CONSTRUCTION OF A SECRETOME MUTANT LIBRARY
OF LACTOBACILLUS REUTERI
By
Javiera Ortiz Villalobos

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Comparative Medicine and Integrative Biology - Master of Science
2013

ABSTRACT
CONSTUCTION OF A MUTANT LIBRARY OF LACTOBACILLUS REUTERI
By
Javiera Ortiz Villalobos
During the last 10 years increased scientific attention has been given to probiotics due
to their promising therapeutic benefits in enteric diseases. In this sense, Lactobacillus reuteri
6475 has emerged as a potential probiotic strain that has demonstrated various probiotic
therapeutic features. These characteristics include the ability of this strain to produce and
secrete the antimicrobial compound reuterin and anti-inflammatory factors.

Because the main probiotic effects of L. reuteri 6475 that have been identified thus far are
secreted, we were interested in developing a mutant library consisting of disruptions of genes
that encode for secreted or cell-wall proteins. To complete this task we employed two different
strategies: the identification of secretome protein sequences by predictive mathematical
methods and the disruption of the protein coding sequences by single-stranded DNA
recombineering. As a result, we have developed a mutant library of 127 secretome genes that
would drive the elucidation of important L. reuteri probiotic mechanisms of action. To
demonstrate the utility of this library, I screened all 127 mutants for the ability to produce
reuterin and found 11 genes that increase reuterin secretion and 3 that eliminate reuterin
secretion when disrupted. Future characterization of these genes will further elucidate the
bacterial pathways that are critical for reuterin production.

Copyright by
JAVIERA ORTIZ VILLALOBOS
2013

This thesis is dedicated to Lyonel, my love, my best friend and my biggest support

iv

ACKOWLEDGEMENTS

First, I would like to acknowledge my mentor Dr. Robert Britton for giving me the opportunity
to join his laboratory, for his advice and help to become a better researcher. Dr Britton has
been a fantastic mentor and I am confident that we will share more experiences and projects
during my future as a PhD student.

Secondly, I would like to thank the professors of my committee thesis: Dr. Shannon Manning,
Dr. Martha Mulks and Dr. Christopher Waters, for all their contributions and suggestions during
the elaboration of my thesis. All of them have been of great help and value.

In third place, I would like to thank Dr. Vilma Yuzbasiyan-Gurkan for helping me during these
last two years. Dr. Vilma has been an extraordinary Director of the Comparative Medicine and
Integrative Program and a wonderful professor. I appreciate very much everything that she has
done to support me, especially during my first months in MSU.

I also would like to thank all the members of the Britton’s Lab for all their help and the great
atmosphere that we have in the laboratory. In particular, I would like to acknowledge my lab
mate and one of my best friends Laura Ortiz. I was extremely lucky to found her in the lab.
Laura is a great co-worker and an incredible friend. Thank you for be there when I most need it
and thank you for all the good moments.

v

I would acknowledge my family for all their love. Although all of them are far away, they have
been present in all this process, wishing me success and cheering up for me.

And finally, I would like to thank my husband Lyonel. Without him anything would have been
possible. You have made me believe that everything is possible and you have made me a better
person. Thank you for all your patience, advice and love. You have been the best partner that I
could ask.

vi

TABLE OF CONTENTS

LIST OF TABLES ................................................................................................................................ ix
LIST OF FIGURES ............................................................................................................................... x
CHAPTER 1: THE PROBIOTIC FEATURES OF LACTOBACILLUS REUTERI
INTRODUCTION ............................................................................................................................... 1
CHARACTERISTICS OF LACTOBACILLUS REUTERI ............................................................................ 3
PREVALENCE ............................................................................................................................. 3
SYMBIOSIS AND COEVOLUTION................................................................................................ 4
MECHANISMS OF ACTION......................................................................................................... 5
Regulation and stimulation of the immune system ........................................................ 6
Anti-inflammatory activity ............................................................................................... 8
Production of antimicrobial compounds ....................................................................... 10
Competitive exclusion.................................................................................................... 11
Enhancement of the epithelial barrier .......................................................................... 12
PROBIOTIC EFFECTS OF LACTOBACILLUS REUTERI IN DISEASE............................................... 14
Diarrhea ......................................................................................................................... 15
Colic ................................................................................................................................ 15
Inflammatory Bowel Disease (IBD) ............................................................................... 16
Allergy ............................................................................................................................ 16
THE PROBIOTIC POTENTIAL OF LACTOBACILLUS REUTERI 6475 .................................................. 18
REFERENCES .................................................................................................................................. 20
CHAPTER 2: CONSTRUCTION OF A SECRETOME MUTANT LIBRARY OF LACTOBACILLUS REUTERI
6475
INTRODUCTION ............................................................................................................................. 28
MATERIALS AND METHODS .......................................................................................................... 33
SIGNALP: A PREDICTIVE SECRETOME TOOL............................................................................ 33
SINGLE-STRANDED DNA RECOBINEERING ............................................................................. 33
RESULTS......................................................................................................................................... 37
DISCUSSION................................................................................................................................... 42
SUPPLEMENTAL INFORMATION ................................................................................................... 47
REFERENCES .................................................................................................................................. 62
CHAPTER 3: EMPLOYING LACTOBACILLUS REUTERI 6475 MUTANT LIBRARY TO SCREEN FOR
GENES THAT ENCODE PROTEINS INVOLVED IN REUTERIN PRODUCTION AND SECRETION
INTRODUCTION ............................................................................................................................. 65
MATERIALS AND METHODS .......................................................................................................... 67
RESULTS......................................................................................................................................... 69
DISCUSSION................................................................................................................................... 74
vii

SUPPLEMENTAL INFORMATION ................................................................................................... 78
REFERENCES .................................................................................................................................. 80
CHAPTER 4: SUMMARY AND SIGNIFICANCE ............................................................................... 83
REFERENCES .................................................................................................................................. 86

viii

LIST OF TABLES

Table 1.1. Mechanisms of action of Lactobacillus reuteri .................................................................. 13
Table 2.1. Predicted secretome genes that were mutated employing single-stranded DNA
recombineering ........................................................................................................................................ 47
Table 2.2. Predicted secretome genes that fail recombineering....................................................... 57
Table 3.1. Reuterin values estimated using a cuvette-colorimetric reuterin assay ........................ 72
Table 3.2. Reuterin values estimated using a 96-colorimetric reuterin assay................................. 78

ix

LIST OF FIGURES

Figure 2.1. MAMA-PCR strategy employed to identify secretome mutant L. reuteri 6475
colonies. The figure shows the MAMA-PCR differences between a wild-type and a mixed
genotype strain. In the wild-type strain, the alignment of the forward and the reverse screening
oligonucleotides will generate a unique 1 Kb PCR fragment (light blue circles). Instead, in the
mixed genotype strain the presence of a 5 consecutive mismatch mutation will allow the
alignment of the MAMA screening oligonucleotide. This new condition will generate a 500 bp
PCR fragment (red circles). For interpretation of the references to color in this and all other
figures, the reader is referred to the electronic version of this thesis......................................... 35

Figure 2.2. Identification of pure genotype L. reuteri 6475 mutant strains. In this example, a PCR
was performed in 23 mixed genotype mutant colonies using forward and reverse screening
oligonucleotides to yield a 1 Kb PCR fragment. The same fragments were digested with a
restriction endonuclease enzyme to screen for 500 bp DNA amplicons. In this case, two L.
reuteri 6475 colonies were identified as pure genotype mutant strains. –C: negative control; Kb:
Kilo bases; bp: base pairs. For interpretation of the references to color in this and all other
figures, the reader is referred to the electronic version of this thesis......................................... 36

Figure 2.3. Recombineering rate of gene HMPREF0536_1129. In this case, the recombineering
efficiency value was estimated as 2%, and it was calculated by dividing the number of mutant
colonies by the total number of screened colonies. M: mutant colony ...................................... 38

Figure 2.4. Distribution of secretome disrupted genes in the L. reuteri F275 chromosome. Blue
line: secretome genes that fail recombineering; red, green and purple lines: secretome
disrupted genes with high >5%, medium 1-5% and low <1% recombineering efficiency levels;
ori: origin of replication; ter: termination site of replication. For interpretation of the references
to color in this and all other figures, the reader is referred to the electronic version of this thesis
....................................................................................................................................................... 39

Figure 2.5. Distribution of attempted (red) and mutated (blue) genes per replichore. The
percentage of disrupted genes in the first replichore is 58.3% and in the second replichore is
75%; ori: origin of replication; ter: termination site of replication. Note the origin and terminus
regions of the two replichores are reversed. For interpretation of the references to color in this
and all other figures, the reader is referred to the electronic version of this thesis ................... 40

x

Figure 2.6. Ratio of mutated genes in the L. reuteri 6475 genome every 200 Kb regions. Square
boxes represent the proportion of genes that were mutated out of the total number of
predicted genes every 200 Kb. ori: origin of replication; ter: termination site of replication ..... 41

Figure 3.1. Reuterin values obtained from secretome mutant strains using a cuvette
colorimetric assay. Reuterin values were obtained at an optical density of 560 nm. The mutant
strain reuterin values were adjusted to the weight of the wild-type L. reuteri 6475 cell pellet to
reflect equivalent number of cells. Mutant strains HMPREF0536_1329, 329 and 972 were
included as controls. Error bars represent calculated standard deviations. Experiments were
repeated at least 3 times independently. *p<0.01 versus WT L. reuteri 6475 reuterin values ... 71

xi

CHAPTER 1
THE PROBIOTIC FEATURES OF LACTOBACILLUS REUTERI

INTRODUCTION
The role of probiotics has generated enormous scientific interest due to their ability to
ameliorate several diseases, including inflammatory bowel disease (IBD) and diarrhea (Bäckhed
et al., 2012). Probiotics are defined as “microorganisms which when administered in adequate
amounts confer a health benefit on the host” (FAO/WHO Group, 2002). The intestinal
microbiota plays a fundamental role in the general health status of animals and humans, and
probiotics can act as therapeutic factors producing different molecules in situ which possibly
will regulate the intestinal microbiome (Ringel and Carroll, 2009; Ringel et al., 2012). Some of
the proposed mechanisms of action of probiotics involved the production of antimicrobial
substances, the modulation of the immune system, and the modification of the intestinal
microbiota (Bermudez-Brito et al., 2012; Marco et al., 2006).

Many probiotic strains are members of the Lactic Acid Bacteria (LAB) group. LAB are a group of
low GC Gram-positive organisms which produce lactic acid, carbon dioxide and other organic
compounds as final products of carbohydrate fermentation. Historically, the production of lactic
acid has played a fundamental role in food conservation through the inhibition of bacterial
growth (Battcock and Azam-Ali, 1998).

1

Currently, the LAB taxonomic classification is composed by several family members including
the Lactobacillaceae family, which is represented by more than 100 cultured species (Makarova
and Koonin, 2007). Because of the safe usage of some of these species in food and beverage
products, different organizations have given the GRAS (Generally Recognized As Safe) status to
many of these strains (FAO/WHO Group, 2002; Guarner et al., 2008).

Within that Lactobacillaceae family, the Lactobacillus genus consists of a group of homo or
heterofermentative rod-shape Gram-positive microorganisms, with low GC content,
nonsporulating and facultative anaerobic respiration (Claesson et al., 2007). Some members of
this genus have been observed to be helpful to the improvement of the intestinal function and
the treatment of different human and animals ailments (de Vrese and Schrezenmeir, 2008), and
thus have been considered as probiotic strains.

Among the Lactobacillus genus, Lactobacillus reuteri is one of the species that has been
employed as a microbial therapeutic agent in numerous clinical trials in animals and humans.
Some of the probiotic effects of this bacterium are related to the amelioration of diarrheal and
allergic diseases, the reduction of infant colic, and the regulation of the immune system
(Kumari et al., 2011; Walter et al., 2011).

In order to further the development of probiotics as effective therapeutics for the abovementioned health conditions, new technology is required in LAB to study the proteins that may
have a significant impact in components and mechanisms that promote probiotic effects. To

2

this end single-stranded (ss) recombineering has been successfully implemented in L. reuteri
and other Gram-positive organisms by our laboratory (van Pijkeren and Britton, 2012; van
Pijkeren et al., 2012). The generation of specific mutations among the whole chromosome
without the need for antibiotic selection is one of the most significant characteristics of this
high throughput system and was implemented in the execution of the work presented in this
thesis.

The present review will describe the general physiological and probiotic characteristics of
Lactobacillus reuteri and the unique characteristics of the specific candidate probiotic strain, L.
reuteri ATCC PTA 6475.

CHARACTERISTICS OF LACTOBACILLUS REUTERI

PREVALENCE
Lactobacillus reuteri is a Gram-positive microbe associated with the gastrointestinal (GI) tract of
humans and other vertebrate animals. In humans, the majority of Lactobacillus species are
considered as allochthonous microorganisms, which are transient inhabitants resulting from
food ingestion or from other established gastrointestinal environments in the same host
(Walter, 2005). In general, relatively high numbers of bacteria are found in the lower GI of
11

humans, achieving concentrations of 10

per gram of feces (Suau et al., 1999). Nevertheless,

molecular probes have shown that the Lactobacillus genus represents 0.01% to 0.6% from the
3

total bacterial fecal population and some of the members of this genus could form microbial
communities by intestinal mucus adherence (Dal Bello et al., 2003; Kimura et al., 1997; Sghir et
al., 2000; Tannock et al., 2000; Vélez et al., 2007). In particular, L. reuteri is considered a
putative autochthonous specie with low prevalence, which has been isolated mainly from ileum
and feces (Reuter, 2001). It has been proposed that L. reuteri communities are poorly
established in humans because of their inability to colonize the upper gastrointestinal tract
(extreme acidic conditions and absence of stratified squamous epithelial layers) and to obtain
essential nutrients in the colon since they are previously reabsorbed in the small bowel
(Macfarlane et al., 2000; Walter, 2008).

Particularly, the upper digestive tracts of pigs, rodents and chickens confer the optimal
conditions to L. reuteri growth and biofilm formation due to the availability of nutrients and the
conformation of epithelial surfaces. These conditions make L. reuteri one of the most abundant
isolated species in these animals (Abbas Hilmi et al., 2007; Fuller and Brooker, 1974; Fuller et
al., 1978; Huey-Chai Line and Savage, 1984; Leser et al., 2002).

SYMBIOSIS AND COEVOLUTION
L. reuteri is a microbe that performs specialized functions in order to adapt to different
vertebrates. Different L. reuteri strains, isolated from a variety of hosts, have shown diverse
characteristics regarding the production of antimicrobial factors and enzymes such as urease
(Walter et al., 2011). This suggests that each particular strain has evolved performing specific

4

biological strategies to maintain their ecological niche and long-term coevolution with its host
(Oh et al., 2010; Walter et al., 2011).

In order to investigate the evolutionary strategy of L. reuteri, Oh et al. (2010) performed a
genetic analysis in 165 different L. reuteri strains derived from human, mouse, rat, pig, chicken
and turkey, to investigate the arrangement of phylogenetic groups. Utilizing a population
genetic approach (amplified fragment length polymorphism and MLSA: MultiLocus Sequence
Analysis) the authors showed that different clusters of L. reuteri are formed depending on the
specific host origin. Consequently, the most probable strategy of L. reuteri evolution was driven
by the host environment, resulting in a specialized host-symbiont relationship. It has also been
proposed by the same authors that long-term coevolution bacteria would have a better
performance as probiotic strains. Therefore, host-origin should be considered as a key attribute
regarding probiotic selection.

MECHANISMS OF ACTION
During the last 10 years, several study groups have proposed different probiotic mechanisms of
actions. Most studies have been focused on the interactions that take place between probiotic
strains or their secreted factors, and host-immune elements. These interactions are directly
related to the generation of anti-inflammatory molecules and the regulation of lymphocyte
populations, which would have a relevant role in the control and amelioration of various
intestinal-inflammatory diseases. Other authors have explored the role of antimicrobial
substances produced by different probiotic strains (Höltzel et al., 2000; Jones and Versalovic,

5

2009; Schaefer et al., 2010; Spinler et al., 2008). The production of growth-inhibitory molecules
by lactobacilli plays a fundamental role in the prevention of pathogen invasions and thus can
shape the composition of the intestinal microbiota. Other probiotic functions such as the
enhancement of the epithelial barrier and the competition for binding sites have also been
investigated. All of these probiotic mechanisms will be discussed in the next paragraphs.

Regulation and stimulation of the immune system
The gut-associated lymphoid tissue (GALT) is composed by the Payer’s patches of the small
intestine, the appendix, and the large-intestinal lymphoid follicles. These organ-structures are
the first defense lines to pathogen invasions within the intestine (Murphy et al., 2008). They
also produce different molecules when external stimulus are received, thus playing a crucial
role in epithelial homeostasis (Hoffmann et al., 2008). Different diseases have been described in
the absence of lymphocyte populations or in anomalous immune responses such as Crohn’s
disease and Ulcerative Colitis (UC). Those conditions suggest a scenario in which the role of
probiotics can ameliorate the symptoms of immune disorders.

In general, the number of gut lymphocytes has increased when humans and animals consume
+

lactobacilli daily. Valeur et al. (2004) found enlarged CD4 and B cell populations in ileal and
duodenal histological samples that were collected from humans who received L. reuteri 55730
for 28 days. These results were in concordance with studies from Hoffmann et al. (2008) who
reported a small-intestinal leucocitary infiltration and a transient increase of proinflammatory
cytokines and chemokines after 6 days of L. reuteri 100-23 supplementation in a RLF
6

(reconstituted Lactobacillus-free) mice model. Moreover, these results were reported without
histological lesions, suggesting an intestinal epithelial activation that ensures the homeostasis
of the intestinal microenvironment.
In addition, Livingston et al. (2010) studied the immune-regulatory effects of murine L. reuteri
by exposing BMD (Bone Marrow Dendritic) cells to heat-killed murine L. reuteri 100-23. Under
these circumstances, the BMD cells enhanced their secretion of IL-10 and TGF (Transforming
Growth Factor)-β. IL-10 is an anti-inflammatory cytokine which is considered a crucial factor to
maintain intestinal homeostasis and a negative regulator of NF-κβ (Nuclear Factor Kappa Beta)
(Iyer and Cheng, 2012; Steidler, 2000; Wang et al., 1995). Samples derived from the spleen and
MLN from L. reuteri 100-23 colonized mice had a higher percentage of Treg cells compared to
non-colonized mice. The most plausible explanation of this phenomenon is the stimulating
Treg-growth effect of TGF-β. Regulatory lymphocytes would generate a tolerant environment in
which L. reuteri would interact with intestinal host cells without generating an inflammatory
response, thus allowing the formation of long-term symbiotic-relationships with the host.

A study by Christensen et al. (2002) agreed with the regulatory effects L. reuteri exerts in mouse
DC (dendritic cells) . They showed that L. reuteri down-regulates IL-12 and IL-6 levels compared
+

to other LAB species. These two cytokines drive the development of CD4 Th2 lymphocyte
populations with IFN-ƴ potential secretory activity (Murphy et al., 2008). Besides, L. reuteri
increases the production of IL-10 and reduces the expression of the B7-2 dendritic cell marker,
which is used to identify dendritic mature cells. It has been suggested that the exerted cytokine
effects and the low B7-2 expression could contribute to a quiescent DC state, in which the
7

recognition of lactobacilli components would prime a Th1 or a Th3 development, but not a Th2
response.

Anti-inflammatory activity
Other authors have focused on the beneficial response that L. reuteri produces by diminishing
inflammatory cytokines and interleukins within the intestine. In 2004, Peña et al. identified two
murine Lactobacillus strains with the ability to reduce macrophage TNF-α (Tumor Necrosis
Factor) levels. The clones L. reuteri 6798 and L. paracasei 1602 were tested in an IBD-colitis
model, characterized by IL-10 deficient mice strains in which colitis was exacerbated through
Helicobacter hepaticus infection. Here pre-colonized animals with Lactobacillus reuteri 6798
and Lactobacillus paracasei 1602 were challenged with H. hepaticus and the cecal transcript
levels of IFN (Interferon)-ƴ, IL-4, TNF-α and mucosal IL-12 were assessed. No significant
differences were observed when compared to basal levels of uninfected animals, indicating a
generally inhibitory effect of pro-inflammatory cytokines by lactobacilli. These results were
supported by histological findings reported on female mice. A reduction of inflammation,
hyperplasia and dysplasia was observed in the animals infected with L. paracasei 1602/L. reuteri
6798 prior H. hepaticus challenge, probably in an estrogen-dependent manner.

The anti-inflammatory effects of L. reuteri were also shown by Ma et al. (2004), who preincubated different concentrations of L. reuteri live bacteria with T84 and HT-29 human cell
lines, which were stimulated with TNF-α and Salmonella enterica to induce IL-8 secretion. At a
concentration of 10 L. reuteri per epithelial cell, inhibition of proinflammatory IL-8 was

8

observed. Considering that TNF-α activates IL-8 production via NF-κβ nuclear translocation, the
authors demonstrated in a Western blot that L. reuteri negatively regulates the degradation of
the IκB (Inhibitor of κB) protein in HeLa cells, thus proposing that L. reuteri is an antiinflammatory candidate. Iyer et al. (2008) has also demonstrated that L. reuteri 6475 downregulates NF-κβ in a TNF-dependent manner. In this experiment, L. reuteri 6475 supernatants
were incubated with human-myeloid cells prior TNF stimulation, suggesting that secreted
compounds could have a relevant role in the modulation of inflammatory host-signaling
pathways.

The TNF-α suppressor effects of the candidate probiotic strain L. reuteri 6475 have also been
investigated (Thomas et al., 2012). The amine histamine was identified as one of the secreted
compounds by which L. reuteri 6475 inhibits the production of TNF-α via signaling through
histamine H2 receptor, a cellular receptor that integrates into the PKA/MAPK signaling cascade.
The PKA/MAPK pathway can modify the status of transcription factors such as AP-1, which can
promote the up-regulation of proinflammatory cytokines (Chi et al., 2006). L. reuteri produces
histamine through the decarboxylation of histidine by the histidine decarboxylase (HdcA)
enzyme. Disruption of the hdcA gene by single-stranded recombineering produces a 60%
increase of TNF levels compared to the wild-type L. reuteri control. These results suggest that
other L. reuteri secreted factors or cell-wall associated compounds could be acting as negative
regulators of TNF production.

9

Production of antimicrobial compounds
Reuterin is an antimicrobial compound produced by L. reuteri from the anaerobic metabolism
of glycerol in a reduced environment. This molecule has been identified as one of the elements
that confers upon L. reuteri the ability to inhibit growth of pathogenic bacteria in the intestine.
Although many bacteria contain the pathway to reduce glycerol, L. reuteri has shown to secrete
reuterin in relatively higher levels (Walter et al., 2011).

Other antimicrobial compounds such as reutericin and reutericyclin have also been reported to
be produced by some strains of L. reuteri, but their antimicrobial effects does not have a range
as broad as reuterin (Spinler et al., 2008) and currently, no studies have reported their
antimicrobial activities in vivo (Vollenweider and Lacroix, 2004).

Different human-derived L. reuteri strains – ATCC 55730, ATCC PTA 6475, ATCC PTA 4659 and
ATCC PTA 5289 – have been characterized for their ability to produce reuterin from glycerol
using HPLC and colorimetric assays. L. reuteri 55730 was characterized as the most efficient
strain, converting 3-fold more glycerol to reuterin than strains 6475, 4659 and 5289. The
observed reuterin yield variation was characterized as a strain-dependent effect, which implies
that L. reuteri strains vary in their ability to convert glycerol to reuterin at the molecular level.
This assumption is supported by microarray data, which have indicated that differences in gene
expression correlate with differences in reuterin production among L. reuteri strains (Spinler et
al., 2008).

10

Various studies have demonstrated how pure reuterin or different reuterin-producer
Lactobacillus reuteri strains (ATCC 55730 and ATCC 6475) can shape the composition of
intestinal microbiota (Cleusix et al., 2008; DeWeirdt et al., 2012). It has been shown that the
addition of L. reuteri 55730 to an established in vitro colonic fermentation system, using
immobilized human fecal microbiota, increases the lactobacilli-enterococci populations and
thus changes the intestinal fermentation pattern. Moreover, the combination of L. reuteri
55730 and different concentrations of glycerol (10mM and 100mM) significantly decreases
Escherichia coli populations. These results suggest an actively in situ production of reuterin
from glycerol fermentation against E. coli strains (Cleusix et al., 2008).

In addition, Spinler et al (2008) observed that four different L. reuteri strains have different
inhibitory growth outcomes against enteric pathogens, like Enterohemorragic E. coli (EHEC),
Enterotoxic E. coli (ETEC), Salmonella enterica, Shigella sonnei and Vibrio cholerae. In this
experiment, L. reuteri 6475 and 4659 demonstrated the most potent inhibitory effects
compared to L. reuteri 5289 and 55730. It is worth to note that although L. reuteri 55730
produces higher amounts of reuterin, it has the weakest inhibitory effect on enteric pathogens,
suggesting that other bacteriocins and secreted or membrane associated proteins could play
also a significant role as antimicrobial compounds.

Competitive exclusion
Intestinal epithelial adhesion is a desirable probiotic feature. It not only inhibits the binding of
pathogenic bacteria, but also it mediates the interactions between host-cell receptors and

11

bacterial membrane structures, which modulates immune-defense and maturation processes
of gastrointestinal cells. In particular, the L. reuteri strains JCM 1081 and JCM 1112 have shown
high epithelial adherence capabilities in an adhesion Caco-2 monolayer assay (Todoriki et al.,
2001). In addition, the recognition of carbohydrate-binding motifs such as asilo-GM1 by the
poultry strains L. reuteri JCM 1081 and TM 105, and by the human isolate JCM 1112 has been
shown by Mukai et al. (2002) in TLC-overlay assays. The importance of these features resides on
the fact that both pathogenic and non-pathogenic strains share the same binding affinity for
host-cell structures (Vélez et al., 2007). Several authors have described the competitive
exclusion capabilities of different L. reuteri strains to outcompete Escherichia coli, Salmonella
typhimurium, Enterococcus faecalis and Helicobacter pylori species (Mukai et al., 2002; Todoriki
et al., 2001). In addition, certain L. reuteri strains would secrete an increased amount of
antimicrobial compounds such as reuterin, once they are adhered to the intestinal epithelium,
promoting a better pathogen exclusion effect (Jones and Versalovic, 2009). Other authors have
shown the reduction in adhesion by aerobic bacteria in the lower-digestive tract, when L.
reuteri was supplemented between 2-16 weeks in an IL-10 mice deficient model. This suggests
the prevention of colitis onset (Madsen et al., 1999).

Enhancement of the epithelial barrier
It has been demonstrated that several probiotic strains of the Lactobacillus and Bifidobacterium
genera promote beneficial effects in the integrity of the gastrointestinal epithelial barrier. The
epithelial structure is composed by cells, mucus, and antimicrobial and IgA secretions, which
play a fundamental role in the correct function and homeostasis of the gastrointestinal tract

12

(Madsen, 2012; Madsen et al., 2001). For instance, Jensen et al. (2012) assessed the epithelial
modifications that 4 different L. reuteri strains – DSM 20016, DSM 17938, 6475, FJ-1– produce
in enterocyte-like Caco-2 cell barrier conformations, using Transepithelial Electrical Resistance
(TER) as an indicator of epithelial barrier function. The study suggests that there is a tendency
to increase the TER Caco-2 ratio after 24 hours of incubation with live L. reuteri, implying a
potential epithelial barrier strengthening. A protective epithelial barrier effect using a
Lactobacillus reuteri cocktail (rat-derived R2LC and JCM 5869 strains, and human-derived ATCC
PTA 4659 and ATCC 55730 strains) has also been suggested by Dicksved et al. (2012) in a DSS
(dextran sodium sulfate) mice colitis model. Here, pre-colonized animals with L. reuteri
developed a mild colitis, compare to those treated with DSS. The decrease in the intestinal
bacteria translocation despite the alteration in microbial composition or the mucus layer,
suggests a general enhancement of the intestinal epithelial barrier. Table 2.1 summarizes the
main mechanisms of action of Lactobacillus reuteri.

Table 1.1. Mechanisms of action of Lactobacillus reuteri.
Mechanisms of action of Lactobacillus reuteri
Functions

Description

Regulation and
stimulation of the
immune system

Increase the number of CD4, B and T reg cells in the
small intestine
Enhanced dendritic cell IL-10 and TGF-β secretion
levels
Anti-inflammatory activity Inhibition of IL-12 and IL-6 secretion levels in
dendritic cells
Reduction of proinflammatory cytokines and
interleukins: IL-4, IL-12, IFN-ƴ in the intestine

13

References
33, 62
7, 33
7
45

Table 1.1 (cont’d)

Production of
antimicrobial compounds

Competitive exclusion

Enhancement of the
epithelial barrier

Reduced IL-8 secretion levels and NF-κβ transcription
levels in human cell lines
Reduction of TNF-α levels in the intestine and in
activated macrophages
Production and secretion of reuterin from glycerol
fermentation. Antimicrobial activity against intestinal
pathogenic bacteria and modification of the
composition of intestinal microbiota using in vitro
models.
Out compete pathogenic bacteria in adhesion-cell
culture assays
Reduction of aerobic bacteria adhesion in a mouse
model
Increased transepithelial electrical resistance in
enterocyte-like cells
Decreased intestinal bacteria translocation in a DSS
mouse model

25, 34
45, 60
10, 13, 55

41, 61
37
26
14

PROBIOTIC EFFECTS OF L. REUTERI IN DISEASE
Animal studies and human-clinical trials have been utilized to evaluate the potential beneficial
effects of different Lactobacillus strains based on in vitro evidence. For example, in Sheil et al.
(2007) more than 20 genetically engineered animal models were reported to study the effects
of probiotic strains in IBD. On the other side, human randomized trials are used to generate
useful information about the efficacy to treat or prevent different diseases, and to identify
possible adverse reactions, thus inferring the potential use of lactobacilli species in the
probiotic market. Some of the main results obtained from these types of studies are briefly
referred in the below paragraphs.

14

Diarrhea
Various studies have shown the amelioration of diarrheal symptoms in children after L. reuteri
supplementation. Francavilla et al. (2012) demonstrated the reduction of the frequency and the
8

duration of acute-watery diarrhea in children from 6 to 36 months. A total of 4 x10 L. reuteri
17938 CFU (colony forming units) were administered daily for one week. By the third day
treatment L. reuteri recipients diminished diarrheal episodes compared to placebo recipients
(46% vs. 73%). Other studies have shown similar results in children that present a rotavirusassociated diarrhea (Shornikova et al., 1997a, 1997b) and in adults with antibiotic-associated
diarrhea (Cimperman et al., 2011).

Colic
Infantile colic is described as a multi-etiological crying status, most commonly described during
the first 3 months of life. Some of the proposed etiological factors are a generalized
gastrointestinal dysfunction, milk’s protein allergy and lactase deficiency, and L. reuteri has
been explored as a possible treatment for these disorders. Specifically, the strains L. reuteri
ATCC 55730 and DSM 17938 have shown the reduction of colicky events after 7 days of
lactobacilli supplementation, compared to simethicone placebo recipients. As a result, L. reuteri
has been proposed as an effective method to treat colic events produced by an intestinal
imbalance (Savino et al., 2007; Szajewska et al., 2010).

15

Inflammatory Bowel Disease (IBD)
IBD is a medical term that describes a group of multi-factorial diseases characterized by a
confined or generalized intestinal inflammation. The main forms of IBD are Ulcerative Colitis
(UC), Crohn’s disease (CD) and pouchitis. Although the etiology of IBD is not clear, it has been
proposed that the gut microbiota can play a fundamental role in the onset of this disease
(Jonkers and Stockbrügger, 2003). The efficacy of the strain L. reuteri 55730 to treat UC in
children has been shown in randomized, placebo-controlled studies. Patients who received an
enema solution of 10 CFU of L. reuteri 55730 showed a significantly clinical remission of the
disease compared to the placebo group (31% vs. 0%). In addition, rectal samples obtained
before and after the trial evaluation were submitted for mucosal-cytokine level analyses. The L.
reuteri group reduced IL-1β, TNF-α and IL-8 levels and increased IL-10 levels (Niv et al., 2005).
Although these results are promising, larger scale studies including double-blind placebo
controlled trials need to be completed to support the use of probiotics in IBD.

Animal studies that involved the study of L. reuteri effects in IL-10 deficient mice, which is a
well-known IBD model, have shown a reduced mucosal inflammation, due in part to a general
reduction in pro-inflammatory cytokines (IL-12, TNF-α, IFN-ƴ, IL-4) (Peña et al., 2005) and to a
possible competitive exclusion against harmful aerobic bacteria (Madsen et al., 1999).

Allergy
Among allergic diseases, eczema is a pathological condition characterized by a non-infectious
chronic dermatitis, with pruritus and inflammation. It has been proposed that genetic and

16

environmental factors could play a major role in eczema development, although dysbiosis has
not been discarded. Families with allergic disease history were subjected to a double-blind,
placebo-controlled trial in which the effect of the probiotic strain L. reuteri 55730 was assessed.
8

Here mothers during the last month of pregnancy consumed 1 x 10 CFU of the probiotic strain,
and new-born babies follow the same treatment for a period of up-2 years. The incidence of
eczema during the first year of treatment was similar in both, the L. reuteri and the placebo
groups. However, during the second year the L. reuteri group shows a considerable decrease in
the incidence of IgE-associated eczema compared to the placebo group (8% vs. 20%). Although
a non-preventive effect was not discarded, L. reuteri 55730 showed an ameliorative effect
during the curse of an atopic allergic dermatitis trial (Abrahamsson et al., 2007).

On the other hand, the antiallergic effects of the human strain L. reuteri ATCC 23272 at
respiratory level have been evaluated by Karimi et al. (2009) in an OVA-sensitized mouse
model. The most important findings of this study revealed the attenuation of the tract
inflammation and the down-regulation of the hyper-responsiveness occurred right after the
inhalation of the antigen challenge. It was hypothesized that the positive response obtained
after the L. reuteri oral supplementation was in part due to the systemic increase in Treg
+

populations and the modulation of the function of CD4 cells. This study provides evidence of
the hypothetical role of L. reuteri in the resolution of respiratory allergic diseases through the
stimulation and regulation of the immune system.

17

THE PROBIOTIC POTENTIAL OF LACTOBACILLUS REUTERI 6475
The commercialization of several probiotic strains in the medical field has grown exponentially
in the last 10 years. There are several advances in the field, and most of the recent studies have
been focused on the effects produced at cellular and intestinal levels. These studies are
concerned with the effects of probiotics in immunity, epithelial barrier enhancement, and
pathogen inhibition due to antimicrobial effects and competition for binding sites. But many
potential roles of probiotics in the treatment and prevention of different ailments still have yet
to be understood. Accordingly, there is an increased need to understand the physiological and
molecular events that govern unexplored mechanisms of actions and their further
characterization using in vivo and in vitro models. Also, the study of surface components seems
to be a key step to generate major advances in the field.

The human Lactobacillus reuteri ATCC PTA 6475 strain is a candidate probiotic isolated from
breast milk, which has demonstrated unique beneficial features at in vitro and in vivo levels
related to the production of the antimicrobial compound reuterin and the inhibition of the
production of TNF-α via H2 receptor. Based on the studies of Thomas et al. (2012) and Iyer et al.
(2008), it has been hypothesized that L. reuteri 6475 secreted factors and/or associated cellwall proteins reduce the secretion of inflammatory molecules such as TNF and decrease the
expression of proinflammatory transcription factors such as NF-κB. In particular, Eaton et al.
(2011) studied the effects of the use of L. reuteri 6475 as a treatment for EHEC-associated
disease using a germfree mouse model. After 3 weeks of L. reuteri suplementation, EHEC
bacterial counts were significantly reduced compared to the counts obtained in EHEC
18

monocolonized mice. One interesting finding of this study was the general reduction of
symptons, such as wight loss, 2 weeks before the pathogen clearance. Even though it was
suggested that L. reuteri 6475 effectively suppressed EHEC colonization in mice, it was
proposed that other probiotic mechanisms might be acting as ameliorative factors despite the
pathogenic colonization state.

Taking all these studies together, we were interested in the development of a L. reuteri 6475
secretome mutant library. The aim of this study is to identify genes that code for proteins that
mediate reuterin release and other secreted beneficial factors (such as anti-TNF activity). The
external proteins can have an important role because of their direct interaction with the
intestinal epithelial barrier. These interactions are crucial to produce host anti-inflammatory
cytokines and to establish probiotic-cell adhesions. On the other hand, transporter proteins
carry out the import and the export of different substances. The importance of nutrients such
as carbohydrates and amino acids allow for normal L. reuteri growth and the subsequent
generation of metabolites that may have beneficial properties. As previously mentioned,
secreted molecules that have not been identified so far can be important for the generation of
probiotic responses and the regulation of host signaling pathways. Finally, those same
characteristics can be employed as biological markers to screen for similar metabolites in other
established or potential probiotic strains.

19

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20

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CHAPTER 2
CONSTRUCTION OF A SECRETOME MUTANT LIBRARY OF LACTOBACILLUS REUTERI 6475

INTRODUCTION

Lactobacillus reuteri strains have been employed as treatments or ameliorative elements in
different clinical trials and animal studies. These studies have shown promising results and
some of these strains are commercialized as probiotics. Currently, L. reuteri strains have been
shown to ameliorate colicky and diarrheal symptoms, to improve general human-health status,
and to contribute to the regulation of intestinal dysfunctions of diverse origin. Although the
employment of L. reuteri as a treatment in different ailments has been effective, mechanisms
of action are just starting to be elucidated and a profound understanding of them is critical to
generate significant advances in the medical field.

Various secreted L. reuteri compounds may be crucial for the development of host beneficial
responses and for the regulation of signaling pathways in eukaryote cells (Sánchez et al., 2010).
In this respect, the research based on the L. reuteri strain 6475 has contributed to the
expansion in the field. Some secreted L. reuteri 6475 factors, such as reuterin and histamine
have been demonstrated to be crucial for the inhibition of pathogenic growth (Eaton et al.,
2011), the regulation of anti-inflammatory responses (Iyer et al., 2008; Thomas et al., 2012) and
the improvement of bone health (Britton and McCabe, unpublished results). On the other hand,
only a small number of L. reuteri surface proteins with probiotic properties have been identified

28

so far. Most of them have been characterized because of their role in biofilm formation,
epithelial adherence and gut persistence (Lebeer et al., 2008).

The identification of important L. reuteri 6475 secreted or cell-wall mediated probiotic
mechanisms of action will be driven by the employment of the secretome mutant library that
was constructed in this study. The main advantage of this library is that it will permit the
elucidation of probiotic features based on a genotype/phenotype systematic approach. Thus,
different phenotypes will lead to the identification of genes involved in the biogenesis and
secretion of probiotics molecules.

The construction of this library is divided into 2 steps: identification of the secretome genes by
the SignalP software followed by the disruption of the genes that encode the secretome
proteins by single-stranded DNA recombineering.

The secretome is defined as “the global study of proteins that are secreted by a cell, tissue or
organism at any given time or under certain conditions”(Hathout, 2007). The main secretion
system in Lactobacillus reuteri is the Sec pathway. This system is based on the recognition of an
amino acid tag, known as signal peptide, which directs the pre-mature protein to the Sec
secretion system on the cytoplasm membrane (Snyder and Champness, 2007).

29

The signal peptide is a region of approximately 15 to 50 amino acids located at the N-terminus
of the global protein sequence, and it is composed of three main regions: n-, h- and c-. The hregion has hydrophobic amino acids and is essential for target and cleavage purposes. This
region is flanked by the n- and the c-region. The n-region contains positively charged amino
acids, and the c-region harbors uncharged and small amino acids at positions -3 and -1 relative
to the cleavage site. These positions are considered essential for a correct signal peptide
cleavage (Martoglio and Dobberstein, 1998).

The presecretory proteins that are targeted to be exported are directed to the SecYEG channel
by the SecB chaperone protein that binds to the signal peptide and prevents the protein from
folding in the cytoplasm. Once the protein passes through the SecYEG channel, the signal
sequence is removed by the Lep protease, and the protein is released into the extracellular
space.

The transmembrane proteins utilize a completely different mechanism compared to the
exported proteins. In the case of the former, the SRP protein complex recognizes the signal
sequence of an emerging protein and stops its translation until it makes contact with the
SecYEG channel. This mechanism occurs mainly because the transmembrane proteins contain
longer signal peptides with high hydrophobic content, compared to the peptides of the
secreted proteins. Thus, the protein precipitation within the cytoplasm is prevented when the
SRP complex translocates those nascent proteins directly into the membrane secretory
machinery (Snyder and Champness, 2007).

30

Most genetic studies that identify genes involved in the physiological characteristics of
lactobacilli have employed different vectors as knockout systems to disrupt or delete genes of
interest (Corr et al., 2007). For decades, these types of tools have governed mutant analyses
and have been effective. However, these older technologies are time-consuming and can yield
unstable mutations. On the other hand, the recent employment of recombineering technology
in lactic acid bacteria has enabled the study of their different proteins, generating stable,
unmarked mutations in a considerably shorter time (van Pijkeren and Britton, 2012; van
Pijkeren et al., 2012).

Recombineering or recombination-mediated genetic engineering, is a high performance tool
which utilizes the λ-phage Red proteins or the rac prophage RecET proteins to allow for the
generation of point mutations, insertions or deletions within bacterial chromosomes. The Red
system is composed by Gam, Exo and Bet, which are three proteins encoded by the λ-phage to
facilitate its own replication and integration into the host DNA during the lysogenic phase. In
recombineering, Gam inhibits the host RecBCD exonuclease system to evade the degradation of
exogenous linear DNA. Then, Exo degrades the 5’ end of double-stranded DNA generating 3’
single-stranded overhangs. Finally, Bet binds to the 3’ end overhangs and anneals the foreign
DNA molecule to homologous regions on the host DNA (Murphy, 1998; Swingle et al., 2010).

Only the Bet protein or another functionally equivalent protein is necessary for single-stranded
recombineering (Sawitzke et al., 2007; Thomason et al., 2005). This technology allows for the
generation of point mutations within the chromosome through the introduction of single-

31

stranded oligonucleotides without the need of antibiotic selection. This system was first
established in Escherichia coli, and since then it has served as the main model to study this new
technique in other microorganisms. In this bacterium, the employment of lagging-strand
oligonucleotides of 40-70 base pairs produces recombineering efficiency levels between 20%
and 50%. In addition, the introduction of oligonucleotides that harbor a C·C mismatch avoids
the Mismatch Repair (MMR) System, allowing the efficient manipulation of the E. coli genome.

In 2012, van Pijkeren and Britton established ssDNA recombineering in some strains of lactic
acid bacteria (van Pijkeren and Britton, 2012; van Pijkeren et al., 2012). In this study,
recombineering was established in the strain L. reuteri 6475 yielding recombineering efficiency
levels of about 1-5%. Although the percentage of recombinant cells appears to be low
compared to the ones obtained in E. coli, these levels are still sufficient to generate mutations
without the need for antibiotic selection. These authors showed that L. reuteri recombineering
requires 100 μg of DNA, which is significantly more than the amount required by E. coli. Also,
complete evasion of the MMR system was achieved using 70-90 bp oligonucleotides harboring
5 consecutive mismatches that anneal to the lagging strand. The validation of this technique
was proved by mutating several genes in different locations around the whole chromosome
and modifying the vancomycin resistance of the strain, which was converted into vancomycin
sensitive.

32

MATERIALS AND METHODS

SIGNALP: A PREDICTIVE SECRETOME TOOL.
In the present project, we used the SignalP software version 3.0 to predict the putative
secreted proteins of the candidate probiotic strain L. reuteri 6475 (Nielsen and Krogh, 1998). To
complete this task the 2,023 open reading frames (ORF) that were identified in the genome of
L. reuteri 6475 were submitted to the SignalP program for signal peptide recognition. Using the
HMM method, we obtained 185 secretome genes, which represent approximately 9% of the
genome sequence. The number of secretome genes located on the first replichore and second
replichore of the chromosomal sequence correspond to 72 and 113, respectively. All the
predicted genes present higher than a 50% probability of being secreted (123 genes ≥ 90%, 29
genes 70-90%, and 32 genes 50-70-%).

SINGLE-STRANDED DNA RECOMBINEERING.
Preparation of recombineering competent bacteria. Lactobacillus reuteri 6475 cells harboring
the RecT expression vector pJP042 were grown in MRS liquid media (BD Difco) under anaerobic
conditions for 18 hrs at 37°C. Erythromycin was added at a concentration of 5 μg/ml. The next
day, cells were sub-cultured under the same broth conditions until achieve an OD600 of 0.55 to
0.65, at which time the expression of the RecT protein was induced for 20 minutes by adding
the induction peptide (MAGNSSNFIHKIKQIFTHR) at a concentration of 10 ng/ml. Cells were kept
on ice for 5 minutes and washed twice with a buffer solution of 0.5 M sucrose and 10% glycerol.
Afterwards, cells were diluted at 1/200 from the total volume of the original sub-culture and
33

distributed in 100 μl aliquots. Aliquots were mixed on ice with 100 μg of recombineering
oligonucleotides and electroporated using 2 mm electroporation cuvettes (BioRad Genepulser
system). After the electroporation, L. reuteri cells were recovered on MRS broth under
anaerobic conditions for 3 hrs at 37°C and plated under non-selective media. Seeded plates
were incubated until the next day using the GasPack EZ Anaerobe Container system at 37°C.
8

The number of cells that survive the electroporation process (viable cells) is estimated in 10
9

CFU/ml, which represent 10% from the initial cell population (10 CFU/ml).

All the recombineering oligonucleotides were designed to replace a 5 nucleotide region of a
secretome gene by 5 consecutive mismatches that generate both a stop codon and a restriction
endonuclease site for screening purposes. The stop codon was designed to occur from the 100
to 300 base pairs of the targeted protein coding sequence.

Each of the 185 secretome transformations were performed using as a control the introduction
of the recombineering oligonucleotide JP577, which was designed to disrupt the L. reuteri 6475
rpoB gene in order to generate a rifampicin resistant phenotype. The mutation of the rpoB gene
generates a recombineering frequency of 1 to 5%, which is calculated dividing the number of
6

mutant cells (Rifampicin resistant colonies) by the total number of viable cells (10 Rif resistant
8

CFU/10 viable cells).

34

Identification of recombineering mutant cells. The identification of recombineering mutants
was completed screening 100 colonies from L. reuteri viable cells by a mismatch amplification
mutation analysis–PCR (MAMA–PCR) (Cha et al., 1992). In this study, the secretome MAMA-PCR
analysis utilizes two screening oligonucleotides to generate a 1 Kb PCR fragment that harbors
the desired mutation in the center. A third oligonucleotide aligns to the mutation and
consequently generates a fragment of 500 bp. The size of the different fragments is detected
using DNA electrophoresis on a 1% agarose gel matrix. The identification of a 1 Kb and a 500 bp
fragment in the same screened colony is called a mixed genotype mutant strain, due to its mix
nature of wild type and mutant bacteria populations, or due to the late insertion of a
recombineering oligonucleotide within the lagging-strand during the L. reuteri replication.
Figure 2.1 shows the details about the MAMA-PCR approach employed in one of the secretome
predicted genes.
Wild-type strain

Mixed genotype mutant strain

fwd oligo

fwd oligo

GAATT
rev oligo

MAMA oligo

rev oligo
100 screened colonies

MAMA oligo

Figure 2.1. MAMA-PCR strategy employed to identify secretome mutant L. reuteri 6475
colonies.
35

Figure 2.1 (cont’d)
The figure shows the MAMA-PCR differences between a wild-type and a mixed genotype strain.
In the wild-type strain, the alignment of the forward and the reverse screening oligonucleotides
will generate a unique 1 Kb PCR fragment (light blue circles). Instead, in the mixed genotype
strain the presence of a 5 consecutive mismatch mutation will allow the alignment of the
MAMA screening oligonucleotide. This new condition will generate a 500 bp PCR fragment (red
circles). For interpretation of the references to color in this and all other figures, the reader is
referred to the electronic version of this thesis.

In order to confirm the generation of the mismatch mutation and to get a pure genotype
mutant strain, the mixed genotype mutant strains are restreaked on a non-selective MRS plate
and incubated at 37°C in anaerobic conditions. On the next day, a PCR reaction is performed
using the flanking screening oligonucleotides to obtain the 1 Kb amplicon described before.
Then, this fragment is digested with a specific restriction endonuclease enzyme, according to
the restriction site previously designed in the recombineering oligonucleotide. The generation
of a unique 500 bp fragment indicates the insertion of the 5 consecutive mutations in both DNA
strands (Figure 2.2). Finally, pure genotype mutants are plasmid cured and stocked at -80°C.
23 screened colonies

Pure genotype mutant strain

Figure 2.2. Identification of pure genotype L. reuteri 6475 mutant strains.

36

-C

Figure 2.2 (cont’d)
In this example, a PCR was performed in 23 mixed genotype mutant colonies using forward and
reverse screening oligonucleotides to yield a 1 Kb PCR fragment. The same fragments were
digested with a restriction endonuclease enzyme to screen for 500 bp DNA amplicons. In this
case, two L. reuteri 6475 colonies were identified as pure genotype mutant strains. –C: negative
control; Kb: Kilo bases; bp: base pairs. For interpretation of the references to color in this and
all other figures, the reader is referred to the electronic version of this thesis.

When mutant strains were not obtained during the first screening round (100 colonies), a
second round was performed in 200 extra colonies. In this case, rather than use a MAMA-PCR
analysis, we employed the restriction endonuclease assay previously described, in order to
discard any false-negatives that are more prevalent in the MAMA-PCR assay.

RESULTS

Generation of a recombineering mutant library. The combination of bioinformatics tools and
single-stranded recombineering has allowed us to develop a systematic mutagenesis strategy to
identify new surface and secreted biocompounds with potential probiotic function in L. reuteri
6475.

After performing recombineering in the 185 secretome coding sequences by the mutational
strategies described in the materials and methods, we have developed a secretome mutant
library composed by 127 knocked-out genes. The total number of mutated genes represents
68% out of the 185 SignalP predicted protein sequences.

37

In particular, each of the targeted genes showed a different recombineering frequency value,
which is calculated as the percentage of bacterial colonies that present the desired mutation
from the total number of screened colonies. As an example, Figure 2.3 shows the
recombineering frequency value calculated for the secretome gene HMPREF0536_1129. In this
case 2 mutant colonies were obtained out of 100 screened colonies, yielding a recombineering
efficiency of 2.0%.

100 screened colonies

M

M

Figure 2.3. Recombineering rate of gene HMPREF0536_1129. In this case, the recombineering
efficiency value was estimated as 2%, and it was calculated by dividing the number of mutant
colonies by the total number of screened colonies. M: mutant colony.

The recombineering rates of the 127 successfully disrupted genes are distributed in 3 groups:
20 show recombineering efficiency levels above 5%; 74 between 1-5%; 25 less than 1% (8 have
no recombineering rate information).

The distribution of disrupted and unsuccessful genes (genes that fail recombineering after
screening a minimum of 300 colonies) is shown in Figure 2.4. The location of the genes has
been established using the SeqBuilder DNASTAR software and employing the chromosomal
38

sequence of the strain L. reuteri 6475 (NCBI Taxon ID 548485). The unsuccessful and
successfully mutated genes (grouped by color according to their recombineering efficiency
levels) are depicted.

200000

L. reuteri 6475
2039.414 kb

500000

1500000

ori

ter
1000000
Figure 2.4. Distribution of secretome disrupted genes in the L. reuteri F275 chromosome. Blue
line: secretome genes that fail recombineering; red, green and purple lines: secretome
disrupted genes with high >5%, medium 1-5% and low <1% recombineering efficiency levels;
ori: origin of replication; ter: termination site of replication. For interpretation of the references
to color in this and all other figures, the reader is referred to the electronic version of this
thesis.

The frequency of successfully mutated genes is different between the chromosomal
replichores. An important difference was observed in relation to the number of disrupted

39

genes out of the total number of genes that were predicted in replichores 1 and 2. The
distribution of genes per replichore is detailed on Figure 2.5 in a linear chromosomal map.
Replichore 1
ori

200000

400000

600000

ter

800000

Replichore 2
ter
1000000

ori
1200000

1400000

1600000

1800000

2000000

Figure 2.5. Distribution of attempted (red) and mutated (blue) genes per replichore. The
percentage of disrupted genes in the first replichore is 58.3% and in the second replichore is
75%; ori: origin of replication; ter: termination site of replication. Note the origin and terminus
regions of the two replichores are reversed. For interpretation of the references to color in this
and all other figures, the reader is referred to the electronic version of this thesis.

The first replichore contains a total of 72 secretome genes of which 42 were successfully
mutated while the second replichore has 113 secretome and 85 were successfully disrupted.
Thus, the ratio of effectively mutated genes on the first and second replichore was 0.58 and
0.75, respectively.

Although the number of secretome genes located on the second replichore is 1.5 times higher
than the number predicted on the first, the number of disrupted genes per replichore indicates
that the probability to mutate a secretome gene that is located on the second replichore is 17%
higher compared to the first replichore.

40

In a more detailed view, Figure 2.6 shows the ratio of mutated genes every 200 Kb within the
genome sequence. In this figure the highest recombineering points throughout the second
replichore, indicating there are no mutational hotspots within the replichores.
Replichore 1
0

200

400

600

1000

800

ori

ter
0.64

0.59

0.56

0.62

0.50

Replichore 2
1000

1200

1400

1600

2000

1800

ter

ori
0.76

0.69

0.76

0.76

0.77

Figure 2.6. Ratio of mutated genes in the L. reuteri 6475 genome every 200 Kb regions. Square
boxes represent the proportion of genes that were mutated out of the total number of
predicted genes every 200 Kb. ori: origin of replication; ter: termination site of replication.

41

DISCUSSION

Variation in the recombineering efficiency levels of knocked-out genes. The generation of a
mutant library of 127 genes has validated the genetic technique single-stranded
recombineering as the most efficient tool to generate mutations in L. reuteri. The fact that
mutant strains were obtained at different recombineering rates was in accordance to the
results reported by van Pijkeren and Britton (2012), in which 11 genes were randomly mutated
at different chromosomal positions and obtained at efficiencies that vary from 0.4% to 19%.

The observed different recombineering rates can be explained because some of the genes can
be essential for growth and some of them are not. The truncation of genes that are essential for
cellular development and survival tend to have detrimental effects on the growth rate of cells.
As a result, the cells that incorporate the mutation are outcompeted by wild-type cells. Thus,
mutated cells are detected during screening at a low rate, if at all. On the contrary, genes that
were mutated at high and medium recombineering rates are considered as non-essential
genes, and the employment of recombineering allowed their mutation at high rates due to the
complete evasion of the MMR system.

Additionally, some mutations might cause the cells to grow so slow that mutant bacteria were
not detected under our experimental conditions. In this case, a higher number of colonies
would need to be screened to increase the chances of obtain a mixed genotype colony. In
conjunction, the survival bacteria can be incubated during 3 through 5 days, leading to the

42

identification of slow growth colonies which would harbor the desired mutation. These types of
colonies would be identified as growth defect colonies based on their late onset and their
smaller size, compared to L. reuteri colonies developed at a normal growth rate and that are
characterized by its regular size and dimension.

The fact that we observe differences between replichores 1 and 2 indicates that there are
genomic factors that affect recombineering efficiency. Genes located on the second replichore
are 17% more likely to be successfully mutated by ss DNA recombineering, compared to the
first replichore. While we cannot rule out that more genes on the first replichore are either
essential for growth or when mutated have a slow growth phenotype (and would have been
missed in our mutagenesis strategy), there does not seem to be any logical reason for this
essential gene distribution. In addition, there does not appear to be any effect of being near the
origin of replication or the terminus in terms of recombineering efficiency. The most
straightforward possibility is the replication forks are not symmetric and there is some
functional difference between the enzymatic complex that replicates replichores 1 and 2. A
good strategy to assess this possibility would be to insert a gene that failed recombineering in
replichore one into the second replichore and evaluate its tendency to be disrupted in this new
position.

Genes that fail recombineering. The number of genes that were difficult to mutate in this study
was 58, representing 31% out of the total of SignalP predicted genes. Although most of these
genes are poorly studied in the Lactobacillus genus, we have hypothesized that some of the

43

proteins coded by these genes could have an essential role in the metabolism, structure or
general functioning of L. reuteri 6475. For example, it has been demonstrated that the proteins
involved in the biogenesis of cell-wall and membrane components are essential to maintain cell
viability (Scheffers and Pinho, 2005). Based on this assumption, we grouped the attempted
1

genes that encode for secretome proteins by their functional category (COG ) and we analyzed
2

their functions using their Pfam (PF ) entries (see Table 3.2).

In relation to cell-wall biogenesis, 5 genes that fail recombineering of this category seem to be
fundamental to maintain the cell viability. The UDP-glucose pyrophosphorylase
(HMPREF0536_1194 PF00483) which catalyzes the conversion of UDP-glucose into UDPgalactose, and the galactosyltransferase (HMPREF0536_0019 PF02397), are required to provide
the backbone carbohydrates for the peptidoglycan formation and their translocation across the
cytoplasm membrane. The penicillin-binding protein 1 (HMPREF0536_1348 PF00905), the
serine D-Ala-D-Ala carboxypeptidase (HMPREF0536_0190 PF00768), and the cell-wall
hydrolases (HMPREF0536_1386 PF01476) are essential proteins required to ensure the correct
assembling and maturation of the peptidoglycan at final stages.

1

COGs: Clusters of Orthologous Groups of proteins, is a protein phylogenetic analysis tool that
classifies a protein according to conserved domain analyses and orthologous gene comparisons
across diverse species.

2

Pfam is a protein database which describes a protein function, based on the analysis of
conserved protein domains by multiple sequence alignments and Hidden Markov Models.
44

Two rod-shape determination proteins were not mutated in the present study. The cell cycle
membrane protein FtsW/RodA/SpoVE (HMPREF0536_1400 PF01098) is fundamental to control
the peptidoglycan production during the cell division and to determine the Gram-positive rodshape (Henriques et al., 1998). The MreC protein (HMPREF0536_1324 PF04085) is also crucial
for the Gram-positive shape-architecture, and it is considered as a cytoskeleton protein (Leaver
and Errington, 2005).

Also, two proteins that had been proposed to be indispensable in prokaryote organisms and
that were difficult to disrupt are the FtsH extracellular protein (HMPREF0536_0621 PF06480),
and the translocase subunit SecY (HMPREF0536_0111 PF00344). The first has been described as
an ATP-dependent protease required for the degradation of unstable proteins, and the second
is crucial for the translocation of premature proteins into the periplasmic or extracellular space,
as described at the beginning of this chapter.

Other proteins that might be essential but are poorly characterized are the biotin synthase
(HMPREF0536_0992 PF02632), which is required as a cofactor in the biosynthesis of fatty acids,
and the N-acetylmuramoyl-L-alanine amidases (HMPREF0536_0648, HMPREF0536_1986
PF01510) that are required for the late peptidoglycan modeling.

In general, the ubiquitous, ancient and conserved character of these proteins across various
bacteria species make us consider that they might also have an essential role in L. reuteri 6475,
and therefore, they were not successfully disrupted.

45

Finally, there are various genes that were unable to be disrupted that code for proteins
involved in the transport of amino acids or carbohydrates. Hypothetically, these substrates
could be employed as backbone sources to construct proteins or carbohydrates of complex
nature. In this case, the supplementation of the recovery and incubation media with different
amino acids and carbohydrates sources can provide a nutritious environment and can represent
a good plan to disrupt these genes. Thus, genes that code for proteins involved in the transport
of these substances could be knocked-out if the nutrient is available on the growth media and is
transported inside the cell by other protein transporters. Since we grow L. reuteri on a rich,
complex medium it is unclear why these genes were not disrupted in the present study.

46

SUPPLEMENTAL INFORMATION
Table 2.1. Predicted secretome genes that were mutated employing single-stranded DNA
recombineering.
MUTATED GENES
Locus Tag

a

HMM

b

Gene product name

c

RE

d

Amino acid transport and metabolism
HMPREF0536_0026

0.61

Amino acid permease-associated protein

2.11

HMPREF0536_0027

0.99

Arginine/ornithine APC family amino acid
transporter

2.20

HMPREF0536_0148

1.00

APC family amino acid-polyamine-organocation
transporter

1.05

HMPREF0536_0161

1.00

Amino acid ABC superfamily ATP binding
cassette transporter, binding protein

19.00

HMPREF0536_0360

0.61

Amino acid permease-associated protein

20.00

HMPREF0536_0644

1.00

APC family amino acid-polyamine-organocation
transporter

2.11

HMPREF0536_0986

1.00

Amino acid ABC superfamily ATP binding
cassette transporter

3.16

HMPREF0536_1176

0.98

APC family amino acid-polyamine-organocation
transporter

1.05

HMPREF0536_1320

0.80

ABC superfamily ATP binding cassette
transporter

4.20

HMPREF0536_1470

0.99

ABC superfamily ATP binding cassette
transporter (spermidine/putrescine)

1.05

HMPREF0536_1471

0.56

ABC superfamily ATP binding cassette
transporter (spermidine/putrescine)

2.11

HMPREF0536_1701

0.65

APC family amino acid-polyamine-organocation
transporter

0.54

47

Table 2.1 (cont’d)
HMPREF0536_1735

0.94

Asparaginase

1.10

HMPREF0536_1859

0.93

APC family amino acid-polyamine-organocation
transporter

0.71

Carbohydrate transport and metabolism
HMPREF0536_0193

1.00

MFS family major facilitator transporter

2.50

HMPREF0536_0528

0.59

pfkB family carbohydrate kinase

8.42

HMPREF0536_0722

1.00

Phosphoglycerate mutase

1.10

HMPREF0536_1083

1.00

MFS family major facilitator transporter

1.05

HMPREF0536_1468

0.84

MFS family major facilitator transporter

1.10

HMPREF0536_1894

0.76

MFS family major facilitator transporter

2.11

Cell division and chromosome partitioning
HMPREF0536_1342

0.98

FtsK/SpoIII family DNA translocase

5.26

Cell envelope and membrane biogenesis
HMPREF0536_0009

1.00

N-acetylmuramoyl-L-alanine amidase

0.70

HMPREF0536_0016

0.78

Capsular polysaccharide biosynthesis protein

1.05

HMPREF0536_0047

0.72

N-acetylmuramoyl-L-alanine amidase

2.80

HMPREF0536_0212

0.51

Glycosyltransferase

1.30

48

Table 2.1 (cont’d)
HMPREF0536_0370

1.00

NLP/P60 family protein

4.21

HMPREF0536_0554

1.00

Substrate binding domain of ABC-type glycine
betaine transport system

ND

HMPREF0536_0602

0.93

D-alanine transfer protein DltD

1.20

HMPREF0536_0633

1.00

Peptidoglycan-binding protein

1.05

HMPREF0536_0877

1.00

Peptidoglycan-binding Lysin protein

9.47

HMPREF0536_0956

1.00

Cell-wall associated hydrolase

0.35

HMPREF0536_0973

0.98

Sortase

3.10

HMPREF0536_1649

1.00

NLP/P60 family protein

0.53

HMPREF0536_1673

1.00

D-alanyl-D-alanine carboxypeptidase

1.00

HMPREF0536_1815

1.00

RND family efflux transporter MFP subunit

0.71

Cell motility and secretion
HMPREF0536_0011

1.00

Muramidase (flagellum-specific)

1.05

HMPREF0536_1101

1.00

Mannosyl-glycoprotein endo-beta-Nacetylglucosamidase

3.16

HMPREF0536_1641

0.68

Mannosyl-glycoprotein endo-beta-Nacetylglucosaminidase

7.37

HMPREF0536_1685

1.00

Muramidase

3.16

49

Table 2.1 (cont’d)

Coenzyme metabolism
HMPREF0536_1546

0.59

Cobalamin biosynthesis protein CbiD

0.70

HMPREF0536_1518

0.88

ABC superfamily ATP binding cassette
transporter (multidrug resistance)

0.35

HMPREF0536_1758

1.00

Penicillin-binding protein

3.30

HMPREF0536_1797

0.61

ABC transporter ATPase

3.60

HMPREF0536_1817

0.66

ABC superfamily ATP binding cassette
transporter

1.10

Defense mechanisms

Energy production and conversion
HMPREF0536_0922

0.00

Nitrate reductase*

ND

HMPREF0536_1329

0.97

ABC superfamily ATP binding cassette
transporter

2.11

HMPREF0536_1662

0.86

NhaC family sodium:proton (Na+:H) antiporter

4.21

Inorganic ion transport and metabolism
HMPREF0536_0056

0.98

Amt family ammonium or ammonia transporter

8.42

HMPREF0536_0178

0.69

Sodium:sulfate symporter transmembrane
region

1.05

HMPREF0536_0219

0.97

Phosphate ABC superfamily ATP binding
cassette transporter, binding protein

0.80

HMPREF0536_0549

1.00

Heavy metal translocating P family ATPase

ND

50

Table 2.1 (cont’d)
HMPREF0536_0563

0.99

NLPA lipoprotein

ND

HMPREF0536_0679

0.92

CPA2 family monovalent cation:proton (H+)
antiporter-2

1.05

HMPREF0536_0934

0.50

MFS family major facilitator transporter,
nitrate:nitrite antiporter

ND

HMPREF0536_0940

1.00

Iron(III) ABC superfamily ATP binding cassette
transporter

0.53

HMPREF0536_1075

0.77

MFS family major facilitator transporter

1.00

HMPREF0536_1535

1.00

Cobalt ABC superfamily ATP binding cassette
transporter

2.30

HMPREF0536_1536

1.00

Cobalamin biosynthesis protein CbiM

0.78

Intracellular trafficking, secretion, and vesicular transport
HMPREF0536_0363

0.99

Oxa 1 family cytochrome oxidase biogenesis
protein

0.35

HMPREF0536_1129

0.76

ABC superfamily ATP binding cassette
transporter EcsB

2.11

HMPREF0536_1282

0.94

Competence protein ComGC

2.11

Nucleotide transport and metabolism
HMPREF0536_0718

1.00

Secreted 5'-nucleotidase

2.50

Posttranslational modification, protein turnover, chaperones
HMPREF0536_0548

0.66

Peptidase M10A and M12B, matrixin and
adamalysin

2.11

HMPREF0536_0980

1.00

Heat shock protein HtpX

6.32

51

Table 2.1 (cont’d)
HMPREF0536_1125

1.00

Peptidylprolyl isomerase

1.05

HMPREF0536_1803

0.91

S1 family peptidase

1.10

Signal transduction mechanisms
HMPREF0536_0875

0.95

yclK: histidine kinase

16.00

HMPREF0536_1940

0.62

Signal transduction histidine kinase

2.11

Translation, ribosomal structure and biogenesis
HMPREF0536_1979

0.92

possible 6-aminohexanoate-cyclic-dimer
hydrolase

0.40

HMPREF0536_0007

1.00

Conserved hypothetical protein

2.11

HMPREF0536_0036

1.00

Conserved hypothetical protein

3.70

HMPREF0536_0042

0.94

Integral membrane protein, interacts with FtsH

0.41

HMPREF0536_0063

0.87

ABC-2 type transport system permease protein

3.50

HMPREF0536_0091

1.00

CamS sex pheromone cAM373 family protein

4.60

HMPREF0536_0157

0.57

Protein of hypothetical function DUF915
hydrolase family protein

1.10

HMPREF0536_0158

0.59

Protein of hypothetical function DUF915
hydrolase family protein

11.80

HMPREF0536_0195

0.81

C4-dicarboxylate anaerobic carrier, arginine
transporter

0.40

Poorly characterized

52

Table 2.1 (cont’d)
HMPREF0536_0200

0.98

PSE family sulfate exporter/Predicted
membrane protein unknown function

0.70

HMPREF0536_0213

0.91

Hypothetical protein

3.16

HMPREF0536_0232

1.00

Conserved hypothetical protein

12.20

HMPREF0536_0240

0.60

Sugar specific permease

ND

HMPREF0536_0263

1.00

Conserved hypothetical protein

0.52

HMPREF0536_0329

1.00

Conserved hypothetical protein

1.05

HMPREF0536_0374

0.54

Membrane protein

11.58

HMPREF0536_0382

0.96

Conserved hypothetical protein

2.11

HMPREF0536_0501

0.55

Conserved hypothetical protein

5.26

HMPREF0536_0658

1.00

Conserved hypothetical protein

1.40

HMPREF0536_0717

1.00

Conserved hypothetical protein

2.11

HMPREF0536_0725

0.99

Conserved hypothetical protein

5.00

HMPREF0536_0827

0.72

Putative transmembrane protein

5.50

HMPREF0536_0843

1.00

Conserved hypothetical protein

1.05

HMPREF0536_0851

0.70

Hypothetical protein

1.40

HMPREF0536_0862

0.95

Cupredoxin family domain protein

0.53

53

Table 2.1 (cont’d)
HMPREF0536_0884

0.64

YbbR family protein

3.16

HMPREF0536_0951

1.00

Conserved hypothetical protein

5.26

HMPREF0536_0957

1.00

Dextransucrase

1.05

HMPREF0536_0972

0.94

Protein of unknown function

6.20

HMPREF0536_0979

0.55

LemA protein

ND

HMPREF0536_1020

1.00

Conserved hypothetical protein

0.53

HMPREF0536_1030

1.00

Possible integral membrane protein

0.70

HMPREF0536_1069

0.86

Conserved hypothetical protein

4.21

HMPREF0536_1070

1.00

Peptidoglycan-binding Lysin protein

1.00

HMPREF0536_1131

1.00

YSIRK Gram-positive signal protein

1.05

HMPREF0536_1155

1.00

Conserved hypothetical protein

1.05

HMPREF0536_1156

1.00

Conserved hypothetical protein

0.35

HMPREF0536_1215

1.00

Surface protein (Peptidoglycan anchored w/o
function)

0.35

HMPREF0536_1269

0.97

Translocase subunit SecD

2.11

HMPREF0536_1273

0.78

Membrane protein

6.32

HMPREF0536_1289

0.92

Conserved hypothetical protein

2.11

54

Table 2.1 (cont’d)
HMPREF0536_1290

0.76

Methyl-accepting chemotaxis family protein

4.21

HMPREF0536_1343

1.00

Conserved hypothetical protein

ND

HMPREF0536_1463

0.93

Conserved hypothetical protein

5.30

HMPREF0536_1469

0.54

Conserved hypothetical protein

2.10

HMPREF0536_1487

1.00

Membrane protein

2.10

HMPREF0536_1503

0.82

Conserved hypothetical protein

3.16

HMPREF0536_1521

0.78

Conserved hypothetical protein

2.11

HMPREF0536_1582

0.90

Permease

0.70

HMPREF0536_1635

1.00

Conserved hypothetical protein

1.05

HMPREF0536_1674

0.66

Conserved hypothetical protein

0.35

HMPREF0536_1675

1.00

Hypothetical cell surface protein

2.11

HMPREF0536_1731

0.94

Membrane protein

5.40

HMPREF0536_1736

1.00

Inosine-5'-monophosphate dehydrogenase

0.53

HMPREF0536_1829

1.00

Conserved hypothetical protein

1.10

HMPREF0536_1880

1.00

DNA/RNA non-specific endonuclease

2.11

HMPREF0536_1905

1.00

Hydrolase family protein of hypothetical
function DUF915

1.05

55

Table 2.1 (cont’d)
HMPREF0536_1908

1.00

Conserved hypothetical protein

1.05

HMPREF0536_1968

0.72

Conserved hypothetical protein

2.11

a Locus Tag accession number provided by Integrated Microbial Genomes (IMG) database.
b Probability of protein secretion predicted by the SignalP 3.0 software Hidden Markov Model
(HMM).
c Gene product name was assigned based on the COG domain analysis provided by the IMG
database.
d Recombineering rate, calculated for each secretome gene according to the number of
mutants colonies obtained per screened colonies.
ND: Not determined
*Gene HMPREF0536_0689 was included in the present study but is not considered a secretome
gene (HMM value=0).

56

Table 2.2. Predicted secretome genes that fail recombineering.
ATTEMPTED GENES
Locus Tag

a

HMM

b

Gene product name

c

Pfam

d

Amino acid transport
HMPREF0536_0309

1.00

ABC superfamily ATP binding cassette
transporter

PF00497

HMPREF0536_0438

0.99

APC family amino acid-polyamine-organocation
transporter

PF00324

HMPREF0536_0838

0.99

APC family amino acid-polyamine-organocation
transporter

PF00324

HMPREF0536_0839

1.00

Arginine/ornithine APC family amino acidpolyamine-organocation transporter, antiporter

PF00324

HMPREF0536_0864

1.00

DMT superfamily drug/metabolite transporter

PF00892

HMPREF0536_1220

1.00

Glutamate ABC superfamily ATP binding cassette
transporter, membrane protein

PF00497

HMPREF0536_1478

0.66

Amino acid permease-associated protein

PF00324

Carbohydrate transport and metabolism
HMPREF0536_1180

0.955

MFS family major facilitator transporter

PF07690

Cell division and chromosome partitioning
HMPREF0536_0710

1

Conserved hypothetical protein/Chromosome
segregation ATPase

HMPREF0536_1400

0.997

FtsW/RodA/SpoVE family cell division protein

57

PF01098

Table 2.2 (cont’d)

Cell envelope and membrane biogenesis
HMPREF0536_0019

0.723

Bacterial sugar transferase/galactosyltransferase

PF02397

HMPREF0536_0190

1

Serine family D-Ala-D-Ala carboxypeptidase

PF00768

HMPREF0536_1194

0.994

UDP-glucose pyrophosphorylase

PF00483

HMPREF0536_1324

0.999

Rod shape-determining protein MreC

PF04085

HMPREF0536_1348

0.642

Penicillin-binding protein 1

PF00905

HMPREF0536_1386

1

Cell-wall associated hydrolase (invasion
associated proteins)

PF01476

HMPREF0536_1670

0.724

Phosphatidylglycerol-membrane-oligosaccharide
glycerophosphotransferase

PF00884

0.78

2-dehydropantoate 2-reductase

PF02558

0.51

ABC superfamily ATP binding cassette
transporter (multidrug resistance)

PF00664

Coenzyme metabolism
HMPREF0536_0962

Defense mechanisms
HMPREF0536_1517

Inorganic ion transport and metabolism
HMPREF0536_0208

0.9475

H(+)-transporting two-sector ATPase

PF02386

HMPREF0536_0237

0.997

ABC superfamily ATP binding cassette
transporter, binding protein

PF03180

HMPREF0536_0247

0.655

Chloride channel protein

PF00654

58

Table 2.2 (cont’d)
HMPREF0536_0388

0.981

ABC superfamily ATP binding cassette
transporter, binding protein

PF03180

HMPREF0536_0587

0.962

ABC superfamily ATP binding cassette
transporter, binding protein

PF01297

HMPREF0536_0589

0.968

Cation ABC superfamily ATP binding cassette
transporter, membrane protein

PF00950

HMPREF0536_1591

0.956

MFS family major facilitator transporter

PF07690

Intracellular trafficking, secretion, and vesicular transport
HMPREF0536_0111

0.991

Preprotein translocase subunit SecY

PF00344

0.743

Phosphatidate cytidylyltransferase

PF01148

Lipid metabolism
HMPREF0536_1134

Posttranslational modification, protein turnover, chaperones
HMPREF0536_0239

0.948

Peptidase M10A and M12B, matrixin and
adamalysin

PF00413

HMPREF0536_0621

0.995

FtsH Extracellular protein

PF06480

Signal transduction mechanisms
HMPREF0536_0359

0.984

Sensor histidine kinase

PF02518

HMPREF0536_1240

0.783

Integral membrane sensor signal transduction
histidine kinase

PF00512

HMPREF0536_1404

0.981

S16 family peptidase

PF05362

59

Table 2.2 (cont’d)

Poorly characterized
HMPREF0536_0008

1

Cell-wall associated hydrolase

HMPREF0536_0017

0.866

Oligosaccharide repeat unit polymerase Wzy

HMPREF0536_0482

0.995

Conserved hypothetical protein

HMPREF0536_0509

0.913

Conserved hypothetical protein

HMPREF0536_0539

0.869

Phosphate-starvation-inducible protein PsiE

HMPREF0536_0593

1

Conserved hypothetical protein

HMPREF0536_0631

1

Peptidoglycan-binding protein

HMPREF0536_0647

0.981

Conserved hypothetical protein

HMPREF0536_0648

1

HMPREF0536_0826

0.661

Putative transmembrane protein

HMPREF0536_0992

0.856

Biotin synthase

HMPREF0536_1034

0.946

Protein of hypothetical function DUF915
hydrolase family protein

HMPREF0536_1039

0.616

Conserved hypothetical protein

HMPREF0536_1066

0.766

Conserved hypothetical protein

HMPREF0536_1116

0.931

Conserved hypothetical protein

N-acetylmuramoyl-L-alanine amidase

60

PF06146

PF01476

PF01510

PF02632

Table 2.2 (cont’d)
HMPREF0536_1178

0.781

Conserved hypothetical protein

HMPREF0536_1501

0.633

Conserved hypothetical protein

HMPREF0536_1588

0.696

Possible hemolysin

HMPREF0536_1667

0.987

Arginine/ornithine APC family amino acidpolyamine-organocation transporter, antiporter

HMPREF0536_1695

0.998

Conserved hypothetical protein

HMPREF0536_1800

0.959

YycH protein

HMPREF0536_1839

0.889

Conserved hypothetical protein

HMPREF0536_1849

0.957

Membrane protein

HMPREF0536_1925

1

Peptidase propeptide and ypeb domain protein

HMPREF0536_1986

1

N-acetylmuramoyl-L-alanine amidase

PF03606

PF07435

PF01510

a Locus Tag accession number provided by Integrated Microbial Genomes (IMG) database.
b Probability of protein secretion predicted by the SignalP 3.0 software Hidden Markov Model
(HMM).
c Gene product name was assigned based on the COG domain analysis provided by the IMG
database
d Pfam accession number provided by the IMG website.

61

REFERENCES

62

REFERENCES
1. Cha, R.S., Zarbl, H., Keohavong, P., and Thilly, W.G. (1992). Mismatch amplification mutation
assay (MAMA): application to the c-H-ras gene. PCR Methods and Applications 2, 14–20.
2. Corr, S.C., Li, Y., Riedel, C.U., O’Toole, P.W., Hill, C., and Gahan, C.G.M. (2007). Bacteriocin
production as a mechanism for the antiinfective activity of Lactobacillus salivarius UCC118.
Proceedings of the National Academy of Sciences of the United States of America 104,
7617–7621.
3. Eaton, K. a, Honkala, A., Auchtung, T. a, and Britton, R. (2011). Probiotic Lactobacillus reuteri
ameliorates disease due to enterohemorrhagic Escherichia coli in germfree mice. Infection
and Immunity 79, 185–191.
4. Hathout, Y. (2007). Approaches to the study of the cell secretome. Expert Review of
Proteomics 4, 239–248.
5. Henriques, A.O., Glaser, P., Piggot, P.J., and Moran, C.P. (1998). Control of cell shape and
elongation by the rodA gene in Bacillus subtilis. Molecular Microbiology 28, 235–247.
6. Iyer, C., Kosters, A., Sethi, G., Kunnumakkara, A.B., Aggarwal, B.B., and Versalovic, J. (2008).
Probiotic Lactobacillus reuteri promotes TNF-induced apoptosis in human myeloid
leukemia-derived cells by modulation of NF-kappaB and MAPK signalling. Cellular
Microbiology 10, 1442–1452.
7. Leaver, M., and Errington, J. (2005). Roles for MreC and MreD proteins in helical growth of
the cylindrical cell wall in Bacillus subtilis. Molecular Microbiology 57, 1196–1209.
8. Lebeer, S., Vanderleyden, J., and De Keersmaecker, S.C.J. (2008). Genes and molecules of
lactobacilli supporting probiotic action. Microbiology and Molecular Biology Reviews :
MMBR 72, 728–764.
9. Martoglio, B., and Dobberstein, B. (1998). Signal sequences: more than just greasy peptides.
Trends in Cell Biology 8, 410–415.
10. Murphy, K.C. (1998). Use of Bacteriophage lambda Recombination Functions To Promote
Gene Replacement in Escherichia coli. J. Bacteriol. 180, 2063–2071.
11. Nielsen, H., and Krogh, A. (1998). Prediction of signal peptides and signal anchors by a
hidden Markov model. Proc Int Conf Intell Syst Mol Biol 6, 122–130.
12. Sánchez, B., Urdaci, M.C., and Margolles, A. (2010). Extracellular proteins secreted by
probiotic bacteria as mediators of effects that promote mucosa-bacteria interactions.
Microbiology (Reading, England) 156, 3232–3242.
63

13. Sawitzke, J. a, Thomason, L.C., Costantino, N., Bubunenko, M., Datta, S., and Court, D.L.
(2007). Recombineering: in vivo genetic engineering in E. coli, S. enterica, and beyond.
Methods in Enzymology 421, 171–199.
14. Scheffers, D.-J., and Pinho, M.G. (2005). Bacterial Cell Wall Synthesis: New Insights from
Localization Studies. Microbiology and Molecular Biology Reviews 69, 585–607.
15. Snyder, L., and Champness, W. (2007). Molecular Genetics of Bacteria (3rd Edition)
(Washington DC, USA).
16. Swingle, B., Markel, E., Costantino, N., Bubunenko, M.G., Cartinhour, S., and Court, D.L.
(2010). Oligonucleotide recombination in Gram-negative bacteria. Molecular Microbiology
75, 138–148.
17. Thomas, C.M., Hong, T., Van Pijkeren, J.P., Hemarajata, P., Trinh, D. V, Hu, W., Britton, R.A.,
Kalkum, M., and Versalovic, J. (2012). Histamine derived from probiotic Lactobacillus reuteri
suppresses TNF via modulation of PKA and ERK signaling. PloS One 7, e31951.
18. Thomason, L., Court, D.L., Bubunenko, M., Constantino, N., Wilson, H., Datta, S., and
Oppenheim, A. (2005). Recombineering : Genetic Engineering in Bacteria Using Homologous
Recombination. Current Protocols in Molecular Biology 1–21.
19. van Pijkeren, J.-P., and Britton, R. a (2012). High efficiency recombineering in lactic acid
bacteria. Nucleic Acids Research 40, e76.
20. van Pijkeren, J.-P., Neoh, K.M., Sirias, D., Findley, A.S., and Britton, R.A. (2012). Exploring
optimization parameters to increase ssDNA recombineering in Lactococcus lactis and
Lactobacillus reuteri. Bioengineered 3, 209–217.

64

CHAPTER 3
EMPLOYING A LACTOBACILLUS REUTERI 6475 MUTANT LIBRARY TO SCREEN FOR GENES THAT
ENCODE FOR PROTEINS INVOLVED IN REUTERIN PRODUCTION AND SECRETION

INTRODUCTION

Many probiotic strains have been characterized by their capacity to generate antimicrobial
substances, which contribute to the clearance and the growth inhibition of pathogenic bacteria
in the intestine. This ability allows probiotic strains to outcompete for binding sites and to
colonize the intestinal epithelia (Ljungh and Wadstrom, 2006; Ventura et al., 2009). In addition,
this capability can be used as a criterion for new probiotic strain selection (FAO/WHO Group,
2002).

In particular, L. reuteri produce various types of antimicrobial substances. These compounds
are represented by low and high-weigh molecular compounds that have been extensively
studied according to their origin, chemical nature and spectrum antimicrobial effect (Lebeer et
al., 2008; Ouwehand, 2009; Šušković and Kos, 2010).

One

of

the

most-studied

low-molecular

weight

compounds

is

reuterin

or

3-

hydroxypropionaldehyde (3-HPA). This neutral and water soluble antimicrobial substance is
produced by several members of the Lactobacillus genus (Cleusix et al., 2007). Reuterin is the
result of a two-step glycerol fermentation process. In the first step, this antimicrobial

65

compound is produced as an intermediary product when glycerol is transformed to 3-HPA
(reuterin) by a cobalamin-dependent glycerol dehydratase enzyme. In the second step, 3-HPA is
then reduced to 1,3-PD by a NADH-oxidoreductase enzyme that allows the recycling of NADH
molecules generated during the fermentation of glucose. Therefore, when NADH cofactors are
produced, they are consumed to reduce 3-HPA to 1,3-PD and can provide a mechanism to
restore the redox balance during carbohydrate fermentation. A limited availability of
carbohydrate would favor the production of 3-HPA and on the contrary, higher levels would
redirect the pathway to the generation of 1,3-PD (Vaidyanathan et al., 2011). L. reuteri is
capable of secreting high levels of 3-HPA into the extracellular medium by mechanisms that are
poorly understood.

The large antimicrobial effect of reuterin against bacteria, yeast, fungi and viruses is explained
by the presence of a highly reactive aldehyde group. It has been demonstrated that the
aldehyde form interacts with thiol groups that form part of small molecules and proteins that
are involved in cell-survival and oxidative stress responses. Thus, reuterin affects the normal
growth and development of other microorganisms inducing an oxidative-stress response and
inhibiting essential enzymes that employ thiol groups in their active sites (Schaefer et al., 2010).

Even though the main mechanisms of action by which reuterin produces its antimicrobial
effects have been elucidated, little is known about other factors involved in its extracellular
secretion. In the current research project, the secretome mutant library of L. reuteri 6475 was
used to screen for genes that code for proteins that are involved in the production or secretion

66

of reuterin. These observed differences could provide hints regarding mechanisms involved in
the secretion and production of reuterin. In addition this study will allow for the exploration of
other factors that could affect the release of this molecule, either increasing or diminishing its
production.

MATERIALS AND METHODS

96-well colorimetric assay. The 96-well colorimetric assay was adapted from colorimetric assay
protocols described by Spinler et al. (2008) and Schaefer et al. (2010). In brief, mutants were
inoculated in 300 μl of MRS broth (BD Difco) in a Costar 96-culture plate flat bottom for 18 hrs
at 37°C using the GasPack EZ Anaerobe Container system. Wild-type L. reuteri 6475 and
negative controls were included. The next day, cells were harvested by centrifugation at 4,000
rpm for 10 minutes and washed twice with 50 mM potassium phosphate buffer pH 7.5.
Afterwards, cells were resuspended in 30 μl of 250 mM glycerol and incubated anaerobically for
2 hours at 37°C. Then, cells were spun down and supernatants were diluted by 10X. A 150 μl
reaction was prepared with 31 μl of 10X supernatant, 24 μl of 10 mM tryptophan solution and
95 μl of concentrated chloride acid. This mix was incubated for approximately 10 minutes
yielding a purple color. Under the presence of chloride acid, the aldehyde group of reuterin
interacts with tryptophan to form a β-carboline chemical compound that oxidizes and yields a
purple pigment. The negative controls were prepared using wild-type L. reuteri 6475 incubated

67

with sterile water as a substitute for glycerol. The colorimetric reactions were measured using
the Perkin Multimode Plate Reader at 560 nm.

Cuvette-colorimetric assay. The cuvette-colorimetric assay is similar to what was previously
described in the 96-well colorimetric assay. Here each recombineering mutant strain was
seeded in 20 ml of MRS media (BD Difco) at 37°C for approximately 18 hrs under anaerobic
conditions. The next day, bacteria cultures were spun down and cell pellets were weighed and
recorded. Cells were washed twice using the 50mM phosphate buffer pH 7.5 and harvested by
centrifugation. Cell pellets were resuspended in 2 ml of 250 mM glycerol and incubated at 37°C
for 2 hours in anaerobic conditions. Then, cells were centrifuged and supernatants were filtered
using a 0.22 μm low-protein binding Millipore-filter unit. To measure the reuterin production of
each recombineering mutant, a 1,5 ml reaction was prepared using 300 μl of filtered
supernatants diluted by 10X, 225 μl of 10 mM tryptophan solution and 900 μl of 12M chloride
acid. Samples were incubated for 8 to 10 minutes and colorimetric reactions were measured at
560 nm using a Genesys 20 Thermo Scientific Spectophotometer.

The mutant strains reuterin values were adjusted to the weight of the wild-type L. reuteri 6475
cell pellet to reflect equivalent bacterial cell densities, using the formula: [wild-type cell pellet
weight/secretome mutant cell pellet weight] x secretome mutant reuterin production.

68

MIC reuterin assay. The Minimum Inhibitory Concentration (MIC) reuterin assay was
performed based on the protocol described by Schaefer et al. (2010). In this experiment, cellfree reuterin supernatants were diluted by two-fold in a Costar 96-culture plate flat bottom to
4

yield a total volume of 100 μl. To determine the MIC assays values 100 μl of 10 Escherichia coli
DH5-α cells/ml were added to each well. E. coli cells were obtained when cultured in 10 ml of
LB broth (BD Difco) at 37°C under continuously aeration for approximately 4 hours until achieve
8

an optical density 0.9-1.0 at 600 nm (10 cells/ml), in which they were diluted to reach a
4

concentration of 104 cells/ml. Positive controls were prepared mixing 10 Escherichia coli DH5α cells/ml with sterile water in equal parts. The 96-plate was incubated at 37°C under aerobic
conditions for 24 hrs. The next day, results were read using the Perkin Multimode Plate Reader
at an optical density of 600 nm. Optical density values that range from 0.00 to 0.05 were
interpreted as negative for E. coli growth. The number of reuterin units was estimated by
calculating the reciprocal of the last sample dilution that inhibited E. coli cell growth.

RESULTS

The mutant library was employed to study the impact of secretome genes involved in the
production and secretion of the antimicrobial compound reuterin.

69

In the first stage, this study used a 96-well reuterin colorimetric assay adapted from the
protocols described by Schaefer et al. (2010) and Spinler et al. (2008) to investigate the
differences between the members of the L. reuteri secretome library in terms of their reuterin
phenotype. In this experiment, the 127 secretome mutants were subjected to reuterin
production by their incubation on a glycerol solution under anaerobic conditions. From the
total number of 127 secretome genes, we identified 25 high producer reuterin strains and 3 low
producer strains, compared to wild-type L. reuteri 6475 reuterin values. The detail of these
results can be found in the supplementary information section of this chapter.

The results of the 96-colorimetric assay presented some variability in the replicates that can be
attributed to the difficulty of handling small volumes and variations in the size of the cell pellet.
In order to confirm the 96-well colorimetric preliminary results we used a second approach
based on a cuvette colorimetric assay. In this experiment, 11 secretome strains were confirmed
out of the 25 putative high producers that were previously identified. The high-producer strains
generated between 66% and 123% more reuterin compared to wild-type L. reuteri values. The 3
low reuterin producers remain unchanged, and were catalogued as non-reuterin producer
strains. The differences between wild-type L. reuteri 6475 and secretome library mutants were
statistically significant with p<0.01. Figure 3.1 and Table 3.1 show the levels of reuterin
produced by each strain that were obtained employing the cuvette colorimetric assay.

Additionally, some of the secretome mutants that showed a different reuterin phenotype in the
colorimetric experiments were investigated using a Minimum Inhibitory Concentration (MIC)

70

Assay to estimate the number of reuterin units produced per milliliter (Schaefer et al., 2010). In
a single experiment, we selected 5 high reuterin producer strains that harbor a knock-out
mutation on genes HMPREF0536_0212, 0219, 0016, 0091 and 0027. The mutant reuterin levels
were compared to wild-type L. reuteri 6475 reuterin levels. This assay revealed that the high
reuterin producer strains showed 2-fold increase in inhibiting Escherichia coli growth, as

560 nm reuterin production

predicted by the colorimetric assays.

18

*

16
14
12

*

*

*

*

*

*

*

*
*

*

10
8
6
4
2
0

Secretome mutant library genes
Figure 3.1. Reuterin values obtained from secretome mutant strains using a cuvette
colorimetric assay. Reuterin values were obtained at an optical density of 560 nm. The mutant
strain reuterin values were adjusted to the weight of the wild-type L. reuteri 6475 cell pellet to
reflect equivalent number of cells. Mutant strains HMPREF0536_1329, 329 and 972 were
included as controls. Error bars represent calculated standard deviations. Experiments were
repeated at least 3 times independently. * p < 0.01 versus WT L. reuteri 6475 reuterin values.

71

Table 3.1. Reuterin values estimated using a cuvette-colorimetric reuterin assay.

a

Secretome gene
Locus Tag

Reuterin production
Average
SD

b
c

Gene product name

Statistically significant higher reuterin producers
HMPREF0536_0232

10.50

1.11

Conserved hypothetical protein

HMPREF0536_1905

10.78

2.38

Hydrolase protein of hypothetical function

HMPREF0536_0212

11.21

0.67

Glycosyltransferase

HMPREF0536_0200

11.39

1.89

PSE family sulfate exporter

HMPREF0536_1069

11.57

2.69

Conserved hypothetical protein

HMPREF0536_0091

11.94

2.54

cAM373 family protein

HMPREF0536_1290

12.74

3.59

Methyl-accepting chemotaxis protein

HMPREF0536_0027

13.06

2.16

Arginine/ornithine APC transporter

HMPREF0536_0219

13.46

1.37

HMPREF0536_1736

13.45

3.11

Phosphate ABC-ATP binding cassette
transporter
Inosine-5'-monophosphate dehydrogenase

HMPREF0536_0016

14.11

1.62

Lipopolysaccharide biosynthesis protein

Non-reuterin producers
HMPREF0536_0973

0.00

Sortase

HMPREF0536_1536

0.00

Cobalamin biosynthesis protein CbiM

HMPREF0536_1535

0.00

Cobalt ABC-ATP binding cassette
transporter

WT L. reuteri 6475 reuterin values
WT L. reuteri 6475

6.30

1.67

WT L. reuteri 6475 H2O

0.00

0.00
72

Table 3.1 (cont’d)
a Integrated Microbial Genomes (IMG) Locus Tag accession number of L. reuteri 6475
secretome genes disrupted by single-stranded DNA recombineering.
b Relative reuterin production measured at an optical density of 560 nm. Experiments were
performed in triplicate.
c Gene product name assigned based on the COG domain analysis provided by the IMG
database.
WT: Wild type L. reuteri 6475; WT H20: Wild type L. reuteri 6475 negative control; SD: Standard
deviation.

Based on the results described above, the functions of the disrupted genes that code for
proteins that influence the secretion of reuterin were explored based on COG and Pfam analysis
(Table 3.1). Among the mutant strains that have shown an increased reuterin production (11),
three of these genes code for proteins involved in the amino acid and ion transport
(HMPREF0536_0027, 0200 and 0219), and another two code for cell-wall and cytoplasm
membrane biogenesis proteins (HMPREF0536_0016 and 0212). The rest of the proteins (6)
have been identified as conserved hypothetical proteins or as proteins which functions have
been poorly characterized among bacteria.

In relation to the secretome mutants that showed deficient reuterin secretion (3), one encodes
for a sortase enzyme (HMPREF0536_0973), and the other two encode for proteins involved in
the transport and metabolism of the vitamin B12) and are located in consecutive positions in
the second replichore (HMPREF0536_1535 and 1536).

73

DISCUSSION

The generation of null mutations in the secretome genes of L. reuteri 6475 using ss DNA
recombineering has allowed the exploration of the role of cell-wall associated proteins and
secreted factors in reuterin production and secretion. Using 96-well and cuvette colorimetric
assays, we were able to screen 127 mutant strains and detect their differences in reuterin
secretion when they were incubated under the presence of glycerol. Through analysis of the
culture supernatants we detected 3 mutant strains that are completely deficient in reuterin
secretion and 11 mutant strains that produce more reuterin when compared to wild-type L.
reuteri 6475 reuterin values.

The three mutant strains that were unable to produce reuterin were disrupted in three
different genes that have an essential role in the secretion of this antimicrobial substance. Two
of these genes are involved in the transport and biosynthesis of cobalamin, and are found in the
same operon. Specifically, the gene HMPREF0536_1535 encodes for the CbiN cobalt ABC-type
protein transporter, which is essential to cobalt import (Pfam PF02553). The gene
HMPREF0536_1536 encodes for the CbiM protein which is essential for cobalamin biosynthesis,
although its exact function has not been elucidated (Pfam PF02553). As mentioned at the
beginning of the chapter, the activity of the glycerol dehydratase enzyme requires the presence
of the cobalamin cofactor in order to produce the 3-hydroxipropionaldehyde (3-HPA) form
(Vaidyanathan et al., 2011).

74

The fact that the genes HMPREF0536_1535 and 1536 encode proteins involved in the
dehydration of glycerol is not unexpected, since they are located in an approximately 46.5 Kb
gene cluster (from HMPREF0536_1520 to 1576) that contain the genes required for the
utilization of glycerol (HMPREF0536_1574) and propanediol (HMPREF0536_1571). Thus, the
utilization of glycerol by L. reuteri 6475 depends directly on cobalamin biosynthesis, and the
production of 3-HPA cannot take place if cobalamin or cobalt are absent. In general the
cobalamin-dependent activity of the glycerol dehydratase enzyme is widely distributed among
bacteria (Daniel et al., 1998), and it also has been reported in various Lactobacillus reuteri
strains such as CRL 1098, F275 and DSM 20016 (Sriramulu et al., 2008; Taranto et al., 2003;
Vaidyanathan et al., 2011).

A third secretome mutant strain that contains a mutation in gene HMPREF0536_0130 also
shows a deficient reuterin phenotype. This gene codes for a sortase protein (SrtA), which is an
enzyme in Gram-positive bacteria responsible for the attachment of external surface proteins
into the peptidoglycan wall. This enzyme recognizes a hydrophobic LPXTG amino acid motif,
which is also known as the sorting signal, on the C-terminus of proteins that have been targeted
for secretion. Once these proteins have been translocated across the cytoplasm membrane, the
sortase recognizes the sorting signal on them and cuts it. Then the sorting signal is linked to an
amino group localized on the cell-wall and the protein remains anchored to the peptidoglycan
structure (Snyder and Champness, 2007).

75

Based on the SrtA function, various L. reuteri 6475 sortase-dependent transport proteins would
remained unanchored if the sortase gene is knocked-out, thus affecting the secretion of
different compounds such as reuterin. To explore this hypothesis, we looked for sorting signals
in genes HMPREF0536_1535 and 1536 which are the only two other genes that produce a
reuterin deficient phenotype, however none of them harbor a sorting signal-like motif.

A reasonable strategy to identify putative reuterin protein transporters would be to search for
sorting signals in the group of proteins that were not able to be disrupted using
recombineering. After that, the genes that encode these proteins can be knocked-out by
recombineering or another mutational strategy, and their reuterin secretion activity can be
assessed by the colorimetric assays described before. In this sense, we would be able to detect
sortase-dependent proteins responsible for the transportation or production of reuterin in L.
reuteri 6475.

We have also identified 11 L. reuteri 6475 secretome strains that showed an increased reuterin
production. Based on the predicted functions of these genes it is not clear how they can affect
the reuterin secretion. From this group of genes, HMPREF0536_0016 and 0212 encode for
proteins related with the cell-wall and cytoplasm membrane biosynthesis, and the
HMPREF0536_0027, 0219, and 0200 are involved in the inorganic ion and amino acid transport.
Because these genes encode for transporters and cell-wall modeling proteins, it is reasonable
to speculate that when disrupted they can alter the fluidity of the membrane, and consequently

76

affect the reuterin secretion. Future work is required to understand how these genes affect
reuterin synthesis and/or secretion.

77

SUPPLEMENTAL INFORMATION

Table 3.2. Reuterin values estimated using a 96-colorimetric reuterin assay.
Secretome Gene
a

Locus Tag

b

Reuterin Production
Average
SD

Classification

HMPREF0536_0016

2.27

0.31

H

HMPREF0536_0027

2.18

0.20

H

HMPREF0536_0056

2.01

0.38

H

HMPREF0536_0091

2.17

0.42

H

HMPREF0536_0200

2.20

0.30

H

HMPREF0536_0212

2.33

0.45

H

HMPREF0536_0213

2.20

0.46

H

HMPREF0536_0219

2.43

0.49

H

HMPREF0536_0232

2.17

0.21

H

HMPREF0536_0329

1.77

0.49

H

HMPREF0536_0554

1.89

0.53

H

HMPREF0536_0658

2.20

0.35

H

HMPREF0536_0679

2.51

0.56

H

HMPREF0536_0934

2.43

0.33

H

HMPREF0536_0972

1.82

0.54

H

HMPREF0536_0973

0.36

0.03

L

HMPREF0536_1069

2.07

0.41

H

HMPREF0536_1269

2.39

0.30

H

HMPREF0536_1290

1.87

0.29

H

HMPREF0536_1329

1.79

0.35

H

HMPREF0536_1468

1.90

0.48

H

HMPREF0536_1535

0.47

0.06

L

HMPREF0536_1536

0.45

0.00

L

78

c

Table 3.2 (cont’d)
HMPREF0536_1736

2.04

0.36

H

HMPREF0536_1815

2.02

0.28

H

HMPREF0536_1880

1.87

0.49

H

HMPREF0536_1905

2.01

0.29

H

HMPREF0536_1908

1.81

0.35

H

WT L. reuteri 6475

1.27

0.39

WT L .reuteri 6475 H2O

0.27

0.06

WT L. reuteri 6475 reuterin values

Total

28

a Integrated Microbial Genomes (IMG) Locus Tag accession number of L. reuteri 6475
secretome genes disrupted by single-stranded DNA recombineering.
b Relative reuterin production measured at an optical density of 560 nm. Measurements were
repeated at least 5 times independently.
c High or low reuterin producer secretome mutants. Results were compared to WT L. reuteri
6475 reuterin values.
H: High producer; L: Low producer; WT: Wild type L. reuteri 6475; WT H20: Wild type L. reuteri
6475 negative control; SD: Standard deviation.

79

REFERENCES

80

REFERENCES
1. Cleusix, V., Lacroix, C., Vollenweider, S., Duboux, M., and Le Blay, G. (2007). Inhibitory
activity spectrum of reuterin produced by Lactobacillus reuteri against intestinal bacteria.
BMC Microbiology 7, 101.
2. Daniel, R., Bobik, T., and Gottschalk, G. (1998). Biochemistry of coenzyme B12-dependent
glycerol and diol dehydratases and organization of the encoding genes. FEMS Microbiology
Reviews 22, 553–566.
3. FAO/WHO Working Group (2002). Guidelines for the Evaluation of Probiotics in Food.
4. Lebeer, S., Vanderleyden, J., and De Keersmaecker, S.C.J. (2008). Genes and molecules of
lactobacilli supporting probiotic action. Microbiology and Molecular Biology Reviews :
MMBR 72, 728–764.
5. Ljungh, A., and Wadstrom, T. (2006). Lactic acid bacteria as probiotics. Current Issues in
Intestinal Microbiology 7, 73–90.
6. Ouwehand, A.C. (2009). Antimicrobial Components from Lactic Acid Bacteria. In Lactic Acid
Bacteria: Microbiological and Functional Aspects, (New York: CRC Press), p. 656.
7. Schaefer, L., Auchtung, T. a, Hermans, K.E., Whitehead, D., Borhan, B., and Britton, R.
(2010). The antimicrobial compound reuterin (3-hydroxypropionaldehyde) induces oxidative
stress via interaction with thiol groups. Microbiology (Reading, England) 156, 1589–1599.
8. Snyder, L., and Champness, W. (2007). Molecular Genetics of Bacteria (3rd Edition)
(Washington DC, USA).
9. Spinler, J.K., Taweechotipatr, M., Rognerud, C.L., Ou, C.N., Tumwasorn, S., and Versalovic, J.
(2008). Human-derived probiotic Lactobacillus reuteri demonstrate antimicrobial activities
targeting diverse enteric bacterial pathogens. Anaerobe 14, 166–171.
10. Sriramulu, D.D., Liang, M., Hernandez-Romero, D., Raux-Deery, E., Lünsdorf, H., Parsons,
J.B., Warren, M.J., and Prentice, M.B. (2008). Lactobacillus reuteri DSM 20016 produces
cobalamin-dependent diol dehydratase in metabolosomes and metabolizes 1,2-propanediol
by disproportionation. Journal of Bacteriology 190, 4559–4567.
11. Šušković, J., and Kos, B. (2010). Antimicrobial activity–the most important property of
probiotic and starter lactic acid bacteria. Food Technology and … 9862, 296–307.
12. Taranto, M.P., Vera, J.L., Hugenholtz, J., De Valdez, G.F., and Sesma, F. (2003). Lactobacillus
reuteri CRL1098 Produces Cobalamin. Journal of Bacteriology 185, 5643–5647.

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13. Vaidyanathan, H., Kandasamy, V., Gopal Ramakrishnan, G., Ramachandran, K., Jayaraman,
G., and Ramalingam, S. (2011). Glycerol conversion to 1, 3-Propanediol is enhanced by the
expression of a heterologous alcohol dehydrogenase gene in Lactobacillus reuteri. AMB
Express 1, 37.
14. Ventura, M., O’Flaherty, S., Claesson, M.J., Turroni, F., Klaenhammer, T.R., Van Sinderen, D.,
and O’Toole, P.W. (2009). Genome-scale analyses of health-promoting bacteria:
probiogenomics. Nature Reviews. Microbiology 7, 61–71.

82

CHAPTER 4
SUMMARY AND SIGNIFICANCE

The anecdotal successes of probiotics in treating diarrhea, colic and intestinal inflammation
(Francavilla et al., 2012; Niv et al., 2005; Savino et al., 2007) has spurred interest to the
exploration of mechanisms of action and the isolation of next-generation probiotics. The
probiotic market is expanding mainly through the use probiotics as supplements to regulate the
immune system and the intestinal function, thus improving the general health conditions. As
reviewed on Chapter 1, the studies in Lactobacillus reuteri have played an important role in the
generation of knowledge to understand various mechanisms of probiotics actions.

Nevertheless, our understanding of how probiotics function is still in its infancy. Several
researchers are attempting to understand probiotic mechanisms of action, but more studies are
necessary to describe the most important differences among probiotic strains and to gain a
deeper knowledge of host-probiotic interactions.

In particular, the Lactobacillus reuteri strain 6475 has demonstrated important probiotic
features that can be essential in the amelioration of different syndromes. For example, it can
reduce the levels of TNF (Thomas et al., 2012) and can decrease the expression of the
transcription factor NFκβ (Iyer et al., 2008). It also produces the antimicrobial compound
reuterin, which can inhibit pathogenic intestinal bacteria (Schaefer et al., 2010). All these

83

changes might positively generate crucial improvements in bone and intestinal health. However
there is little evidence showing these functions are critical for probiotic effects in vivo.

Considering that both anti-inflammatory compounds and reuterin are secreted, I have directed
the construction of a secretome mutant library of L. reuteri 6475 that will be a useful tool to
screen for proteins that might be essential in the secretion and production of these substances.
In particular, the selection of the secretome genes was engineered employing the SignalP
software (Nielsen and Krogh, 1998), which predicted a total number of 185 protein sequences
are secreted.

From this list of 185 proteins, 127 genes encoding these proteins were successfully disrupted
using single-stranded DNA recombineering. One possible reason that 58 genes were not
disrupted is because these genes may encode for proteins that are indispensable for the growth
of L. reuteri. Other possibilities reside in the fact that mutations could cause the bacteria to
grow so slowly that we were not able to detect these mutant colonies under our experimental
conditions.

An interesting observation found during the construction of this library is the difference among
replichores in the success of recombineering successfully disrupting genes. Although these
observations are not completely understood, the genes located on the second replichore had a
higher success rate of being disrupted (75% vs. 58% for the first replichore). At this point we do
not understand the reason for the replichore bias.

84

In Chapter 3, the secretome mutant library was employed to explore the role of secreted and
cell-wall associated proteins in the secretion and production of the antimicrobial compound
reuterin. In this study 11 mutant strains showed a 2-fold increased production of reuterin
compared to wild-type L. reuteri reuterin levels. Because several of these mutants encode for
proteins involved in the transport and cell-wall modeling is that it has been proposed that their
disruption could affect the integrity of the bacterial cell-wall and thus the fluidity of reuterin
secretion.

In addition, 3 secretome mutant strains were completely deficient for reuterin production. Two
of these proteins were identified as cobalamin precursors and one of them as a sortase.
Proteins involved in cobalamin production are essential for reuterin production, since this
compound is produced by a glycerol dehydratase cobalamin-dependent enzyme. The fact that a
sortase mutant was non-reuterin producer provides evidence that a secretome protein(s) that
carry LPXTG motifs have relevant function in relation to the secretion of this molecule.

Finally, we forecast that the employment of this mutant library will be essential to identify new
secreted molecules and cellular-anchored proteins that could have a relevant role in
colonization, inhibition of pathogenic bacteria and regulation of immune responses in the host.
In a close future these characters can be employed as biological markers to identify similar
marks in other probiotic bacteria.

85

REFERENCES

86

REFERENCES
1. Francavilla, R., Lionetti, E., Castellaneta, S., Ciruzzi, F., Indrio, F., Masciale, A., Fontana, C., La
Rosa, M.M., Cavallo, L., and Francavilla, A. (2012). Randomised clinical trial: Lactobacillus
reuteri DSM 17938 vs. placebo in children with acute diarrhoea-a double-blind study.
Alimentary Pharmacology & Therapeutics 36, 363–369.
2. Iyer, C., Kosters, A., Sethi, G., Kunnumakkara, A.B., Aggarwal, B.B., and Versalovic, J. (2008).
Probiotic Lactobacillus reuteri promotes TNF-induced apoptosis in human myeloid
leukemia-derived cells by modulation of NF-kappaB and MAPK signalling. Cellular
Microbiology 10, 1442–1452.
3. Nielsen, H., and Krogh, A. (1998). Prediction of signal peptides and signal anchors by a
hidden Markov model. Proc Int Conf Intell Syst Mol Biol 6, 122–130.
4. Niv, E., Naftali, T., Hallak, R., and Vaisman, N. (2005). The efficacy of Lactobacillus reuteri
ATCC 55730 in the treatment of patients with irritable bowel syndrome--a double blind,
placebo-controlled, randomized study. Clinical Nutrition (Edinburgh, Scotland) 24, 925–931.
5. Savino, F., Pelle, E., Palumeri, E., Oggero, R., and Miniero, R. (2007). Lactobacillus reuteri
(American Type Culture Collection Strain 55730) versus simethicone in the treatment of
infantile colic: a prospective randomized study. Pediatrics 119, e124–30.
6. Schaefer, L., Auchtung, T. a, Hermans, K.E., Whitehead, D., Borhan, B., and Britton, R.
(2010). The antimicrobial compound reuterin (3-hydroxypropionaldehyde) induces oxidative
stress via interaction with thiol groups. Microbiology (Reading, England) 156, 1589–1599.
7. Thomas, C.M., Hong, T., Van Pijkeren, J.P., Hemarajata, P., Trinh, D. V, Hu, W., Britton, R.A.,
Kalkum, M., and Versalovic, J. (2012). Histamine derived from probiotic Lactobacillus reuteri
suppresses TNF via modulation of PKA and ERK signaling. PloS One 7, e31951.

87