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Frontiers in Microbiology 01 frontiersin.org
Eect of NaCl stress on
exoproteome profiles of Bacillus
amyloliquefaciens EB2003A and
Lactobacillus helveticus EL2006H
JudithNaamala , SowmyalakshmiSubramanian ,
LeviniA.Msimbira and DonaldL.Smith *
Department of Plant Science, McGill University, Montreal, QC, Canada
Salt stress can aect survival, multiplication and ability of plant growth promoting
microorganisms to enhance plant growth. Changes in a microbe’s proteome
profile is one of the mechanisms employed by PGPM to enhance tolerance of
salt stress. This study was focused on understanding changes in the exoproteome
profile of Bacillus amyloliquefaciens EB2003A and Lactobacillus helveticus
EL2006H when exposed to salt stress. The strains were cultured in 100 mL M13 (B.
amyloliquefaciens) and 100 mL De man, Rogosa and Sharpe (MRS) (L. helveticus)
media, supplemented with 200 and 0 mM NaCl (control), at pH 7.0. The strains
were then incubated for 48 h (late exponential growth phase), at 120 rpm and
30 (B. amyloliquefaciens) and 37 (L. helveticus) °C. The microbial cultures were
then centrifuged and filtered sterilized, to obtain cell free supernatants whose
proteome profiles were studied using LC–MS/MS analysis and quantified using
scaold. Results of the study revealed that treatment with 200 mM NaCl negatively
aected the quantity of identified proteins in comparison to the control, for both
strains. There was upregulation and downregulation of some proteins, even up to
100%, which resulted in identification of proteins significantly unique between the
control or 200 mM NaCl (p ≤ 0.05), for both microbial species. Proteins unique
to 200 mM NaCl were mostly those involved in cell wall metabolism, substrate
transport, oxidative stress tolerance, gene expression and DNA replication and
repair. Some of the identified unique proteins have also been reported to enhance
plant growth. In conclusion, based on the results of the work described here,
PGPM alter their exoproteome profile when exposed to salt stress, potentially
upregulating proteins that enhance their tolerance to this stress.
KEYWORDS
PGPM, Lactobacillus helveticus, Bacillus amyloliquefaciens, salt stress, exoproteome
profile
1. Introduction
Plant growth promoting microorganisms (PGPM) and their derivatives are key technology
sources for sustainable agriculture, especially with the urgent need to slow climate change and
its adverse eects (Naamala and Smith, 2020). For centuries, use of PGPM based inoculants to
sustainably enhance plant growth and increase yield, under stressed and ideal conditions has
been practiced in dierent parts of the world (Babalola and Glick, 2012; Bashan etal., 2014;
García-García etal., 2020; Naamala etal., 2023). e ability of PGPM and or their derivatives
OPEN ACCESS
EDITED BY
Woo-Suk Chang,
University of Texas at Arlington, UnitedStates
REVIEWED BY
Do-Won Jeong,
Dongduk Women’s University, Republic of
Korea
Pushp Sheel Shukla,
Dalhousie University, Canada
Klára Kosová,
Crop Research Institute, Czechia
*CORRESPONDENCE
Donald L. Smith
donald.smith@mcgill.ca
RECEIVED 17 April 2023
ACCEPTED 31 July 2023
PUBLISHED 28 August 2023
CITATION
Naamala J, Subramanian S, Msimbira LA and
Smith DL (2023) Eect of NaCl stress on
exoproteome profiles of Bacillus
amyloliquefaciens EB2003A and Lactobacillus
helveticus EL2006H.
Front. Microbiol. 14:1206152.
doi: 10.3389/fmicb.2023.1206152
COPYRIGHT
© 2023 Naamala, Subramanian, Msimbira and
Smith. This is an open-access article distributed
under the terms of the Creative Commons
Attribution License (CC BY). The use,
distribution or reproduction in other forums is
permitted, provided the original author(s) and
the copyright owner(s) are credited and that
the original publication in this journal is cited,
in accordance with accepted academic
practice. No use, distribution or reproduction is
permitted which does not comply with these
terms.
TYPE Original Research
PUBLISHED 28 August 2023
DOI 10.3389/fmicb.2023.1206152
Naamala et al. 10.3389/fmicb.2023.1206152
Frontiers in Microbiology 02 frontiersin.org
to enhance plant growth is associated with their ability to exude in
their growth environment, proteins and metabolites with plant growth
promoting characteristics (Prithiviraj etal., 2003; Gray etal., 2006b;
Schwinghamer etal., 2016; Piechulla etal., 2017).
Salinity stress is a major global constraint to crop production,
aecting both plant yield quality and quantity. Although PGPM can
mitigate the eects of salinity stress on plants, it can also aect the
ability of PGPMs to enhance plant growth and may lead to microbial
death in case of exposure to levels beyond those tolerated (Zahran,
1997; Soussi etal., 2001; Nadeem etal., 2015; Naamala etal., 2022,
2023). Some microbes have developed mechanisms for surviving at
high salt concentrations. e ability of microbes to tolerate saline
conditions is in part dependent on their ability to regulate salt
concentration in their cytoplasm, in relation to that of their growth
environment. Mechanisms employed to regulate salt concentration
within the microbe include accumulation of osmolytes such as
glutamate and proline in their cytoplasm through de novo synthesis or
uptake from their growth environment (Zahran, 1997; Soussi etal.,
2001; Oren, 2008; Bojanovic etal., 2017), upregulation of iron uptake
mechanisms such as production of siderophores (Bojanovic etal.,
2017), alteration of their cell membrane composition (Bojanovic etal.,
2017; Hachicho et al., 2017), and maintenance of a high KCl
concentration in their cytoplasm to match that of their growth
medium (Oren, 2002; Oren, 2008). Eecting these mechanisms may
necessitate the microbe to make changes to its genome, proteome, and
metabolome proles, most probably upregulating those components
of each essential for enhancing salt tolerance mechanisms.
Protein expression occurs when genes are transcribed into
messenger RNA (mRNA), which is then translated into proteins, which
are major constituents of microbial cells (Karpievitch et al., 2010;
Zhang etal., 2010). Protein expression and secretion are usually in
response to either internal or external stimuli such as exposure to biotic
and abiotic stress (Zhang etal., 2010; Armengaud etal., 2012; Schoof
etal., 2022). e microbial proteome loosely translates to all proteins
associated with a given microbe. e microbial exoproteome refers to
proteins found in the immediate extracellular milieu of a microbe,
arising from active cellular secretion, passive excretion and or cell lysis
(Desvaux etal., 2010; Armengaud etal., 2012; Rubiano-Labrador etal.,
2015; Schoof et al., 2022). For microbes cultured in laboratories,
microbial exoproteome would refer to total proteins in spent media
aer removal of all microbial cells through centrifugation and ltration.
Exoproteome composition reects a microbe’s physiological state at a
given time and can provide insight into a microbe’s interactions with
its surroundings (Armengaud etal., 2012). Abiotic stresses such as
salinity, acidity and alkalinity aect the quantity and quality of proteins
synthesized and expressed by a microbe at a given time (Singleton
etal., 1982; Soussi etal., 2001; Msimbira etal., 2022). Exploring the
exoproteome of a microbe exposed to salt stress can provide insight
into a set of proteins expressed in response to salt stress, which could
then enhance our understanding of salt tolerance mechanisms in
microbes (Rubiano-Labrador etal., 2015). In general, the ‘omics’
studies of biological systems have resulted in better understanding of
microbes and their environment (Karpievitch etal., 2010). Advances
in technology, such as invention of high through put tandem mass
spectrometry and liquid chromatography have allowed for easy
identication, analysis, classication, and function annotation of
complex protein samples (Listgarten and Emili, 2005; Zhang etal.,
2010; Armengaud, 2013; Kucharova and Wiker, 2014; Msimbira etal.,
2022). It is interesting to note that while some PGPM may lose their
ability to enhance plant growth following exposure to salt stress, others
may gain or bemore eective at enhancing plant growth, aer exposure
to some level of stress (Subramanian et al., 2021). erefore,
understanding how microbial exoproteome proles change with
changes in salt stress can improve utilization of CFS as plant growth
biostimulants, and enhance our elucidation of mechanisms employed
to enhance plant growth and or tolerate salt stress, given that some
proteins such as enzymes play a vital role in stress tolerance and plant
growth stimulation (Ahmad etal., 2010).
Bacillus amyloliquefaciens are rod shaped endospore forming gram
positive bacteria from the genus Bacillus and family Baciliaceae
(Woldemariam etal., 2020; Ngalimat etal., 2021). B. amyloliquefaciens is
widely used in the food, pharmaceutical and agricultural sectors
(Woldemariam etal., 2020). B. amyloliquefaciens and its derivatives have
been reported to enhance plant growth under stressed and ideal
conditions (Cappellari and Banchio, 2020; Cappellari et al., 2020; Duan
etal., 2021; Kazerooni etal., 2021; Naamala etal., 2022). Lactobacillus
helveticus is a gram positive facultative anaerobic lactic acid bacterium
(LAB) that is widely used in the food processing industry. However,
L. helveticus and its derivatives have also been reported to enhance plant
growth (Naamala etal., 2023). is study was focused on understanding
changes in exoproteome proles of B. amyloliquefaciens EB2003A and
L. helveticus EL2006H, at 0 mM NaCl and 200 mM NaCl. Results from
previous studies have shown that CFSs of both strains, when exposed to
200 mM NaCl, enhanced germination and radicle length of soybean, and
corn, as well as growth variables of potato (Naamala etal., 2022, 2023).
Wetherefore point out some of the proteins identied in this study, that
have been reported to enhance plant growth, as well as some of the
mechanisms plants employ to support growth.
2. Materials and methods
2.1. Obtaining protein samples
B. amyloliquefaciens EB2003A and L. helveticus EL2006H, which
were generously provided by EVL Inc., were cultured in 100 mL M13
and 100 mL De man, Rogosa and Sharpe (MRS) media, respectively,
supplemented with 200 and 0 mM NaCl (control), at pH 7.0. ey
were incubated for 48 h (late exponential growth phase), at 120 rpm
and a temperature of 30 and 37°C, for B. amyloliquefaciens EB2003A
and L. helveticus EL2006H, respectively. Four replicates per treatment
per species were cultured. Microbial cultures were then centrifuged,
using a Sorvall Biofuge Pico (Mandel Scientic, Guelph, ON, Canada),
for 10 min, at 10,000 rpm (15,180× g; SLA-1500) and 4°C, to pellet the
microbial cells and separate them from the cell-free supernatant (CFS)
(Gray etal., 2006a; Subramanian etal., 2021). e CFS was further
vacuum ltered using 0.22 μm nylon lters to ensure that all bacterial
cells were removed. Trichloroacetic acid (TCA; T9151, Sigma Aldrich)
precipitation was used to extract total proteins from the obtained CFS
replicates. e CFS was mixed with 100% (w/v) TCA, in 250 mL
conical asks to create a 25% working solution of TCA. e mixture
was incubated at −20°C for 1 h, and transferred to an orbital shaker
(MBI, Montreal Biotech Inc., Canada) with shaking speed of 90 rpm,
in a cold room, at a temperature of- 4°C, for protein precipitation,
overnight. is was followed by a 10 min centrifugation at 4°C and
10,000 rpm, to pellet the protein. e obtained protein pellet was then
Naamala et al. 10.3389/fmicb.2023.1206152
Frontiers in Microbiology 03 frontiersin.org
washed with ice-cold acetone, air-dried under a laminar ow hood,
and dissolved in 2 M urea (U4883, Sigma Aldrich). e protein
obtained from the four replicates of each treatment were pooled to
form one sample per species. e experiment was repeated four times
to get the appropriate biological replicates. Concentration of proteins
obtained from the four experiments was determined, using the Lowry
method (Lowry etal., 1951).
2.2. LC–MS/MS protein profiling
Aer determining the concentration of the obtained proteins,
10 μg protein per sample was dissolved in 20 μL of 2 M urea and sent
to Montreal Clinical Research Institute (IRCM), for liquid
chromatography mass spectrometry (LC–MS/MS) analysis. Total
proteins were digested using trypsin enzyme and injected into an LC–
MS/MS equipped with Linear Trap Quadrupole Velos Orbitrap
(ermo Fisher Waltham, MA, UnitedStates). e data set obtained
from the mass spectra were searched against Bacillus spp. and
Lactobacillus spp. databases, using Mascot soware (Matrix Science,
London, UnitedKingdom). Scaold Soware (version 5.1.2, Proteome
Soware Inc., Portland OR) was used to validate the obtained MS/MS
based peptides and proteins, using an equal to or greater than 95%
acceptance of protein probability, with a minimum of two peptides
and 95% peptide probability (Keller etal., 2002).
2.3. Quantitative data analysis
Proteomic data for identied proteins obtained from the LC–MS/
MS analysis was quantitatively analyzed, based on spectra count values,
using Scaold 5 (Scaold Soware for MS/MS Proteomics). Spectra
count values were normalized and subjected to analysis of variance, at
the 5% signicance level, using a Benjamini-Hochberg multiple test
correction, to detect signicant dierences between treatments.
Signicance was based on both sher’s exact test (p ≤ 0.05) and fold
change of more than or equal to 1.2. FASTA les generated from
Scaold 5 were analyzed using OmicsBox for functional annotation
and interpretation of the protein sequences. Volcano plots were created
using OriginPro soware (OriginPro learning edition, version 2023
learning edition) while Venn diagrams were generated using Scaold
soware for MS/MS proteomics. e LC–MS/MS proteomic data are
available in the Mass Spectrometry Interactive Virtual Environment
(MassIVE) at doi:10.25345/C5PG1HZ4M and PXD041778 for Bacillus
amyloliquefaciens EB2003A, and doi:10.25345/C54B2XF6V and
PXD041177 for Lactobacillus helveticusel2006h.
3. Results
3.1. Exoproteome analysis for Bacillus
amyloliquefaciens EB2003A
Based on scaold and OmicsBox analyzes of the LC–MS data,
there were variations in identied proteins for CFS of
B. amyloliquefaciens EB2003A cultured at 0 mM NaCl and 200 mM
NaCl, as shown in Table1 and Supplementary Table S1. In general,
NaCl lowered the quantity of identied proteins, total unique spectra,
and total unique peptides, as shown in Figure1, visualized at 95%
protein threshold, 2 minimum peptides and 0.00% decoy FDR. A total
of 1,295 proteins, 9,718 total peptides and 15,283 total spectra were
identied. Out of the observed proteins, 1,024 were shared between
both salt levels while 197 were unique to 0 mM NaCl, and 74 proteins
were unique to 200 mM NaCl.
Further quantitative analysis of the LC–MS/MS data output using
scaold showed a signicant decrease in the quantity of identied
proteins at 200 mM NaCl in comparison to 0 mM NaCl, at p ≤ 0.05
(Fisher’s exact test). Several proteins were upregulated or down
regulated at both salt levels as shown in Supplementary Table S1.
Likewise, a number of proteins were unique to either 0 or 200 mM
NaCl. Analysis with a ≥ 1.2 fold change also showed signicant
variations in proteins identied for 0 and 200 mM NaCl, as shown in
Figure 2. Supplementary Table S3 shows OmicsBox data for
B. amyloliquefaciens EB2003A.
3.2. Exoproteome analysis for Lactobacillus
helveticus EL2006H
Based on scaold and OmicsBox analyzes of the LC–MS/MS data,
there were variations in identied proteins for L. helveticus EL2006H
cultured at 0 mM NaCl and 200 mM NaCl, as shown in Table2 and
Supplementary Table S2. Two hundred mM NaCl greatly aected
identied proteins, with the majority downregulated, even to 100%.
Figure3 shows a comparison of the quantity of total proteins, total
unique peptides, and total unique spectra for 0 and 200 mM NaCl,
visualized at 95% protein threshold, 2 minimum peptides and 0.00%
decoy FDR. A total of 317 proteins, 1,628 peptides and 2,307 spectra
were observed. Out of the observed proteins, 136 were shared between
both salt levels while 178 were unique to 0 mM NaCl, and 3 were
unique to 200 mM NaCl, as shown in Figure3.
Further quantitative analysis of the LC–MS/MS output, using
scaold showed a signicant decrease in identied proteins at 200 mM
NaCl, in comparison to 0 mM NaCl, at p ≤ 0.05 (Fisher’s exact test).
e majority of the proteins were signicantly downregulated at
200 mM NaCl (Supplementary Table S2), with only seven upregulated
proteins, namely, a cluster of hypothetical proteins GFB61_00500, a
cluster of SLAP domain-containing proteins, a cluster of peptide ABC
transporter substrate-binding proteins, surface proteins, bronectin
type III domain-containing proteins, a cluster of Stk1 family PASTA
domain containing Ser/r kinases, and a cluster of metal ABC
transporter substrate binding proteins. Based on the fold analysis (1.2-
fold change and above), only three proteins namely, bronectin type
III domain-containing protein, cluster of hypothetical proteins and
cluster of metal ABC transporter, were signicantly upregulated as
shown in Supplementary Table S2. Supplementary Table S4 shows
OmicsBox data for L. helveticus 2006H. Figure4 is a volcano plot
illustrating the distribution of identied proteins for
L. helveticus EL2006H.
3.3. Functional annotation of proteins of
Bacillus amyloliquefaciens CFS observed at
0 and 200 mM NaCl
Using gene ontology (GO) enrichment analysis, identied
proteins were grouped into four groups, based on their functions,
namely, enzyme code distribution, cellular components, biological
Naamala et al. 10.3389/fmicb.2023.1206152
Frontiers in Microbiology 04 frontiersin.org
processes, and molecular functions, as shown in Table1. ere was a
variation in the eect of 200 mM NaCl on the proteins performing
various functions, majority being downregulated while some were
upregulated, yet others were unique to 200 mM NaCl. Worthy noting
is that all proteins involved in the various listed biological processes
were downregulated while proteins related to the extracellular region
cellular component were unique to 200 mM NaCl. Proteins involved
in catalyzing proteins and nucleic acids were also upregulated by 18.8
and 17.5%, respectively. Under the enzyme code distribution, all
enzymes were downregulated except for Translocases which were
upregulated by 20.6%.
3.4. Functional annotation of proteins of
Lactobacillus helveticus EL2006H CFS
observed at 0 and 200 mM NaCl
Using gene ontology (GO) enrichment analysis, identied
proteins were grouped in four sets, namely, biological processes,
cellular components, molecular functions, and enzyme code
distribution, as shown in Table2. ere was a variation in the eect of
200 mM NaCl on the proteins performing various functions; the
majority were downregulated although a few were upregulated.
Proteins in some functional groups were unique to 200 mM NaCl
(Table2). For example, under biological processes, transmembrane
transport and cell adhesion were unique to 200 mM NaCl while under
cellular components, transporter complex and membrane protein
complex were unique to 200 mM NaCl. Under molecular function,
peptidoglycan muralytic activity, protein binding and structural
constituents of cell walls were unique to 200 mM NaCl. Notably, all the
upregulated/unique proteins perform functions related to cell wall
metabolism or substrate transportation.
4. Discussion
Uncontrollable changes in microbial environments, especially
under eld conditions, requires PGPM to adapt to the changes in
order to survive (Gao etal., 2007; Galperin, 2010). Salinity stress is a
leading global abiotic stress aecting crops and PGPM proliferation
(Liu etal., 2023). When a microbe is exposed to stress, it may alter its
proteome prole, upregulating proteins essential for enhancing
tolerance to the stress while down regulating those that are likely not
so essential (Galperin, 2010; Msimbira et al., 2022). As a result,
proteome proles of a microbe grown in dierent environmental
conditions may vary signicantly. e current study compared
exoproteome proles of B. amyloliquefaciens EB2003A and
L. helveticus EL2006H exposed to 0 and 200 mM NaCl. Results of the
study showed variations in total proteins identied for both strains at
the two salt levels. Some of the identied proteins were unique to
either 0 or the 200 mM NaCl. Among the proteins unique to 200 mM
NaCl were cell wall metabolic enzymes, transcription/translation
regulators, potential virulence factors, phage proteins, antibiotics
resistance proteins, solute transporter proteins and, of course,
hypothetical proteins. ese ndings are to some extent like those of
Pumirat et al. (2009) and Rubiano-Labrador et al. (2015) who
examined the exoproteome of Burkholderia pseudomallei and Tistlia
consotensis exposed to salinity stress.
e cell-wall is the outermost layer of a bacterial cell, that acts as
a stress barrier and maintains cell shape (Mueller and Levin, 2020).
erefore, maintaining the integrity of the cell wall is a mechanism for
stress tolerance in bacteria. is may explain why, based on GO
function analysis, the dierent functional groups identied in the
current study, protein classes performing functions related to the cell
wall and the extracellular region were upregulated at 200 mM NaCl,
in both B. amyloliquefaciens EB2003A and L. helveticus
EL2006H. Peptidoglycan is the major component of the cell wall,
TABLE1 Comparing the distribution of B. amyloliquefaciens EB2003A
proteins to the dierent functional groups according to GO enrichment
analysis, at 0 mM NaCl (control) and 200 mM NaCl (treatment).
# Sequences
Functional group 0 mM NaCl 200 mM NaCl
Biological process
Organic substance metabolic process 1,202 1,028 (↓14.5%)
Primary metabolic process 1,061 919 (↓13.4%)
Cellular metabolic process 1,100 912 (↓17.1%)
Nitrogen compound metabolic process 939 852 (↓9.3%)
Biosynthetic process 637 493 (↓22.6%)
Small molecule metabolic process 631 490 (↓22.3%)
Catabolic process 204 193 (↓5.4%)
Cellular components
Intracellular anatomical structure 500 395 (↓21%)
Cytoplasm 468 363 (↓22.4%)
Membrane 162 232 (↑30.2%)
Cell periphery 99 164 (↑39.6%)
Intrinsic component of membrane 110 159 (↑30.9%)
Extracellular region 0 19 (↑100%)
Molecular function
Ion binding 821 706 (↓14.0%)
Organic cyclic compound binding 701 620 (↓11.6%)
Heterocyclic compound binding 701 620 (↓11.6%)
Hydrolase activity 663 579 (↓12.7%)
Small molecule binding 580 463 (↓20.2%)
Oxidoreductase activity 438 331 (↓24.4%)
Transferase activity 481 354 (↓26.4%)
Carbohydrate derivative binding 350 296 (↓15.4%)
Catalytic activity, acting on a protein 177 218 (↑18.8%)
Catalytic activity, acting on a nucleic acid 160 194 (↑17.5%)
Ligase activity 54 0 (↓100%)
Enzyme code distribution
Hydrolases 618 537 (↓13.1%)
Isomerases 114 90 (↓21.1%)
Ligases 182 148 (↓18.7%)
Lyases 134 102 (↓23.9%)
Transferases 463 344 (↓25.7%)
Translocases 54 68 (↑20.6%)
Oxidoreductases 418 310 (↓25.8%)
e bold values represent proteins upregulated at 200 mM NaCl.
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Frontiers in Microbiology 05 frontiersin.org
whose synthesis, polymerization, modication and turn over
contribute to maintaining cell wall integrity (Popham and Young,
2003; Sauvage etal., 2008; Shin etal., 2020). In this study, proteins,
such as LytR family transcriptional regulator, Amidases, peptidoglycan
endopeptidases and penicillin binding proteins (PBPs), SLAP domain-
containing protein and surface proteins were uniquely produced by
either B. amyloliquefaciens EB2003A or L. helveticus EL2006H,
exposed to 200 mM NaCl. ey all play vital roles in peptidoglycan
metabolism and maintenance of the cell wall. For example, LytR
family transcriptional regulator proteins, also known as LytR-
CpsA-Psr (LCP) family proteins, are in fact enzymes involved in the
attachment of Glycopolymers, such as wall teichoic acids on the
peptidoglycan (Kawai etal., 2011; Molloy, 2011; Gale etal., 2017;
Siegel etal., 2019). Previously, this family of proteins was reported to
play a transcription regulation role (Gao etal., 2007; Galperin, 2010),
although in later studies, Kawai and co-authors disagreed (Kawai
etal., 2011), suggesting that these regulatory roles could bebacterial
genus, species, or strain specic. Bacterial amidases play a vital role in
bacteria cell wall metabolism because, especially under stressful
conditions, they are involved in the remodeling, turnover, recycling,
and metabolism of peptidoglycan (Park, 1995; Weber etal., 2013;
Senzani etal., 2017; Mueller and Levin, 2020). Endopeptidases play a
major role in maintaining bacterial cell integrity and shape, through
processes such as peptidoglycan turnover and modication (Shin
et al., 2020). Penicillin binding protein PBP4, a peptidoglycan
endopeptidase was reported to enhance tolerance of B. subtilis to salt
stress by modifying the peptidoglycan (Palomino et al., 2009).
B. subtilis was reported to recycle its peptidoglycan toward the end of
its exponential growth, entering stationary phase, which could enable
prolonged survival of the bacteria during stationary phase (Borisova
etal., 2016). Surface proteins, also known as the glycoprotein layer or
S layer proteins (Engelhardt, 2007; Hynönen and Palva, 2013), and
surface layer associated proteins (SLAP), in the current study, were
unique to L. helveticus treated with 200 mM NaCl. Expression of
surface proteins has been linked to the ability of some Lactobacilli
species to tolerate changes in the human gastrointestinal tract
conditions, such as bile and low pH (Sengupta etal., 2013). It should
benoted that, not all prokaryotes produce surface proteins and that
their role varies from one group to another, leaving no universal
function of surface proteins in species that do possess them
(Engelhardt, 2007). Upregulation of surface proteins was also observed
in Lactobacillus acidophilus IBB 801 exposed to dierent abiotic
stresses such as NaCl, bile salt and high temperature (Grosu-Tudor
etal., 2016). Deletion of IgdA SLAP resulted in a mutant that was more
sensitive to salt stress and had a visibly disrupted cell surface when
compared to strains with the protein (Klotz etal., 2020). However, the
mechanisms through which surface proteins and SLAP enhance
tolerance to salt stress is yet to beveried. Likewise, Stk1 family
PASTA domain-containing Ser/r kinase also upregulated in
L. helveticus exposed to 200 mM NaCl have been reported to play a
role in bacteria cell wall metabolism and cell division (Janczarek etal.,
2018). Its expression was reported to enhance tolerance of
Streptococcus suis serotype 2 to oxidative stress (Zhu etal., 2014).
Exposure to stress may bea trigger for bacteria to reprogram their
gene expression, consequently resulting in new gene products that
could beessential for stress tolerance. In the current study, proteins
such as MarR family transcriptional regulators and rRNA
pseudouridine synthase were upregulated at 200 mM NaCl. e MarR
FIGURE1
Comparison of total proteins, total peptides and total spectra identified at 0 and 200 mM NaCl, for B. amyloliquefaciens EB2003A (p ≤ 0.05).
FIGURE2
Volcano plots showing the distribution of identified proteins for B.
amyloliquefaciens EB2003A as −log10 (Benjamini–Hochberg-adjusted
p-values) plotted against log2 (fold change) for 0 vs. 200 mM NaCl.
The two blue dotted vertical lines represent a ± 1.2-fold change, while
the pink dotted horizontal line indicates the significance threshold
(before logarithmic transformation) p ≤ 0.02425.
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family transcriptional regulators constitute a prominent family of
transcription factors involved in the reprogramming of gene
expression in response to stress conditions, such as oxidative stress
(Pérez-Rueda etal., 2004; Grove, 2013; Deochand and Grove, 2017).
MarR are involved in metabolism and antibiotic resistance of some
bacteria (Will and Fang, 2020). e enzyme rRNA pseudouridine
synthase catalyzes the synthesis of RNA pseudouridine from uracil,
the most common modied nucleoside in rRNA that plays a role in
gene expression (Ofengand, 2002; Zhao et al., 2018). Sulfate
Transporter and Anti-Sigma factor antagonist (STAS) domain-
containing proteins are produced in multiple species, including
bacteria and mammals (Moy and Seshu, 2021). In bacteria, they are
associated with stress tolerance among other factors, by regulating the
large family of sigma factors (ρ) that bind to RNA polymerase to
confer transcriptional target gene specicity (Moy and Seshu, 2021).
For instance, sporulation in B. subtilis, which is a response to stress
involves anti-anti-sigma factors, or anti-anti-ρ, which are STAS
domain proteins (Sharma etal., 2011). Changes in gene products may
also aect cellular biochemical composition and cellular processes, as
observed in the current study.
Salt stress leads to osmotic, oxidative, and ionic stress in microbes,
which, depending on their severity could cause damage to cellular
components such as the cell membrane, nucleic acids, enzymes, and
other proteins. A high concentration of salt ions such as Na+ and Cl−
in the microbe’s extracellular environment may cause loss of water
from the microbial cell, leading to loss of cell turgor pressure as well
as reduction in cellular processes such as metabolism (Krämer, 2010;
Tsuzuki etal., 2011; Rubiano-Labrador etal., 2015). To counter act
such eects, microbes develop mechanisms that enhance microbial
tolerance to oxidative, osmotic, or ionic stress. In the current study, at
200 mM NaCl, proteins that enhance bacteria’s tolerance to oxidative
stress were unique to 200 mM NaCl. For example, ioredoxins play
a major role in bacterial response to oxidative stress by quenching
singlet oxygen, scavenging hydroxyl radicals and donating hydrogen
to peroxidases (Chae etal., 1994; Das and Das, 2000; Zeller and Klug,
2006; Lu and Holmgren, 2014; Cheng etal., 2017). Members of the
xenobiotic response element (XRE) family transcriptional regulators,
among other functions, have been reported to enhance oxidative stress
tolerance in dierent bacteria species such as Streptococcus suis and
Corynebacterium glutamicum (Hu etal., 2019; Si etal., 2020; Zhang
etal., 2022). Flavodoxin family proteins were reported to enhance
tolerance of plant growth promoting rhizobacteria, such as
Pseudomonas uorescens Aur6 and Ensiifer meliloti, to oxidative stress
(Coba de la Peña etal., 2013). Heme A synthase catalyzes the synthesis
of heme A from heme O (Lewin and Hederstedt, 2016). Heme A is
particularly a co-factor of terminal oxidase enzymes involved in
oxygen reduction during aerobic respiration (Hederstedt, 2012; Choby
# Sequences
Functional group 0 mM NaCl 200 mM NaCl
Isomerases 33 3 (↓90.1%)
Translocases 9 6 (↓33.3%)
Lyases 18.5 0 (↓ 100%)
Ligases 21.75 0 (↓100%)
e bold values represent proteins upregulated at 200 mM NaCl.
TABLE2 (Continued)TABLE2 Comparing the distribution of L. helveticus EL2006H CFS
proteins to the dierent functional groups according to GO enrichment
analysis, at 0 mM NaCl (control) and 200 mM NaCl (treatment).
# Sequences
Functional group 0 mM NaCl 200 mM NaCl
Biological process
Organic substance metabolic process 320 50 (↓84.4%)
Primary metabolic process 294 44 (↓85.0%)
Nitrogen compound metabolic process 282 46 (↓83.6%)
Cellular metabolic process 239 12 (↓94.9%)
Biosynthetic process 149 0 (↓100%)
Small molecule metabolic process 76 5 (↓93.4%)
Catabolic process 60 4 (↓93.3%)
Establishment of localization 62 51 (↓17.7%)
ATP metabolic process 8 0 (↓100%)
Transmembrane transport 0 40 (↑100%)
Cell adhesion 0 7 (↑100%)
Cellular components
membrane 170 94 (↓44.7%)
intracellular anatomical structure 161 0 (↓100%)
intrinsic component of membrane 147 81 (↓44.8%)
cell periphery 110 75 (↓31.8%)
cytoplasm 92 0 (↓100%)
organelle 72 0 (↓100%)
extracellular region 17 24 (↑29.2%)
external encapsulating structure 29 24 (↓17.2%)
Transporter complex 0 19 (↑100%)
Membrane protein complex 0 19 (↑100%)
Molecular function
Hydrolase activity 189 76 (↓59.7%)
Organic cyclic compound binding 200 7 (↓96.5%)
Heterocyclic compound binding 200 7 (↓96.5%)
Ion binding 138 14 (↓89.8%)
Catalytic activity, acting on a protein 89 39 (↓56.2%)
Small molecule binding 89 3 (↓96.6%)
Transferase activity 86 0 (↓100%)
Structural constituent of ribosome 67 0 (↓100%)
Carbohydrate derivative binding 53 0 (↓ 100%)
Isomerase activity 9 0 (↓100%)
ATP hydrolysis activity 18 0 (↓100%)
Peptidoglycan muralytic activity 0 9 (↑ 100%)
Protein binding 0 16 (↑ 100%)
Structural constituent of cell wall 0 6 (↑ 100%)
Enzyme code distribution
Oxidoreductases 38 4 (↓89.5%)
Transferases 86 4 (↓95.3%)
Hydrolases 167 76 (↓54.5%)
(Continued)
Naamala et al. 10.3389/fmicb.2023.1206152
Frontiers in Microbiology 07 frontiersin.org
and Skaar, 2016). High levels of intracellular heme have been reported
to activate Hap1p which subsequently induces the transcription of
genes involved in oxidative stress response (Martínez etal., 2016).
Since salt stress can result in damage to essential microbial
constituents such as enzymes, and nucleic acids, DNA and RNA, it is
important that damaged components are repaired or replaced with
new ones. Upregulation of proteins that are directly or indirectly
involved in synthesis and repair of cellular proteins and nucleic acids
was observed in the current study. e enzyme 2′,3′-cyclic-nucleotide
2′-phosphodiesterase plays a major role in the metabolism of purines
and pyrimidines, the building blocks of DNA and RNA, and provide
energy and co-factors important in cell-division (Yin etal., 2018). is
is because it contains cyclic phosphodiesterase and 3′-nucleotidase
activity and catalyzes the hydrolysis of 2′,3′-cyclic nucleotides to yield
nucleotides and phosphate. erefore, the enzyme plays a role in DNA
and RNA synthesis and repair through provision of building blocks.
e enzyme m
1
A22-tRNA methyltransferase (TrmK) catalyzes N
(1)-adenosine methylation to N1 of adenine 22 of bacterial tRNA
(Roovers etal., 2008; Sweeney etal., 2022). Addition of a methyl group
plays a role in maintaining stability of tRNA (Roovers etal., 2008).
Stability of tRNA is essential in protein synthesis since they bridge the
gap between mRNA and amino acids during translation. e enzyme
thioredoxin plays a major role in protein repair and DNA synthesis by
donating hydrogen that reduces ribonucleotide reductase and
methionine sulfoxide reductase which catalyze the process (Zeller and
Klug, 2006; Lu and Holmgren, 2014). e upregulation of such
enzymes may also explain the high frequency of protein classes whose
function annotation involves catalytic activity, acting on nucleic acid,
and catalytic activity acting on proteins, that was observed in
B. amyloliquefaciens EB2003A, at 200 mM NaCl.
When exposed to stress, its paramount that microbes maintain an
even ow of substrates from their environment to the inside of the cell,
and vice versa. is ensures availability of carbon sources for energy as
well as metabolites required to serve purposes such as osmoregulation,
enzyme co-factors, synthesis of proteins and nucleic acids. e microbe
requires energy for channeling to survival mechanisms (Yan etal.,
2015; Msimbira et al., 2022). Microbes respond to osmotic stress
through intracellular accumulation of inorganic ions such as K+ and
organic solutes such as proline (Soussi etal., 2001; Bojanovic etal.,
2017). Although some of these osmo-protectants can besynthesized de
novo, it is less energy ecient than sourcing them from outside of the
cell (Zahran, 1997; Oren, 2008; Zhang etal., 2015; Bojanovic etal.,
2017; Lycklama etal., 2018). erefore, maintaining adequate substrate
transport systems is essential for microbial tolerance to stress. In this
study, weobserved upregulation of a number of proteins associated
with various substrate transport systems, such as the ATP binding
cassette (ABC) transporter, the major facilitator superfamily (MFS)
transporter and the phosphotransferase system (PTS) fructose
transporter subunit IIC in B. amyloliquefaciens EB2003A and
L. helveticus EL2006H. e ABC transporter facilitates transportation
of a wide range of substrates such as sugars, amino acids, and metals
from the microbe’s external environment (Du etal., 2011; Lycklama
et al., 2018; Teichmann et al., 2018). Among the observed ABC
transporter proteins were the amino acid ABC transporter substrate-
binding protein, multispecies: ABC transporter substrate-binding
protein, and multispecies: zinc ABC transporter substrate-binding
protein. e MFS transporter is one of the oldest protein families, a
group of secondary active transporters involved in selective
FIGURE3
Comparison of total proteins, total peptides and total spectra identified at 0 and 200 mM NaCl for L. helveticus EL2006H (p ≤ 0.05).
FIGURE4
Volcano plots showing the distribution of identified proteins for L.
helveticus EL2006H as −log10 (Benjamini–Hochberg-adjusted
p-values) plotted against log2 (fold change) for 0 vs. 200 mM NaCl.
The two dotted black vertical lines represent a ± 1.2-fold change,
while the blue dotted horizontal line indicates the significance
threshold (before logarithmic transformation) p ≤ 0.01935.
Naamala et al. 10.3389/fmicb.2023.1206152
Frontiers in Microbiology 08 frontiersin.org
transportation of substrates such as carbohydrates, amino acids, and
lipids, to ions, peptides and nucleosides, across microbial membranes
and plays a role in other microbial physiological processes, such as
resistance to toxic compounds like antibiotics and salicylic acid, and
enhanced salt tolerance (Yan, 2013; Lee etal., 2016; Pasqua etal., 2019).
For instance, MFS eux pumps VceCAB were reported to enhance the
tolerance of E. coli to bile salts (Woolley etal., 2005). e PTS system
is involved in uptake and phosphorylation of carbohydrates as well as
signal transduction (Bernhard, 2012). e enzyme IIC component
selectively transports sugar molecules across microbial membranes
(Jahreis etal., 2008; Jason etal., 2014). is allows microbes such as
bacteria to eciently utilize carbohydrate sources of their choice, at a
given time (Jahreis etal., 2008). e fructose family is a subfamily and
the oldest of the glucose superfamily of PTS. e ability of an organism
to utilize various carbon sources enables them to survive in varying
environmental conditions. As a result, upregulation of sugar uptake
systems has been linked to microbe response to stress, because
microbes require nutrients and osmo-protectants for survival under
stressful conditions (Pittman etal., 2014).
When exposed to stress, some members of the genus Bacillus,
B. amyloliquefaciens EB2003A inclusive, form spores which are
essential for survival under stressful conditions for long periods of
time (Setlow, 2014; Ghosh etal., 2018). Once favorable conditions are
restored, the spores germinate, giving rise to new microbial cells. e
germination of spores is triggered by amino acids such as L-alanine,
L- valine and L- asparagine (Setlow, 2014; Ghosh etal., 2018). In the
current study, alanine containing proteins: cation symporter family
protein and asparagine synthetase B were unique to the 200 mM NaCl
exoproteome of B. amyloliquefaciens EB2003A. e enzyme
asparagine synthase catalyzes the synthesis of asparagine from
aspartate and glutamine (Lomelino etal., 2017; Zhu etal., 2019). e
alanine cation symporter family protein is a transporter protein that
transports alanine but no other amino acids (Ma etal., 2019).
In addition to the proteins with known functions, several
hypothetical proteins were also unique to 200 mM NaCl treatment.
Proteins are classied as hypothetical if a corresponding mRNA
sequence is available in the data base, but there is no similar protein
sequence, hence, insucient information concerning their possible
functions. However, it’s possible that such proteins play a role in
enabling the microbe’s survival in growth conditions under which
they are produced.
In previous studies, CFS of B. amyloliquefaciens EB2003A and
L. helveticus EL2006H exposed to 200 mM NaCl enhanced
germination and radicle length of corn and soybean and growth
variables of potato grown under NaCl stress and normal conditions
(Naamala etal., 2022, 2023). e ability of a microbe to enhance
plant growth is related to its ability to exude into their growth
environment, bioactive substances with ability to enhance plant
growth. Among the proteins upregulated at 200 mM NaCl, in the
current study, are those that have been reported to enhance plant
growth under stressed and ideal conditions. Whether these proteins
were in part responsible for the bioactivity observed in our previous
study needs to beinvestigated further. However, application of
exogenous heme has been reported to enhance plant tolerance to
stress such salt stress (Woodson etal., 2011; Zhang etal., 2013; Wu
etal., 2022). e heme precursor 5-aminolevulinic acid (ALA) was
reported to enhance growth of plants exposed to salt stress (Hui
etal., 2006; Daneshmand and Oloumi, 2015; Genişel and Erdal,
2016; Wu etal., 2022). Exogenous application of ALA, resulted in
an increase in heme content, an indication that heme is involved in
the role of ALA in alleviating salinity stress (Wu etal., 2022). Heme
is involved in the transformation of superoxide anions in the
antioxidant system, hence, potentially playing a pivotal role in
mitigating the eects of oxidative stress on plants (Wu etal., 2022).
Esterases have been reported to play a role in plant growth and
development, involved in such crucial stages as seed germination,
pollen development, lateral root, and overall root development
(Takahashi etal., 2010; Clauss etal., 2011; Dolui and Vijayaraj,
2020; Zhang etal., 2020; Ursache etal., 2021; Shen etal., 2022). In
fact, esterases are believed to have played a role in the evolutionary
colonization of land by plants, through the conservation of water in
a desiccating environment (Niklas etal., 2017; Philippe etal., 2020).
MarR homologs were involved in symbiotic plant microbe
interactions. For example, the MarR homolog ExpG Sinorhizobium
meliloti activates transcription of three exp. operons that are
involved in the production of galactoglucan, which it needs for
plant root nodulation (Becker etal., 1997; Bartels et al., 2003).
Proteins such as the MFS eux pumps were reported to beinvolved
in the interaction of plants and symbiotic microbes, such as rhizobia
through enhancing nodulation and enhancing tolerance to
avonoids (Pasqua et al., 2019). ioredoxin, another protein
(enzyme) that was unique to 200 mM NaCl exoproteome of
B. amyloliquefaciens EB2003A is also found in higher plants where
it is classied as a disulde regulatory protein, belonging to a
complex of regulatory proteins consisting of types f, m, x, y, h, and
o (Meng etal., 2010). ioredoxin proteins play major roles in the
regulation of carbon metabolism, embryogenesis, chloroplast
development and mobilization of seed reserves, in plants (Jedelská
etal., 2020). ey also play a role in plant responses to biotic and
abiotic stresses through protection from reactive oxygen species
(Dos Santos and Rey, 2006; Meyer etal., 2012; Geigenberger etal.,
2017). ioredoxin h ortholog Trx h9, was reported play a role in
the germination of wheat (Li etal., 2009). It is believed that Trx h
regulates seed germination by reducing the disulde proteins stored
in the dry seed to the sulydryl state, following the addition of
water (Maeda etal., 2003, 2005; Rhazi etal., 2003; Zahid et al.,
2008). Meng et al. (2010) observed chlorotic leaves, short and
smaller roots in Arabidopsis thaliana Trx h9 mutants. Furthermore,
mutant plants were dwarf, with small and irregular mesophyll cells,
as well as lower chloroplast numbers and less chlorophyll, in
comparison to the wild type control, suggesting that Trx h plays a
role in plant growth (Meng et al., 2010). High expression of
thioredoxin h8 was observed in tobacco plants whose growth was
enhanced when treated with Bacillus aryabhattai (Xu etal., 2022).
Amidases are involved in the biosynthesis of indole acetic acid, a
phytohormone that plays major roles in plant growth and
development (Spaepen et al., 2007). Other PGPM such as
Pseudomonas putida have also been reported to produce amidase
(Chacko et al., 2009). Because they are involved in nitrogen
metabolism, amidases increase nitrogen use eciency in plants,
which subsequently enhances plant growth under both stressed and
non-stressed conditions (Unkefer etal., 2023).
ere are several mechanisms through which the identied
proteins can enhance plant growth. ese include regulation of the
anti-oxidant system, regulation of the photosynthetic system, ion
balance, hydrolysis of compounds that aect plant quality,
Naamala et al. 10.3389/fmicb.2023.1206152
Frontiers in Microbiology 09 frontiersin.org
mobilization of nutrients during seed germination, biosynthesis of
phytohormones involved in metabolic pathways such as nitrogen
metabolism and maintenance of plant fertility (Spaepen etal., 2007;
Takahashi etal., 2010; Clauss etal., 2011; Dolui and Vijayaraj, 2020;
Zhang etal., 2020; Unkefer etal., 2023).
Based on these studies, it’s possible that some of the proteins
upregulated at 200 mM NaCl stress were responsible for enhancing
radicle length, germination of corn and soybean, and growth variables
of potato, as observed in our previous studies.
5. Conclusion
Salinity stress aects survival, growth, and ability of PGPM to
enhance plant growth. However, some PGPM have developed
mechanisms of tolerating high levels of salt, altering their exoproteome
prole being one. In the current study, B. amyloliquefaciens EB2003A
and L. helveticus EL2006H, exhibited unique proteins when exposed
to 200 mM NaCl, some of which have also been reported to enhance
plant growth. Results of the study are in line with previous reports that
when exposed to stress, microbes alter their exoproteome prole. To
the best of our knowledge, this is the rst study to report on the eect
of NaCl on B. amyloliquefaciens EB2003A and L. helveticus EL2006H
exoproteome proles. Findings of this study will expand knowledge
regarding mechanisms through which Bacillus spp. and Lactobacillus
spp. tolerate salt stress at the protein level.
Data availability statement
e datasets presented in this study can be found in online
repositories. e names of the repository/repositories and accession
number(s) can be found in the Mass Spectrometry Interactive Virtual
Environment (MassIVE) online repository, at doi:10.25345/
C5PG1HZ4M and PXD041778 for Bacillus amyloliquefaciens
EB2003A, and doi:10.25345/C54B2XF6V and PXD041177 for
Lactobacillus helveticusEL2006H.
Author contributions
JN set up the experiments, was involved in data analysis and wrote
the manuscript. SS advised on experimental set up, data analysis, and
manuscript editing. LM helped with data analysis and manuscript
review and editing. DS provided funding, the intellectual environment
and did extensive manuscript editing. All authors have read and
agreed to the nal version of the manuscript.
Funding
is work was funded through a grant from Consortium de
recherche et innovations en bioprocédés industriels au Québec,
number CRIBIQ 2017-034-C30, with support from synagri and
EVL inc.
Conflict of interest
e authors declare that the research was conducted in the
absence of any commercial or nancial relationships that could
beconstrued as a potential conict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors and
do not necessarily represent those of their aliated organizations, or
those of the publisher, the editors and the reviewers. Any product that
may be evaluated in this article, or claim that may be made by its
manufacturer, is not guaranteed or endorsed by the publisher.
Supplementary material
e Supplementary material for this article can befound online
at: https://www.frontiersin.org/articles/10.3389/fmicb.2023.1206152/
full#supplementary-material
SUPPLEMENTARY TABLE S1
Comparison of exoproteome for B. amyloliquefaciens EB2003A, at 0 and
200 mM NaCl.
SUPPLEMENTARY TABLE S2
Comparison of exoproteome for L. helveticus El2006H, at 0 and 200
mM NaCl.
SUPPLEMENTARY TABLE S3
B. amyloliquefaciens EB2003A OmicsBox data.
SUPPLEMENTARY TABLE S4
L. helveticus EL2006H OmicsBox data.
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