Strict and Facultative Anaerobes: Medical and Environmental Aspects Chapter Abstracts
Chapter 1: The Phylogeny and Classification of Anaerobic Bacteria
Erko Stackebrandt
Abstract
Anaerobic prokaryotes have dominated life on the planet Earth for possibly more than a billion years before the
increasing oxygen content forced them to retreat into niches, hostile
for oxygenic organisms. These environments were provided by
geological conditions and by oxygenic microorganisms. Eukaryotic
species in concert with aerobic bacteria provided additional
environmental facilities in which anaerobic forms could evolve. The
diversity of morphologies, chemical and physiological traits
represented among the recent anaerobic organisms are found in
phylogenetic lineages that branched off early in the tree of
ribosomal RNA gene sequences, which indicates that genetic diversity
was already high 3.8 billion years ago. The majority of archaeal and
bacterial lineages embrace anaerobic prokaryotes, though only few of
them are defined solely by obligate and strictly anaerobic forms.
This chapter will provide a summary on the diversity of anaerobes of
the domain Bacteria, emphasising the classification at levels above
the genus rank.
Chapter 2: An Overview of Anaerobic Metabolism
Michael J. McInerney and Lisa M. Gieg
Abstract
In many anaerobic ecosystems, a consortium of microorganisms rather than a single species is the catalytic unit
responsible for biodegradation. Interspecies hydrogen and formate
transfer are critical in regulating the flow of carbon and electrons
in many anaerobic ecosystems. Anaerobes use novel approaches such as
the addition of fumarate or a carboxyl group to activate
hydrocarbons, the hydrolysis of ATP to provide low potential
electrons to reduce aromatic rings, and oxidation-reduction or
cofactor B12-mediated reactions to generate free radicals
for dehydration reactions. In addition to fermentative metabolism
where ATP production occurs mainly by substrate-level
phosphorylation, anaerobes can use diverse inorganic or organic
compounds as electron acceptors. Anaerobic respiratory chains use
redox loops, redox-driven pumps, and the separation of
proton-consuming and proton-producing reactions across the membrane
to generate a chemiosmotic potential. Some anaerobes can generate a
chemiosmotic potential by the electrogenic efflux of compounds across
the membrane. The dependence of anaerobes on low potential and free
radical biochemistry makes them very sensitive to oxygen. The degree
to which an anaerobe depends on oxygen-sensitive systems relative to
its ability to detoxify reactive oxygen compounds determines its
oxygen tolerance.
Chapter 3: Redox (Oxygen)-Dependent Gene Regulation in Facultative Anaerobes
R. Gary Sawers and Michiko M. Nakano
Abstract
Our goal in this chapter is to review recent and current themes in the area of redox-dependent gene regulation.
Over the last few years there has been an explosion in research
looking at mechanisms of oxygen and redox sensing, transmission of
these signals to the genome and their consequences on metabolism.
These cover Gram-positives and -negatives, facultative anaerobes as
well as obligate aerobes and phototrophs. We focus on recent aspects
of redox sensing and gene regulation only and highlight new areas of
research in the field. This research field is flourishing and
revealing some very surprising andexciting new science.
Chapter 4: Anaerobic Metabolism by Pseudomonas aeruginosa in Cystic Fibrosis Airway Biofilms: Role of Nitric Oxide, Quorum Sensing, and Alginate Production
Daniel J. Hassett, Sergei V. Lymar, John J. Rowe, Michael J. Schurr, Luciano Passador, Andrew B. Herr, Geoffrey L. Winsor, Fiona S. L. Brinkman, Sang Sun Yoon, Gee W. Lau, and Sung Hei Hwang
Abstract
The goal of this review is to highlight new developments that could lead to better treatment strategies for
complications associated with chronic Pseudomonas aeruginosa infections in cystic fibrosis (CF) airway disease. Recent data
suggest that P. aeruginosa grows anaerobically in "biofilms" enmeshed in the thick airway mucus of CF patients. The
most energy-efficient form of anaerobic growth by P. aeruginosa is via respiration using nitrate
(NO3-) or nitrite (NO2-)
as a terminal electron acceptor. Both of these nitrogen oxides are
amply present in CF airway surface liquid. In this review, we discuss
how the anaerobic biofilm mode of growth actually benefits P. aeruginosa in the context of CF airway disease. We will also
describe in detail the anaerobic respiratory pathway and how
enzymatic production and disposal of a gaseous by-product of
anaerobic growth, nitric oxide (NO), is tightly regulated and
critical for anaerobic survival during respiration. We next describe
how the process of intercellular communication known as quorum
sensing is necessary for anaerobic survival of P. aeruginosa. Finally, when P. aeruginosa shifts to a mucoid, alginate-overproducing form, the anaerobic biofilm mode of growth and
anaerobic metabolism is further promoted. Thus, it is our belief that
new therapeutic strategies aimed at targeting anaerobic gene products
and determining the effectiveness of various anti-P. aeruginosa antibiotics under anaerobic conditions need to be
developed to help combat these infections.
Chapter 5: Oral Microbial Communities Genetic Analysis of Oral Biofilms
Howard K. Kuramitsu
Abstract
Dental plaque represents one of the most complex bacterial biofilms that exist in nature. These bacterial
communities represent an ideal system to investigate the interactions
between different members of a heterogeneous population. The
utilization of both molecular genetic as well as various microscopic
approaches has suggested that biofilm formation represents a
genetically regulated developmental program. Genes involved in
attachment to inert surfaces, extracellular polysaccharide synthesis,
quorum sensing, detachment, as well as those involved in microcolony
interactions have all been demonstrated to play a role in biofilm
development in bacteria. Some of these genes have also been shown to
be important in biofilm development by both gram-positive facultative
anaerobes involved in supragingival plaque formation and
gram-negative obligate anaerobic bacteria present in subgingival
plaque. In addition, evidence for cell-cell communication by means
of gene transfer as well as signaling molecules has also been
demonstrated. This information may be useful in designing new
strategies to regulate dental plaque formation and subsequently
dental caries and periodontitis.
Chapter 6: The Gut Microflora
Rodrigo Bibiloni, Jens Walter and Gerald W. Tannock
Abstract
The gut of monogastric animals is colonised in the distal regions (ileum and colon) by a complex bacterial community
in which anaerobic bacteria predominate numerically. Analysis of the
composition of this community (generally referred to as the gut
microflora) by the use of nucleic acid-based methods has revealed
that many of the bacterial inhabitants have not yet been cultivated
in the laboratory. Despite this handicap, information about the
overall impact of the bacterial community on the host has been
obtained by comparing the characteristics of germfree and
conventional animals. In studying host-microflora relationships,
however, it is essential to work with bacterial species that
establish and persist within (colonise) the gut ecosystem rather than
species that are merely transient. Differentiating between
autochthonous and allochthonous species is therefore critical in
investigations aimed at revealing the bacterial traits that are
essential for life in the gut. Members of the genus
Lactobacillus are ideal model bacteria with which to carry out such investigations because they predominate in the proximal regions
of the gut of mice, poultry and pigs. It is estimated that hundreds
of autochthonous bacterial species reside in the gut, yet the
antigenic load associated with their cells does not stimulate a
marked inflammatory response in the gut mucosa. In contrast, the
presence of gut pathogens results in stimulation of the innate and
adaptive immune systems and the eventual destruction of the
pathogenic cells. Investigations to resolve the question as to how
the mucosal immune system differentiates gut microflora from
pathogenic species suggest that Toll-like-receptors, oral tolerance
mechanisms, and the production of secretory IgA molecules that coat
the cells of members of the gut microflora, are involved.
Chapter 7: The Bacillus Holds its Breath - Latency and the Hypoxic Response of Mycobacterium tuberculosis
David R. Sherman and David M. Roberts
Abstract
Tuberculosis (TB) has plagued mankind for millennia, and with more than two million deaths annually it remains
in the upper-most echelon of infectious killers. Central to the
pathogenic success of Mycobacterium tuberculosis (MTB) is its ability to persist within humans for long periods without causing any
overt disease symptoms. A positive tuberculin skin test and/or chest
X-ray indicative of MTB infection in the absence of disease symptoms
define the clinical syndrome known as latent tuberculosis. Current
models state that oxygen tension within the host is critical to the
balance between latent TB and active disease, but several aspects of
these models including the role of hypoxia are still untested. In
this chapter we summarize and critically review the data accumulated
over many decades associating TB with oxygen tension. We highlight
both the strong evidence linking oxygen levels with MTB growth and
disease as well as the substantial gaps in our understanding of TB
latency.
Chapter 8: Molecular Basis For Aerotolerance of the Obligately Anaerobic Bacteroides Spp.
Anthony D. Baughn and Michael H. Malamy
Abstract
Obligate anaerobes cannot grow in the presence of atmospheric concentrations of oxygen. It is likely that this
deficit is due, in part, to the presence of oxygen-labile targets
within the cell. Despite the inability to grow under aerobic
conditions, many obligate anaerobes can survive transient exposure to
O2 and reactive oxygen species (ROS). In the presence of
O2 and ROS, obligately anaerobic Bacteroides species elicit a coordinated response that is essential for survival
during periods of oxidative stress. This oxidative stress response
(OSR) includes expression of ROS quenching enzymes, such as
superoxide dismutase and multiple peroxidases, as well as the DNA
protective protein Dps. Similar to other eubacteria, expression of
Bacteroides OSR proteins is subject to both OxyR-dependent and OxyR-independent regulation. In addition to the ability to quench
ROS, B. fragilis can consume O2 via a cytochrome bd oxidase respiratory chain. In the presence of nanomolar
concentrations of O2, this respiratory chain can function
in energy metabolism, indicating that the response of this bacterium
to O2 is more complex than was previously thought.
Chapter 9: Clostridial And Bacteroides Toxins Structure and Mode of Action
Michel R. Popoff
Abstract
Clostridia are the bacteria, which produce the highest number of toxins, and are involved in severe diseases in man
and animals. Most of the clostridial toxins are pore-forming toxins
responsible for gangrenes and gastro-intestinal diseases. Among them,
perfringolysin has been largely studied and it is the paradigm of the
cholesterol binding cytotoxins, whereas Clostridium septicum alpha toxin, which is related to aerolysin, is the prototype of
several clostridial toxins forming small pores. Other toxins active
on the cell surface possess an enzymatic activity such as
phospholipase C and collagenase and are involved in the degradation
of specific cell membrane or extracellular matrix components. Three
groups of clostridial toxins have the ability to enter cells: large
clostridial toxins, binary toxins, and neurotoxins. The binary and
large clostridial toxins alter the actin cytoskeleton by
enzymatically modifying the actin monomers and the regulatory
proteins from the Rho family, respectively. Clostridial neurotoxins
proteolyse key components of the neuroexocytosis system. Botulinum
neurotoxins inhibit neurotransmission at the neuromuscular junctions,
whereas tetanus toxin targets the inhibitory interneurons of the
central nervous system. The high potency of clostridial toxins result
from their specific targets, which have an essential cellular
function, and from the type of modification that they induce.
Chapter 10: Toxin Gene Regulation in Clostridium
Michiko M. Nakano, Peter Zuber, and Abraham L. Sonenshein
Abstract
Recent findings have greatly increased our understanding of toxin gene regulation in Clostridium. The VirR/VirS
two-component regulatory proteins regulate transcription of genes
encoding extracellular toxins in Clostridium perfringens. Some genes are directly activated by the VirR response regulator that
interacts with the target DNA. In other cases, a VirR/VirS-dependent
regulatory RNA is required for gene expression via an as yet
unidentified mechanism. The transcription of the enterotoxin gene in
C. perfringens appears to be regulated by mother cell-specific sigma factors, sE, and sK, which are present and active only
during sporulation. Transcriptional regulation utilizing alternative
sigma factors has also been uncovered in recent studies of toxin gene
regulation in Clostridium difficile, Clostridium tetani, and Clostridium botulinum. Continued genome sequence determination of Clostridium spp., as well as transcriptome and proteome analyses, will greatly contribute to
furthering our understanding of how toxin genes are regulated by
various regulators and environmental factors.
Chapter 11: Use of Anaerobic Bacteria for Cancer Therapy
J. Martin Brown, and Shie-chau Liu
Abstract
This chapter describes the power of genetically engineered bacteria in cancer therapy. In the applications we will
be considering, the bacteria are genetically engineered to carry a
specific gene into tumors. This is not what is classically thought of
as gene therapy, which is defined as the introduction of a gene, or
part of a gene, into the cancer cells (or normal cells). In other
words bacteria are not vectors for the introduction of genes into
mammalian cells. However, anaerobic bacteria (and other types of
bacteria) can and do concentrate in tumors by various means and can
carry a gene of interest to produce a protein of choice in tumors.
This can be a powerful adjunct to cancer therapy. In this chapter we
will consider necrosis-targeted therapy, of which species of the
obligate anaerobe Clostridium are the prototypical agent.
Chapter 12: Bioremediation Processes Coupled to the Microbial Reduction of Fe(III)Oxides in Sedimentary Environments
Robert T. Anderson
Abstract
In recent years the contribution of anaerobic processes to in situ contaminant transformation has been recognized as an increasingly important if not dominant process
during the natural attenuation of groundwater contaminants. Microbial
processes coupled to Fe(III) reduction have been viewed as of
particular importance because upon the depletion of dissolved oxygen,
Fe(III) is generally the most abundant potential electron acceptor
within subsurface sediments. A wide diversity of Fe(III)-reducing
bacteria are known but relatively few are found to be important for
in situ contaminant transformation. Members of the Geobacteraceae have been detected in situ in association with contaminant degradation consistent with the known
physiology of these organisms. Geobacteraceae are known to utilize aromatic hydrocarbons as growth substrates, remove important
metal contaminants from groundwater and have been associated with
halogenated solvent degradation. Geobacter species are also known to utilize electrodes as electron acceptors, a consequence of
respiring solid phase Fe(III). Recent insights into the mechanism of
metal reduction in Geobacter species may explain the prevalence of this group in sediments. Further genome-enabled studies
of Geobacteraceae physiology are likely to lead to gene expression assays for monitoring in situ metabolism and suggest ways to promote contaminant degradation in situ.
Chapter 13: Prokaryotic Arsenate and Selenate Respiration
Joanne M. Santini1 and John F. Stolz
Abstract
The oxyanions of arsenic and selenium can be used by prokaryotes as terminal electron acceptors in anaerobic
respiration. Prokaryotes that respire these semi-metals are
phylogenetically diverse and have been isolated from both pristine
and contaminated environments. A total of 33 different species of
prokaryotes have been isolated representing eight different
prokaryotic phyla. These organisms are not obligate arsenate or
selenate respirers as they can utilise a number of different terminal
electron acceptors and also use a variety of different electron
donors. The mechanisms by which these organisms respire with
arsenate and selenate are slowly becoming uncovered. The enzymes
responsible for the reduction are either periplasmic or
membrane-bound terminal reductases linked to electron transport
chains involved in energy generation. To date, all the respiratory
arsenate and selenate reductases characterised are members of the
DMSO reductase family of molybdenum-containing enzymes.
Chapter 14: Molecular and Cellular Biology of Acetogenic Bacteria
Volker Müller, Frank Imkamp, Andreas Rauwolf, Kirsten Küsel, and Harold L. Drake
Abstract
Acetogenic bacteria are acetate-producing anaerobes that utilize CO2 as a terminal electron
acceptor. The reductive pathway by which acetogens reduce
CO2 is termed the acetyl-coenzyme A (acetyl-CoA)
"Wood-Ljungdahl" pathway and yields acetate as a catabolic end
product. In addition to being a terminal electron-accepting process,
the acetyl-CoA pathway also provides the cell with a mechanism for
the fixation of CO2 under autotrophic conditions.
Pathways that are biochemically very similar to the acetyl-CoA
pathway are utilized by other prokaryotes for the autotrophic
fixation of CO2 and the oxidation of acetate. Thus, the
acetyl-CoA pathway and processes that are biochemically very similar
to it serve a variety of functions in nature. The main objectives of
this chapter are to examine the (a) diverse metabolic features of
acetogens that allow them to colonize diverse habitats, (b)
regulatory and molecular aspects of specialized processes by which
acetogens reduce CO2, synthesize acetate, and conserve
energy, and (c) in situ consequences of the physiological capabilities of acetogens.
Chapter 15: Anaerobic Oxidation of Inorganic Nitrogen Compounds
Ingo Schmidt and Mike S. M. Jetten
Abstract
The biological nitrogen cycle is a complex interplay between many microorganisms catalyzing different reactions.
The ammonia and nitrite oxidizing bacteria that were thought to be
chemolithoautotrophic were placed into the family
Nitrobacteraceae. For a long time, the oxidation of the inorganic nitrogen compounds ammonia and nitrite by nitrifiers was
thought to be restricted to oxic environments, and the metabolic
flexibility of these organisms seemed to be limited. The discovery of
a novel pathway for anaerobic ammonia oxidation by
Planctomyces (anammox) and the finding of an anoxic metabolism by "classical" nitrifiers showed that these assumptions are no longer
valid.
Chapter 16: Reductive Dehalogenation of Polychlorinated Benzenes and Dioxins
Lorenz Adrian and Ute Lechner
Abstract
Highly chlorinated aromatic compounds like chlorobenzenes, polychlorinated dibenzo-p-dioxins, dibenzofurans and biphenyls are toxic, poorly water-soluble
compounds, which are persistent in the environment and tend to
accumulate in food chains. They are resistant to most aerobic
biodegradation processes. Under anaerobic conditions, however, they
can serve as electron acceptors in a respiratory process called
dehalorespiration, thereby undergoing a reductive dehalogenation. In
this review we summarize the current knowledge about the reductive
dechlorination of chlorobenzenes and polychlorinated dioxins. A
variety of pathways of microbial reductive dechlorination of dioxins
and chlorobenzenes are described suggesting the involvement of
different microbes in the observed dechlorination processes. The
first described anaerobic bacterium able to respire with chlorinated
benzenes and dioxins belongs to the genus Dehalococcoides, forming an isolated cluster within the green non-sulfur bacteria.
Therefore, this review also summarizes physiological and biochemical
properties of Dehalococcoides, including the remarkable specialization to dehalorespiration as a lifestyle and the capability
to use a broad spectrum of halogenated compounds as electron
acceptors. Biostimulation of dehalogenating bacteria present in
contaminated anaerobic habitats can be a potentially useful approach
to enhance natural attenuation processes and is discussed with
respect to the versatility of Dehalococcoides species.
Chapter 17: Biotransformation of Carbon Tetrachloride by Facultative Anaerobic Bacterium Pseudomonas stutzeri
Andrzej Paszczynski, Jonathan Sebat, Daniel Erwin, and Ronald L. Crawford,
Abstract
Carbon tetrachloride (CCl4) and its dechlorination products are toxic and/or carcinogenic in mammals.
Thus, their environmental fate is of concern to both environmental
scientists and government regulators. A unique CCl4 dechlorination
mechanism has been discovered in cultures of iron-limited
Pseudomonas stutzeri strain KC. This mechanism is characterized by extensive hydrolysis to give CO2 as a
major product, with low or undetectable levels of chloroform. A
low-molecular-weight metal chelator promotes this route of
CCl4 decomposition. That agent is
pyridine-2,6-bis(thiocarboxylic acid) (pdtc). Although pdtc synthesis
is regulated by iron stress, preliminary results from 59Fe
uptake experiments suggest that pdtc is not the bacterium's primary
siderophore. Unlike other siderophores, pdtc chelates copper(II),
cobalt(III), and iron(III) with similar, very high affinity (Kd =
1034). The affinity decreases dramatically
(1012) when iron(III) is reduced to iron(II), suggesting
that pdtc functions as a siderophore towards iron in P. stutzeri but is not required for growth of the bacterium in
low-iron media. Pdtc chelates many transition metals, some heavy
metals, and also some lanthanides. Pdtc has antimicrobial activity,
and this may be its main physiological function in nature. Pdtc is
produced by P. stutzeri during both anaerobic and aerobic growth.
P. stutzeri is a classic facultative anaerobic bacterium (denitrifier) and can be used for CCl4 degradation in either the
presence or absence of oxygen. The reaction of pdtc with
CCl4 does not require oxygen since it forms thiophosgene
and this intermediate hydrolyzes to CO2, H2S
and HCl. Current research is elucidating the genetic basis of pdtc
biosynthesis and resistance to it by its producers. This knowledge
should allow the ultimate use of pdtc or pdtc-producing
microorganisms for in situ bioremediation of CCl4 and/or heavy metals and radionuclides in locations such as anaerobic
aquifers.
Chapter 18: Solventogenesis by Clostridia
Peter Dürre
Abstract
Clostridium acetobutylicum and C. beijerinckii are able to produce industrially important solvents such as acetone, butanol, and 2-propanol. The respective enzymes are
induced shortly before the transition from exponential to stationary
growth phase. The bacteria thus counteract the deleterious effects of
butyric and acetic acids that had been synthesized during active
growth. Regulation of solventogenesis is closely coupled to that of
sporulation, a developmental program that guarantees long-time
survival. Five operons are meanwhile known that are essential for
acetone and butanol synthesis. Their regulation is complex, involving
probably several transcription factors (among them Spo0A, the master
regulator of sporulation), RNA processing, and co- or
posttranslational modification of the gene product. DNA supercoiling
plays an important role in signal transduction. Solventogenesis is
also coupled to the stress response and a number of other metabolic
reactions, as revealed by RNA analyses, two-dimensional gel
electrophoresis, and DNA microarrays. Characterization of the genes
and the still growing understanding of their regulation has allowed
the metabolic engineering of recombinant strains with improved
solvent formation ability and with clostridial genes for the
production of commercially important polyesters.
Chapter 19: The Clostridial Cellulosome
Anne Belaich, Chantal Tardif, Henri-Pierre Fierobe, Sandrine Pagès, and Jean-Pierre Belaich
Abstract
The cellulosomes of five cellulolytic Clostridia have been investigated. Cellulosomes are large molecular complexes which efficiently hydrolyze crystalline cellulose
and where the catalytic subunits are anchored onto a non-enzymatic
protein called scaffoldin. Many components of the various
cellulosomes, produced as recombinant proteins in Escherichia coli, have been studied from a biochemical and structural point
of view. Special attention was devoted to the cellulosome of a sixth
Clostridium: Clostridium acetobutylicum. This bacterium is non-cellulolytic but recent sequencing of its genome revealed that
this species harbours all the genes coding for cellulosomal
components. In four of the six Clostridia, the genes encoding the
major components of the cellulosome are organized in large clusters
on the chromosome. The study of a spontaneous mutant of
Clostridium cellulolyticum, affected in cellulolysis, provided interesting information on the regulation of the genes belonging to
these clusters. Previously established genetic tools, now available
for the two mesophilic Clostridia, C. cellulolyticum and C. acetobutylicum, will allow new approaches for studying the cellulosomes "in clostridio".
Chapter 20: Microbial Community Structure and Functions in Methane Fermentation Technology for Wastewater Treatment
Yuji Sekiguchi and Yoichi Kamagata
Abstract
To date, anaerobic (methanogenic) fermentation technology has been widely applied to the treatment of municipal and
industrial wastes and wastewaters. A number of anaerobic processes
have been intensively developed, and the application of these
processes is being expanded to low-strength wastewaters, wastes and
wastewaters under extreme temperature conditions, and more complex
wastewaters containing anthropogenic compounds and/or compounds that
are recalcitrant to biodegradation. The recent development of
molecular techniques in the field of microbial ecology has allowed us
to explore the microbial diversity and community structure of those
anaerobic processes. As a result of the development and application
of these techniques, we now have better insight into the community
composition and architecture of anaerobic sludge, which can be
adapted to treat a variety of waste/wastewaters under different
operation conditions. Importantly, the community was found to be
composed in large part of various yet-to-be cultured microorganisms,
some of which were often found to play significant roles in those
anaerobic processes. To reveal the function of the community
constituents, numerous efforts have been made to isolate relevant
microbes in the anaerobic processes, and the information on the
functions of the microbes in anaerobic sludge is accumulating at an
encouraging rate. In this chapter, the state-of-the-art anaerobic
waste/wastewater treatment technologies, the microbial community
structure in anaerobic sludge, and the functions of individual
populations are summarized.
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