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MICROBIOLOGY RESEARCH ADVANCES

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 MICROBIOLOGY RESEARCH ADVANCES

METAGENOMICS
METHODS, APPLICATIONS
AND PERSPECTIVES

CAMILLA BENEDETTI
EDITOR

New York
Copyright © 2014 by Nova Science Publishers, Inc.

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 CONTENTS
Preface vii
Chapter 1 Potential and Limitations of Metagenomic Functional Analyses 1
Laura Terrón-González, Olga Genilloud and Eduardo Santero
Chapter 2 Metagenomics: Library Construction and Screening Methods 45
Roberto S. Dias, Lívia C. F. Silva, Monique R. Eller,
Valéria M. Oliveira, Sergio O. DE Paula and Cynthia C. Silva
Chapter 3 The Use of Ion Torrent PGM for Bacterial Diversity Analyses:
The Study Case of Five Brazilian Hydroelectric Reservoirs 67
Diego Assis das Graças, Rafael Azevedo Baraúna,
Luciano Chaves Franco, Tiago Ferreira Leão,
Pablo Henrique Caracciolo Gomes de Sá,
Adonney Allan de Oliveira Veras, Adriana Ribeiro Carneiro,
Jaqueline Meireles, Kenny da Costa Pinheiro,
Artur Luiz da Costa da Silva and Rommel Thiago Jucá Ramos
Chapter 4 Metagenomic Profiling for Assessing Environmental Healthy 87
Felipe H. Coutinho, João Victor R. Ferreira, Andressa S. Silva,
Ida Carolina N. Direito, Denise M. M. Pessoa
and Alexander M. Cardoso
Chapter 5 Mining Novel Genes and Enzymes of Uncultured Eukaryotic
Microorganisms by an RNA-Based Approach 99
Nobutada Kimura
Chapter 6 Developments in the Retrieval of Novel Biocatalysts
by Metagenomic Approaches 115
Digvijay Verma and T. Satyanarayana
Chapter 7 Isolation of Soil Metagenomic DNA: Challenges and Solutions 137
Sonia Sharma, Kailash Narayan Bhardwaj, Sangeeta Pandey
and Ramesh Chander Kuhad
Chapter 8 Microbial Exploration in Extreme Conditions: Metagenomic
Analysis and Future Perspectives 157
S. Ramganesh, A. T. Maredza and M. Tekere
Index 183
 PREFACE
This book discusses metagenomics' methods, applications and perspectives.
Chapter 1 – Metagenomic analysis has extraordinary potential to improve our
understanding of microbial populations in their natural environment and identify novel genes
of interest. The key feature of such analyses is that they are performed using metagenomic
libraries constructed from total DNA isolated from a particular niche rather than a laboratory
culture. Thus, metagenomic analyses potentially allow access to all the genetic resources
present in an environment, regardless of whether or not they belong to microorganisms that
can be cultured in the laboratory. Sequence-based metagenomic analyses rely on comparisons
with databases of known genomic sequences whilst functional analyses rely on screening
libraries on the basis of the phenotypes cloned DNA can confer to host bacteria. Therefore,
functional analysis allows the identification of novel genes with functions that could not have
been predicted from their DNA sequence. However, a number of factors currently limit
access to the full potential offered by functional metagenomic analyses. One major restriction
is that despite the development of many procedures, indicators and genetic tools, we still lack
effective screening methods for many activities. Another major limitation is the inefficient
expression of some metagenomic genes in the host bacteria used for screening. Many
metagenomic genes are derived from bacteria with highly divergent physiologies and gene
expression machineries that are absent from the surrogate host. This review focuses on the
main problems that limit the potential of functional analyses and on approaches that can be
used to, at least partially, circumvent these problems and have allowed the identification of a
large number of different activities from metagenomic libraries.
Chapter 2 – In the last decade, metagenomics has created a great revolution in microbial
ecology. Metagenomics allows the study of uncultured microorganisms from the total DNA
of environmental samples. This is an important approach to elucidate the structure of
microbial communities and understand the functions that occur in complex environmental
samples. At first, metagenomics covered only the construction of metagenomic libraries by
cloning large fragments of DNA in appropriated vectors. Currently, with high-throughput
DNA sequencing technologies, the metagenomic approach involves the massive sequencing
of total DNA or total RNA, without the cloning step, of environmental samples. This chapter
is a review of different strategies that can improve the library construction or massive
sequencing of environmental samples and consequently increase the chances of finding a
positive hit. It will also describe screening methods currently based on sequence and function
to look for positive clones and interesting genes in the metagenomic library.
viii Camilla Benedetti

Chapter 3 – Characterizing the microbial community is the first step towards the
understanding of the ecological aspects of the hydroelectric reservoirs, since those
environments are highly complex and heterogeneous. One reason for this complexity is that
foundations of Hydroelectric Power Stations (HPS) flood a wide area in order to create the
reservoir, trapping a variety of organic matters from the vegetation around and sheltering a
rich fauna of fish and other animals. It‘s estimated that all area inundated by hydroelectric
reservoirs around the globe is equivalent to the area of the German territory.
Those engineered environments arouse the curiosity of the scientific community, since they
are considered a source of greenhouse effect gases (like methane and carbon dioxide),
interfering on the life cycle from (micro)organism. This chapter consists of an original
research that aimed to assess the microbial diversity of five HPS‘s reservoirs at the Brazilian
territory. The authors used semiconductor sequencing by Ion Torrent Personal Genome
Machine (PGM) to determine the bacterial diversity of five reservoirs from plants already in
operation – Xingó, Itaipú, Três Marias, Balbina and Funil. They collected one liter of water
from a series of points in the reservoirs to extract the total DNA, joining samples in a pool
according to the HPS. The authors performed a polymerase chain reaction to amplify the 16S
rRNA bacterial gene using universal primers with barcodes and sequencing adaptors to each
pool of sample. The authors sequenced the amplicons in a single run at Ion Torrent PGM,
chip 318. The sequencing resulted in 2,900,226 reads with quality value greater than 20 and
length greater than 100 base pairs, originating around 581 Mbp of highly reliable genetic
information. They submitted the sequences to alpha diversity and taxonomic analyses using
RDP pipeline and Mothur, which we consider robust and highly reproducible. The most
abundant bacterial phyla were Actinobacteria, Proteobacteria, Bacteroidetes, Verrucomicrobia
and Cyanobacteria. Other less representative phylum found were OD1, Planctomycetes, TM7,
Acidobacteria, Firmicutes, Nitrospira, Gemmatimonadetes, Chlamydiae, Chlorobi,
Fusobacteria, Deinococcus-Thermus, WS3, Armatimonadetes, Chloroflexi, OP11,
Lentisphaerae, SR1 and Thermotogae. The differences of microbial communities found in the
reservoirs illustrate the vast variability between the HPS‘s reservoir, in spite of that the most
abundant bacteria were assigned to the orders Burkholderiales, Rhizobiales and
Actinomycetales, but photosynthetic and methanotrophic bacteria were also significant in all
reservoirs.
Chapter 4 – Metagenomics provides new opportunities in environmental science and
technology. Next-generation sequencing (NGS) technologies, coupled with advanced
bioinformatics tools, have enabled rapid progress in microbial ecology and discovery of novel
genes. In this chapter, an overview of the current state of analysis of metagenomic data and
their possible applications for bioremediation of pesticides, strategies to combat global
climate change and development of novel biomarkers for assessing water quality in light of
currently available resources and tools are presented.
Chapter 5 – Analyses of microbial genome sequences have revealed numerous number of
gene sequences, with the potential to code the enzymes for using an industrial, agricultural
and pharmaceutical application. Metagenomics have identified a number of novel genes and
enzymes in the environment. However, eukaryotic microorganisms are not being used to be a
target of functional metagenomics. Since functional genes of eukaryotic microorganisms are
separated by intron, it is very difficult to screen functional genes of eukaryotic
microorganisms by DNA-based method. Metatranscriptomic is mRNA-based functional
community analyses method based on expressed genes is a more suitable means to identify
 Preface ix

eukaryotic genes and enzymes in the environment, because metagenomic analysis based on
DNAs cannot determine the structural genes whose introns are excluded, let alone detect
ecologically relevant active functions. An RNA-based metatranscriptomic approach can
circumvent the recurrent problems in the conventional metagenomic approach, and 3‘ poly-A
tails-specific purification and subsequent reverse transcription lead to construction of a cDNA
library, allowing comprehensive analyses of the eukaryotic genes specifically expressed. This
chapter summarizes the methods that have been developed for exploring the genetic and
functional diversity of eukaryotes by applying a metatranscriptomic approach to target genes
encoding an industrial application.
Chapter 6 – The search for ideal biocatalysts for specific applications is in progress all
over the world. Although spectacular advancements have been made in improving the
properties (stability to high pH and temperatures, affinity and activity) of biocatalysts using
protein engineering and directed evolution, there is still a huge gap between what are
available and those needed to be functional in extreme industrial process conditions. The
majority of enzymes that are in use today have been sourced from mere 0.1% of the
culturable microbes. There is a possibility of obtaining novel biocatalysts from the major
portion of non-culturable microbial diversity. The recently emerged culture–independent
approach, metagenomics, allows accessing genes encoding novel biocatalysts from the major
portion of non-culturable microbial diversity. The emerging field of metagenomics has truly
boosted the chances of discovering novel biocatalysts which will revolutionize biocatalysis.
Metagenomics helps in understanding culturable and non-culturable microbial diversity in the
environment besides discovering novel biocatalysts and other metabolites.
The recent developments in the retrieval of biocatalysts from a great variety of
environmental samples employing metagenomics approaches will be reviewed in this chapter.
First step in metagenomics is the extraction of humus-free DNA from the environmental
samples. This has been achieved by treatment of the extracted DNA with activated charcoal
and polyvinylpolypyrridine as well as using commercial kits. The second obstacle is the
screening libraries obtained from metagenomes, which is labor intensive. Robot-assisted
systems are now being used for this purpose. Sequence and activity driven analyses are the
two approaches for screening clones obtained through metagenomic libraries. Sequence
driven analysis is independent of expression of the cloned gene, while activity based
screening relies on the expression of the gene. As activity based screening has nothing to do
with already reported sequences in the database, the probability to access novel genes is more
as compared to the sequence based analysis that fully depends on existing sequences for the
respective genes. The function based approach always gives the full length gene of the
expressed clone, while the sequence based approach primarily retrieve the partial sequences.
Functional based approach is, therefore, more promising to reveal the hidden Pandora‘s box
of the inaccessible microbiota.
Several starch, cellulose, xylan, protein, lipid, chitin, phytate and pectin hydrolyzing
enzymes, and nitrilase, nitrile hydratase, and amidases have been discovered from
metagenomes of a great variety of environmental (normal and extreme) samples.
The discovery of enzymes by culture-independent metagenomic approaches is no more a
concept, but a reality. The Verenium Corporation, USA has commercialized several enzymes
including phytase developed through metagenomics. It is surprising that several highly
important enzymes like phytase, amylopullulanase, urease, superoxide dismutase, asperginase
and carbonic anhydrase represent the class of biocatalysts that are either not touched or
x Camilla Benedetti

properly exploited using this unconventional approach. Only one xylanase has been retrieved
by metagenomic approach that can withstand extreme conditions prevailing in paper and pulp
industries. Metagenomics is being peeled but extensive efforts are needed to understand the
mechanisms involved in finding the association of microorganisms with their habitat and their
unculturability on plates. Managing the metadata generated through metagenomics is another
challenge. This selfish technology must continue exploration of novel biocatalysts and other
biological products. The chapter focuses on recent developments made in the retrieval of
biocatalysts from a variety of environmental genomes employing metagenomic approaches.
Chapter 7 – Metagenomics, enabling the study of majority of unculturable microbes, has
opened new vistas of understanding the microbial diversity. Among various natural
environments being studied by the microbiologists, soil is probably the most challenging
reservoir with respect to microbial community size and diversity of species. Soils harbour a
large number of microorganisms, of which only 0.1-1.0% have been reported to be culturable
under standard conditions. Therefore, culture independent approach would play a major role
in unravelling the hidden microbial flora not only from soil but from other environmental
samples as well. The success of any soil metagenomic investigation is crucially dependent on
method(s) used for isolation of metagenomic DNA from soil samples. An ideal protocol
should enable an efficient in extraction of high molecular weight DNA free from inhibitors
and amenable to biotechnological manipulations. High yield of DNA is also an important
criterion for accessing the effectiveness of a protocol for isolation of the environmental DNA.
Also, the extracted DNA should be an unbiased representation of the microbial community of
the soil sample, i.e. the protocol should be able to extract DNA from hard to lyse cells and
rare species efficiently. Several protocols for isolation of soil metagenomic DNA have been
developed and these protocols have been grouped into two categories- strategies that consist
of direct extraction of nucleic acids from soil through in situ lysis and the second approach is
based on the separation of bacteria from the soil particles followed by the extraction of DNA.
This chapter focuses on an overview of the challenges encountered during extraction of soil
DNA and the strategies that can be adopted to achieve this challenging objective.
Chapter 8 – Over the past few decades microbial life in extreme environments has
attracted broad scientific interests. Extreme environments harbor microorganism that
represent the oldest inhabitants on Earth, and whose high adaptability has continued to
challenge our understanding of biochemistry, biology and evolution. The majority of
microorganisms cannot be cultivated using established laboratory methods thus requiring
alternative approaches for their characterization. Metagenomics is a culture-independent
genomic analysis, divided into sequence-based and function-driven analyses. These two
branches of metagenomics address the challenge of studying microbial communities and
functions in environments that are as yet unculturable and that represent more than 99% of
the organisms in extreme environments. This new approach expands our understanding of the
ecology and evolution of organisms, and aids in the discovery of diverse members of
previously undefined classes of microorganisms. Metagenomics, does not need any selection
(e.g. cultivation/enrichment) and greatly reduces technical biases often encounter in pure
culture selection. This review will compare methods, highlight progress, discusses
opportunities, challenges and perspectives in metagenomics with special reference to genome
discoveries in extreme hot environments.
In: Metagenomics ISBN: 978-1-61122-358-3
Editors: Camilla Benedetti © 2014 Nova Science Publishers, Inc.

Chapter 1

POTENTIAL AND LIMITATIONS OF METAGENOMIC

FUNCTIONAL ANALYSES

Laura Terrón-González1, Olga Genilloud2 and Eduardo Santero1

1
Centro Andaluz de Biología del Desarrollo
University Pablo de Olavide/CSIC/Junta de Andalucía, Sevilla, Spain
2
Fundación Medina, Parque Tecnológico de Ciencias de la Salud
Granada, Spain

ABSTRACT
Metagenomic analysis has extraordinary potential to improve our understanding of
microbial populations in their natural environment and identify novel genes of interest.
The key feature of such analyses is that they are performed using metagenomic libraries
constructed from total DNA isolated from a particular niche rather than a laboratory
culture. Thus, metagenomic analyses potentially allow access to all the genetic resources
present in an environment, regardless of whether or not they belong to microorganisms
that can be cultured in the laboratory. Sequence-based metagenomic analyses rely on
comparisons with databases of known genomic sequences whilst functional analyses rely
on screening libraries on the basis of the phenotypes cloned DNA can confer to host
bacteria. Therefore, functional analysis allows the identification of novel genes with
functions that could not have been predicted from their DNA sequence.
However, a number of factors currently limit access to the full potential offered by
functional metagenomic analyses. One major restriction is that despite the development
of many procedures, indicators and genetic tools, we still lack effective screening
methods for many activities. Another major limitation is the inefficient expression of
some metagenomic genes in the host bacteria used for screening. Many metagenomic
genes are derived from bacteria with highly divergent physiologies and gene expression
machineries that are absent from the surrogate host. This review focuses on the main
problems that limit the potential of functional analyses and on approaches that can be
used to, at least partially, circumvent these problems and have allowed the identification
of a large number of different activities from metagenomic libraries.


Corresponding author: Eduardo Santero ([email protected]).
2 Laura Terrón-González, Olga Genilloud and Eduardo Santero

1. MICROBIAL DIVERSITY
Microorganisms are the most abundant and diverse living creatures on Earth. They have
been able to colonize almost all natural environments despite the extremely harsh conditions
of many of them. The main way of establishing phylogenetic relatedness is through
comparison of 16S ribosomal RNA sequences (or 18S sequences in eukaryotes).
Identification of 16S ribosomal RNA sequences from many different environments has
revealed the enormous bacterial diversity on the planet, which have been classified into 52
different phyla. This type of analysis has also revealed that more than 99% of bacterial
sequences belong to unknown bacteria that have not yet been cultured in the laboratory, and
that a significant fraction of phyla do not contain a single cultured representative (Rappé &
Giovannoni, 2003). This means that our knowledge of the microbial world, including the
enormous reservoir of genetic and metabolic diversity, the structure of bacterial populations
in different niches and the interrelationships among them, is extraordinarily limited. Access to
this vast and diverse repository of information is an extraordinary challenge but its systematic
characterization is of great interest to microbiologists in general and to microbial ecologists
and biotechnologists in particular. Unraveling the mysteries of this unknown microbial world
will provide insights into microbial communities and their interactions with different habitats,
as well as information on new functionalities and access to the genetic resources of these
uncultured microbes (Ferrer et al., 2009), but will require the development of new techniques
and genetic tools to take advantage of the resources offered by uncultured microbes.
Although enormous efforts are being made to cultivate different kinds of bacteria from
complex habitats, a promising complementary approach is to access the genetic information
of these habitats through culture independent techniques. The idea of collecting information
from a habitat by directly isolating and subsequently sequencing DNA present in
environmental samples was pioneered by Norman Pace (Pace et al., 1985). Since then,
numerous studies have forced us to acknowledge the extent of our ignorance of the microbial
world. The enormous potential of this technique prompted many scientists to explore
uncultured microbes, thus creating a new discipline called metagenomics, a term coined by
Handelsman et al. (1998).
Initial metagenomic analyses were targeted small-scale projects aimed at identifying
microbial diversity in the environment, where metagenomic libraries were constructed with
amplicons of 16S rDNA (Hugenholtz et al., 1998). The advent of next generation sequencing
(NGS) enabled the undertaking of very large-scale shotgun metagenomic projects (Venter et
al., 2004; Tyson et al., 2004; Qin et al., 2010; Census, http://www.coml.org/; Earth
microbiome, http://www.earthmicrobiome.org/). These and other projects have permitted
huge advancements in microbial ecology since they allow functions to be assigned to
taxonomic groups and have the potential to help us understand not only specific ecological
niches but also their extended habitats as a whole (Gilbert et al., 2011).
Information gained from metagenomics also gives access to new biomolecules and
biocatalysts (enzymes), whose identification and application to medicine and different types
of industries is rapidly growing because of their novel properties, improved performance,
reduced costs or lower environmental burden in industrial processes.
Large metagenomic shotgun sequencing does not require the construction of
metagenomic libraries. However, the identification and exploitation of new bioproducts, so-
 Potential and Limitations of Metagenomic Functional Analyses 3

called bioprospecting, relies on the construction of large metagenomic libraries that are
maintained in surrogate hosts (Simon & Daniel, 2011).

2. OVERVIEW OF METAGENOMIC LIBRARY CONSTRUCTION

A key issue is the representativeness of information obtained from a metagenomic
library. Representativeness depends on the size of the library, sample biodiversity and the
bias generated during library construction and analysis.
A metagenomic analysis can completely cover the genomic diversity of sites representing
specialized niches with very low diversity, such as biofilms grown on acid mine drainage
contaminated water (Tyson et al., 2004). However, the representativeness of libraries from
more diverse environments, such as soils where up to 105 different genomes can be found, is
generally low (Vieites et al., 2009). This is because the number of independent clones and the
average size of metagenomic DNA inserted into each clone, which define the size of the
library, are limited. Therefore, one has to keep in mind that a metagenomic library is a
collection of genome fragments collected from a particular site, at a particular moment and
should be considered a partial representation of the site analyzed because of our limited
analysis capacity. In general, the most represented diversity in the library will be that of the
most abundant microorganisms in the sample and, therefore, we may miss a significant
number of low abundance species from the site.
Construction of a metagenomic library involves isolation of DNA from an environmental
sample and cloning in appropriate vectors that will be subsequently maintained in surrogate
bacterial hosts. In addition to our own limited analysis capacity, representativeness of the
sample may be even lower due to biases introduced at different steps during metagenomic
library construction and analysis. Several protocols for metagenomic library construction
have been published recently (Taupp et al., 2009; Simons & Daniel, 2010. Martínez &
Osburne, 2013; Sabehi & Béjà, 2013). It is not the aim of this section to go into the details of
these procedures but rather to highlight some general considerations relevant to functional
screening and the identification of bioproducts.

2.1. Sampling

A critical first step for successfully bioprospecting for particular functions or enzymatic
activities is to predict which sites may potentially be enriched for the activity of interest.
Constructing metagenomic libraries from these sites may increase the odds of obtaining
positive hits. For instance, if interested in plant polymer hydrolyzing activities, appropriate
sites for isolating microbiota would be the rumen of different animals (Ferrer et al., 2007;
Gruninger et al., 2014) or the guts of wood-feeding insects (Warnecke et al., 2007; Scully et
al., 2013). Similarly, if interested in xenobiotic contaminant degradation activities, the
sampling sites should be contaminated with the compound of interest or related compounds
(Brennerova et al., 2009). Alternatively, constructing libraries from complex and less studied
habitats, such as marine environments, may increase the chances of identifying completely
4 Laura Terrón-González, Olga Genilloud and Eduardo Santero

novel types of enzyme but may also reduce the overall number of positive hits (Kennedy et
al., 2011).
In many instances, it is desirable that enzymes have some particular extremophilic
characteristic such as activity at extreme temperatures, pHs, salt concentrations or pressures.
Halophilic bacteria and bacteria tolerant to extreme pHs have natural adaptations that enable
them to maintain more temperate conditions in their intracellular environments. This means
that their intracellular enzymes themselves may not be extremophilic. However, most of the
metagenomic enzymes identified by functional screening are extracellular enzymes that have
to be active under extreme environmental conditions. Therefore, if interested in enzymes with
particular extremophilic characteristics, the metagenomic DNA should be isolated from the
corresponding extreme environment. Nevertheless, there are a number of reports identifying
clones with extremophilic characteristics from non-extreme environments such as salt-
tolerant enzymes from pond water and the human gut (Kapardar et al., 2010; Culligan et al.,
2012), or thermostable or cold-adapted enzymes from soils (Faoro et al., 2012; Ko et al.,
2012).
One option for increasing the odds of identifying enzymes with an activity of interest is
to perform laboratory enrichment cultures under conditions that aim to increase the proportion
of target genes in the bacterial community of the sample. This deliberate introduction of bias
reduces the original biodiversity because the sample is enriched for bacteria that grow faster
under the imposed laboratory conditions.
As a consequence, it is possible that some genes or activities linked to bacteria that are
not favored under such conditions will be missed. On the other hand, this strategy may
significantly increase the chances of detecting genes or activities from low abundance
bacteria in the original sample.
A recent study has shown drastic changes to the bacterial community of a soil after chitin
enrichment (Jacquiod et al., 2013). As indicated by Daniel (2004), a comparison of positive
hits for alcohol oxidoreductases using two metagenomic libraries constructed from the same
environmental sample, one without prior enrichment (Knietsch et al., 2003a) and the other
after enrichment for polyol-fermenting microorganisms (Knietsch et al., 2003b), showed that
screening half of the clones from the enriched library resulted in the same number of positive
clones. However, 50% of the sequences from the unbiased library were unrelated to
previously known sequences, compared to only 10% for the biased library.
In spite of the potential loss of biodiversity, enrichment is common practice and several
recent reports have shown its beneficial effects (Tan et al., 2013; Costa et al., 2014).
Enrichment may be particularly suitable for samples with low bacterial density (Ferrer et al.,
2005a), when searching for particular enzyme characteristics, such as high thermostability, if
it is possible to set up appropriate enrichment conditions (Chow et al., 2012), or when
samples come from complex environments where the targeted activity may not be particularly
enriched, for instance, when searching for hydrolyzing activities in soil samples (Gabor et al.,
2004a; Jacquiod et al., 2013).
In addition, enrichment may avoid the problems caused by the presence of inhibitory
substances when extracting DNA from environmental samples (see below).
 Potential and Limitations of Metagenomic Functional Analyses 5

2.2. Isolation and Purification of DNA from Bacterial Samples

There are four important characteristics that define the suitability of DNA obtained from
an environmental sample for construction of metagenomic libraries: purity, yield,
representativeness and fragment size. Many different protocols have been published for
extracting DNA but their suitability depends on the objective of the metagenomic library and
the origin of the sample. Soils are particularly difficult environments for isolating DNA
samples because of their physicochemical properties that lead to uneven distribution of
microbes and to contamination with humic and fulvic acids that inhibit subsequent enzymatic
reactions (Daniel, 2005). Lombard et al. (2011) have recently reviewed the advantages and
drawbacks of different procedures for sampling and isolating soil DNA in terms of quality
and quantity. The different procedures can be divided into two broad categories: direct
extraction of DNA from the environmental sample, and indirect extraction, which involves
the isolation of cells prior to lysis (Gabor et al., 2003; van Elsas et al., 2008a). Direct
extraction may lead to a less biased DNA sample but also implies isolating DNA from all
eukaryotic and prokaryotic organisms present in the sample. Procedures involving the
separation of cells by physical (blending) or chemical means followed by gradient
centrifugation or size filtration to remove large eukaryotic cells may enrich the sample for
bacteria while reportedly maintaining functional diversity (Delmont et al., 2011). It may also
remove inhibitory contaminants from the environmental sample, although yield may also be
reduced (Gabor et al., 2003; Daniel, 2005; Sabehi and Bèjá, 2013).
Cell lysis may be gentle or harsh. Harsh lysis involving a combination of mechanical
methods, such as agitation in the presence of glass beads, and chemical/enzymatic methods is
the most efficient way of isolating DNA.
Some protocols estimate that up to 90% of the bacterial cells in a given soil are lysed
(Howeler et al., 2003). However, the resulting DNA is sheared and unsuitable for the
construction of large fragment metagenomic libraries (Gabor et al., 2003). On the other hand,
gentle lysis based on chemical reagents clearly yields higher quality large DNA fragments.
However, the major drawback is that species resistant to chemical attack, such as Gram
positive bacteria, may be underrepresented (Lombard et al., 2011).

2.3. Cloning into the Right Vector

Vector selection is a critical part of metagenomic library construction. The choice of

vector influences the size of inserts that can be cloned and the likelihood that metagenomic
genes will be expressed. There are four general types of vector: expression plasmids, lambda
vectors, cosmids and fosmids, and bacterial artificial chromosomes (BACs). Examples of
each vector class suitable for building metagenomic libraries, along with their characteristics,
are described in Table 1.
Standard plasmids can be used to construct small insert libraries (smaller than 10 kb).
Depending on their replication systems, these plasmids can be maintained in a number of
different bacterial species, although all of them can replicate in high copy numbers in E. coli,
the bacteria normally used to host metagenomic libraries. While much larger fragments can
be cloned into these vectors, this would reduce transformation frequency, therefore, the
number of clones, and clone stability due to inefficient replication of such large replicons.
 Table 1. Vectors used for metagenomic libraries. Abbreviations: Ap: ampicillin; Hy: hygromycin; Tc: tetracycline; Cm:
chloramphenicol; Km: kanamycin; Am: apramycin; Er: erythromycin; Gm: gentamicin. *1 When the vector used for the construction
of the metagenomic library was previously described, the vector reference is indicated in parenthesis. *2 Used for genomic libraries and
proposed to be used for metagenomic libraries. *3 Vectors integrate into the chromosome but cannot replicate in those bacteria. *4
Vectors have the cos site but they were used as BACs and the libraries were not packed in lambda heads; the DNA inserts in some of
them are larger than those that could accommodate if packaged in lambda

Insert size; marker;

Vector Replicon Copy number Host range Vector promoter Reference(s)*1
other features
Lambda
ZAP II E. coli 5 kb; ApR Lee et al., 2012a
ZAP Express E. coli plac 5 kb; KmR Rees et al., 2003; Ferrer et al., 2005b
Plasmids
p18GFP pUC Veryhigh E. coli 7 kb; ApR; promoter trap Uchiyama et al., 2005
pBGR1 E. coli 5-10 kb; ApR; promoter trap (Park & Kim, 2010); Hwang et al., 2012
pBluescript II SK / pUC Very high E. coli 3-8 kb; ApR Waschkowitz et al., 2009; Guane t al., 2007
KS(+)
pCR-XL-TOPO pUC Very high E. coli 4 kb; KmR Simon et al., 2009
pCR2.1-TOPO pUC Veryhigh E. coli plac 2-5 kb; KmR, ApR Waschkowitz et al., 2009
pJOE930 pUC Very high E. coli plac 3-8 kb; ApR (Altenbuchner et al., 1992); Lämmle et al.,
2007
pUC18 pUC Very high E. coli plac 2-9 kb; ApR Peng et al., 2011
pZerO-2 pUC Very high E. coli plac 5.5 kb; KmR van Hellemond et al., 2007
pETDuet1 ColE1 High E. coli pT7 1-3 kb; ApR Owen et al., 2012
pGEM-3Zf(+) pUC Very high E. coli pT7 1-15 kb; ApR Jiang et al., 2010
pCF430 RK2 Low Broad Gram (-) para 5-10 kb; TcR (Newman & Fuqua, 1999); Allen et al., 2009
pHT01 ColE1 High E. coli, Bacillus 9-20 kb; ApR, CmR Biver et al., 2013b
BACs
pBAC-1003 F Low E. coli, Streptomyces*3 100 kb; AmR Ouyang et al., 2010
pMBD14 F Low E. coli, Streptomyces*3, Up to 85 kb; CmR, Martínez et al., 2004
Pseudomonas*3 AmR; oriT
pPAC-S1*2 F Low E. coli,Streptomyces*3 Up to 140 kb; KmR Sosio et al., 2000
pGNS-BAC F and Low E. coli and 80 kb; GmR, CmR; oriT Kakirde et al., 2011
RK2 Broad Gram (-)
Cosmids
pJC8 RK2 low Broad Gram (-) 37 kb; TcR, GmR; oriT; Cheng et al., 2014
Gateway®
 Insert size; marker;
Vector Replicon Copy number Host range Vector promoter Reference(s)*1
other features
pJC24 RK2 low Broad Gram (-) TcR; oriT; Gateway® Cheng et al., 2014
pWE15 ColE1 High E. coli pT7 25-40 kb; ApR Voget et al., 2003
pWEB ColE1 High E. coli pT7 ApR Brady et al., 2004
Supercos I pUC Veryhigh E. coli pT7 ApR Brady et al., 2004
pJWC1 RK2 Low Broad Gram (-) TcR Craig et al., 2009; Craig et al., 2010
pKS13S RK2 Low Broad Gram (-) 25 kb; TcR; oriT Ono et al., 2007
pLAFR3 RK2 Low Broad Gram (-) 25 kb; TcR; oriT (Staskawicz et al., 1987); Wexler et al., 2005
pRK7813 RK2 Low Broad Gram (-) 33 kb; TcR; oriT (Jones & Gutterson, 1987); Wang et al., 2006
pMM436 pUC Veryhigh E. coli, Streptomyces*3 35 kb; AmR; oriT; Easy McMahon et al., 2012
insert recovery
pOS700I ColE1 High E. coli, Streptomyces*3 50 kb; ApR, HyR Courtois et al., 2003
pFX583 pMB1 and High E. coli, Streptomyces pT7 35-45 kb; KmR; oriT Lussier et al., 2010; Lussier et al., 2011
pJV1
pEBP18*2 ColE1 High E. coli, Pseudomonas, pT7and KmR, CmR Troeschel et al., 2012
(min+) Bacillus*3 p(Xyl)
pEBP41*2 ColE1 High Broad Gram (-), Bacillus pT7 and p(Xyl) KmR, GmR Troeschel et al., 2012
pBBR1
pUB110
Fosmids
pEpiFOS-5 F Low E. coli 35 kb; CmR Lee et al., 2004; Lim et al., 2005
pSMART BAC*4 F and Low and High E. coli >50 kb; ApR Gong et al., 2013
RK2 inducible
pBeloBAC11*4 F Low E. coli pT7 36 kb; CmR (Kim et al., 1996); Rondon et al., 2000
pCC1FOS / F and Low and High E. coli pT7 33 kb; CmR Suenaga et al., 2007; Silva et al., 2013
pCC2FOS RK2 inducible
pUvBBAC*2*4 F and Low E. coliand Broad Gram Up to 178 kb; CmR, ErR Hain et al., 2008
pIP501 (+)
pCT3FK*2 F and Low and High E. coli, pT7 35 kb; CmR, KmR Angelov et al., 2009
RK2 inducible Thermusthermophilus*3
R
pRS44-pTA44 F and Low and High Broad Gram (-) pT7 35 kb - up to 200 kb; Cm , Aakvik et al., 2009
RK2 inducible Dependent of TnRS48 KmR; oriT
pMPO579 F and Low and High E. coli pT7 and Psal 35 kb; CmR; oriT; promoter Terrón-González et al., 2013
RK2 inducible trap; N antitermination
8 Laura Terrón-González, Olga Genilloud and Eduardo Santero

On the other hand, the smaller the insert, the greater the number of clones required to
cover the same length of metagenomic DNA. In addition, if the insert is too small, only
functions/activities encoded by a single gene can be identified. Another caveat is that high
copy number plasmids with strong promoters may drive the overexpression of genes, with
potentially deleterious effects to the host bacteria.
On the other extreme are BACs, which possess the replication and partition functions of
the very stable single copy F plasmid. It has been shown that vectors based on the F plasmid
can accommodate and stably replicate very large DNA inserts of up to 300 kb (Shizuya et al.,
1992), which represents 10-15% of the several megabases that make up a typical bacterial
genome. An additional advantage is that the low copy number prevents the overexpression of
potentially toxic genes. However, the inherent technical difficulty of working with very large
DNA fragments is a major disadvantage (Sabehi and Bèjá, 2013). In practice, this means that
the usual insert size in BAC-based metagenomic libraries is significantly smaller than 300 kb
(Aakvik et al. 2011) (see Table 1). Additional disadvantages that reduce representativeness
include the low number of transformants obtained, which reduces coverage, and the bias due
to the gentle cell breakage that is required to obtain large DNA inserts. These vectors are
suitable for metagenomic analyses of bacterial communities because large inserts facilitate
the assembly of individual genomes. The main disadvantages for functional screening are that
libraries can only be screened in E. coli and that the expression of metagenomic genes relies
on their own expression capacity inside the surrogate E. coli host. BAC vectors carrying
additional replication systems or able to integrate into the bacterial chromosomes seem
promising since they expand the range of potential host bacteria for functional screening to
different Gram positive and Gram negative bacteria (Table 1).
The common feature of the remaining types of vector is that they allow metagenomic
library clones to be packaged into lambda phage heads. This advance increases the number of
clones of a metagenomic library due to more efficient transfection-based introduction of
foreign DNA into E. coli. The size of the DNA that can be packaged is limited to
approximately 50-52 kb, a little over the whole lambda phage genome. Lambda phage
vectors, which produce infection plaques on a lawn of E. coli, accommodate small insert
DNA fragments with an average size of 5-6 kb. For obvious reasons, lambda phage vectors
cannot be directly used for selective functional screening (see below) although functional
screens of metagenomic libraries using lambda phage vectors have been successful in
identifying a number of different enzyme activities (Ferrer et al., 2005a; Ferrer et al., 2005b;
Wang et al., 2009). Since phages lyse the host cells, they may be appropriate for functional
screening of intracellular enzymes that are difficult to detect by direct colony screening. On
the other hand, functional screening of some activities on phage plaques appears to be less
sensitive than that on grown colonies (Cottrell et al., 1999), probably because of the lower
enzyme-producing cell biomass.
Cosmids are plasmid vectors that bear the lambda cos site, which allows packaging of
DNA molecules into lambda heads. A number of cosmids have broad host range replication
systems that enable them to maintain the metagenomic library in different bacterial hosts, thus
allowing functional screening to be performed in a wider variety of species (Table 1). Fosmid
vectors combine the advantages of BACS and cosmids. Since they possess the very stable F
plasmid replication and partition systems, they can be used as BACs to construct very large
insert libraries, or alternatively, they can be used as cosmids for efficient transfection of E.
coli with smaller insert libraries. Cosmids and fosmids can accommodate DNA inserts of up
 Potential and Limitations of Metagenomic Functional Analyses 9

to 40 kb, depending on the size of the plasmid vector. This size is sufficiently large to allow
the cloning of complete gene clusters whose combined activity may be required for some
functions. In our view these types of vector represent a suitable balance between insert size,
ease of use and cloning efficiency. Fosmids have the disadvantage that most fosmid
metagenomic libraries can only be maintained in E. coli although fosmids bearing additional
broad host range replication systems have now been developed (Table 1).
One of the major limitations of functional metagenomic screens is the difficulty in
achieving efficient heterologous expression of library clones in the surrogate bacterial host.
A number of different types of vectors incorporate features that aim to, at least partially, solve
this problem. These features will be further analyzed in Section 5.

2.4. Host Bacteria

Once metagenomic DNA fragments of an appropriate size have been cloned into the
vector of choice, they have to be introduced and maintained in a surrogate bacterial host.
Essentially, all vectors can replicate in E. coli and most of the advantageous features of
vectors for constructing metagenomic libraries, such as stable replication of large inserts in
BACs or the high packaging efficiency of lambda heads for subsequent transfection, are only
applicable to E. coli. In addition, a number of mutant E. coli strains are available to increase
the number of clones that can be obtained, and DNA can be very easily recovered from E.
coli strains. All these features make E. coli by far the most common host for constructing
metagenomic libraries.
Most metagenomic analyses are directly performed using E. coli. However, when
functionally screening for activities of interest, in order to eliminate the limitations of a single
host, it is often desirable to be able to screen the library using alternative host species. In
addition to E. coli, other Gram negative bacteria such as Pseudomonas, Sphingomonas,
Burkholderia, Agrobacterium, Xanthomonas and Rhizobium (Taupp et al., 2011), and Gram
positive species including Bacillus, Lysteria and Streptomyces (Aakvik et al., 2011) have
been successfully used for such screens. Features of vectors designed for functional screening
in heterologous hosts are further analyzed in Section 5.

3. METAGENOMIC LIBRARY ANALYSES

3.1. Sequence-Driven Analysis

Sequence-driven analysis of metagenomes is mainly aimed at identifying the major

bacterial populations present in a particular ecological niche and to understand the ecological
relevance of different functional metabolisms. However, this approach may also lead to the
identification of genes coding for functions or activities of biotechnological interest.
Identification of these genes may be achieved through a number of techniques, including gene
targeting through hybridization, PCR amplification and direct sequencing of metagenomes.
Colony hybridization using probes specific for domains conserved among proteins of
interest followed by sequencing of the positive clones, has been a successful approach for the
10 Laura Terrón-González, Olga Genilloud and Eduardo Santero

isolation of genes of interest (Demanèche et al., 2009a; Demanèche et al., 2009b). In order to
screen a large number of clones on a single membrane it is necessary to plate a high density
of colonies, which may limit the sensitivity of the screen, or require performing many
different screens, which makes this approach very labor intensive. An alternative to standard
colony hybridization is to use metagenomic microarrays, which can screen for a large number
of clones on a single slide (Park et al., 2008; Pathak & Gärtner, 2010). This approach has the
inherent limitation that it only identifies metagenomic sequences that are sufficiently similar
to the selected probe. Identification of a positive hit will depend on the degree of similarity
between the target and the probe, and the stringency of the hybridization conditions used.
An alternative to hybridization is PCR amplification. This involves the multialignment of
many known gene sequences from different bacteria, and identification of the most conserved
regions. These conserved regions are used to design degenerate primers that would amplify
similar regions from metagenomic library clones. PCR amplification allows the identification
of positive clones, which can then be sequenced from the flanking regions (Kotik, 2009;
Tuffin et al., 2009). This approach has been successful in identifying particularly interesting
genes within bacterial communities, such as nitrogen fixing genes in acid mine drainage
metagenomic libraries (Dai et al., 2014), enzymes of industrial relevance such as
oxidoreductases, lipases and esterases (Eschenfeldt et al., 2001; Bell et al., 2002), and also
genes encoding biologically active molecules such as polyketide synthases with antimicrobial
activity (Courtois et al., 2003; Feng et al., 2011), and anticancer and immunosuppressive
compounds (Owen et al., 2013). However, it is a labor intensive approach since it implies
manipulating individual library clones. Of course, gene identification is restricted to those
genes that are sufficiently similar to the chosen primers for correct hybridization and DNA
amplification.
In general, metagenomic libraries containing very large DNA inserts are most suited for
gene targeted screens, since it allows the screening of more DNA with lower clone numbers
and also facilitates the isolation of other linked genes that may be involved in the same
function.
The development of NGS technologies has allowed the sequencing of metagenomic
clones either separately or in pools, and the development of large-scale shotgun sequencing
projects that do not require the prior construction of metagenomic libraries. These large
sequencing projects have provided a huge amount of information on the genomes of
uncultured bacteria. Although this information has not yet been sufficiently exploited, data
mining of sequence databases allows the identification of metagenomic genes encoding
proteins of interest. These genes can be amplified by PCR if DNA samples are still available
or, alternatively, can be chemically synthesized. This approach named ―synthetic
metagenomics‖ has been successfully applied to isolate methyl halide transferase enzymes
(Bayer et al., 2009) and glycoside hydrolases (Allgaier et al., 2010).
Since sequence-based screening relies on sequence similarity to previously known
sequences, it has the inherent limitation that identified genes are unlikely to be strongly
divergent from already known genes and, therefore, precludes the identification of completely
different genes that may have evolved activities of interest by functional convergence.
However, sequence-based screening approaches may be particularly suited for the
identification of small biologically active molecules, which require a large number of genes
that are not normally expressed in surrogate hosts (Piel, 2011; Culligan et al., 2014).
This method is also suitable when searching for extremophilic enzymes such as cold or heat
The Project Gutenberg eBook of My four
weeks in France
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Title: My four weeks in France

Author: Ring Lardner

Illustrator: Wallace Morgan

Release date: February 27, 2024 [eBook #73059]

Language: English

Original publication: Indianapolis: The Bobbs-Merrill Company,

1918

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*** START OF THE PROJECT GUTENBERG EBOOK MY FOUR WEEKS

IN FRANCE ***
 Transcriber’s Note
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MY FOUR WEEKS IN FRANCE
A password was what he wanted, and Mr. Poincaré had forgotten to
give me the correct one
MY FOUR WEEKS
IN FRANCE
By
RING W. LARDNER
AUTHOR OF

Gullible’s Travels, Etc.

ILLUSTRATED BY

WALLACE MORGAN
INDIANAPOLIS
THE BOBBS-MERRILL COMPANY
PUBLISHERS
 Copyright 1918
The Bobbs-Merrill Company

PRESS OF
BRAUNWORTH & CO.
BOOK MANUFACTURERS
BROOKLYN, N. Y.
 CONTENTS
CHAPTER PAGE
I Dodging Submarines to Cover the Biggest Game of All 9
II I Get to Paris and Encounter Some Strange Sights 30
III I Try to Get to the American Camp—But Meet Disaster 54

IV Finally I Get to the American Camp; What I Find There 76

V My Adventures at the British Front 100
VI How I Didn’t Drive Major Blank’s Car to Camp Such-
and-Such 128

VII I Start Home, with a Stop-Over at London 146

VIII Back in Old “O Say”; I Start Answering Questions 171
 MY FOUR WEEKS IN FRANCE

I
DODGING SUBMARINES TO COVER THE
BIGGEST GAME OF ALL

Wednesday, July 18. A Lake Michigan Port.

I kept an appointment to-day with a gentleman from Somewhere
in Connecticut.
“How,” said he, “would you like to go to France?”
I told him I’d like it very much, but that I was thirty-two years
old, with a dependable wife and three unreliable children.
“Those small details,” he said, “exempt you from military duty.
But we want you as a war correspondent.”
I told him I knew nothing about war. He said it had frequently
been proved that that had nothing to do with it. So we hemmed and
we hawed, pro and con, till my conscientious objections were all
overruled.
“In conclusion,” said he, “we’d prefer to have you go on a
troopship. That can be arranged through the War Department.
There’ll be no trouble about it.”
 Monday, July 30. A Potomac Port.
To-day I took the matter up with the War Department, through
Mr. Creel.
“Mr. Creel,” I said, “can I go on a troopship?”
“No,” said Mr. Creel.
There was no trouble about it.

Wednesday, August 1. An Atlantic Port.

The young man in the French Consulate has taken a great fancy
to me. He will not visé my passport till I bring him two more
autographed pictures of myself.
George W. Gloom of the steamship company said there would be
a ship sailing Saturday.
“Are we convoyed through the danger zone?” I inquired.
“We don’t guarantee it,” said he. “There has never been an
accident on this line,” he added.
“What I was thinking about,” said I, “wouldn’t be classed as an
accident.” Further questioning developed the comforting fact that the
ship I am taking has never been sunk.
I told him I wanted a cabin to myself, as I expected to work.
“You will be in with two others,” he said.
“I would pay a little more to be alone,” said I.
This evidently was not worth answering, so I asked him how
long the trip would take.
“I know nothing about it,” said he.
“I believe that,” said I when I was well out of his ear-shot.
 Wednesday, August 8. At Sea.
We left port at ten last night, a mere three and a half days
behind schedule. The ship and I should be very congenial, as we are
about the same age.
My roommates are a young man from Harvard and a young man
from Yale, but so far I have managed to keep the conversation
neutral. We suspect that they made ours a first-class cabin by
substituting the word 1ère for 2ème on the sign, and I am very
certain that my berth was designed for Rabbit Maranville.
Our passenger list includes a general, a congressman, a lady
novelist and her artist husband, French; a songbird, also French; two
or three majors, a Thaw, and numerous gentlemen of the consular
service. The large majority on board are young men going into
American Ambulance and Y. M. C. A. work.
After breakfast this morning there was life-boat drill, directed by
our purser, who is permanently made up as Svengali. He sent us
down to our cabins to get our life-belts and then assigned us to our
boats. Mine, No. 12, is as far from my cabin as they could put it
without cutting it loose from the ship, and if I happen to be on deck
when that old torpedo strikes, believe me, I’m not going to do a
Marathon for a life-belt. Shoes off, and a running hop, step and jump
looks like the best system. Moreover, I’m going to disobey another of
the rules, which is that each passenger must remain calm.
Next we had to fill out a form for the enlightenment of Svengali
as to our destination, business, home address, foreign address,
literary tastes, etc. One item was “the names of relatives or friends
you lofh.” This was unanswered, as nobody aboard seemed to know
the meaning of the verb.
In the fumoir this afternoon a young American wanted a match.
He consulted his dictionary and dug out “allumette.” But he thought
the t’s were silent and asked Auguste for “allumay.” Auguste
disappeared and returned in five minutes with a large glass of
lemonade. The cost of that little French lesson was two francs.
I am elected to eat at the “second table.” Our bunch has
luncheon at twelve-thirty and dinner at seven. The first table crowd’s
hours are eleven and five-thirty. Breakfast is a free-for-all and we sit
where we choose. My trough mates at the meals are two Americans,
a Brazilian, and four Frenchmen. Ours is a stag table, which
unfortunate circumstance is due to the paucity of women, or, as they
are sometimes called, members of the fair sex. The Brazilian speaks
nine or ten languages, but seems to prefer French. The two
Americans are always engaged in sotto voce dialogue, and the four
Frenchmen race with the Brazilian for the conversational speed
championship of the high seas. This leaves me free to devote all my
time to the proper mastication of food.

Thursday, August 9. Completely at Sea.

A gentleman on board is supplied with one of these newfangled
one hundred dollar safety suits. The wearer is supposed to be able
to float indefinitely. It is also a sort of thermos bottle, keeping one
warm in cold water and cool in hot. I do not envy the gent. I have
no ambition to float indefinitely. And if I didn’t happen to have it on
when the crash came, I doubt whether I could spare the time to
change. And besides, if I ever do feel that I can afford one hundred
dollars for a suit, I won’t want to wear it for the edification of mere
fish.
When Svengali isn’t busy pursing, he is usually engaged in chess
matches with another of the officers. The rest of the idle portion of
the crew stand round the table and look on. Sometimes they look on
for an hour without seeing a move made, but they never seem to
lose interest. Every little movement brings forth a veritable torrent of
français from the spectators. I can understand the fascination of
chess from the player’s end, but could get few thrills from watching,
especially when there was standing room only.
Far more fascinating to look at is the game two of my French
trough mates play at breakfast. The rules are simple. You take a
muffin about the size of a golf ball. You drop it into your cup of
chocolate. Then you fish for it, sometimes with a spoon, but more
often with your fingers. The object is to convey it to your mouth
without discoloring your necktie. Success comes three times in five.
The players are about evenly matched. One of them I suspect, is
not in the game for sport’s sake, but has a worthier object. Nature
supplied him with a light gray mustache, and a chocolate brown
would blend better with his complexion. If the muffins hold out, his
color scheme will be perfect before we reach port.
The discovery has been made that there’s a man on board who
plays the cornet, so if we are subbed it will not be an unmitigated
evil.

Friday, August 10.

Every morning one sees on the deck people one never saw
before, and as we have not stopped at any stations since we started,
the inference is that certain parties have not found the trip a
continuous joy ride.
A news bulletin, published every morning, sometimes in English
and sometimes in French, keeps us right up to date on thrilling
events, thrillingly spelled. I have copied a sample:

It is now the tim for the final invaseon of the west by the
eastren american league teams and before this clash is over it
will be definitively known wether the two sox teams are to fight
it out in a nip and tuk finish or wether the Chicago sox will have
 a comfortable margen to insure a world series betwean the two
largest American citys Chicago and New York.

The French news deals exclusively with the developments in the

world series Over There, which is, perhaps, almost as important.
A new acquaintance made to-day was that of the Gentleman
from Louisiana. He introduced himself to scold me and another guy
for not taking sufficient exercise. We told him we found little
pleasure in promenading the deck.
“That’s unnecessary,” he said. “Get yourselves a pair of three-
pound dumb-bells and use them a certain length of time every day.”
So we are constantly on the lookout for a dumb-bell shop, but
there seems to be a regrettable lack of such establishments in mid-
ocean.
The Gentleman from Louisiana says he is going to join the
Foreign Legion if they’ll take him. He is only seventy years old.
“But age makes no difference to a man like I,” says he. “I
exercise and keep hard. All my friends are hard and tough. Why, one
of my friends, an undertaker, always carries a razor in his boot.”
Presumably this bird never allows psychological depression in his
business.
The Gentleman from Louisiana continues:
“I’ve got a reputation for hardness, but I’m only hard when I
know I’m right. I used such hard language once that they injected
me from a committee. I was state senator then. But in all the time I
held office I never talked more than two minutes.”
We expressed polite regret that he was not a state senator still.
And we asked him to have a lemonade.
“No, thank you. Even the softest drinks have a peculiar effect on
me. They make my toes stick together.”
 We guaranteed to pry those members apart again after he had
quenched his thirst, but he would not take a chance.
On the way cabinward from this fascinating presence, I was
invited into a crap game on the salle à manger floor. The gentleman
with the dice tossed a hundred-franc note into the ring and said:
“Shoot it all.” And the amount was promptly oversubscribed. So I
kept on going cabinward.

Samedi, 11 Août.
The man back there in the steamship office can no more
truthfully say: “There has never been an accident on this line.”
I awoke at three-thirty this morning to find the cabin insufferably
hot and opened the port-hole which is directly above my berth. The
majority of the ocean immediately left its usual haunts and came
indoors. Yale and Harvard were given a shower bath and I had a
choice of putting on the driest things I could find and going on deck
or drowning where I lay. The former seemed the preferable course.
Out there I found several fellow voyagers asleep in their chairs
and a watchman in a red-and-white tam-o’-shanter scanning the
bounding main for old Hans W. Periscope.
I wanted sympathy, but the watchman informed me that he ne
comprended pas anglais, monsieur. So we stood there together and
scanned, each in his own language.
My garçon de cabine promises he will have me thoroughly bailed
out by bedtime to-night.
I sat at a different breakfast table, but there was no want of
entertainment. At my side was a master of both anglais and français,
and opposite him an American young lady who thinks French is
simply just impossible to learn.
 “Mademoiselle,” says he, “must find it difficult to get what she
likes to eat.”
“I certainly do,” says she. “I don’t understand a word of what’s
on the menu card.”
“Perhaps I can help mademoiselle,” says he. “Would she like
perhaps a grapefruit?”
She would and she’d also like oatmeal and eggs and coffee. So
he steered her straight through the meal with almost painful
politeness, but in the intervals when he wasn’t using his hands as an
aid to gallant discourse, he was manicuring himself with a fork.

The majority of the ocean immediately left its usual haunts and
came indoors
 This afternoon they drug me into a bridge game. My partner was
our congressman’s secretary. Our opponents were a Standard Oil
official and a vice-consul bound for Italy. My partner’s middle name
was Bid and Mr. Oil’s was Double. And I was too shy to object when
they said we’d play for a cent a point.
At the hour of going to press, Standard Oil had practically all the
money in the world. And my partner has learned that a holding of
five clubs doesn’t demand a bid of the same amount.

Sunday, August 12.

The boat seems to be well supplied with the necessities of life,
such as cocktails and cards and chips, but it is next to impossible to
obtain luxuries like matches, ice-water and soap.
Yale and Harvard both knew enough to bring their own soap, but
my previous ocean experiences were mostly with the Old Fall River
Line, on which there wasn’t time to wash. Neither Yale nor Harvard
ever takes a hint. And “Apportez-moi du savon, s’il vous plaît,” to the
cabin steward is just as ineffectual.
All good people attended service this morning, and some bad
ones played poker this afternoon.
In a burst of generosity I invited a second-class French young
lady of five summers to have some candy. She accepted, and her
acceptance led to the discovery that the ship’s barber is also its
candy salesman.
This barber understands not a syllable of English, which fact has
added much to young America’s enjoyment. The boys, in the midst
of a hair cut, say to him politely: “You realize that you’re a damn
rotten barber?” And he answers smilingly: “Oui, oui, monsieur.”
Yesterday, I am told, a young shavee remarked: “You make me sick.”
The barber replied as usual, and the customer was sick all last night.
 To-morrow afternoon there is to be a “concert” and I’m to speak
a piece, O Diary!

Monday, August 13.

The concert was “au profit du Secours National de France.
Œuvre fondée pour répartir les Secours aux Victimes de la Guerre.”
Ten minutes before starting time they informed me that I was to
talk on “The American National Game,” and I don’t even know how
the White Sox came out a week ago to-morrow.
The afternoon’s entertainment opened with a few well-chosen
remarks by our congressman. The general, designated on the
program as “chairman,” though his real job was toastmaster, talked a
while about this, that and the other thing, and then introduced the
cornet player, using his real name. This gentleman and I blew at the
same time, so I have no idea what he played. I got back in time for
some pretty good harmonizing by three young Americans and a boy
from Cincinnati. Then there was a Humorous Recitation (the
program said so) by a gent with a funny name, and some really
delightful French folk songs by the lady novelist. After which came a
Humorous Speech (the program forgot to say so) by myself,
necessarily brief, as I gave it in French. The French songbird
followed with one of those things that jump back and forth between
Pike’s Peak and the Grand Cañon, and a brave boy played a ukelele,
and the quartette repeated. In conclusion, we all rose and attempted
La Marseillaise.
Some of the programs had been illustrated by the lady novelist’s
artist husband, and these were auctioned off after the show. I made
my financial contribution indirectly, through better card players than
myself. My bridge partner, I noticed, had recovered from his attack
of the Bids.
 Tuesday, August 14.
The concert, by the way, was given in the salon de conversation,
which, I think, should be reserved for the Gentleman from Louisiana.
He has now told me two hundred times that he won his election to
the State Senate by giving one dollar and a half to “a nigger.”
One of our young field-service men spoiled the forenoon poker
game with a lecture on how to catch sharks. His remarkable idea is
to put beefsteak on a stout copper wire and troll with it. He has
evidently been very intimate with this family of fish, and he says
they are simply crazy about beefsteak. Personally, I have no desire
to catch sharks. There are plenty aboard. But I do wish he had not
got to the most interesting part of his theory at the moment the
dealer slipped me four sixes before the draw. Everybody was too
busy listening to stay.
We have discovered that the man behind the gun in the fumoir
bears a striking resemblance to Von Hindenburg, but no one has
been found who will tell him so.
There was a track meet this afternoon, and the author of this
diary was appointed referee. But the first event, a wheelbarrow race,
was so exciting that he feared for his weak heart and resigned in
favor of our general. There didn’t seem to be much else to the meet
but ju-jutsu, the sport in which skill is supposed to triumph over
brawn. I noticed that a two-hundred-and-thirty-pound man was the
winner.
We are in that old zone, and the second table’s dinner hour has
been advanced to half past six so that there need be no lights in the
dining-room. Also, we are ordered not to smoke, not even to light a
match, on deck after dark. The fumoir will be running for the last
time, but the port-holes in it will all be sealed, meaning that after
thirty-five smokers have done their best for a few hours the
atmosphere will be intolerable. We can stay on deck smokeless, or
we can try to exist in the airless fumoir, or we can go to bed in the
dark and wish we were sleepy. And the worst is yet to come.

Wednesday, August 15.

The rules for to-night and to-morrow night provide for the
closing of our old friend, the fumoir, at seven o’clock, and that
witching hour is on you long before you expect it, for they jump the
clock fifteen minutes ahead every time it’s noon or midnight. The
ship will not be lit up. The passengers may, if they do their shopping
early.
There was another life-boat “drill” this afternoon. Every one was
required to stand in front of his canoe and await the arrival of
Svengali. When that gent appeared, he called the roll. As soon as
you said “Here” or “Present,” your part of the “drill” was over. When
the time comes I must do my drifting under an alias, as Svengali
insists on designating me as Monsieur Gardnierre. But No. 12 is at
least honored with two second-class ladies. Many a poor devil on the
ship is assigned to a life-boat that is strictly stag.
The Gentleman from Louisiana to-day sprang this one:
“You know when I part my hair in the middle I look just like a
girl. Well, sir, during the Mardi Gras, two years ago, I put on a page’s
costume and parted my hair in the middle. And you know girls under
a certain age must go home at nine o’clock in the evening. Well, sir,
a policeman accosted me and told me I had to go home. I gave him
the bawling out of his life. And maybe you think he wasn’t
surprised!”
Maybe I do think so.
The Gentleman strayed to the subject of Patti and wound up
with a vocal imitation of that lady. He stopped suddenly when his
voice parted in the middle.
 We have seen no periscopes, but when I opened my suit-case
this morning I met face to face one of those birds that are house
pets with inmates of seven-room flats at twenty-five dollars per
month. I missed fire with a clothes brush, and before I could aim
again he had submerged under a vest. Looks as if the little fellow
were destined to go with me to Paris, but when I get him there I’ll
get him good.

Thursday, August 16.

Great excitement last night when a small unlighted boat was
sighted half a mile or so off our port. Our gunners, who are said to
receive a bonus for every effective shot, had the range all figured
out when the pesky thing gave us a signal of friendship. It may have
been part of the entertainment.
To-day we persuaded the Gentleman from Louisiana to part his
hair in the middle. The New Orleans policeman is not guilty.
It develops that while first- and second-class passengers were
unable to read or smoke after dark, the third-class fumoir is running
wide open and the Greeks have their cigarettes, libations and card
games, while the idle rich bore one another to death with
conversation.
Un Américain aboard is now boasting of the world’s
championship as a load carrier. It was too much trouble for him to
pay Auguste for each beverage as it was served, so he ran a two
days’ charge account. His bill was one hundred and seventy-eight
francs, or thirty-five dollars and sixty cents.
“Who got all the drinks?” he asked Auguste.
“You, monsieur,” that gent replied.
“And what do you charge for a highball?”
“One franc, monsieur,” said Auguste.
 Which means, if Auguste is to be believed, that one hundred and
seventy-eight highballs went down one throat in two days. And the
owner of the throat is still alive and well. Also, he says he will
hereafter pay as you enter.
As an appetizer for dinner to-night the captain told everybody to
remain on deck, fully dressed and armed with a life-belt, this
evening, until he gave permission to retire.
We’re all on deck, and in another minute it will be too dark to
write.
To-morrow night, Boche willing, we will be out of the jurisdiction
of this Imp of Darkness.
 II
I GET TO PARIS AND ENCOUNTER SOME
STRANGE SIGHTS

Friday, August 17. A French Port.

In obedience to the captain’s orders we remained on deck last
night, fully dressed, till our ship was past the danger zone and in
harbor. There was a rule against smoking or lighting matches, but
none against conversation.
The Gentleman from Louisiana and a young American Field
Service candidate had the floor. The former’s best was a report of
what he saw once while riding along beside the Columbia River. An
enormous salmon jumped out of the water and raced six miles with
the train before being worn out. Whether the piscatorial athlete flew
or rode a motorcycle, we were unable to learn.
The Gentleman from Louisiana yielded to his younger and
stronger countryman. Some one had spoken of the lack of convoy.
“Don’t you think we haven’t a convoy,” the kid remarked.
I scanned the sea in all directions and saw nothing but the dark
waters. “Where is it?” I inquired.
“There’s one on each side of us,” said Young America. “They’re
about twenty miles from the ship.”
“I should think,” said somebody, “that a very slender submarine
might slip in between our side kicks and us and do its regular job.”
 “No chance,” the youth replied. “The convoy boats are used as
decoys. The sub would see them first and spend all its ammunition.”
A little later he confided in me that the new American war-ships
were two hundred and forty-five thousand horsepower. I had no idea
there were that many horses left to measure by.
We spotted a shooting star. “That was a big one,” I said.
“Big! Do you know the actual size of those things? I got it
straight from a professor of astronomy. Listen. They’re as small as a
grain of sand.”
“Why do they look so big?”
“Because they’re so far away and they travel so fast.”
Round ten o’clock, beckoning lights ashore told us we were close
to safety. But the French gunners remained at their posts two hours
longer. The captain’s shouted order, relieving them from duty, was
music to our ears.
After midnight, however, we turned a complete circle, and at
once the deck was alive with rumors. We had been hit, we were
going to be hit, we were afraid we would be hit, and so on. The fact
was that our pilot from ashore was behind time and we circled round
rather than stand still and be an easy target while awaiting him. We
were in harbor and anchored at three. Many of us stayed up to see
the sun rise over France. It was worth the sleep it cost.
They told us we would not dock until six to-night. Before retiring
to my cabin for a nap, I heard we had run over a submarine and
also that we had not. The latter story lacked heart interest, but had
the merit, probably, of truth. Submarines have little regard for traffic
laws, but are careful not to stall their engines in the middle of a
boulevard.
I was peacefully asleep when the French officers came aboard to
give us and our passports the Double O. They had to send to my
cabin for me. I was ordered to appear at once in the salon de
conversation. A barber hater addressed me through his beard and
his interpreter: “What is Monsieur Laudanum’s business in France?”
I told him I was a correspondent.
“For who?”
“Mark Sullivan.”
“Have you credentials from him?”
“No, sir.”
“Your passport says you are going to Belgium. Do you know
there are no trains to Belgium?”
“I know nothing about it.”
“Well, there are no trains. How will you go there?”
“I’ll try to get a taxi,” I said.
“Are you going from here to Paris?”
“Yes.”
“And where are you going from Paris?”
“I don’t know.”
“Please explain that answer.”
“I will go wherever the authorities permit me to go.”
“That is not a satisfactory answer.”
“I’m sorry.”
“What is your real business in France?”
“To write.”
“I’m afraid we’ll have to keep your passport. You will appear to-
morrow morning at nine o’clock at this address.”
And they handed me a scary-looking card.
On the deck I met our congressman and told him my troubles.
 “I know these fellows very well,” he said. “If you like, I can fix it
for you.”
“No,” I replied proudly. “I’d rather do my own fixing.”
At the dock I got into a taxi and asked to be taken to the ——
Hotel. Not to my dying day will I forget that first ride in a French
taxi. Part of the time we were on the right side of the street, part of
the time on the left, and never once were we traveling under a
hundred and fifty miles an hour. We turned twenty corners and
always on one ear. We grazed dozens of frightened pedestrians,
many of them men crippled in the war, or by taxis, and women too
old to dodge quickly. We aimed at a score of rickety horse-drawn
vehicles, but our control was bad and we bumped only one. In front
of the hostelry we stopped with a jerk.
“Comme beaucoup?” I asked the assassin.
“Un franc cinquante,” he said.
Only thirty cents, and I thought I knew why. When they get
through a trip without killing any one, they feel they have not done
themselves justice nor given you a square deal.
I found myself a seat at a sidewalk table and ordered
sustenance. The vial they brought it in was labeled “Bière Ritten,”
but I suspect the adjective was misspelled.
Till darkness fell I watched the passing show—street-cars with
lady motormen and conductors; hundreds of old carts driven by old
women, each cart acting as a traveling roof for an old dog; wounded
soldiers walking or hobbling along, some of them accompanied by
sad-faced girls; an appalling number of women in black; a lesser
number of gayly garbed and extremely cordial ones, and whole
flocks of mad taxis, seeking whom they might devour.
By using great caution at the street crossings, I succeeded in
reaching the telegraph office where I wrote a message informing
Paris friends of my arrival. I presented it to the lady in the cage, who
handed it back with the advice that it must be rewritten in French. I
turned away discouraged and was starting out again into the gloom
when I beheld at a desk the songbird of the ship. Would she be kind
enough to do my translating? She would.
The clerk approved the new document, and asked for my
passport. I told her it had been taken away. She was deeply grieved,
then, but without it monsieur could send no message. Bonne nuit!
Back at the hotel I encountered the Yankee vice-consul, a
gentleman from Bedford, Indiana. I told him my sad plight, and he
said if matters got too serious his office would undertake to help.
With his assurances to comfort me, I have retired to my room to
write, to my room as big as Texas and furnished with all the modern
inconveniences.

Saturday, August 18. Paris.

It is Saturday night and they have hot water, but before I take
advantage of it I must recount the thrilling experiences of the day.
After a sidewalk breakfast of “oofs” and so-called café in
Bordeaux, I went to keep my engagement at court. It was apparent
that I was not the only suspect. The walk outside and the room
within were crowded with shipmates, most of them from the second
cabin, all looking scared to death.
I stood in line till I realized that I must make it snappy if I
wanted to catch the eleven-five for Paris; then I butted my way into
the august presence of Him of the Beard.
He recognized me at once and told me with his hands to go up-
stairs. In a room above I found the English-speaking cross-examiner,
with the accent on the cross.
He waved me to a chair and began his offensive.