Michael E. Ford

Origins of Highly Mosaic Mycobacteriophage Genomes

Bacteriophages are the most abundant organisms in the biosphere and play major roles in the ecological balance of microbial life. The genomic sequences of ten newly isolated mycobacteriophages suggest that the bacteriophage population as a whole is amazingly diverse and may represent the largest unexplored reservoir of sequence information in the biosphere. Genomic comparison of these mycobacteriophages contributes to our understanding of the mechanisms of viral evolution and provides compelling evidence for the role of illegitimate recombination in horizontal genetic exchange. The promiscuity of these recombination events results in the inclusion of many unexpected genes including those implicated in mycobacterial latency, the cellular and immune responses to mycobacterial infections, and autoimmune diseases such as human lupus. While the role of phages as vehicles of toxin genes is well established, these observations suggest a much broader involvement of phages in bacterial virulence and the host response to bacterial infections.

Exploring the Mycobacteriophage Metaproteome: Phage Genomics as an Educational Platform

Bacteriophages are the most abundant forms of life in the biosphere and carry genomes characterized by high genetic diversity and mosaic architectures. The complete sequences of 30 mycobacteriophage genomes show them collectively to encode 101 tRNAs, three tmRNAs, and 3,357 proteins belonging to 1,536 “phamilies” of related sequences, and a statistical analysis predicts that these represent approximately 50% of the total number of phamilies in the mycobacteriophage population. These phamilies contain 2.19 proteins on average; more than half (774) of them contain just a single protein sequence. Only six phamilies have representatives in more than half of the 30 genomes, and only three—encoding tape-measure proteins, lysins, and minor tail proteins—are present in all 30 phages, although these phamilies are themselves highly modular, such that no single amino acid sequence element is present in all 30 mycobacteriophage genomes. Of the 1,536 phamilies, only 230 (15%) have amino acid sequence similarity to previously reported proteins, reflecting the enormous genetic diversity of the entire phage population. The abundance and diversity of phages, the simplicity of phage isolation, and the relatively small size of phage genomes support bacteriophage isolation and comparative genomic analysis as a highly suitable platform for discovery-based education.

The Generalized Transducing Salmonella Bacteriophage ES18: Complete Genome Sequence and DNA Packaging Strategy

The generalized transducing double-stranded DNA bacteriophage ES18 has an icosahedral head and a long noncontractile tail, and it infects both rough and smooth Salmonella enterica strains. We report here the complete 46,900-bp genome nucleotide sequence and provide an analysis of the sequence. Its 79 genes and their organization clearly show that ES18 is a member of the lambda-like (lambdoid) phage group; however, it contains a novel set of genes that program assembly of the virion head. Most of its integration-excision, immunity, Nin region, and lysis genes are nearly identical to those of the short-tailed Salmonella phage P22, while other early genes are nearly identical to Escherichia coli phages lambda and HK97, S. enterica phage ST64T, or a Shigella flexneri prophage. Some of the ES18 late genes are novel, while others are most closely related to phages HK97, lambda, or N15. Thus, the ES18 genome is mosaically related to other lambdoid phages, as is typical for all group members. Analysis of virion DNA showed that it is circularly permuted and about 10% terminally redundant and that initiation of DNA packaging series occurs across an approximately 1-kbp region rather than at a precise location on the genome. This supports a model in which ES18 terminase can move substantial distances along the DNA between recognition and cleavage of DNA destined to be packaged. Bioinformatic analysis of large terminase subunits shows that the different functional classes of phage-encoded terminases can usually be predicted from their amino acid sequence.

The pKO2 Linear Plasmid Prophage of Klebsiella oxytoca

Temperate bacteriophages with plasmid prophages are uncommon in nature, and of these only phages N15 and PY54 are known to have a linear plasmid prophage with closed hairpin telomeres. We report here the complete nucleotide sequence of the 51,601-bp Klebsiella oxytoca linear plasmid pKO2, and we demonstrate experimentally that it is also a prophage. We call this bacteriophage phiKO2. An analysis of the 64 predicted phiKO2 genes indicate that it is a fairly close relative of phage N15; they share a mosaic relationship that is typical of different members of double-stranded DNA tailed-phage groups. Although the head, tail shaft, and lysis genes are not recognizably homologous between these phages, other genes such as the plasmid partitioning, replicase, prophage repressor, and protelomerase genes (and their putative targets) are so similar that we predict that they must have nearly identical DNA binding specificities. The phiKO2 virion is unusual in that its phage lambda-like tails have an exceptionally long (3,433 amino acids) central tip tail fiber protein. The phiKO2 genome also carries putative homologues of bacterial dinI and umuD genes, both of which are involved in the host SOS response. We show that these divergently transcribed genes are regulated by LexA protein binding to a single target site that overlaps both promoters.

Genome organization and characterization of mycobacteriophage Bxb1

Mycobacteriophage Bxb1 is a temperate phage of Mycobacterium smegmatis. The morphology of Bxb1 particles is similar to that of mycobacteriophages L5 and D29, although Bxb1 differs from these phages in other respects. First, it is heteroimmune with L5 and efficiently forms plaques on an L5 lysogen. Secondly, it has a different host range and fails to infect slow‐growing mycobacteria, using a receptor system that is apparently different from that of L5 and D29. Thirdly, it is the first mycobacteriophage to be described that forms a large prominent halo around plaques on a lawn of M. smegmatis. The sequence of the Bxb1 genome shows that it possesses a similar overall organization to the genomes of L5 and D29 and shares weak but detectable DNA sequence similarity to these phages within the structural genes. However, Bxb1 uses a different system of integration and excision, a repressor with different specificity to that of L5 and encodes a large number of novel gene products including several with enzymatic functions that could degrade or modify the mycobacterial cell wall.

Complete genomic sequence of the virulent Salmonella bacteriophage SP6.

We report the complete genome sequence of enterobacteriophage SP6, which infects Salmonella enterica serovar Typhimurium. The genome contains 43,769 bp, including a 174-bp direct terminal repeat. The gene content and organization clearly place SP6 in the coliphage T7 group of phages, but there is approximately 5 kb at the right end of the genome that is not present in other members of the group, and the homologues of T7 genes 1.3 through 3 appear to have undergone an unusual reorganization. Sequence analysis identified 10 putative promoters for the SP6-encoded RNA polymerase and seven putative rho-independent terminators. The terminator following the gene encoding the major capsid subunit has a termination efficiency of about 50% with the SP6-encoded RNA polymerase. Phylogenetic analysis of phages related to SP6 provided clear evidence for horizontal exchange of sequences in the ancestry of these phages and clearly demarcated exchange boundaries; one of the recombination joints lies within the coding region for a phage exonuclease. Bioinformatic analysis of the SP6 sequence strongly suggested that DNA replication occurs in large part through a bidirectional mechanism, possibly with circular intermediates.

Corrected Sequence of the Bacteriophage P22 Genome

We report the first accurate genome sequence for bacteriophage P22, correcting a 0.14% error rate in previously determined sequences. DNA sequencing technology is now good enough that genomes of important model systems like P22 can be sequenced with essentially 100% accuracy with minimal investment of time and resources.

Pancreatic cholesterol esterase, ES-10, and fatty acid ethyl ester synthase III gene expression are increased in the pancreas and liver but not in the brain or heart with long-term ethanol feeding in rats.

Introduction Chronic alcohol consumption predisposes susceptible individuals to both acute and chronic pancreatitis. Aims Our hypothesis was that alcohol increases the risk of pancreatitis by disrupting defense mechanisms and/or enhancing injury-associated pathways through altered gene expression. Hence, we studied the expression of pancreatic genes in rats chronically exposed to ethanol. Methodology Male Wistar rats were pair-fed liquid diets without and with ethanol for 4 weeks. Total RNA was extracted from rat pancreas and other organs. The mRNA expression patterns among pancreatic samples from ethanol-fed rats and controls were compared with use of mRNA differential display. The differentially expressed cDNA tags were isolated, cloned, and sequenced. Results One cDNA tag that was overexpressed in the pancreas showed 99% sequence homology to a rat pancreatic cholesterol esterase mRNA (CEL; Enzyme Commission number [EC] 3.1.1.13). The differential expression was confirmed by realtime PCR. Gene expression was also increased in the liver but not in the heart or brain of the alcohol-fed rats. Because CEL has fatty acid ethyl ester (FAEE)-generating activity and FAEEs play a major role in acute alcoholic pancreatitis, we determined the expression of other genes encoding for FAEE-generating enzymes and showed similar organ-specific expression patterns. Conclusion Our results demonstrate that chronic ethanol consumption induced expression of FAEE-related genes in the pancreas and liver. This upregulation may be a central mechanism leading to acinar cell injury.

Chapter 12 – Evolutionary Relationships Among Diverse Bacteriophages and Prophages: All The World's a Phage

Publisher Summary This chapter discusses evolutionary relationships between diverse bacteriophages and prophages. The reported DNA and predicted protein sequence similarities, implying homology, between the genes of dsDNA bacteriophages and prophages spanning a broad phylogenetic range of host bacteria. By comparing the genomes of several newly characterized phages and cryptic prophages, it appears that the vast majority of dsDNA tailed-phages have common ancestry, and that they undergo profuse exchange of functional genetic elements drawn from a large shared pool. The classification of bacteriophages by their host range, morphology or available life-cycles has led to conflicting conclusions regarding their origins and evolution. Phages can be found in virtually all places where their bacterial hosts exist. As phages with near-identical genomes are rarely isolated from independent sources in nature, the term “species” is of limited use in describing relationships among phages. The complete genomic sequences of several phages closely related to phage lambda have been determined, and shown to have similar genomic organizations.

Analysis of the hereditary pancreatitis-associated cationic trypsinogen gene mutations in exons 2 and 3 by enzymatic mutation detection from a single 2.2-kb polymerase chain reaction product.

BACKGROUND Hereditary pancreatitis is associated with at least 2 mutations in the cationic trypsinogen gene. The purpose of the present study is to test the utility of T4 endonuclease VII for the detection of cationic trypsinogen R117H mutations. In addition, the possibility of screening for R117H, N21I, and A8V mutations in a single 2.2-kb polymerase chain reaction (PCR) product using T4 endonuclease VII was investigated. METHODS Twenty-nine DNAs from control patients and patients with known cationic trypsinogen R117H, A8V, or N21I mutations were selected from the ongoing hereditary pancreatitis study of the Midwest Multicenter Pancreatic Study Group. The samples were coded and randomized, and a 911-bp sequence containing exon 3 or a 2,212- bp sequence containing exons 2 and 3 were amplified by PCR using fluorescent- labeled primers. The PCR products were digested with T4 endonuclease VII and screened for mutations on an automated DNA sequencer. RESULTS In all cases with a mutation, a cleavage fragment on the direct and/or complementary DNA strand could easily be visualized, and its approximate size correlated with the predicted location of the known mutations within the PCR product. When the code for affected status was broken, there was 100% correlation between previous DNA sequence or restriction fragment length polymorphism findings and the T4 endonuclease VII digestion results for all 29 DNAs. CONCLUSION T4 endonuclease VII accurately identified the known cationic trypsinogen gene mutations in exons 2 and 3. Enzymatic mutation detection appears to be an accurate and useful method for screening individuals for known trypsinogen gene mutations and may be useful in identifying previously unidentified mutations within large regions of interest.