Isolation, characterization and functional analysis of a bacteriophage targeting Culex pipiens pallens resistance-associated Aeromonas hydrophila.

BACKGROUND: Culex pipiens pallens is a well-known mosquito vector for several diseases. Deltamethrin, a commonly used pyrethroid insecticide, has been frequently applied to manage adult Cx. pipiens pallens. However, mosquitoes can develop resistance to these insecticides as a result of insecticide misuse and, therefore, it is crucial to identify novel methods to control insecticide resistance. The relationship between commensal bacteria and vector resistance has been recently recognized. Bacteriophages (= phages) are effective tools by which to control insect commensal bacteria, but there have as yet been no studies using phages on adult mosquitoes. In this study, we isolated an Aeromonas phage vB AhM-LH that specifically targets resistance-associated symbiotic bacteria in mosquitoes. We investigated the impact of Aeromonas phage vB AhM-LH in an abundance of Aeromonas hydrophila in the gut of Cx. pipiens pallens and its effect on the status of deltamethrin resistance. METHODS: Phages were isolated on double-layer agar plates and their biological properties analyzed. Phage morphology was observed by transmission electron microscopy (TEM) after negative staining. The phage was then introduced into the mosquito intestines via oral feeding. The inhibitory effect of Aeromonas phage vB AhM-LH on Aeromonas hydrophila in mosquito intestines was assessed through quantitative real-time PCR analysis. Deltamethrin resistance of mosquitoes was assessed using WHO bottle bioassays. RESULTS: An Aeromonas phage vB AhM-LH was isolated from sewage and identified as belonging to the Myoviridae family in the order Caudovirales using TEM. Based on biological characteristics analysis and in vitro antibacterial experiments, Aeromonas phage vB AhM-LH was observed to exhibit excellent stability and effective bactericidal activity. Sequencing revealed that the Aeromonas phage vB AhM-LH genome comprises 43,663 bp (51.6% CG content) with 81 predicted open reading frames. No integrase-related gene was detected in the vB AH-LH genome, which marked it as a potential biological antibacterial. Finally, we found that Aeromonas phage vB AhM-LH could significantly reduce deltamethrin resistance in Cx. pipiens pallens, in both the laboratory and field settings, by decreasing the abundance of Aeromonas hydrophila in their midgut. CONCLUSIONS: Our findings demonstrate that Aeromonas phage vB AhM-LH could effectively modulate commensal bacteria Aeromonas hydrophila in adult mosquitoes, thus representing a promising strategy to mitigate mosquito vector resistance.

Phenotypic and Genetic Characterization of Aeromonas hydrophila Phage AhMtk13a and Evaluation of Its Therapeutic Potential on Simulated Aeromonas Infection in Danio rerio.

Phage therapy can be an effective alternative to standard antimicrobial chemotherapy for control of Aeromonas hydrophila infections in aquaculture. Aeromonas hydrophila-specific phages AhMtk13a and AhMtk13b were studied for basic biological properties and genome characteristics. Phage AhMtk13a (Myovirus, 163,879 bp genome, 41.21% CG content) was selected based on broad lytic spectrum and physiologic parameters indicating its lytic nature. The therapeutic potential of phage AhMtk13a was evaluated in experimental studies in zebrafish challenged with A. hydrophila GW3-10 via intraperitoneal injection and passive immersion in aquaria water. In experimental series 1 with single introduction of AhMtk13a phage to aquaria water at phage-bacteria ratio 10:1, cumulative mortality 44% and 62% was registered in fish exposed to phage immediately and in 4 h after bacterial challenge, correspondingly, compared to 78% mortality in the group with no added phage. In experimental series 2 with triple application of AhMtk13a phage at ratio 100:1, the mortality comprised 15% in phage-treated group compared to the 55% in the control group. Aeromonas hydrophila GW3-10 was not detectable in aquaria water from day 9 but still present in fish at low concentration. AhMtk13a phage was maintained in fish and water throughout the experiment at the higher concentration in infected fish.

Molecular Characterization of Ahp2, a Lytic Bacteriophage of Aeromonas hydrophila.

Aeromonas hydrophila is an opportunistic pathogen that infects fish, amphibians, mammals, and humans. This study isolated a myophage, vB_AhyM_Ahp2 (Ahp2), that lytically infects A. hydrophila. We observed that 96% of the Ahp2 particles adsorbed to A. hydrophila within 18 min. Ahp2 also showed a latent period of 15 min with a burst size of 142 PFU/cell. This phage has a linear double-stranded DNA genome of 47,331 bp with a GC content of 57%. At least 20 Ahp2 proteins were detected by SDS-polyacrylamide gel electrophoresis; among them, a 40-kDa protein was predicted as the major capsid protein. Sequence analysis showed that Ahp2 has a genome organization closely related to a group of Aeromonas phages (13AhydR10RR, 14AhydR10RR, 85AhydR10RR, phage 3, 32 Asp37, 59.1), which infect Aeromonas hydrophila and Aeromonas salmonicida. The tail module encompassing ORF27-29 in the Ahp2 genome was present in all Aeromonas phages analyzed in this study and likely determines the host range of the virus. This study found that Ahp2 completely lyses A. hydrophila AH300206 in 3.5 h at a MOI of 0.0001 and does not lysogenize its host. Altogether, these findings show that Ahp2 is a lytic Aeromonas phage and could be a candidate for therapeutic phage cocktails.

Isolation, characterization and genomic analysis of vB-AhyM-AP1, a lytic bacteriophage infecting Aeromonas hydrophila.

AIMS: Aeromonas hydrophila is a zoonotic pathogen displaying resistance to multiple antibiotics. Here, we aim to develop a candidate biocontrol agent against A. hydrophila. METHODS AND RESULTS: In this study, we isolated and characterized the phage vB-AhyM-AP1 from sewage. It showed lytic activity against A. hydrophila strains. One-step growth curve revealed that the latent period lasted for 40 min. The burst size of one lytic cycle was 1413 PFU per infected cell. Temperature stability studies showed that the phage vB-AhyM-AP1 was active over temperatures ranging from 4 to 45°C for 1 h. pH stability studies indicated that the phage remained active within a pH range of 5-10 after 24 h of incubation. Stability tests in salt solutions showed that the phage was stable at salinities ranging from 0·1 to 2%. The phage also showed stabilities in organic solvents when incubated for 10 min. The Illumina Hiseq sequencing of its genome indicated that the phage vB-AhyM-AP1was a jumbo phage with a genome size of 2, 54 490 bp and GC content of 40·3%. The phylogenetic analysis of the terminase large subunit and major capsid protein indicated that the phage closely clustered with other Tevenvirinae phages. The genome encoded 455 ORFs and 22 tRNAs. The phage resulted in a reduction of 0·8 log units of viable A. hydrophila cells in biofilms grown on PVC coupons maintained in a low nutrient medium for 10 days. CONCLUSIONS: The phage showed lytic activity against planktonic and biofilm cells of A. hydrophila. Genome-based prediction showed it to be a strictly lytic phage without any virulence or antibiotic resistance genes indicating safety for environmental and clinical applications. SIGNIFICANCE AND IMPACT OF THE STUDY: The multidrug-resistant strains of A. hydrophila pose a significant health risk to both cultured fishes and consumers leaving few options for treatment. Phage vB-AhyM-AP1 may be used as a candidate biocontrol agent against A. hydrophila strains.

Use of Bacteriophage to Control Experimental Aeromonas hydrophila Infection in Tilapia (Oreochromis niloticus).

BACKGROUND AND OBJECTIVE: Antibiotics have been used to treat Aeromonas hydrophila infections in fish farming. However, their extensive uses can cause many negative effects including the development of drug-resistant bacterial strains. The main objective of this study was to find an alternative to antibiotics to inhibit A. hydrophila both in vitro and in vivo. MATERIALS AND METHODS: A bacteriophage infecting A. hydrophila was isolated from a fish a pond water sample. It was classified based on its genome type studied by enzymatic digestion and morphology investigated by transmission electron microscopy. Its ability to control experimental A. hydrophila infection in tilapia (Oreochromis niloticus) was examined by feeding tilapia with fish diets supplemented with different titers of the bacteriophage. RESULTS: A bacteriophage specific to Aeromonas hydrophila UR1 designated PAh4 was isolated and classified as a member of the family Myoviridae. When tilapia experimentally infected with A. hydrophila at the median lethal dose (3.16×105 CFU per fish) were fed the fish diets supplemented with the bacteriophage PAh4 at doses ranging from 105-108 PFU g-1 of diet, the diets could reduce the mortality rate of infected tilapia in a dose-dependent manner. CONCLUSION: The bacteriophage PAh4 can be used as an alternative to antibiotics to control A. hydrophila infection in tilapia.

Complete genome sequence of a novel lytic phage infecting Aeromonas hydrophila, an infectious agent in striped catfish (Pangasianodon hypophthalmus).

The bacteriophage vB_AhM_PVN02 (PVN02), infecting Aeromonas hydrophila, was isolated from a striped catfish pond water sample in Can Tho City, Vietnam. The phage had high lytic activity with a latent period and burst size of approximately 20 min and 105 plaque-forming units per cell, respectively. Observation of the phage by transmission electron microscopy indicated that PVN02 belongs to the family Myoviridae. The genome of PVN02 is a double-stranded linear DNA with a length in 51,668 bp and a content of 52% GC. Among the 64 genes, 16 were predicted to encode proteins with predicted functions. No virulence or antibiotic resistance genes were found in the genome, suggesting it would be a useful biocontrol agent. Classification of the phage based on sequence comparisons, phylogenetic analysis, and gene-sharing networks was carried out, and it was found to be the first representative of a new species within a previously undefined genus in the family Myoviridae. This study confirmed that PVN02 is a novel lytic phage that could potentially be used as an agent to control Aeromonas hydrophila in striped catfish in the Mekong Delta, Vietnam.

Isolation and characterization of bacteriophages against virulent Aeromonas hydrophila.

BACKGROUND: Aeromonas hydrophila is an important water-borne pathogen that leads to a great economic loss in aquaculture. Along with the abuse of antibiotics, drug-resistant strains rise rapidly. In addition, the biofilms formed by this bacterium limited the antibacterial effect of antibiotics. Bacteriophages have been attracting increasing attention as a potential alternative to antibiotics against bacterial infections. RESULTS: Five phages against pathogenic A. hydrophila, named N21, W3, G65, Y71 and Y81, were isolated. Morphological analysis by transmission electron microscopy revealed that phages N21, W3 and G65 belong to the family Myoviridae, while Y71 and Y81 belong to the Podoviridae. These phages were found to have broad host spectra, short latent periods and normal burst sizes. They were sensitive to high temperature but had a wide adaptability to the pH. In addition, the phages G65 and Y81 showed considerable bacterial killing effect and potential in preventing formation of A. hydrophila biofilm; and the phages G65, W3 and N21 were able to scavenge mature biofilm effectively. Phage treatments applied to the pathogenic A. hydrophila in mice model resulted in a significantly decreased bacterial loads in tissues. CONCLUSIONS: Five A. hydrophila phages were isolated with broad host ranges, low latent periods, and wide pH and thermal tolerance. And the phages exhibited varying abilities in controlling A. hydrophila infection. This work presents promising data supporting the future use of phage therapy.

Characterization and genome sequencing of three Aeromonas hydrophila-specific phages, CF8, PS1, and PS2.

Aeromonas hydrophila is an important finfish pathogen, besides being an opportunistic human pathogen. In the present study, the genomes of three A. hydrophila-specific phages, CF8, PS1, and PS2, were isolated, characterized and sequenced. Transmission electron microscopy showed that all three phages had typical Myoviridae morphology. The linear dsDNA genomes of CF8, PS1, and PS2 were 238,150 bp, 237,367 bp, and 240,447 bp in length, with a GC content of 42.2%, 38.8%, and 38.8%, respectively. The low sequence similarity (67.6% - 69.8% identity with 27.0% - 29.0% query coverage) to other phage genomes in the NCBI database indicated the novel nature of the CF8, PS1, and PS2 genomes. A total of 244, 247, and 250 open reading frames (ORFs) were predicted in the CF8, PS1, and PS2 genome, respectively. During the annotation process, functional predictions were made for 28-31 ORFs, while the rest were classified as "hypothetical proteins" with yet unknown functions. Genes for tRNAs were also detected in all phage genomes. As all three phages in the present study had a very narrow host range with lytic activity against only one strain of A. hydrophila, these phages could be good candidates for phage typing applications. Moreover, the endolysin- and lytic-transglycosylase-encoding genes could be used for recombinant cloning and expression of anti-microbial proteins.

Isolation and characterization of phage AHP-1 and its combined effect with chloramphenicol to control Aeromonas hydrophila.

To develop an alternative bio-control measure for multi-drug resistant pathogenic Aeromonas hydrophila, which causes motile Aeromonas septicemia in fish, novel virulent phage (AHP-1) was isolated from carp tissues. Morphological analysis by transmission electron microscopy revealed that AHP-1 belongs to Myoviridae family. AHP-1 displayed 81% of moderate adsorption by 25 min, and latent period of 40 min with burst size of 97 PFU mL-1 at an optimal multiplicity of infection (MOI) 0.1. AHP-1 was stable over a broad range of pH (4-11), temperature (4-50 °C), and salinity (0.1-3.5%). Both time and MOI dependent in vitro A. hydrophila growth inhibition was observed with AHP-1. AHP-1 (10 MOI) showed higher growth inhibition against A. hydrophila than chloramphenicol (5 µg mL-1), and combined treatment was more promising than individuals. Immune gene expression analysis of zebrafish upon continuous bath exposure to AHP-1 resulted significantly higher (il-6 and sod-1) or slight induction (tnf-α, il1-ß, il-10, and cxcl-8a) than controls at beginning of the phage exposure, but those lowered to basal level by day 12 post-phage exposure. It suggests no adverse immune responses have occurred for the AHP-1 dose that used, and have potential for the phage therapy. Further detailed in vivo studies are needed to confirm the protective efficacy of newly isolated AHP-1 against A. hydrophila infection.

Genomic characterization of a novel virulent phage infecting the Aeromonas hydrophila isolated from rainbow trout (Oncorhynchus mykiss).

The virulent bacteriophage MJG that specifically infects Aeromonas hydrophila was isolated from a water sample from a river in Harbin, China. The genome of phage MJG was a double-stranded linear DNA with 45,057 bp, possessing 50.11% GC content. No virulence or resistance genes were found in the phage genome. Morphological observation, genomic characterization, and phylogenetic analysis indicated that MJG was closely related to phages belonging to the genus Sp6virus in the Podoviridae family. This phage is a novel member within Sp6virus that could infect and lyse A. hydrophila. This study could serve as a genomic reference of A. hydrophila phages and provide a potential agent for phage therapy.

Selection of Phages to Control Aeromonas hydrophila - An Infectious Agent in Striped Catfish.

Striped catfish (Pangasianodon hypophthalmus) farming in the Mekong Delta Vietnam (MKDVN) importantly contributes to national aquaculture export. Currently, however, diseases occur more frequently across the entire MKDVN region. One of the most common types is hemorrhagic septicemia caused by Aeromonas hydrophila. In this study, isolation and selection of the phages for control in vitro Aeromonas hydrophila were conducted. 24 phages were isolated from 100 striped catfish pond water samples. Next, lytic activity of these phages was clarified. Four phages with short latent period (about 25 to 40 min) and/or high burst size (about 67 to 94 PFU/ cell) were selected to evaluate their infection activity to different phage-resistant A. hydrophila strains. Two phages termed as TG25P and CT45P were subjected to the phage cocktail to inactivate A. hydrophila. Re-growth of the host bacteria appeared about eight hours after treatment. Usage of the phage cocktail that attach different host bacterial receptors is not always much effective than usage of single phage. This is the first report about phage therapy to control A. hydrophila isolated from striped catfish. Some challenges in the phage cocktail were shown to achieve strategies in prospective studies in the context of high antibiotic resistance of A. hydrophila.

Immunostimulation of Cyprinus carpio using phage lysate of Aeromonas hydrophila.

Over the last 50 years, various approaches have been established for the development of antigens for immunostimulation. We used phage lysate (PL), composed of inactivated antigens by the lytic bacteriophage pAh 6-c for Aeromonas hydrophila JUNAH strain to develop a vaccine for the prevention of A. hydrophila infection in Cyprinus carpio (common carp). We also assessed the poly D,L lactide-co-glycolic acid (PLGA) microparticles encapsulation method to increase the efficiency of the vaccine. Six groups of vaccines involving encapsulated by PLGA, formalin killed cells, or phage lysate at low or high concentration were prepared for intraperitoneal injection in C. carpio. Blood specimens and head kidney samples were collected at various time points for bacterial agglutination assay and to assess relative expression of immune-related genes interleukin-1 beta (IL-1ß), tumor necrosis factor alpha (TNF-α), lysozyme C, and serum amyloid A (SAA). The vaccine groups using high dose phage lysate antigen showed significantly higher agglutination titers than all other groups at 4- and 6-weeks post vaccination (wpv), with the titer of the PLGA encapsulated vaccine group being highest from 10 wpv to the end of the experiment. The survival rate of fish immunized with the phage lysate vaccines were higher than that of fish immunized with the formailin killed cells vaccine in the challenge experiment conducted 6 wpv. Additionally, the PLGA-encapsulated high dose phage lysate antigen vaccinated groups showed the best protective efficacy in the challenge experiment 12 wpv. Vaccines using the phage lysate antigen also showed higher IL-1ß and lysozyme C gene expression at 7 days post vaccination (dpv) and 2 wpv, and higher TNF-α gene expression was seen at 7 dpv. Higher SAA gene expression was seen in these groups at 1 dpv. These results suggest that phage lysate antigen has the potential to induce robust immune responses than formalin killed cells-based vaccines, and could be more effective as a novel inactivated antigen in preventing A. hydrophila infection in C. carpio.

Characterization and genomic analyses of Aeromonas hydrophila phages AhSzq-1 and AhSzw-1, isolates representing new species within the T5virus genus.

In this study, two bacteriophage isolates, AhSzq-1 and AhSzw-1 that specifically infect Aeromonas hydrophila strain KT998822, were isolated from seawater and characterized. One-step growth curves showed that the latent period of AhSzq-1 and AhSzw-1 are 50 min and 60 min, respectively. The sequence similarities between AhSzq-1 and AhSzw-1 were 88% at the DNA and 83% at the protein level, suggesting that these two phages are representatives of two different species. The virion morphology, DNA genome size and terminal repeats of these two phages are similar to those of viruses classified as T5virus phages. Both phylogenetic analyses and proteomic comparison show that AhSzq-1 and AhSzw-1 group with members of the T5virus genus. We thus propose these two phages as representative isolates of two new species within the T5virus genus.

Genomic Characterization of the Novel Aeromonas hydrophila Phage Ahp1 Suggests the Derivation of a New Subgroup from phiKMV-Like Family.

Aeromonas hydrophila is an opportunistic pathogenic bacterium causing diseases in human and fish. The emergence of multidrug-resistant A. hydrophila isolates has been increasing in recent years. In this study, we have isolated a novel virulent podophage of A. hydrophila, designated as Ahp1, from waste water. Ahp1 has a rapid adsorption (96% adsorbed in 2 min), a latent period of 15 min, and a burst size of 112 PFU per infected cell. At least eighteen Ahp1 virion proteins were visualized in SDS-polyacrylamide gel electrophoresis, with a 36-kDa protein being the predicted major capsid protein. Genome analysis of Ahp1 revealed a linear doubled-stranded DNA genome of 42,167 bp with a G + C content of 58.8%. The genome encodes 46 putative open reading frames, 5 putative phage promoters, and 3 transcriptional terminators. Based on high degrees of similarity in overall genome organization and among most of the corresponding ORFs, as well as phylogenetic relatedness among their DNAP, RNAP and major capsid proteins, we propose a new subgroup, designated Ahp1-like subgroup. This subgroup contains Ahp1 and members previously belonging to phiKMV-like subgroup, phiAS7, phi80-18, GAP227, phiR8-01, and ISAO8. Since Ahp1 has a narrow host range, for effective phage therapy, different phages are needed for preparation of cocktails that are capable of killing the heterogeneous A. hydrophila strains.

Isolation of a lytic bacteriophage against virulent Aeromonas hydrophila from an organized equine farm.

A bacteriophage (VTCCBPA6) against a pathogenic strain of Aeromonas hydrophila was isolated from the sewage of an organized equine breeding farm. On the basis of TEM analysis, phage belonged to family Myoviridae. PCR amplification and sequence analysis of gp23 gene (encoding for major capsid protein) revealed phylogenetic resemblance to T4 like virus genus. Protein profiling by SDS-PAGE also indicated its resemblance to T4 like phage group. However, the comparison of its gp23 gene sequence with previously reported phages showed similarity with T4-like phages infecting Enterobacteriaceae instead of Aeromonas spp. Thus, to our knowledge, this report points toward the fact that a novel/evolved phage might exist in equine environment against A. hydrophila, which can be potentially used as a biocontrol agent.

Implication of lateral genetic transfer in the emergence of Aeromonas hydrophila isolates of epidemic outbreaks in channel catfish.

To investigate the molecular basis of the emergence of Aeromonas hydrophila responsible for an epidemic outbreak of motile aeromonad septicemia of catfish in the Southeastern United States, we sequenced 11 A. hydrophila isolates that includes five reference and six recent epidemic isolates. Comparative genomics revealed that recent epidemic A. hydrophila isolates are highly clonal, whereas reference isolates are greatly diverse. We identified 55 epidemic-associated genetic regions with 313 predicted genes that are present in epidemic isolates but absent from reference isolates and 35% of these regions are located within genomic islands, suggesting their acquisition through lateral gene transfer. The epidemic-associated regions encode predicted prophage elements, pathogenicity islands, metabolic islands, fitness islands and genes of unknown functions, and 34 of the genes encoded in these regions were predicted as virulence factors. We found two pilus biogenesis gene clusters encoded within predicted pathogenicity islands. A functional metabolic island that encodes a complete pathway for myo-inositol catabolism was evident by the ability of epidemic A. hydrophila isolates to use myo-inositol as a sole carbon source. Testing of A. hydrophila field isolates found a consistent correlation between myo-inositol utilization as a sole carbon source and the presence of an epidemic-specific genetic marker. All epidemic isolates and one reference isolate shared a novel O-antigen cluster. Altogether we identified four different O-antigen biosynthesis gene clusters within the 11 sequenced A. hydrophila genomes. Our study reveals new insights into the evolutionary changes that have resulted in the emergence of recent epidemic A. hydrophila strains.

Complete Genome Sequence of Aeromonas hydrophila Phage CC2.

Aeromonas hydrophila is one of the major pathogenic bacteria for fish and people. To develop an effective antimicrobial agent, we isolated a bacteriophage from sewage, named CC2, and sequenced its genome. Comparative genome analysis of phage CC2 with its relatives revealed that phage CC2 has higher sequence homology to A. salmonicida phage 65 than to A. hydrophila phage Aeh1. Here, we announce the complete genome sequence of CC2 and report major findings from the genomic analysis.

An RNA hairpin sequesters the ribosome binding site of the homing endonuclease mobE gene.

Previous transcript mapping of the bacteriophage Aeh1 nrd operon revealed a predicted RNA hairpin upstream of the homing endonuclease mobE gene. We enzymatically mapped the hairpin, showing that the mobE ribosome binding site is sequestered. Cloning of the hairpin upstream of lacZ resulted in reduced beta-galactosidase activity, consistent with translational regulation.

Multiple controls regulate the expression of mobE, an HNH homing endonuclease gene embedded within a ribonucleotide reductase gene of phage Aeh1.

Mobile genetic elements have the potential to influence the expression of genes surrounding their insertion site upon invasion of a genome. Here, we examine the transcriptional organization of a ribonucleotide reductase operon (nrd) that has been invaded by an HNH family homing endonuclease, mobE. In Aeromonas hydrophila phage Aeh1, mobE has inserted into the large-subunit gene (nrdA) of aerobic ribonucleotide reductase (RNR), splitting it into two smaller genes, nrdA-a and nrdA-b. This gene organization differs from that in phages T4, T6, RB2, RB3, RB15, and LZ7, where mobE is inserted in the nrdA-nrdB intergenic region. We present evidence that the expression of Aeh1 mobE is regulated by transcriptional, posttranscriptional, and translational controls. An Aeh1-specific late promoter drives expression of mobE, but strikingly the mobE transcript is processed internally at an RNase E-like site. We also identified a putative stem-loop structure upstream of mobE that sequesters the mobE ribosome binding site, presumably acting to down regulate MobE translation. Moreover, our transcriptional analyses indicate that the surrounding nrd genes of phage Aeh1 are expressed by a different strategy than are the corresponding phage T4 genes and that transcriptional readthrough is the only mechanism by which the promoterless Aeh1 nrdB gene is expressed. We suggest that the occurrence of multiple layers of control to limit the expression of mobE to late in the Aeh1 infection cycle is an adaptation of Aeh1 to reduce any effects on expression of the surrounding nrd genes early in phage infection when RNR function is critical.