This document summarizes research on stress responses in Lactic Acid Bacteria (LAB) like Lactobacillus that are used as probiotics. It discusses how probiotics must survive multiple stresses to confer health benefits. Major stress response mechanisms in LAB are regulated by proteins like HrcA and CtsR. Studies have identified genes involved in responses to heat, cold, acidity, oxidation, starvation and other stresses. Improving stress responses through genetic engineering, like overexpressing stress genes, could produce more robust probiotic cultures better able to deliver health benefits.

1. STRESS RESPONSES IN LACTOBACILLI:
IMPLICATIONS IN PROBIOTICS
Diwas Pradhan
Dairy Microbiology Division
ICAR-National Dairy Research Institute
April, 2012

2. INTRODUCTION
• By definition,
probiotics must
reach their target
site alive to
confer a health
benefit to the
host
• A plethora of
stresses needs to
be encountered

3. • LaCOG analyses employed to identify a set of LAB genes associated with
various stress responses.
• HrcA involved in the control of the heat-shock protein expression
• Heat-shock proteins commonly under HrcA control and performing
chaperonin-like functions (GroELS, DnaJK, GrpE) are universally
conserved.
• The involvement of CtsR in the regulation of class III stress proteins,
including the Clp proteases and related functions.
• The corresponding Clp proteases appear to be universally present in
these LAB genomes.
• Majority of stress-tolerance genes are conserved among strains of a
particular species
• strain-specific survival capacities depend on their relative levels of expression
rather than their presence or absence.
Lactic Acid Bacteria Genomics

4. • The major HSPs:
o The classical chaperones DnaK, GroEL, and GroES (participate
in protein folding, protein translocation, and possibly higher-
order protein assembly)
o The Clp family of proteins (disposing heat damaged proteins)
play an indispensable role in protein quality
o Regulated by HrcA and CtsR regulon
• HtrA, FtsH, sHSPs, RecA, and Other Important Factors
Responses to Heat Stress
Varmanen, P. and Savijoki, K., 2011

5. • A number of CSPs are induced
• Function as transcriptional and translational regulators
• Also act as molecular chaperones
• Three csp genes (cspL, cspC, and cspP) identified in
Lactobacillus plantarum NC8. Overproduction causes distinct
phenotypic effects
• CspL overproduction transiently alleviated the cold-shock impairment
of growth
• CspP overproduction enhanced the cryotolerance
• CspC overproduction improves growth adaptation at optimal
temperatures
Responses to Cold Stress
Capozzi, V., 2011

6. • Also induced heat-responsive genes sHSP
suggesting a cross-talk between the heat-
and cold-stress responses
• An increase in C16:0 and C18:2 fatty acids in
Lactobacillus acidophilus grown at low
temperature
Responses to Cold Stress
Capozzi, V., 2011

7. Cold-shock Response In Bacillus
subtilis
• Biological modifications
related to temperature
perception and signal
transduction (red)
• Membrane adaptation
(yellow)
• Nucleoid structure and
transcription (blue)
• CSPs and translation
apparatus (green)
• Metabolism, protein
folding, and cell
differentiation (violet)

8. • Oxygen, Superoxide, Hydrogen Peroxide and the
Hydroxyl Radical(HO˙)
• SOD, thioredoxin reductase, peroxidase, glutathione
reductase and RecA
• Incorporation of high concentrations of manganese
by Lb. plantarum
• SOD or manganese produce H2O2 from O2˙−.
Responses to Oxidative Stress
Cesselin, B., et al., 2011

9. Lb. plantarum, jonsohnii and casei
• Encodes cydABCD genes.
• Require heme and a menaquinone to activate respiration metabolism
A Respiratory Chain In LAB

10. • Activity and pH optima of the proton translocating
(H)-ATPase
• Acid tolerance response(ATR)
a. Decrease fluidity of CM- increase the concentration of
saturated FAs or cyclopropane FA(C19:0)
b. Saturated to unsaturated FAs ratio- from 0.4 to 4.9
• Malate and Histidine contribute to acid adaptation
• His contributes to intracellular buffering
Acid stress
Broadbent, J. R., et. al., 2010

11. • Two-component regulatory systems (2CRS) and
ABC-type oligopeptide transport proteins
– (Opp) known to function as sensors for
environmental change
• Differential expression of transposase genes
• Metabolism, Information storage, processing and
Cellular processes genes were significantly
altered
Acid stress

12. Model for Physiological Responses of GG to Bile
Stress.
Koskenniemi, K. et al., 2011

13. • Self-Immunity: Immunity proteins on their cytoplasmic
membrane for protection against the produced
bacteriocin.
• NisI and SpaI- lipoproteins anchored to the membrane surface
via a lipid-modified N-terminal cysteine residue.
• ABC transporter(LanFEG)
• The two-component systems (TCSs) and extracellular
sigma factors are involved in the intensive action toward
cell wall active antibiotics.
Responses to Bacteriocins and Other Antimicrobials
Asaduzzaman, S. M. and Sonomoto, K., 2011

14. • Carbohydrate exhaustion ushers LAB into the NC state
• Resuscitation has not been demonstrated yet.
• Switch to the catabolism of amino acids using
aminotransferases (ATases)
• Many of the regulatory or quorum-sensing molecules are not
produced.
• The accumulation of compatible molecules enables cellular stability
• Loss of the ability to produce colonies
• Accompanied by the production of Met and Ser into the medium
Responses to Starvation
Weimer, B. C., 2011

15. • Majority of analyses have generally focused on
one particular stress
• Need for network reconstruction based on
multiple stress-induced transcriptome profiles
• Not been reported for LAB to date
• Will help reveal the regulatory networks and
complete regulons involved.
Assessment of Multiple Stress
Responses
and Regulatory Network
Reconstruction
Bron, P. A., 2011

16. 1. In Vitro approaches to identify Robustness
Genes in Lactic Acid Bacteria
• Comparative Genomic Hybridization
• DNA microarray technology
2. In Vivo Expression Technology
Functional Genomics Approaches to
Unravel Lactic Acid Bacteria Stress
Responses
Bron, P. A. et. al., 2011

17. Zomer, A. and Sinderen, D. van., 2010
Stress Gene Regulatory Network Of
B. breve UCC2003

18. • The CGH approach employs
one-directional comparison
of gene-content profiles per
strain using genome wide
microarrays that are
designed on the basis of the
genome of a single strain.
• Enables the construction of
high-resolution genome
wide presence-absence
patterns for each of the
strains that is analyzed.
Comparative Genomic
Hybridization
genome sequence
of a robust strain
DNA of a robust
strain
DNA of the
strain
Sequence present in both (grey)
Sequence present in robust strain
but normal strain (black)

19. 19
Widely used technology
to identify several of the
(conserved) genetic
factors regulated during
stress imposed on LAB
DNA microarray technology

20. Proteomics

21. • Powerful method that
allow the genome
wide identification of
in vivo induced (ivi)
promoters and their
corresponding genes
utilizing a promoter
trapping system.
In Vivo Expression
Technology(IVET)
Rediers, H. et al., 2005

22. Strategic Approach To Identify And Validate
Stress Tolerance Associated Genes And
Functions

23. Pregenomics Approaches
 Additives
 Pre-adaptation
 Cross-Protection
Postgenomics Strategies
 Overexpression of Stress Regulon Members by Genetic
Modification
 Targeted Mutagenesis of Stress Regulators
 Fermentation-Enhanced Probiotic Function
Improving Robustness
Bron, P. A., et. al., 2011

24. ADDITIVES
A straightforward way to improve stress response
Glucose, fructose, Tween-80, betaine, gum acacia etc.
PREADAPTATION
LAB have developed several conserved stress
responses
The CtsR and HrcA regulators
Overlap in different stress regulons

25. Overexpression of Stress Regulon Members
by Genetic Modification
Alteration of expression levels of
genes encoding stress regulon members or regulators
prior to stress exposure
Targeted Mutagenesis of Stress Regulators
Manipulation of complete stress regulons by
targeting the canonical stress regulator CtsR

26. • Exploits stress response systems of pathogenic bacteria.
• Heterologous expression of BetL from L. monocytogenes in
the Lactobacillus salivarius UCC118 resulted in a strain with
improved resistance to numerous stresses.
• A novel bile resistance mechanism- BilE
1. Operates by excluding bile from the cell
2. Not many homologues of the bilE operon have been identified in
any of the genomes of the probiotic organisms
Pathobiotechnology
Mills, S., et. al., 2011

27. Fermentation-Enhanced Probiotic
Function

28. Stress in the Context of
Microbial Communities
Stress and the Cell
Cycle
Stress and DNA
Damage Control
Stress-Induced
Mutagenesis
Stress in the Context
of the Single Cell
Sensing and Signaling
Stress
Papadimitriou, K., and Kok, J., 2011

29.  The ability of probiotic bacteria to survive the harsh
environments has been a major factor in their selection
criteria.
 Induction of the probiotic stress response through pre-
adaptation strategies- not always successful.
 Developing a molecular toolbox (through
pathobiotechnology, targeting indigenous defense
strategies), should ensure that the most functionally
active strains can be confidently selected for probiotic
development.
CONCLUSIONS

30.  Apprehensions on the use of Genetically Modified
organisms.
 Studies which evaluate the safety of engineered
probiotics are crucial if the technology is to gain
acceptance.
 In this way next-generation probiotic cultures will be
better equipped to face technological and
gastrointestinal challenges as well as meeting medical
demands
Cont.