Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Aggregatibacter actinomycetemcomitans (Aa) é uma bactéria associada à Periodontite Agressiva (PA). Ela invade tecidos moles, com ocorrência de lisogenia e bacteriófagos presentes em até 69% das subespécies. Estudos in vitro sugerem que a indução do bacteriófago (Aa17) ocorre numa co-cultura de Aa lisogênico com fibroblastos humanos. Se esta interação ocorre in vivo, com liberação do vírus, uma reação imunológica contra o Aa17 aconteceria. O objetivo deste estudo é constatar se anticorpos (AC) contra proteínas do Aa17 existem e estão associados à doença periodontal. Um objetivo adicional foi testar a resposta de AC contra os sorotipos do Aa. 52 indivíduos participaram: 31 com PA, 5 com Periodontite Crônica (PC) e 16 com Periodonto Saudável (PS). Soro foi coletado após a classificação clínica. As proteínas do Aa17 foram obtidas de preparações purificadas. As subespécies do Aa utilizadas para amostras de proteínas através de sonicação foram: 43717(American Tissue Culture Collection - ATCC) sorotipo A, 43718 (ATCC) sorotipo B, 33384 (ATCC) sorotipo C, IDH781 sorotipo D, NJ9500 sorotipo E and CU1000 sorotipo F. As proteínas foram separadas em géis de poliacrilamida e transferidas para membranas de nitrocelulose. As reações de Western-blotting ocorreram com o AC primário sendo o soro de cada indivíduo. Todas as membranas foram lidas pelo sistema Odyssey que captura sinais no AC secundário (antihumano). A resposta de AC contra ao menos uma proteína do Aa17, assim como pelo menos um sorotipo do Aa foi observado em todos, com exceção de dois indivíduos (com PS), participantes. Um indivíduo do grupo PC e três do PA tiveram resposta de AC contra alguns, mas não todos os sorotipos do Aa. A resposta de AC contra todos os sorotipos foi o achado mais comum nos grupos PA (28/31), PS (14/16) e PC (4/5). A resposta de AC contra o complexo de proteínas do Aa17 foi observado em 7 indivíduos com PA, 2 com PC e 6 com PS. A presença de AC contra qualquer proteína do Aa17 tem significância estatística (p= 0,044), assim como a resposta de AC contra o sorotipo C (p= 0,044). Reações intensas foram vistas quando o soro reagiu contra proteínas do sorotipo C; em alguns casos um sinal tão forte que cobriu a maioria da faixa. Essa resposta intensa esteve presente em 17, 3 e 1 dos indivíduos com PA, PC e PS e tem significância estatística entre os grupos PA e PS (p= 0,001). A resposta de AC contra uma proteína do Aa17 ou seu complexo foi observado em todos os grupos. Esse achado sugere que a indução in vitro do Aa17 poderia também ocorrer in vivo, embora não sendo necessariamente associada à periodontite. A resposta de AC contra vários sorotipos do Aa foi um achado comum e não associado com a doença. Entretanto, a presença e a intensidade da resposta de AC contra o sorotipo C está associada à PA. / Aggregatibacter actinomycetemcomitans (Aa) is a bacteria associated with Aggressive Periodontitis (AP). It invades soft tissues, with occurrence of lysogeny and bacteriophage presence up to 69% of Aa subspecies. In vitro studies suggested that bacteriophage (Aa17) induction occurs upon co-culture of Aa lysogens subspecies with human fibroblasts. If such an in vivo interaction resulted in Aa17 induction and release of virions, an immunologic reaction to Aa17 proteins could ensue. The purpose of this investigation was to learn whether serum antibodies (AB) to Aa17 proteins are found in human sera, and whether they are associated with periodontal disease. An additional purpose was to test the AB response against known Aa serotypes.52 individuals took part: 31 with AP, 5 with Chronic Periodontitis (CP) and 16 with a Healthy Periodontium (HP). Serum was collected after clinical classification. Aa17 proteins were obtained from purified Aa17 preparations. The Aa strains used for protein sampling through sonication were: 43717(American Tissue Culture Collection - ATCC) serotype A, 43718 (ATCC) serotype B, 33384 (ATCC) serotype C, IDH781 serotype D, NJ9500 serotype E and CU1000 serotype F. Proteins were separated by SDS-PAGE gels and then transferred to nitrocellulose membranes. Western-blotting reactions were carried out with the primary AB being each subjects serum. All membranes were read through the Odyssey system which captures signals from a dye in a secondary (antihuman) AB. AB response against at least one Aa17 protein, as well as a response to at least one Aa serotype, was observed in all but two individuals (with HP) who participated in the study. Serum from one individual from the CP group and three from the AP group had AB response to some, but not all Aa serotypes. AB response against all Aa serotypes was the most common finding in AP (28/31), HP (14/16) and CP (4/5) groups. AB response to the full complex of Aa17 proteins was observed in 7 individuals with AP, 2 with CP and 6 with HP. Chi-square tests comparing the AP with HP groups indicated that the presence of AB against any Aa17 protein is statistically significant (p= 0,044), as well as AB response against serotype C (p= 0,044). Intense reactions were seen when sera reacted with proteins from serotype C; in some cases the signal was so strong that it covered much of the lane. This intense response was present in 17, 3, and 1 of the AP, CP and HP subjects, respectively and was statistically significant (p=0.001) between the AP and HP groups. AB response against a Aa17 protein or its whole complex was observed in all groups. This finding suggests that the in vitro Aa17 induction could also be occurring in vivo, although it is not necessarily associated with periodontitis. AB response against multiple Aa serotypes was a common finding and is not associated with disease. However, the presence and intensity of AB response against Aa serotype C, is associated with AP.

The increasing incidence of infections due to antibiotic resistant bacteria has led to renewed interest in bacteriophages (= phages) and phage therapy. Although phage therapy has been applied to control bacterial diseases in plants, poultry, livestock and humans, its application in aquaculture is still relatively limited. The emergence of phage-resistant bacterial mutants has been considered to be one of the major limitations of phage therapy. This study aimed to (i) isolate and characterise phages; (ii) select phages and their bacterial hosts to set up in vivo phage therapy models with aquaculture animals, and estimate the efficiency of phage therapy; (iii) investigate the generation and characteristics of phage-resistant mutants, and thus estimate the consequence of applying phage therapy when phage-resistant mutants emerge; and (iv) discuss the prospects for application of phages in aquaculture. Two Vibrio isolates and their phages were isolated from a Scottish marine fish farm. Based on the results of conventional phenotype testing and 16S rRNA gene sequencing analysis, the two vibrios, V9 and V13, were identified as Vibrio splendidus and Vibrio cyclitrophicus, respectively. The bacterial characteristics including morphology, temperature and salinity range of growth, production of extracellular enzymes, and the possession of virulence genes were examined. According to the morphological characteristics observed using transmission electron microscopy by negative staining, phage PVS9 of V. splendidus V9 was identified as a myophage, while phage PVC13 of V. cyclitrophicus V13 was identified as a siphophage. The phages could only lyse one bacterial host strain and their genomic DNA was double stranded with a size of ~46 kb. The two Vibrio isolates were found to be non- or of low virulence to rainbow trout, goldsinny wrasse and Artemia in pathogenicity experiments. Thus an in vivo phage therapy model could not be set up using these Vibrio isolates and their phages. Two phages pAS-3 and pAS-6 were isolated using the Aeromonas salmonicida subsp. salmonicida Hooke strain as the host. Phages pAS-3 and pAS-6 had a similar genome size of ~50 kb, and the same relatively narrow host range within A. salmonicida subsp. salmonicida strains. The siphophage pAS-3 formed clear plaques and inhibited A. salmonicida Hooke growth in vitro completely for at least 18 hours when using MOI = 1,000, whereas the podophage pAS-6 formed turbid plaques and weakly inhibited Hooke growth. Rainbow trout exposed by intraperitoneal injection with 0.1 mL of the raw phage preparations at a concentration of 108 PUF mL-1 showed no adverse effects over 14 days. In the phage therapy trial, fish were firstly injected with 1 x 102 CFU fish-1 of A. salmonicida Hooke, then immediately injected with phage preparations of pAS-3 and pAS-6, respectively, using MOI = 10,000. Compared with the control group (which did not receive phage treatment), phage treated groups showed a delay in the time to death, and lower mortalities. However, the mortalities and time to death between phage treated and non-treated groups were not significantly different. Phage-resistant mutants of pathogenic A. salmonicida strain Hooke were induced by repeatedly challenging with phage pAS-3. One of the mutants, termed HM, was chosen to compare the characteristics with the parental wild-type strain Hooke. Test results including the formation of ‘smooth’ colonies on TSA, autoagglutination negative, the formation of creamy colonies on Coomassie Brilliant Blue agar, and the degradation of a thick/furry layered structure on the cell surface indicated a deficiency of the A-layer in the phage-resistant mutant HM. Therefore, it was deduced that the A-layer either directly acted as the receptor of A. salmonicida phage pAS-3, or was affected indirectly by the change of an unknown phage receptor. The greater wax moth larvae model was used to compare the virulence of the phage-resistant mutant HM and the parental wild-type strain Hooke, as it is an ethically acceptable animal model, which has the advantages of being low cost and convenient for injection, and is also a recognised alternative model for bacterial pathogens of fish. The results showed that virulence of the phage-resistant mutant HM did not decline in the greater wax moth larvae model compared with that of the parental wild-type strain Hooke. In conclusion, different approaches were used to isolate and characterise phages from different aquaculture environments for potential use in phage therapy. A rainbow trout model was set up using pathogenic A. salmonicida strain Hooke and two A. salmonicida phages pAS-3 and pAS-6. The use of phage treatment led to lower cumulative mortalities and delay to the time of death, although the differences between the groups were not significant, futher work is required to determine if these phages have potential in phage therapy. The consequence of applying phage therapy when phage-resistant mutants emerge was estimated based on their characteristics and virulence, and no decline in virulence of the phage-resistant mutant from this study indicates the importance of fully testing the virulence of phage-resistant mutants before carrying out large scale field trials of phage therapy. It appears feasible to use phage therapy as an alternative approach to control bacterial infections in aquaculture, but further studies are required to focus on improving effectiveness, and also to overcome the concrete limitations and hurdles in application and commercialisation. Moreover, a broader range of applications of phages in aquaculture should be explored.

No description available.

Initiation of transcription is a major step in the regulation of gene expression. A dominant theme in regulation of gene expression lies in understanding the mechanism involved in selective expression of the genes in response to external or internal stimuli. Gene regulatory proteins bind DNA at specific sites either cognate to the promoters they act upon or at a distance, thereby exerting their effect by turning on (activation) or turning off (repression) the genes. Response of these factors to the environmental signals is further achieved by the DNA binding affinity of the transcription factors that can be modulated by small ligands, concentrations of which may fluctuate in response to nutrient availability and stress. Bacteriophages achieve a high degree of efficiency in gene expression by evolving elegant strategies of transcriptional control. mom gene of enterobacteriophage Mu serves as an excellent model to understand this elaborate regulation of gene expression. The gene encodes a unique DNA modification function that confers an anti-restriction phenotype to the phage genome. Though dispensable for phage growth, it is fascinating in two respects (i) a novel modification; (ii) regulation follows a complex scheme without precedence in prokaryotes. mom is the last gene to be expressed during the phage lytic life cycle. Premature expression of the gene is deleterious to both host and phage and hence it is under a complex regulatory network. Dam methylase, a host encoded protein acts as a positive regulator of gene expression, an example where methylation has been shown to play a positive role in regulating tranascription. OxyR, another host encoded protein negatively regulates mom gene expression. Dam methylation prevents the binding OxyR to its site located in the mom regulatory region. The regulatory interplay also involves two phage encoded proteins. C, a middle gene product is essential for transcriptional switch from middle to late genes and Com, a late gene product, for enhancing translation of mom mRNA. Thus, C and Com serve as transcriptional and translational activators of mom gene expression. Pmom is a weak promoter with both -10 and -35 elements away from consensus and a sub-optimal 19 bp spacer element encompassing a stretch of 6T residues that act as negative elements. ‘T stretch’ is known to induce a kink in the DNA. The sub-optimal spacer region makes the promoter elements out of phase and RNAP by itself cannot bind at mom promoter. C protein exerts its effect in activation in a multistep mechanism. The protein binds DNA as a dimer overlapping the promoter and unwinds the DNA, realigning the promoter elements, thus recruiting the RNAP. In the next step, it enhances the promoter clearance by the enzyme, thus enhancing the rate of productive transcription. With this prevailing knowledge on C mediated mom gene expression, the present thesis work describes the experiments carried out to further understand the molecular mechanism of second step activation at Pmom. Genetic and biochemical analysis were carried out to identify the interacting surface of C protein on RNAP. Subsequently, studies have been extended to understand the C mediated transactivation at other late promoters- lys, I, P, which encode for the lysis and morphogenetic functions of the phage. Finally, Mg2+ coordinating residues in C protein were identified to decipher the ligand induced conformational changes in the activator protein required for its transactivator function. Chapter I, a general introduction to the thesis, deals with the detailed discussion on gene expression and its regulatory mechanisms. RNA polymerase (RNAP) being the central molecule of gene expression (transcription) its organization and assembly are discussed. With the availability of the high resolution crystal structures of bacterial RNAP, an in-depth review on RNAP structure in terms of its potential regulatory targets, conformational changes associated with the formation of a functional holoenzyme, and during its transition from initiation to elongation processes have been described. Regulation of transcription with an emphasis on activation mechanism, ligand mediated allosteric transitions in regulatory proteins and the polymerase-activator interactions are discussed citing a few examples. The chapter concludes by introducing bacteriophage Mu and mom gene and its regulation by C. The objectives of the thesis form the concluding section of the chapter. Activators are capable of resurrecting defective promoters in response to cellular demands. The unusual, multistep activation of mom promoter (Pmom) by C protein involves activator mediated promoter unwinding to recruit RNA Polymerase (RNAP) and subsequent enhanced promoter clearance of the enzyme. The first step of transactivation is an interaction independent step, while the later might involve a transient interaction between C and one of the subunits of RNAP. Previous studies pointed out β′ subunit to be the most probable interaction partner. Chapter II comprises the genetic and biochemical studies carried out to confirm this observation. Employing a genetic screen mutations in rpoC gene (encoding the β′ subunit of RNAP), were isolated which result in the defective RNAP. The mutant RNAPs were assayed for their C specific activity by in vivo transactivation assays. Such mutants have been purified and characterized to understand their effect at different steps of C mediated mom gene expression during transcription initiation. The mutant RNAP had normal transcription activity with typical σ70 promoters but exhibited reduced productive transcription and enhanced abortive initiation on C-dependent Pmom. Experiments carried out to probe the interaction between C and mutant RNAP revealed that the physical interaction per se is not disrupted between the two proteins. Post C-mediated recruitment of RNAP to the promoter, transient interactions between the two proteins appears to induce subtle conformational changes in RNAP leading to an enhanced promoter clearance. Transactiavtor protein C is essential for the expression of other late genes lys, I, P apart from mom during the phage life cycle. Although the mechanism of multistep activation at Pmom has been elucidated, little is known on the transactivation from lys, I and P promoters. Chapter III includes studies carried out to understand the process of activation at these promoters. Owing to the differences in their C-binding site and promoter architecture it was important to investigate the differential effect of C, if any at lys, I , P promoters compared to that at Pmom. Activators in prokaryotes are shown to stimulate different steps of transcription initiation pathway ranging from the polymerase binding to the promoters to the post recruitment steps of isomerization and promoter clearance. Effect of C at different steps of transcription initiation pathway was analysed. The results indicate that C is absolutely essential for transcription from lys, I and P promoters similar to mom. However, at these promoters C exerts its effect at the step of Isomerisation from closed complex to open complex formation. Thus, C acts at a single step here and the mode of activation is different from that observed at Pmom. C dimer binds DNA with high affinity and sequence specificity, to an interrupted palindromic sequence overlapping the -35 element of mom promoter. Mg2+ mediated conformational transitions in C protein are essential for its DNA binding and transactivation functions. Chapter IV deals with the identification of the Mg2+ coordinating residues in C protein. Primary sequence analyses lead to the identification of a putative metal coordinating motif (EXDXD) towards the N-terminus of the protein. These residues were subjected to site directed mutagenesis to infer their role in Mg2+ coordination, its associated allosteric transition required for specific interaction with DNA. Mutants showed an altered Mg2+ induced conformation, compromised DNA binding and reduced levels of transcription activation when compared to C protein. Though Mg2+ is widely used in various DNA transaction reactions, this study provides the first insights on the importance of metal-ion induced allosteric transitions in regulating transcription factor function.

No description available.

Bacteriophages (hereafter referred as phages) can translate their mRNAs efficiently by maximizing the use of codons decoded by the most abundant tRNAs of their bacterial hosts. Translation efficiency directly influences phage fitness and evolution. Reengineered phages find application in controlling their host population in both health and industry. The objective of this thesis work is to examine the factors shaping codon choices of single stranded DNA (ssDNA) and double stranded DNA (dsDNA) Escherichia coli phages. In chapter two, we employed two indices, rRSCU (correlation in relative synonymous codon usage between phages and their hosts) and CAI (codon adaptation index) to measure codon adaptation in phages. None of the analyzed ssDNA phages encode tRNAs while some dsDNA phages encode their own tRNAs. Both rRSCU and CAI are negatively correlated with number of tRNA genes encoded by these dsDNA phages. We observed significantly greater rRSCU for dsDNA phages (without tRNAs) than ssDNA phages. In addition, we propose that ssDNA phages have evolved a novel codon adaptation strategy to overcome the disruptive effect of their high C→T mutation rates in codon adaptation with host. In chapter three, we formulated an index phi to measure selection by host translation machinery and to present explicit linear and nonlinear models to characterize the effect of C→T mutation and host-tRNA-mediated selection on phage codon usage. The effect of selection (phi) on codon usage is detectable in most dsDNA and ssDNA phage species. C→T mutations also interfere with nonsynonymous substitutions at second codon positions, especially in ssDNA phages. Strand asymmetry along with the accompanying local variation in mutation bias can significantly affect codon adaptation in both dsDNA and ssDNA phages.

As antibiotic resistant bacterial strains are becoming more prevalent in healthcare settings, it is necessary to find alternative methods of detecting and treating these infections. One of the antibiotic resistant strains of interest is the carbapenem-resistant Enterobacteriaceae (CRE). CREs have the ability to evade some of the most potent antibiotics currently in use and employ carbapenemases to negate the effect of antibiotics. The three most common carbapenemase genes, found in carbapenem-resistant Enterobacteriaceae along with a gene found only in Escherichia coli were chosen to create a qPCR assay for rapid detection of resistant infections. The carbapenemase genes are KPC, VIM and NDM and the E. coli gene is uidA, a β-glucuronidase gene. Consensus sequences were obtained from each of the genes to account for the many variants of each gene. We were able to triplex the assay and test it against a library for twenty isolates varying by which gene they contain. Additional research has been conducted on the library of carbapenem-resistant Enterobacteriaceae using bacteriophages or phage. The Phage Hunters class isolated and identified twenty phage that infect K. pneumoniae. Out of the twenty phage, seven phage were able to effectively infect carbapenem-resistant K. pneumoniae.

Conventional wastewater treatment plants are able to reduce contaminant loads within regulations but do not take into account emerging contaminants. Antibiotic resistance genes and antibiotic resistant bacteria have been shown to survive wastewater treatment and remain detectable in effluents. The safety of treated wastewaters is crucial, otherwise unregulated and unmitigated emerging contaminants pose risks to public health and impede wastewater reuse. This dissertation aimed to further understanding of emerging microbial threats, and tested two natural and low-cost tools for their mitigation: sunlight, and bacteriophages. A wastewater bacterial isolate, named E. coli PI-7, which is highly antibiotic resistant, carries the novel antibiotic resistance gene New Delhi metallo-beta-lactamase NDM-1 gene, and displays pathogenic traits, was chosen to model responses to the treatments. Results found that solar irradiation was able to achieve a 5-log reduction in E. coli PI-7 numbers within 12 hours of exposure. However, the results also emphasized the risks from emerging microbial contaminants since E. coli PI-7, when compared with a non-pathogenic strain E. coli DSM1103 that has less antibiotic resistance, showed longer survival under solar irradiation. In certain instances, E. coli PI-7 persisted for over 6 hours before starting to inactivate, exhibited complex stress resistance gene responses, and activated many of its concerning pathogenicity and antibiotic resistance traits. However, upon solar irradiation, gene expression results obtained from both E. coli strains also showed increased susceptibility to bacteriophages. Hence, bacteriophages were coupled with solar irradiation as an additional mitigation strategy. Results using the coupled treatment found reduced cell-wall and extracellular matrix production in E. coli PI-7. DNA repair and other cellular defense functions like oxidative stress responses were also impeded, rendering E. coli PI-7 more susceptible to both stressors and successfully hastening the onset of its inactivation. Overall, the dissertation is built upon the need to develop strategies to further mitigate risks associated with emerging microbial contaminants. Solar irradiation and bacteriophages demonstrate potential as natural and low-cost mitigation strategies. Sunlight was able to achieve significant log-reductions in tested E. coli numbers within a day’s exposure. Bacteriophages were able to overwhelm E. coli PI-7’s capacity to resist solar inactivation while not affecting the indigenous microbiota.

Phages are the most abundant components of the marine environments and can control host abundances. The severity of viral infections may depend on whether phages are lytic, lysogenic, or chronic, which can be influenced by host activity and by environmental conditions. Lysogeny remains the least understood process. Knowledge of virioplankton dynamics and their life strategies in the Red Sea remain unexplored. In this Ph.D. research we aimed to quantify virioplankton abundance, the variability on viral and bacterial dynamics, and to investigate the occurrence of lytic and lysogenic phages in the Red Sea. Accordingly, we used the flow cytometric technique to enumerate viral and bacterial abundances in the coastal pelagic area during two years of sampling and in the coastal lagoon waters for one year, together with water column distribution in open Red Sea waters. We conducted incubations of natural microbial communities in the laboratory to induce lysogenic bacteria by using the chemical mutagenic mitomycin C. We also explored the influence of host abundance, temperature, and ultraviolet radiation on viral dynamics and lysogeny. Our results showed that abundances of virses in the Red Sea ranged from 106 to 107 virus-like particles per mL, and bacteria ranged from 104 to 105 cells per mL. We observed a large variability i the values of virus-to-bacterium ratios, and lower values of viral production to those for temperate coastal waters and relatively close to values reported in other oligotrophic areas. Although the lytic phase was prevalent, lysogeny was detected when bacterial abundances decreased. We determined inducible lysogenic bacteria from undetectable to ~56% in the coastal Red Sea, although we found a lower maximum of 29.1% at a eutrophic coastal lagoon. The decay rates of viruses were influenced by UVB exposure, suggesting their susceptibility to solar radiation. Exposure to UVB radiation-induced prophage varied between 4 and 34%. Our findings identified the significant role of viral infections in controlling bacterial abundance and the importance of both lytic and lysogenic phases in the Red Sea waters. This study contributes to the understanding of lysogeny in marine phages.

Bacterial viruses, also known as phages, are intrinsic components of the Earth’s Critical Zone (CZ). Together with diverse communities of bacteria and fungi, they occupy habitats of the CZ extending from the vegetation canopy, through the soils and into the aquifers. In this thesis, I aimed to study the transport of phages in the upper CZ and their interactions with non-host soil bacteria and fungi to reveal their role in regulating the CZ microbial life. To reflect transport processes of CZ-inhabiting phages in soil tracers are highly useful. Thus, in the first study, I evaluated the transport efficiency and particle intactness of marine tracer phages passing through soil. Marine phages were selected as tracer, because they are non-pathogenic, non-toxic, naturally absent and thus non-multiplying in the terrestrial subsurface. I found that the marine phages PSA-HM1 and PSA-HS2 retained high phage particle intactness in contrast to commonly used Escherichia virus T4. This suggests their potential as particle tracers to mimic the transport of (bio-) colloids of similar traits in soil. Soil in the CZ is often unsaturated and restricts mobility of microorganisms. Fungi bridge unsaturated zones in soil and hence provide network for microbial transport. In the second study, I developed a hyphosphere model system mimicking unsaturated soil environment, and reported on the ability of hyphal-riding bacteria to co-transport lytic phages and to utilize phages as “weapons” for improved colonization of water-unsaturated habitats. As the findings emphasize the importance of hyphal transport of bacteria and associated phages, in the third study, I developed a hyphae-assisted approach and isolated five soil bacteria able to co-transport phages. In analogy to invasion frameworks in macroecology, the hyphosphere model system with bacteria and co-transported phages can be useful models to simulate processes of biological invasion at micro-scale. In the fourth study, I investigated dormant phages (i.e. prophages) that are widespread in the CZ and can be induced under environmental stress. I found that volatile fungal metabolites can act as triggers for prophage induction and may exert long-distance manipulation of prophage activity thereby affecting microbial community and nutrient cycling in soil. Altogether, the findings may help to elucidate transport processes of phages in the CZ and to reveal the role of phages in the CZ microbial ecosystem. Approaches (e.g. phage as tracers) and findings (e.g. phage-bacterial co-transport) may also serve as useful tools for testing hypothesis in other disciplines, such as hydrogeology, invasion ecology and chemical ecology.