally occurring co-culture composed of heterotrophic bacteria, Halomonas sp. HL-48 and Marinobacter sp. HL- 58, to ask two fundamental scientific questions: 1) how do the phenotypes of two naturally co-existing species respond to partnership as compared to axenic growth? and 2) how do growth and molecular phenotypes of these species change with respect to competitive and commensal interactions? We hypothesized – and confirmed – that co-cultivation under glucose as the sole carbon source would result in competitive interactions. Similarly, when glucose was swapped with xylose, the interactions became commensal because Marinobacter HL-58 was supported by metabolites derived from Halomonas HL- 48. Each species responded to partnership by changing both its growth and molecular phenotype as assayed via batch growth kinetics and global transcriptomics. These phenotypic responses depended on nutrient availability and so the environment ultimately controlled how they responded to each other. This simplified model community revealed that microbial interactions are context-specific and different environmental conditions dictate how interspecies partnerships will unfold

Phenotypic responses to interspecies competition and commensalism in a naturally derived microbial co-culture

The fundamental question of whether different microbial species will co-exist or compete in a given environment depends on context, composition and environmental constraints. Model microbial systems can yield some general principles related to this question. In this study we employed a naturally occurring co-culture composed of heterotrophic bacteria, Halomonas sp. HL-48 and Marinobacter sp. HL- 58, to ask two fundamental scientific questions: 1) how do the phenotypes of two naturally co-existing species respond to partnership as compared to axenic growth? and 2) how do growth and molecular phenotypes of these species change with respect to competitive and commensal interactions? We hypothesized – and confirmed – that co-cultivation under glucose as the sole carbon source would result in competitive interactions. Similarly, when glucose was swapped with xylose, the interactions became commensal because Marinobacter HL-58 was supported by metabolites derived from Halomonas HL- 48. Each species responded to partnership by changing both its growth and molecular phenotype as assayed via batch growth kinetics and global transcriptomics. These phenotypic responses depended on nutrient availability and so the environment ultimately controlled how they responded to each other. This simplified model community revealed that microbial interactions are context-specific and different environmental conditions dictate how interspecies partnerships will unfold

Metagenomic Analysis of Lysogeny in Tampa Bay: Implications for Prophage Gene Expression

Phage integrase genes often play a role in the establishment of lysogeny in temperate phage by catalyzing the integration of the phage into one of the host's replicons. To investigate temperate phage gene expression, an induced viral metagenome from Tampa Bay was sequenced by 454/Pyrosequencing. The sequencing yielded 294,068 reads with 6.6% identifiable. One hundred-three sequences had significant similarity to integrases by BLASTX analysis (e≤0.001). Four sequences with strongest amino-acid level similarity to integrases were selected and real-time PCR primers and probes were designed. Initial testing with microbial fraction DNA from Tampa Bay revealed 1.9×107, and 1300 gene copies of Vibrio-like integrase and Oceanicola-like integrase L−1 respectively. The other two integrases were not detected. The integrase assay was then tested on microbial fraction RNA extracted from 200 ml of Tampa Bay water sampled biweekly over a 12 month time series. Vibrio-like integrase gene expression was detected in three samples, with estimated copy numbers of 2.4-1280 L−1. Clostridium-like integrase gene expression was detected in 6 samples, with estimated copy numbers of 37 to 265 L−1. In all cases, detection of integrase gene expression corresponded to the occurrence of lysogeny as detected by prophage induction. Investigation of the environmental distribution of the two expressed integrases in the Global Ocean Survey Database found the Vibrio-like integrase was present in genome equivalents of 3.14% of microbial libraries and all four viral metagenomes. There were two similar genes in the library from British Columbia and one similar gene was detected in both the Gulf of Mexico and Sargasso Sea libraries. In contrast, in the Arctic library eleven similar genes were observed. The Clostridium-like integrase was less prevalent, being found in 0.58% of the microbial and none of the viral libraries. These results underscore the value of metagenomic data in discovering signature genes that play important roles in the environment through their expression, as demonstrated by integrases in lysogeny

Molecular and genomic studies of temperate phages from Halomonas aquamarina and Bacillus spp. isolates from the Gulf of Mexico

Viruses are the most abundant biological entities in the ocean and are believed to contribute to nutrient cycling, bacterial diversity, and horizontal gene exchange. However, little is known about the relationship between temperate phages and their hosts in marine environments. In this thesis, phage-host systems from the Gulf of Mexico were used to study the influence of temperate phages in bacteria. PhiHAP-1 is a temperate myovirus induced with mitomycin C from Halomonas aquamarina isolate. The genome of this phage was 39,245 nucleotides long and contained 46 predicted genes. Besides genes involved in lysogeny, PhiHAP-1 contained a protelomerase, which is responsible for resolution of telomeric ends in linear plasmid-like phages. Hybridization studies and PCR analysis indicated not only a lack of integration of the prophage in the host chromosome, but differences in genome arrangement between the prophage and virion forms of PhiHAP-1. These results suggest that PhiHAP-1 exists as a non-integrating linear phage with telomeric ends. Eleven pigmented Bacillus spp. isolates were examined for the occurrence of lysogeny and sporulation through induction with mitomycin C and decoyinine, respectively. The results from these experiments suggested a variety of interactions can occur between phages and their hosts, some of which may influence sporulation. The lysogenic strain B14905 had high frequency of sporulation and was selected for further analysis. The genome of B14905 contained 4 prophage-like regions, one of which was independently sequenced from an induced lysate. PCR and TEM analysis of a mitomycin C induced lysate indicated that two of these regions were inducible prophage, one was a defective phage, and one was a non-inducible phage remnant. One of the inducible prophages contained a transcriptional regulator that is hypothesized to be involved in regulation of host sporulation. The diversity of prophage and prophage-like elements found in B14905 suggest that the genetic diversity of phages in the oceans is vast. The studies of the temperate phages from H. aquamarina and Bacillus spp. isolates illustrates that integration of molecular, genomic, and function studies can be used to provide insight into the influence of prophage on host bacteria

The Temperate Marine Phage ΦHAP-1 of Halomonas aquamarina Possesses a Linear Plasmid-Like Prophage Genome ▿

A myovirus-like temperate phage, ΦHAP-1, was induced with mitomycin C from a Halomonas aquamarina strain isolated from surface waters in the Gulf of Mexico. The induced cultures produced significantly more virus-like particles (VLPs) (3.73 × 1010 VLP ml−1) than control cultures (3.83 × 107 VLP ml−1) when observed with epifluorescence microscopy. The induced phage was sequenced by using linker-amplified shotgun libraries and contained a genome 39,245 nucleotides in length with a G+C content of 59%. The ΦHAP-1 genome contained 46 putative open reading frames (ORFs), with 76% sharing significant similarity (E value of <10−3) at the protein level with other sequences in GenBank. Putative functional gene assignments included small and large terminase subunits, capsid and tail genes, an N6-DNA adenine methyltransferase, and lysogeny-related genes. Although no integrase was found, the ΦHAP-1 genome contained ORFs similar to protelomerase and parA genes found in linear plasmid-like phages with telomeric ends. Southern probing and PCR analysis of host genomic, plasmid, and ΦHAP-1 DNA indicated a lack of integration of the prophage with the host chromosome and a difference in genome arrangement between the prophage and virion forms. The linear plasmid prophage form of ΦHAP-1 begins with the protelomerase gene, presumably due to the activity of the protelomerase, while the induced phage particle has a circularly permuted genome that begins with the terminase genes. The ΦHAP-1 genome shares synteny and gene similarity with coliphage N15 and vibriophages VP882 and VHML, suggesting an evolutionary heritage from an N15-like linear plasmid prophage ancestor

Effects of Elevated Carbon Dioxide and Salinity on the Microbial Diversity in Lithifying Microbial Mats

Atmospheric levels of carbon dioxide (CO2) are rising at an accelerated rate resulting in changes in the pH and carbonate chemistry of the world’s oceans. However, there is uncertainty regarding the impact these changing environmental conditions have on carbonate-depositing microbial communities. Here, we examine the effects of elevated CO2, three times that of current atmospheric levels, on the microbial diversity associated with lithifying microbial mats. Lithifying microbial mats are complex ecosystems that facilitate the trapping and binding of sediments, and/or the precipitation of calcium carbonate into organosedimentary structures known as microbialites. To examine the impact of rising CO2 and resulting shifts in pH on lithifying microbial mats, we constructed growth chambers that could continually manipulate and monitor the mat environment. The microbial diversity of the various treatments was compared using 16S rRNA gene pyrosequencing. The results indicated that elevated CO2 levels during the six month exposure did not profoundly alter the microbial diversity, community structure, or carbonate precipitation in the microbial mats; however some key taxa, such as the sulfate-reducing bacteria Deltasulfobacterales, were enriched. These results suggest that some carbonate depositing ecosystems, such as the microbialites, may be more resilient to anthropogenic-induced environmental change than previously thought

Metagenomic Analysis of Lysogeny in Tampa Bay: Implications for Prophage Gene Expression

Phage integrase genes often play a role in the establishment of lysogeny in temperate phage by catalyzing the integration of the phage into one of the host\u27s replicons. To investigate temperate phage gene expression, an induced viral metagenome from Tampa Bay was sequenced by 454/Pyrosequencing. The sequencing yielded 294,068 reads with 6.6% identifiable. One hundred-three sequences had significant similarity to integrases by BLASTX analysis (e≤0.001). Four sequences with strongest amino-acid level similarity to integrases were selected and real-time PCR primers and probes were designed. Initial testing with microbial fraction DNA from Tampa Bay revealed 1.9×107, and 1300 gene copies of Vibrio-like integrase and Oceanicola-like integrase L−1 respectively. The other two integrases were not detected. The integrase assay was then tested on microbial fraction RNA extracted from 200 ml of Tampa Bay water sampled biweekly over a 12 month time series. Vibrio-like integrase gene expression was detected in three samples, with estimated copy numbers of 2.4-1280 L−1. Clostridium-like integrase gene expression was detected in 6 samples, with estimated copy numbers of 37 to 265 L−1. In all cases, detection of integrase gene expression corresponded to the occurrence of lysogeny as detected by prophage induction. Investigation of the environmental distribution of the two expressed integrases in the Global Ocean Survey Database found the Vibrio-like integrase was present in genome equivalents of 3.14% of microbial libraries and all four viral metagenomes. There were two similar genes in the library from British Columbia and one similar gene was detected in both the Gulf of Mexico and Sargasso Sea libraries. In contrast, in the Arctic library eleven similar genes were observed. The Clostridium-like integrase was less prevalent, being found in 0.58% of the microbial and none of the viral libraries. These results underscore the value of metagenomic data in discovering signature genes that play important roles in the environment through their expression, as demonstrated by integrases in lysogeny

Lysogeny and Sporulation in Bacillus Isolates from the Gulf of Mexico▿ †

Eleven Bacillus isolates from the surface and subsurface waters of the Gulf of Mexico were examined for their capacity to sporulate and harbor prophages. Occurrence of sporulation in each isolate was assessed through decoyinine induction, and putative lysogens were identified by prophage induction by mitomycin C treatment. No obvious correlation between ability to sporulate and prophage induction was found. Four strains that contained inducible virus-like particles (VLPs) were shown to sporulate. Four strains did not produce spores upon induction by decoyinine but contained inducible VLPs. Two of the strains did not produce virus-like particles or sporulate significantly upon induction. Isolate B14905 had a high level of virus-like particle production and a high occurrence of sporulation and was further examined by genomic sequencing in an attempt to shed light on the relationship between sporulation and lysogeny. In silico analysis of the B14905 genome revealed four prophage-like regions, one of which was independently sequenced from a mitomycin C-induced lysate. Based on PCR and transmission electron microscopy (TEM) analysis of an induced phage lysate, one is a noninducible phage remnant, one may be a defective phage-like bacteriocin, and two were inducible prophages. One of the inducible phages contained four putative transcriptional regulators, one of which was a SinR-like regulator that may be involved in the regulation of host sporulation. Isolates that both possess the capacity to sporulate and contain temperate phage may be well adapted for survival in the oligotrophic ocean

Daylight-driven carbon exchange through a vertically structured microbial community

Interactions between autotrophs and heterotrophs are central to carbon (C) exchange across trophic levels in essentially all ecosystems and metabolite exchange is a frequent mechanism for distributing C within spatially structured ecosystems. Yet, despite the importance of C exchange, the timescales at which fixed C is transferred in microbial communities is poorly understood. We employed a stable isotope tracer combined with spatially resolved isotope analysis to quantify photoautotrophic uptake of bicarbonate and track subsequent exchanges across a vertical depth gradient in a stratified microbial mat over a light-driven diel cycle. We observed that C mobility, both across the vertical strata and between taxa, was highest during periods of active photoautotrophy. Parallel experiments with 13C-labeled organic substrates (acetate and glucose) showed comparably less exchange of C within the mat. Metabolite analysis showed rapid incorporation of 13C into molecules that can both comprise a portion of the extracellular polymeric substances in the system and serve to transport C between photoautotrophs and heterotrophs. Stable isotope proteomic analysis revealed rapid C exchange between cyanobacterial and associated heterotrophic community members during the day with decreased exchange at night. We observed strong diel control on the spatial exchange of freshly fixed C within tightly interacting mat communities suggesting a rapid redistribution, both spatially and taxonomically, primarily during daylight periods