John H. Paul

Functional metagenomic profiling of nine biomes

Microbial activities shape the biogeochemistry of the planet and macroorganism health. Determining the metabolic processes performed by microbes is important both for understanding and for manipulating ecosystems (for example, disruption of key processes that lead to disease, conservation of environmental services, and so on). Describing microbial function is hampered by the inability to culture most microbes and by high levels of genomic plasticity. Metagenomic approaches analyse microbial communities to determine the metabolic processes that are important for growth and survival in any given environment. Here we conduct a metagenomic comparison of almost 15 million sequences from 45 distinct microbiomes and, for the first time, 42 distinct viromes and show that there are strongly discriminatory metabolic profiles across environments. Most of the functional diversity was maintained in all of the communities, but the relative occurrence of metabolisms varied, and the differences between metagenomes predicted the biogeochemical conditions of each environment. The magnitude of the microbial metabolic capabilities encoded by the viromes was extensive, suggesting that they serve as a repository for storing and sharing genes among their microbial hosts and influence global evolutionary and metabolic processes.

Pathogenic Human Viruses in Coastal Waters

SUMMARY This review addresses both historical and recent investigations into viral contamination of marine waters. With the relatively recent emergence of molecular biology-based assays, a number of investigations have shown that pathogenic viruses are prevalent in marine waters being impacted by sewage. Research has shown that this group of fecal-oral viral pathogens (enteroviruses, hepatitis A viruses, Norwalk viruses, reoviruses, adenoviruses, rotaviruses, etc.) can cause a broad range of asymptomatic to severe gastrointestinal, respiratory, and eye, nose, ear, and skin infections in people exposed through recreational use of the water. The viruses and the nucleic acid signature survive for an extended period in the marine environment. One of the primary concerns of public health officials is the relationship between the presence of pathogens and the recreational risk to human health in polluted marine environments. While a number of studies have attempted to address this issue, the relationship is still poorly understood. A contributing factor to our lack of progress in the field has been the lack of sensitive methods to detect the broad range of both bacterial and viral pathogens. The application of new and advanced molecular methods will continue to contribute to our current state of knowledge in this emerging and important field.

Prophages in marine bacteria: dangerous molecular time bombs or the key to survival in the seas?

Bacteriophages are realized to be numerous and important components of oceanic food webs principally because of their lytic capabilities. The subtle changes that temperate phages impart to their hosts in the oceans are far less understood. Occurrences of lysogeny in the oceans correlate well with conditions unfavorable for rapid host growth. In coliphage λ, phage encoded repressors have been shown to modulate host metabolic gene expression and phenotype, resulting in economizing host energy expenditure. Comparison of lysogenized marine bacteria to the uninfected hosts indicated that prophage acquisition is correlated with host metabolic gene suppression. Screening 113 marine bacterial genomes for prophages yielded 64 prophage-like elements, 21 of which strongly resembled gene transfer agents (GTAs). The remaining 39 putative prophages had a relatively high incidence of transcriptional regulatory and repressor-like proteins (∼2/40 kb prophage sequence) compared to lytic marine phages (∼0.25/40 kb phage sequence). Here, it has been hypothesized that marine prophages directly contribute to host survival in unfavorable environments by suppression of unneeded metabolic activities. It has been further suggested that such metabolic downshifts are the result of phage-encoded repressors and transcriptional regulators acting directly on host genes. Finally, the widespread occurrence of GTAs may be an efficient mechanism for horizontal gene transfer in the oceans.

High Frequency of Horizontal Gene Transfer in the Oceans

Viruslike particles enable lateral gene transfer among marine microorganisms. Oceanic bacteria perform many environmental functions, including biogeochemical cycling of many elements, metabolizing of greenhouse gases, functioning in oceanic food webs (microbial loop), and producing valuable natural products and viruses. We demonstrate that the widespread capability of marine bacteria to participate in horizontal gene transfer (HGT) in coastal and oceanic environments may be the result of gene transfer agents (GTAs), viral-like particles produced by α-Proteobacteria. We documented GTA-mediated gene transfer frequencies a thousand to a hundred million times higher than prior estimates of HGT in the oceans, with as high as 47% of the culturable natural microbial community confirmed as gene recipients. These findings suggest a plausible mechanism by which marine bacteria acquire novel traits, thus ensuring resilience in the face of environmental change.

Detection of Viral Pathogens by Reverse Transcriptase PCR and of Microbial Indicators by Standard Methods in the Canals of the Florida Keys

In order to assess the microbial water quality in canal waters throughout the Florida Keys, a survey was conducted to determine the concentration of microbial fecal indicators and the presence of human pathogenic microorganisms. A total of 19 sites, including 17 canal sites and 2 nearshore water sites, were assayed for total coliforms, fecal coliforms, Escherichia coli, Clostridium perfringens, enterococci, coliphages, F-specific (F(+)) RNA coliphages, Giardia lamblia, Cryptosporidium parvum, and human enteric viruses (polioviruses, coxsackie A and B viruses, echoviruses, hepatitis A viruses, Norwalk viruses, and small round-structured viruses). Numbers of coliforms ranged from <1 to 1, 410, E. coli organisms from <1 to 130, Clostridium spp. from <1 to 520, and enterococci from <1 to 800 CFU/100 ml of sample. Two sites were positive for coliphages, but no F(+) phages were identified. The sites were ranked according to microbial water quality and compared to various water quality standards and guidelines. Seventy-nine percent of the sites were positive for the presence of enteroviruses by reverse transcriptase PCR (polioviruses, coxsackie A and B viruses, and echoviruses). Sixty-three percent of the sites were positive for the presence of hepatitis A viruses. Ten percent of the sites were positive for the presence of Norwalk viruses. Ninety-five percent of the sites were positive for at least one of the virus groups. These results indicate that the canals and nearshore waters throughout the Florida Keys are being impacted by human fecal material carrying human enteric viruses through current wastewater treatment strategies such as septic tanks. Exposure to canal waters through recreation and work may be contributing to human health risks.

Gene deletion as the cause of α thalassaemia: The severe form of α thalassaemia is caused by a haemoglobin gene deletion

Two independent groups show that the absence of all or part of the globin α-chain gene is the origin of the homozygous α thalassaemia.

Seasonal Variation in Lysogeny as Depicted by Prophage Induction in Tampa Bay, Florida

ABSTRACT A seasonal study of the distribution of lysogenic bacteria in Tampa Bay, Florida, was conducted over a 13-month period. Biweekly water samples were collected and either were left unaltered or had the viral population reduced by filtration (pore size, 0.2 μm) and resuspension in filtered (pore size, 0.2 μm) water. Virus-reduced and unaltered samples were then treated by adding mitomycin C (0.5 μg ml−1) to induce prophage or were left untreated. In order to test the hypothesis that prophage induction was phosphate limited, additional induction experiments were performed in the presence and absence of phosphate. Induction was assessed as an increase in viral direct counts, relative to those obtained in controls, as detected by epifluorescence microscopy. Induction of prophage was observed in 5 of 25 (20%) unaltered samples which were obtained during or after the month of February, paralleling the results from a previous seasonal study. Induction of prophage was observed in 9 of 25 (36%) of the virus-reduced samples, primarily those obtained in the winter months, which was not observed in a prior seasonal study (P. K. Cochran and J. H. Paul, Appl. Environ. Microbiol. 64:2308-2312, 1998). Induction was noted in the months of lowest bacterial and primary production, suggesting that lysogeny was favored under conditions of poor host growth. Phosphate addition enabled prophage induction in two of nine (22%) experiments. These results indicate that prophage induction may occasionally be phosphate limited or respond to increases in phosphate concentration, suggesting that phosphate concentration may modulate the lysogenic response of natural populations.

Global-scale processes with a nanoscale drive: the role of marine viruses

Viruses, the smallest and most numerous of all biotic agents, represent the planets largest pool of genetic diversity. The sheer abundance of oceanic viruses results in ~1029 viral infections per day, causing the release of 108–109 tonnes of carbon per day from the biological pool (Suttle, 2007). Still, how and to what extent virus-mediated nanoscale processes are linked to global-scale biodiversity and biogeochemistry is poorly defined.

Quantification of Human Polyomaviruses JC Virus and BK Virus by TaqMan Quantitative PCR and Comparison to Other Water Quality Indicators in Water and Fecal Samples

ABSTRACT In the United States, total maximum daily load standards for bodies of water that do not meet bacterial water quality standards are set by each state. The presence of human polyomaviruses (HPyVs) can be used as an indicator of human-associated sewage pollution in these waters. We have developed and optimized a TaqMan quantitative PCR (QPCR) assay based on the conserved T antigen to both quantify and simultaneously detect two HPyVs; JC virus and BK virus. The QPCR assay was able to consistently quantify ≥10 gene copies per reaction and is linear over 5 orders of magnitude. HPyVs were consistently detected in human waste samples (57 of 64) and environmental waters with known human fecal contamination (5 of 5) and were not amplified in DNA extracted from 127 animal waste samples from 14 species. HPyV concentrations in sewage decreased 81.2 and 84.2% over 28 days incubation at 25 and 35°C, respectively. HPyVs results were compared to Escherichia coli, fecal coliform, and enterococci concentrations and the presence of three other human-associated microbes: Bacteroidetes, Methanobrevibacter smithii, and adenovirus. HPyVs were the most frequently detected of these in human and contaminated environmental samples and were more human specific than the Bacteroidetes (HF183) or M. smithii. HPyVs and M. smithii more closely mimicked the persistence of adenovirus in sewage than the other microbes. The use of this rapid and quantitative assay in water quality research could help regulatory agencies to identify sources of water pollution for improved remediation of contaminated waters and ultimately protect humans from exposure to pathogens.

Significance of Lysogeny in the Marine Environment: Studies with Isolates and a Model of Lysogenic Phage Production

A bstractThe importance of lysogeny in marine microbial populations is just beginning to be understood. To determine the abundance of lysogens in bacterial populations, we studied the occurrence of lysogenic bacteria among bacterial isolates from a variety of marine environments. More than 116 bacteria isolated on artificial seawater nutrient agar plates were tested for the presence of inducible prophage by mitomycin C and UV radiation. Induction was determined as a decrease in culture absorbance at 600 nm, after treatment with inducing agents. Samples in which optical density decreased or remained the same after induction were further examined by transmission electron microscopy, for the presence of virus-like particles. More than 40% of the bacterial isolates contained inducible prophage, as determined by mitomycin C induction. A higher percentage of lysogenic bacteria was found in isolates from oligotrophic environments, compared to coastal or estuarine environments. These studies suggest that lysogenic bacteria are important components in marine microbial populations. However, a mathematical model based on viral and bacterial abundance and production rates suggests that, under normal conditions, lysogenic viral production contributes less than 0.02% of total viral production. Therefore, lysogens in the marine environment may serve as a source of viruses and only contribute significantly to viral production during natural induction events.