Abstract
BARRANGOU, RODOLPHE. Functional genomic analyses of carbohydrate utilization by Lactobacillus acidophilus. (Under the direction of Professor Todd R. Klaenhammer).
Carbohydrates are a primary source of energy for microbes. Specifically, lactic acid bacteria have the ability to utilize a variety of nutrients available in their respective habitats. For probiotic microbes inhabiting the human gastrointestinal tract, the ability to utilize sugars non-digested by the host plays an important role in their survival.
Lactobacillus acidophilus is a probiotic organism which can utilize a variety of mono-, di- and poly-saccharides, including prebiotic compounds such as fructooligosaccharides and raffinose. However, little information is available about the mechanisms and genes involved in carbohydrate utilization by lactobacilli. The transport and catabolic
machinery involved in utilization of glucose, fructose, sucrose, FOS, raffinose, lactose, galactose and trehalose was characterized using global transcriptional profiling.
Microarray hybridizations were carried out using a round-robin design and data analyzed using a two-stage mixed model ANOVA. Genes differentially expressed between
machinery of the Leloir pathway. Insertional inactivation of genes encoding sugar
transporters and hydrolases confirmed microarray results. Quantitative RT-PCR was also used to confirm differential gene expression. Additional transcription experiments
showed specific induction of genes encoding sugar transporters and hydrolases, and transcriptional repression by glucose. Collectively, microarray data revealed coordinated and regulated transcription of genes involved in sugar utilization based on carbohydrate availability, likely via carbon catabolite repression.
The relationships between gene expression level, codon usage, chromosomal location and intrinsic gene parameters were investigated globally. Gene expression levels correlated most highly with GC content, codon adaptation index and gene size. In
contrast, gene expression levels did not correlate with GC content at the third codon position. Perhaps the high correlation between GC content and gene expression is due to the low genomic GC composition of L. acidophilus. Analysis of variance was used to investigate the impact of chromosomal location on gene expression after data was segregated into four groups, by strand and orientation relative to the origin and terminus of replication. Results showed genes on the leading strand were more highly expressed. Also, genes pointing toward the terminus of replication showed higher expression levels. This preference allows for co-directional replication and transcription. Collectively, results showed a strong influence of chromosomal architecture, GC content and codon usage on gene transcription.
Globally, analysis of gene expression in Lactobacillus acidophilus revealed orchestrated transcription, and adaptation to environmental conditions. Specifically, dynamic adaptation to carbohydrate sources available in the environment might
FUNCTIONAL GENOMIC ANALYSES OF CARBOHYDRATE
UTILIZATION BY
LACTOBACILLUS ACIDOPHILUS
by
RODOLPHE BARRANGOU
A dissertation submitted to the Graduate Faculty of North Carolina State University
in partial fulfillment of the requirements for the Degree of
Doctor of Philosophy
FUNCTIONAL GENOMICS
Raleigh
2004
APPROVED BY:
________________________________ _________________________________
Dr. Todd R. Klaenhammer Dr. Greg Gibson
Chairman of Advisory Committee
________________________________ _________________________________
Biography
Rodolphe Barrangou, the son of Charles Barrangou-Poueys and Roseline Helie, was born
on July 20, 1975 in Caen, France and raised in Paris, France. He attended the University
of Rene Descartes, Paris V (France) between 1994 and 1996 where he obtained a degree
in Life Sciences. He also attended the University of Technology of Compiegne (France)
between 1996 and 1998 where he obtained a M. S. degree in Biological Engineering. In
January 1999, he began working towards a Master of Science in Food Science at North
Carolina State University (USA) in the Vegetable Fermentation Laboratory
(USDA-ARS) under the direction of Dr. Henry P. Fleming and Dr. Todd R. Klaenhammer. In
January 2001, he began working towards a Ph. D. in Functional Genomics at North
Carolina State University (USA) in the Southeast Dairy Foods Research Center under the
direction of Dr. Todd R. Klaenhammer.
Acknowledgements
First and foremost, I would like to thank my advisor, Dr. Todd R. Klaenhammer for
giving me the opportunity to pursue another graduate degree at NC State, for his time,
supervision, guidance, availability and support throughout my graduate education. I also
wish to acknowledge Dr. Greg Gibson, Dr. Robert M. Kelly, and Dr. Dahlia Nielsen, for
serving on my advisory committee, giving me time outside of committee meetings, and
insightful discussions. Also, I would like to acknowledge all my co-workers and
collaborators within the “Klaenhammer lab”, especially Evelyn Durmaz, Dr. Andrea
Azcarate Peril, Dr. Eric Altermann, and Tri Duong, for technical help, sharing their
expertise and suggestions. I would also like to thank my other collaborators on campus, at
the GRL (Dr. Bryon Sosinski and Regina Brierley), for providing help with microarray
printing and scanning; in the Microbiology Department (Dr. Jose Bruno-Barcena and Dr.
Hosni Hassan), for proving help with Q-PCR; and my collaborators in the Bioinformatics
Program, namely Shannon Conners and Joshua Starmer for collaborating with me. I
would also like to acknowledge Dr. Barbara Sherry and Dr. Stephanie Curtis for their
leadership in the Functional Genomics program. I would like to dedicate my work to my
whole family for teaching me everything that I need to know, and for understanding my
need to go overseas. I would also like to acknowledge my friends Tri and Mike for
making my experience in the lab (and beyond) particularly enjoyable. Finally, I would
like to give a very special and personal thank you to my wife Lisa, for her patience,
understanding, and permanent support throughout my graduate career, for helping me
make the right decisions, understand what is important, and sharing everything in my life.
Table of contents
LIST OF TABLES. ___________________________________________________VII
LIST OF FIGURES. _________________________________________________ VIII
LIST OF ABBREVIATIONS. ___________________________________________ X
CHAPTER I – LITERATURE REVIEW: TRANSPORT SYSTEMS IN LACTIC ACID BACTERIA. _________________________________________________ 1
1.1 INTRODUCTION. __________________________________________________ 2 1.2 THE LACTIC ACID BACTERIA. ______________________________________ 4 1.3 GENOMICS OF LACTIC ACID BACTERIA. ____________________________ 8 1.4 FERMENTATION CAPABILITIES OF LACTIC ACID BACTERIA. ________ 11 1.5 ABC TRANSPORTERS. _____________________________________________ 13 1.6 PTS TRANSPORTERS. _____________________________________________ 17 1.7 OTHER TRANSPORTERS. __________________________________________ 19 1.8 REGULATION AND CARBON CATABOLITE REPRESSION. ____________ 22 1.9 CONCLUSIONS AND PERSPECTIVES. _______________________________ 26 1.10 REFERENCES. ___________________________________________________ 29
CHAPTER II – FUNCTIONAL AND COMPARATIVE GENOMIC ANALYSES OF AN OPERON INVOLVED IN FRUCTOOLIGOSACCAHRIDE
UTILIZATION BY LACTOBACILLUS ACIDOPHILUS. _________________ 42
2.1 ABSTRACT. ______________________________________________________ 43 2.2 INTRODUCTION. _________________________________________________ 44 2.3 MATERIALS AND METHODS. ______________________________________ 45 2.3.1 Bacterial strain and media used in this study. ________________________ 45 2.3.2 Computational analysis of the putative msm operon. ___________________ 46
2.3.3 RNA isolation and analysis. ______________________________________ 46
2.3.4 Comparative genomic analyses. ___________________________________ 47
2.3.5 Phylogenetic trees. _____________________________________________ 48
2.3.6 Gene inactivation. ______________________________________________ 48
2.4 RESULTS. ________________________________________________________ 49 2.4.1 Computational analysis of the msm operon. __________________________ 49 2.4.2 Sugar induction and co-expression of contiguous genes. ________________ 50
2.4.3 Mutant phenotype analysis. ______________________________________ 51
2.4.4 Comparative genomic analyses and locus alignments. _________________ 51
2.4.5 Phylogenetic trees. _____________________________________________ 52
2.4.6 Catabolite response elements (cre) analysis. _________________________ 53 2.5 DISCUSSION. _____________________________________________________ 54 2.6 REFERENCES. ____________________________________________________ 60
CHAPTER III – GLOBAL ANALYSIS OF CARBOHYDRATE UTILIZATION
AND TRANSCRIPTIONAL REGULATION IN LACTOBACILLUS
ACIDOPHILUS USING WHOLE-GENOME cDNA MICROARRAYS._____ 77
3.1 ABSTRACT. ______________________________________________________ 78 3.2 INTRODUCTION. _________________________________________________ 80 3.3 MATERIALS AND METHODS. ______________________________________ 82 3.3.1 Bacterial strain and media used in this study. ________________________ 82
3.3.2 RNA isolation. _________________________________________________ 82
3.3.3 Microarray fabrication. _________________________________________ 83
3.3.4 cDNA target preparation and microarray hybridization. ________________ 83 3.3.5 Microarray data collection and analysis. ____________________________ 84
3.3.6 Real-Time Quantitative RT-PCR. __________________________________ 86
3.4 RESULTS. ________________________________________________________ 86 3.4.1 Differentially expressed genes. ____________________________________ 86
3.4.2 Real-Time Quantitative RT-PCR. __________________________________ 91
3.5 DISCUSSION. _____________________________________________________ 92 3.6 REFERENCES. ____________________________________________________ 98
CHAPTER IV – GLOBAL CHARACTERIZATION OF THE LACTOBACILLUS ACIDOPHILUS TRANSCRIPTOME AND ANALYSIS OF RELATIONSHIPS BETWEEN GENE EXPRESSION LEVEL, CODON USAGE,
CHROMOSOMAL LOCATION AND INTRINSIC GENE
CHARACTERISTICS._____________________________________________ 115
4.1 ABSTRACT. _____________________________________________________ 116 4.2 INTRODUCTION. ________________________________________________ 118 4.3 MATERIALS AND METHODS. _____________________________________ 120
4.3.1 Genome and microarray data. ___________________________________ 120
4.3.2 Gene intrinsic parameters. ______________________________________ 121
4.3.3 Codon adaptation index. ________________________________________ 122
4.3.4 Ribosome binding site identification. ______________________________ 123 4.3.5 Statistical analyses. ____________________________________________ 123 4.4 RESULTS. _______________________________________________________ 124
4.4.1 Distribution patterns. __________________________________________ 124
4.4.2 Correlation analyses. __________________________________________ 126
4.4.3 Chromosomal location. _________________________________________ 127
4.5 DISCUSSION. ____________________________________________________ 130 4.6 REFERENCES. ___________________________________________________ 142
APPENDIX I – FUNCTIONAL AND COMPARATIVE GENOMIC ANALYSES OF AN OPERON INVOLVED IN FRUCTOOLIGOSACCAHRIDE
UTILIZATION BY LACTOBACILLUS ACIDOPHILUS. ________________ 157
List of tables
Chapter I
1. Genomes of lactic acid bacteria and other probiotic species. _______________ 36 2. Carbohydrate utilization profiles for select lactic acid bacteria. ____________ 37 3. Transmembrane domains in L. acidophilus transporters. __________________ 38
Chapter II
1. Catabolite responsive elements sequences. ______________________________ 64 2. Primers used in this study. ___________________________________________ 65 3. Genes and proteins used for comparative genomic analyses. _______________ 66
Chapter IV
1. Codon usage table. _________________________________________________ 145 2. Correlation analyses. _______________________________________________ 146 3. Analysis of variance between chromosomal locations. ____________________ 147 4. Correlation analyses, by chromosomal location. ________________________ 148
List of figures
Chapter I
1. Phylogenetic tree of lactic acid bacteri and select microbial species. _________ 39 2. Transporters commonly found in lactic acid bacteria. _____________________ 40 3. Transmembrane domains in ABC, PTS and GPH transporters in L. acidophilus.
__________________________________________________________________ 41
Chapter II
1. Operon layout. _____________________________________________________ 68 2.Sugar induction and repression. _______________________________________ 69 3. Growth curves. _____________________________________________________ 70 4. Operon architecture analysis. _________________________________________ 71 5. Neighbor-joining phylogenetic tree. ____________________________________ 72 6. Co-expression of contiguous genes. ____________________________________ 73 7. Mutant growth on select carbohydrates. ________________________________ 74 8. Motifs highly conserved amongst repressors and fructosidases. _____________ 75 9. Biochemical pathways. ______________________________________________ 76
Chapter III
1. Round-robin microarray hybridization design. _________________________ 102 2. Hierarchical clustering analyses of gene expression patterns. ______________ 103 3. Hierarchical clustering analyses of gene expression patterns for select genes and
operons. _________________________________________________________ 104 4. Volcano plot comparison of gene expression between FOS and raffinose. ____ 105
5. Contour plot comparison of gene expression between FOS, raffinose and trehalose.
_________________________________________________________________ 106 6. Global differential gene expression. ___________________________________ 107 7. Gene fold induction. ________________________________________________ 108 8. RT-Q-PCR analysis of differentially expressed genes. ____________________ 109 9.Genetic loci of interest. ______________________________________________ 110
10.Lactose locus in select lactobacilli. ___________________________________ 111 11. Catabolite responsive elements sequences. ____________________________ 112 12. Carbohydrate utilization in L. acidophilus. ____________________________ 113 13. Expression of glycolysis genes. ______________________________________ 114
Chapter IV
1. Gene distribution over select parameters. ______________________________ 149 2. Chromosomal locations. ____________________________________________ 150 3. Correlations between gene expression level and intrinsic genes parameters. _ 151 4. Analysis of variance, by chromosomal location. _________________________ 153 5. Correlations between gene expression level and intrinsic genes parameters, by
chromosomal location. _____________________________________________ 154 6. Gene distribution over select parameters, by chromosomal location. _______ 156
x List of abbreviations
ABC ATP Binding Cassette
ANOVA ANalysis Of Variance
CAI Codon Adaptation Index
CCR Carbon Catabolite Repression
CH CHaperone proteins
CRE Catabolite Responsive Element
DNA Deoxyribo Nucleic Acid
EC Enzyme Commission
FOS Fructo Oligo Saccharides
GIT Gastro Intestinal Tract
GPH Galactoside Pentose Hexuronide
LaOT Lagging strand, between the Origin and Terminus LaTO Lagging strand, between the Terminus and Origin LeOT Leading strand, between the Origin and Terminus LeTO Leading strand, between the Terminus and Origin
LGT Lateral Gene Transfer
LSM Least Squares Means
MSM Multiple Sugar Metabolism
NCFM North Carolina Food Microbiology
NDO Non Digestible Oligosaccharides
ORF Open Reading Frame
PCR Polymerase Chain Reaction
PEP Phospho Enol Pyruvate
PHX Predicted Highly eXpressed
PTS Phoshoenolpyruvate Transferase System
RBS Ribosome Binding Site
RNA Ribo Nucleic Acid
RP Ribosomal Proteins
RSCU Relative Synonymous Codon Usage
SD Shine Dalgarno
1.1 Introduction
Bacteria are a dominant and diverse life form on earth. Molecular comparisons
between life forms divide organisms into three groups, namely eubacteria, archaebacteria
and eukaryotes (Woese et al., 1990). At the molecular level, those three groups are based
on differences within the ribosomal RNA (rRNA) structure and sequence (Woese et al.,
1990). This triad-nomenclature includes the eukaryote-prokaryote dichotomy, which is
based on presence / absence of a nucleus. Specifically, life on earth is divided into three
“domains”, namely Bacteria (replacing eubacteria), Archaea (replacing archaebacteria)
and Eucarya (replacing eukaryotes) (Woese et al., 1990; Embley et al., 1994), wherein
there are six “kingdoms”, bacteria, fungi, plantae, animalia, protoctista (protozoa) and
chromista (Embley et al., 1994; Margulis, 1996; Cavalier-Smith, 2004). Both archaea and
bacteria are monohomogenomic, with no nucleus, whereas eucarya are
polyheterogenomic and contain a nucleus (Margulis, 1996).
The importance of microbes for all life-forms has been illustrated recently. Recent
phylogenetic analyses suggest the eukaryotic genome actually resulted from the fusion of
an archaeal genome with a bacterial genome (Margulis, 1996; Rivera and Lake, 2004),
consequently changing the tree of life into the ring of life (Rivera and Lake, 2004). This
recent theory emphasizes the historical and evolutionary importance of the bacterial
kingdom.
Prokaryotic diversity and predominance illustrate the physiological flexibility of
microbes, as well as their adaptability to many environments. A recent metagenomic
oceanic study investigating microbial genome diversity within a water community
illustrates our limited knowledge and comprehension of microbial diversity and
physiological properties (Venter et al., 2004), although measures of microbial diversity
have previously shown our limited knowledge of microbial diversity as well (Curtis et al.,
2002; Curtis and Sloan, 2004). The limited extent of microbial diversity is well
documented, and most environmental studies end up uncovering novel species and
lineages (Embley et al., 1996; Cavalier-Smith, 2004). Recent “conservative” assumptions
estimate microbial diversity at over 1030 individuals representing over 107 species
(Embley et al., 1994; Curtis et al., 2002; Curtis and Sloan, 2004), although estimates of
microbial diversity may be inaccurate. Nevertheless, recent advances in microbial
genomics have shown microbial diversity at many levels, especially for microbes that can
be cultured. Specifically, microbial diversity is visible both within and between species,
including differences in genome size, genome content, GC content, codon usage, mobile
genetic elements, cell shape, occurrence in the environment, growth conditions
(temperature, oxygen level, energy sources), and many others.
From a genomic standpoint, microbial diversity is visible through genome size,
GC content (ranging between 25% and 75%; Muto and Osawa, 1987), codon usage
(Grantham et al., 1980), genome content, and occurrence of bacteriophage and plasmids.
Although the differences can be overwhelming and represent a large proportion of the
genome, even within a given species, those differences illustrate physiological adaptation
to various environmental conditions. Specifically, microbes tend to adapt to their
environment via evolutionary pressures, in order to optimize their survival and
competitiveness.
Interestingly, genes encoding sugar transporters and carbohydrate hydrolases can
represent a large proportion of strain-specific genes, with ABC transporters reported to
the highest horizontal gene transfer frequency in Thermotoga maritima (Nesbo et al.,
2002). Similarly, it has been suggested that genes involved in catabolic properties of B.
longum (Schell et al., 2002) and sugar uptake genes in L. plantarum (Kleerebezem et al.,
2003) have been acquired via horizontal gene transfer, as part of the adaptation process of
these bacteria to their respective environments.
Understanding how microbes modulate their genomes to acquire physiological
properties and phenotypic traits that further their ability to withstand environmental
conditions and utilize resources available in their various habitats is important.
Specifically, for lactic acid bacteria, this review illustrates how various transport systems
contribute to their ability to utilize a diversity of energy sources available in a number of
habitats.
1.2 The lactic acid bacteria
Lactic acid bacteria (LAB) are a heterogeneous family of microbes which can
ferment a variety of nutrients (Poolman, 2002) primarily into lactic acid. LAB are mainly
Gram-positive, non-sporulating, acid tolerant, anaerobic bacteria divided in two subsets,
the low GC taxa, and the high GC taxa. Biochemically, lactic acid bacteria include both
homofermenters and heterofermenters. The former produce primarily lactic acid, while
the latter yield also a variety of fermentation by-products, including mostly acetic acid,
ethanol, carbon dioxide and formic acid (Hugenholtz et al., 2002; Kleerebezem and
Hugenholtz, 2003). Although their primary contribution consists of the rapid formation of
lactic acid, which results in acidification of food products, they also contribute to flavor,
texture and nutrition in a variety of food products (Kleerebezem and Hugenholtz, 2003).
Environmentally, LAB reside in a variety of habitats, including human cavities
such as the gastrointestinal tract (Lactobacillus plantarum, Lactobacillus acidophilus,
Lactobacillus johnsonii, Bifidobacterium longum, Streptococcus agalactiae,
Enterococcus faecalis), the oral cavity (S. mutans, B. longum), the respiratory tract (S.
pneumoniae) and the vaginal cavity (B. longum, S. agalactiae) (Tannock, 1999;
Ouwehand et al., 2002; Vaughan et al., 2002). Additionally, lactic acid bacteria are
naturally found in a variety of environmental niches including dairy, meat, vegetable and
plant environments (Kleerebezem et al., 2003).
The two driving forces behind the tremendous amount of work performed in lactic
acid bacteria are their use in fermentation processes and as probiotics. Specifically, a
diversity of microbial strains is used as starter cultures in the food industry, primarily in
dairy applications, although Lactococcus lactis is by far the best characterized lactic acid
bacterium (Bolotin et al., 1999). Additionally, select strains are used as health-promoting
probiotics in food product and dietary supplements (Gibson and Roberfroid, 1995; Reid,
1999).
In fermentation processes, lactic acid bacteria are used as starter cultures.
Although they are used in fermentation of meats, vegetables and wine, they are primarily
used in dairy processes. Specifically, they are widely used in cheese and yogurt
manufacturing. As a result, Lactococcus lactis is perhaps the most extensively studied
species among LAB, and a variety of genetic tools have been developed therein (Bolotin
et al., 1999; Hugenholtz et al., 2002; Kleerebezem and Hugenholtz, 2003).
Probiotics are generally defined as “live microorganisms which, when
administered in adequate amounts, confer a health benefit on the host” (Reid et al., 2003).
Probiotic microbes promote health via their presence and sometimes residence in the
human gastrointestinal tract, and interaction with the intestinal flora and host tissue. As a
result, phenomena such as adherence to human epithelial cells, survival at low pH,
resistance to acids, survival in the presence of bile salts, and competition with other
commensals all contribute to their ability to survive and promote human health (Sanders
and Klaenhammer, 2001). However, those functionalities rely on the survival and
competitiveness of the strain, which is dependent upon its ability to efficiently use
nutrient sources available in the intestinal environment. As a result, transporters are a key
factor involved in probiotic functionality. Lactic acid bacteria generally harbor a
significant number of transporters for acquisition of a diverse set of carbohydrates and
amino-acids.
Similarly, organisms used in fermentation applications need to use energy sources
available in their environment in order to carry out the desired metabolic processes. As a
result, uptake of nutrients, particularly carbohydrates is essential for fermentative LAB.
Therefore, identification and characterization of their transport systems is essential to
develop our understanding of the physiological processes involved in their
functionalities.
Although a large diversity of microbes produce lactic acid, only select members
of the lactic acid bacteria are widely used in fermentation processes and probiotic
applications. The primary genera employed are: Lactococcus, Lactobacillus,
Streptococcus, Bifidobacterium, and to a lesser extent Leuconostoc, and Oenococcus.
Additionally, within those genera, most of the work has focused on only a few select
species, as shown in Table 1.
A large and diverse microbial community resides in the human gastrointestinal
(Tannock, 1999). In particular, the complex microbial population in the intestine includes
beneficial bacteria such as bifidobacteria and lactobacilli (Tannock, 1999; Ouwehand et
al., 2002; Vaughan et al., 2002). Although they are not dominant microbes, probiotics are
important organisms that can promote health in a variety of mucosal locations, including
the human intestine. In humans, lactobacilli and bifidobacteria in particular, are perceived
as exerting health-promoting properties (Gibson and Roberfroid, 1995; Ouwehand et al.,
2002). Lactobacilli have been associated with a variety of health-promoting
functionalities, widely documented for humans, specifically in the case of Lactobacillus
species (Reid, 1999; Sanders and Klaenhammer, 2001). The large intestine in particular is
the most heavily colonized region of the human digestive tract (Gibson and Roberfroid,
1995). The colonic microbiota feeds on the unabsorbed remains of the diet, which
primarily consist of non-digestible sugars (Alles et al., 1996). Even though microbes have
a limited capacity to utilize substrates present in the environment, some bacteria have a
diverse genomic makeup shaped by evolution and adaptation that is selectively fashioned
to utilize and catabolize a wide range of nutrients present in their environmental niche.
Consequently, a wide carbohydrate catabolic potential likely allows microbes to compete
and survive in environmental niches where sugar molecules are scarce, as previously
suggested for Lactobacillus plantarum (Kleerebezem et al., 2002), Lactobacillus
acidophilus (Barrangou et al., 2003; Altermann, 2004), Lactobacillus johnsonii
(Pridmore et al., 2004) and Bifidobacterium longum (Schell et al., 2004). The ability of
select intestinal microbes to utilize intestinal nutrients, including substrates non-digested
by the host plays an important role in their ability to successfully survive and colonize the
mammalian intestinal tract. Whether they are fermentative organisms, or
health-promoting probiotics, microbial growth is primarily dependent upon energy sources such
as carbohydrates.
1.3 Genomics of lactic acid bacteria
In the recent past, substantial progress has been achieved in microbial genomics,
particularly in genome sequencing. To date, over 193 complete microbial genomes have
been published (NCBI website, www.ncbi.nlm.nih.gov/genomes/MICROBES/
complete.html), including 174 bacteria and 19 archaea, covering a wide diversity of
taxonomic groups. Early microbial genome analyses suggest that genome content reflects
adaptation to environmental conditions, specifically genes involved in transport and
catabolism of nutrients, since microbes shape their genomes to efficiently utilize
available resources and adapt to their habitats, according to temperature, levels of
oxygen, toxic compounds, and other factors.
The genome sequences of several lactic acid bacteria have been published,
including Lactococcus lactis (Bolotin et al., 1999), S. mutans (Ajdic et al., 2002), S.
pneumoniae (Tettelin et al., 2001), S. agalactiae (Tettelin et al., 2002), S. pyogenes
(Ferretti et al., 2001), Bifidobacterium longum (Schell et al., 2002), Lactobacillus
plantarum (Kleerebezem et al., 2003), L. johnsonii (Pridmore et al., 2004) and L.
acidophilus (Altermann et al., 2004). Several more are underway (Klaenhammer et al.,
2002; Siezen et al., 2004). For these LAB, probiotic organisms and other intestinal
microbes, genome features are presented in Table 1.
Lactic acid bacteria are low GC organisms (lactobacilli, streptococci, lactococci)
and high GC organisms (bifidobacteria, brevibacteria) (Table 1). LAB genomes vary
widely in size (between 1.8 and 4.4 Mbp), although most genomes are between 1.8 and
2.5 Mbp (Table 1). Genetically, LAB are diverse, as illustrated in Figure 1, including
high GC genera such as Bifidobacterium and Brevibacteria, and distinct low GC genera
Leuconostoc and Oenococcus seems distant from other LAB (Figure 1). In contrast,
streptococci and lactococci appear closely related, as well as lactobacilli and pediococci
(Figure 1).
Recent genome analyses have shown that bifidobacteria, streptococci and
lactobacilli possess specialized saccharolytic potentials which reflect the nutrient
availability in their respective environments (Tettelin et al., 2001; Ajdic et al., 2002;
Schell et al., 2002; Kleerebezem et al., 2003; Altermann et al., 2004; Pridmore et al.,
2004). Analysis of the L. plantarum genome revealed a variety of transporters, suggesting
a broad capacity to adapt to varying environmental conditions (Kleerebezem et al., 2003).
In particular, a “lifestyle adaptation island” bearing genes involved in sugar transport and
metabolism was defined on the chromosome (Kleerebezem et al., 2003). Similarly, the
diversity of transporters in S. mutans and S. pneumoniae have been associated with an
increased ability to utilize nutrient sources present in their environments, namely the oral
cavity and respiratory tract (Tettelin et al., 2001; Ajdic et al., 2002). The L. acidophilus
NCFM genome was also recently determined, and further substantiates these
observations (Altermann et al., 2004). Early analyses indicate that the genome contents of
bifidobacteria and lactobacilli reflect their habitats, particularly with regards to transport
systems able to utilize a variety of carbohydrates. In silico analyses of the genes encoded
in these genomes provide insight as to their fermentative and uptake capabilities. In
particular, a variety of putative carbohydrate transporters have been identified, suggesting
a wide saccharolytic potential for most of these microbes, especially with regards to
mono- and di-saccharides. However, most of the substrates for ABC transporters, and
some of the substrates for PTS transporters remain unknown (Altermann et al., 2004).
This is not uncommon, since a large portion of the content of microbial genomes remains
obscure, even for model organisms, consisting of unknown ORFs and conserved genes
encoding hypothetical proteins.
Within LAB. a diverse saccharolytic potential has previously been associated with
microbial ability to establish residency in specific environmental niches, in particular
adaptation of Bifidobacterium longum to the human gastro-intestinal tract (GIT) (Schell
et al., 2002), cariogenic activity of Streptococcus mutans in the oral cavity
(Vadeboncoeur and Pelletier, 1997; Ajdic et al., 2002), and the incidence of Lactobacillus
plantarum in a variety of environmental niches (Kleerebezem et al., 2003). Perhaps a
diverse catabolic potential is derived from environmental pressures, in response to
competition for scarce nutrients in the intestinal ecosystem (Schell et al., 2002;
Barrangou et al., 2003) and in the mouth cavity (Vadeboncoeur and Pelletier, 1997; Ajdic
et al., 2002). Although energy sources in the environment are vital for survival, the
capacity to uptake them efficiently can result in a competitive advantage. Therefore,
understanding the transportomes of microbes is expected to provide insight into their
respective abilities to survive and compete within their natural habitats. The classification
of transporter families encoded within a genome, and the identification of the uptake
systems provides a platform for understanding which resources are used by a specific
microorganism. Although there are only a few families of transporters well characterized
in prokaryotes, within each family, there are a diverse number of uptake systems with
varying substrate specificities.
This overview will describe the main families of transporters identified in lactic
acid bacteria, categorize within each family the uptake systems that are well
characterized, and investigate the diversity of transport capabilities within and between
organisms. Specifically, the capability of LAB to utilize a variety of carbohydrates via
the PTS and ABC transporter super families of transporters will be reviewed.
1.4 Fermentation capabilities of lactic acid bacteria
There are different means by which carbohydrates are utilized by bacteria: either
they are hydrolyzed outside of the cell into readily fermentable sugars and transported
into the cell thereafter, or they are transported into the cell and then catabolized. Either
way, carbohydrates have to be transported into the cell in order to be catabolized and
used as an energy source.
Although early genome analyses of LAB genomes have specifically looked at
utilization of carbohydrates, the actual substrates for the majority of the transporters
identified remain unknown. Additionally, the classification of transporters into specific
families, and attribution of a specific substrate, derived from in silico analyses, remains
largely putative. Nevertheless, the comparison of both fermentation patterns and genomic
content provides substantial insight into the transport abilities of LAB.
The fermentation profiles for L. acidophilus, L. johnsonii, L. gasseri and L.
plantarum are shown in Table 2. Additionally, detailed transporter annotations are
available for S. mutans, S. pneumoniae, L. lactis and L. acidophilus (Table 2). It appears
that most LAB have the ability to utilize a variety of mono- and di-saccharides,
specifically, hexoses such as fructose, glucose, galactose and mannose, and disaccharides
such as cellobiose, lactose, maltose, sucrose and trehalose (Table 1). In contrast,
utilization discrepancies are observed between LAB for pentoses, oligosaccharides, sugar
alcohols, deoxysugars and modified sugars (Table 2). Globally, it appears that LAB are
specialized for utilization of hexoses and disaccharides, and select species have gained
the ability to utilize more complex carbohydrates individually. This is consistent with
previous findings in LAB suggesting that L. plantarum, L. johnsonii and L. lactis appear
to ferment mainly mono-, di- and tri-saccharides (Siezen et al., 2004).
For the intestinal LAB, a limited number of species have the ability to transport
undigested complex carbohydrates, including prebiotics. Prebiotics are defined as
“non-digestible substances that provide a beneficial physiological effect on the host by
selectively stimulating the favorable growth or activity of a limited number of indigenous
bacteria” (Reid et al., 2003). These compounds include non-digestible plant
oligosaccharides such as FOS and raffinose (Van Laere et al., 2000; Rycroft et al., 2001).
Among LAB, L. acidophilus, L. plantarum, L. casei, S. thermophilus and a variety of
bifidobacteriahave the ability to utilize FOS (Kaplan and Hutkins, 2000).
There are three primary families of transporters in LAB that have been identified
for sugar transport: (i) secondary active transport via the major facilitator superfamily
(MFS); (ii) the phosphoenolpyruvate transferase system (PTS); and (iii) the ATP binding
cassette (ABC) transport system (Paulsen et al., 1998; Paulsen et al., 2000; Saier, 2000;
Kaplan and Hutkins, 2003).
1.5 ABC transporters
The ABC superfamily (TC #3.1) is a diverse family of transporters which include
both inwardly importers and outwardly exporters (Saier, 2000; Davidson and Chen,
2004), whereby substrate translocation is coupled with adenosine tri phosphate (ATP)
hydrolysis (Locher et al., 2002). TC numbers represent categories of the Transport
Commission classification (Saier, 2000). ABC transporters are a dominant transporter
superfamily in bacteria (Paulsen et al., 2000), and, they are the most abundant class of
primary transport systems in lactic acid bacteria (Poolman, 2002). ABC transporters are
the most dominant transporter family in L. plantarum, wherein 57 complete systems were
annotated (Kleerebezem et al., 2003), in S. mutans where over 60 ABC transporters are
hypothetically present (Ajdic et al., 2002) and in S. pneumoniae where over 30% of
transporters are predicted to be sugar transporters. Although ABC transporters recognize
a variety of substrates, in LAB, ABC uptake transporters primarily recognize
carbohydrates. In contrast, in B. longum, most of the 25 ABC transporters seem to have
specificity for oligopeptides and amino acids (Schell et al., 2003). For most LAB,
members of the ATP binding cassette (ABC) family of transporters include uptake
proteins identified primarily for the transport of mono-, di-, tri- and poly- saccharides.
Specifically, ABC transporters have been characterized for the transport of maltose,
trehalose, lactose, arabinose, ribose, glucose, fucose, raffinose, and a variety of peptides.
ABC transporters usually consist of several subunits, namely the nucleotide
binding domains (NBDs), the membrane spanning domains (MSDs), and substrate
binding proteins (SBPs) (Quentin et al., 1999; Braibant et al., 2000; Poolman, 2002). The
minimum “complete” ABC transporter must include both a nucleotide binding domain
and a membrane spanning domain. Importers are usually pentameric, including two
NBDs, two MSDs and one SBP, whereas exporters are tetrameric, including two NBSs
and two MSDs (Braibant et al., 2000) (Figure 2).
There are many sub-families of ABC transporters, which are classified by the
nature of the substrate being translocated, including peptides, amino-acids, drugs,
antibiotics, iron, ions, and carbohydrates (Braibant et al., 2000). For importers, ABC
transporters involved in the uptake of carbohydrates are a key sub-family. Specifically,
most carbohydrate ABC transporters are similar to MalEFGK (Paulsen et al., 2000),
whereby MalE is a periplasmic substrate/solute binding protein (pfam 01547), MalFG are
two membrane-spanning permeases (pfam 00528), and MalK is a cytoplasmic
nucleotide-binding protein (pfam 00005), characteristic of the four subunits of a typical
ABC transport system (Quentin et al., 1999). In prokaryotes, the various elements of
ABC transporters are usually encoded by genes in the same operon, or locus, as
illustrated by the malEFGK and msmEFGK operons (Russel et al., 1992; Quentin et al.,
1999; Braibant et al., 2000; Barrangou et al., 2003).An anchoring motif similar to LPxTG
is usually present at the N-terminus of the substrate binding protein, allowing attachment
of this protein to the cell wall via a hydrophobic lipid extension (Quentin et al., 1999;
Braibant et al., 2000). However, this anchoring motif can vary between organisms, as
shown in L. plantarum, where the anchoring consensus sequence is LPQTxE
(Kleerebezem et al., 2003). Each permease usually contains four to eight transmembrane
α-helices, with most MSDs containing six trans-membrane regions (Table 3, Figure 3),
which form a trans-membrane channel allowing transport of the substrate across the
membrane, into the cell cytoplasm.
For the nucleotide binding protein, which is responsible for the hydrolysis of ATP
associated with transport of each molecule into the cell, there are several well conserved
motifs typical of ABC transporters. Specifically, genome-wide analyses of ABC
transporters in prokaryotes have shown that four motifs within the NBDs are highly
conserved between and within species, namely: Walker A (P loop), Walker B, Linton and
Higgins, and the ABC signature sequence (Linton and Higgins, 1998; Quentin et al.,
1999; Braibant et al., 2000; Locher et al., 2001; Davidson and Chen, 2004). The Walker
A motif has a GxxGxGKST / [AG]xxxxGK[ST] consensus, Walker B has a hhhhDEPT /
DExxxxxD consensus, the Linton and Higgins has a hhhhH+/- consensus, and ABC
signature sequence has a LSGG / LSGGQ consensus, whereby h and +/- represent
hydrophobic and charged residues, respectively (Linton and Higgins, 1998; Quentin et
al., 1999; Braibant et al., 2000; Davidson and Chen, 2004).
Perhaps the best characterized sugar ABC transporter in LAB is the MsmEFGK
transport system (Russell et al., 1992). It was originally described in S. mutans (Russell et
al., 1992; McLaughlin et al., 1996), and homologs were found in S. pneumoniae
(Rosenow et al., 1999) and L. acidophilus (Barrangou et al., 2003; Altermann et al.,
2004). MsmEFGK is involved in uptake of multiple sugars, including melibiose,
raffinose, isomaltotriose and FOS (Russell et al., 1992; Barrangou et al., 2003; Kaplan
and Hutkins, 2003).
Also, in B. longum, MalEFGK-like ABC transporters seem to be involved in the
transport of plant oligosaccharides such as arabinoglycans and arabinoxylans, which is
consistent with the presence of endoarabinosidases and endoxylanases in the genome
(Schell et al., 2003). A similar combination has also been found in E. faecalis (Paulsen et
al., 2003).
Most exporters are involved in transport of components toxic to the cell, such as
drugs and antibiotics (Poolman, 2002), whereas most importers are involved in transport
of energy sources and building blocks. Multidrug ABC transporters are commonly found
in LAB genomes. In particular, the LmrA multidrug ABC transporter has been well
characterized in Lactococcus lactis (van Veen et al., 1999). LmrA has the ability to
export anthracyclines, vinca-alkaloids, antibiotics and cytotoxic agents such as ethydium
bromide (van Veen et al., 1999). Multidrug ABC transporters are part of the mechanisms
developed by microbes in response to the occurrence of toxic compounds in their natural
habitats.
Overall, ABC transporters involved in carbohydrate uptake seem to have affinity
primarily for tri- and poly-saccharides. The substrate specificity is determined by the
substrate binding protein, although one specific SBP can recognize more than one
substrate, as illustrated by the msm operon in S. mutans (Russell et al., 1992). In
environments whereby tri- and poly-saccharides are present, such as the lower
gastro-intestinal tract, ABC transport systems are expected to provide a competitive advantage
by expanding the organism’s access to the pool of available substrates.
1.6 PTS transporters
Members of the phosphoenolpyruvate:sugar phosphotransferase system family of
transporters include uptake proteins identified primarily for the transport of mono- and
di-saccharides. The PTS is characterized by a phosphate transfer cascade involving
phosphoenolpyruvate (PEP), enzyme I (EI), HPr, and various EIIABCs, whereby a
phosphate originating from PEP is ultimately transferred to the carbohydrate substrate
(Vadeboncoeur and Pelletier, 1997; Siebold et al., 2001; Poolman, 2002; Warner and
Lolkema, 2003). Specifically, PTS transporters (TC # 4.1 - 4.6) have been characterized
for the transport of glucose, mannose, fructose, cellobiose, sucrose, trehalose (Table 2). It
was previously suggested the PTS system is the primary sugar transport system of
Gram-positive bacteria (Ajdic et al., 2002; Warner and Lolkema, 2003). Although PTS
transporters are not found in archaea or eukarya, they are present in most bacteria
(Paulsen et al., 2000; Saier, 2000). The PTS consists of three (EIIA, B and C) or four
domains (EIIA, B, C and D) (Saier and Reizer, 1992). The hydrophilic chains bearing the
first and second phosphorylation sites are EIIA and EIIB, respectively, while the
transmembrane channel and sugar binding site consist of EIIC (Saier and Reizer, 1998).
The number of predicted transmembrane spanning domain is usually 10 in PTS
transporters (Table 3, Figure 3), which is different from ABC transporters. When
applicable, EIID is the hydrophobic protein of the splinter group (Saier and Reizer,
1992). The range and specificity of substrates transported by each PTS transporter is
determined by the range of the EII complex.
In streptococci, PTS transporters are important in carbohydrate uptake and
regulation (Vadeboncoeur and Pelletier, 1997). Specifically, in Streptococcus salivarius,
Streptococcus mutans and Streptococcus sobrinus, PTS transporters involved in uptake of
a variety of mono- and di- saccharides have been identified (Vadeboncoeur and Pelletier,
1997). In contrast, only one PTS transporter is present in B. longum (Schell et al., 2003).
In lactobacilli, a variety of PTS transporters have been identified, including 13, 16
and 25 complete PTS transporters in L. lactis, L. johnsonii and L. plantarum, respectively
(Bolotin et al., 1999; Schell et al., 2003; Kleerebezem et al., 2003). In streptococci, a
variety of PTS transporters have also been identified, including 21 complete PTS
transporters in S. pneumoniae (Tettelin et al., 2001).
Since there is a correlation between the genomic association of genes and
functional interaction of the proteins they encode (Snel et al, 2002), catabolic enzymes
are expected to be encoded in the vicinity of the genes encoding transporters of their
substrates. Similarly, transcriptional regulators are also commonly found in the vicinity
of the genes they control. As a result, for carbohydrate loci, transcriptional regulators,
transporters and sugar hydrolases are usually found in operons and loci.
Perhaps the best characterized PTS transporters in LAB are the sucrose and
glucose/mannose transport systems (Luesink et al., 1999b; Cochu et al., 2003). The
sucrose PTS locus has been described in L. lactis (Luesink et al., 1999b), L. plantarum
(Naumoff and Livshits, 2001) and P. pentosaceus (Naumoff and Livshits, 2001). The
glucose/mannose PTS EIIABCDMan transporter was recently characterized in S.
thermophilus (Cochu et al., 2003). Both PTS transporters have also been found in
recently sequenced LAB genomes (see table 2).
A number of lactic acid bacteria uptake glucose and mannose via a PTS
transporter. Specifically, the EIIMan PTS transporter has the ability to uptake both
mannose and glucose (Cochu et al., 2003). The glucose-mannose PTS transporter has
been well characterized in S. thermophilus (Cochu et al., 2003). The glucose PTS has
been identified in a variety of streptococci, namely S. mutans, S. sobrinus and S.
thermophilus (Vadeboncoeur and Pelletier, 1997).
Several PTS loci have been well characterized in LAB, especially the glucose,
fructose and sucrose loci, which contain the mannose / glucose PTS transporter
EIIABCDMan, the fructose PTS transporter EIIABCFru, and the sucrose PTS transporter
EIIBCASuc. Additionally, the trehalose locus, including a trehalose PTS transporter
EIIABCTre has been well characterized in L acidophilus (Duong et al., 2004). Putative
PTS transporters have also been identified in a variety of LAB (Table 2), but the
annotation is based on similarity to other non-LAB transporters, and most have not been
substantiated by functional analyses. In streptococci, PTS activity has been shown for
glucose, fructose, mannose, lactose, mannitol, sorbitol, maltose, sucrose, trehalose, and
xylitol (Vadeboncoeur and Pelletier, 1997).
Overall, PTS transporters involved in carbohydrate uptake appear to have affinity
primarily for mono- and di-saccharides. The substrate specificity is determined by the
EIIA, EIID or EIIC substrate binding protein, although one specific SBP can recognize
more than one substrate, as illustrated by the mannose / glucose EIIABCDMan. In
environments whereby mono- and di- saccharides are present, such as the upper
gastrointestinal tract, PTS transport systems might provide efficient carbohydrate
utilization and potentially a competitive advantage.
1.7 Other transporters
Lactic acid bacteria possess a variety of transport systems (Saier, 2000; Konings,
2002). In addition to ABC and PTS transporters, the main transporter families include the
F0F1 ATPase, the uniport / symport / antiport systems, and the protein secretion / export
system (Konings, 2002; Figure 2).
The secondary transport system, including uniport / symport / antiport complexes
is involved primarily in transport of amino acids, ions, and acids (Konings, 2002).
Specifically, amino-acid transporters have been well characterized in Lactococcus lactis
(Bolotin et al., 1999; Konings, 2002).
The F0F1-ATPase (TC 3.1, Paulsen et al., 1998) has been well characterized in
several LAB, including lactobacilli (Kullen and Klaenhammer, 1999; Sievers et al.,
2003), bifidobacteria (Ventura et al., 2004), oenococci (Sievers et al., 2003), pediococci
(Seivers et al., 2003) and Lactococcus lactis (Bolotin et al., 1999; Konings, 2002). The
operon has been well characterized in L. acidophilus (Kullen and Klaenhammer, 1999)
and B. lactis (Ventura et al., 2004), whereby atpBEFHAGDC encode the a, c, b, δ, α, γ,
β, and ε subunits of the F0F1-ATPase, respectively. This transport system is an important
element in the response and tolerance to low pH, which is instrumental for resistance to
acid stress in the human gastrointestinal tract. This is another typical example of how
genomes of intestinal microbes include specific transporters which allow them to exist in
various environments. Similarly, members of the Oenococcus and Leuconostoc genera
used in wine fermentation also have a F0F1-ATPase which confers resistance to low pH
(Sievers et al., 2003).
The major facilitator superfamily (MFS) includes a variety of transporters (TC
#2.1-2.2). Specifically, the glycoside-pentoside-hexuronide (GPH):cation symporter
family is associated with transport of carbohydrates, including galactosides (Saier, 2000).
This family includes 12 transmembrane domains (Saier, 2000), which is different from
PTS transporters, and similar to the two combined MSDs in each ABC transporter (Table
3, Figure 3).
With regard to drug resistance, in addition to multidrug ABC transporters, LAB
have also developed secondary transporters which export drugs and toxic compounds
(van Veen et al., 1999). Specifically, in L. lactis, LmrP mediates the extrusion of drugs,
such as antibiotics, in antiport with protons (van Veen et al., 1999). This system is also
part of the major facilitator superfamily (Saier, 2000).
Member of the LacS subfamily of galactoside-pentose-hexoronide subfamily of
translocators have been identified for the uptake of lactose and galactose in Lactobacillus
bulgaricus (Leong-Morgenthaler et al., 1991), Leuconostoc lactis (Vaughan et al., 1996),
S. thermophilus (van den Bogaard et al., 2000; Vaillancourt et al., 2002), S. salivarius
(Vaillancourt et al., 2002; Lessard et al., 2003), L. delbrueckii (Lapierre et al., 2002) and
L. helveticus (Fortina et al., 2003). A similar GPH transporter, LacY, is present in L.
lactis (Bolotin et al., 1999).
Although LacS contains a PTS EIIA at the carboxy-terminus, towards the
cytoplasmic side of the protein (Vaughan et al., 1996; Lessard et al., 2003), it is not a
member of the PTS family of transporters. Also, LacS contains 12 transmembrane
domains, which differs from PTS transporters (Table 3, Figure 3). LacS has been reported
to have the ability to take up both galactose and lactose in select organisms (Vaughan et
al., 1996; van den Bogaart et al., 2000), although the specificity varies between
organisms, and depends on the presence of alternative galactoside transporters in the
organism.
A LacS-LacY homolog was also identified in L. brevis (Djordjevic et al., 2001).
Although it is a member of the GPH family (TC # 2.2), it did not include a PTS IIA
domain, indicating dependence upon a different regulatory network than that of the PTS
and other GPH transporters (Djordjevic et al., 2001).
The gene encoding the GPH transporter is usually associated with ORFs encoding
enzymes involved in the metabolism of galactosides. Specifically, saccharolytic enzymes
likely involved in the metabolism of galactosides include the enzymatic machinery of the
Leloir pathway, although operon organization is variable and unstable among LAB
(Lapierre et al., 2002; Vaillancourt et al., 2002; Boucher et al., 2003; Fortina et al., 2003;
Grossiord et al., 2003; Pridmore et al., 2004). The Leloir pathway allows catabolism of
both lactose and galactose into substrates of glycolysis (Grossiord et al, 2003).
Alternatively, the tagatose pathway may also metabolize galactosides (de Vos, 1996;
Boels et al., 2003).
1.8 Regulation and carbon catabolite repression
To understand how microbes utilize carbohydrates, we must determine the genetic
and biochemical bases for sugar transport into the cell, and identify the regulatory
networks involved in transcription of genes encoding transporters. Carbohydrate transport
and catabolism are well orchestrated in LAB, so as to utilize carbohydrate sources
optimally. The regulatory mechanism for global carbohydrate utilization is carbon
catabolite repression (CCR).
Carbon catabolite repression (CCR) is a mechanism widely distributed amongst
Gram-positive bacteria, usually mediated in cis by catabolite response elements (cre)
(Weickert and Chambliss, 1990; Miwa et al., 2000), and in trans by repressors of the
LacI family, responsible for transcriptional repression of genes encoding unnecessary
saccharolytic components (Weickert and Chambliss, 1990; Viana et al., 2000;
Muscariello et al, 2001; Titgemeyer and Hillen, 2002; Warner and Lolkema, 2003). Cre
sequences (Weickert and Chambliss, 1990) are well conserved amongst Gram-positive
bacteria and found in most LAB in the promoter-operator of many genes involved in
carbohydrate utilization (Barrangou et al., 2003), including: L. plantarum (Muscariello et
al., 2001), L. pentosus (Mahr et al., 2000). CCR controls transcription of proteins
involved in transport and catabolism of carbohydrates (Miwa et al., 2000), as to
transcribe genes encoding the transport and enzymatic machinery of a particular
substrate, exclusively when it is present in the environment. This regulatory system
allows cells to coordinate the utilization of diverse carbohydrates, as to focus primarily
on preferred energy sources (Poolman, 2002). Understanding carbon catabolite repression
is critical to describing how microbes adapt their uptake machinery to changing nutrients
in their environment.
CCR is able to control both PTS, ABC and GPH transporters. Specifically, ABC
transporters of the MsmEFGK family have been shown to be repressed by glucose in a
manner consistent with CCR, in S. pneumoniae (Rosenow et al., 1999; Barrangou et al.,
2003). Similarly, genes of the galactose operon seem to be regulated via CCR in S.
salivarius (Vaillancourt et al., 2002).
The L. acidophilus genome encodes a large variety of genes related to
carbohydrate utilization. In particular, many members of the ABC and PTS families of
transporters were found. Additionally, the members of the general carbohydrate
utilization regulatory network were identified, namely HPr (ptsH), E1 (ptsI), CcpA
(ccpA) and HPrK/P (ptsK). Similarly, all those genes were identified in S. pneumoniae
(Tettelin et al., 2001). Those genes are involved in an active regulatory network based on
sugar availability. The regulatory networks involved in sugar utilization are not well
documented in lactobacilli and bifidobacteria, whereas they have been characterized in
streptococci (Vadeboncoeur and Pelletier, 1997). Nevertheless, previous work has
indicated involvement of CcpA in repression of specific operons in L. casei, and L.
plantarum (Viana et al., 2000; Muscariello et al., 2001) and L. pentosus (Mahr et al.,
2000). Specifically, the pepQ-ccpA locus has been identified in L. pentosus, L.
delbrueckii, L. casei, S. mutans and L. lactis (Mahr et al., 2000), and in most cases, a cre
sequence is found in the promoter-operator region of ccpA. The PTS is characterized by a
phosphate transfer cascade involving PEP, EI, HPr, and various EIIABCs, whereby a
phosphate is ultimately transferred to the carbohydrate substrate (Saier, 2000; Titgemeyer
and Hillen, 2002; Warner and Lolkema, 2003). HPr is a key component of CCR, which is
regulated via phosphorylation by enzyme I (EI) and HPr kinase/phosphatase (HPr K/P).
While HPr is the primary regulator of CCR, HPr K/P is the sensor enzyme of CCR in
Gram positive bacteria (Nessler, et al., 2003). HPrK/P has been found in a variety of
LAB, including L. casei, L. brevis, L. delbrueckii, L. gasseri, L. acidophilus, L. lactis,
Streptococcus bovis, S. mutans, S. salivarius, S. pneumoniae, S. pyogenes, S. agalactiae
and Leuconostoc mesenteroides (Warner and Lolkema, 2003; Altermann et al., 2004).
Similarly, HPr has also been found in a variety of LAB, including L. casei, L. sakei, L.
acidophilus, L. gasseri, L. brevis, L. mesenteroides, L. lactis. E. Faecalis, S. mutans, S.
salivarius, S. bovis, S. pyogenes, S. pneumoniae, S. thermophilus, S. agalactiae and
Oenococcus oeni (Warner and Lolkema, 2003; Altermann et al., 2004). The HPr-HPrK/P
complex has been characterized structurally (Fieulaine et al., 2002). When HPr is
phosphorylated at His15, the PTS is on (Poolman, 2002), and carbohydrates transported
via the PTS are phosphorylated via EIIABCs. In contrast, when HPr is phosphorylated at
Ser46, the PTS machinery is not functional (Vadeboncoeur and Pelletier, 1997;
Mijakovic et al., 2002,; Nessler et al., 2003). HPr-Ser46 acts as a co-repressor by binding
to CcpA (Fieulaine et al., 2002; Nessler et al., 2003). Ultimately, CcpA binds to cre
sequences in the promoter-operator region of operons encoding carbohydrate transporters
and hydrolases, and prevents their transcription (Hueck and Hillen, 1995; Poolman,
2002).
HPr has been identified in E. faecalis (Vadeboncoeur and Pelletier, 1997), S.
pyogenes (Deutscher and Saier, 1983; Vadeboncoeur and Pelletier, 1997), and L. lactis
(Luesink et al., 1999a).
CcpA-dependent repression and activation is well documented in a variety of
LAB, including enterococci, lactobacilli, lactococci and streptococci, especially with
regard to repression of the genes involved in utilization of galactosides (Titgemeyer and
Hillen, 2002).
The interaction between HPr and LacS has been shown in S. salivarius (Lessard et
al., 2003). It happens between HPr-His and EIIALacS, although LacS is not a member of
the PTS system. Since HPr is the primary regulator of CCR, the interaction between HPr
and LacS illustrates the likely regulation of the GPH system by CCR. In S. thermophilus,
the control of LacS by CCR has been illustrated, likely via interaction between CcpA and
two cre sequences found in the promoter-operator region of the lacSZ. Operon (van den
Bogaard et al., 2000).
Although the phosphorylation cascade suggests regulation at the protein level,
studies in LAB report both transcriptional modulation and constitutive expression of
ccpA and ptsHI. Specifically, in S. thermophilus, CcpA production is induced by glucose
(can den Bogaard, 2000). Similarly, in other bacteria, the carbohydrate source modulates
ptsHI transcriptional levels (Luesink et al., 1999a). In contrast, expression levels of ccpA
in L. pentosus (Mahr et al., 2000) and of ptsHI in S. thermophilus (Cochu et al., 2003) did
not vary in the presence of different carbohydrates.
Carbon catabolite repression is likely present in L. acidophilus, since all the
necessary regulatory proteins are encoded within its genome, cre-like sequences are
present in the promoter-operator regions of several carbohydrate loci (Barrangou et al.,
2003), and transcription of operons involved in utilization of non-preferred carbohydrates
is repressed by glucose (Barrangou et al., 2003).
Carbon catabolite repression illustrates how lactic acid bacteria adapt dynamically
to the diverse carbohydrate sources available in their various habitats.
1.9 Conclusions and perspectives
Although a variety of putative carbohydrate transporters have been identified in
LAB genomes recently published, little information is available regarding their biological
functions and expression profiles. Specifically, the substrate specificity of most PTS and
ABC transporters remains unclear, as illustrated in the incomplete annotation of most
PTS transporters in L. plantarum, L. acidophilus, L. johnsonii and S. pneumoniae
(Kleerebezem, 2003; Altermann, 2004; Schell et al., 2003; Tettelin et al., 2001). As a
result, in silico analyses must be confirmed and complemented by transcriptional and
biological analyses.
Surveys of carbohydrate uptake systems revealed greater diversity in prokaryotes
than eukaryotes. Specifically, eukaryotic carbohydrate transport is dominated by the
MFS, whereas that of prokaryotes involved both the MFS, PTS and ABC superfamilies
of transporters (Saier, 2000).
Recent advances in high throughput technologies, primarily genome sequencing
and microarrays have yielded global data that provide insight into the physiology of
microbes. Particularly, LAB genome analyses have illustrated the breadth and importance
of carbohydrate transporters in lactobacilli and bifidobacteria. Similarly, global
transcriptome analyses, similar to those carried out in Escherichia coli (Beloin et al.,
2004), Bacillus subtilis (Blencke et al., 2003), Vibrio cholerae (Meibom et al., 2003),
Thermotoga maritima (Chhabra et al., 2003; Pysz et al., 2004a; Pysz et al., 2004b) and
Pyrococcus furiosus (Shockley et al., 2003), applied to carbohydrate utilization
investigation in LAB will provide further insight into the transporters and metabolic
pathways involved in adaptation of LAB to their various environmental conditions.
Ultimately, genetic engineering of LAB could allow development of better starter
cultures and probiotic strains, optimized for utilization of specific carbohydrate sources,
and competition with other commensals. Genetic engineering in LAB is now possible,
following the development of molecular biology tools, including food-grade systems (de
Vos, 1996; Russell and Klaenhammer, 1998; Boucher et al., 2002; Kleerebezem and
Hugenholtz, 2003).
Overall, the combination of a diverse saccharolytic enzymatic machinery with a
polyvalent transport system, consisting primarily of ABC and PTS transporters, allows
lactic acid bacteria to utilize a variety of nutrient resources efficiently and dynamically
adapt its transcriptome to environmental conditions, ultimately rending these microbes
more competitive in their respective environments.
1.10 References
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