Summary
The collaborative project aimed at setting up a laboratory of
functional bacterial genomics at Hong Kong University in association
with the Institut Pasteur in Paris, combining in vivo and in
vitro experiments to in silico analysis of the
genomes. The Vice-Chancellor of the University of Hong Kong viewed
the Centre as the Microbiology pole of the Genome Centre to be
created at the University, for the development of genomics in Hong
Kong and the region. In order to demonstrate its feasability, the
project used two model bacteria Escherichia coli and Bacillus
subtilis, together with poorly known counterparts that are
important for the control of pathogens and human health and
environment, Photorhabdus luminescens and Bacillus
cereus (anthracis). These latter bacteria were in
addition chosen for their appropriate behaviour under laboratory
conditions.
Many publications came out of this programme
(they can be seen as publications
marked with a star at the site of the Genetics of Bacterial
Genomes Unit) and several genome programmes were completed during
this programme as a collaborative effort: Leptospira
interrogans
(with the Genome Centre in Shanghai), Staphylococcus
epidermidis (a Firmicute, in collaboration with Fudan
University in Shanghai) and, naturally, the genome of Photorhabdus
luminescens (see also our databases,
that are continuously evolving).
Details of the research planned
1. Introduction
Louis Pasteur used to say that "Scientific knowledge is the heritage
of mankind". Having as a final goal the development of an
infrastructure based on the creation and usage of normalised genomic
data sets, namely genome sequences and their functional annotation, the
involvement of a broad community, in particular at places that have been
recently more or less kept out of the main stream of scientific
production was perceived, at the onset of the Centre, as a critical
issue. Furthermore, it was clear, following the remarks of Louis Pasteur
himself, that on should never speak of applied research, but, rather of
application of the discoveries made by scientists involved in research:
applications first need discoveries. This meant that, to study agents
causing diseases, for example, one needed to study first appropriate
models that would be amenable to in-depth experimental investigation.
Two classes of bacteria were important in this respect, the Firmicutes
(the model of which is Bacillus subtilis, and which comprise the
deadly agent Bacillus anthracis) and the enterobacteria (the
model of which is Escherichia coli, the best known organism, and
which comprise, as we shall see later on, Photorhabdus luminescens,
an insect pathogen that would be extremely interesting against the fight
against mosquitoes, that might soon plague Hong Kong again, because of
general environment changes).
Indeed we recognized that the added-value of the resulting concepts,
experimental results and products will be directly proportional to our
capacity to stimulate the production and the exchanges of very
heterogeneous data sets (physiology, functional analysis, structures,
…). This required the promotion of a standard at an international scale.
China, with its ancient civilisation, and its huge population was
certainly a place that first deserved consideration in this respect.
More specifically, to allow easy and efficient interaction with
laboratories everywhere in the world, it was clear that a place already
well connected with the rest of the world — in particular in terms of
connection to the Internet — was needed. Hong Kong, with its century old
University, seemed remarkably suited for the startpoint of a project in
genomics meant to have important developments in the future. It should
be emphasised at this point that this project was conceived as an
integrative and collaborative project, that would be of interest for a
variety of different laboratories at HKU and elsewhere in Hong Kong and
in China in general.
2. State of the art (as seen at the beginning
of year 2000, see our databases)
Genome sequencing, now a readily available technology, provides us with
an immense variety of genes and gene products. Unfortunately, knowing
the genome text only does not generally bring about the real knowledge
that we wish to possess on the function of individual genes, nor on the
way they collectively interact. As a matter of fact, it becomes
necessary to annotate the genome text, i.e. correlate the text to the
corresponding biological knowledge. Identification of gene functions
requires the combination of several domains of experiments. This
research activity is usually named functional analysis (or functional
genomics). It makes use both of computers (in silico analysis,
for short, Danchin et al. 1991), and molecular genetics and biochemistry
(in vivo and in vitro experiments). Genome sequencing
projects combined with functional analysis produces a flow of data which
puts a particular focus on relationships between the biological
sequences. The need for models, methods and tools to manipulate in
vivo, in vitro and in silico sequence networks or
clusters is becoming more and more compelling. Indeed, the current lack
of such infrastructure obviously creates a bottleneck for the
exploration and exploitation of the enormous amount of biological
information thus made available. The objective of the current research
programme is precisely to illustrate, with model cases, how such
research could be organised and rapidly rewarding. This should trigger a
broad discussion to initiate a further effort to build larger
infrastructures, that would readily place Hong Kong and China among the
leading few in functional genomics.
Until recently, there existed only a few major projects of functional
analysis of model genomes, among which one for B. subtilis
(Harwood and Wipat 1996), the other for S. cerevisiae (Eurofan).
A project for the best known model, E. coli, is being started in
Japan, where, in parallel, functional analysis of the model of
cyanobacteria Synechocystis PCC 6803 is being carried out (at
the Kazusa Institute). In these projects, each gene of the organism is
disrupted, one at a time, and fused to reporter genes, with the hope to
discover phenotypes that may help to identify the corresponding
functions. To our knowledge we do not possess heuristics permitting
direct access to unknown functions, and apart from preliminary studies
by two of the participating applicants there does not exist similar in
silico work in the world. However, an excellent illustration of
the concept of neighborhood has been developed with the software Entrez,
created by D. Lipman and colleagues at the NCBI. The discovery of a
major function of a long known enzyme, polynucleotide phosphorylase, is
a direct illustration of the outcome from combining different
neighborhoods (Danchin 1997, Nitschké
et al. 1998) . Significant development has been achieved on the
experimental side of technology. However, this has been much slower than
initially expected, and protein identification by mass spectrometry,
although efficient, is slower than usually assumed. The same is true for
global analysis of the transcription products. The results obtained at
the end of this work will therefore be, in any case, a useful
contribution to our basic knowledge of bacteria.
3. Genomics of Photorhabdus luminescens and Bacillus
cereus (anthracis)
Two contrasted images depict genomes: at first sight genes appear to be
distributed randomly along the chromosome. In contrast, their
organisation into operons or pathogenicity islands suggests that, at
least locally, related functions are in physical proximity. We proposed,
to try and understand genome organisation, to explore in vivo, in
vitro and in silico, the distribution of genes along the
chromosome, generalising the concept of neighborhood to many more types
of vicinities than succession in the genomic text. Our first
observations suggested that this order is far from random, but is indeed
linked to the function of genes in relation with the cell's architecture
(Guerdoux-Jamet et al.
1997, Danchin
& Hénaut, 1997). These results were fragmentary, but they now
have been experimentally validated (see Publications
with the Centre). In collaboration with the newly created Unit of
Genetics of Bacterial
Genomes and the Laboratory of Genomics of Microbial Pathogens at
the Institut Pasteur, we combine in silico analysis of the
genomes (bioinformatics) of two organisms Photorhabdus luminescens
and Bacillus cereus (or its neighbour B. anthracis),
related respectiveley to two model organisms, Escherichia coli
and Bacillus subtilis, to their study in vivo (reverse
genetics and physiological biochemistry, in particular using
two-dimensions protein electrophoresis coupled to mass spectrometry (in
the Laboratory of Structural Chemistry of Macromolecules, but also at
Pasteur Institute's Genopole Technological Platforms), with biochemical
and structural analyses using optical and electron microscopy, to
further explore this hypothesis.
Needless to say, even if our working hypothesis is partially
falsified, our approach is both precise and original enough to allow us
to propose new hypotheses and discover new functions of orphan genes,
for which the usual "functional analysis" approaches are ineffective.
Indeed we could systematically propose explicit hypotheses about gene
functions, and not, as unfortunately this must be generally the case,
give phenomenological descriptions of disrupted mutants… As a specific
outcome of this work we constructed a variety of quantitative
two-dimensions gel electrophoresis proteins maps of B. subtilis,
E. coli and P. luminescens in several metabolic and
genetic conditions. In particular, we concentrated on a part of
metabolism that has been long studied, then completely overlooked,
despite its major importance in the control of gene expression
(metabolism of polyamines and related molecules, Sekowska
et al. 1998). We discovered new protein complexes in the cell,
that were a priori unexpected, comprising proteins with
apparently unrelated activities. As for 2-D gel protein spots, the
proteins in the complexes were identified by mass spectroscopy intially
through a collaboration with the Ecole
Supérieure de Physique et Chimie Industrielles, and at the
Technology Platform 3 of the Institut Pasteur. When possible, maps were
systematically related to morphological analyses of the cell using GFP
fusions and fluorescent microscopy. We choosed functions identified as
of prime importance for linking the chromosome structure to the cell's
architecture among those that might be linked to pathogenicity. Data are
being made available via specialised databases on the World-Wide Web.
The sequencing of the first genomes revealed an astonishingly high
proportion of genes of unknown function (up to 50 % of the genes). This
suggests that the observations made under laboratory conditions cover
only a small part of the natural environmental conditions encountered by
living organisms. It is fundamental to consider of primary importance
the biotope of the organism, and to study this organism under conditions
which reflect as much as possible those of the biotope. It is also
interesting to choose organisms which play a role in human health and
the food industry. Another point of major importance, which will be
documented below, is that knowledge increases in an immense way when one
becomes able to compare genomes with counterparts that are not too
distant, phylogenetically speaking. This is often the only way to
identify the elusive signals that control gene expression. Finally, it
was useful to compare the data in the process of acquisition with those
previously available for the organism.
The particular feature in the study of bacteria is the systematic
practice of pure cultures, either in liquid cultures or on Petri dishes.
As is well known, model organisms for bacteria are Escherichia coli,
and - to a lesser extent Bacillus subtilis (Kunst
et al. 1997) —, and only those. But it is also well-known that, in
the wild, E. coli does not generally make pure cultures, but,
rather, interacts with many other organisms. In a way the situation in
the laboratory is an experimental artefact… This is less so for B.
subtilis which is often found as a pure culture, in the process of
flax retting for instance. It was therefore of the utmost importance to
study a parent of E. coli that would, in its normal environment,
make pure cultures.
Enterobacteriaceae of the genus Xenorhabdus and Photorhabdus
are potent insect pathogens. These are highly pathogenic after
inoculation but usually not after ingestion, in contrast with Bacillus
thuringiensis (a very close parent of B. cereus). Their
natural vectors are nematodes belonging to the families of of
Steinernematidae and Heterorhabditidae. In nature, Photorhabdus
bacteria live as symbionts in the guts of nematodes that invade insects.
Once inside an insect host, the bacteria are released from the nematode,
kill their host, and set up rounds of bacterial and nematode
reproduction digesting the entire insect, which makes food for large
numbers of nematodes. Curiously, the insect corpses, which do not rot,
left behind glow in the dark as the microbe produce light in addition to
potent insecticides.
After numerous tests, carried out world-wide, these nematode-bacteria
couples are considered as efficient helpers in the fight against insect
scavengers of cultures. They have broad targets, the most sensitive ones
being Coleoptera and Lepidoptera while Diptera are more resistant. It is
therefore important to explore their potential as vectors against
mosquitoes, which spread the deadly Dengue fever in SouthEast Asia.
Their harmlessness for vertebrates is another advantage of these
nematode-bacteria couples which are already tested in research and
development, and commercialised (Biosys, Koppert NL.). Little is known
about their genetics, beside their strong kinship to E. coli,
the model organism for Enterobacteriaceae.
From our point of view, it is remarkable that these bacteria form pure
cultures both in the nematode (where in fact they do not multiply, but
where they are found almost
exclusively of other bacteria) and in the insect they kill. This
indicates an original mode of life where many factors have to be
accounted for: cooperation between cells (quorum sensing), the synthesis
of many antibiotics (antibacterial agents and fungicides, used for the
killing of potential competitors), and the synthesis of insecticides.
Moreover, these bacteria possess an efficient general protein secretion
system. These bacteria are in symbiotic interaction with nematodes that
are strongly related to the model nematode (the genome of which is now
completed) Caenorhabditis elegans. This is a further positive
factor to understand symbiotic interaction at the genetic level.
Finally, luminescence in Photorhabdus is a convenient marker for
virulence, allowing to follow easily certain aspects of this complex
process.
After completion of their genome sequence by the Laboratory of
Genomics of Microbial Pathogens at the Pasteur Institute (see GOLD for
monitoring genome programmes) in Paris from a strain isolated by the
Laboratory of Comparative Pathology in Montpellier, these bacteria
constitute an efficient tool for the study of unknown functions in
Enterobacteriaceae (in particular the process of colony formation), but
also the synthesis of toxins, the secretion of proteins, and the
synthesis of antibiotics. Finally, these bacteria seem to be an ideal
paradigm for the study of nematode-bacteria interactions, allowing one
to test new genetic methods, applicable afterwards to more complicated
models such as mammals, or in general to pathogenic host-vector
interactions.
An attempt to create a group devoted to the study of this organism has
therefore been set up at the Centre, initially with the help of a
scientist (Christian Tendeng) employed by the French Ministry of Foreign
Affairs. Later on a senior scientist was recruited to organize and
develop work on this topic.
What is known about Photorhabdus
and Xenorhabdus
These bacteria present numerous advantages since they synthesise
metabolites and proteins of interest for the agro-food and
pharmaceutical industry:
Insecticides
Photorhabdus has at least one
insecticidal protein complex. These toxins are active after
ingestion and injection. Some of their targets seem to be the epithelium
of the insect gut.
Antimicrobial agents
As expected from their life cycle, the synthesis of antimicrobial
agents is a characteristic feature of both bacterial genera. Different
antibiotic families are represented (including sulfur containing
molecules): xenorhabdines and indol derivatives are found in Xenorhabdus.
Photorhabdus produces two other chemical families: hydroxylbenes
and polyketides (the latter correspond to colored pigments,
antraquinones). These are large-spectrum antibiotics especially active
on Gram-positive bacteria but also on Gram-negative bacteria, while
others act as fungicides or immunomodulators. Many genes corresponding
to these families are visible in the genome sequence (preliminary
data).
At least one bacteriocidal
protein (bacteriocin) has been characterised, it corresponds to a
phage tail with an antibiotic activity restricted to two
entomopathogenic genera and to Proteus sp.
Pathology
The majority of the strains are highly pathogenic and represent, among
entomopathogenic bacteria, the group which is the most pathogenic by
injection. A single cell is indeed sufficient for the killing of Galleria
mellonella (greater wax moth). However, an X. poinarii
species exists with no larvicidal activity. In addition,as most
enterobacteria, P. luminescens is subject to phase variation.
These observations are consistent with the presence of pathogenicity
islands in these bacteria. A few studies reported human infection with
bacteria from this group.
Symbiosis
At the end of the parasitic cycle, the bacteria cause septicemia, and
trigger the development, sexual differentiation and reproduction of the
nematodes. However, it is not known which bacterial metabolites or
enzymes are involved in this co-metabolism. These bacterial products may
constitute one of the key factors for the simplified mass production of
nematodes. For this reason it is extremely interesting to study
intermediary metabolism of these bacteria, and to compare it to its
counterpart in the E. coli model.
Exoenzymes
These bacteria secrete numerous enzymes in large amount during their
stationary phase of growth. Some
of these enzymes have been partially characterised at the
biochemical level: a Photorhabdus alkaline metalloprotease, a
chitinase, a Tween 80-lipase, and a Xenorhabdus lecithinase-like
protein. An exonuclease activity has also been observed for many Photorhabdus
and Xenorhabdus strains but no further characterisation has been
carried out.
Quorum-sensing and bioluminescence (see the bioluminescence Web
page)
It has long been known that bacteria form colonies on agar plates. If
the medium is appropriate these colonies give rise to bacterial
swarming. In the late 1960s it was observed that cultures of Vibrio
fischeri (a luminescent Gram negative bacterium that colonizes
squid) remained non luminescent during the first hours of growth, during
which time the number of cells increased. Luminescence appeared when the
population reached a significant density, at a moment when bacteria ran
out of nutrients. This collective behavior meant that a bacterial
function was expressed at a certain cell density; the organisms in the
population were sensing each other. This was termed "quorum sensing".
This is the best studied function since these genes are used as reporter
genes in many studies. Photorhabdus is the sole
bioluminescent bacterium living in soil (while many photoluminescent
bacteria are found in water, in particular sea water).
Bacterial physiology
Certain strains of the genus Photorhabdus are psychrotrophs
(adapted to low temperatures). The causes for this adaptation are not
known and deserve investigation. Related enterobacteria will be studied
to clarify some of the issues raised by adapation to low temperature.
However, other strains can grow at temperatures up to 39 °C, in
particular tropical strains.
What is known about Bacillus spp.
The following uses in part an entry written by AD for the Encyclopedia
of Genetics (Academic Press).
Bacilli form an homogeneous class of endosprore forming bacteria that
are generally positive for the Gram coloration test. These bacteria are
chemoorganotrophs, and usually aerobic or facultatively anaerobic. The
best known model is Bacillus subtilis, the genome of which has
been sequenced (Kunst
et al. 1997). The genome of the pathogen B. anthracis is
being sequenced at The Institute for Genomic Research (TIGR), and parts
of the genome of the entomopathogen B. thuringiensis have
already been published. Unlike most other bacterial species,
endospore-forming bacteria are highly resistant to the letal effects of
heat, drying, many chemicals and radiation. In fact, one fashionable
hypothesis of the origin of life on Earth by panspermia (Sven Arrhenius,
and more recently Francis Crick) rests on the notion that bacterial
spores such as thoses of B. subtilis could travel through space
and survive for millions of years. Despite its nice appeal to wild
imagination, this hypothesis essentially puts the investigation of the origin
of life out of our reach…
Bacillus cereus is widely distributed in nature. It is
commonly found in soil, herbaceous plants, milk and dried foodstuff. It
is occasionally the cause of gastroenteritis. Bacillus thuringiensis
is an insect pathogen obtained from diseased larvae. It can be
considered as a subtype of B. cereus, from which it differs
mainly by the presence of parasporal endocytoplasmic toxin crystals.
These bacteria share many common features.
The envelope of the vegetative cell
Gram positive bacteria have complex envelopes comprising one bilayer
lipid membrane separating the cytoplasm from the exterior of the cell.
The membrane is part of a very complex structure that comprises many
layers (up to 40) of murein, or peptidoglycan, a complex of peptides
containing D-amino acids (in particular meso-diaminopimelic acid) and
aminosugars. The cell envelope also has several layers of teichoic acid.
The possible existence of a periplasm
in Bacilli in a distinct cell compartment surrounded by the
cytoplasm membrane and the cell wall is a controversial issue.
Cytoplasm, membrane, and protoplast supernatant fractions were prepared
from protoplasts generated from phosphate-limited cells. The protoplast
supernatant fraction was found to include cell wall-bound proteins,
exoproteins in transit, and contaminating cytoplasmic proteins arising
through leakage from a fraction of protoplasts. By this operational
definition, 10% of the proteins can be considered "periplasmic".
Sporulation
Upon starvation, B. subtilis stops growing and initiates
sporulation. This developmental process involves the differentiation
into two cell types. The process begins with a reorganization of the
cell cycle that leads to the production of cells whose size and
chromosome content is appropriate for the developmental process. The
formation of the two cell types, a forespore and a mother cell twice as
large as the forespore, with differing developmental fates is the first
morphological indication of the early stages of sporulation. Endospore
formation is a multistep process that is common among Bacilli. This
seemingly simple structure is the product of a very complex network of
interconnected regulatory pathways that become activated during late
growth in response to unbalanced nutritional shifts and cell cycle
related signals. Sporulation starts with stage 0 (vegetative growth).
Symmetrical cell division, characteristic of vegetative growth, is
blocked. Instead, the cell divides asymmetrically to produce a small
polar prespore cell and a much larger mother cell. During stage I,
asymmetrical preseptation starts. The cellular DNA takes the shape of an
axial filament. At stage II septation proceeds and the daughter
chromosomes are separated. Spore development follows at stage III
(engulfment of the forespore and complete separation of the spore
membrane from that of the mother cell). Stage IV involves formation of
the spore cortex. In stage V spore coat proteins are synthesized and
assembled. At stage VI the spore becomes highly refractile under the
microscope, and it acquires heat and stress resistance. Finally, the programmed
death of the mother cell occurs, leading to lysis and release of
the mature spore (stage VII).
The spore coat is a complex envelope comprising several layers of
spore coat proteins that protect the almost entirely desiccated interior
of the spore, where DNA is compacted and protected from harmful
influence of the environment. Under conditions of appropriate moisture,
in media that contain alanine, glucose and minerals, spores can
germinate. This process involves swelling and a complex lytic process
that opens and sometimes degrades the coating envelope during which time
metabolism is initiated. Cells then resume normal vegetative growth.
Quorum-sensing and chemotaxis
A variety of processes are regulated in a cell-density- or
growth-phase-dependent manner in Gram-positive bacteria. In the early
1990s quorum sensing
was discovered in B. subtilis. It was certainly linked to
sporulation (to swarm or to sporulate, that is the question), but the
functional reason(s) for the existence of the process are not yet known.
Most bacteria that use quorum sensing systems inhabit an animal or a
plant. The microorganisms benefit from the process, but the host
organism may or may not. Each bacterium produces small diffusible
molecules that allow cell to cell communication. As the population of
bacteria increases, so does the concentration of the signalling
molecules. Sensors recognize these molecules. Once the local
concentration in the medium has reached a threshold value, the sensor
proteins transmit a signal to a transcriptional regulator. Examples of
such quorum-sensing modes are the development of genetic competence in B.
subtilis and Streptococcus pneumoniae, the virulence
response in Staphylococcus aureus, and the production of
antimicrobial peptides by several species of Gram-positive bacteria
including lactic acid bacteria. It seems likely that similar processes
exist in B. cereus and B. thuringiensis.
Bacterial populations coordinate their activities.
Cell-density-dependent regulation in these systems appears to follow a
common theme. First, the signal molecule (a post-translationally
processed peptide-pheromone) is secreted by a dedicated
ATP-Binding-Cassette (ABC) exporter. The role of the secreted peptide
pheromone is to function as the input signal for a specific sensor
component of a two-component signal-transduction system. Co-expression
of the elements involved in this process results in self-regulation of
peptide-pheromone production. Peptides are secreted and processed, under
various conditions that are further recognized by the cell. Next, in
response to the pheromone, cells swim in a coordinated fashion, thereby
forming a kind of wall surrounding rings of bacteria having the same
exploration behavior.
In the same way, B. cereus is a motile bacterium, endowed with
a complex flagellar machinery. This permits cells to swim towards from
nutrients or away from repellents.
Protein secretion
Bacillus subtilis is one of the organisms of choice in the study
of protein secretion.
Many fundamental aspects of this process are not yet understood. At
least two systems enable proteins to be inserted into the membrane
and/or to be located outside of the membrane or secreted into the
surrounding medium. In B. subtilis, the Sec-dependent pathway
(one that recognizes signal peptides) has at least five different signal
peptide peptidases. Proteins that are periplasmic in Gram negative
bacteria are also found in B. subtilis, presumably as
lipoproteins (i.e. possessing a specific signal peptide, cleaved
upstream of a cysteine residue that is covalently coupled to the outer
lipid layer of the cell membrane upon cleavage). Comparison with the
genome of B. cereus will help better understanding of this
important process.
Metabolism
In addition to the need for compartmentalization, living cells must
chemically transform some molecules into others. Metabolism
is the hallmark of life. Cells can be in a dormant state — this is
the case of spores, for example — but one cannot be sure that they are
living organisms unless, at some point, they initiate metabolism. In
general, one distinguishes between primary metabolism (the
transformation of molecules that support cell growth and energy
production), and secondary metabolism (transactions involving molecules
that are not necessary for survival and multiplication, but assist in
the exploration and occupation of biotopes (e.g. antibiotic synthesis)).
Carbon, oxygen, nitrogen, hydrogen, sulfur and phosphorus are the core
atoms of life. Electron transfers and catalytic processes, as well as
the generation of electrochemical gradients, require many other atoms,
in the form of ions. Metabolic processes allow the cell to concentrate,
modify and excrete ions and molecules that are necessary to energy
management, growht and cell division.
Nutrients and ions are transported into cells by a number of more or
less specific permeases, most of which belong to the ABC permease
category. In Gram positive bacteria, these permeases generally comprise
a binding lipoprotein responsible for part of the specificity, located
at the external surface of the membrane, an integral membrane channel
made of proteins of two different types, and a dimeric, membrane bound
cytoplasmic complex, that binds and hydrolyzes ATP as the energy source.
For positively charged ions, selectivity is the most important feature
of the permease, because the electrochemical gradient is oriented
towards the interior of the cell (negative inside). Ions must be
concentrated from the environment until they reach the concentration
required for proper activity, but must not reach inhibitory levels.
Apart from iron (which is scavenged from the environment with highly
selective siderophores synthesized in response to iron limitation),
manganese is the most important transition metal ion for Bacilli. It is
required for many enzyme activities (such as superoxide dismutase,
agmatinase, phosphoglycerate mutase, pyrophosphatase, etc.). Copper is
important for electron transfer. Cobalt is required by the important
recycling protein methionine aminopeptidase. Nickel is needed by urease…
Zinc is a cofactor of polymerases and dehydrogenases, magnesium is
involved in catalytic complexes with substrate in about one third of
enzyme reactions. Potassium is needed to construct the electrochemical
gradient of the cell’s cytoplasm, and is a likely cofactor in many
reactions. Calcium is perhpas needed in major reactions during the
division cycle, but the importance of this ion still remains a mystery.
Anions are also important. They have to be imported against a strong
electrochemical gradient. Phosphate in particular requires a set of
highly involved transport systems. Sulfate is the precursor of many
important coenzymes in addition to cysteine and methionine, but not much
is yet known about its transport and metabolism, except by comparison
with the counterparts known to be present in E. coli. The study
of sulfur metabolism
is therefore a priority.
Carbon and nitrogen metabolism in Bacilli follow the general rules of
intermediary metabolism in aerobic bacteria, with a complete glycolytic
pathway and a tricarboxylic acid cycle. Electron transfer to oxygen is
mediated by a set of cytochromes and cytochrome oxidases, allowing
efficient respiration. Bacillus cereus is able to grow
anaerobically with appropriate electron acceptors.
As in other living organisms, the ubiquitous polyamines putrescine and
spermidine play a fundamental but still enigmatic role. They arise via
the decarboxylation of arginine to agmatine, coupled to a
manganese-containing agmatinase, and not from decarboxylation of
ornithine, as in higher eucaryotes.
Pathogenicity
Bacillus subtilis is generally recognised as safe (GRAS). It
is much used at the industrial level for both enzyme production or for
food supply fermentation. Riboflavin is derived from genetically
modified B. subtilis using fermentation techniques. Traditional
techniques (e.g. random mutagenesis followed by screening; ad hoc
optimization of poorly defined culture media) are important and will
continue to be utilized in the food industry, but biotechnology must now
include genomics to target artificial genes that follow the sequence
rules of the genome at precise position, adapted to the genome
structure, as well as to modify intermediary metabolism while complying
to the adapted niche of the organism, as revealed by its genome. As a
complement to standard genetic engineering and transgenic technology,
knowing the genome text has opened a whole new range of possibilities in
food product development, in particular allowing "humanization" of the
content of food products (adaptation to the human metabolism, and even
adaptation to sick or healthy conditions). These techniques provide an
attractive method to produce healthier food ingredients and products
that are presently not available or are very expensive. B. subtilis will
remain a tool of choice in this respect.
Most Bacilli are innocuous, but Bacillus cereus is the
etiological agent of several types of infections, including fulminant
ophtalmitis. Its kins
B. thuringiensis and B. anthracis are highly
pathogenic bacteria (the former for insects, the latter for animals and
man). Bacillus anthracis is the agent of the deadly anthrax
disease. Louis Pasteur discovered the bacteria responsible for anthrax.
This led to an immunization protocol using a weakened strain of the
bacteria. All his research led Pasteur to crusade for sterilization and
hygiene to prevent the spread of infectious diseases. The etiology of
the disease has been remarkably analysed by Robert Koch. Several studies
suggest that the agent has been used for warfare (see Centre for
Nonproliferation Studies), and although it is not clear how much
propaganda there is in the general reports and talks about this organism
(see Not
every truth is good to say), it is clear that it is important to
understand its biology.
4. A work plan: the exploration of
"neighbourhoods"
Because P. luminescens (resp. B. cereus) is related to
E. coli (resp. B. subtilis) we have started its study by
annotating genes in comparison with E. coli (resp. B.
subtilis). This required us to study in priority genes that are
present in both organisms, but do not yet possess an assigned function.
As an initial step we have set up specialised databases that allow one
to manipulate cognate genomes in parallel. This asked us to elaborate
a new data structure.
In silico exploration of neighborhoods
To proceed rapidly towards understanding of the function we used the concept
of neighbourhood, for which we have developed an efficient tool,
available on the World-Wide Web (Nitschké et al. 1998). The first
neighbourhood (relationship) between genes highlighted by genome
projects is the proximity on the chromosome. From the gene point of
view, this relationship represents its physical neighbourhood. The
existence of operons and pathogenicity islands shows, at least for
prokaryotes, that genes physically neighbours from each other can be
functionally related. Formally, this means that, for these genes, a
relationship exists between two types of neighbourhood: physical and
functional.
The definition of "Neighbourhood" can be generalised to many more
types of vicinities. We have just introduced physical and functional
neighbourhoods and another interesting one is certainly the structural
similarity between genes or gene products. In fact, one can use most
attributes of the sequences to define clusters reflecting a particular
type of neighbourhood. For example, a simple property as the
isolelectric point, which often gives a first idea of a gene product
compartmentalisation, can be used (Moszer
et al., 1995). More complex neighbourhoods have proved to produce
particularly revealing results: two genes may be considered as
neighbours because they use the genetic code the same way.
Remarkably, results of functional analysis perfectly and naturally fit
in the neighbourhood model. This is obvious for metabolic pathway
identification (Sekowska
et al. 2000), and it is also true for differential expression
studies, using 2D electrophoresis of proteins or DNA array technology,
which produce network of co-expressed sequences that can be seen as
another type of neighbourhood. In fact, the neighbourhood concept is
very well suited to represent biological knowledge from the sequence
standpoint. When several genes are identified as being involved in a
given biological function, as soon as this result is published, one can
find neighbours in the literature because they appear in the same book
or article. This means that most of the knowledge concerning functional
neighbourhood is almost immediately available in the literature. Systematic
extraction of this bibliographic vicinity is an amazingly rich and
powerful resource.
Neighbourhood, as a formal model, offers a unified representation of
available data: it handles functional knowledge as well as any
structural or physico-chemical properties. Heterogeneous types of
information are projected as networks of sequence relationships.
Comparing or bringing together data of very various types becomes
possible. The most promising consequence is the possibility to imagine,
design and develop tools which will support an inductive exploration of
biological data, organised in appropriate database structures (Médigue
et al. 1993, Moszer
et al. 1995, Bocs
et al. 2002). Each neighbourhood is meant to shed specific light
on a gene and bringing together the objects of the neighbourhood is a
way to look for its function. This induction, as opposed to the usual
deductive reasoning, results in fundamentally new findings.
Identification of relationships between different types of
neighbourhood is a result of this exploration. Systematic and
statistical analysis of these relationships can subsequently be used to
derive predictive methods. This is precisely what is done currently when
sequence similarity (structural neighbourhood) is used to transfer
functional information between sequences (functional neighbourhood). The
great advantage of using other types of neighbourhood is on the one
hand, to get functional hints is absence of sequence similarity and on
another hand, to bring complementary evidence when sequence similarity
does not provide clear-cut answers.
This approach is of great benefit for the growing field of genome
comparison. First because sequences with similar functions can be
identified with more accuracy. Second, because neighbourhood will help
to address the problem of discrimination between orthologs and paralogs.
Finally, because the concepts and tools developed to bring together and
to compare different neighbourhoods can be used and extended to compare
a given neighbourhood in different organisms
In addition to an important contribution in the understanding of the
general gene organisation and function in enterobacteria (including the
model organism E. coli) the study of Photorhabdus is
expected to lead to a large number of applications, in particular in the
following domains:
• characterisation of insecticidal toxins, in particular in the fight
against mosquitoes (that cause dangerous diseases such as dengue fever
in the region)
• production of antibiotics (including fungicides)
• protein secretion (including potential secretion of products of high
pharmaceutical value).
Despite its limited human resources in this program, the role of the
Centre in Hong Kong has been to provide some in silico, in
vivo, and in vitro annotation required to set up the stage
for these studies to start in depth investigation of genomes, at
appropriate public or private places, preferably in an interactive way.
Polyamines and sulfur
metabolism
Among the genes revealed by the in silico analysis described
above some have been inactived or expressed in a controlled manner by
homologous recombination. Our work was restricted to robust fragments of
metabolism or control, in which hypotheses can be validated with strong
certainty. As a first step, we have been concentrating on the metabolism
of polyamines (because it is ancestral, and universal, but quite
diverse, and linked to nucleic acids structures). As an example we have
discovered in B. subtilis arginine decarboxylase, S-adenosyl-methionine
decarboxylase, and the putrescine
metabolism pathways. Generally, we studied operons having a
fragmented structure in one of the models and being continuous in the
other (this is the case of arginine and polyamine metabolism). In the
same way, we inactivated control genes having a specific effect on DNA
bending and supercoiling. These genes were chosen because they strongly
differ in Gram+ and Gram– bacteria. At this step, appropriate cassettes
were used to allow visualisation in optical and electron microscopy (GFP
and histidine-tails hybrids). Sulfur metabolism is intimately linked to
oxygenases (mono- and dioxygenases), and we are devoting a special
emphasis on this class of enzymes, in collaboration with laboratories at
HKU and IP Paris.
We are systematically studying heterologous enzyme complexes (focusing
on "impurities" during purification). Furthermore, we analyse oligomer
organisation of the proteins looking for structures that permit the
formation of plane
layers. We continuously annotate - back to in silico
analysis - the genomic text as a function of these results and study the
distribution of genes corresponding to common distinctive features of
their products. When needed, we raise antibodies against these proteins,
and purify their complexes on immuno-affinity columns, but in general we
combine the approach of reverse genetics permitting us to construct GFP
fusions, to purification and immunodetection.
Among the physical principles that may organise the cell's
architecture we hypothesise that plane layers play a major role. This is
being investigated at the ultrastructural level.
Global functional genomics: the transcriptome and the proteome
To benefit from the knowledge of the whole genome sequences we
initiated global studies of gene expression. This makes use of
techniques permitting one to analyse transcription of the whole set of
genes (expression profiling, or transcriptome analysis) as well as of
the whole set of gene products (proteome analysis).
The number of genes to be considered is of the order of 4,000-5,000.
This number is perfectly compatible with filters (membranes) technology
using PCR products (DNA arrays). As a first step, we preferred this
technology as a first step, to the microchips technique, that is more
expensive and do not allow reusing the same slide for several
experiments, hence lackinjg a crucial control. Expression profiling
technology is more difficult to develop for procaryotes than for
eucaryotes, because mRNAs are not polyA-tailed. In addition, RNA
extraction comprises a major amount of ribosomal RNA, leading to a
background level when hybridisation is made directly with the RNA
preparation (see discussion).
We have therefore created a new process for extracting RNA from
bacteria, and preparing the corresponding cDNA. We used primers internal
to the genes and reverse transcriptase, leading one cDNA copy per RNA
molecule present in the RNA preparation. This cDNA preparation is
hybridised to the filter, and quantitated using a phosphorimager. We are
in parallel collaborating with IP Paris to the development of a database
structure to correlate with the genome databases (Colibri
and SubtiList, of the "GenoList" class, see below). The
corresponding specialized database SubScript, is built
with the BACELL consortium.
The mutants are characterised at the protein level using 2-D gel
electrophoresis (with training of scientists and research assistants at
the IP Paris). Protein spots are identified by mass spectrometry
(MALDI-TOF and Nano-Electrospray: 90% can be identified using
proteolytic peptides maps and genome databases), and used to enrich our
databases. We are improving our electrophoresis techniques along three
main lines: gels for large scale purification of certain protein spots,
building up and enrichment of 2-D gel databases coupled to genome
databases, and identification of low concentration spots by MS (in
collaboration with the Laboratory of Neurobiology and Cell diversity at
the ESPCI in Paris).
5. Bacterial sequence management and genome annotation
Functional genomics
requires, in parallel with in vivo and in vitro
experiments, an important effort in bioinformatics, in silico
analysis. We are therefore organising data and annotation of the Photorhabdus
luminescens and Bacillus cereus genomes in such a way as
to help to manage sequence data, and help to predict and discover gene
functions, taking into account the knowledge of the genome sequences as
they are being collected.
To construct the P. luminescens
and B. cereus specialised databases it is necessary to introduce
in the GenoList engine sequence data and their corresponding
annotations. This input required construction of a generic procedure
(elaborated within the process).
Genome annotation is being developed at the Genetics of Bacterial
Genomes Unit through the collaborative effort of
We expect that our study will improve our knowledge of enterobacteria, in
particular in relation with their reactions to stressful conditions. This
will be extremely useful because these bacteria form a large cohort of
human and animal pathogens.
In this respect, knowing more about their sulfur and polyamine
metabolism has helped in defining new targets for specifically designed
chemicals (synthetic artificial antibiotics). It should indeed be noticed
at this point that a lead was followed by several companies in this
direction, but that inaccurate experimental results diverted their effort
to other leads.
A first one is the study of bacterial genes having counterparts in the
human genome. For example the cyaY gene of E. coli (the
counterpart of which may be existing in P. luminescens, a much
better model than E. coli for the study of complex interactions)
is the homolog of the frataxin gene, causing Friedreich ataxia, a
completely not understood disease. We could of course start to make an
analysis of the corresponding function in our models (remembering in
particular that mitochondria are the progeny of symbiotic bacteria). Other
example exists, in particular in metabolism, that may be worth analysing.
The most promising avenue for collaboration between human and bacterial
genomics might stem from the unraveling of pathways in bacteria. Indeed,
much remains to be known in these organisms, and we have chosen to
investigate the situation of polyamines and sulfur metabolism precisely
for this reason of lack of sufficient knowledge. But there is a
fundamental reason that links bacteria to higher eucaryotes: mitochondria
have bacterial ancestors. And, in fact, because of the deleterious effect
of Muller's ratchet (accumulation of deleterious mutations in the absence
of sex), most bacterial genes found in the original symbiote had to
migrate to the host nucleus.
