Re: Borrelia genetics
a_weisman_at_yahoo.com
Date: 02/27/05
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Date: 27 Feb 2005 06:28:17 -0800
eugeneshapiroisapig wrote:
> Hey guys, I could spend a lot of time at the library looking this up
on
> my own, or I could save time and ask here. What I am interested in
now
> are basic facts about the molecular biology of Borrelia species other
> than Borellia burgdorferi. By which I mean hermsii, etc., you know
the
> stuff that causes relapsing fever and things like this. In
particular,
> I am interested now in the genetic organization of these bugs.
> Burgdorferi has I believe 9 circular plasmids, 12 linear plasmids,
and
> a linear chromosome. What about other borrelia? Also interested in
> worldwide distribution of hermsii. Any other information about
> comparative cell biology or evolution would also be appreciated.
Thanks.
Thought you might be interested in the following:
Book Reviews: The Spirochetes: Molecular and Cellular Biology
http://www.horizonpress.com/hsp/revs/revsspi.html
The Spirochetes: Molecular and Cellular Biology
Book Reviews
This page contains excerpts from published reviews. Full details of our
books are available, including how to buy.
From: International Microbiology (2002) 5: 43-44
Although invisible to the naked eye, prokaryotes are an essential
component of the Earth's biota, including plants and animals.
Spirochetes form a cohesive phylogenetic group in Bacteria, and they
are cosmopolitan in distribution, in aquatic environments and in
animals. Spirochetes are also of significant medical interest as they
cause a variety of diseases in humans, some quite prevalent: Syphilis
is a major health problem in several parts of the world; leptospirosis
is the most common worldwide waterborne zoonosis; Lyme disease is the
most prevalent arthropod-borne disease in the United States; and
periodontal disease, which is the major cause of tooth loss, is among
the most frequent human infections. Spirochetes have occasionally been
confused with spirilla and also with protozoan infection. Fritz
Schaudinn (18711906) discovered that a spirochete caused syphilis and
thought that it might be related to the trypanosome. The 606 compound
(Salvarsan) that Paul Ehrlich (18541915) synthesized proved to be
effective in curing the trypanosome (protozoan) infection, so he
proceeded to test 606 as a cure for syphilis. It was remarkably
successful and thus the ''magic bullet'' against the dreaded syphilis
had been found. Spirochetes had already been observed by direct
microscopy by Antonie van Leeuwenhoek (1632-1723), the discoverer of
both protists and bacteria. Van Leeuwenhoek's ''animalcules'' drawings
of his own mouth presumably corresponded to many oral treponemes. Hideo
Noguchi (1876-1928) recognized that the chancres manifested early in
the course of syphilis were replete with tiny spirochetes. He named
them Treponema pallidum (''pale turning threads'') because of their
appearance and failure to take up stain.
The book edited by Saier and García-Lara contains 18 chapters that
summarize important overall aspects of spirochetes, such as motility
and chemotaxis (2 chapters); genetics (10 chapters);
spirochete-specific bacteriophage (1 chapter) and host-parasite
interactions (5 chapters). Each chapter is written in a scientific
journal- like review format by specialists in this field. For more
information, there is an updated bibliography. Figures, schemes and
tables contribute additional information to the text.
The phylogenetic foundation of spirochetes based on comparative
analysis of 16S rRNA sequences is discussed in Chap. 1. Spirochetes are
presently classified as belonging to the Class Spirochaetes in the
order Spirochetales and are divided into three major phylogenetic
groupings. The first family, Spirochaetaceae, contains species of the
genera Borrelia, Brevinema, Cristispira, Spirochaeta, Spironema and
Treponema. The second named ''Serpulinaceae'' contains the genus
Brachyspira (Serpulina). The third, Leptospiraceae, contains species of
the genera Leptonema and Leptospira. Novel spiro- chetal species have
been identified from the human oral cavity, the termite gut, and other
host-associated or freeliving sources. Spirochetes have received much
attention not only because of their infectious potential, but also
because of their interesting morphological and motility features.
Spirochetes have a unique structure and, as a result, their motility
differs from that of other bacteria. Chapter 2 explains spirochete
gyrations, rotations and their periplasmic flagella; to understand
spirochete motility at a molecular level, the proteins and genes
involved in motility are being analyzed. Motile bacteria are generally
chemotactic, which allows the organism to perform directed movements in
response to various environmental signals. Enteric bacteria such as
Escherichia coli or Salmonella respond with movement towards nutrients.
Chapter 3 reveals the role of chemotaxis in the pathogenesis of
spirochetes. Note that the size of the chromosome of both Borrelia
burgdorferi and Treponema pallidum barely exceeds 1 Mb and lacks genes
for many metabolic functions. However, motility and chemotaxis genes
take up more than 6% of the chromosome in T. pallidum and about 5% in
B. burgdorferi. Chapter 4 describes the characterization of
bacteriophage FBB-1 of Lyme disease agent B. burgdorferi.
Spirochete-specific bacteriophages have coevolved with their hosts over
evolutionary time. For this reason, the study of phages can provide
clues to the ecology and molecular biology of their host organisms.
Chapter 5 focuses on the genetics of lipopolysaccharide (LPS) in
Leptospira. LPS is the dominant antigen on the surface of Leptospira
and Brachyspira, in complete contrast to T. pallidum and B. burgdorferi
which have no LPS. Chapter 6 discusses the triangle of parasitic
interactions between the spirochete and its vertebrate host, and the
spirochete and its tick vector using Borrelia spirochete as a model.
Understanding the key events at the tick vector-host interface will
provide a better understanding of the epidemiology and ecology of these
important human pathogens.
In the last few years, the genomes of many bacteria, including B.
burgdorferi (1997) and T. pallidum (1998), have been sequenced,
Chapters 7, 8 and 9 provide a comprehensive genomic analysis of these
two spirochetes. With the availability of genomic data, it is possible
to draw their metabolic maps (Chap. 7). Chapter 8 analyzes membrane
transporters, and finally, Chap. 9 describes the housekeeping genes of
Borrelia. All examined Borrelia species contain numerous, small
circular and linear extrachromosomal DNA molecules that may exceed half
the size of the linear chromosome. Why are these bacteria driven toward
a minimalist (1 M b) chromosome, while at the same time they maintain
large numbers of extrachromosomal elements? Chapter 10 focuses on the
most obvious example of sequence repetition found in Borrelia species,
the cp32 plasmid family. Chapter 11 discusses the current repertoire of
antibiotic markers that are useful for spirochetal genetic
manipulation. Chapter 12 reviews the methods that have been used in
spirochetes for gene inactivation and DNA exchange, with a primary
focus on B. burgdorferi. Chapter 13 offers strategies for constructing
shuttle vectors in Treponema pallidum. Genome analysis renders a
prediction of gene function, and it may also be used to identify DNA
elements involved in transcription, replication, recombination and
other cellular processes. Chapter 14 describes the role of genomics in
approaching the study of Borrelia DNA replication.
Characterization of mechanisms governing the expression of the outer
membrane protein (OMP) genes in Leptospira are described in Chap. 15;
these genes should provide insight into host-parasite interactions.
Furthermore, recent advances in heterologous expression of Leptospiral
OMP genes are opening new avenues of vaccine development. Chapter 16
discusses several genetic regulatory mechanisms adopted by B.
burgdorferi to sense and adapt to different host and environmental
conditions either in vitro or in vivo. Chapter 17 focuses on the
environmental transitions that spirochetes undergo during their life
cycles and the mechanisms of transcriptional regulation that might
possibly mediate spirochetal adaptations to such changes. Chapter 18
analyzes the aspects of the biology of T. pallidum in the context of a
century of experimental studies and the recently determined genome
sequence. T. pallidum and a group of closely related pathogenic
spirochetes have evolved to become highly invasive, persistent
pathogens with little toxigenic activity and an inability to survive
outside the mammalian host. The genome sequence will provide useful raw
data for additional functional studies on the structure, metabolism and
pathogenesis of these organisms.
Compared to what we know about other pathogenic bacteria, our
information concerning spirochetes is quite minimal. This book is
devoted to the structural, molecular, physiological and evolutionary
aspects of spirochetes. The chapters contain summaries of recent
accomplishments and exciting new genetic tools for studying several
spirochete species. It is truly an interesting time in the discipline
of spirochetology! This book is especially recommended for advanced
students, senior researchers, physicians and dentists; but all
microbiologists have the exceptional opportunity for extending their
understanding of unusual prokaryotic characteristics, such as linear
chromosomes, a cytoskeleton and the periplasmic flagella that confer
rapid motility and uncommon chemotactic capabilities.
Chapter Abstracts: The Spirochetes: Molecular and Cellular Biology
http://www.horizonpress.com/hsp/abs/absspi.html
The Spirochetes: Molecular and Cellular Biology
Chapter Abstracts
How to buy this book
--------------------------------------------------------------------------------
Introduction to Spirochete Evolution, Genome Analyses and Physiology
M.H. Saier, Jr.
This book is devoted to structural, molecular, physiological and
evolutionary aspects of spirochetes. The spirochetes are a small
cohesive group of chemoheterotrophic bacteria, readily distinguishable
from other bacteria on the basis of their unique cell structures.
Spirochete cells are generally long and narrow, helical in form, and
surrounded by delicate flexible walls. All known spirochetes can swim
actively in liquid media by virtue of their axial fibrils: periplasmic
flagelli that underlie the cell wall. The cells often bend or flex
during movement, and they can attain remarkable directed velocities and
even swim through highly viscous solutions (see chapter by Li, Motaleb,
Sal, Goldstein and Charon). The motile behavior of these bacteria
allows them to respond to chemical and physical stimuli in their
environments (see chapter by Lux, Moter and Shi). These organisms
comprise a relatively close-knit group of organisms that, based on 16S
RNA analyses, diverged from other bacteria during early evolutionary
history. They exhibit unusual prokaryotic characteristics such as
linear chromosomes and a cytoskeleton. They also are the targets of
spirochete-specific bacteriophage which have co-evolved with their
hosts over evolutionary time. For this reason, the study of phage can
provide clues to the ecology and molecular biology of their host
organisms (see chapter by Eggers, Casjens, Hayes, Garon, Damman, Oliver
and Samuels).
Spirochetes are currently divided into nine genera (see chapter by
Paster and Dewhirst). While some of these genera include free living
organisms of bright colors and unusual physiological properties, others
are pathogens of humans and animals, the causative agents of syphilis,
Lyme disease, relapsing fever, leptospirosis and peridontitis. The
pathogens responsible for these diseases exhibit tremendous structural
and physiological variability. For example, although they are
considered Gram-negative bacteria with a double envelope membrane, many
spirochetes including Treponema and Borrelia lack outer membrane
lipopolysaccharides (LPS). Others including Leptospira and Brachyspira
do synthesize LPS, and LPS is the principle surface antigen displayed
by these organisms, of considerable importance to diagnostics and
immunity. The chapter by Bulach, Kalambaheti, de la Peña-Moctezuma and
Adler discusses these important antigens from structural and
biosynthetic standpoints. The importance of vector-host interactions,
particularly with respect to tick-borne borreliae, is discussed by
Nuttall, Paesen, Lawrie and Wang. The pathogenic properties of many
spirochetes have provided much of the scientific impetus for their
study.
Genome analyses of two spirochetes, Treponema pallidum and Borrelia
burgdorferi, the bacterial determinants of syphilis and Lyme disease,
respectively, have revealed a wide range of protein structures. Most
protein folds identified in these two organisms have been shown to be
common to many other organisms, but a few spirochete-specific folds
have been identified (see chapter by Das, Hegyi and Gerstein).
Additionally, the metabolic capabilities of these two spirochetes are
limited. For example, they rely primarily on glycolysis for energy
generation. Transport systems in these two pathogens are highly
restricted, showing patterns of transporter types that differ from
those found in all other bacteria for which complete genomes are
currently available (see chapter by Saier and Paulsen). Information
obtained by analyzing the genome of T. pallidum is analyzed in terms of
the known biological properties of this important pathogen. Genome
analyses of Borrelia species have revealed features unusual to
bacteria, often more frequently associated with eukaryotes (see chapter
by Casjens). Thus, housekeeping genes are maintained primarily on the
large chromosome of B. burgdorferi while the numerous circular and
linear plasmids (or small chromosomes) of this organism bear
spirochete-specific genes that may prove to be important for
pathogenesis. They may be rapidly evolving as compared to the
chromosomal genes. These plasmids are greatly enriched for repetitive
sequences, the most obvious of which are found in the family of cp32
plasmids (see chapter by Stevenson, Zückert and Akins).
Hypervariability within these plasmids as well as the presence of
multiple paralogues undoubtedly has survival value for the bacteria in
their host organisms. These molecular features may provide mechanisms
for avoiding the animal's immune system.
This book thus summarizes important overall aspects of spirochetes as a
phylogenetic group of organisms, but it also surveys a few key
representative pathogenic spirochetes in greater detail with respect to
specific traits. To what extent the specific findings reported will
prove to be relevant to other less well studied spirochetes has yet to
be determined. However, genome sequences have for the first time
provided detailed information about the life processes of two of these
important and interesting microorganisms, and genome sequencing
projects for other spirochetes that will allow comparative studies are
currently in progress. It is an exciting time in the discipline of
spirochetology!
--------------------------------------------------------------------------------
Introduction to Genetic Development in Spirochetes
J. García-Lara
Diverse and ubiquitous, the morphologically captivating spirochetes
include the causative agents of various important diseases, i.e.
syphilis, periodontitis, Lyme disease, Leptospirosis and swine
dysentery. The advancement of our knowledge in regard to the biology of
this bacterial family has been hampered by the absence of suitable
genetic tools. This limitation has been of special significance in the
understanding of the mechanism of disease, because the few pathogenic
spirochetes that can be cultured in vitro are also particularly
intractable genetically. The advent of the new millennium is witnessing
a revolution of the genetic methodologies available to study
spirochetes. This book presents the relevant milestones of this
process.
Besides genetic engineering, the manipulation of DNA needed in
investigations of gene regulation and protein function requires the
ability to transfer DNA to the desired bacterial recipient. Tilly and
coworkers exhaustively discuss the methods available for DNA exchange
in spirochetes, and their succesful application to gene inactivation.
Obviously, it is necessary to be able to discriminate those spirochetes
carrying the recombinant DNA. Addressing this issue, Hardham and Rosey
comprehensively cover in their chapter the selective and screening
markers proven to be useful in spirochetes, as well as suggest
potential alternative antibiotic resistances to be tested.
Although, the transient presence of exogenous DNA fragments has been
sufficient to attain homologous recombination and to carry out studies
of gene expression, the efficiency, convenience and versatility of
these systems is limited when compared with that provided by stable
vectors. A remarkable effort has been carried out by a number of
laboratories towards obtaining stable episomes capable of shuttling DNA
between genetically amenable bacteria and spirochetes. Saint Girons et
al. describe the recent advances in this area for Treponema denticola,
Leptospira biflexa and Borrelia burgdorferi.
The strategies used to obtain stable maintenance of exogenous DNA have
been based on broad host range plasmids or on the random cloning of
endogenous sequences containing a native replicon from the spirochetal
genome. In addition, an innovative approach guided by genome analysis
to locate spirochetal origins of replication is extensively elaborated
in the chapter by García-Lara et al. Besides its application to the
development of shuttle vectors, this methodology has provided the first
origins of replication from the unusual linear chromosome and linear
plasmids of Borrelia burgdorferi. It has also unveiled a collection of
candidate genes to be involved in replication and/or partitioning.
The contribution of genomics to spirochetal research has been certainly
catalyzed by the sequencing of the complete genome of Treponema
pallidum and Borrelia burgdorferi. Similarly, the work on outer
membrane proteins of Borrelia or the treponemes has evolved at a more
rapid pace than that of other genres, and it has patterned, for
instance, much of the investigations in Leptospira. The resulting
cross-talk generated by the findings obtained from various spirochetal
species, based both, on predictive genomics and experimental results,
has rendered exciting observations. For example, the definition of a
lipobox for spirochetal lipoproteins, presented by Zuerner and
coauthors in their chapter. They also describe various aspects of
leptospiral research that lead the pack of the spirochetes, such as
genetic complementation analysis and on studies pertaining to
repetitive DNA sequences in the genome.
A complete genetic system for a bacterial species needs to consider the
technology required to study the differential gene expression that will
take place as the microorganism encounters different environments. In
the case of bacterial pathogens, this would include their corresponding
hosts. Seshu and Skare provide a thorough presentation of the
methodologies and findings on in vivo gene expression as it relates to
Borrelia burgdorferi.
The current knowledge on transcriptional regulators in spirochetes and
on the DNA elements that mediate their action is reviewed by Indest et
al. Amongst the various mechanisms of gene regulation addressed by
these authors, they discuss one of the spotlights of current
microbiological research, regulation by quorum sensing. The outcome of
studies on differential gene expression impacts the basic understanding
of the disease process, the search for therapeutic targets, and the
design of biotechnological tools.
As the methodologies presented in this book further develop and the
validity of other proposed alternatives is assessed, additional
contributions of unquestionable value such as DNA microarray technology
will assist in unraveling the mysteries of the biology of spirochetes.
--------------------------------------------------------------------------------
Chapter 1
Phylogenetic Foundation of Spirochetes
B.J. Paster and F.E. Dewhirst
The spirochetes are free-living or host-associated, helical bacteria,
some of which are pathogenic to man and animal. Comparisons of 16S rRNA
sequences demonstrate that the spirochetes represent a monophyletic
phylum within the bacteria. The spirochetes are presently classified in
the Class Spirochaetes in the order Spirochetales and are divided into
three major phylogenetic groupings, or families. The first family
Spirochaetaceae contains species of the genera Borrelia, Brevinema,
Cristispira, Spirochaeta, Spironema, and Treponema. The proposed second
family "Serpulinaceae" contains the genus Brachyspira (Serpulina). The
third family Leptospiraceae contains species of the genera Leptonema
and Leptospira. Novel spirochetal species, or phylotypes, that can not
be presently cultivated in vitro, have been identified from the human
oral cavity, the termite gut, and other host-associated or free-living
sources. There are now over 200 spirochetal species or phylotypes, of
which more than half is presently not cultivable. It is likely that
there is still a significant unrecognized spirochetal diversity that
should be evaluated.
--------------------------------------------------------------------------------
Chapter 2
Gyrations, Rotations, Periplasmic Flagella: The Biology of Spirochete
Motility
C. Li, Md. A. Motaleb, M. Sal, S.F. Goldstein, and N.W. Charon
Spirochetes have a unique structure, and as a result their motility is
different from that of other bacteria. They also have a special
attribute: spirochetes can swim in a highly viscous, gel-like medium,
such as that found in connective tissue, that inhibits the motility of
most other bacteria. In spirochetes, the organelles for motility, the
periplasmic flagella, reside inside the cell within the periplasmic
space. A given periplasmic flagellum is attached only at one end of the
cell, and depending on the species, may or may not overlap in the
center of the cell with those attached at the other end. The number of
periplasmic flagella varies from species to species. These structures
have been shown to be directly involved in spirochete motility, and
they function by rotating within the periplasmic space. The mechanics
of motility also vary among the spirochetes. In Leptospira, a motility
model developed several years ago has been extensively tested, and the
evidence supporting this model is convincing. Borrelia burgdorferi
swims differently, and a model of its motility has been recently put
forward. This model is based on analyzing the motion of swimming cells,
high voltage electron microscopy of fixed cells, and mutant analysis.
To better understand spirochete motility on a molecular level, the
proteins and genes involved in motility are being analyzed. Spirochete
periplasmic flagellar filaments are among the most complex of bacterial
flagella. They are composed of the FlaA sheath proteins, and in many
species, multiple FlaB core proteins. Allelic exchange mutagenesis of
the genes which encode these proteins is beginning to yield important
information with respect to periplasmic flagellar structure and
function. Although we are at an early stage with respect to analyzing
the function, organization, and regulation of many of the genes
involved in spirochete motility, unique aspects have already become
evident. Future studies on spirochete motility should be exciting, as
only recently have complete genome sequences and tools for allelic
exchange mutagenesis become available.
--------------------------------------------------------------------------------
Chapter 3
The Role of Chemotaxis in Pathogenesis of Spirochetes
R. Lux, A. Moter, and W. Shi
Chemotaxis is an important feature of motile organisms that allows
navigation through various environments. It enables them to detect
nutrients and to avoid unfavorable or dangerous conditions. Motility
and chemotaxis are widely acknowledged as important virulence factors
for pathogenic bacteria. In this chapter, we try to explore the role of
chemotaxis in the pathogenesis of spirochetes. Chemotaxis might be
involved in tissue identification and penetration, and represents a
possible mechanism for evasion of the host's immune defense. The recent
development of genetic tools for pathogenic spirochetes and "tracking"
techniques, employing fluorescent in situ hybridization (FISH), could
revolutionize our understanding of the importance of chemotaxis for
infection and persistence of these bacteria in their host.
--------------------------------------------------------------------------------
Chapter 4
Bacteriophages of Borrelia burgdorferi and Other Spirochetes
C.H. Eggers, S. Casjens, and D.S. Samuels
A number of bacteriophage-like particles have been observed in
association with members of the bacterial order Spirochetales, the
spirochetes. Several spirochete bacteriophages have been isolated and
characterized at the molecular level. We have characterized a
bacteriophage, designated fBB1, of the Lyme disease agent, Borrelia
burgdorferi. Here we review the history of the association between the
spirochetes and their bacteriophages, with a particular emphasis on
fBB1 and its prophage, the 32-kb circular plasmid family of B.
burgdorferi.
--------------------------------------------------------------------------------
Chapter 5
The Genetics of Lipopolysaccharide Biosynthesis in Leptospira
D.M. Bulach, T. Kalambaheti, A. de la Peña-Moctezuma and B. Adler
Lipopolysaccharide (LPS) is the major surface antigen of Leptospira.
Variation in LPS structure is the basis for the more than 200 serovars
that have been identified. Despite the importance of this antigen in
immunity and diagnostics, there is relatively little known about the
genetics and chemistry of leptospiral LPS, as compared to some members
of the Enterobacteriaceae. The nucleotide sequence of the locus
encoding enzymes for the biosynthesis of the O-antigen component of
leptospiral LPS (rfb locus) has been determined for three serovars
namely, L.interrogans serovar Pomona, L.interrogans serovar Hardjo
subtype Hardjoprajitno and L.borgpetersenii serovar Hardjo subtype
Hardjobovis. In the absence of data relating to the chemical structure
or genetic tools to construct isogenic mutants in Leptospira,
similarity analysis has been used to provide insight into the
mechanisms by which the leptospiral O-antigen is assembled by
comparison with characterized systems from other bacteria. In addition,
comparison of the gene layout in each of the serovars provides an
indication of the genetic basis for serovar diversity.
--------------------------------------------------------------------------------
Chapter 6
Tick-Host Interactions in Spirochete Transmission
P.A. Nuttall, G.C. Paesen, C.H. Lawrie, V. Hajnická, N. Fuchsberger,
and H. Wang
Tick-borne spirochetes include borreliae that cause Lyme disease and
relapsing fever in humans. They survive in a triangle of parasitic
interactions between the spirochete and its vertebrate host, the
spirochete and its tick vector, and the host and the tick. Until
recently, the significance of vector-host interactions in the
transmission of arthropod-borne disease agents has been overlooked.
However, there is now compelling evidence that the pharmacological
activity of tick saliva can have a profound effect on pathogen
transmission both from infected tick to uninfected host, and from
infected host to uninfected tick. The salivary glands of ticks provide
a pharmacopoeia of anti-inflammatory, anti-haemostatic and anti-immune
molecules. These include bioactive proteins that control histamine,
bind immunoglobulins, modulate cytokines, and inhibit the alternative
complement cascade. The effect of these molecules is to provide a
privileged site at the tick-host interface in which borreliae and other
tick-borne pathogens are sheltered from the normal innate and acquired
host immune mechanisms that combat infections. Understanding the key
events at the tick vector-host interface, that promote spirochete
infection and transmission, will provide a better understanding of the
epidemiology and ecology of these important human pathogens.
--------------------------------------------------------------------------------
Chapter 7
Genome Analyses of Spirochetes: A Study of the ProteinStructures,
Functions
and Metabolic Pathways in Treponema pallidum and Borrelia burgdorferi
R. Das, H. Hegyi, and M. Gerstein
We perform a comprehensive genome analysis on two spirochetes, T.
pallidum and B. burgdorferi. First, we focus on the occurrence of
protein structures in these organisms. We find that there are only a
few spirochete-specific folds, relative to those in other types of
bacteria. The most common fold, by far, in the spirochetes is the
P-loop NTP hydrolase, followed by the TIM barrel. These folds also
happen to be amongst the most multifunctional of the known folds. We
also survey the membrane-protein structures in T. pallidum and find a
notable large family with twelve transmembrane (TM) helices, reflecting
the prevalence of 12-TM transporters in bacteria. Then we move to
analysis of the metabolic pathways and overall metabolism in the
spirochetes, using the metabolic-flux-balancing method. We find that
the lipid biosynthesis pathway is absent from the spirochetes. This
strongly limits the degree to which these organisms can metabolize
NADPH. In turn, we find that the spirochetes distribute flux
disproportionately through the glycolytic pathway instead of the
NADPH-providing pentose phosphate pathway. Further information is
available at <a target="w1"
href="http://bioinfo.mbb.yale.edu>bioinfo.mbb.yale.edu</a>
--------------------------------------------------------------------------------
Chapter 8
Whole Genome Analyses of Transporters in Spirochetes: Borrelia
burgdorferi and Treponema pallidum
M.H. Saier, Jr. and I.T. Paulsen
The completely sequenced genomes of two spirochetes, Borrelia
burgdorferi (Bbu) and Treponema pallidum (Tpa) were analyzed for the
distribution of transporter types. Both organisms exhibited fewer
proteins with >7 a-helical transmembrane spanners (TMSs), and fewer
identified transport systems per megabase pair of DNA than most other
prokaryotes analyzed. Each organism exhibits one recognizable ion
channel protein of the MscS family. Tpa has twice as many primary
carriers as Bbu but lacks PTS permeases that are plentiful in Bbu. Tpa
is the only prokaryote so far sequenced which has two F-type ATPases.
Large families of secondary nutrient uptake carriers (MFS and APC) that
are prevalent in other organisms are essentially lacking in
Spirochetes. The largest Spirochete secondary carrier families consist
of efflux systems. While both Bbu and Tpa exhibit an unusual degree of
transporter diversity, major differences in specificity exist between
these two organisms.
--------------------------------------------------------------------------------
Chapter 9
Borrelia Genomes
S. Casjens
All analyzed members of the spirochete genus Borrelia contain a linear
chromosome about 910 kbp long. The complete sequence of the B.
burgdorferi B31 genome predicts that its chromosome carries essentially
all of this organism's housekeeping genes. In accordance with these
bacterial species' obligatory parasitic lifestyle, its genes encode
enzymes that are capable of only a minimal metabolism, in which all
nucleotides, amino acids, fatty acids and enzyme cofactors must be
scavenged from the host. In addition to the chromosome, all Borrelia
isolates examined carry multiple linear and circular plasmids with
lengths between 5 and 200 kbp. The plasmids, which account for over 600
kbp in isolate B31, carry very few genes with homology to genes outside
of the Borrelia genus. But they do carry numerous predicted lipoprotein
genes, many of which are have been shown to be or are expected to be
outer surface proteins. Ten of the linear plasmids have strikingly low
protein coding potential for bacterial DNA. These plasmids have enjoyed
numerous past duplicative rearrangements, which have resulted in the
presence of a substantial fraction of the DNA that appears to be
currently undergoing mutational decay, presumably because it is no
longer under selection for function.
--------------------------------------------------------------------------------
Chapter 10
Repetition, Conservation, and Variation: the Multiple cp32 Plasmids of
Borrelia Species
B. Stevenson, W.R. Zückert and D.R. Akins
Members of the spirochete genus Borrelia contain large numbers of
extrachromosomal DNAs. Sequence analysis of the B. burgdorferi strain
B31 genome indicated that its many plasmids contain large quantities of
repeated sequences, the most obvious of which are the cp32 plasmid
family. Individual spirochetes may carry nine or more different, but
homologous, cp32 plasmids. Every other species of Borrelia examined
thus far also contains multiple plasmids related to the B. burgdorferi
cp32s. These plasmids are arguably the best characterized of all the
borrelial plasmids, and epitomize the apparent redundancy evident in
the many plasmids carried by these bacteria. Despite their extensive
similarities, cp32 plasmids contain some open reading frames whose
sequences often vary between plasmids, and which encode proteins
synthesized by the bacteria during vertebrate infection. In this
chapter, we analyze the hypervariable and conserved regions of the cp32
plasmid family, and discuss possible reasons why borreliae harbor
multiple gene paralogs.
--------------------------------------------------------------------------------
Chapter 11
Antibiotic Selective Markers and Spirochete Genetics
J.M. Hardham and E.L. Rosey
Until very recently, the pathogenic spirochetes have been refractory to
genetic manipulation. This has been due, in part, to difficulties with
in vitro growth and the genetic distance that spirochetes are from
typical Gram-negative and Gram-positive organisms. Insertional
mutagenesis and other genetic techniques are now possible in some of
the pathogenic spirochetes such as Borrelia burgdorferi, Brachyspira
(Serpulina) hyodysenteriae, Leptospira sp., and Treponema denticola.
However, organisms such as Treponema pallidum, which cannot be grown in
vitro, are still not amenable to genetic manipulation. These recent
advances have paved the way for more detailed genetic studies of
transcriptional regulation, protein function, protein localization,
metabolic capabilities, motility, and pathogenic nature of this group
of spirochetes. This chapter will discuss the current repertoire of
antibiotic markers that are useful for spirochetal genetic
manipulation. Further advances in selectable markers and shuttle
vectors will allow researchers to complete Koch's molecular hypothesis
for various virulence genes of the pathogenic spirochetes and increase
the overall understanding of these challenging bacteria.
--------------------------------------------------------------------------------
Chapter 12
DNA Exchange and Insertional Inactivation in Spirochetes
K. Tilly, A.F. Elias, J.L. Bono, P. Stewart, and P. Rosa
Spirochetes have complex life cycles and are associated with a number
of diseases in humans and animals. Despite their significance as
pathogens, spirochete genetics are in their early stages. However, gene
inactivation has been achieved in Borrelia burgdorferi, Brachyspira
hyodysenteriae, Leptospira biflexa, and Treponema denticola. Here, we
review methods that have been used in spirochetes for gene inactivation
and DNA exchange, with a primary focus on B. burgdorferi. We also
describe factors influencing electrotransformation in B. burgdorferi.
In summary, optimal transformation frequencies are obtained with log
phase bacteria, large amounts of DNA (up to 50 mg per transformation),
and high field strength (12.5-37.5 kV/cm). Infectious B. burgdorferi
isolates transform with frequencies 100-fold lower than those found for
high passage, non-infectious strains. Surface characteristics of the
bacteria, which often correlate with infectivity, are among the
obstacles to effective transformation by electroporation.
--------------------------------------------------------------------------------
Chapter 13
Shuttle Vectors for Spirochetes
I. Saint Girons, B. Chi, and H. Kuramitsu
Constructions of Escherichia coli-spirochete shuttle vectors are based
on naturally occurring plasmids, broad host range plasmids or
bacteriophages. This chapter primarily focuses on genetic tools for
Treponema denticola which is associated with periodontal diseases. The
T. pallidum FlaA protein, E. coli b-galactosidase, and the green
fluorescent protein were successfully expressed in T. denticola from a
shuttle vector system. Shuttle vectors for Borrelia burgdorferi and
Leptospira biflexa were reported, opening the way to further genetic
manipulation.
--------------------------------------------------------------------------------
Chapter 14
The Role of Genomics in Approaching the Study of Borrelia DNA
Replication
J. García-Lara, M. Picardeau, B.J. Hinnebusch, W.M. Huang, and S.
Casjens
The identification of chromosomal and episomal origins of replication
in the genome of the causative agent of Lyme disease, the spirochete
Borrelia burgdorferi, has been greatly facilitated by genomics.
Analysis of genome features, including strand compositional
asymmetries, organizational similarities to other bacterial origins of
replication, and the presence of homologues of genes involved in
replication and partitioning, have contributed to the identification of
a collection of putative origins of replication within the Borrelia
genome. This analysis has provided the basis for the experimental
verification of origins in the linear chromosome and in the linear
plasmid lp28-2. Information generated during the study of these origins
will significantly contribute to the understanding of the mechanisms of
replication and partitioning in Borrelia.
--------------------------------------------------------------------------------
Chapter 15
Technological Advances in the Molecular Biology of Leptospira
R. Zuerner, D. Haake, B. Adler, and R. Segers
Pathogenic members of the genus Leptospira have been refractory to
genetic study due to lack of known mechanisms of genetic exchange. To
bypass this limitation, several techniques have been useful for
Leptospira gene discovery, including heterologous complementation of
Escherichia coli mutants, screening of DNA libraries with probes, and
random sequence analysis. Construction of combined physical and genetic
maps revealed the presence of two circular chromosomal replicons. The
organization of the L. interrogans genome is quite variable, with
genetically similar strains differentiated by many rearrangements.
These rearrangements likely occur through recombination between
repetitive DNA elements found scattered throughout the genome. Analysis
of intervening sequences and genes encoding LPS biosynthetic enzymes
provide evidence of lateral transfer of DNA between Leptospira spp. We
have also gained insight into the biology of these bacteria by
analyzing genes encoding LPS and outer membrane proteins (OMPs). Some
of these OMPs are differentially expressed. Characterization of
mechanisms governing the expression of the OMP genes should provide
insight into host-parasite interactions. Furthermore, recent advances
in heterologous expression of leptospiral OMP genes are opening new
avenues of vaccine development.
--------------------------------------------------------------------------------
Chapter 16
The Many Faces of Borrelia burgdorferi
J. Seshu and J.T. Skare
In this chapter we describe several genetic regulatory mechanisms
adopted by the agent of Lyme disease, Borrelia burgdorferi, to sense
and adapt to different host and environmental conditions either in
vitro or in vivo. This regulation results in the increased or decreased
synthesis of several proteins whose levels are believed to play key
roles in the ability of B. burgdorferi to cycle between both arthropod
and mammalian hosts. Moreover, the differential synthesis of these
proteins serves to modulate the response of B. burgdorferi to signals
in the requisite host and may also, in some cases, function as
virulence determinants of this spirochete. Elucidation of these
mechanisms will help in the understanding of the pathogenicity of B.
burgdorferi as well as aid in identifying proteins that are important
during different stages of infection.
--------------------------------------------------------------------------------
Chapter 17
Transcriptional Regulation in Spirochetes of Medical Importance
K.J. Indest, R. Ramamoorthy, and M.T. Philipp
Spirochetes belong to a widely diverse family of bacteria. Several
species in this family can cause a variety of illnesses including
syphilis and Lyme disease. Despite the fact that the complete genome
sequence of two species, Borrelia burgdorferi and Treponema pallidum,
have been deciphered, much remains to be understood about spirochetal
gene regulation. In this chapter we focus on the environmental
transitions that spirochetes undergo during their life cycles and the
mechanisms of transcriptional regulation that might possibly mediate
spirochetal adaptations to such changes.
--------------------------------------------------------------------------------
Chapter 18
Biology of Treponema pallidum: Correlation of Functional Activities
With Genome Sequence Data
Steven J. Norris, David L. Cox, and George M. Weinstock
Aspects of the biology of T. pallidum subsp. pallidum, the agent of
syphilis, are examined in the context of a century of experimental
studies and the recently determined genome sequence. T. pallidum and a
group of closely related pathogenic spirochetes have evolved to become
highly invasive, persistent pathogens with little toxigenic activity
and an inability to survive outside the mammalian host. Analysis of the
genome sequence confirms morphologic studies indicating the lack of
lipopolysaccharide and lipid biosynthesis mechanisms, as well as a
paucity of outer membrane protein candidates. The metabolic
capabilities and adaptability of T. pallidum are minimal, and this
relative deficiency is reflected by the absence of many pathways,
including the tricarboxylic acid cycle, components of oxidative
phosphorylation, and most biosynthetic pathways. Although
multiplication of T. pallidum has been obtained in a tissue culture
system, continuous in vitro culture has not been achieved. The balance
of oxygen utilization and toxicity is key to the survival and growth of
T. pallidum, and the genome sequence reveals a similarity to lactic
acid bacteria that may be useful in understanding this relationship.
The identification of relatively few genes potentially involved in
pathogenesis reflects our lack of understanding of invasive pathogens
relative to toxigenic organisms. The genome sequence will provide
useful raw data for additional functional studies on the structure,
metabolism, and pathogenesis of this enigmatic organism.
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