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 Bioinformatics and Data
Analysis in Microbiology
Edited by
  

Caister Academic Press

 Contributors | vii

Karen E. Nelson Vijay Shankar

The J. Craig Venter Institute Boonshoft School of Medicine
Rockville, MD Wright State University
USA Dayton, OH
USA
kenelson@jcvi.org
shankar.5@wright.edu
Oleg Paliy
Boonshoft School of Medicine Paul Stothard
Wright State University Department of Agricultural, Food and Nutritional
Dayton, OH Science
USA University of Alberta
Edmonton, AB
oleg.paliy@wright.edu
Canada
Oleg N. Reva stothard@ualberta.ca
Department of Biochemistry, Bioinformatics and
Computational Biology Unit Sebastian Szpakowski
University of Pretoria The J. Craig Venter Institute
Pretoria Rockville, MD
South Africa USA
oleg.reva@up.ac.za shpakoo@gmail.com

Larisa A. Safronova Özlem Taştan Bishop

Department of Antibiotics Research Unit in Bioinformatics (RUBi)
Institute of Microbiology and Virology NAS of Department of Biochemistry and Microbiology
Ukraine Rhodes University
Kiev Grahamstown
Ukraine South Africa
safronova_larisa@ukr.net ozlem.tastanbishop@gmail.com

Marketa Sagova-Mareckova Angel Valverde

Crop Research Institute Centre for Microbial Ecology and Genomics
Prague Department of Genetics
Czech Republic University of Pretoria
Pretoria
sagova@vurv.cz
South Africa
Cathal Seoighe angel.valverde@up.ac.za
School of Maths, Applied Maths and Statistics
College of Science Gary Van Domselaar
NUI Galway National Microbiology Laboratory
Ireland Public Health Agency of Canada
Winnipeg, MB
cathal.seoighe@nuigalway.ie
Canada
gary.vandomselaar@phac-aspc.gc.ca
 Current books of interest

Microarrays: Current Technology, Innovations and Applications 2014

Metagenomics of the Microbial Nitrogen Cycle: Theory, Methods and Applications 2014
Proteomics: Targeted Technology, Innovations and Applications 2014
Biofuels: From Microbes to Molecules 2014
Human Pathogenic Fungi: Molecular Biology and Pathogenic Mechanisms 2014
Applied RNAi: From Fundamental Research to Therapeutic Applications 2014
Halophiles: Genetics and Genomes 2014
Phage Therapy: Current Research and Applications 2014
The Cell Biology of Cyanobacteria 2014
Pathogenic Escherichia coli: Molecular and Cellular Microbiology 2014
Campylobacter Ecology and Evolution 2014
Burkholderia: From Genomes to Function 2014
Myxobacteria: Genomics, Cellular and Molecular Biology 2014
Next-generation Sequencing: Current Technologies and Applications 2014
Omics in Soil Science 2014
Applications of Molecular Microbiological Methods 2014
Mollicutes: Molecular Biology and Pathogenesis 2014
Genome Analysis: Current Procedures and Applications 2014
Bacterial Toxins: Genetics, Cellular Biology and Practical Applications 2013
Bacterial Membranes: Structural and Molecular Biology 2014
Cold-Adapted Microorganisms 2013
Fusarium: Genomics, Molecular and Cellular Biology 2013
Prions: Current Progress in Advanced Research 2013
RNA Editing: Current Research and Future Trends 2013
Real-Time PCR: Advanced Technologies and Applications 2013
Microbial YZ%) Pumps: Current Research 2013
Cytomegaloviruses: From Molecular Pathogenesis to Intervention 2013
Oral Microbial Ecology: Current Research and New Perspectives 2013
Bionanotechnology: Biological Self-assembly and its Applications 2013
Real-Time PCR in Food Science: Current Technology and Applications 2013
Bacterial Gene Regulation and Transcriptional Networks 2013
Bioremediation of Mercury: Current Research and Industrial Applications 2013
Neurospora: Genomics and Molecular Biology 2013
Rhabdoviruses 2012

Full details at www.caister.com


We are at the door of an exciting future for micro-
biology. The rapid advancement of sequencing
techniques, coupled with the new methodologies
of bioinformatics to handle large scale data analy-
sis, are providing exciting opportunities for us to
understand microbial communities from a variety
of environments beyond previous imagination.
Data analysis is extremely important for a deeper
knowledge of microbes and their habitats, and
for many applications of microbiology ranging
from understanding the basis of diseases or host
pathogen interactions so as to design drugs and
develop vaccines, to many other biotechnology
applications, including barcoding, microbial bio-
remediation and bio-fuel production.
This book aims to present up-to-date and
detailed information on various aspects of bio-
informatics data analysis with applications to
microbiology. It describes a number of different
useful bioinformatics tools, highlights the links
to some wet-lab techniques, explains different
approaches to tackle a problem, talks about cur-
rent challenges and limitations, demonstrates
the applications, and discusses future trends.
A brief summary of each chapter is as follows:
Chapter 1 provides a review of microbes and
their importance in the context of ecosystems,
and an overview of the methods applied to study
microbes within ecosystems. It aims to give an
introduction to newcomers to the fields of micro-
biology and bioinformatics. Chapter 2 reviews
sequencing technologies and discusses the
challenges of assembly and ways of tackling the
problems. Chapter 3 highlights the scope of micro-
bial variations and explores the importance of
accurate genome assembly for structural variation
Preface
and single nucleotide polymorphism analysis.
Chapter 4 deals with genome annotation, and
reviews computational methods that have been
developed for annotation of bacterial and archaeal
genomes. Chapter 5 explores the methods for
comparative analysis of microbial genomes, and
focuses on the analysis of Mycobacteria as a case
study. Microbial community profiling is the topic
of Chapter 6, in which fingerprinting techniques
and construction of phylogenetic trees are interro-
gated. Metagenomics, one of the fastest advancing
fields of microbiology, is discussed in Chapter 7
with examples of the Human Microbiome Project
and a Baltic Sea study. Chapter 8 further inter-
rogates human microbiome analysis and presents
the pros and cons of using 16S rRNA based
sequencing studies. Chapter 9 takes us to another
interesting topic, phylogenetic microarrays, in
which 16S rRNA sequences are used to determine
the composition of microbial communities, yet
from the microarray aspect. The last chapter is
about genetic barcoding with its applications to
microbiology and biotechnology. Overall, this
is an essential book for researchers, lecturers
and students involved in microbiology and/or
different aspects of bioinformatics including next-
generation sequencing, sequence data analysis,
comparative genome analysis, metagenomics and
more.
The book has been peer reviewed. Each chap-
ter of the book has been reviewed by at least one
reviewer and by the editor. The reviewing process
was undertaken by other authors contributing
to the book, and I believe the constructive com-
ments of the reviewers significantly improved
the quality of the book. I thank the authors of
x | Preface
the book chapters for their participation in this
process. My special thanks go to Tim Downing
who was always available for the reviewing pro-
cess, and always responded exceptionally fast. I
am especially grateful to Gary Van Domselaar,
Morag Graham and Paul Stothard who agreed to
write an extra chapter in a very short time, when
another author dropped out. I also would like to
thank Fourie Joubert for his initial contribution
in discussing the chapter topics and suggesting
some potential authors. My deepest thanks to the
Sabanci University, Istanbul-Turkey, for hosting
me and providing a friendly working environment
during my sabbatical which made it possible to
finalize the book. Last but not least, I would like
to thank my husband, Nigel T. Bishop, for his con-
tinuous support in my first time as an editor.
Özlem Taştan Bishop
Understanding the Unseen Majority
Around Us: An Overview of
&'!' ' 
Meesbah Jiwaji, Gwynneth F. Matcher and Rosemary A. Dorrington
Abstract
Of all the living organisms on the planet, microor-
ganisms are the most numerically abundant and
diverse in nature. Despite their ubiquity, research-
ers have only begun to understand the diversity
profiles, metabolic functioning and potential
economic value of these organisms.
Classical investigation of microorganisms
involves the culture and study of selected
microbes in the laboratory setting. While this
approach has yielded much information, there
are two major drawbacks. Firstly, most microbes
present in the environment are unculturable using
currently available media/methodologies and,
secondly, they focus on one, often attenuated,
isolate/species and/or a set of genes at a time.
To overcome these problems, researchers have
focussed on the development of new technolo-
gies that yield large, reliable and robust datasets
in fields that include genomics, transcriptomics
and proteomics. Importantly, the development
of high-throughput sequencing technologies has
dramatically advanced the analysis of microbial
species diversity and their functioning within
ecosystems. The large volumes of information-
rich data require intelligent, and often repetitive,
computational analysis, stressing the need for
development of suitable bioinformatics analy-
sis tools. This chapter provides an overview of
microbes and the importance of why we need to
understand them, as well as the methods applied
to studying microbiota within ecosystems.
1
Introduction
The field of microbiology is undergoing a renais-
sance. The discipline has changed dramatically
since Antonie van Leeuwenhoek, the man who
invented the microscope, watched bacteria that
he recovered from his own teeth through his
homemade microscope (van Leeuwenhoek,
1677). Traditional microbiology has focussed
on the study of individual organisms, but now,
while microorganisms and their processes still
need to be understood at the level of the indi-
vidual, increasingly, the aim is to understand the
cumulative influence of microorganisms on the
functioning of biological systems. As a conse-
quence, microbiology has progressed from the
study of individual genomes to that of microbial
populations in ecosystems. Scientific observations
at the subcellular level are now being interpreted
at rapidly increasing levels of complexity in order
to gain a better understanding of the complex
metabolic pathways within cells as well as their
interactions in the context of the ecosystem. In
order to gain understanding of system organiza-
tion on such a large scale, new methodologies
are constantly being developed, most of which
generate extremely large sets of raw data which
need to be curated and analysed using the tools of
bioinformatics.
What is a microorganism?
The term ‘microorganism’ is used to describe any
small organism, typically an entity that has a mass
of less than 10–5 g and a length of less than 500 μm,
with a largest dimension of 100–150 μm (Hughes
Martiny et al., 2006; Karl, 2007). It is important to
2 | Jiwaji et al.
note that currently organisms are assigned to the
‘micro’ class, based only on their size. This ignores
any differences in their evolutionary histories and
metabolic capabilities. Given time, and with the
increasing awareness of the diversity of microor-
ganisms, this method of classification may need to
be revisited.
The classification of organisms involves placing
them within collections/groups of related species.
Traditionally, microorganisms were classified
within the Domains Prokarya or Eukarya based
on their morphology, the environment they were
isolated from, the means by which they generated
energy, their nutrient requirements and their
mode of replication (Woese et al., 1990; Karl,
2007; Schleifer, 2009). Since the advent of molec-
ular biology and the collection of sequence data,
microorganisms are being classified into three
Domains; the Bacteria, Archaea and Eukarya (Fig.
1.1) (Woese et al., 1990). Bacteria and Archaea are
grouped together as prokaryotes based on several
cellular characteristics. These include the absence
of a membrane surrounding the genome, the lack
of introns in the encoded genes, the absence of
intracellular organelles (e.g. chloroplasts, mito-
chondria, etc.) and ribosomal subunits of 30S
and 50S (eukaryotic subunits are larger at 60S and
40S). While Archaea and Bacteria do share several
similarities, significant differences do exist. For

  


 



 
  !
Figure 1.1    \    !=         7
&   Y%.  \     !     !!%  ! \
example, bacterial cell walls contain peptidogly-
can whilst archaeal cell walls do not and bacterial
cell membranes contain ester links whilst those of
Archaea have ether links. While Archaea and Bac-
teria are both classified as prokaryotes, Archaea
do in fact share a number of morphological char-
acteristics and DNA sequence similarities with
that of Eukaryotes prompting the speculation
that Archaea and Eukarya diverged from bacteria
before they diverged from one another (Fig. 1.1)
(Zillig, 1991; Schleifer, 2009).
Microbes in the world around
us – the unseen majority
Microorganisms are very old; they are thought to
have inhabited the Earth for more than 3.5 billion
years as evidenced by their presence in fossils, for
example the Burgess Shale in Canada (Schopf,
2001). It is interesting that through their evolu-
tionary history, microorganisms have remained
simple and small and that their classification is
often expressed in terms of their physiology and
metabolism rather than their morphologies.
Microorganisms are integral components of all
ecosystems and there is no environment stud-
ied to date in which microorganisms have not
been found. From the extreme environments
where temperatures exceed 100°C or fall below
  


  # 
   

"  
#
 
$

0°C, pHs that range from below 2 to over 10, or
pressures over 100 MPa, to every conceivable
environment in between (Ulukanli and Digrak,
2002; Baker-Austin and Dopson, 2007; Moyer
and Morita, 2007; Fang et al., 2010; Synowiecki,
2010). In the polar regions to the deserts, from
the shallow to the deep oceans, from exotic envi-
ronments to one’s own backyard, there exists an
unexpected abundance and diversity of microor-
ganisms (Pace, 1997). In terms of abundance, for
example, one gram of soil may harbour up to 10
billion microorganisms potentially representing
thousands of different species (Roselló-Mora
and Amann, 2001). It is now well recognized that
microorganisms are both taxonomically diverse
and metabolically complex. In fact, microorgan-
isms dominate their environments in terms of
biomass, diversity and metabolic activity and are
thus truly the ‘unseen majority’ (DeLong and
Karl, 2005).
Of particular interest is the role of the microor-
ganism in ecosystem biology. Ecosystems consist
of interconnected systems whose components
interact over a broad range of physical and bio-
logical states (DeLong, 2007). While several
physico-chemical parameters may be similar
between different ecosystems, the microbial com-
munities within these ecosystems differ. Thus,
while microorganisms are ubiquitous, their distri-
bution is not uniform and their diversity profiles
differ significantly between ecological niches. A
strong body of evidence shows that the individual
environment selects, and is partly responsible for,
the spatial variation in the diversity and popula-
tion structure of microorganisms (Hughes
Martiny et al., 2006). In addition to the effect of
a wide range of physico-chemical conditions on
microbial diversity profiles, factors such as inter-
species competition and predation also play a role
in effecting relative abundances and distributions
of microbial species (DeLong, 2007; Hibbing et
al., 2010).
Microorganisms form a considerable propor-
tion of the total biomass in all ecosystems and
so are supremely important in the functioning
of global ecosystem processes. For example,
in marine ecosystems, it is estimated that the
microbes are responsible for up to 98% of the
primary productivity and the mediation of all
Microbes: An Unseen Majority Around Us | 3
biogeochemical processes (Sogin et al., 2006).
Microorganisms harvest and convert solar energy,
catalyse important biogeochemical transforma-
tions of free nutrients and trace elements to sustain
ecosystems, they form crucial links in the carbon
and nitrogen cycles, and they both produce and
consume greenhouse gases including carbon
dioxide (CO2), nitrous oxide (N2O) and methane
(CH4) (McGrady-Steed et al., 1997; Naeem and
Li, 1997; van der Heijden et al., 1998; Cavigelli
and Robertson, 2000; Horz et al., 2004; Bell et
al., 2005). In addition, cellular microorganisms
are pivotal in driving the sulfur and phosphorous
cycles, the production of secondary metabolites
including vitamins and co-factors and bioac-
tive compounds such as antibiotics that confer
selective advantages in the highly competitive
environmental niches (Fagerbakke et al., 1996;
Demain, 2007).
In the past, the majority of microbial genetic
data generated has been defined by the researcher’s
individual areas of interest and has often focused
on individual species of microorganisms. For
example, the study of Acidithiobacillus ferrooxidans
and its application to bioremediation (Umrania,
2006). More recently, there has been a shift in the
approach to studies and there has been increasing
interest in the study of environmental ecosystems
as a whole. As examples, Desai et al. (2010), Xu et
al. (2010) and Petrić et al. (2011) examined the
microbial communities in xenobiotic-contami-
nated soils during bioremediation.
Microorganisms and climate
change
The study of climate change includes green-
house-gas-induced warming, and in the case of
water bodies, water acidification (Doney, 2006).
Both of these are destructive processes that
are likely to affect the macro-faunal and -floral
community structure and dynamics as well as
extinction of species. To date, the effect of cli-
mate change on microbial species distribution
and abundances has not been shown. However,
considering the crucial contribution of microbial
metabolism to ecosystem processes, a shift in
microbial population dynamics is likely to have
severe implications.
4 | Jiwaji et al.
Microorganisms both consume and produce
major greenhouse gases, thus they have central
roles both as effectors and monitors of global
climate change (DeLong, 2007). The effect of
climate change on microorganisms is easiest to
monitor in the Antarctica, the coldest, windi-
est, driest and most isolated continent on Earth.
Due to the environment, Antarctic food webs are
relatively simple, and the absence of insect and
mammalian herbivores means that most energy
and biomass is channelled into a detritus trophic
pathway that is dominated by microorganisms
(Davis, 1981). Thus, soil microorganisms have a
disproportionate importance in nutrient cycling
and other ecosystem processes in the ice-free ter-
restrial Antarctic ecosystems. The simplicity of the
Antarctic ecosystem makes it particularly vulner-
able to environmental perturbations like global
warming and the Antarctic Peninsula is among the
most rapidly warming regions on the planet. This
has been demonstrated by significant increases in
the abundance of fungi and bacteria and particu-
larly in the Alphaproteobacteria-to-Acidobacteria
ratio, which is indicative of higher soil nutrient
availability (Thomson et al., 2010). The observed
shifts are consistent with increased turnover of
carbon and nitrogen in the soil upon warming of
the environment (Thomson et al., 2010; Yergeau
et al., 2012). In the oceans, phytoplankton can
also be used as monitors of climate change. These
eukaryotic microorganisms influence the biologi-
cal pump which cycles carbon, removing it from
the upper ocean and transporting it to the deep sea.
Falkowski and Oliver (2007) used the behaviour
of phytoplankton to predict the potential effects
of climate change on the microorganism commu-
nity structure. As these microorganisms are major
participants in oceanic primary production, any
changes to the phytoplankton community have
knock-on effects and implications for fishery pro-
ductivity as well as global ocean ecology.
The impact of microorganisms
in industry
From a health and economic point of view,
microorganisms are of great interest and they
may impact our lives both positively and nega-
tively. For example, microbes may be pathogenic
and can cause serious diseases (Karlen, 1995).
Many microbial species are responsible for the
degradation of food produce and can result in
significant economic losses. On the positive side,
for example, microorganisms are indispensable
for their symbiotic roles in the human digestive
tract (Eckburg et al., 2005). In addition, they can
be harnessed for the production of fuels, chemical
compounds, animal feeds, human food, antibiot-
ics and pharmaceuticals (Zhu et al., 2012).
Microorganisms also represent a vast and
dynamic reservoir of genetic variability thus the
harnessing of this genetic diversity has allowed
scientists to unlock the economic potential of
microorganisms. Some of the important meta-
bolic processes that microorganisms are involved
in include:
Nitrogen fixation
Nitrogen fixation, the assimilation of atmospheric
nitrogen (N2) into ammonia, is an economically
and environmentally important natural microbial
function, which yields hundreds of tonnes of bio-
logically available nitrogen and is worth billions
of dollars to global agriculture (Herridge et al.,
2008).
Bioremediation
Bioremediation, the use of microorganisms to
sequester or remove pollutants, is increasing both
as a concept and as an economically viable appli-
cation. Non-biological processes are estimated to
cost ten-fold more to remediate known hazard-
ous waste sites whereas bioremediation would
cost less and could occur in the same time frame.
This is a developing area and has the potential of
becoming economically important. For example,
hydrocarbon cold seeps and ecologically devas-
tating oil spills have led to the development of
marine microorganisms that will utilize alkanes as
carbon sources. These bacteria and their enzymes
have a potential role in bioremediation and oil
processing (Xu et al., 2008; Wasmund et al., 2009;
Augustinovic et al., 2012).
Bioprospecting
Bioprospecting, defined as the discovery and
subsequent commercialization of useful products
from environmental isolates, has been central in

the search for novel pharmaceuticals and com-
pounds of industrial importance (Dionisi et al.,
2012). More than 75% of all antibacterial com-
pounds and approximately 50% of all anticancer
compounds that are in use clinically are either
natural products or derivatives thereof (Newman
and Cragg, 2007). The percentage of biologically
active natural products is much higher than that
of synthetic compounds primarily due to the fact
that natural products have evolved bioactivity in a
biological context (Firn and Jones, 2003).
Since Alexander Fleming’s observation of the
antibacterial activity of penicillin in 1928, and its
subsequent application to treating bacterial infec-
tions, a large number of microbe-derived natural
products have been used in the pharmaceutical
sector for a diverse range of medical applications.
This includes biologically active compounds like
the antibiotics cephalosporins, tetracyclines, ami-
noglycosides, rifamycins and chloramphenicol,
the mycotoxin asperlicin, the immunosuppres-
sant cyclosporine and the cholesterol-lowering
agent lovastatin (Pan et al., 2010). In addition to
the production of pharmaceutically important
metabolites, microorganisms also represent
a rich source of enzymes with applications as
biocatalysts in the production of a wide range of
industrially useful compounds. While the pro-
duction of these compounds in most instances
can be achieved via chemical processes, bio-
catalysts are an attractive alternative due to the
lower temperatures and neutral pH at which
they function, as well as the production of fewer
toxic by-products (Azerad, 1995; Koeller and
Wong, 2001). Furthermore, biocatalysts often
exhibit specific regioselectivity thereby negating
the requirement of chemical synthesis pathways
for substrate functional-group protection which
involves addition of several steps in the synthetic
pathway to block and unblock substituents
(Schmid et al., 2001).
Research into interesting enzymes with
potential industrial applications, for example
psychrophilic enzymes, is incredibly competitive
and lucrative (Struvay and Feller, 2012; Feller,
2013). The range of enzymes with unique abilities
includes lipases (de Pascale et al., 2008; Jeon et
al., 2009), cellulases (Ekborg et al., 2007; Shan-
mughapriya et al., 2009), chitinases (Cottrell et
Microbes: An Unseen Majority Around Us | 5
al., 1999; Hobel et al., 2005), proteases, alkane
hydroxylase genes (Xu et al., 2008; Wasmund et
al., 2009), esterases with high tolerances for salt,
organic solvents, cold and high pressure (Aurilia et
al., 2008; Chu et al., 2008), and metalloproteases
with high temperature optima (Lee et al., 2007).
Lipases have wide-ranging applications in the
food, detergent, and pharmaceutical industries
(de Pascale et al., 2008; Jeon et al., 2009). Cel-
lulases have been the subject of intense study
because of their role in the generation of biofuels
from renewable cellulosic substrates (Ohkuma,
2003; Ekborg et al., 2007; Shanmughapriya et
al., 2009). Esterases have great commercial value
because of their use in industrial biotransforma-
tions (Aurilia et al., 2008; Chu et al., 2008).
Some of these enzymes have been isolated
using sequence-based approaches by screening for
genes with known bioactivity. These methods rely
on similarity to known protein families. Others
have been isolated using functional screens for
enzyme activity (Ferrer et al., 2005). The bio-
technological and industrial microbiological
potential for new natural products and processes
arising from mining microbial diversity is set to
increase in the coming years, especially with the
application of newly developed techniques, and
in particular high-throughput robotic screening
technology (Bornscheuer et al., 2012).
Assessing microbial
populations
Traditionally, study of microorganisms has been
conducted by staining to visualize the organisms,
microscopy and subsequent image analysis. At
first, stains were used to quantify the abundance
of microorganisms and their biomass (Francisco
et al., 1973; Porter and Feig, 1980). Visualization
of stained cells was then enhanced by powerful
image analysis (Psenner, 1990). More recently,
stains have been developed to visualize microor-
ganisms like viruses (Ortmann and Suttle, 2009)
and to quantify aspects of microbial metabolism
(including respiration and enzyme degradation).
In addition, radioisotopes (Simon and Azam,
1989) and fluorescent stains (Cotner et al., 2001)
have been applied to the measurement of bacterial
growth rates.
6 | Jiwaji et al.
In addition to direct visualization of micro-
bial populations, microorganisms have been
grown under laboratory conditions, often as
pure cultures, and been used for sequence or
function-based biological studies. Advances in
environmental chemistry (including stable iso-
topes, ultrafiltration, measurement of dissolved
organic carbon (DOC) and dissolved inorganic
carbon (DIC), high performance liquid chro-
matography (HPLC) and mass spectrometry
(MS) have also enhanced our ability to study
microorganisms and their interaction with their
environment (Cotner and Biddanda, 2002; Han-
delsman, 2004).
Up until the 1980s, identification of micro-
organisms was achieved by culturing individual
isolates in the laboratory, viewing the organisms
microscopically and subjecting these cultures to
a variety of biochemical tests. While much valu-
able data has been generated by this approach,
there are several major limitations. Firstly, rep-
lication of the complex environment in which
microorganisms exist in a laboratory setting is
often extremely difficult and it is estimated that
less than 0.1% of microorganisms are currently
culturable (Stres, 2007). Furthermore, these
approaches are time-consuming, labour-intensive
and often subjective. Added to this, microorgan-
isms that are abundant and/or those that can be
cultured under some environmental conditions
may change into dormant or possibly uncultur-
able forms under other conditions (Hattori et al.,
1997). The severity of the problem of relying on
culture-dependent studies has been highlighted
by cultivation-independent surveys where a
major discrepancy can be observed between
viable plate count technology and direct methods
such as epifluorescence microscopic counts and
ssRNA phylogenetic analysis. For example, the
observation that marine bacterioplankton are
infected by huge viral numbers (tens of billions
of phage per litre) and that this phage predation
is remarkably specific was undetected primarily
because of a lack of representative pure cultures
(DeLong, 2007).
Viruses, as technically ‘non-living’ entities,
cannot be studied with the same methods used to
study other microorganisms. Classical methods
used for the identification of viruses include in
vitro viral amplification followed by cell culture
followed by visual observation of cytopathic
effects. Virus discovery can also be based on
specific nucleic acid hybridization and antigenic
cross-reactivity, for example on DNA or antibody
microarrays. This can be followed by visualiza-
tion using electron microscopy or by testing for
immunological cross-reactivity using panels of
sera, which is a powerful and rapid method for the
identification of the unknown viral agents. Tenta-
tive identification of the virus allows the use of
more specific molecular approaches like the use of
PCR with primers that target the likely viral group
for definitive genetic characterization (Delwart,
2007; Mokili et al., 2012).
The application of molecular methods bypass
the drawbacks presented by culture-dependent
methodologies and have led to an improved and
deeper understanding of the microbial compo-
nents of ecosystems. These new approaches have
made apparent our lack of knowledge about envi-
ronmental biodiversity. It is estimated that there
are ca. 1.5 million taxa that have been described
at the species level (de Meeus and Renaud, 2002)
yet this represents only a small proportion of the
estimated diversity. Currently, there is also a large
discrepancy between the microorganism diversity
that we detect in biological samples and what is
actually present. Molecular ecology studies sug-
gest that ca. 1–5% of microbial species have been
isolated (Floyd et al., 2005; Hughes Martiny et al.,
2006). And mycologists estimate that there are 1.5
million species of fungi despite the fact that only
72,000 species have been isolated or described
(Hawkswerth, 1997).
Molecular approaches to investigating micro-
bial processes can focus either on individual
isolates or on the microbial population as a whole
within an ecosystem. Either way, a key aim in
the molecular biological study of microorgan-
isms is to determine the genetic information
present within the cell followed by correlation
of the encoding genetic material to the complex
biological processes which occur within the cell.
Bioinformatics analyses are a crucial require-
ment, not only in curating and analysing large
scale sequence data but also in bridging the gap
between genetic code and the encoded function-
ality of genes.

DNA sequence determination
Up until recently, sequence analysis of DNA
has been achieved by Sanger sequencing. In this
method, DNA polymerase-dependent synthesis
of a complementary DNA strand occurs in the
presence of both natural 2′-deoxynucleotides
(dNTPs) and 2′,3′-dideoxynucleotides (ddNTPs),
which function as irreversible terminators of syn-
thesis (Sanger et al., 1977). The DNA synthesis
reaction is terminated at random whenever a
ddNTP is added to a growing oligonucleotide
chain. This results in products of varying lengths
with an appropriate ddNTP at the 3′ terminus.
These truncated products are separated based on
size, using capillary electrophoresis, and the ter-
minal ddNTPs used to reveal the DNA sequence
of the DNA template strand. This technology has
been in use since 1977 and is well established.
However, in addition to being lengthy and labour
intensive, this methodology suffers from the limi-
tation that only one sequence can be generated
per capillary which in turn impacts on the amount
of genetic information that can be generated.
Furthermore, amplification of DNA fragments
is required before they can be sequenced. This
amplification can be achieved either in vivo, by
clonal amplification, or in vitro by PCR, and con-
sequently is susceptible to both host-related and
PCR biases (Hall, 2007).
To deal with the shortcomings of Sanger
sequencing, faster, cheaper, and simpler methods
for sequencing that bypass the cloning bias and
time- and labour-intensive nature of the Sanger
method have been developed. These new tech-
nologies differ in their approach to generating
sequence data, the average read length generated,
and the error rate distribution (Shendure and Ji,
2008) thus the appropriate sequencing method
needs to be selected based on the application.
A brief description of three of these sequencing
technologies, namely pyrosequencing, reversible
terminator sequencing chemistry and ligation
based sequencing chemistry will be covered in
this chapter. Detailed information on sequencing
techniques can be found in Chapter 2.
Pyrosequencing is a sequencing-by-synthesis
technique whereby enzyme driven biochemical
reactions and chemiluminescence are used to
generate sequence data. In this method, the DNA
Microbes: An Unseen Majority Around Us | 7
template is sheared and the resultant DNA frag-
ments immobilized on beads at a ratio such that
a single DNA molecule is immobilized per bead.
Individual DNA fragment-carrying beads are then
captured into separate emulsion droplets and these
droplets act as individual amplification chambers
which are capable of producing 107 clonal copies
of a unique DNA template per bead (Margulies et
al., 2005). Each template-containing bead is then
transferred into a well of a picotitre plate along
with smaller packing beads containing the immo-
bilized enzymes ATP sulfurylase and luciferase.
The use of the picotitre plate allows hundreds of
thousands of pyrosequencing reactions to be car-
ried out in parallel, thus increasing the sequencing
throughput (Margulies et al., 2005). Once the
clonally amplified bead-immobilized DNA frag-
ments have been deposited into the picotitre plate
wells, the picotitre plate is flooded with dNTP
solutions in a sequential manner. As a result of
the action of the polymerase, inorganic pyrophos-
phate (PPi) is released whenever a complementary
nucleotide is incorporated into the growing DNA
strand. This PPi is converted to ATP by the sul-
furylase enzyme and the luciferase in turn utilizes
the ATP to convert luciferin to oxyluciferin with
light as a by-product. The number of bases incor-
porated into the extending DNA strand is directly
proportional to the amount of pyrophosphate
released which in turn is reflected in the amount
of light generated. This chemiluminescent signal
is detected by an extremely sensitive camera and
interpreted as sequence data (Margulies et al.,
2005; Rothberg and Leamon, 2008). Pyrose-
quencing is valuable for sequencing regions of
DNA that are technically difficult due to strong
secondary structure or high GC content as well as
for sequencing regions that are resistant to cloning
in Escherichia coli (Goldberg et al., 2006). One of
the drawbacks of pyrosequencing is the reduced
reading accuracy over homopolymeric stretches
of identical nucleotides (Ansorge, 2009).
The 454 GS FLX+ Platform, which utilizes
pyrosequencing chemistry and is currently mar-
keted by Roche Applied Science, is capable of
generating 700 Mb of sequence with a consensus
accuracy of 99,997% and a read length of up to
1000 bp (http://454.com/products/gs-flx-sys-
tem/index.asp, April 2013). The data generated
8 | Jiwaji et al.
on the 454 platform correlates well with other well
established gene expression profiling technologies
including microarrays (Torres et al., 2008).
An alternative to pyrosequencing is the reversi-
ble terminator chemistry (Bennett, 2004; Bennett
et al., 2005; Bentley, 2006) which involves what is
termed solid-phase bridge amplification of single-
molecule DNA templates. Here, one terminal of
a single DNA molecule hybridizes to a comple-
mentary adaptor which is immobilized on a solid
surface, known as a flowcell. With the adapter
functioning as a primer, the polymerase extends
the DNA fragment thereby generating a copy of
the original template. This copy is attached to
the flowcell via the adaptor/primer which is now
incorporated into the DNA strand. Following
denaturation, the flowcell is rinsed to remove the
original template leaving the immobilized DNA
copy behind. This DNA fragment then bends over
and the free terminal end hybridizes to a nearby
complementary adaptor forming a DNA ‘bridge’.
Once again, the polymerase extends the adaptor/
primer generating a new copy of the DNA which
is also attached to the flowcell. This process is
repeated until a cluster of ~1000 clonal copies of
a single template molecule is formed. After ampli-
fication, a flowcell with approximately 40 million
clusters of DNA amplicons is then subjected to a
DNA sequencing-by-synthesis methodology that
uses a custom DNA polymerase that can incor-
porate specialized reversible terminators with
removable fluorescent moieties into growing oli-
gonucleotide chains. The terminators are labelled
with fluorophores of four different wavelengths
to distinguish between the different nucleotide
bases and the sequence of the template in each
cluster is determined by detecting the wavelength
generated at each successive nucleotide addition
step. While this methodology is more effective
at sequencing homopolymeric stretches than
pyrosequencing, the sequence reads are shorter
(Bennett et al., 2005; Bentley, 2006; Ansorge,
2009). Also, the custom DNA polymerases and
the use of the reversible terminators do result in a
higher number of substitution errors (Hutchison,
2007). The HiSeq 2500/1500 platform, which
utilizes reversible terminator chemistry, is cur-
rently marketed by Illumina. Depending on the
run mode (i.e. Rapid-Run Mode or High-Output
Run Mode), the HiSeq generates between 95 to
600 Gb of sequence with read lengths of between
35 and 150 bp (http://www.illumina.com/sys-
tems/hiseq_2500_1500.ilmn, April 2013).
The third approach to high-throughput
sequencing utilizes ligation based chemistry. This
approach differs from other next generation tech-
nologies in that it is based on hybridization and
ligation of fluorescently labelled oligonucleotides
rather than polymerase-dependent incorporation
of nucleotides. For this approach, an emulsion
PCR single-molecule amplification step similar to
the one used in the pyrosequencing technique is
conducted. The amplification products are then
transferred onto a glass surface and sequence
analysis occurs via sequential rounds of hybridi-
zation and ligation with 16 oligonucleotide
octamers labelled with four different fluorescent
dyes. After addition, ligation, and fluorescent
detection of the complementary octamer, the last
two nucleotides with the fluorescent dye attached
are cleaved off and the next octamer flowed over
the immobilized template DNA. Once the entire
length of the DNA fragment has been covered, it
is stripped and the ligation of the octamers begins
anew from one nucleotide back (n–1) from where
the previous hybridizations occurred. As a result,
each position on the template is effectively probed
twice, and the identity of the nucleotide is deter-
mined by analysing the colour that results from
two successive ligation reactions. Most impor-
tantly, this two-base encoding scheme allows the
differentiation between a sequencing error and a
sequence polymorphism because an error would
be detected in one particular ligation reaction
but a polymorphism would be detected in both
reactions. This methodology is carried out on the
supported oligonucleotide ligation and detection
system (SOLiD) supplied by Applied Biosystems.
The SOLiD 4™ instrument is capable of generating
80–100 Gb of sequence data per run in 35–50 bp
reads per 8 to 13 day sequencing run (http://
www.appliedbiosystems.com/absite/us/en/
home/applications-technologies/solid-next-gen-
eration-sequencing/next-generation-systems/
solid-4-system.html, April 2013).
The availability of these high-throughput
sequencing technologies has democratized
genomics by substantially reducing the cost of

the technology. In addition, they have removed
the need for in vivo cloning by clonal amplifica-
tion of spatially separated single molecules using
either emulsion PCR or bridge amplification on
a solid surface. As these methodologies use single
molecule templates, they allow for the detection
of heterogeneity in a DNA sample thus provid-
ing a powerful advantage over Sanger sequencing
(Bentley, 2006; Thomas et al., 2006).
Bioinformatics analysis of
sequence data
Part of bioinformatics research involves the man-
agement and analysis of large scale sequence data
that has been generated, and is a rapidly growing
field of science that incorporates aspects of biol-
ogy, mathematics and computer science. Once
sequence data have been generated, bioinformat-
ics analysis is required as explained in detail in
Chapters 2, 3 and 4. Depending on the application,
input template, and platform utilized, this initial
processing would include removal of substandard
reads and the alignment of reads into contigs (in
the case of whole genome sequencing). The final
role played by bioinformaticists is the curation of
the huge datasets generated by next generation
sequencing technologies.
Determination of the nucleotide sequence
of a target organism or population of organisms’
genetic material on its own is relatively uninform-
ative. Defining how this nucleotide sequence is
responsible for structural and metabolic function-
ality is more important. Bioinformatics forms the
bridge between the sequence data and the biologi-
cal functioning in an organism/organisms. Once
the nucleotide sequence has been determined,
the first step in the bioinformatics analysis of the
sequence is gene prediction by detecting potential
open reading frames (ORFs). This is achieved by
identifying conserved sequences responsible for
the initiation and termination of transcription
as well as the site for the initiation of translation.
Once a potential ORF is identified, the next step is
to annotate the putative gene by assigning a func-
tion to the sequence. By comparing the encoded
query protein sequence with a database of proteins
with known functions, putative proteins with suf-
ficient homology to the known proteins can then
Microbes: An Unseen Majority Around Us | 9
be assigned the corresponding function. If the
query protein sequence does not show high levels
of similarity with known proteins, annotation by
function can be carried out whereby the putative
proteins domains can be assigned a function. For
example, a particular arrangement of hydrophobic
regions within a protein may indicate a membrane
protein. While assignment of function based
on similarity to other proteins does provide an
extremely valuable starting point when correlat-
ing DNA sequence to cellular metabolism, it is
important to keep in mind that subsequent bio-
logical validation is required for confirmation.
Application of molecular
approaches to the study of
microorganisms
The genomes of living organisms are essentially
barcodes and contain sufficient information to
both identify the microorganism as well as outline
its physiological functionality (Blaxter, 2003;
Blaxter and Floyd, 2003). Genomes contain
areas of high and low identity with the differences
between the corresponding genes typically clus-
tered in sections such as the third (wobble) bases
of codons, intronic, and intergenic DNA. As sig-
nificant stretches of the genome are maintained by
selection to be identical or near-identical between
members within a taxon, but which vary between
taxa, these segments can be applied to both iden-
tification and taxonomy. In addition, as these
sequences evolve, they represent both specific
and systematic data (Woese, 1987). This makes
sequence-based methods incredibly powerful,
and this field has revolutionized the ways that we
both classify and study microorganisms in their
ecosystems, as well as how we screen for novel
products and processes.
Whole genome sequencing
The first microbial genome to be sequenced to
completion was that of the human pathogen Hae-
mophilus influenzae (Fleischmann et al., 1995). In
the same year, the smallest genome of a free-living
microorganism, the pathogenic microorganism
Mycoplasma genitalium, was also sequenced (Fraser
et al., 1995). Since then, the complete sequences
of more 4328 microbial genomes have been
10 | Jiwaji et al.
elucidated (complete and permanent draft) of
which 187 are archaeal, 3958 are prokaryotes and
183 are eukaryotic (www.genomesonline.org,
April 2013). Initially, genome sequence projects
focused on sequencing pathogenic bacteria, now
biotechnological consortia are catching up: cur-
rently 47% of all bacterial-sequencing projects
deal with microorganisms that have industrial
applications, 52% of the projects are focused on
sequencing pathogens and approximately 1%
have targeted exotic organisms (Bode and Muller,
2005; www.genomesonline.org, May 2013; www.
tigr.org, May 2013).
Knowledge of the genome of a given organism
provides tremendous biological insight into cellu-
lar processes that may not be evident when using
classical culturing/assay techniques which are
limited to the study of phenotypic characteristics
under the culture/assay conditions. This allows
for the identification of genes encoding for poten-
tially economically useful metabolites or proteins
which may not be produced or expressed under
current culture/assay conditions. Furthermore,
by increasing our understanding of cellular pro-
cesses and mechanisms of gene regulation within
target organisms, the ability to optimize microbial
metabolism for enhanced application in industry
is increased (Xu et al., 2013). In addition to the
information generated when sequencing a single
genome, the large number of genome sequences
which are currently publicly available on databases
allows for comparative studies between genomes
of different organisms. Such comparative studies
can provide valuable information with respect to
the encoded function of genes particularly when
genomes of closely related strains with differing
phenotypes are compared against one another. In
addition to novel species, multiple isolates of the
same species are also being sequenced. This is due
to the fact that even well-known species such as
Escherichia coli show large levels of heterogene-
ity between strains. For example, comparison of
E. coli genomes sequenced to completion show
a discrepancy in size from 4.6 to 5.5 Mbp. This
means that there are close to one million nucleo-
tides worth of sequence data that is present in
one strain but absent in another (Binnewies et
al., 2006). Comparative genomics is discussed in
detail in Chapter 5.
Currently, the most widely utilized approach
to whole genome sequencing is termed ‘shotgun
sequencing’ (see Chapter 2). In this technique, the
genome of a chosen microbe is randomly sheared
into millions of DNA fragments which are then
sequenced. Owing to the random nature of the
DNA shearing, many of these fragments will over-
lap in terms of sequence data. By aligning these
overlapping fragments against one another, it is
possible to assemble a larger contiguous sequence
(contig). If there are regions of the genome which
are not represented in the sequenced fragment
library, this will result in contigs which do not
overlap and can therefore not be joined together
to form a full length sequence of the genome. In
this instance, targeted re-sequencing of the miss-
ing region is then done by amplifying this region
of the genome using primers specific to the termi-
nal sequence in the contig. Once the genome has
been assembled, the annotation of the genome is
begun where the structural and functional features
of the genome are identified using bioinformatics
tools (discussed in detail in Chapter 4).
Microbial species diversity
Microbial species diversity in a given environment
is determined by the physico-chemical nature of
the environment as well as by changes that are
caused by the metabolic activities of the microor-
ganisms within the community (see Chapter 6).
Ideally, a study designed to analyse the biodiver-
sity in an environmental sample would examine
all the species within the community as well as the
size of these species populations. As more envi-
ronments are being sampled and analysed, it is
evident that the majority of the microbes have yet
to be cultured (Rappe and Giovannoni, 2003).
This is driving the need for technologies to cul-
tivate and enrich formerly ‘not-yet-culturable’
organisms (Stewart, 2012). This is a laborious
and time-consuming process and so techniques
that circumvent the cultivation of organisms but
still allow the exploration of diversity are being
explored.
When selecting specific genetic regions for
analysis of microbial populations, factors such a
ubiquity (the target gene must be present in all
species), species sequence conservation (to allow
for species identification) and evolution-induced

interspecies variability (to allow for differentia-
tion between species as well as to infer taxonomic
relatedness) need to be considered (Petti, 2007).
The sequences that have been applied to molecu-
lar barcoding include the:
1 nuclear small subunit ribosomal RNA gene
(SSU, also known as 16S rRNA in prokary-
otes, and 18S rRNA in most eukaryotes);
2 nuclear large-subunit ribosomal RNA gene
(LSU, also known as 23S rRNA and 28S
rRNA gene);
3 internal-transcribed spacer section of the
ribosomal RNA cistron (ITS, separated by
the 5S ribosomal RNA gene into ITS1 and
ITS2 regions);
4 mitochondrial cytochrome c oxidase 1 (CO1
or COX1) gene; and
5 chloroplast ribulose bisphosphate
carboxylase large subunit (rbcL) gene
(Blaxter, 2004).
Analysis of the 16S rRNA gene is discussed
in detail in Chapter 8 and genomic barcoding is
covered further in Chapter 10.
Each gene target has both its benefits and
pitfalls and these have to be weighed up when
selecting the most appropriate gene. The useful-
ness of each of these genes is determined by (i)
the ease with which it can be isolated from the
sample, (ii) the level of variation between indi-
viduals, (iii) the ease with which the amplified
sequences can be aligned and analysed and (iv)
the availability and number of other sequences
from known and identified specimens. It should
be kept in mind that sequence-based analyses can
be adversely affected if the DNA has extreme base
composition biases, polynucleotide runs or very
stable secondary structures thus this needs to be
taken into account when the targets are selected
for analysis. In addition, with a sequence-based
system, it is essential that a repository of sequence
information, whether these sequences are housed
by annexes to databases like EMBL/GenBank or
in a freestanding effort, is available (Hebert et al.,
2003).
For these targets, the PCR can amplify suf-
ficient quantities of DNA for study and there are
numerous approaches to analyse these amplified
Microbes: An Unseen Majority Around Us | 11
gene targets. For example, randomly amplified
polymorphic DNA (RAPD), terminal restriction
fragment length polymorphism (TRFLP), or
denaturing gradient gel electrophoresis (DGGE)
allow for the assessment of microbial diversity
as well as comparison of community structure
between different ecosystems or over time (Liu
et al., 1997). While these approaches are rapid
and provide useful information in terms of shifts
in microbial population dynamics, their major
disadvantage lies in the fact that identification
of the individual microbes cannot be carried
out. In order to identify the species, analysis of
the nucleotide sequence must be done. Up until
recently, this has been achieved by Sanger-based
sequencing of individual clones representing
PCR-amplified genes from a few hundred repre-
sentatives in a microbial population. The major
limitation of this is that only the dominant
genotypes within the target population would be
sampled. By contrast, next-generation sequencing
technologies generate several thousand sequences
per sample which consequently allows for the
detection of rare biosphere within a given ecosys-
tem community. This rare biosphere is important
in terms of ecosystem functioning as they may
represent critical components of complex con-
sortia or be dormant vestiges of a past ecological
setting with the potential to become dominant
should environmental conditions shift in favour
of their growth. An additional advantage of next-
generation sequencing technologies is that, due to
the large number of sequences generated, a more
accurate representation of the relative abundances
of the bacterial phylotypes within a target ecosys-
tem can be provided (Sogin et al., 2006; Huse et
al., 2008).
The first studies of bacterial diversity using
culture-independent methods have completely
changed our view of the biosphere. Many new
prokaryotic divisions have been described based
on culture-independent studies (Giovannoni et
al., 1990; Schmidt et al., 1991; Barns et al., 1994).
While some of the sequences were close to known
cultured taxa, others showed deeply divergent
lineages with no known cultured members. Many
of these newly identified lineages are now known
to be widespread in many different environments
and are sometimes dominant, be it numerically
12 | Jiwaji et al.
or ecologically. While it is true that some globally
distributed ecosystems have a low bacterial diver-
sity (Hentschel et al., 2002), most environments,
including some of the most inhospitable regions
of our planet, showed an unexpected and deep
diversity (Pace, 1997; Rothschild and Mancinelli,
2001; Furlong et al., 2002; Venter et al., 2004).
However, one must bear in mind that there
are pitfalls in assessing microbial diversity using
PCR-based techniques as PCR amplification may
introduce a bias with each physical, chemical
and biological step resulting in a distorted view
of the actual environment. This may be caused
by the choice of the primers, the conditions of
PCR, potential inhibitors that are present in the
reactions and variable amplification efficiencies of
target genes between species to name a few poten-
tial issues (Wintzingerode et al., 1997).
Metagenomics
Metagenomics provides a gene-based explora-
tion of the microbial community as a whole on
the basis of genetic material (DNA or RNA) and
it returns high resolution data rich information
(Moore et al., 2011). The value of this information
has been enhanced by the availability of sequence
data on a large number of genomes. This topic is
discussed in detail in Chapter 7.
Metagenomic analysis involves isolating DNA
from an environmental sample and analysing
the totality of the DNA. As a consequence, the
DNA libraries contain the genetic information of
all organisms present at a specific location at the
sampling time (Daniel, 2004; Streit et al., 2004;
Grant et al., 2006). Previously, this DNA would
be cloned into suitable vectors, the clones trans-
formed into an appropriate host bacterium, and
the resulting transformants screened. The clones
could be screened for phylogenetic markers, for
conserved genes, for expression of specific traits
such as enzyme activity or antibiotic production
or they could be sequenced via Sanger sequencing.
In the case of sequencing data, these sequences
can be used to query databases allowing for the
inference of phylogeny or the identification of
putative functional genes. With the availability
of next generation sequencing technologies,
metagenomes can be analysed without the need
for time-consuming and labour intensive cloning
steps. Instead, DNA isolated from environmental
samples can be sequenced directly (Tuffin et al.,
2009). This approach has been successfully applied
to terrestrial and aquatic environments resulting
in the discovery of genes for antibiotics, antibiotic
resistance and industrial enzymes (D’Costa et al.,
2007; Lammle et al., 2007; Suenaga et al., 2007).
Examples of enzymes isolated from microorgan-
isms using a functional metagenomics approach
include lipases, esterases, amylases, amidases and
chitinases (Hardeman et al., 2007; Lee et al., 2007;
Chu et al., 2008; Xu et al., 2008). Thus the applica-
tion of metagenomics has paved the way for the
discovery of new genes, proteins and biochemi-
cal pathways (Prakash and Taylor, 2012). This
technology has also been very important for the
identification of new biocatalysts that have been
developed by nature, isolated by bioprospecting
and optimized by directed evolution (Fernández-
Arrojo et al., 2010; Yeh et al., 2011; de Pascale et
al, 2012). Viral metagenomics studies have shown
that up to 60% of the sequences in a viral prepara-
tion are unique, these virus sequences represent
unknown viral species that would be missed by
traditional Sanger sequencing approaches but are
detected by the application of next generation
sequencing technologies (Delwart, 2007; Mokili
et al., 2012).
The full potential of metagenomics is yet to
yet be fully realized. This can be attributed to the
inability of some metagenomic clones to produce
active enzymes. Also, functional metagenomic
approaches rely on E. coli as an expression host
for metagenome-encoded proteins. While this
may occur for a large number of genes, others
from more distantly related organisms may not
be expressed because of differences in the gene
promoters, or the levels of expression may be det-
rimentally affected due to differences in the codon
usage. If transcription and translation of foreign
genes results in the production of protein, it is
possible that the limitations of E. coli to post-trans-
lationally modify or export the protein may result
in the lack of detectable active protein. The availa-
bility of suitable hosts for heterologous expression
remains a barrier to efficient mining of functional
metagenomic data (Handelsman, 2004). It is pos-
sible to couple functional screening approaches
to other approaches like substrate-induced gene

expression screening (SIGEX) to enhance data
mining (Uchiyama et al., 2005; Uchiyama and
Watanabe, 2007). SIGEX was initially developed
to detect metagenomic clones that expressed
catabolic genes of interest in the presence of
appropriate substrates. The time-consuming and
labour-intensive nature of functional screens can
also be alleviated by using robotic instrumenta-
tion to screen large clone libraries for functional
activities in a high-throughput manner (Kennedy
et al., 2008).
Metagenomics, in addition to being a powerful
technique in itself, can be paired with metatran-
scriptomics and metaproteomics to generate
complementary datasets for a more thorough
analysis of the microorganisms in a community
(Ram et al., 2005; Frias-Lopez et al., 2008) includ-
ing the human microbial biome (discussed in
Chapter 8). Despite the recent exciting research
advances involving next generation sequenc-
ers, it should be noted that the application of
these methods is still in its infancy. Efficient and
rigorous data analysis pipelines need to be imple-
mented and further studies are required to verify
the robustness of these techniques as well as the
correlation of these results with those obtained by
previous methods.
Metatranscriptomics and
metaproteomics
Adaptive responses are driven by changing levels
of transcription in the cell as well as changes
in the levels of translation. Metatranscriptom-
ics, and by extension metaproteomics, focuses
on microbial gene expression within complex
natural habitats, allowing for culture-independent
whole-genome expression profiling of complex
microbial communities (Moran, 2009; Sorek and
Cossart, 2010; Gosalbes et al., 2011; Mader et al.,
2011). However, mining the ‘transcriptome’ and
the ‘proteome’, which represent the collection of
transcribed sequences and the translated proteins
respectively, poses a significant challenge, particu-
larly when it comes to comparing data generated
on different ‘omics’ platforms. There are both tech-
nical and biological hurdles to overcome. Efficient
techniques to isolate total environmental RNA
and protein are still being developed; however,
the inherent complexities of RNA and proteins
Microbes: An Unseen Majority Around Us | 13
means that these techniques lag behind those that
have been developed for DNA. The relationship
between RNA and protein is complex; thus, it is
important to be aware of biases in the techniques,
for example the differential lifetimes of mRNA
and protein. This requires the analysis of temporal
changes in transcript and protein levels. The chal-
lenge with transcriptomic and proteomic datasets
remains the identification of true mRNA–protein
concordance and discordance (Hack, 2004; Fang
et al., 2008). For this reason, metatranscriptom-
ics and metaproteomics are fields that are still
being developed, particularly the ability to study
gene expression and protein translation in natural
environments, which hold special promise for
studying microorganism function in ecosystems.
The transcriptome refers to coding RNA
(mRNA) and noncoding RNA (including rRNA,
tRNA, structural RNA, regulatory RNA and
other RNA species). In addition, when discuss-
ing RNA species, it is important to differentiate
between de novo synthesized RNA (capped pri-
mary transcripts) and post-transcriptionally
modified (uncapped secondary) transcripts as
only the processed RNAs will be represented
in the proteome of the microorganism. Under-
standing transcriptome dynamics is essential for
a clearer understanding of the functional output
of the genome and can provide valuable insight
into gene expression patterns, gene function and
regulation (van Vliet, 2010).
Early studies of transcriptional activity in
microbial cells relied on the sequence analysis
of cDNA libraries. Currently, microarrays are
the most widely used technique for studying
transcriptomes. Arrays provide specific and
relative quantification of gene expression free of
the bias that is associated with cloning and they
allow high-throughput analysis of relative tran-
script levels (Hinton et al., 2004). The technique
involves the conversion of cellular RNA into
labelled cDNA, which in turn is used for hybridi-
zation to short oligonucleotides that represent the
coding sequences within a genome. To analyse
the transcriptome, annotated genome sequences
have been used to construct microarrays that
represent the majority of all of the predicted genes
in a genome. For example, the transcriptomes of
Mycoplasma pneumonia, Halobacterium salinarum,
14 | Jiwaji et al.
Caulobacter crescentus, Bacillus subtilis, Escherichia
coli and Listeria monocytogenes have all been ana-
lysed on arrays (Selinger et al., 2000; McGrath
et al., 2007; Güell et al., 2009; Koide et al., 2009;
Rasmussen et al., 2009; Toledo-Arana et al., 2009;
Wurtzel et al., 2009).
Recently there have been dramatic advances
enabling the extension of DNA microarray tech-
nology applications to studying environmental
transcriptomics (Parro et al., 2007; Moreno-Paz
et al., 2010; Pinto et al., 2011). Microarrays that
target the 16S rRNA gene have been used to study
the population structure of microbial communi-
ties (Brodie et al., 2006). Functional gene arrays,
for example the Geochip array (He et al., 2007),
have been used to study nitrogen and carbon
metabolism in Antarctic and PCB contaminated
soils (Leigh et al., 2007; Yergeau et al., 2007)
and the cellular processes in microbial biofilms
(Duran-Pinedo et al., 2011). Phylogenetic micro-
arrays are discussed in detail in Chapter 9.
While arrays have been instrumental in
developing the current understanding of tran-
scriptomes, we have started to reach the limits of
the applicability of this technology (Bloom et al.,
2009). Arrays, like other hybridization-depend-
ent techniques, have a relatively limited dynamic
range for the detection of the levels of transcripts
due to background, saturation and spot density
and quality. Arrays need to include sequences
that cover multiple strains as mismatches
negatively affect hybridization efficiency and the
design of appropriate probes is critical to avoid
a high background due to nonspecific- or cross-
hybridization. The comparison of transcription
levels between experiments is challenging and
usually requires complex normalization methods
(Hinton et al., 2004). Finally, the sensitivity of
scanning instruments determines the quality
of data that is collected. As probe design limits
detection to sequences that are already known,
microarrays are more appropriate when they are
applied to characterize ecosystems rather than to
discover new genes and functions. Also important
is the difficulty in differentiating between de novo
synthesized transcripts and modified transcripts.
While there are techniques that will allow the
differentiation of the two sets of transcripts
(Hashimoto et al., 2009), the alternative is to
couple array-based transcript analysis to a high-
throughput sequencing of cDNA libraries (Hoen
et al., 2008), resulting in increased transcriptomic
data that has been validated by independent plat-
forms (Roh et al., 2010).
All next generation sequencing technologies
can be used for transcriptome sequencing. In this
case, total RNA is extracted from the organism and
converted into cDNA by reverse transcription.
Because prokaryotic mRNAs lack the poly(A)
tail that is typically used for reverse transcription
priming in eukaryotic RNA-seq applications,
alternative priming approaches are used. These
include random hexamer priming (Passalacqua
et al., 2009), oligo(dT) priming from artificially
polyadenylated mRNAs (Frias-Lopez et al., 2008)
and priming from a specific RNA probe ligated
to mRNAs (Wurtzel et al., 2009). Sequencing
platforms have been used to detect genes, intron
usage and alternative initiation codons in yeast
(Nagalakshmi et al., 2008; Sorek et al., 2010; van
Vliet, 2010; Mader et al., 2011). The application
of these platforms is incredibly powerful. In a
recent article, 512 new genes were predicted for
the nitrogen-fixing plant symbiont Sinorhizobium
meliloti after analysis of its transcriptome (Mao et
al., 2008).
There are, however, technical challenges with
metatranscriptomics approaches, which include
the need for the standardization of protocols such
that datasets can be compared. With environmen-
tal samples, technical choices can dramatically
affect results and molecular techniques are prone
to artefacts and biases no matter what platforms
are chosen for sample analysis and data collection
(Raz et al., 2011). The reproducible recovery of
mRNA and proteins from natural environments
is technically challenging as bacterial mRNA
often has a very short half-life and hence can
be highly unstable (Deutscher, 2003; Condon,
2007). Furthermore, obtaining sufficient mRNA
for replicated studies with environmental samples
is often difficult. It is essential that future stud-
ies consider how to meet accepted standards for
independent replication, as this will allow robust
statistical analyses of changes or differences in
expression patterns (Moran, 2009). In these cases
it is necessary first to amplify the RNA. Cao et
al. (2010) performed a comparison of several
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Leah (in Shakespeare’s Merchant of Venice), viii. 296.
Leantio and his Mother (in Middleton’s Women, beware of Women),
v. 215.
Lear (Shakespeare’s), i. 17, 23, 163, 176, 179, 186, 200, 233, 257, 293,
392; ii. 80; iii. 168, 192; v. 4, 5, 8, 52, 56, 145, 188, 225, 244; vi.
273–4, 409, 425, 456; vii. 341, 342; viii. 24, 31, 174, 185, 215, 249,
302, 305, 427–9, 430, 440, 445, 447, 449–50; ix. 421; x. 82–3, 112
n., 117, 156; xi. 451, 491, 533; xii. 33, 198.
Learning, Advancement of (Lord Bacon’s), iv. 200 n.; v. 328; ix. 186;
x. 258; xii. 35 n.
Leatherhead (in Moore’s The Blue Stocking), viii. 239.
Lechery (in Spenser), v. 39.
Leda with her Swan (a picture), iv. 103.
Leddi, Ben (mountain), ii. 318.
Lee, Nathaniel, v. 357; viii. 159; x. 205.
Leech-gatherer, The (Wordsworth’s), v. 122 n., 156; xi. 512.
Leeds, ii. 65; ix. 302.
Lefebre, Robert, xi. 242.
Legion Petition, The (Defoe’s), x. 360.
Leibnitz, G. W., i. 410; iv. 216; vii. 306; xi. 94, 166, 168; xii. 35.
Leicester Fields, ii. 1; vi. 296 n.; xi. 242.
—— Sir John, vi. 376.
Leicestershire, ii. 14; vii. 184.
Leigh, Anthony, i. 157; viii. 258.
—— Miss, viii. 467, 469.
—— Hunt. See Hunt, J. Henry Leigh.
Leipsic, iii. 177.
Leith Walk, ix. 98.
Leland, John, iv. 204 n.
Lely, Sir Peter, vi. 39, 398; vii. 107; viii. 68; ix. 38, 39, 397; xi. 517;
xii. 168.
Lemnos (island), v. 14.
Lenitive (in Hoare’s The Prize), vi. 417; viii. 388; xii. 24.
Lennox, Lady Sarah, vii. 211.
Lennoxes, The, vi. 460.
Lenthall, William, iii. 398.
Leo X., i. 49; vi. 378; x. 190, 206.
—— —— (Raphael’s), ix. 226, 366.
Leominster, ii. 66, 196.
Leon (in Beaumont and Fletcher’s Rule a Wife and Have a Wife), viii.
49, 233; xi. 317.
Leon, Madame, xi. 300.
Leonard (in Holcroft’s The Noble Peasant), ii. 110.
—— (in Cumberland’s Word for Nature), ii. 206.
Leonidas, x. 255.
Leonardo da Vinci, i. 142; ii. 199, 402; iv. 365; vi. 11, 12, 321, 347,
455; vii. 61; viii. 148; ix. 26, 35, 41, 104, 120, 225, 278, 381–3, 417,
482; x. 341; xi. 214, 237, 240 n.; xii. 36, 37, 189, 277.
Leonore (in Molière’s Ecole des Maris), xi. 356.
Leontes (in Shakespeare’s Winter’s Tale), i. 155; v. 257; viii. 376; xi.
206.
Leopold, Prince, xii. 250.
—— of Austria, x. 55.
—— Peter and the President du Paty (Landor’s), x. 247.
Lepidus (in Ben Jonson’s Sejanus), v. 264.
Leporello (in Shadwell’s The Libertine), viii. 371, 462; xi. 308.
Les Plaideurs (Racine’s), x. 107.
Lessing, Gotthold Ephraim, iv. 218; v. 362; x. 119, 274.
Lethbridge, Sir Thomas, xii. 202.
Letitia Hardy (in The Belle’s Stratagem), xi. 404.
Letter-Bell, The, xii. 235.
—— to Bedford, Duke of (Burke’s), i. 427; iii. 210, 335; vii. 11, 115 n.,
118, 228, 257, 275; x. 212.
—— to * * * * on the Rev. W. L. Bowles’s Strictures on the Life and
Writings of Pope (Byron’s), xi. 486 n.
—— to the Dilettanti Society (Barry’s), ix. 422.
—— to a Dissenter, etc., A (Halifax’s), x. 368.
—— to Dunning (Horne Tooke’s), iv. 238, 240.
—— to the Editor of My Grandmother’s Review (Byron’s), iv. 258.
—— to a Friend in London, The (Shelley’s), x. 267.
—— to Mon Prince, The (Lord Castlereagh’s), ix. 315.
—— to a Noble Lord (Burke’s). See Letter to Bedford, Duke of.
—— on Reform (Duke of Richmond’s), vi. 156.
—— to William Smith, Esq., M.P., from Robert Southey, Esq., iii. 210,
218, 224.
Letters (Burke’s), iii. 257.
—— (Farquhar’s), viii. 89.
—— (Gray’s), v. 118.
—— (to and from Holcroft), ii. 240 et seq.
—— (of Junius). See Junius.
—— from Correspondents (Dr Johnson’s), viii. 101.
—— on Ireland (Kendall’s), vi. 394.
—— in Answer to Malthus, iv. 1.
Lettres de Cachet, i. 388.
Leverian Museum, ii. 212.
Leviathan, Hobbes’s, ii. 400 n.; iii. 292; viii. 19 n.; xi. 30, 31, 32, 33,
35, 46, 129, 173 n.; xii. 413.
Levis, Duke of, iii. 181.
Levite, The, vii. 365.
Leviticus, The Book of, xi. 506.
Lewes (town), iii. 414.
Lewis, Lee, ii. 264; xii. 24.
Lewis, William Thomas, ii. 122, 219; vi. 232, 275; viii. 386, 454; xi.
366.
Lexiphanes (by Campbell), vi. 421.
Liaisons Dangereux, Les (Ch. de Laclos), ii. 115.
Liar, The (by Samuel Foote), viii. 11.
Liber Amoris; or, The New Pygmalion, ii. 283.
—— Veritatis (Claude’s), xi. 213 n., 394 n.
Liberal, The (the newspaper), i. xxx; iii. 442; iv. 258, 414, 431; vii.
378–9; ix. 246; xi. 7; xii. 241, 253, 259, 275, 285.
Liberal Snake (Disraeli’s Vivian Grey), xii. 339.
Libertine, The (Shadwell’s), viii. 370; also referred to in viii. 54; xi.
316, 397.
Liberty, On (Cowley), viii. 58, 60.
—— Poem on (Thomson’s), v. 91.
—— and Necessity, On, xi. 48, 50.
Library (Crabbe’s), xi. 606.
Licinio, Giovanni Antonio. See Pordenone.
Liege (town), ii. 280.
Lieutenant Bowling (Smollett’s Roderick Random), x. 35.
—— Worthington (in The Poor Gentleman), xi. 376.
Life’s Decay (Shakespeare’s Sonnet), i. 361.
Liffey, The (river), ii. 61; ix. 416.
Light, Hymn to (Cowley’s), viii. 58.
—— of Nature Pursued (Tucker’s), iv. 369; vi. 327; vii. 355 n.; xi. 85,
178 n.; xii. 358.
Ligny, Godfrey de, x. 57.
Lille, ix. 302.
—— Count de, iii. 290.
Lillie, Charles, viii. 497.
Lilliputians (Swift’s Gulliver’s Travels), v. 15, 112; xi. 483.
Lillo, George, i. 194; ii. 212; v. 6, 359; viii. 268.
Lillys, The, iii. 420.
Lily [Lilly], William, iii. 141.
—— of St Leonard’s (Scott’s), xi. 531, 556.
Limberham, Mr; or, The Kind Keeper (Dryden’s), viii. 393.
Lincoln’s Inn, iii. 86, 126; iv. 282, 284; vii. 447, 448, 449, 452 n.; viii.
8.
Lincolnshire, ii. 14; iii. 396; x. 310.
Lingo (O’Keefe’s Agreeable Surprise), iii. 233; vi. 417; viii. 167, 319,
387; xii. 24.
Lingua, v. 289, 292.
Linley, Thomas, ii. 102, 114.
Linnæus, Carl von, v. 24.
Linton (a town), x. 416; xii. 272, 273.
Lion’s Head (in London Magazine), viii. 479.
Lipsius, Justus (Rubens’s portrait of), ix. 226.
Lisbon Job (Canning’s), iii. 301.
Lismahago (in Smollett’s Humphry Clinker), viii. 117; x. 35; xii. 253.
Lissardo (in Mrs Centlivre’s The Wonder), viii. 156; xi. 402.
Liston, John, i. 154, 247; ii. 368; v. 120; vi. 417; vii. 133, 300, 508;
viii. 140, 159–60, 177, 193, 196, 227, 233, 254, 273, 275, 283, 292,
315, 353, 371, 385, 391, 392, 413, 428, 443, 462, 465–6, 469, 475,
507, 526, 529, 536; ix. 15, 174; xi. 252, 303–4, 316, 367, 376–8,
387–8, 404; xii. 23, 24, 365, 366.
Liston’s Cloten, viii. 540.
—— Mrs, viii. 195, 261.
Litchfield, ii. 14, 15, 166.
Literary Character, On the, i. 131.
—— Examiner (newspaper), ix. 186; xi. p. vii; 540.
—— Gazette, The, vii. 123.
Literary Remains (Hazlitt’s), xi. 596.
Literature of the South (Sismondi’s), x. 44.
Little, Thomas, vii. 368.
—— Baddington (a town in Fielding’s Tom Jones), viii. 113.
—— Filcher, The. See Captive Bee, The.
—— French Lawyer, The (Beaumont and Fletcher), v. 261.
—— Hunchback, The (in Arabian Nights), viii. 12.
—— Offsprings, The; or, Little Offerings (a farce), xi. 369.
—— Pickle (in The Spoilt Child), viii. 470; xii. 24.
—— Red Riding Hood (Fairy Tale), iv. 93; xii. 122.
Littleton, Edward, i. 80.
Liverati, Mons. (a musician), xi. 388.
Livernois (Monsieur), ix. 108.
Liverpool, ii. 55; iv. 320, 341; vi. 58, 103, 153, 156, 190, 203, 204 n.,
387; vii. 28; ix. 302; xi. 480 n.
—— Lord, iii. 48, 59, 75, 76; iv. 225; vii. 268; xi. 480; xii. 275.
Lives of British Poets (Dr Johnson’s), v. 46; viii. 58 n.
Livia (in Middleton’s Women, Beware Women), v. 215.
—— (in Jonson’s Fall of Sejanus), v. 265.
Living in London, viii. 242.
—— to One’s Self, On, vi. 90.
—— Poets, On the, v. 143.
Livy, iv. 283; vi. 13.
Llangollen, vi. 34, 186.
—— (Wilson’s), xi. 199.
—— Vale, xii. 268.
Lloyds, The, iii. 206.
Lloyd’s, ii. 176.
Lochiel (Scott’s), xi. 531.
Lock, Matthew, xi. 404.
Locke, John, i. 425; ii. 133; iii. 296; iv. 45, 212, 285, 377; v. 108; vi.
31, 64, 337, 360, 432; vii. 21, 33, 88, 224, 371, 373, 454 n.; viii. 18
n.; x. 134, 176, 232, 249, 361; xi. 1, 29, 30, 42, 44–5, 47, 58, 59, 62,
64, 74, 126, 127, 129, 165–6, 168 n., 171, 174, 176, 178–9, 181–4 et
seq., 578; xii. 26, 27, 35, 313, 403.
Locke, Mr, a great Plagiarist, xi. 284.
Locke’s Essay on Human Understanding, On, xi. 74.
Lockhart, John Gibson, vi. 498; viii. 478 n.; x. 407, 411.
Lockitt. See Lucy Lockitt.
Locksley (Scott’s Ivanhoe), vi. 81; viii. 424.
Locrine (? Shakespeare), i. 357.
Locusta Poisoning a Young Slave (Figalon’s), ix. 128.
Lodon, The River, v. 121.
Lodovico (in Webster’s White Devil), v. 241, 245.
Lofft, Capel, viii. 241.
Loftus (brother-in-law of Rev. W. Hazlitt), vii. 502.
Logan, John, ii. 328; v. 122.
Loggia in the corridors of the Vatican (Raphael’s), ix. 240.
Logic, xii. 350.
—— Condillac’s, xi. 173 n.
Logos, i. 52.
Loiter (in Kenney’s The World), viii. 229.
Lombard, Peter, i. 332.
—— Street, vi. 113.
Lombardy, ix. 264.
Lomond, Ben, ii. 328, 329.
—— Loch, ii. 329.
—— —— (Hofland’s), xi. 242.
London, Account of (Pennant’s), vii. 69.
—— Views of (a book), vi. 429.
—— Bridge, ii. 242; xi. 352.
—— Description of the Morning in (Swift’s), v. 109.
—— Institution, vi. 199; xii. 76.
—— Magazine, vi. 469, 483, 484, 494; vii. 481, 496, 498–9, 502–4;
viii. 383, 477, 479; ix. 18, 439 et seq., 466, 468; x. 223; xi. p. viii,
464, 481, 486, 508, 521, 531, 537.
London Prodigal, The (? Shakespeare’s), i. 357.
—— Wall, iv. 365; vii. 69, 254.
—— Weekly Review, xii. 296, 297, 301, 306, 311, 316, 321, 328, 330,
336.
Londoners and Country People, On, vii. 66.
Long-Acre, xii. 120.
Long, Charles, i. 379.
—— Robinson (a cricketer), xii. 17.
Long’s (16 New Bond St.), iv. 259; vi. 202; xi. 344, 385, 486.
Longford Castle, ix. 55, 56.
Longhena, Baldassare, ix. 269.
Longinus, i. 401; xii. 168.
Longman, Mr (publisher), vii. 378.
—— Messrs, iv. 312.
Longus, x. 14.
Look of a Gentleman, On the, vii. 209.
Lopez Banos (in Landor), x. 251.
—— de Vega, vi. 49; x. 118.
Lord Alton (in Godwin’s Cloudesley), x. 392.
—— Avondale (in Merton’s School of Reform), viii. 315.
—— Clamourcourt (in Jameson’s Living in London), viii. 242, 243.
—— Danvers (in Godwin’s Cloudesley), x. 386, 392.
—— Duberly (Liston’s), vi. 417.
—— Foppington (in Vanbrugh’s Relapse), i. 12; vi. 275, 444; viii. 9,
36, 37, 82, 83, 151, 304, 328, 465; xi. 309, 439.
—— and Lady Froth (Congreve’s), viii. 72.
—— Glenallan (in Scott’s Antiquary), viii. 413; ix. 202.
—— Grizzle (in Fielding’s Mock Doctor), viii. 159, 540; xi. 377; xii.
365.
Lord Lovell (in Massinger’s A New Way to Pay old Debts), v. 267; vii.
274, 277, 304.
—— of the Manor, The (Burgoyne’s), xi. 316.
—— Mayor’s Procession, The (Hogarth’s), viii. 142.
—— —— Show, viii. 18.
—— Ogleby (G. Colman the elder’s The Clandestine Marriage), vii.
210; viii. 154.
—— Peter (in Swift’s Tale of a Tub), iii. 136; iv. 245; v. 112; vii. 192.
—— Sands (in Shakespeare’s King Henry VIII.), viii. 387; xii. 24.
—— Townley (in Vanbrugh’s The Provoked Husband), vi. 453; viii.
465; xi. 346.
—— Trinket (in G. Colman the elder’s Jealous Wife), viii. 317, 505.
Lord’s Cricket-ground, xii. 17, 233, 373.
Lords, On the Conversation of, xii. 38.
Lorenzo (in Shakespeare’s Merchant of Venice), vi. 279.
Loretto (a town), x. 304.
Lorraine, Claude. See Claude.
Loss of The Royal George (Cowper’s), v. 95.
Lot and his Family (West’s), xi. 190.
Lothario (in Rowe’s The Fair Penitent), i. 12; ii. 59; viii. 151, 288.
Lothbury, x. 310.
Loudon Hill, iv. 247.
Loughborough, Baron, ii. 99; vi. 438.
Louis IX., Saint, ix. 175.
—— XIII., ix. 110.
—— XIV., iii. 100, 160, 258, 307, 311; v. 106; vi. 419; vii. 185, 308,
323, 346; viii. 251; ix. 14, 23, 150, 165; x. 233, 250, 303; xi. 275,
354–5; xii. 122.
Louis XIV. taking leave of his Grandchild (Madame Hersent’s), ix.
124.
—— XV., i. 388; v. 114; vi. 349; xii. 287.
—— XVI., iii. 32 n., 290; vii. 268.
—— XVIII., iii. 101, 106, 158, 175, 228, 240, 290, 319 n., 448; vi. 360;
viii. 267, 275 n., 340; ix. 94, 108, 124, 125; xi. 413, 417, 551; xii. 141,
 356, 448.
Lounger, The (newspaper), viii. 105.
Loutherbourg, P. J., i. 149; ii. 185; vii. 95.
Louvet, Jean-Baptiste Louvet de Couvray, vi. 102.
Louviers (a town), ix. 101, 102, 103, 104.
Louvre, The, i. 45, 145, 163; iii. 169, 421; iv. 324; vi. 15, 17, 93, 174,
237, 319; vii. 24, 274, 280, 281, 285, 291, 314; viii. 148, 443; ix. 31,
53, 59, 106, 108, 112, 113, 120, 126, 129, 147, 160, 165, 224, 225,
226, 232, 237, 241, 270, 271, 273, 301, 302, 352, 359, 365, 366,
372, 385, 388, 472, 475, 491; xi. 196, 197, 213, 222, 237, 273, 352;
xii. 189, 190, 198, 209, 216, 322.
Lovatt, Lord, iii. 285 n.
Love, Miss, viii. 464; xi. 377.
—— and a Bottle (Farquhar’s), viii. 89.
—— of the Country, On the, i. 17.
—— and Gout (? Jameson), viii. 242, 322.
—— Law and Physic (Kenney’s), viii. 159, 193.
—— of Life, On the, i. 1.
—— in Limbo (? Millingen), viii. 227.
—— for Love, (Congreve’s), viii. 278;
also referred to in ii. 84; vii. 127; viii. 71, 72, 77.
—— of Power or Action as a Main Principle in the Human Mind, as
Sensibility to Pleasure or Pain, xi. 263.
Love in a Riddle (Cibber’s), viii. 162.
—— and Toothache (a play), viii. 536.
—— in a Tub (Etherege’s), viii. 68.
—— in a Village (Bickerstaffe’s), ii. 301; vi. 293, 352, 382; viii. 163,
330, 341, 532; xi. 317, 366.
—— in a Wood (Wycherley’s), viii. 78; xi. 573.
Love’s Catechism (in Farquhar’s The Beaux’ Stratagem), xii. 122.
Love’s Consolation (Shakespeare’s Sonnet), i. 360.
—— Deity (Donne’s), viii. 52.
—— Frailties (Holcroft’s), ii. 159, 161.
—— Labour’s Lost (Shakespeare’s), i. 332;
also referred to in v. 128; xi. 360, 416.
—— Last Shift (Cibber’s), viii. 162.
—— Sacrifice (Ford’s), v. 270.
Loves of the Angels (Moore’s), iv. 258; vii. 134; ix. 73.
—— of the Gods (Titian’s), ix. 73.
—— of Persiles and Sigismunda (Cervantes), viii. 110.
Lovegrove, Thomas, viii. 250, 253.
Lovelace (in Richardson’s Clarissa Harlowe), i. 12; ii. 128; vii. 227 n.;
viii. 120, 151, 561; x. 38, 39; xii. 63, 435.
Loveless (in Vanbrugh’s Relapse), viii. 79.
Lovell, Robert, ii. 279.
Lover’s Complaint, The (Shakespeare’s), i. 360.
—— Melancholy, The (Ford’s), v. 270, 318.
Lovers’ Vows (Mrs Inchbald’s adaptation of Kotzebue), viii. 249;
also referred to in ii. 196, 198; v. 360; viii. 335; xi. 362.
Lovibond, Edward, v. 122.
Lowe, Sir Hudson, vii. 83; x. 227.
—— Mauritius, ii. 191.
Lowth, Robert, Bishop of London, iv. 238, 391.
Lowther Estate, iii. 421.
Loyola, Ignatius, vi. 303; ix. 43.
Lubin Log (in Kenney’s Love, Law and Physic), viii. 159, 193, 416,
428, 540; xi. 377; xii. 23.
Lucan, iii. 222; x. 13.
Lucca, ix. 213.
Lucetta (in Shakespeare’s Two Gentlemen of Verona), i. 319.
Lucian, v. 199; viii. 28; x. 17.
Luciani, Sebastiano. See Piombo, S. del.
Lucien Buonaparte’s Collection, etc., xi. 237.
Lucifer, v. 279.
Lucinda (in Bickerstaffe’s Love in a Village), viii. 329.
Lucio (in Marston’s Antonio and Mellida), v. 225.
Lucio (in Shakespeare’s Measure for Measure), i. 391; viii. 283, 284.
Lucius (in Shakespeare’s Julius Cæsar), i. 199.
Lucretia (in Fielding’s Joseph Andrews), vii. 223.
—— Borgia (portrait of), xii. 36.
Lucretius, xi. 492.
Lucy (in Wycherley’s Love in a Wood), viii. 78.
—— (in Sheridan’s Rivals), viii. 508.
—— Bertram (in Scott’s Guy Mannering), iv. 248 n.; viii. 292.
—— Lockitt (in Gay’s Beggar’s Opera), i. 66; iii. 156; v. 108; viii. 194,
255–6, 268, 315, 324, 470; xi. 373, 533.
Ludgate Hill, ii. 215; vii. 275.
Ludlow, ii. 66, 196.
Ludovico (in Mrs Radcliffe’s Castle of Otranto), viii. 126.
—— (in Othello), viii. 221.
Luini, Bernardino, ix. 224, 278.
Luke (in Massinger’s City Madam), xii. 142.
—— (in Sir J. B. Burgess’s Riches), viii. 208.
Luppino, Miss, viii. 244, 535.
Lusiad (Camoens), i. 33.
Luss (a town), ii. 329.
Lust’s Dominion; or, The Lascivious Queen, v. 207.
Lutea Alanson (Suckling’s), viii. 56.
Luther, Martin, iv. 250; vi. 147; viii. 297; xi. 216; xii. 195, 348.
Lutrin, The (Boileau), v. 73.
Lutterworth, ii. 14, 166.
Luttrel, Hon. Temple, iii. 422.
Luxembourg, The, ix. 23, 110, 123, 129, 157, 159.
Lyceum, The, v. 147; viii. 239, 243, 244, 314, 412, 463, 471; xi. 381.
Lycidas (Milton’s), i. 31, 36, 94; iii. 433; v. 59, 98, 315, 371; vii. 160;
viii. 232, 233.
See also Milton.
Lydgate, John, v. 34.
Lydia (in Lyly’s Mother Bombie), v. 198.
Lydia Languish (in Sheridan’s The Rivals), viii. 509.
Lydia Melford (in Smollett’s Humphry Clinker), viii. 410.
Lying Valet, The (Holcroft’s), ii. 80.
Lyly, John, v. 192;
also referred to in v. 193, 197, 201 et seq.
Lynn, iii. 405.
Lynton, iii. 149.
Lyonnais, The, Diligence, ix. 177.
Lyons, i. 90; ii. 275; vi. 384; ix. 154, 176, 178, 181, 182, 183, 184, 193.
Lyrical Ballads, The (Wordsworth’s, etc.), i. 92; iii. 168; iv. 271, 273,
275, 313; v. 129, 131, 146, 156, 162, 164; vi. 44; vii. 226; viii. 420; x.
135, 142; xi. 311, 335 n., 457, 512; xii. 269, 273, 329.
Lystra, Sacrifice of (Raphael’s), xi. 211.
Lyttelton, George, Lord, iii. 414.
—— Thomas, Lord, iii. 423; vii. 350.
 M.

M——, Lord, vi. 380.

M——, Mr (? Malthus), iv. 241.
M——, (? Tom Moore), vi. 358.
M—— (T.), vi. 454.
MacAdam, John Loudon, iv. 250; ix. 94.
MacAlpine, Mr, xii. 379.
—— Mrs, xii. 379.
—— Miss, viii. 275.
Macartney, Lord, vi. 455.
Macauley, Elizabeth Wright, iv. 223.
Macbeth (Shakespeare’s), i. 186;
also referred to in i. 138, 179, 200, 201, 238, 293, 300, 311, 394,
395; iii. 168; v. 10, 52, 56, 188, 218, 220, 223; vi. 39, 392, 394,
409, 410; viii. 31, 49, 185, 199, 203, 208–9, 249, 272, 305, 314,
378, 472, 518; ix. 401, 474; x. 81, 82, 111, 117; xi. 192, 315, 404,
451, 482, 506, 601; xii. 33, 365.
Macbriar (Scott’s Old Mortality), iv. 247; viii. 129; xi. 531; xii. 277.
Maccabees, The Book of, xi. 321.
Macclesfield, ii. 12, 14, 18.
Macculloch, John Ramsay, ii. 415; xii. 131, 141, 320, 345, 361, 412.
Macdonald, Chevalier, ii. 107.
Macduff (in Shakespeare’s Macbeth), v. 48; vi. 39; viii. 333; xi. 316.
MacFane (in Holcroft’s Anna St Ives), ii. 128.
Macflecknoe (Dryden’s), v. 80.
M’Gibbon, Mrs, viii. 192, 459.
Macgregor, Sanders, viii. 105.
Machiavelli, Niccolo, iv. 283; v. 186; vii. 28; xi. 424.
Macintosh, Sir James, iv. 279;
also referred to in ii. 196; iii. 86; iv. 211, 319; vi. 205 n.; vii. 447,
448, 451 n., 460; ix. 490; xi. 465, 467, 468; xii. 264, 275, 347.
Macirone, Francis, Interesting Facts relating to the Fall and Death of
Joachim Murat, etc., iii. 177, 183.
MacIvor, Fergus, ix. 367.
Mackenzie, Henry, ii. 195, 200; iv. 367; vii. 227; x. 399; xi. 546 n.; xii.
67.
Macklin, Charles, i. 157, 158; ii. 56, 57, 58, 59, 60, 61, 62, 63, 109 n.;
viii. 166, 351.
Maclean, Dr, ii. 232.
Macready, William Charles, ii. 302; vi. 277, 278; viii. 334, 335, 337,
338, 356, 368, 391, 426, 440, 442, 457, 465, 534; xi. 315, 391.
Macready’s Macbeth, Mr, xi. 315.
—— Othello, viii. 338.
Macullamores, The, xii. 255.
Mad Tom (in Shakespeare’s King Lear), i. 260, 268; viii. 302, 440,
441.
Madame Centaur (in Ben Jonson’s Silent Woman), viii. 43.
—— d’Orbe (in Rousseau’s New Eloise), ix. 146.
—— Haughty (in Ben Jonson’s Silent Woman), viii. 43.
—— Mavis (in Ben Jonson’s Silent Woman), viii. 43.
—— Valmont (in Tyran Domestique), xi. 356.
Madamira (song in Shadwell’s Libertine), viii. 372.
Madge Wildfire (Scott’s Heart of Midlothian), iv. 248; vii. 342; xi.
388.
Madison, James, iii. 172 n.
Madman, The (Hogarth’s), xii. 242.
Mad Mother, The (Wordsworth’s), xii. 270.
Madoc (Southey’s), iv. 265.
Madonna, The (Correggio’s), xii. 356.
—— Guido’s, ix. 34.
—— Raphael’s, ix. 261, 433.
—— of Foligno, The (Raphael’s), x. 191.
—— and Child (Cranach’s), ix. 354.
—— of the Crown, or of the Garland (Raphael’s), ix. 67; xi. 485.
Madonna and Infant Christ (Vandyke’s), ix. 21.
—— Pia, ix. 252.
—— della Seggia (Raphael’s), ix. 226.
Madras System, Southey’s Tract on, iii. 149.
Madrid, i. 123; iii. 119.
Maeviad (Gifford’s), i. 379 n., 380, 385, 396; iv. 304, 309; vi. 221.
Maffei, Counts, x. 303.
Magdalen (Dolci’s), ix. 41.
—— (Guido’s), ix. 57.
—— (Titian’s), ix. 269, 270.
Magellan, v. 187; xi. 601.
Maggiore, Lago, ix. 278.
Magna Charta, iv. 93; vi. 155.
Magnus Troil (in Scott’s The Pirate), xi. 534, 536.
Magpie; or, Maid of Palisseau (? Dibdin’s version), xi. 304, 381.
Mahomet, xi. 472 n.
—— (Voltaire’s), vi. 383.
Mahomet’s Coffin, iii. 138.
Maid’s Tragedy, The (Beaumont and Fletcher’s), v. 251.
Maid and Magpie (Arnold’s version), viii. 244;
also referred to in vii. 339; viii. 280; ix. 175; xi. 304, 381.
—— of Orleans (Shakespeare’s 1st Henry VI.), i. 292.
—— of The Vale (Holcroft’s adaptation), ii. 86.
Maiden Queen (Dryden’s), i. 195.
Maidstone, ii. 186.
Main Chance, The, xii. 78.
Maintenon, Madame de, vi. 419; ix. 179.
Maitland, General, ii. 218, 222.
Maître Jacques (a play), xi. 380.
Major Bath (in Fielding’s Joseph Andrews), vii. 223.
—— Dumpling (in Jones’s The Green Man), viii. 468.
—— Galbraith (Scott’s), iv. 248.
—— Oakley (in G. Colman the elder’s Jealous Wife), viii. 532.
—— O’Flaherty (in The West Indian), ii. 83; viii. 511.
Major Sturgeon (Foote’s Mayor of Garratt), viii. 167, 168, 246, 317,
318, 392; xi. 366, 368, 396.
Makins, Mr (in Amory’s John Buncle), i. 54; iii. 142.
Malades, Mont des, ix. 98.
Malatesta, Anthony, ix. 218.
Malbecco (Spenser’s Faëry Queen), iii. 55; v. 42, 43.
Malcolm (in Shakespeare’s Macbeth), v. 48.
Malcontent, The (Marston’s), v. 226, 228, 229, 271 n., 272.
Malebranche, Nicolas, iv. 216; vii. 144, 146; xi. 286, 288.
Malevole (in Marston’s The Malcontent), v. 225, 228, 230.
Malibran (de Beriot, Maria Felicita), xii. 384.
Mall in St James’s Park, View of (Gainsborough’s), vi. 437.
Mallet, David, v. 375.
Malmesbury, x. 143.
Malone, Edmond, ii. 184; vi. 366, 510, 511; x. 172, 173.
Malta, ii. 173, 175; iii, 3, 5, 7.
Malthus, Thomas Robert, iv. 287;
also referred to in iii. 95; iv. 241; vi. 269; vii. 193; x. 403; xii. 40,
141.
Malthus’s Doctrines, An Examination of: (1) the Geometrical and
Arithmetical Series, iii. 356.
—— Essay, On the Originality of, iii. 361.
—— Principle to the Poor Laws, On the application of, iii. 374.
—— Reply to the Essay on Population, by Rev. T. R., iv. 1;
also referred to in iii. 462; xii. 412.
Malthuses, The, xii. 255.
Malvil (Murphy’s), viii. 164.
Malvolio (in Shakespeare’s Twelfth Night), iii. 213; v. 97; viii. 12, 32,
164, 302.
Mambrino (in Cervantes’ Don Quixote), viii. 108; x. 27.
Man, The, in the Louvre (Titian’s), ix. 273.
Man, Aphorisms on, xii. 209.
—— The Essay on (Pope’s), v. 76, 373; xi. 491; xii. 31.
—— in Black, portrait (in the Louvre), xii. 192.
—— of Business (G. Colman, the elder), ii. 109 n.
—— of Feeling, The (Mackenzie’s), ii. 336; vii. 227; viii. 105; xii. 67.