University of Hertfordshire Hatfield, Hertfordshire, UK
For further volumes: http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexed in PubMed.
Bacteriophages
Methods and Protocols
Edited by Ebenezer Tumban
School of Veterinary Medicine, Texas Tech University, Amarillo, TX, USA
Editor
Ebenezer Tumban
School
of Veterinary Medicine
Texas Tech University
Amarillo, TX, USA
ISSN 1064-3745ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-0716-3548-3ISBN 978-1-0716-3549-0 (eBook) https://doi.org/10.1007/978-1-0716-3549-0
This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use.
The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature.
The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.
Paper in this product is recyclable.
Preface
Many bacteriophages or phages (also known as viruses that infect bacteria) have been identified over the last seven decades. Some of these bacteriophages have been identified using metagenomics approaches (a lot is not known about these bacteriophages), while others have been isolated/propagated in culture. They have been isolated from bacteria that grow at normal conditions (temperature, pH, salinity) as well as from those that grow in extreme environments such as high or low temperatures, low or high pH, high or low salinity, etc. Many of these bacteriophages have been characterized; we now know the structures, genetic make-up/sizes, as well as the functions of most genes in bacteriophages that have been isolated up to date. Some of these genes, including bacteriophages in general, have been exploited to develop novel products/tools for many applications in biomedical science. This volume is not intended to cover uncharacterized bacteriophages nor provide protocols for the isolation of bacteriophages from all possible environments given the fact that the protocols are similar regardless of the sources of samples; the book provides protocols for the isolation and enumeration of bacteriophages from different types of samples as well as sequencing, and characterizing their genomes. The book concludes with a summary on the use of bacteriophages/bacteriophage-derived products in biomedical sciences.
This volume begins with an overview of the diversity of bacteriophages in terms of size, structure and morphology, genomic make-up and sizes, as well as the diversity of host ranges (Chap. 1 by Bert Ely and colleagues). The chapter is followed by reviews on the diversity of bacteriophages in the human gut (Chap. 2 by Amanda Carroll-Portillo and colleagues), oceans/polar regions (Chap. 3 by Janina Rahlff and colleagues), and in hot springs (Chap. 4 by Timothy Marks and colleague).
The second part of the book focuses on protocols for the isolation of bacteriophages for characterization. As mentioned above, bacteriophages are present wherever their bacterial host may be present. Some of these may be in remote areas with limited resources. Chapter 5 (by Ayesha Hassim and colleague) describes a protocol to isolate bacteriophages from soil sample in a poorly equipped lab in Kruger National Park. Chapter 6 (by Dennis Nielsen et colleagues) presents a protocol to separate and concentrate bacteriophages and other viruses from fecal samples, which can be used to isolate bacteriophages. It is worth mentioning that, at times, bacteriophages of interest (to be isolated) may be very low in sample source (for example water), thus making it very challenging to isolate rare bacteriophages. Chapter 7 (by Jacques Mathieu and colleagues) outlines a protocol to isolate rare bacteriophages using an enrichment step. The protocol also highlights strategies that make the isolation of larger or jumbo phages as well as lipid-containing phages inclusive compared to traditional isolation protocols, which exclude jumbo phages and inactivate lipid-containing phages. Following the isolation of bacteriophage(s), their genomic sequence(s) have to be sequenced for further characterization. Chapter 8 (by Jason Gill and colleagues) delineates a protocol to determine the complete sequence of a bacteriophage genome from DNA extraction to the assembly of sequence reads.
The third part of the book is devoted to enumeration of bacteriophages in a sample. Chapters 9 and 10 (by Diana E. Waturangi and by Lorena Rodriguez Rubio & colleagues) describe a simple and efficient method, plaque assay, to quantify bacteriophages in a sample. In addition to this, Chap. 9 provides strategies to make difficult-to-see small plaques, especially those formed by from jumbo bacteriophages, more visible. Chapter 10 (by Lorena Rodriguez Rubio and colleagues) goes further to describe how to quantify bacteriophages using molecular techniques such as real-time polymerase chain reaction; this technique has an advantage over plaque assay in quantifying bacteriophages in samples that have not yet been isolated/cultured. Chapter 11 (by Elena A. Dlusskaya and colleague) presents a flowcytometer-based approach (virometry) to calculate the concentration of bacteriophages in a sample. Last but not the least, Chap. 12 (by Douwe van Sinderen and colleagues) reports a metagenomics approach to enumerate the diversity of bacteriophages in an environment (specifically in fermentation settings) with a virome extraction method that is tailored towards bacteriophages.
The fourth part of the book is dedicated to the characterization of bacteriophages. After determining the sequences of isolated bacteriophages, described in early chapters (Chaps. 8 and 12), the genomes of the phages have to be annotated and their evolutionary relations have to be analyzed in order to determine open reading frames and their putative functions. Chapter 13 (by He ´ ctor Ricardo Morbidoni and colleague) describes a protocol to annotate and determine the phylogenic relation of a bacteriophage. Chapter 14 (by Gino Cingolani and colleagues) details a protocol (localized reconstruction) to reconstruct and visualize cryogenic-electron microscopy images of the tail of a bacteriophage. Chapter 15 (by Andrea I. Moreno-Switt and colleagues) provides an overview of how molecular interactions of bacteriophages with their bacterial hosts lead to resistance and counter-resistance and consequently, co-evolution. In the following chapter (Chap. 16), Sittinan Chanarat and colleagues present a protocol to assess physical interactions between a bacterial host and its bacteriophage/bacteriophage-derived proteins. Knowledge from such studies can be used to choose bacteriophages (for phage therapy) and/or to develop phage-derived antimicrobial enzymes that are tailored towards specific bacteria without the fear of lysogeny. Chapter 17 (by John Chen and colleague) outlines a protocol to transfer a plasmid or chromosomal DNA to a bacterium using a bacteriophage. This technology can be used to introduce foreign genetic elements to bacterial strains, which normally are not competent under natural conditions, in order to assess gain of functions such as antibiotic resistance, virulence, and phage therapy resistance.
The fifth part of the book highlights the application of bacteriophages and bacteriophage-derived components in biomedical sciences. Bacteriophages have a plethora of applications in biomedical sciences. Chapter 18 (by Alaa A A Aljabadli and colleagues) reviews the literature on using bacteriophages to deliver therapeutics to specific cells and bacteria. Chapter 19 (by Jianmin Gao and colleagues) provides a technique, using a cysteine cross-linker, to make a bacteriophage library displaying non-natural cyclic peptides. Chapter 20 (by Johan Robben and colleagues) describes a method to apply deep sequencing to assess the quality of bacteriophage protein libraries and enrichments during phage display. Chapter 21 (by John Aviator and colleagues) delineates a method to detect/identify bacteria in a solution or blood samples using bacteriophage and photoacoustic flow cytometry. This approach does not require bacteria to be cultured nor its nucleic acid to be amplified. The
subsequentchapter(Chap. by Alicja Wegrzyn and colleagues) outlines a protocol to isolate,propagate,andcharacterize a bacteriophage library for phage therapy, as well as assessthetherapeuticefficacy of phages/cocktail in an insect model. Chapter (by Elina Laanto)providesanoverview on how bacteriophage resistance in bacteriophage therapy can beovercome.Thebook concludes with Chap. (by Ebenezer Tumban). The chapter providesasummaryonthe application of virus-like particles, derived from bacteriophages, forvaccinedesign.
TX, USA
Amarillo,
Ebenezer Tumban
PART IAN OVERVIEW OF THE DIVERSITY OF
1 Structural and Genomic Diversity of Bacteriophages
Bert Ely, Jacob Lenski, and Tannaz Mohammadi
2 The Diversity of Bacteriophages in the Human Gut
Amanda Carroll-Portillo, Derek M. Lin, and Henry C. Lin
3 Breaking the Ice: A Review of Phages in Polar Ecosystems
Mara Elena Heinrichs, Gonc¸alo J. Piedade, Ovidiu Popa, Pacifica Sommers, Gareth Trubl, Julia Weissenbach, and Janina Rahlff
4 The Diversity of Bacteriophages in Hot Springs
Timothy J. Marks and Isabella R. Rowland
PART II ISOLATION OF BACTERIOPHAGES
5 Isolation of Bacteriophages from Soil Samples in a Poorly Equipped Field Laboratory in Kruger National Park
Ayesha Hassim and Kgaugelo Edward Lekota
6 Purification and Up-Concentration of Bacteriophages and Viruses from Fecal Samples
Frej Larsen, Rasmus Riemer Jakobsen, Xiaotian Mao, Josue Castro-Mejia, Ling Deng, and Dennis S. Nielsen
7 Isolation of Enterococcus Bacteriophages from Municipal Wastewater Samples Using an Enrichment Step
Cory Schwarz and Jacques Mathieu
8 Phage DNA Extraction, Genome Assembly, and Genome Closure
Justin Boeckman, Mei Liu, Jolene Ramsey, and Jason Gill
PART III ENUMERATION OF BACTERIOPHAGES
9 Enumeration of Bacteriophages by Plaque Assay
Diana Elizabeth Waturangi
10 Detection and Quantification of Bacteriophages in Wastewater Samples by Culture and Molecular Methods
Laura Sala-Comorera, Maite Muniesa, and Lorena Rodrı ´ guez-Rubio
11 Flow Virometry: A Fluorescence-Based Approach to Enumerate Bacteriophages in Liquid Samples .
Elena A. Dlusskaya and Rafik Dey
12 A Metagenomics Approach to Enumerate Bacteriophages in a Food Niche 185
Kelsey White, Giovanni Eraclio, Gabriele Andrea Lugli, Marco Ventura, Jennifer Mahony, Fabio Dal Bello, and Douwe van Sinderen
PART IV CHARACTERIZATION OF BACTERIOPHAGES
13 Bioinformatic Analysis of Staphylococcus Phages: A Key Step for Safe Cocktail Development 203 Soledad Telma Carrasco and He´ctor Ricardo Morbidoni
14 Use of Localized Reconstruction to Visualize the Shigella Phage Sf6 Tail Apparatus
Chun-Feng David Hou, Fenglin Li, Stephano Iglesias, and Gino Cingolani
15 Bacteriophage–Host Interactions and Coevolution
Diana M. A ´ lvarez-Espejo, Da ´ cil Rivera, and Andrea I. Moreno-Switt
16 Unraveling Physical Interactions of Clostridioides difficile with Phage and Phage-Derived Proteins Using In Vitro and Whole-Cell Assays
Wichuda Phothichaisri, Tanaporn Phetruen, Surang Chankhamhaengdecha, Tavan Janvilisri, Puey Ounjai, Robert P. Fagan, and Sittinan Chanarat
17 Phage Transduction of Staphylococcus aureus
Melissa-Jane Chu Yuan Kee and John Chen
PART VAPPLICATION OF BACTERIOPHAGES AND BACTERIOPHAGE-DERIVED COMPONENTS
18 The Next Generation of Drug Delivery: Harnessing the Power of Bacteriophages
Alaa A. A. Aljabali, Mohammad B. M. Aljbaly, Mohammad A. Obeid, Seyed Hossein Shahcheraghi, and Murtaza M. Tambuwala
19 Construction of Nonnatural Cysteine-Cross-Linked Phage Libraries
Brittney Chau, Kristi Liivak, and Jianmin Gao
20 Application of Deep Sequencing in Phage Display 333 Vincent Van Deuren, Sander Plessers, Rob Lavigne, and Johan Robben
21 The Application of Bacteriophage and Photoacoustic Flow Cytometry in Bacterial Identification
Robert H. Edgar, Anie-Pier Samson, and John A. Viator
22 Propagation, Purification, and Characterization of Bacteriophages for Phage Therapy
Katarzyna Kosznik-Kwas´nicka, Gracja Topka, Jagoda Mantej, Łukasz Grabowski, Agnieszka Necel, Grzegorz We˛grzyn, and Alicja We˛grzyn
23 Overcoming Bacteriophage Resistance in Phage Therapy
Elina Laanto
24 Bacteriophage Virus-Like Particles: Platforms for Vaccine Design
Ebenezer Tumban
Contributors
ALAA A. A. ALJABALI • Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy, Yarmouk University, Irbid, Jordan
MOHAMMAD B. M. ALJBALY • Faculty of Pharmacy, Jordan University of Science and Technology, Irbid, Jordan
DIANA M. A ´ LVAREZ-ESPEJO • Escuela de Medicina Veterinaria, Pontificia Universidad Catolica de Chile, Santiago, Chile
FABIO DAL BELLO • Sacco S.r.L, Cadorago, CO, Italy
JUSTIN BOECKMAN • Center for Phage Technology, Texas A&M University, College Station, TX, USA; Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX, USA
SOLEDAD TELMA CARRASCO • Laboratorio de Microbiologı ´ a Molecular, Facultad de Ciencias Me´dicas, Universidad Nacional de Rosario, Rosario, Argentina
AMANDA CARROLL-PORTILLO • Division of Gastroenterology and Hepatology, University of New Mexico, Albuquerque, NM, USA
JOSUE CASTRO-MEJIA • University of Copenhagen, Frederiksberg C, Denmark
SITTINAN CHANARAT • Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand; Laboratory of Molecular Cell Biology, Center for Excellence in Protein and Enzyme Technology, Faculty of Science, Mahidol University, Bangkok, Thailand
SURANG CHANKHAMHAENGDECHA • Department of Biology, Faculty of Science, Mahidol University, Bangkok, Thailand
BRITTNEY CHAU • Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, MA, USA
JOHN CHEN • Infectious Diseases Translational Research Programme, Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
MELISSA-JANE CHU YUAN KEE • Infectious Diseases Translational Research Programme, Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
GINO CINGOLANI • Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA
LING DENG • University of Copenhagen, Frederiksberg C, Denmark
RAFIK DEY • School of Public Health, University of Alberta, Edmonton, AB, Canada
ELENA A. DLUSSKAYA • Faculty of Arts, Business & Science, University College of the North, The Pas, MB, Canada
ROBERT H. EDGAR • Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA
BERT ELY • Department of Biological Sciences, University of South Carolina, Columbia, SC, USA
GIOVANNI ERACLIO • Sacco S.r.L, Cadorago, CO, Italy
ROBERT P. FAGAN • School of Biosciences, Florey Institute, University of Sheffield, Sheffield, UK
JIANMIN GAO • Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, MA, USA
JASON GILL • Center for Phage Technology, Texas A&M University, College Station, TX, USA; Department of Animal Science, Texas A&M University, College Station, TX, USA
ŁUKASZ GRABOWSKI • Laboratory of Phage Therapy, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Gdansk, Poland
AYESHA HASSIM • Department of Veterinary Tropical Disease, Faculty of Veterinary Science, University of Pretoria, Pretoria, South Africa
MARA ELENA HEINRICHS • Institute for Chemistry and Biology of the Marine Environment, Carl von Ossietzky University, Oldenburg, Germany
CHUN-FENG DAVID HOU • Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA
STEPHANO IGLESIAS • Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA
RASMUS RIEMER JAKOBSEN • University of Copenhagen, Frederiksberg C, Denmark
TAVAN JANVILISRI • Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand
KATARZYNA KOSZNIK-KWAS ´ NICKA • Department of Medical Microbiology, Faculty of Medicine, Medical University of Gdansk, Gdansk, Poland
ELINA LAANTO • Department of Biological and Environmental Science and Nanoscience Center, University of Jyvaskyla, Jyv € askyl € a, Finland; Molecular and Integrative Biosciences Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland
FREJ LARSEN • University of Copenhagen, Frederiksberg C, Denmark
ROB LAVIGNE • Animal and Human Health Engineering (A2H), Leuven (Arenberg), KU Leuven, Leuven, Belgium
KGAUGELO EDWARD LEKOTA • Unit for Environmental Sciences and Management, Microbiology, North West University, Potchefstroom, South Africa
JACOB LENSKI • Department of Biological Sciences, University of South Carolina, Columbia, SC, USA
FENGLIN LI • Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA
KRISTI LIIVAK • Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, MA, USA
DEREK M. LIN • Biomedical Research Institute of New Mexico, Albuquerque, NM, USA
HENRY C. LIN • Division of Gastroenterology and Hepatology, University of New Mexico, Albuquerque, NM, USA; Medicine Service, New Mexico VA Health Care System, Albuquerque, NM, USA
MEI LIU • Center for Phage Technology, Texas A&M University, College Station, TX, USA; Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX, USA
GABRIELE ANDREA LUGLI • Laboratory of Probiogenomics, Department of Chemistry, Life Sciences, and Environmental Sustainability, University of Parma, Parma, Italy
JENNIFER MAHONY • School of Microbiology & APC Microbiome Ireland, University College Cork, Cork, Ireland
JAGODA MANTEJ • Univentum Labs Ltd., The UG SPV, Gdansk, Poland
XIAOTIAN MAO • University of Copenhagen, Frederiksberg C, Denmark
TIMOTHY J. MARKS • Department of Pharmaceutical and Clinical Sciences, Campbell University, Buies Creek, NC, USA
JACQUES MATHIEU • Department of Civil and Environmental Engineering, Rice University, Houston, TX, USA; Sentinel Environmental, Houston, TX, USA
TANNAZ MOHAMMADI • Department of Biological Sciences, University of South Carolina, Columbia, SC, USA
HE ´ CTOR RICARDO MORBIDONI • Laboratorio de Microbiologı ´ a Molecular, Facultad de Ciencias Me´dicas, Universidad Nacional de Rosario, Rosario, Argentina
ANDREA I. MORENO-SWITT • Escuela de Medicina Veterinaria, Pontificia Universidad Catolica de Chile, Santiago, Chile
MAITE MUNIESA • Departament de Gene`tica, Microbiologia i Estadı ´ stica, Universitat de Barcelona, Barcelona, Spain
AGNIESZKA NECEL • Department of Medical Microbiology, Faculty of Medicine, Medical University of Gdansk, Gdansk, Poland
DENNIS S. NIELSEN • University of Copenhagen, Frederiksberg C, Denmark
MOHAMMAD A. OBEID • Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy, Yarmouk University, Irbid, Jordan
PUEY OUNJAI • Department of Biology, Faculty of Science, Mahidol University, Bangkok, Thailand
TANAPORN PHETRUEN • Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand; Laboratory of Molecular Cell Biology, Center for Excellence in Protein and Enzyme Technology, Faculty of Science, Mahidol University, Bangkok, Thailand
WICHUDA PHOTHICHAISRI • Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand
GONC¸ ALO J. PIEDADE • Department of Marine Microbiology and Biogeochemistry, NIOZ Royal Netherlands Institute for Sea Research, ‘t Horntje, The Netherlands; Institute for Biodiversity and Ecosystem Dynamics (IBED), University of Amsterdam, Amsterdam, The Netherlands
SANDER PLESSERS • Department of Biochemistry, Molecular and Structural Biology, KU Leuven, Leuven, Belgium
OVIDIU POPA • Institute of Quantitative and Theoretical Biology Heinrich-Heine University Duesseldorf, Duesseldorf, Germany
JANINA RAHLFF • Centre for Ecology and Evolution in Microbial Model Systems (EEMiS), Department of Biology and Environmental Science, Linnaeus University, Kalmar, Sweden; Aero-Aquatic Virus Research Group, Friedrich Schiller University Jena, Jena, Germany
JOLENE RAMSEY • Center for Phage Technology, Texas A&M University, College Station, TX, USA; Department of Biology, Texas A&M University, College Station, TX, USA
DA ´ CIL RIVERA • Escuela de Medicina Veterinaria, Universidad Andres Bello, Santiago, Chile
JOHAN ROBBEN • Department of Biochemistry, Molecular and Structural Biology, KU Leuven, Leuven, Belgium
LORENA RODRI ´ GUEZ-RUBIO • Departament de Gene`tica, Microbiologia i Estadı ´ stica, Universitat de Barcelona, Barcelona, Spain
ISABELLA R. ROWLAND • Department of Pharmaceutical and Clinical Sciences, Campbell University, Buies Creek, NC, USA
LAURA SALA-COMORERA • Departament de Gene`tica, Microbiologia i Estadı ´ stica, Universitat de Barcelona, Barcelona, Spain
ANIE-PIER SAMSON • Department of Engineering, Duquesne University, Pittsburgh, PA, USA
CORY SCHWARZ • Department of Civil and Environmental Engineering, Rice University, Houston, TX, USA; Sentinel Environmental, Houston, TX, USA
xviContributors
SEYED HOSSEIN SHAHCHERAGHI • Department of Medical Genetics, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
PACIFICA SOMMERS • University of Colorado Boulder, Boulder, CO, USA
MURTAZA M. TAMBUWALA • Lincoln Medical School, Brayford Pool Campus, University of Lincoln, Lincoln, UK
GRACJA TOPKA • Univentum Labs Ltd., The UG SPV, Gdansk, Poland
GARETH TRUBL • Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, CA, USA
EBENEZER TUMBAN • School of Veterinary Medicine, Texas Tech University, Amarillo, TX, USA
VINCENT VAN DEUREN • Department of Biochemistry, Molecular and Structural Biology, KU Leuven, Leuven, Belgium
DOUWE VAN SINDEREN • School of Microbiology & APC Microbiome Ireland, University College Cork, Cork, Ireland
MARCO VENTURA • Laboratory of Probiogenomics, Department of Chemistry, Life Sciences, and Environmental Sustainability, University of Parma, Parma, Italy
JOHN A. VIATOR • Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA; Department of Engineering, Duquesne University, Pittsburgh, PA, USA
DIANA ELIZABETH WATURANGI • Department of Master in Biotechnology, Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia, Jakarta, Indonesia
ALICJA WE˛GRZYN • Phage Therapy Laboratory, University Center for Applied and Interdisciplinary Research, University of Gdansk, Gdansk, Poland
GRZEGORZ WE˛GRZYN • Department of Molecular Biology, Faculty of Biology, University of Gdansk, Gdansk, Poland
JULIA WEISSENBACH • Centre for Ecology and Evolution in Microbial Model Systems (EEMiS), Department of Biology and Environmental Science, Linnaeus University, Kalmar, Sweden
KELSEY WHITE • School of Microbiology & APC Microbiome Ireland, University College Cork, Cork, Ireland
Part I
An Overview of the Diversity of Bacteriophages
Chapter 1
Structural and Genomic Diversity of Bacteriophages
Bert Ely, Jacob Lenski, and Tannaz Mohammadi
Abstract
Bacteriophage diversity is a relatively unknown frontier that is rapidly being explored, leading to a wealth of new information. New bacteriophages are being discovered at an astounding rate via both phage isolation studies and metagenomic analyses. In addition, a nucleotide sequence-based viral taxonomic system has been developed to better handle this wealth of new information. As a result of these developments, phage scientists are transitioning from knowing that there must be huge numbers of diverse kinds of phage particles in natural environments to identifying the actual abundance and phage diversity that is present in specific environments. This review documents the beginning of this transition, offering a glimpse into the magnitude of change unfolding in the field. It stands as a testament to the expanding frontiers of phage research, illuminating the remarkable progress made in unraveling the intricate world of bacteriophage diversity and advancing our understanding of these enigmatic viral entities.
The study of bacteriophages is enjoying a renaissance due to the development of inexpensive DNA sequencing technologies and the increasing need to combat bacterial antibiotic resistance. This review will focus on the genomics of environmental bacteriophages since large numbers of phage genomic sequences have become available due to the recent isolation of numerous individual phages from environmental sources as well as the abundance of metagenomic nucleotide sequence data derived from environmental samples.
Metagenomic nucleotide sequence data are obtained when total DNA is extracted from environmental samples (for extraction details, see Chaps. 5, 6, 7, 8, and 10), and the resulting DNA is sequenced using high-throughput nucleotide sequencing systems (see Chaps. 8 and 12 for details). The nucleotide sequence data obtained from the sequencing analysis reflect the DNA of all the
microorganisms that were present in the sample along with environmental DNA. Environmental DNA is free DNA that is present in the sample and is not associated with any organisms that are present in the sample. For example, metagenomic data obtained from sediment samples from a 2-million-year-old ecosystem were recently shown to contain environmental DNA from mastodons, caribou, hares, rodents, and geese [1]. Although the samples from this study were not analyzed for DNA from microorganisms, microorganisms are abundant in contemporary environmental samples, and nucleotide sequences from these samples are a rich source of phage nucleotide sequences. In fact, since bacteriophages are thought to be 10 times more numerous than bacteria, huge numbers of bacteriophage genome sequences can be found in nucleotide sequences derived from a single environmental sample. These phage genome sequences can then be compared to the phage genome sequences present in the NIH National Center for Biotechnology Information (NCBI) database to identify those genomes that are similar to previously described phage genomes as well as those that have never been seen before.
This wealth of new viral genome information has provided an opportunity to update the system used to classify viruses. Traditionally, viruses have been categorized by their shape, size, and the kind of nucleic acid that contains their genomic information. Viral genomes can consist of DNA or RNA, and these nucleic acids can be single-stranded (ss) or double-stranded (ds). However, distantly related phages can have a similar size and physical structure. Therefore, the International Committee for the Taxonomy of Viruses (ICTV) has begun the process of developing a new classification system that uses genome sequence data as an important basis for taxonomic classification [2].
2 Bacteriophage Taxonomy
As indicated above, the ICTV has added nucleotide sequence homology as a primary component of the viral classification system [2]. For example, for two phage isolates to be considered members of the same bacteriophage species, their nucleotide sequences must be at least 95% identical across the length of their entire genomes in a pairwise comparison (percent identity × query coverage). Similarly, phages considered to be in the same genus must have at least 70% nucleotide identity across the length of their entire genomes in a pairwise comparison. Because of their evolutionary diversity, bacteriophages are widely spread throughout the current viral taxonomy and can have dsDNA, ssDNA, dsRNA, or ssRNA genomes (Table 1). Thus, bacteriophages with similar morphology and genome nucleic acids can be further classified based on the similarity of their genome nucleotide sequences.
Picobirnavirus a RNA ds Bisegmented/ unsegmented Spherical Lytic dsRNA transcription model ss single strand, ds double strand a Picobirnavirus has only recently been thought to infect prokaryotes rather than mammals. Thus, information on the method of replication and life cycle may be inaccurate
3
dsDNA Phages
Bacteriophages within the phylum Uroviricota are dsDNA phages that have tails and represent nearly half of all known phage genera. The tails vary in length and can be flexible or rigid, contractile, or noncontractile (Fig. 1). The phage heads can be isometric, prolate, or elongated (Fig. 1). Some phages with prolate or elongated heads have head filaments [3]. Genome sizes vary from 18 to 500 kb, and individual phage can have either lytic or lysogenic lifestyles. During phage particle assembly, the phage genomes are loaded into a preformed prohead, which then is processed into the mature head. Tails can be assembled separately and attached by a connector, or they can be built directly onto the mature phage head. In March 2023, the phylum Uroviricota contained 4 orders, 47 families, 98 subfamilies, 1197 genera, and 3601 species. Thus, this phylum contains most of the known dsDNA bacteriophages. In contrast to the Uroviricota, the bacteriophages of the order Kalamavirales are nonenveloped, tailless, icosahedral phages with spikes at their fivefold vertices (Fig. 2). Most are lytic phages that replicate their 15 kb dsDNA genomes using a protein-primed replication method, and their genomes are surrounded by an internal membrane [4]. These membranes can produce tail-like tubes for injecting their DNA into the host cell. In March 2023, this order contained 1 family, 5 genera, and 12 species with phage PRD1, the best-studied phage in this order. Similarly, phages in the family Autolykiviridae also are nonenveloped, icosahedral phages, with linear dsDNA. However, they have double jelly-roll capsid proteins and can infect marine bacteria and archaea [5]. Most of these phages have 10 kb genomes and a relatively broad host range. In March 2023, this family included 2 genera and 5 species.
Fig. 1 (a) A phage particle with an icosahedral head and a rigid, contractile tail with tail fibers at the end. (b) A phage particle with an icosahedral head and a rigid, noncontractile tail with tail fibers at the end. (c) A phage particle with an elongated head and a flexible tail. (d) A phage particle with a prolate head and a flexible tail
Fig. 2 (a) A phage particle with an icosahedral head and spikes at the fivefold vertices. (b) A phage particle with an icosahedral head that is surrounded by a membrane
Vinaverales is an order of dsDNA phages that includes a single genus, Corticovirus. Phages in this genus are nonenveloped, tailless, and icosahedral with protein spikes at the fivefold vertices. Their 10 kb genomes are supercoiled dsDNA enclosed in a lipid membrane [6]. A second order of viruses with either circular or linear dsDNA genomes, Halopanivirales, includes viruses that infect archaea as well as phages that infect thermophilic bacteria. They have tailless, icosahedral heads, and their genomes are also enclosed by a lipid membrane [7]. The order Halopanivirales includes two genera that infect archaea and one genus that infects Thermus,a thermophilic genus of bacteria.
Finally, the Plasmaviridae family has not been assigned to a larger taxonomic group. It consists of a single species that is characterized by temperate, enveloped virions (Fig. 2) that release their progeny through budding from the cell membrane of their cell wall-less Acholeplasma hosts [8]. Conversely, they enter their hosts by fusion of the phage and host membranes. Once inside, the supercoiled 12 kb dsDNA phage genome can be transcribed to produce new phage particles, or it can integrate into the host genome by site-specific recombination. The resulting lysogenic bacteria produce few, if any, phage particles [9]. However, phage production can be induced by treatment with mitomycin C or by ultraviolet light (for details about phage induction, see Chap. 17). Once mature phage particles are assembled, they are released from the cell one at a time by budding from the cell membrane without killing the host cell. Consequently, the infected host cell can continue to grow and divide, but at a much slower rate due to the burden of producing new virus particles. Thus, in contrast to the plaques produced by lytic phages, the observed Plasmaviridae phage plaques are due to reduced bacterial growth rather than the lysis of the host cells.
4
ssDNA Phages
The phylum Hofneiviricota includes the filamentous ssDNA phages that extrude phage particles from the host cell membrane without lysis. These phages can integrate into host genomes or exist extrachromosomally. In March 2023, this phylum contained 3 families, 31 genera, and 51 species. The three families include Inoviridae (25 genera, 43 species), Paulinoviridae (2 genera, 2 species), and Plectroviradae (4 genera, 6 species). Inoviridae adsorb to pili on Gram-negative cells and mature phages are extruded from the host cell [10]. Their genomes consist of 5–11 kb supercoiled ssDNA, and the phage DNA can integrate into the host genome or remain extrachromosomal. Phage M13 is a well-studied example. The Paulinoviridae include only two phages: the Bifilivirus, which infects Propionibacteria, and Thomixvirus, which infects Thermus bacteria. Phages classified as Plectroviradae infect bacteria in the phylum Mycoplasmatota. They have 4.5–8 kb circular genomes, and the newly assembled virions are also secreted without killing the cell.
Phages in the phylum Phixviricota are nonenveloped, icosahedral phages with circular ssDNA genomes that replicate through a rolling circle mechanism. The genome sizes range from 4.5 to 6 kb and code for 11 genes. In March 2023, this phylum contained 1 family, 2 subfamilies, 7 genera, and 22 species including the well-studied PhiX174 phage. Most members of the Microviridae family are lytic, but a few may be lysogenic.
The Finnlakeviridae family has not been assigned to a larger taxonomic group. Interestingly, the single species of Finnlakeviridae has a circular ssDNA genome, and the phage particle includes an internal lipid membrane, the first ssDNA phage to have one [11]. They infect and eventually lyse bacteria in the genus Flavobacteruim.
5 RNA Phages
The class Vidaverviricetes includes enveloped, icosahedral phages with tri-segmented dsRNA genomes. There is a single genus, Cystovirus, that includes seven species. The Cystovirus particles enter the bacterial host by first adsorbing to a bacterial pilus and then fusing with the cell membrane when the pilus is retracted. Phage progenies are released by lysing the host cell. In March 2023, the Vidaverviricetes included one genus and seven species including the Phi6 phage discovered by Ann Vidaver [12]
In contrast, Leviviricetes is a class of positive-strand ssRNA phages that was discovered more than 50 years ago and has recently been greatly expanded through metagenomics (see below and
Fig. 3 (a) A filamentous ssDNA phage particle. (b) An icosahedral ssDNA or RNA phage particle. (c) A spherical RNA phage particle
[13, 14]). As of March 2023, the ICTV website included 2 orders, 6 families, 271 genera, and 882 species in the class Leviviricetes Not much is known about the majority of the phages that have been identified through metagenomic analyses since only nucleotide sequence data are available, and the actual phage particles that would be produced by these genomes were never isolated or observed. However, the well-studied ssRNA phages are nonenveloped and require bacterial pili to attach to their host. The ssRNA phage MS2 was the first phage to have its genome sequenced [15].
The family Picobirnaviridae are small, nonenveloped spherical viruses (Fig. 3) with dsRNA genomes that occur in two segments totaling 4.4 kb. Recent studies have proposed that they infect prokaryotes rather than mammals [16, 17]. Thus, information about their method of replication and life cycle may be inaccurate.
6 Phage Discovery
For the past two decades, the SEA-PHAGES program has encouraged undergraduates to isolate new bacteriophages from environmental sources, resulting in the isolation and genome nucleotide sequencing of more than 800 phages that infect mycobacterial hosts [18, 19]. These efforts have been complemented by the efforts of numerous other laboratories to isolate phages from natural sources, resulting in a great increase in the number of isolated phages and sequenced phage genomes.
More recently, the availability of vast amounts of nucleotide sequence information coupled with the new nucleotide sequencebased virus classification system has led several research groups to focus on specific kinds of phage genomes that are present in metagenomic datasets. For example, Yutin et al. [20] focused on dsDNA phage genome sequences in human gut metagenomes. Since the ends of most dsDNA phage genomes recombine to form circles after entry into the host cell, they searched 4752 human gut metagenomic DNA sequences for circular DNAs and found 4907 complete phage genomes. They analyzed a subset of these phage genomes that had homology to the crAss phages that infect bacteria
of the phylum Bacteroidetes and showed that they are the most common phages in the human gut, comprising 12% of the 4907 phage genomes (for more information about the diversity of phages in the human gut, see Chap. 2). These crAss phage genomes were grouped into five families including two that had not been identified previously. A follow-up study of additional dsDNA phages that infect bacteria of the phylum Bacteroidetes proposed three more families of phages that infect bacteria of the phylum Bacteroidetes and have not been described previously [21]. A third study analyzed metagenomic nucleotide sequence information from brine samples to identify additional dsDNA, ssDNA, and ssRNA phages that infect halophilic bacteria [22]. Thus, these three studies illustrate the potential of metagenomic studies to completely revolutionize our understanding of bacteriophage diversity.
Using a different approach, Weinheimer and Aylward [23] analyzed Tara Oceans’ metagenomic data to identify 85 jumbo phages with genome sizes that ranged from 200 to 500 kb. Comparisons of these phage genomes resulted in five clusters based on their gene content and proposed replication strategies. Thus, they nearly doubled the number of known jumbo phages, while the five genetically distinct clusters show that jumbo size evolved multiple times. In addition, Gregory et al. [24] used the same dataset to identify 195,728 populations of DNA viruses in the world’s oceans. For details about the diversity of phages in ocean/polar ecosystems and hot springs, see Chaps. 3 and 4
Metagenomic nucleotide sequence data are usually thought of as dsDNA sequences as in the examples above. However, Callanan et al. [13] used data from 82 publicly available metatranscriptomic datasets to identify ssRNA phage genomes. Prior to their study, only 29 ssRNA phage genomes were present in the GenBank database. However, they were able to identify 1015 near-complete genomes, increasing the number of available ssRNA phage genome sequences 60-fold. In addition, comparisons of the predicted amino acid sequences of the capsid proteins indicated the presence of eight independent lineages comparedwith the two previously identified ssRNA phage lineages. A complementary study based on the identification of RNA-directed RNA polymerase genes among the 28 terrabases of Global Ocean RNA sequences led to the identification of new phyla of ssRNA viruses, suggesting that ssRNA viruses are much more diverse than previously recognized [25].
As indicated above, some phages can establish a lysogenic relationship by integrating their genomes into the genome of the host bacterium. Therefore, Touchon et al. [26] surveyed the 2110 complete bacterial genomes present in GenBank in November 2013 and identified 2246 prophage elements that were present in these genomes. Seven years later, Johnson et al. [27] performed a similar analysis, but due to the vast increase in the available data,
they limited their search to the genus Pseudomonas and examined more than 5000 Pseudomonas genome sequences present in the GenBank database. They were able to identify more than 6700 integrated phage genomes, and nearly half of these phages shared no significant nucleotide sequence similarity to the genomes of any known phages. Thus, they represent a wealth of previously unknown lysogenic phages. However, integrated phage genomes are subject to the same mutational pressures as bacterial genomes. Therefore, they can accumulate single-base mutations as well as insertions and deletions. Since metagenomic analyses cannot test integrated phage genomes for the ability to produce phage particles that can infect a new host, we cannot discriminate between those genomes that represent contemporary phage lineages and those that represent extinct phage lineages. Nevertheless, these lysogenic phage genomes provide a wealth of information about the evolution and diversity of lysogenic phages that infect members of the genus Pseudomonas, and it would be interesting to perform similar analyses of other bacterial genera to see what additional kinds of bacteriophages could be identified.
7 Bacteriophage Host Range
Host range describes the ability of bacteriophages to infect one or more kinds of bacteria. Some phages can infect one or a few bacterial species, while other phages can infect many bacterial species as well as bacteria from different genera. Host range is determined by several factors (for additional information, see Chaps. 15 and 16). First, the phage must get its genome into the cell. Thus, the first step is binding to a potential host cell often via a binding protein that the phage uses to attach to a particular molecule on the surface of the host cell. Obviously, the phage particle could only bind to a cell that has the appropriate receptor. However, some broad host-range phage particles have multiple binding proteins to increase the number of potential hosts. Once the phage genome gets inside the host cell, the infection can still be aborted by host defense mechanisms or by the absence of compatible host proteins that are needed for phage genome transcription or replication.
To date, most well-studied phages have narrow host ranges [28]. However, it is not clear whether a narrow host range is the norm or whether it represents a sampling bias. For example, enrichments that use several different host strains simultaneously may facilitate the isolation of broad host-range phages [29–35]. In contrast, a more recent study concluded that having multiple hosts during the isolation procedure does not increase the chances of finding broad host-range phages [36]. Thus, finding broad hostrange bacteriophages might be a function of effort and careful analyses.
Nevertheless, a host-range extension protocol was developed to extend the host range of phages that infect Pseudomonas aeruginosa. Mapes et al. [37] combined mixed cultures of four phages with a mixture of 16 different host strains and then purified individual phage strains by plaque isolation after multiple passages of the phage mixture on a fresh host mixture. Over 30 cycles, the host range of individual phages expanded, as shown by spot testing on the 16 host strains as well as 10 additional P. aeruginosa strains. Thus, host range appears to be a rapidly evolving bacteriophage trait.
Since most of the newly discovered bacteriophages are known to exist only by the presence of their genomes in metagenomic studies, host-range experiments cannot be performed. However, analyses of the CRISPR spacers present in the bacterial genomes that are present in the metagenomic sequence data where the phage genomes were identified allowed the researchers to predict potential hosts for many of the corresponding phages [21].
8 Phage Genome Comparisons
The ease and low cost of sequencing of phage genomes have facilitated studies of multiple closely related phage genomes. For example, a study of 12 Dolichocephalovirinae (previously called type B3 or CbK-like phages) showed that 10 of the 12 phage genomes studied were sufficiently similar to each other that they could be considered members of a single species of the genus Shapirovirus using the current ICTV criteria [38]. A follow-up study [39] of the two outliers and four additional members of the Dolichocephalovirinae subfamily with larger genomes resulted in the identification of three new genera designated Colossusvirus, Bertelyvirus, and Poindextervirus. In addition, the original representatives of these new genera are sufficiently different from each other that they should be considered different species since they have less than 95% genome identity in pairwise comparisons. Recently, environmental sampling by our laboratory has identified a third species of Bertelyvirus, with eight closely related phage genomes identified to date (Ely, “unpublished”). A second study of 17 members of the Autographiviridae family resulted in the identification of seven new genera (Ely, “unpublished”). Similarly, Baltinovic et al. [40] investigated phages previously clustered in the genus Kuravirus and showed that this genus should be split into four genera. Thus, there are so many kinds of phages remaining to be discovered that small individual studies of newly isolated phages can result in the identification of new phage genera.
9 Phage Genome Structure
In addition to identifying new genera and species of bacteriophages, genome comparisons of related phages can provide insights into phage genome evolution. For example, in all 17 genomes in the Autographiviridae family comparison described in the previous section, the shared genes are in the same order in the same location in each genome (Ely, “unpublished”). A similar phenomenon was observed in the Dolichocephalovirinae subfamily comparison where gene order was conserved among the diverse genera even though the Bertelyvirus genomes shared only 54% of their genomes with the other three genera in the subfamily [39]. Despite the extent of the disparity, the genome organization was sufficiently conserved that we were able to detect a 23 kb inversion in the Bertelyvirus genomes relative to the gene order in the other three genera. Thus, conserved gene order can be used to show genetic relationships among bacteria from different genera. Similarly, gene order patterns were used to categorize T4-related phage into subfamilies of the Straboviridae family [41]. Thus, conserved gene order can be used to show genetic relationships among bacteria within subfamilies as well.
Within a genus, whole-genome comparisons can be used to identify gene insertions and deletions. For example, when five Kronosvirus isolates were compared, a small number of speciesspecific genes were identified (Mohammadi et al., submitted for publication). These results suggest that gene insertions and deletions are relatively rare since only a small number were observed among the five Kronosvirus genomes analyzed in this study. However, the origin of these inserted genes has yet to be determined since no other match is present in the GenBank database. Recently, Gosselin et al. [42] described a novel way that genome insertions can occur. They showed that intein-mediated recombination has resulted in the presence of conserved methylase genes among divergent phage groups, an unusual form of horizontal gene transfer. Thus, more studies are needed to better understand the gene insertions that are observed in phage genomes.
A common feature of many of the phage genomes described in this section is that the head, tail, and genome replication genes are present in three clusters, which are located close to each other in the phage genomes (Fig. 4). In addition, each type of phage genome also includes large clusters of hypothetical genes with no known function. Within-species comparisons indicate that these
Fig. 4 A typical phage genome organization of genes that code for proteins of related function
hypothetical genes are conserved within a species. However, they accumulate more mutations than the genes with known functions, suggesting that they can accommodate more variation than the genes coding for enzymes and structural proteins (Mohammadi et al., submitted for publication). When Wilson and Ely [39] compared the genes present in the genomes of the Dolichocephalovirinae subfamily, they found significant nucleotide sequence homology among members of the same genus, but little nucleotide sequence homology among the different genera despite the conservation of gene order. In addition, they found no examples of genes being transferred from one genus to another. Thus, despite sharing a common host bacterial strain, these four genera of the Dolichocephalovirinae subfamily have evolved independently since they last shared a common ancestor.
10 Conclusions
Phage studies are enjoying a renaissance. In recent years, large numbers of previously unknown phages have been described, and we can expect the process to continue. In addition, as additional phage genome sequences are determined, the nucleotide sequencebased taxonomic system developed by the ICTV [2] will provide a method for organizing the newly discovered phages based on genetic relatedness. Thus, there will be a great expansion in our knowledge of bacteriophage taxonomy. In addition, there will be a wealth of new information about phages and their interactions with their hosts and each other that will undoubtedly lead to new concepts about phage genome evolution and the role of phages in natural environments. Given the rapid pace of advancements in phage studies, it is highly likely that within a few years, this review will become outdated. Future students will undoubtedly marvel at the limited understanding we possess about phages in 2023, highlighting the dynamic nature of scientific exploration and the continuous pursuit of knowledge in the field of phage studies.
References
1. Kiaer KH, Pedersen MW, De Sanctis B, de Cahsan B, Korneliussen TS et al (2022) A 2-million-year-old ecosystem in Greenland uncovered by environmental DNA. Nature 612:283 – 291. https://doi.org/10.1038/ s41586-022-05453-y
2. Turner D, Kropinski AM, Adriaenssens EM (2021) A roadmap for genome-based phage taxonomy. Viruses 13:506. https://doi.org/ 10.3390/v13030506
3. Guerrero-Ferreira RC, Viollier PH, Ely B, Poindexter JS, Georgieva M, Jensen GJ, Wright ER (2011) A novel mechanism for
bacteriophage adsorption to the motile bacterium Caulobacter crescentus. Proc Natl Acad Sci U S A 108:9963–9968. https://doi.org/10. 1073/pnas.1012388108
4. Stro ¨ msten NJ, Bamford DH, Bamford JK (2005) In vitro DNA packaging of PRD1: a common mechanism for internal-membrane viruses. J Mol Biol 348(3):617–629. https:// doi.org/10.1016/j.jmb.2005.03.002
5. Kauffman K, Hussain F, Yang J, Arevalo P, Brown JM, Chang WK et al (2018) A major lineage of non-tailed dsDNA viruses as unrecognized killers of marine bacteria. Nature 554:
7. Demina TA, Pietila MK, Svirskaite ˙ J, Ravantti JJ, Atanasova NS, Bamford DH, Oksanen HM (2017) HCIV-1 and other tailless icosahedral internal membrane-containing viruses of the family Sphaerolipoviridae. Viruses 9:32. https://doi.org/10.3390/v9020032
8. Krupovic M, ICTV Report Consortium (2018) ICTV virus taxonomy profile: Plasmaviridae. J Gen Virol 99(5):617–618. https:// doi.org/10.1099/jgv.0.001060
9. Dybvig K, Maniloff J (1983) Integration and lysogeny by an enveloped mycoplasma virus. J Gen Virol 64:1781–1785. https://doi.org/ 10.1099/0022-1317-64-8-1781
11. Mantynen S, Laanto E, Sundberg LR, Poranen MM, Oksanen HM, Report Consortium I (2020) ICTV virus taxonomy profile: Finnlakeviridae. J Gen Virol 101(9):894–895. https://doi.org/10.1099/jgv.0.001488
12. Vidaver AK, Koski RK, Van Etten JL (1973) Bacteriophage Φ6 a lipid-containing virus of Pseudomonas phaseolicola. J Virol 11(15): 799–805. https://doi.org/10.1128/jvi.11.5
13. Callanan J, Stockdale RS, Shkoporov A, Draper LA, Ross RP, Hill C (2020) Expansion of known ssRNA phage genomes: from tens to over a thousand. Sci Adv 6:eaay5981. https:// doi.org/10.1126/sciadv.aay5981
14. Callanan J, Stockdale SR, Adriaenssens EM, Kuhn JH, Rumnieks J, Pallen MJ, Shkoporov AN, Draper LA, Ross RP, Hill C (2021) Leviviricetes: expanding and restructuring the taxonomy of bacteria-infecting single-stranded RNA viruses. Microb Genom 7(11):000686. https://doi.org/10.1099/mgen.0.000686
15. Fiers W, Contreras R, Duerinck F, Haegeman G, Iserentant D, Merregaert J et al (1976) Complete nucleotide sequence of bacteriophage MS2 RNA: primary and secondary structure of the replicase gene. Nature 260: 500 – 507. https://doi.org/10.1038/ 260500a0
16. Krishnamurthy SR, Wang D (2018) Extensive conservation of prokaryotic ribosomal binding sites in known and novel picobirnaviruses. Virology 516:108–114. https://doi.org/10. 1016/j.virol.2018.01.006
17. Boros A ´ , Polga ´ r B, Pankovics P, Fenyvesi H, Engelmann P, Phan TG, Delwart E, Reuter G (2018) Multiple divergent picobirnaviruses with functional prokaryotic Shine-Dalgarno ribosome binding sites present in cloacal sample of a diarrheic chicken. Virology 525:62–72. https://doi.org/10.1016/j.virol.2018. 09.008
18. Hatfull GF, Pedulla ML, Jacobs-Sera D, Cichon PM, Foley A, Ford ME et al (2006) Exploring the Mycobacteriophage metaproteome: phage genomics as an educational platform. PLoS Genet 2(6):e92. https://doi.org/ 10.1371/journal.pgen.0020092
19. SEA-PHAGES|Home.Availableonline: https://seaphages.org/.Accessedon 20 Mar 2023
20. Yutin N, Benler S, Shmakov SA, Wolf YI, Tolstoy I, Rayko M et al (2021) Analysis of metagenome-assembled viral genomes from the human gut reveals diverse putative CrAsslike phages with unique genomic features. Nat Commun 12:1044. https://doi.org/10. 1038/s41467-021-21350-w
21. Benler S, Yutin N, Antipov D, Rayko M, Shmakov S et al (2021) Thousands of previously unknown phages discovered in wholecommunity human gut metagenomes. Microbiome 9:78. https://doi.org/10.1186/ s40168-021-01017-w
22. ZeinEldin RA, Ahmed MM, Hassanein WS, Elshafey N, Sofy AR, Hamedo HA, Elnosary ME (2023) Diversity and distribution characteristics of viruses from soda lakes. Genes 14(2):323. https://doi.org/10.3390/ genes14020323
23. Weinheimer AR, Aylward FO (2022) Infection strategy and biogeography distinguish cosmopolitan groups of marine jumbo bacteriophages. ISME J 16:1657–1667. https://doi. org/10.1038/s41396-022-01214-x
24. Gregory AC, Zayed AA, Conceicao-Neto N, Temperton B, Bolduc B et al (2019) Marine DNA viral macro-and microdiversity from pole to pole. Cell 177:1109–1123.e14. https://doi. org/10.1016/j.cell.2019.03.040
25. Zayed AA, Wainaina JM, Dominguez-HuertaG, Pelletier G, Guo J et al (2022) Cryptic and abundant marine viruses at the evolutionary origins of earth’s RNA virome. Science 376: 156–162. https://doi.org/10.1126/science. abm5847
26. Touchon M, Bernheim A, Rocha E (2016) Genetic and life-history traits associated with the distribution of prophages in bacteria. ISME J 10:2744–2754. https://doi.org/10.1038/ ismej.2016.47
Another random document with no related content on Scribd:
XI.
SECOND TERM IN CONGRESS.
R. BLAINE reached home weary in body, but fresh in spirit, from the great political war in Massachusetts, New York, and Pennsylvania, just in time to cast his ballot the last time for Abraham Lincoln. He had stumped his own state from “Kittery to Houlton,” which are the extreme points in Maine, and had put in about fifty speeches in the other states,—between one and two hundred in all. He had confidence in the result, for he had been near the people and got their temper and knew the purpose of their sovereign will in the matter, and so it came, but with it the reflection that they were only about five years off from the Dred Scott decision, and every free state but one voting solid in the electoral college for the great abolition president, Abraham Lincoln.
How dark and infamous, and mysterious, too, looked the repeal of the Missouri Compromise; the war with Mexico; the Kansas and Nebraska bill; the proposition to purchase Cuba for purposes of slavery, and all the political paltroonery and truckling of honored public men, the trimmers and time-servers!
But what ruin strewed the pathway to such triumph! There was not a slave in all the land now, according to the proclamation, emphatically endorsed, and the rebellion well-nigh crushed. The effort had been, it is thought, for the South to hold out until after the presidential election, and hope for the defeat of Mr. Lincoln. The war was over six months after his re-election.
In less than a month after election day, Mr Blaine was in his seat in congress (December 5th), and there, also, with a knowledge of the fact that not only had Mr. Lincoln been re-elected president, but he himself, also, had been re-elected to congress, for the election took place a year before each term expired. How could he be otherwise than happy regarding the political outlook of either himself or the nation. He need have little thought for himself; he had surely caught at the flood that tide which leads on to greatness. He was not a coming man, but one who had already come. His record of the former session had made him more widely known, and known in a larger sense. Indeed, he was every way a larger man; beloved at home, respected and admired abroad in other states, and where his great life-work had so auspiciously begun—in congress.
The principle of evolution was at work upon him in its only true sense, just as it operates in tree and flower, where heaven and earth in all their vital forces are made tributary to Nature’s laws of unfolding in the deep processes of growth upward to perfection.
There had been a wondrous involution from centuries of great history, according to subtle, silent laws of hereditary inheritance, in very blood and life, of tone, and quality, and temper, and now there is evolved, evoked, just that of power which tells of kinship with those who have gone before.
It should not cause surprise that Nature keeps her treasures, or that the right, the good, the true, live to confront the wrong, the false, the bad, with just those elements of a nobler life that no power can resist.
The people everywhere were singing,—
“Our God is marching on.”
And so he was, in all of truth and right maintained, in all of good performed.
Never were the good and true remembered in such hosts as when the nation struggled with her foes. What mighty ones stepped out of the chaos of a dismal past into splendid life with her! Their name is legion; grand in every sphere of greatness, and great in every realm
of grandeur. They thought out the nation first; fought out and forged it in battle-heat, and hurled it like a thing of life, upon its great career. It never loses its power to go, to be, and conquer, bringing ever to the birth, and upward into strong, armed life those whose great abilities are her own; her own for defense; her own for war, living in their lives, powerful in their strong right arms,—one with them in destiny. Among that number now, though reckoned with a multitude, was James G. Blaine.
He surveyed the field for but a single day after the second session of his first congress opened,—the thirty-eighth,—and then undid the mischief of another. It was called the “Gold bill” in the House, and had simply been offered and referred to the committee of Ways and Means, by a Mr. Stevens, of Pennsylvania.
Its substance was, that a dollar note issued by the Government, declared lawful money and legal tender, is declared of equal value for all purposes as gold and silver coin of like denominations. A contract made payable in coin may be payable in legal tender, and anyone should be imprisoned who received a greenback for less than gold coin was worth, and fined as well.
Gold went up in Wall Street within twenty-four hours after the bill was presented, twelve per cent. Mr Blaine saw it and moved a reconsideration of it, sections two, three, five, and six being the objectional features of the bill. His speech in support of his motion did not occupy ten minutes. The author of the bill, Mr. Stevens, said,
“My friend from Maine (Mr. Blaine) has an intuitive way of getting at a great national question, one that has exercised the thoughts of statesmen of several countries for many years.” This in opening; and in closing his speech, he said,—
“How the gentleman from Maine, by his intuitive knowledge of these things comes to understand at once what the ablest statesmen of England took months to mature, I cannot very well understand. It is a happy inspiration.”
Had he a knowledge of his long years of study, that it was then twenty-five years since he finished reciting Plutarch, and but little less than twenty since his graduation, had he a knowledge of the strong, determined spirit of mastery which characterized him in all his work, could he have read over at that moment the long list of volumes over which he had poured, had he known these things, he would not have felt that a genius of intuition who got at things by inspiration merely, sat before him, but one with a genius for the hardest kind of a student’s work, with intuitions born of high intelligence and inspiration that comes from conscious strength. No wonder he was an enigma, a man beyond his years and place, yet master of the situation.
Mr. Stevens’ motion to table the motion of Mr. Blaine, failed, fifty-one to sixty-eight, and then the motion of Mr. Blaine regarding the bill of Mr. Stevens, carried, seventy-three to fifty-two. It is interesting to notice, that though the gentleman did not call up his bill for a solid month,—not until after the holidays,—and then came in with an elaborate argument showing the financial course of England in her war with France in 1793, and then in her war finally with the whole of continental Europe, though he seemed to have made a careful study of his subject, and of England’s financial policy, he closed with this sentence:—
“I feel that England never had so absurd a law as to pay one part of her war-debt in gold and another part in Bank of England notes.” He said “I feel,” he did not know. But Mr. Blaine knew, and so he asked him whether the bonds negotiated by England upon the continent were not payable in gold.
“I do not know,” was the answer.
Then Mr. Blaine stated, “Every one of them negotiated upon the continent was payable in gold, both principal and interest. Every one negotiated at the Hague, at Frankfort-on-the-Main, and elsewhere upon the continent, was negotiated upon the gold basis exclusively.”
This was no contest to win, but simply to bring out financial intelligence in a semi-official way, for the benefit of the country. It was a most sensitive subject. Gold was up to two hundred and fifty, that
is, a hundred dollars in gold cost two hundred and fifty dollars in greenbacks, and Mr. Stevens had endeavored in a wrong way, as Mr. Brooks showed, to correct gambling in gold, but Mr. Blaine could furnish him with deficiencies of knowledge, and manifest the acumen of a statesman upon a subject so great.
Mr. Blaine had his magnetic power then, and Mr. Stevens refers to it, and his great power over the House in securing so promptly the passage of his motion. He said,
“The House, partaking of the magnetic manner of my friend from Maine, became alarmed, and immediately laid the bill on the table.”
It was his power of quick, thrilling action; of feeling strongly, and making others feel as he did; of casting upon them the glow of his own brilliancy; of charming them with the rhapsody of his own genius; of piercing them with the energy of his own thinking, and so shutting them up to his conclusions by the force of his own arguments; it was thus by methods the fairest and most honorable to his abilities, that he carried all before him. And one can but see in his repeated control of the House, the power of his friendships.
Cox, Pendleton, Brooks, and others of the opposition would show him the greatest courtesies in debate. Randall, even, in his first session, gave him time out of his own hour for an entire speech, and Cox encouraged him in the midst of his Gold bill speech, by saying he was with him on it.
When the Naval Academy bill was before the House, he moved to repeal a section relating to cadets “found deficient.” If they had a hundred demerit marks in six months they would be expelled. Mr. Blaine had visited the academy in 1861, as a member of the “Board of Visitors,” and while there a young man was dismissed, not for any fault of scholarship, for he was among the brightest and best in his class.
Becoming deeply interested in the cause of the young man, he went to Washington and successfully interceded with the secretary of war, and he was restored. He subsequently graduated very high in classrank, and since his entrance upon active service has distinguished
himself as an officer of great merit, serving with efficiency and distinction as ordnance-officer on General Sheridan’s staff in that splendid, victorious campaign in the valley of the Shenandoah.
The demerits were given for singularly small offences, as: “floor out of order near wash-stand, four demerits,” etc., etc.
Mr. Blaine insisted that to the secretary of war and the president be restored the power that was taken from them at the last session,—to pardon any cadet discharged for any of these offences.
General Schenck joined him, and the amendment was adopted.
There is a little section of his speech on the Military Academy bill which shows his admiration for the telling power of manhood, and his utter scorn of sacrificing great ability, for which the nation was so loudly calling then, to little, simple things, good in themselves, but not of first importance, that we cannot forbear to give it. Here it is, verbatim, as he delivered it in congress:—
“Many of the cadets, sir, who have been very precise and decorous in their conduct in matters of petty discipline at the academy, and manage to pass through smoothly, often graduating with high rank obtained by very strict attention to ‘folding beds by 10 . .,’ and ‘drawing curtains by at precisely 6.45 . .’ (academy rules), are unfortunately never heard from afterwards. Their names do not always figure in the record of our bloody battles, and they have achieved no distinction in this war, with all its thousand opportunities, while on the other hand not a few of the graduates at the academy who at the Point had the ‘odor of tobacco in their rooms,’ and whose ‘floors were out of order near the wash-stand,’ have blazoned their names high on the roll of fame for conduct as gallant and skill as great as ever graced the battle-fields of any age country.”
Efficiency has ever been the test with him in his own work, and this he applies to others; as one has said, “We measure others in our own half-bushel; of course we do, we have no other.”
Early in the session he had a running debate which tried his metal, with Thayer, of Pennsylvania; Justin S. Morrill, of Vermont; James S.
Wilson, of Iowa; General Schenck, of Ohio, and S. S. Cox, of Ohio yielding the floor for the purpose.
It was not only a proof of his knowledge, but also of his ability to use it on demand, and he showed himself equal to the exigency, and showed that he was generally found away on the lead in his discussion of constitutional measures and application of principles. It is possible for a man to go over, in a long-winded speech, a vast amount of ground, which has been tramped as bare as the campground of a brigade of soldiers, by a multitude of debaters; ground which has been surveyed, and staked out, and pre-empted, and owned for a century or more, and concerning which, as concerning the constitution there is no question. Such speeches as these wearied the progressive spirit of advanced ones, and made them restless when the fate of great interests hung on the decision of a few hours’ discussion. No one watched more closely the utterances of men upon the floor, or held them to a stricter account.
In presenting a minority report on amendment of rules for the government of the House, Mr. Morrill had placed some undue restriction upon the powers of congress, and courteously waiting until he had finished a long speech of ten or eleven columns, Mr. Blaine asked him whether the power of impeachment would not extend to cabinet officers, and so their attendance upon the sittings of the House be compelled, a point Mr. Morrill had denied.
There had been little demand for this power slumbering in the constitution,—power which was used upon a president shortly afterward,—but brought prominently to the attention of the House, and much light thrown on it by the answers tersely given to near a score of questions, members were pleased to ask Mr. Blaine, and while he was ready with abundant answers; clear and strong, and packed with knowledge of the highest legal type, he was ready as well if there was hint of an assailant in manner or tone, to thrust out a sharp, rising question which would almost take the breath of the man who might be after him. When General Schenck asked him if the secretary of war was a civil officer, his quick reply was, “I do not think that a ‘civil’ question.” Neither was it, for as member of the cabinet of
course he was a civil officer, as much so as the president himself, who was by virtue of his office “Commander-in-chief of the armies of the Union.”
But Mr. Blaine had great respect for age and learning, and allowed no opportunity to show it to pass by unimproved. His early intercourse with his Grandfather Gillespie had developed largely veneration both for gray hairs and scholarly attainments, a veneration which had matured by associations with his teachers and great men of the nation whom he had met in his youthful days, and those whom he had since come to know and honor.
When Mr. Henry Winter Davis came on with his great naval speech, Mr. Blaine heard him with special pleasure, and had some very complimentary things to say of “the caustic, scathing, truthful, and deserved criticism of the naval department in building,” as Mr. Blaine said, “twenty iron-clad vessels, at a cost of ten millions of dollars, that will not stay on top of water.”
Mr. Pike had just taken him to task for this last statement, when the “hammer fell,” and Mr. Davis, showing his appreciation of the courtesies of Mr. Blaine, arose and said, “I ask unanimous consent that the gentleman from Maine may be permitted to proceed.” This was indeed a consideration which young members seldom received from the veterans of the House, and especially from one with a national reputation for scholarly attainments. But as “the debate in Committee of the Whole was closed by order of the House,” the Chair could not grant the request, and just here Mr. Blaine’s shrewdness and intimate knowledge of parliamentary rules showed itself. “I move,” he said, “to amend the amendment, by striking out the first line; that will entitle me to the floor for a few minutes longer.”
Then he went on to give an official fact, as he called it, and he knew well the value of such things; there was nothing “fine-spun” about them, but strong and stubborn, and full of power to convince. “Out of ninety British steamers,” he said, “caught within a given period in attempting to run the blockade, only twelve were caught by vessels built by the present administration of the navy department; while seventy-eight were caught either by purchased vessels, or vessels
inherited from the old navy I submit, sir, that this fact bears with crushing force on the practical question of the speed and efficiency of vessels of the new navy.” It is bad enough to swindle the government at any time, and in any thing, but in times of war to swindle her in the construction of iron-clad vessels that will not float, yet needed at once for active service, and produce twenty of them at half a million dollars apiece, was enough to arouse the indignation not only of the older member, Mr. Davis, but also of the younger man, Mr. Blaine.
And this now gave him a new, fresh start, untrammeled by crutch or cane, casting him wholly upon his own resources, and placing him where he must put forth all the power in him, or utterly fail.
“When the Jeannette went down, crushed and sunken by the ice,” writes Lieutenant Danenhouser, “we started with our boats southward, dragging them over the ice, broken and piled in every conceivable shape. We accomplished seven miles the first week, only to find, by taking observations, that the ice-floe had drifted us back to the northward twenty-seven miles, and so placing us twenty miles to the rear of the spot where we had started, and our ship had sunk.” They had intrepid spirits, but no firm ground; he had both the intrepid spirit and the firm ground on which to stand, and his victory was swift and certain.
Mr. Blaine never lost an opportunity to do a favor, or make a friend. Doing duty was his delight; getting hold of strong, plain, practical facts, and presenting them in a way that showed a constant, abiding interest in his constituency, that he was living and toiling for them, and had their best interest, and those of the entire state of Maine, and the whole country at heart.
Here is one of his plain, practical statements, showing his loyalty to home interests, as well as the business interests of the country. A vessel from his district had been chartered to government to carry a cargo of four hundred and fifty tons of coal from Philadelphia to New Orleans, for six thousand dollars. Upon her return her disbursements had been six thousand, two hundred and thirty-eight dollars and five cents. She received six thousand dollars in certificates of
indebtedness from the government, then selling them at ninety-four, which made but five thousand six hundred and forty dollars in cash, showing a net cash loss of five hundred and ninety-eight dollars and five cents, besides the interest on advance, about two hundred dollars more.
“And now, sir,” said Mr. Blaine, “after this melancholy experience the tax-collector came forward and demanded of the owner of the vessel, two and one-half per cent. on the six thousand dollars which the government paid, as above, and on top of all losses already incurred actually compelled him to pay one hundred and fifty dollars under that section of the internal-revenue law, which we are seeking to amend.
“A man’s profit in business,” he goes on to say, “affords a fair basis of taxation, but it is a cruel mockery of one’s misfortune to assess a tax upon losses.”
He further plead that “as commercial men of the country, who do so much to sustain our finances and our honor, they should be relieved from its oppressive exactions.”
There were no mists or fogs about him to conceal him or his methods, and what he said stood out in the clear light of day. In this case he was able to catch up from memory, a better argument for the repeal of the oppressive section of the law than had come to the House in a lengthy written memorial from a company doing business on the Schuylkill Canal in Pennsylvania, and who could make sitings net them four hundred and ten dollars, while in the case cited by Mr. Blaine, one trip was made at a loss of nine hundred and forty-eight dollars and five cents.
Seldom did he cite his own opinion. It was the bludgeon of hard, solid facts with which he did his best execution. Others might theorize, and imagine, and conceive, and spin web after web of sophistry, like the spider, out of themselves, to be full as flimsy when the storm of debate beat upon it, but not he. He evidently kept up a living acquaintance with those to whom he was responsible, and this, with an ever vigilant correspondence, enabled him to know, and not simply think and feel, but actually to know their adverse experiences
where the operations of the machinery of government affected them, and with reasonable and apparent facts in hand he could easily procure the remedy. This lively interest, so practical and so potent as well, was with him a constant element of power.
He lost no opportunity to familiarize himself with business enterprises, great and small, and get the best authority on all questions of finance and trade, and as a result he could speak with pertinency, and from a mind prolific of the freshest data on the practical questions as they were constantly coming before the House, and especially in the old war-days, when the vexed questions of internal revenue, with all its myriad details regarding the nature and value of taxable articles, were being adjusted.
At one time when he first entered congress, nearly every article that entered into the construction of a ship was taxed, and then upon her tonnage, and then, beside, upon the gross receipts for carrying the cargo. He saw to it at once that those matters were attended to.
But a fresh call was out for troops, and it was a final call. They were getting ready for the great opening of the spring campaign which was to speedily end in crushing the Rebellion, and annihilating the Confederacy. There was a flaw in the enrollment law passed the last session, which Mr Blaine had discovered, and sought to remedy It permitted recruiting in the rebel states, and credits for previous naval enlistments. “From these two sources have arisen the gigantic and wide-spread evil of filling quotas of towns without adding troops to the army.” He had offered an amendment which was designed to bring back recruiting to “an honest, meritorious, and patriotic effort to fill the ranks of our gallant army with men, and not with shadowy fictions which pass under the name of ‘paper credits.’” The quotas of entire cities, districts, and possibly states, had been thus filled “without adding a single man or musket to the effective military force of the nation. There was fraud, and he would so change the law that it could not be perpetuated.”
There were substitute-brokers, who, in some mysterious way, would get hold of these “credits,” as they were called, and sell them, much as torn scrip is sold.
“We can deal just by the government,” he said, “in its struggle for existence. It calls for men, and it is worse than madness to answer this call with anything else than men.
“In conclusion,” and his words reveal a genuine patriotism and zeal of affection for the soldier, “nothing so discourages the brave men at the front as the belief that proper measures are not adopted at home for re-enforcing and sustaining them.
“After four years of such patriotic and heroic effort for national unity as the world has never witnessed before, we cannot now afford to have the great cause injured, or its fair fame darkened by a single unworthy incident connected with it. The improper practices of individuals cannot disgrace and degrade the nation, but after these practices are brought to the attention of congress, we shall assuredly be disgraced and degraded if we fail to apply the remedy. Let us, then, in this hour of the national need, do our duty here, our duty to the troops in the field, our duty to our constituents at home, and our country; above all, to our country, whose existence has been in such peril in the past, but whose future of greatness and glory seems now so assured, and so radiant.”
Few utterances of those long, dark years, breathed a spirit of more devoted loyalty than is found even in these few sentences, and they were uttered when they would do the most good, and secure just those re-enforcements that would gladden the hearts of veterans, and hasten the end of the struggle.
Mr. Blaine had a keen eye for fraud, and made it his business to detect it; and he was just fearless enough to hold it up to the light of day. Wherever he unearthed it he would point out the individual, and point his finger at him and say, with a boldness known only to invective and scorn, “Thou art the man!”
He never seemed to take care of his popularity, but of his constituents and of his country. Enemies abounded, and evil, and wrong; and to these he paid effective attention, rightly judging that no course is safer, or accords with fuller satisfaction, than the right course. With him, character was the citadel of strength and
influence; and so we find him knowing and trusting himself, reaching for wrong in all of its strongholds.
And there was much to encourage now Sherman had reached the sea; Columbia, S. C., was captured; Charleston was evacuated; the old flag was again flying over Fort Sumter, and Washington’s Birthday was to be celebrated, by order of the secretary of war, E. M. Stanton, by a “national salute at West Point, and at every fort, arsenal, and army head-quarters of the United States, in honor of the event.” This twenty-second day of February was a long, busy day in congress. It was a quarter past five before the House adjourned. Mr. Blaine was in his seat all day long, voting steadily for the right and against the wrong. The conquered states, cut off from the Rebellion and rescued to liberty and lawful authority, were left without government, and must be provided, as Tennessee had been in the person of Andrew Johnson, now vice-president, with provisional governors. Much legislation was requisite. Every man in congress who had ever had any pro-slavery proclivities, was in his place contesting every step of progress with men who had never breathed aught but the air of freedom and known only loyal heart-beats.
One bill granted citizenship to all colored men who had served in the army and navy.
Right royal work, this, for such a man to be doing on a day so sacred; helping into citizenship the colored man, ever loyal, ever true.
This seemed to be the great feature of all the great bills before the House that day. It came up in the bill to encourage enlistments, and the worth and dignity of being an American citizen was held up before the negro as a prize for him to win; as something in store for him in the future; and so as giving to the colored troops, and all who united with them, this personal interest in relation to the government. But it takes time to get such thoughts adjusted to minds struggling with the fact of Emancipation, and so little is done but give the bills a hearing and pass them to another reading. Coming events had cast their shadows before them. It was, however, but the shadow of a passing cloud, and told of a great, bright sun shining in the heavens
yonder, which would soon dissipate all clouds and shadows, and the long night of bondage ended, give a glorious day, in which the world might see in the poorest black man of the South an American citizen, possessed of certain inalienable rights, among which are life, liberty, and the pursuit of happiness.
To the happy consummation of a task so grand, whose inspiration comes from that free and holy place where “all are one,” Mr. Blaine had set his hand, only to remove it when the chaplet of America’s proudest, noblest glory was on the black man’s brow.
That life is most divine which is most in line with Providence, and has the most of uplifting power in it, which stands the highest up, and can reach the farthest down, is many-handed in its helpfulness, and strong-handed as well, to unshackle humanity in body, in soul, and in spirit, and tell the fallen or sunken ones how to get upward toward God and heaven.
Opening the gates of heaven means unlocking the gates of earth, and to this latter task the statesmen of the nation stood pledged from that day, since numbered among the nation’s holidays. A close student of Mr. Blaine’s congressional career will be impressed with the fact that it seems planned and determined before-hand. There are no surprises in it. He seems to have determined upon his course before entering it, and gives his strength to certain measures, and does not fritter it away upon every resolve, or amendment, or motion, that happens to be before the House, affecting some far away interest of a day-dreamer.
He recognizes the fact fully that he is one of a great body of men, each one of whom is charged with interests of an important character to their state or district, and many heavily weighted with special and peculiar measures of national importance. These must all have their opportunity. Less than ninety working-days usually comprise the session, and there are but four of these in a congress, —from March to adjournment, and from December to March, and then repeated, constitutes a congressional term, with eight of them in a presidential term, or two a year for the four years. Beside, it takes so long a time to get measures through congress that the successful
man finds it necessary to devote himself with great carefulness to the few measures of importance he would have adopted, and become law organic or otherwise.
Very soon after Mr. Blaine entered congress he presented a resolution instructing the Committee on Judiciary to inquire into the expediency of amending the constitution so as to allow congress to levy an export tax. But the session closed, and it is not reported, and now his second session is closing, and still it is not forthcoming. Why not? He will know the reason why! And so there comes a day near the session’s close, only the day before Mr. Lincoln’s second inauguration, when he arises and states “a little grievance.” He states the resolution, its being offered at the last session, and now again at the present session. It had been to the Ways and Means Committee, to which it had been transferred. Evidently he had been ready to grapple with the subject for some time, and proceeded to do so. It involved an amendment to the constitution, and one “essential to the financial success of the government, and to the agricultural, commercial, and manufacturing prosperity of the country in all future time.”
It was stated that the measure would have been presented by the committee, if they had supposed time would have permitted of its consideration. It presented a subject that was discussed at length in the Convention of 1787. The “Madison Papers” give a synopsis of the constitutional debates of that convention, and show that many of the strongest men of that body, the really far-sighted ones, opposed the insertion of the clause prohibiting a tax on exports. The vote was not a very decisive one, nor did its advocacy come from the Southern or “staple states,” and opposition from Northern states.
He proceeds to deliver what is his great speech, if not the great speech of the session. It was probably not over an hour long, but he had not proceeded far before it became apparent that he had thoroughly studied the subject, and was investing it with a new interest.
A great debt of more than two billion eight hundred million was on the nation. Mr. Blaine’s amendment was looking towards its
liquidation. It was the wise, strong look far ahead. He saw in it several hundred millions of revenue in the export of cotton, tobacco, and naval stores, without affecting the demand for them, and also in petroleum, and numberless articles, still more of revenue. France was taxing her wines and brandies, and countries having peculiar commodities taxed them.
Cotton which sold in Liverpool at eleven and three-quarters pence per pound in December, 1861, sold for twenty-four and one-half pence per pound in just one year from that date. The three million two hundred thousand bales of five hundred pounds each, this country had exported, were missed there.
“Whoever as secretary of the treasury shall undertake and succeed in paying the debt,” he argues in closing, “must have open to him the three great avenues of taxation, namely, the tariff, the excise system, and the duties on exports, and must be empowered to use each in its appropriate place, by congressional legislation.”
And so he closed the first half of his second congressional year, with the same policy of questions with which he began, aiming still at thoroughness and mastery, still the guiding stars of his history, the moulding powers and the prominent features of his great career.
XII.
CONTINUED WORK IN CONGRESS.
T is Inauguration Day in Washington. Not McClellan,—he is in Europe,—but Lincoln is to be inaugurated. It is a day of wondrous glory to him, and to the nation, but one so oppressed with the cares of state has but little joy in it. There is no retiring president to sign all the tardy bills of an expiring congress. He must do it all, and then go from the realizations of the past to the unknown of the new. There was no instant of rest for him between laying off the armor and putting it on anew.
Of all the many thousand eyes that looked on him that day, none were more brilliant with the look of praise, none gleamed with a soullight more fervent, none took in the scene with deeper thoughts of the hour or the future, oppressive with interest, than Mr. Blaine.
Little did he dream of twenty years to come. He had thought to scale the centuries as they stood like silent statues in the sombre, shadowy past, and read out the hieroglyphics of their history. But just as the rebellion was broken, shattered, staggering to its fall, and seemed certain, and was scarce hung about with doubt, so now to faith the future is bright and clear, while hope is strong and almost gay with vivid anticipations.
Mr Blaine was profoundly impressed with the religious character of Abraham Lincoln, as exemplified in the tone of his public documents. He says: “Throughout the whole period of the war he constantly directed the attention of the nation to dependence on God. It may
indeed be doubted whether he omitted this in a single state paper In every message to congress, in every proclamation to the people, he made it prominent. In July, 1863, after the battle of Gettysburgh, he called upon the people to give thanks because ‘it has pleased Almighty God to hearken to the supplications and prayers of an afflicted people, and to vouchsafe signal and effective victories to the army and navy of the United States,’ and he asked the people ‘to render homage to the Divine Majesty and to invoke the influence of His Holy Spirit to subdue the anger which has produced and so long sustained a needless and cruel rebellion.’”
“On another occasion,” writes Mr. Blaine, “recounting the blessings which had come to the Union, he said: ‘No human counsel hath devised, nor hath any mortal hand worked out these great things. They are the gracious gifts of the Most High God, who, while dealing with us in anger for our sins, hath nevertheless remembered mercy.’
Throughout his entire official career, attended at all times with exacting duty and painful responsibility, he never forgot his own dependence unto the same authority, or the dependence of the people upon a Higher Power.” And then he quotes those words of the great man, uttered reverently to the people assembled in crowds to congratulate him upon the return of peace: “In the midst of your joyous expressions, He from whom all blessings flow must first be remembered.”
His last inaugural, delivered but a little while before this final utterance, was in keeping with it. It was a deeply religious document, referring to no political measure or material interest, and in six days after the people crowd about him, full of joy at the close of the war, the bullet of the assassin is in his brain! What a week was that in which the war closed, and the great Lincoln was murdered! And what a summer was that, when the broken armies came marching home, halting in Washington for the great review!
But a campaign is on Mr. Blaine, and he hurries home. For the third time Samuel Coney is elected governor, and Mr. Blaine has again done his work well. Autumn passes, and he is in his place at the opening of the thirty-ninth congress. With his usual unforgetfulness, he resumes connection with a bill presented by him in the early part
of the previous congress, for reimbursing the loyal states for warexpenses in response to the president’s call for troops. His bill is very explicit, and shows that during the long delay he had perfected it in its details. No flaw is found in it, no amendment is made to it, but it is at once referred, upon his motion, to a select committee of seven, and upon his motion he demands the previous question, so that the matter shall be attended to at once. The bill was read a first and second time, and so referred.
Mr. Blaine is of course upon the committee, and by his motion members are added to it, and they are empowered to hire a clerk. What a work to examine and pass upon all the war-debts of the loyal states! A grave question soon makes its appearance in congress. In undoing the legislation of years, enacted in the interests of slavery, they have come to the basis of representation. The slave is not yet a citizen, and if the basis is population and not suffrage, the South will have an immense advantage,—indeed an advantage similar to that enjoyed before the war, when, though slaves were expressly recognized as chattels, and according to the Dred Scott decision, “a black man had no rights a white man was bound to respect,” yet, according to slavery law five of them gave their master three extra votes.
But the ratio of voters to population varied from nineteen to fifty-eight per cent. in different states, as, for example, California had two hundred and seven thousand voters out of a population of three hundred and fifty-eight thousand one hundred and ten, while Vermont had but eighty-seven thousand voters out of a population of three hundred and fourteen thousand three hundred and sixty-nine, and each had three representatives in congress; that is, eightyseven thousand voters in Vermont sent three congressmen, while two hundred and seven thousand voters in California sent but the same number.
There were more women and children in Vermont, two to one, than in California, and so in the latter state there were more than twice as many voters in the same population.
It was with such arguments as the above,—a mathematical argument, without sophistry, and that cannot be impeached,—that he opposed a constitutional amendment making suffrage and not population the basis of representation, and so reserving an argument to use in framing the citizenship of the freemen.
Mr. Blaine has long been noted for the great rapidity with which he works.
He very soon has an immense report from his committee of nine, to pay the loyal states their war-claims. In it twenty-six states, five territories, and the District of Columbia have their war-claims adjusted, and they are to receive all the way from nine thousand nine hundred and fifty-five dollars, as in the case of the territory of Dakota for enlisting one hundred and eighty-one men, up to twenty million nine hundred and ninety-three thousand two hundred and eighty dollars, as in the case of New York for enlisting three hundred and eighty-one thousand six hundred and ninety-six men; and it is a peculiarity with him to know for himself, by careful computation, the exact truth of the statistics he employs.
One day it so happened that he used the calculations of a distinguished member who was chairman of a prominent committee, —that of ways and means,—and they were called in question; but soon after he was able to affirm publicly that they were correct.
There was such a charm in being right and knowing it, despite all contradiction, that he could not forego the pleasure, the very confidence and self-respect, even at the expense of perplexing effort. A point of order was raised against him one day; his instant reply was, “That point was raised exactly ten years ago and overruled,” and the chair ruled in harmony with his remembrance.
His great love for mathematics, and the position he was in requiring it, he was led to make an extensive study of the history of finance, and in a speech of great length, by which he supported his report to pay the vast war-claims of the loyal states, he clearly shows the wide range of his acquaintance with the subject. He shows great familiarity with the policy and utterances of Alexander Hamilton, his exceeding common-sense methods, which he quotes with so great
