Editorial overview: Novel technologies in microbiology:Recent advances in techniques in microbiologyEmmanuelle Charpentier and Luciano A Marraffini

Current Opinion in Microbiology 2014, 19:viii–x

For a complete overview see the Issue

Available online 10th July 2014

http://dx.doi.org/10.1016/j.mib.2014.06.012

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History has shown that microbiology has frequently been at the forefront of

the discovery and development of novel technologies. A wide range of

techniques from classical genetics to more sophisticated biophysics and

systems biology have been developed and applied to understand the life

of microbes starting from model microorganisms grown in flasks to more exotic

microbes cultivated in simulated natural environment in interaction with their

hosts and predators. A substantial number of molecular principles of micro-

biology have also been valuable sources to discover new or advance further

technologies for a range of application beyond the microbiology field, revo-

lutionizing biotechnology and biomedicine. In recent years, the field of

microbiology is experiencing a rebirth owing to the development and appli-

cation of various novel technologies aiming at a deeper understanding of

microorganisms. How many new microbial species are to be discovered? How

to improve fast and accurate identification of pathogens in the hospital setting?

How to identify links between microorganisms and disease? How bacteria

help to keep us healthy? How pathogenic and non-pathogenic microbes

evolve in response to their environment? How new antibiotic resistances

develop? How microbes communicate and exchange genetic material? How

microbes defend themselves from and adapt to their environment and hosts?

What are the specific regulations involved in homogenous and heterogeneous

populations of cells at the single molecule and single cell levels? How many

new key regulators and effectors are yet to be identified? What are the

interactomes in play? How to visualize subcellular structures and processes?

In this issue, Current Opinion in Microbiology has selected a series of articles

highlighting some of the most recent technological developments and their

applications in microbiology. These include high throughput sequencing for

the analysis of genomes and transcriptomes, mass spectrometry for the

identification of bacterial pathogens in the clinical setting and of metabolite

production within complex microbial communities, and novel approaches

for antimicrobial discovery and for the development of fuel-producing

microorganisms. The continuous effort to combine innovative and sophis-

ticated technologies has already advanced the analysis of microbes at an

unprecedented level of detail and will be critical for researchers of the future

to discover new fascinating concepts of biology.

High Throughput Sequencing (HTS) has revolutionized microbiology

revealing new insights into bacterial evolution, epidemiology, and patho-

genesis. In their review, McAdam, Richardson and Fitzgerald summarize

selected recent studies that have applied HTS to address fundamental

questions in the biology of infectious diseases. For example, HTS has

enabled high-resolution phylogenetic analyses of bacterial populations,

Emmanuelle Charpentier1,2

1 Helmholtz Centre for Infection Researchand Hannover Medical School, 38124Braunschweig, Germany2 The Laboratory for Molecular Infection

Medicine Sweden, Umea University, Umea,

Sweden

e-mail: emmanuelle.charpentier@helmholtz-

hzi.de

Emmanuelle Charpentier is Professor at theHelmholtz Centre for Infection Research and

Hannover Medical School in Germany and

Umea University in Sweden. Her maininterests lie in the understanding of the

mechanisms of regulation in bacterial

infection and immunity. Her research

programs aim to identify new RNAs andproteins and decipher their biogenesis,

functions, and modes of action at the

molecular and cellular level. Application of

her research in biotechnology and medicineis well exemplified by the recent discovery of

the CRISPR-Cas9 tool now broadly used for

genome engineering in cells and organisms.

Luciano A MarraffiniThe Rockefeller University, 1230 YorkAvenue, New York, NY 10065, USAe-mail: Luciano.Marraffini@rockefeller.edu

Luciano A. Marraffini is an AssistantProfessor and Head of the Laboratory of

Bacteriology at The Rockefeller University.

His research focuses on understanding the

molecular mechanisms of CRISPR-Casimmunity and their role in the control of

horizontal gene transfer between bacteria.

Available online at www.sciencedirect.com

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Current Opinion in Microbiology 2014, 19:viii–x www.sciencedirect.com

Editorial overview: Novel technologies in microbiology Charpentier and Marraffini ix

providing a better understanding of bacterial evolution

during infection and more precise tracing of origins and

transmissions of outbreaks. HTS has revealed that bac-

terial pathogens can undergo considerable diversification

during infection processes, and has offered a considerable

improvement for global gene expression profiling studies.

The combination of HTS and transposon mutagenesis

has led to the development of a series of powerful

approaches that facilitated the identification of the genes

required for the survival of pathogens in their host and of

other microbes in other environments. Future techno-

logical advances in HTS are likely to have a profound

impact on the microbiology field. With the development

of platforms capable of single-molecule sequencing with

ever increasing read lengths, the technology already

offers the possibility to assemble accurately individual

species within a microbial community. Applications of

HTS to non-cultivable organisms will aid the investi-

gation of infectious diseases of unknown aetiology. The

combination of novel culture-free methodologies and

HTS approaches should also facilitate the rapid diagnosis

and in silico determination of sensitivity profiles of patho-

gens in the clinical setting.

While HTS of genomic DNA has pushed forward our

understanding of bacterial evolution and speciation, HTS

of bacterial transcripts (RNA-sequencing or RNA-seq)

provided genome-wide gene expression profiling and

transcript annotations at a single nucleotide resolution,

allowing the identification of a large number of novel

small regulatory RNAs and antisense RNAs. Sharma and

Vogel discuss the development and recent applications of

the differential RNA-seq (dRNA-seq) method. dRNA-

seq offers the additional feature to differentiate primary

from processed RNA populations. The technology has

initially been applied to describe the primary transcrip-

tome of the gastric pathogen Helicobacter pylori, and has

since been widely used to generate global maps of start

sites of transcripts in various species, providing new

insights into processing events of mRNAs and RNAs

with regulatory functions. Describing selected examples,

this review illustrates how the technology enabled new

biological insights in bacterial gene regulation. The

authors also comment on refinements and further im-

provement of the technology to be expected in the near

future and suggest three main research areas where the

technology could be valuable: single-cell RNA-seq, meta-

transcriptomics, and simultaneous RNA expression pro-

filing of bacterial pathogens together with their eukar-

yotic hosts.

Three reviews cover recent applications of advanced mass

spectrometry (MS) technologies. Moore, Caprioli and

Skaar review our current knowledge of advanced MS

technologies that have been applied for the study of

microorganisms. The authors highlight the potential of

these biophysical methods as clinical tools for both the

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diagnosis of pathogens in laboratories and the discovery of

novel targets for therapeutic intervention. Matrix-assisted

laser desorption/ionization mass spectrometry (MALDI

MS) is used to determine molecular profiles of small cell

populations for rapid identification of microbes in diag-

nostic analysis. Histology-directed MS analyses allow the

direct profiling of molecules from patient sera and tissues

and the identification of biomarkers in discrete areas of

infected tissues. With advanced mass spectrometry tech-

nologies, molecular profiling across tissue sections can

also be achieved in a more systematic fashion and enable

measurements of spatial distributions of molecules and

analytes in specific regions in situ in both two-dimensional

and three-dimensional analyses. The authors underline

that challenges in the improvement of more advanced

MS-based analytical instrumentations lie in part in the

relative low abundance of microbial proteins and the

small size of most microorganisms. Nevertheless, the

authors highlight some additional recent developments

such as advanced laser optics enabling the imaging of

single cells and advanced electronics in new mass spec-

trometers facilitating the rapid acquisition of data for large

3D imaging data sets. The authors also mention the

development of ionic matrices to improve the ionization

of proteins or MALDI-compatible surfaces to help cap-

ture bacteria from biological samples. Aldridge and Rhee

present an overview of the so-called metabolic technol-

ogies. Advances in NMR-based and MS-based methods

have enabled the detection and quantitation of cellular

metabolites. The application of these technologies to the

microbiology field has begun to reveal an unexpected

diversity of composition, structure and regulation of

metabolic networks in microbes with substantial changes

in metabolic needs triggered in different environments or

growth conditions. The authors highlight the technologi-

cal developments in this area, describing applications to

precisely measure metabolite concentration and subcel-

lular localization, to assign metabolic functions of

unknown genes and to study the structure and regulation

of metabolic networks. In a related review, Fang and

Dorrestein summarize MS technologies that enable the

direct detection and analysis of specialized metabolites

produced by microbial colonies and communities. There

is an increasing need to understand the detailed chem-

istry involved in microbial behavior and develop tech-

nologies that could benefit strain identification for clinical

use. Over the last years, substantial efforts have been

directed towards the development of more modern and

sensitive instrumentations that integrate compatible

microbial and mass spectrometry workflows. The authors

describe MS techniques that have so far been mostly

applied to microbiology. For example, imaging mass

spectrometry and real-time mass spectrometry enable

two-dimensional and three-dimensional visualization of

metabolite distribution with little or no sample prep-

aration. Recent developments make it now possible to

map microbial molecules spatially, visualize the

Current Opinion in Microbiology 2014, 19:viii–x

x Novel technologies in microbiology

molecules produced by living microbial colonies at the

single cell level. The authors conclude that future

advances towards the combination of MS with new mol-

ecular visualization tools and informatics approaches will

improve the level of characterization of microbes and

their chemical repertoire.

The dramatic increase in infections caused by multi-resist-

ant bacteria and the shortage in effective antibiotics has

resulted in a renaissance in bacteriophage-based therapy

and in the developing of novel approaches for the discovery

of antimicrobials. In this issue, Citorik, Mimee and Lu

discuss recent advances in bacteriophage-based technol-

ogies and the recent introduction of synthetic biology

methods in this field. The authors have selected some

examples demonstrating how synthetic biology has

enabled the engineering of modified phages resulting in

innovative next-generation bacteriophage-based tools for

the study and treatment of infectious diseases. Phage

display has been extensively used in this regard and for

the development of novel therapeutics, and phage lysins

have been investigated in recent years as potential anti-

microbials. Bacteriophage components constitute a core

set of parts in the synthetic biology toolbox. Phage-derived

enzymes and technologies have improved genome engin-

eering techniques for the tailoring of strains in specific

applications, to generate genetic diversity and to study

accelerated evolution. Charlop-Powers, Milshteyn and

Brady focus their review on recent advances in metage-

nomic approaches for antibiotic discovery. Capture of

DNA from the environment (eDNA) and subsequent

identification and expression of biosynthetic gene clusters

in heterologous hosts can provide means to decipher

unexplored biosynthetic pathways encoded by the gen-

omes of environmental bacteria and thus bridge biosyn-

thetic diversity to drug discovery pipelines. The authors

explain in detail the sequence-based methods that inter-

rogate the biosynthetic content of metagenomic samples,

identify lead targets, and allow the recovery of complete

biosynthetic pathways from eDNA libraries. Activation of

gene cluster expression is then followed by the production

and discovery of small molecules. In contrast, function-

based methods will identify clones that are already bio-

synthetically active in a heterologous host by detecting a

clone-induced phenotype in a host organism. The authors

explain that novel technologies such as the Nanopore-

based sequencing or single-cell and microdroplet-based

methods have a clear potential to extend the power of

whole genome sequencing to metagenomes. Robinson,

Adolfsen and Brynildsen take on a different approach

Current Opinion in Microbiology 2014, 19:viii–x

for the discovery of new antimicrobials: the rational

design of inhibitors of enzymes and biochemical path-

ways essential for bacterial survival that are absent from

human cells. While this is a classical methodology for the

discovery of antibiotics, the authors review efforts toward

a novel target pathway: nitric oxide (NO) detoxification

and repair. NO is a potent antimicrobial compound that

immune cells produce to fight pathogens. Thus, to estab-

lish an infection, pathogens depend on pathways that

neutralize NO radicals and repair the damage they exert

on different biomolecules. Inhibitors of these pathways

are under investigation as next-generation antibiotics. In

particular, the authors focus on the use of quantitative

kinetic modeling to improve the analysis and under-

standing of NO stress (and other broadly reactive anti-

microbials) at systems level.

In addition, this issue of Current Opinion in Microbiologyincludes two reviews in some of the most exciting areas of

biotechnology: production of microbial biofuels and

CRISPR-based genome editing technologies. Endophy-

tic fungi have the property to produce volatile organic

compounds (VOCs) with hydrocarbon-like properties

when agricultural wastes are used as substrates. VOCs

are identical or closely related to compounds found in

diesel fuels and thus have the potential to be used as

‘green chemicals’ and fuels, also referred to as Mycodie-

sel. In his review, Strobel presents the history of the

production of Mycodiesel by fungi, describes examples of

fungi that produce VOCs and highlights some of the new

methodologies that have been developed specifically for

the study of fungal production of hydrocarbons. In prin-

ciple, the microbe is isolated and identified, the compo-

sition in VOCs is determined and sequence information

of the gene cluster responsible for VOC production is

obtained, which is used to genetically manipulate the

microbe to enhance production. There are also efforts to

determine the ideal conditions for hydrocarbon pro-

duction by fungi with the aim to produce ‘superprodu-

cing’ fungal strains. Strobel presents his views on the

impact that these promising green chemicals and fuels

may have in the chemical industry for a variety of indus-

trial, medicinal, and household purposes. CRISPR-Cas

systems are reviewed by Charpentier and Marraffini.

They take on the history of these prokaryotic adaptive

immune systems, the recent advances in our understand-

ing of their mechanisms of action, and the exciting possi-

bilities for using CRISPR-associated (Cas) RNA-guided

nucleases for the precise genetic manipulation of bacterial,

fungal, insect, plant and mammalian organisms.

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