Archives of Microbiology (2022) 204:687

https://doi.org/10.1007/s00203-022-03290-1

ORIGINAL PAPER

Novel antimicrobial activity of protein produced by Streptomyces

lividans TK24 against the phytopathogen Clavibacter michiganensis
Fernanda J. Calderón‑de la Sancha1   · Ulises Carrasco‑Navarro1   · Gerardo Santander1   ·
Javier Barrios‑González1   · Armando Mejía1 

Received: 25 May 2022 / Revised: 14 October 2022 / Accepted: 17 October 2022

© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022

Abstract
Antimicrobial proteins and peptides are an alternative to current antibiotics. Here, we report an antimicrobial activity in
a low-molecular-weight protein secreted naturally by Streptomyces lividans TK24 when glucose or glycerol were used as
carbon sources. The antimicrobial activity was demonstrated against Bacillus subtilis, Bacillus cereus, Kokuria rhizophila,
Clostridium sporogenes and Clavibacter michiganensis, causal pathogen of tomato bacterial canker; one of the most destruc-
tive bacterial diseases of this crop. The protein fraction with antimicrobial activity was identified and quantified by LC–MS/
MS. From a total of 155 proteins, 11 were found to be within the range of 11.3–13.9 kDa of which four proteins were selected
by functional analysis as possibly responsible for the antimicrobial activity. Protein fractionation, correlation analysis between
antimicrobial activity and abundance of selected proteins, as well as transcriptional expression analysis, indicate that 50S
ribosomal protein L19 is the main candidate responsible for antimicrobial activity.

Keywords  Antimicrobial protein · Antimicrobial activity · Moonlighting protein · 50S ribosomal protein L19 ·
Streptomyces lividans · Clavibacter michiganensis

Introduction permeability through interaction with the cell membranes,

At the moment, the emergence of pathogenic microorgan-
isms resistant to antimicrobials is a global problem that
threatens both the health and agricultural sectors (Davies
2006). There is an undeniable need to find new antimicrobial
molecules that help attack microbial pathogens, without the
disadvantages of those in current use, such as toxicity, high
rates of antibiotic resistance, pollutants, etc. (Butler et al.
2017). The use of antimicrobial peptides/proteins both natu-
ral and synthetic are an alternative for pathogenic bacteria
that are resistant to existing antibiotics (Jenssen et al. 2006;
Sierra et al. 2017). These molecules exhibit antibacterial,
antifungal, and anti-parasitic activities through membrane
disruption, pore-forming, and enhancing the membrane
Communicated by Erko Stackebrandt.
* Armando Mejía
ama@xanum.uam.mx
1
Departamento de Biotecnología, Universidad Autónoma
Metropolitana Unidad Iztapalapa, Av. San Rafael Atlixco
186, Col. Vicentina, Iztapalapa, 09340 Mexico City, Mexico
which leads to cell death (Hancock and Diamond 2000; Jens-
sen et al. 2006; Yaghoubi et al. 2021). The first reported pep-
tide was Nisin from Streptococcus lactis, a Gram-positive
bacterium, Nisin belongs to a group of bacteriocins called
lantibiotics that are part of a family of highly modified pep-
tides that are secreted (de Vos et al. 1995; Rodríguez 1996).
Later, the antimicrobial peptide Colistin from the Gram-
positive bacteria Bacillus polymyxa, was discovered in 1947
(Karaiskos and Giamarellou 2014). In 2014 Pyocin JU-Ch
1 was reported as an antimicrobial protein with a molecular
weight of approximately 30 kDa produced by Pseudomonas
aeruginosa, a Gram-negative bacterium (Grewal et al. 2014).
In 2008, a 63 kDa molecular weight antimicrobial protein
produced by Streptomyces fulvissimus was reported (Malik
et al. 2008).
There are proteins that exhibit more than one physiologi-
cally relevant biochemical or biophysical function, this type
of protein is called moonlighting protein (Jeffery 2017).
Such is the case of ribosomal proteins whose conventional
role is ribosome assembly and protein translation but also
involved in various physiological and pathological processes
(Zhou et al. 2015). Interestingly, some ribosomal proteins

13
Vol.:(0123456789)
 687   Page 2 of 12
also show antimicrobial activity (Hurtado-Rios et al. 2022).
The first report featured the antimicrobial peptide cecro-
pin, which was isolated from Hyalophora cecropia. It was
mapped to the N-terminal region of the 50S ribosomal pro-
tein L1 (Putsep et al. 1999). Later, the antimicrobial activity
of ribosomal proteins L27 and L30 secreted by Lactoba-
cillus salivarius SGL 03, a Gram-positive bacterium, was
identified (Pidutti et al. 2017). Ribosomal protein S23 and
ribosomal protein S15 from Branchiostoma japonicum were
recently identified with antimicrobial activity, both proteins
are components of the 40S subunit of eukaryotes whose
homolog in prokaryotes are S12 (RPS12) and S19 (RPS19)
of the 30S subunit, respectively (Ma et al. 2020; Qu et al.
2020).
In the present study, we identified the 50S ribosomal pro-
tein L19 of Streptomyces lividans TK24 (slRPL19) as a new
antimicrobial protein. Currently, there are no reports of anti-
microbial proteins produced naturally by S. lividans TK24;
however, our research group found that, when grown in a
medium with glycerol or glucose as the only carbon sources,
it produces a low molecular weight antimicrobial protein.
We also showed that slRPL19 acts against phytopathogenic
bacterium Clavibacter michiganensis, which is responsible
for bacterial canker in tomatoes, considered one of the most
destructive diseases, with presence in practically all the
tomato-producing regions of the world.
Materials and methods
Conservation and storage of strains
The S. lividans TK24 strain was grown on SFM agar and was
preserved in 40% (v/v) glycerol and 0.1% (v/v) Tween (1:1
ratio) (Kieser et al. 2000). The stock was stored at −20 °C.
Escherichia coli DH5α, Bacillus cereus QAM11, Kokuria
rhizophila EGI111 and Bacillus subtillis ATCC 6633 were
grown on Luria–Bertani (LB) media (Kieser et al. 2000).
Clostridium sporogenes QAM-C12 on Tryptic Soy Broth.
Clavibacter michiganensis QAM-C01 and Xanthomonas
campestris QAM-X07. The QAM strains were provided by
the company QUÍMICA AGRONÓMICA DE MÉXICO
S.A. de C.V. All strains were grown at 30 °C, preserved in
40% (v/v) glycerol, and stored at −20 °C.
Bacterial growth and fermentation
The production process started with a pre-culture of S. liv-
idans TK24, aliquots of 60 μl bacterial suspension were
mixed 50 ml of Phage medium (10 g/l glucose, 5 g/l Bacto-
tryptone, 5 g/l yeast extract, 5 g/l Lab Lemco powder, 0.74
g/l ­CaCl2 ­2H2O, 0.5 g/l M
­ gSO4 ­7H2O, pH 7.2) contained in
250 ml baffle Erlenmeyer flasks. The incubation occurred at
13
Archives of Microbiology (2022) 204:687
30 ℃ with shaking at 180 rpm for 48 h. Twenty percent (v/v)
of pre-culture was transferred to 250 ml baffle Erlenmeyer
flasks containing 100  ml of production medium UPgly
(10 g/l glycerol, 5 g/l Bacto-tryptone, pH 7.2). The flasks
were shaken at 180 rpm and 30 ℃ for 48 h. The culture was
centrifuged (Beckman Allegra 64R centrifuge, 4 ℃, 15 min,
8500 g) and the pellet was discarded. Supernatant fraction
was filtered through 0.22 μm filters (Millipore) to obtain the
biomass-free extract (BFE).
Tested carbon sources
Pre-culture was performed according to the previous section.
Twenty percent (v/v) of the pre-culture was transferred to
250 ml baffle Erlenmeyer flasks containing 100 ml of modi-
fied production medium. The carbon source was changed
to glucose (UPglu), xylose (UPxyl) and fructose (UPfru),
independently, maintaining the same C/N ratio of the UPgly
production medium. The positive control was the culture
with UPgly. All cultures were grown at 30 °C, 180 rpm for
144 h. Biomass production and antimicrobial activity tests
were performed every 24 h. The cultures were centrifuged
(Beckman Allegra 64R centrifuge, 4 °C, 15 min, 8,500 g).
Antibacterial activity assay
The antibacterial activity of the BFE from a culture with
UPgly was determined by two methods, the disc diffusion
method and the agar well diffusion method, both using
20 ml of 1% LB agar plates embedded with 20 µl bacterial
cultures (density of 1 × ­109 cells/ml) of B. subtilis ATCC
6633, B. cereus QAM11, C. sporogenes QAM-C12, K.
rhizophila EGI111, C. michiganensis QAM-C01, E. coli
DH5α, X. campestris QAM-X07, respectively. For the first
method, sterile paper discs (6 mm) impregnated with 30 µl
of a sample containing 2–3 µg of total protein were placed
on the agar plate, which contained the test strain; for the
second method, aliquots (200 µl) containing 14–15 µg of
total protein were poured into the 9 mm holes of the LB agar
plates, which contained the test strain. The plates were kept
in refrigeration (4 °C, 30 min) and then incubated (30 °C,
16 h). The plates were then examined for clean zones, indic-
ative of antibacterial activity and reported as the measure of
diameter (mm).
Molecular weight fractionation
To obtain fractions with different molecular weights of BFE
from a culture with UPgly, ultrafiltration was performed with
membrane sizes of 30 kDa, 10 kDa and 3 kDa. Ultrafil-
tration units (Ultra-15 amicon, NMWL) were used. 15 ml
of the BFE were placed in the 30 kDa ultrafiltration unit,
then the fraction obtained below 30 kDa was transferred to
Archives of Microbiology (2022) 204:687 Page 3 of 12  687
the 10 kDa ultrafiltration unit. Finally, the fraction obtained
below 10 kDa was transferred to a 3 kDa ultrafiltration unit.
Centrifugation conditions were 8,000 g, 4 °C for 20 min.
The antimicrobial activity of each fraction was evaluated and
sterile UPgly medium was considered as a negative control.
SDS‑PAGE coupled with antimicrobial activity test
SDS-PAGE was made under non-denaturing conditions
of the samples obtained in the fractionation of molecular
weight; 20 µl of the sample were mixed with 6 µl of load
buffer. The mixtures were injected, respectively, into the
wells of the gel (12% Mini-PROTEAN® polyacrylamide gel
TGX™, Bio-Rad). The electrophoresis was carried out using
the electrophoresis Cell Mini-PROTEAN® (300 V, 20 min).
The gel was washed four times with sterile distilled H ­ 2O
and then the gel was divided into two fragments of the same
size to separate the duplicates. One of the gel fragments
was fixed with 25% (v/v) isopropanol and 10% (v/v) glacial
acetic acid for 4 h, then washed with sterile distilled H ­ 2O
until the acetic acid smell was no longer perceived. The gel
was stained with Coomassie blue overnight with constant
agitation at room temperature. Finally, a water–methanol
solution was used to fade the gel and it was developed in a
Gel Doc EZ imager (Bio-Rad) gel documentation system.
For the antimicrobial activity test, the other gel fragment was
placed in a sterile Petri dish, then covered with 1.5% LB agar
containing the indicator microorganism. The plate was kept
at 4 °C for 30 min and after incubating at 30 °C overnight.
Molecular weight estimation
The location of the clear zone was compared to the bands
on the stained gel to determine which band was responsible.
Then the determination of protein molecular weight in SDS-
PAGE was estimated by the method described by Matsumoto
et al. 2019, which considers an experimental error of 10%.
Dual Color Standard molecular weight marker (Bio-Rad)
was used for the calibration curve.
Effect of proteases on the antimicrobial activity
of BFE
The effect of proteolytic digestion on antimicrobial activity
was determined by incubating BFE with Trypsin (Sigma-
Aldrich), α-chymotrypsin (Sigma-Aldrich) and proteinase
K (Promega), respectively. A final concentration of 1 µg/µl
was used. Reactions containing filter-sterilized enzyme solu-
tion and BFE were incubated at 37 °C for 3, 6, 9, and 24 h.
Subsequently, the reaction was stopped by heating at 100 °C
for 10 min. Antimicrobial activity was determined by a well
diffusion test, using B. subtilis as indicator microorganism.
Antimicrobial protein(s) purification
The BFE obtained from the culture with the UPgly produc-
tion medium was subjected to a series of purification steps.
Ammonium sulphate precipitation
Proteins present in 1 L of the BFE were precipitated by the
addition of ­(NH4)2SO4 (80% saturation; 4 °C), then collected
by centrifugation (Beckman Allegra 64R centrifuge, 4 °C,
20 min, 11,000 g). The precipitated proteins were resus-
pended in 10 ml of buffer A (10 mM Tris–HCl, pH 8.0).
Salts were removed when using ultrafiltration tubes with
3 kDa membranes (Ultra-15 amicon, Millipore) since one
of their applications is just that: Desalting, buffer exchange
and protein dialysis.
Then, molecular weight cut-off was performed using
10 kDa membranes (Ultra-15 amicon, Millipore). Ultrafil-
tration conditions were 8,000 g at 4 °C for 20 min. The frac-
tion > 10 kDa was collected and the fraction was called PP.
The antimicrobial activity of the PP fraction was tested using
B. subitilis as an indicator microorganism.
Ion exchange chromatography (IEC)
Two ml of the PP fraction were injected into a Bio-Scale
Mini UNOsphere Q column, 40 × 12.6 mm inner diameter
(Bio-Rad); elution was performed with a linear gradient of
NaCl, from 0.1 M to 1 M; dilutions were performed with
30 mM Tris–HCl buffer, pH 7.9. All the fractions obtained
were tested for antimicrobial activity using the indicator
microorganism. The fraction that presented antimicrobial
activity was called the IEC fraction.
Hydroxyapatite chromatography (HAC)
Two ml of the IEC fraction were injected into a 5 ml Bio-
Scale Mini CHT cartridge, Type II, 40 µm media (Bio-Rad).
Elution was performed with a linear gradient of sodium
phosphate, 5 to 500 mM. All the fractions obtained were
tested for antimicrobial activity using the indicator micro-
organism. The fraction that presented antimicrobial activity
was called HAC.
All fractions showing antimicrobial activity from each
purification step were quantified by the Bradford method
and loaded on a 12% Mini-PROTEAN® polyacrylamide
gel TGX™, Bio-Rad. Electrophoresis was performed as
described in the section “SDS-PAGE coupled with antimi-
crobial activity test”, using the Mini-PROTEAN Tetra Cell
electrophoresis system (300 V, 20 min).
13
 687   Page 4 of 12 Archives of Microbiology (2022) 204:687

Protein identification by mass spectrometry
Protein identification was performed by liquid chromatog-
raphy-tandem mass spectrometry (LC–MS/MS). The BFE
samples obtained from 48 h of the cultures with different
carbon sources (glucose, glycerol, fructose and xylose) were
subjected to LC–MS/MS analysis. In addition, the band
cut from an SDS-PAGE gel of approximately a molecular
weight of 12.6 ± 1.26 kDa was also subjected to LC–MS/
MS analysis, this band corresponded to a sample obtained
from the HAC. All the samples were digested with trypsin
(20 ng/ml) and incubated at 37 °C overnight. The superna-
tant was recovered after centrifugation, respectively. Subse-
quently, the peptides were purified by solid phase extraction
(C18Zip-Tips, Millipore). The peptides were analyzed by
nano LC–MS/MS using a Finnigan MicroAS autosampler
and Surveyor MS pumping system coupled to an LTQ-Orbit-
rap mass spectrometer (Thermo Fisher Scientific).
Resource of protein sequence and functional
information
Mass spectra (MS/MS) were analyzed using Mascot (Matrix
Science, London, UK; Mascot version in Proteome Discov-
erer 2.3.0.523). Mascot was configured to search for Refseq_
Streptomyces_Lividans_TK24. The search was performed
with an ionic mass tolerance fragment of 0.60 kDa. Cysteine
carbamidomethyl was specified in Mascot as a fixed modi-
fication. Methionine oxidation was specified in Mascot as a
variable modification.
Criteria for the identification of proteins
Scaffold (Proteome Software, Inc., Portland, OR 97,219,
Oregon, United States) was used to validate protein identi-
fications derived from MS/MS sequencing results. Scaffold
verifies peptide identifications assigned by the Mascot search
engine. Peptide identifications were accepted if they could
be established at a probability greater than 95.0%, as per
the Peptide Prophet algorithm (Keller et al. 2002). Protein
identifications were accepted if they could be established at
a probability greater than 99.0%. Protein probabilities were
assigned by the Protein Prophet algorithm (Nesvizhskii et al.
2003).
RNA extraction and cDNAs synthesis total
Biomass obtained at 48 h of culture with different carbon
sources (glycerol, fructose, xylose and glucose) was sepa-
rated from the liquid medium by centrifugation and immedi-
ately pulverized in a mortar with the help of liquid nitrogen.
The resulting powder was resuspended in the lysis buffer of
the AURUM TM Total RNA Mini Kit (Bio-Rad), for fur-
ther extraction according to the manufacturer’s instructions.
RNA integrity was observed by electrophoresis with the Gel
Doc EZ imager gel documentation system (Bio-Rad). RNA
and cDNA concentration were determined spectrophotomet-
rically using NanoDrop™ One (Thermo Fisher Scientific).
3.5 ng of RNA was used to synthesize cDNA, for which
SuperScript™ III (Invitrogen™) reverse transcriptase, Ran-
dom Primers (Invitrogen™), and dNTPs (Promega) were
used.
Real‑time PCR (qRT‑PCR)
The primers used to measure the expression of each gene
(slRPL19, slSP, slHFP y slDrsEdcP) (Table  1), were
designed from the sequence of slRPL19 gen (GenBank
Accession No. EFD66468.1), slSP gen (GenBank Acces-
sion No. EFD64487.1), slHFP gen (GenBank Accession
No. EFD69344.1), and slDrsEdcP gen (GenBank Acces-
sion No. EFD67876.1). Three biological replicates of each
condition were used to perform qRT-PCR, with the com-
mercial mix SsoAdvanced™ Universal SYBR Green Super-
mix (Bio-Rad). The qRT-PCR reactions were carried out in
a GENTIER 48 Real-time PCR System (Tianlong Science

Table 1  Primers used for qRT- Primers Sequences (5′- 3′) Amplicon length Source
PCR in this study (bp)

slRPL19 Forward TCC​GCA​AGG​TCT​CCT​TCT​ 127 This study

slRPL19 Reverse AGC​TCG​CGC​AGG​TAGTA​ This study
slSP Forward GCT​TTC​CGC​AGT​TCT​CCT​ 142 This study
slSP Reverse GAT​CGT​TCC​GTA​GCC​CTT​ This study
slHFP Forward CCA​AGG​CGC​ACC​ACAAG​ 137 This study
slHFP Reverse GCT​GCC​GGT​GTT​GAAGA​ This study
slDrsEdcP Forward CTG​CTG​GAC​TCG​CTCCT​ 130 This study
slDrsEdcP Reverse CCT​CCT​GCA​CGA​AGA​CCT​ This study
rpoA Forward AAG​GGC​AAG​CTG​GAG​ATG​ 127 This study
rpoA Reverse TGA​GAA​CCG​GCG​AGT​AGA​ This study

13
Archives of Microbiology (2022) 204:687 Page 5 of 12  687

and Technology). Relative expression was quantified by the
­2−ΔΔCt method (Livak and Schmittgen 2001), using rpoA as
a reference gene for the analysis.
Results
Antimicrobial activity of BFE
The antimicrobial compound was found in the extracellular
medium (BFE) of S. lividans TK24. The highest antimicro-
bial activity occurred at 48, 48, 96 and 144 h when glucose,
glycerol, fructose, and xylose were used, respectively. The
treatment with glycerol as a carbon source presented higher
activity with respect to the other treatments (Fig. 1). For
further experiments, it was decided to collect the supernatant
after 48 h of growth at 30 °C.
To find the fractions that present antimicrobial activity,
a fractionation by a molecular weight of the BFE was car-
ried out with UPgly medium. We observed that the frac-
tion greater than 10 kDa presented a microbial inhibition
halo, the fraction less than 3 kDa also had an inhibition
halo, however, it was 3 times smaller than the previous
one (Fig. 2).
The fraction greater than 10 kDa was exposed against
some Gram-positive and Gram-negative bacteria. The
results showed antimicrobial activity against B. subtilis
ATCC 6633, B. cereus QAM11, C. sporogenes QAM-C12,
K. rhizophila EGI111 and C. michiganensis QAM-C01, all
are Gram-positive (Table 2). Figure 3 shows antimicro-
bial activity assays with inhibition halos against B. subtilis
(positive control) and C. michiganensis (phytopathogenic
bacteria).

Fig. 1  A Growth kinetic of S. lividans TK24 using different carbon source. B Antimicrobial activity using B. subtilis ATCC 6633 as indicator
microorganism. Data are expressed as the mean ± error of three independent experiments

Fig. 2  Antimicrobial activity against Bacillus subtilis, of each fraction obtained by ultrafiltration. The BFE-gly was the positive control

13
 687   Page 6 of 12 Archives of Microbiology (2022) 204:687

Table 2  Antimicrobial activity of proteins in biomass-free extract of Table 3  Effect of proteolytic enzymes on the antimicrobial activity of
S. lividans TK24 BFE
Type Target microorganism Antimi- Treatment Residual antimicrobial activity (%)
crobial
­activitya 3 h 6 h 9 h 24 h

Gram-positive Bacillus subtilis ATCC 6633  +  Trypsin
Bacillus cereus QAM11  +  α-Chimotrypsin
Clostridium sporogenes QAM-C12  +  Proteinase K
Kokuria rhizophila EGI111  + 
Clavibacter michiganensis QAM-C01  + 
Gram-negative Escherichia coli DH5α –
Xanthomonas campestris QAM-X07 –
a
  + antimicrobial activity, – absence of antimicrobial activity
Effect of proteases on the antimicrobial activity
of the protein fraction
The effect of proteolytic enzymes was determined using the
B. subtilis strain as an indicator microorganism. The results
showed that, upon exposure of BFE with proteases, the anti-
microbial activity did not decrease (Table 3).
Antimicrobial protein purification
In this study, we performed 3 purification steps, consisting of
ammonium sulfate precipitation, ion exchange chromatogra-
phy (IEC) and hydroxyapatite chromatography (HAC) (see
Materials and methods). During each step, the fractions that
showed antimicrobial activity were selected. The PP fraction
obtained after precipitation with ammonium sulfate was the
fraction that showed antimicrobial activity. In the case of
IEC, antimicrobial activity was observed from fraction 12
to 20 and in fraction 12 a very evident peak was detected in
the chromatogram (Fig. S1). In HAC, antimicrobial activity
100 100 100 100
100 100 100 100
100 100 100 100
was observed in the first four fractions; a peak in the chro-
matogram was also observed (Fig. S2). Finally, an SDS-
PAGE of the samples that showed activity in each purifica-
tion step was performed. The SDS-PAGE gel coupled to an
antimicrobial activity test showed a zone of inhibition in
the lanes corresponding to the PP and HAC samples. The
bands responsible for the zone of inhibition corresponded
to a molecular weight between 11.3 and 13.9 kDa (Fig. 4).
Protein identification by LC–MS/MS
In this study, the BFE-gly sample and a purified sample
(band cut from an SDS-PAGE) were analyzed. In the BFE-
gly, 2041 proteins were identified and in the cut band, 209
proteins were identified (data not shown in the manuscript),
which allows to demonstrate that the purification steps
helped to purify 89.7% of the total proteins found in the
BFE. For the selection of possible antimicrobial proteins,
we searched for proteins identified in both samples. From
a total of 155 proteins found in both samples, we limited
ourselves to select those that corresponded to a molecu-
lar weight between 11.3 and 13.9 kDa (Table S1). Then,
through functional analysis, we selected 4 proteins as the
best candidates to possess the antimicrobial activity: DrsE
domain-containing protein (slDrsEdcP), Hit-family protein

Fig. 3  Agar plates of Bacil-

lus subtilis ATCC 6633 and
Clavibacter michiganensis
QAM-C01 exposed to BFE-
gly > 10 kDa

13
Archives of Microbiology (2022) 204:687 Page 7 of 12  687
Fig. 4  Samples obtained in
the purification steps of the
antimicrobial protein produced
by S. lividans TK24. SDS-
PAGE stained with Coomassie
blue (left side); direct detection
of antimicrobial proteins from
gel SDS-PAGE covered with B.
subtilis ATCC 6633 as indicator
microorganism (right side).
Lane 1: Standard molecular
weight marker (250–10 kDa).
Lane 2: Fraction > 10 kDa of
BFE-gly was used (18 µg/lane).
Lane 3: PP (18 µg/lane). Lane
4: HAC (18 µg/lane)
(slHFP), 50S ribosomal protein L19 (slRPL19) and secreted
protein (slSP), (Table 4). The 4 selected proteins were also
identified in the BFE of cultures with UPglu, UPxyl and
UPfru by LC–MS/MS (data not shown in the manuscript).
Correlation analysis
From the BFEs obtained from the different cultures, both
antimicrobial activity tests and LC–MS/MS were performed.
To find a relationship between the abundance of selected
proteins obtained through the number of normalized spectra
and the antimicrobial activity measured through the diameter
of the microbial inhibition zone, a correlation analysis was
performed. The results showed that there was no correlation
between the abundance of slSP, slDrsEdcP, slHFP proteins
and antimicrobial activity (Fig. 5A–C). However, a posi-
tive correlation was observed between the abundance of the
slRPL19 protein and the antimicrobial activity (Fig. 5D).
This analysis indicates that the 50S ribosomal protein
L19 (slRPL19) could be responsible for the antimicrobial
activity.
Gene expression of S. lividans TK24 under different
culture conditions
To measure the transcriptional level of genes slSP,
slDrsEdcP, slHFP and slRPL19 under 4 different culture
conditions, a real-time PCR was performed. We found that
the transcriptional level of the slSP gene of S. lividans TK24
was significantly higher with the UPfru medium culture than
with UPgly, UPglu and UPxyl, and the latter three did not
differ significantly from each other (Fig. 6A). In the case of
the slDrsEdcP gene, there was a significant increase in its
transcription in the UPxyl medium culture relative to the
UPgly, UPglu, and UPfru medium cultures (Fig. 6B). The
transcriptional level of the slHFP gene was higher when
grown in the UPfru medium relative to expression in the
UPgly, UPglu and UPxyl medium cultures (Fig. 6C). Inter-
estingly, a significant increase in the transcription of the
slRPL19 gene was observed in the UPglu and UPgly cul-
tures, while in the UPxyl and UPfru cultures the transcript
levels were decreased (Fig. 6D). This result supports the
idea that the 50S ribosomal protein L19 is responsible for
the antimicrobial activity.
Discussion
The need for new antimicrobials is indisputable. Species
of the genus Streptomyces are known to be the most vital
sources of natural compounds with a pragmatic history of
producing new bioactive molecules, including several com-
mercially important drugs (Miao and Davies 2010; Das et al.
2018). Here, we found that S. lividans TK24 secretes an
antimicrobial protein. There is a possibility that the protein
found in the medium is due to cell lysis, however, given that
the strain never reached the death phase during the BFE
collection time, instead, growth was maintained, and the
antimicrobial activity decreased (Fig. 1), we consider that
it is likely that the protein is secreted into the extracellular
medium. On the other hand, the results showed that, upon
exposure of BFE with proteases, the antimicrobial activ-
ity did not decrease. Our results are in agreement with that
reported by Pidutti et al. 2017, who find that ribosomal pro-
teins (L27, L30) from L. salivarius SGL 03 have antimicro-
bial properties. Moreover, like us, they were localized in the
extracellular medium and their activity is also maintained in
the presence of trypsin, alpha-chymotrypsin, proteinase K,
13
 687   Page 8 of 12 Archives of Microbiology (2022) 204:687

Table 4  Selected proteins with possible antimicrobial activity secreted by S. lividans TK24

Protein Entry name (UniProtKB) Molecular Exclusive Sequence Sequence
weight unique coverage
(kDa) peptide (%)

DrsE domain-containing protein D6EK37_STRLI 12.55 5 64 MAKKLVIKVTAGADAPERC-

Hit-family protein
50S ribosomal protein L19
Secreted protein
SQAFTVAAVAVASGVDVS-
LWLTGESAWFAVPGRAAE-
FELPHAAPLPDLLDSLLAG-
GRVTLCTQCAARRDLTEKD-
VIEGVRIAGAQVFVQEALAD-
DTQALVY
D6EE98_STRLI 12.44 5 59 MAGEPQDDCLFCKIVAGQIPA-
TIVRETDTTVAFRDINPQAPTH-
VLVIPKAHHKDAAALAAE-
APQLAADVLRETQAVAD-
DEKLDSYRTVFNTGSGAGQT-
VWHAHAHVLGGRGLEWPPG
D6ERH4_STRLI 13.14 5 44 MSHLLDSVDAASLRSDVPAFRPG-
DTVNVHVRVIEGNRSRVQQFK-
GVVIRRQGAGVRETFTVRKVS-
FSVGVERTFPVHTPIVEKI-
ELVTRGDVRRAKLYYLRELRG-
KAAKIKEKRDS
D6ESN1_STRLI 13.74 3 43 MKKLASALLSAVLLG-
GMSVAAAPAASAVTAP-
GATASAAPGCVTETK-
EVSKGYGTITVCVNEDG-
SARAYGDLHCTAFGAVLVM-
WEIGWETASGVKVSTVATA​GWG​
DDDIEFDVDPSAGTDGIKGITG-
VDEIYLATIYM

lysozyme and pepsin (Pidutti et al. 2017). Another example
is the antimicrobial protein JU-Ch 1 produced by P. aerugi-
nosa was exposed to Proteinase K and Lipase, and main-
tained its activity at 90%. Several bacteria known to produce
cyclic peptides with an unusual arrangement of amino acids
show resistance to many proteases (Grewal et al. 2014). The
same case of an antimicrobial protein produced by S. ful-
vissimus that also maintained antimicrobial activity in the
presence of proteinase K and α-chymotrypsin. The authors
suggest that such enzymes at least do not cleave portions
of the protein that are necessary for biological activity and
exemplify another pathogenic protein from bovine spongi-
form encephalopathy that confers resistance to proteases
(Malik et al. 2008). In the same sense, it has been shown that
specific amino acid sequences of a protein are responsible
for antimicrobial activity. For example, residues 47–67 of
ribosomal protein S15, residues 67–84 of ribosomal pro-
tein S23, and residues 2–27 and 23–46 of ribosomal protein
L30 of Branchiostoma japonicum are part of core regions
conferring antimicrobial activity (Ma et al. 2020; Qu et al.
2020; Chen et al. 2021). Based on this, it is possible that the
antimicrobial activity of our protein should be attributed not
to the complete amino acid sequence but to a part of it.
A partial purification of the biomass-free extract was car-
ried out, then using an SDS-PAGE coupled with an antimi-
crobial activity assay we found that the molecular weight
of the antimicrobial protein corresponded to a range of
11.3–13.9 kDa. Subsequently, by mass spectrometry and a
proteomic analysis, four proteins were selected (Table 4).
Theoretically, all are intracellular proteins; however, it has
been shown there are proteins that have a cytoplasmic func-
tion, and also actively participate in processes of the biologi-
cal environment with a different function are called moon-
lighting proteins (Jeffery 1999, 2017).
The first protein is the DrsE domain-containing protein
(UniProtKB accession number: D6EK37_STRLI) called the
slDrsEdcP protein in this document; a soluble cytoplasmic
protein involved in intracellular sulfur oxidation, encoded in
the Dsr locus of Chromatium vinosum (Pott and Dahl 1998;
Dahl et al. 2005). This protein is found in several species of
the genus Streptomyces; however, it has not been character-
ized and has an unknown function. The second protein is the
Hit-family protein (UniProtKB accession number: D6EE98_
STRLI) called the slHFP protein in this work. These pro-
teins are ubiquitous, however, their function has only been
reported in the cytoplasm of eukaryotes, where it has been
characterized as an intracellular purine ribonucleotide

13
Archives of Microbiology (2022) 204:687 Page 9 of 12  687
Fig. 5  Correlation analysis between two response variables: antimicrobial activity (zone of inhibition, mm) and protein abundance (#normalized
total spectra). A slSP protein; B slDrsEdcP protein; C slHFP protein; D slRPL19 protein
receptor (Brenner 2002). Its function in prokaryotes is not
yet clearly defined, however, Mollicutes, small bacteria that
lack a cell wall, have shown to be linked to membrane pro-
teins, while in Chlamydiaceae, a family of Gram-negative
bacteria, they are genetically and physically associated with
cytoplasmic proteins (Hopfe et al. 2005). The third protein,
a Secreted protein (UniProtKB accession number: D6ESN1_
STRLI) referred to in this work as slSP, has a signal peptide,
and is reported with unknown functions in the databases.
Finally, the fourth protein is a 50S ribosomal protein L19
(UniProtKB accession number: D6ERH4_STRLI) called the
slRPL19 protein in this work. This protein has a theoretical
molecular weight of 13.14 kDa, is located at the 30S-50S
ribosomal subunit interface and may play a role in the struc-
ture and function of the aminoacyl-tRNA binding site.
Considering culture conditions that show antimicrobial
activity and culture conditions that do not show antimicro-
bial activity, a couple of experiments were essential to attrib-
ute the antimicrobial activity to a single protein. By cor-
relation analysis between the number of normalized spectra
(LC–MS/MS) of each of the four proteins and the antimicro-
bial activity, there was a positive correlation only with the
slRPL9 protein. It was shown that the higher the abundance
of the slRPL19 protein, the higher the antimicrobial activity
in the UPgly cultures, followed by the UPglu culture. In the
case of the UPfru and UPxyl cultures, where the abundance
of slRPL19 protein decreased, there was no antimicrobial
activity (Fig. 5). On the other hand, from a transcriptional
analysis (qRT-PCR) of the genes encoding the 4 selected
proteins, it was found that the slDrsEdcP gene had a higher
relative expression when cultured with UPxyl; the slHFP
gene was more highly expressed when cultured with UPfru;
in the case of the slSP gene, the level of transcription was
higher when grown with UPfru; finally, the expression levels
of the slRSL19 gene were higher in the UPgly and UPglu
cultures (Fig. 6). The results indicate that 50S ribosomal
protein L19 is responsible for the antimicrobial activity
because our results agree with those reported by Ishihama
et al. 2008, they found that there are significant associations
between the abundance of a protein and its properties and
functions in the cell, they also observed a significant correla-
tion between protein and mRNA abundance in E. coli cells
(Ishihama et al. 2008). In the same sense, Sidders et al. 2007
showed a correlation between the level of transcription and
the biological importance of proteins applying this analy-
sis to the prokaryote Mycobacterium tuberculosis (Sidders
et al. 2007). Based on the above, we tried to find which of
the genes was expressed under the conditions with activity
and whether it was no longer expressed under the conditions
without antimicrobial activity.
Currently, there are studies of ribosomal proteins that
have antimicrobial activity at the extracellular level, such
13
 687   Page 10 of 12
Fig. 6  The relative expression level of four genes of S. lividans TK24
in response to carbon source modification in UP production medium
(UPfru, UPxyl, UPglu and UPgly). A Relative expression level of
slSP gene. B Relative expression level of slDrsEdcP gene. C Rela-
as L27 and L30 proteins. Both are ribosomal and are
secreted into the extracellular medium by L. salivarius
SGL 03. These are low molecular weight (9.94 kDa and
6.55 kDa, respectively) proteins and act against Strepto-
coccus pyogenes, Streptococcus uberis and Enterococcus
faecium (Pidutti et al. 2017). It is interesting to mention
that the 50S ribosomal protein L36, with a theoretical
molecular weight of 5 kDa, was identified in a superna-
tant with antimicrobial activity from Pediococcus acidi-
lactici (García-Cano et al. 2019). Another study reports
a 60S ribosomal protein L29, with a molecular weight of
6.48 kDa, isolated from the acidic extract of Pacific oyster
gills, Crassostrea gigas. This protein acts against Gram-
positive and Gram-negative bacteria (Seo et al. 2017).
Interestingly, many ribosomal proteins are secreted by
Lactobacillus sakei subsp. sakei 2a and have antimicrobial
activity, for example, the 30S ribosomal protein S21 with
a molecular weight of 6.8 kDa, acts against Enterococ-
cus faecalis ATCC 19,483, Listeria innocua Li7, Listeria
monocytogenes Scott A and Staphylococcus epidermis (De
Carvalho et al. 2010). Recently, the mechanism of anti-
microbial activity of a ribosomal protein has been identi-
fied, which occurs through the interaction of the protein
with the bacterial membrane, causing its depolarization,
13
Archives of Microbiology (2022) 204:687
tive expression level of slHFP gene. D Relative expression level of
slRPL19 gene. All expression results were normalized relative to
the rpoA gene by the 2­ −ΔΔCt method. Error bars represent the stand-
ard deviation of the mean value
followed by the intracellular production of reactive oxygen
species (ROS). In the same study, the region of the protein
responsible for antimicrobial activity was identified, cor-
responding to the sequence RRFSRGLKRKHLALIKKL-
RKAKK (Qu et al. 2020). It is interesting to observe that
this peptide presents a similarity of 72.7% with a segment
in the sequence of the 50S ribosomal protein L19 identi-
fied in our study (Fig. S3).
Our results showed that S. lividans TK24 produces a pro-
teinaceous compound with antimicrobial activity against the
phytopathogen C. michiganensis and this activity is not due
to any previously reported secondary metabolite but to a low
molecular weight protein. Protein fractionation, correlation
analysis between antimicrobial activity and abundance of
selected proteins, as well as transcriptional expression analy-
sis, indicate that the 50S ribosomal protein L19 is the main
candidate responsible for antimicrobial activity.
Supplementary Information  The online version contains supplemen-
tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 00203-0​ 22-0​ 3290-1.
Acknowledgements  This work was supported by “Programa Especial
de Apoyo a la Investigación” Rectoría General, UAM, I21-2019. Fer-
nanda J Calderón-de la Sancha thanks Consejo Nacional de Ciencia y
Tecnología (CONACYT-México) for the scholarship (No. 718101).
Archives of Microbiology (2022) 204:687 Page 11 of 12  687
We thank Dr. Denis Faubert at the Proteomics Facility of the Institut
de Recherches Cliniques de Montreal, Canada, for his assistance in the
LC–MS/MS analysis. Finally, to the Divisional Laboratory of Molec-
ular Biology (LDBM) of the Universidad Autónoma Metropolitana-
Iztapalapa, México.
Author contributions  FJCS and AM participated in the study concep-
tion. Material preparation, data collection and analysis were performed
by FJCS, UCN and GS. FJCS and UCN had full access to all the data
in the study and take responsibility for the integrity of the data and
accuracy of the data analysis. FJCS, UCN, JBG and AM drafted and
revised the paper. All authors read and approved the manuscript.
Funding  This work was supported by “Programa Especial de Apoyo a
la Investigación” Rectoría General, UAM, I21-2019. Fernanda J Cal-
derón-de la Sancha has received the doctoral scholarship No. 718101
from Consejo Nacional de Ciencia y Tecnología (CONACYT-Mexico).
Data availability  The datasets generated during and analyzed during
the current study are available from the corresponding author on rea-
sonable request.
Declarations  11. https://​doi.​org/​10.​1016/​S0968-​0004(98)​01335-8
Conflict of interest  The authors declare that they have no conflict of
interest.
References
Brenner C (2002) Hint, Fhit, and GalT: Function, structure, evolution,
and mechanism of three branches of the histidine triad super-
family of nucleotide hydrolases and transferases. Biochemistry
41:9003–9014. https://​doi.​org/​10.​1021/​bi025​942q
Butler MS, Blaskovich MA, Cooper MA (2017) Antibiotics in the clini-
cal pipeline at the end of 2015. J Antibiot 70:3–24. https://d​ oi.o​ rg/​
10.​1038/​ja.​2016.​72
Chen Y, Yao L, Wang Y et al (2021) Identification of ribosomal pro-
tein L30 as an uncharacterized antimicrobial protein. Dev Comp
Immunol 120:104067. https://​doi.​org/​10.​1016/j.​dci.​2021.​104067
Dahl C, Engels S, Pott-Sperling AS et al (2005) Novel genes of the
dsr gene cluster and evidence for close interaction of Dsr pro-
teins during sulfur oxidation in the phototrophic sulfur bacterium
Allochromatium vinosum. J Bacteriol 187:1392–1404. https://​doi.​
org/​10.​1128/​JB.​187.4.​1392-​1404.​2005
Das R, Romi W, Das R et al (2018) Antimicrobial potentiality of act-
inobacteria isolated from two microbiologically unexplored forest
ecosystems of Northeast India. BMC Microbiol 18:1–16. https://​
doi.​org/​10.​1186/​s12866-​018-​1215-7
Davies J (2006) Where have all the antibiotics gone? Can J Infect Dis
Med Microbiol 17:287–290. https://d​ oi.o​ rg/1​ 0.1​ 155/2​ 006/7​ 07296
De Carvalho KG, Bambirra FHS, Kruger MF et al (2010) Antimicro-
bial compounds produced by Lactobacillus sakei subsp. sakei 2a,
a bacteriocinogenic strain isolated from a Brazilian meat product.
J Ind Microbiol Biotechnol 37:381–390. https://​doi.​org/​10.​1007/​
s10295-​009-​0684-y
de Vos WM, Kuipers OP, Roelof Van Der Meer J, Siezen RJ (1995)
Maturation pathway of nisin and other lantibiotics: post-trans-
lationally modified antimicrobial peptides exported by Gram-
positive bacteria. Mol Microbiol 17:427–437. https://​doi.​org/​
10.​1111/j.​1365-​2958.​1995.​mmi_​17030​427.x
García-Cano I, Rocha-Mendoza D, Ortega-Anaya J et  al (2019)
Lactic acid bacteria isolated from dairy products as potential
producers of lipolytic, proteolytic and antibacterial proteins.
Appl Microbiol Biotechnol 103:5243–5257. https://​doi.​org/​10.​
1007/​s00253-​019-​09844-6
Grewal S, Bhagat M, Vakhlu J (2014) Antimicrobial protein pro-
duced by Pseudomonas aeruginosa JU-Ch 1, with a broad
spectrum of antimicrobial activity. Biocatal Agric Biotechnol
3:332–337. https://​doi.​org/​10.​1016/j.​bcab.​2014.​04.​006
Hancock REW, Diamond G (2000) The role of cationic antimicrobial
peptides in innate host defences. Trends Microbiol 8:402–410.
https://​doi.​org/​10.​1016/​S0966-​842X(00)​01823-0
Hopfe M, Hegemann JH, Henrich B (2005) HinT proteins and
their putative interaction partners in Mollicutes and Chlamy-
diaceae. BMC Microbiol 5:1–14. https://​d oi.​o rg/​1 0.​1 186/​
1471-​2180-5-​27
Hurtado-Rios JJ, Carrasco-Navarro U, Almanza-Pérez JC, Ponce-
Alquicira E (2022) Ribosomes: the new role of ribosomal proteins
as natural antimicrobials. Int J Mol Sci 23:9123. https://​doi.​org/​
10.​3390/​ijms2​31691​23
Ishihama Y, Schmidt T, Rappsilber J et al (2008) Protein abundance
profiling of the Escherichia coli cytosol. BMC Genomics 9:102.
https://​doi.​org/​10.​1186/​1471-​2164-9-​102
Jeffery CJ (1999) Moonlighning proteins. Trends Biochem Sci 24:8–
Jeffery CJ (2017) Protein moonlighting: what is it, and why is it impor-
tant? Phil Trans R Soc B 373:20160523. https://​doi.​org/​10.​1098/​
rstb.​2016.​0523
Jenssen H, Hamill P, Hancock REW (2006) Peptide antimicrobial
agents. Clin Microbiol Rev 19:491–511. https://​doi.​org/​10.​1128/​
CMR.​00056-​05
Karaiskos I, Giamarellou H (2014) Multidrug-resistant and extensively
drug-resistant Gram-negative pathogens: current and emerging
therapeutic approaches. Expert Opin Pharmacother 15:1351–
1370. https://​doi.​org/​10.​1517/​14656​566.​2014.​914172
Keller A, Nesvizhskii AI, Kolker E, Aebersold R (2002) Empirical
statistical model to estimate the accuracy of peptide identifications
made by MS/MS and database search. Anal Chem 74:5383–5392.
https://​doi.​org/​10.​1021/​ac025​747h
Kieser T, Bibb MJ, Buttner MJ, et al (2000) Practical Streptomyces
genetics. The John Innes Foundation, Norwich
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression
data using real-time quantitative PCR and the 2-ΔΔCT method.
Methods 25:402–408. https://​doi.​org/​10.​1006/​meth.​2001.​1262
Ma Z, Qu B, Yao L et al (2020) Identification and functional charac-
terization of ribosomal protein S23 as a new member of antimi-
crobial protein. Dev Comp Immunol 110:103730. https://​doi.​org/​
10.​1016/j.​dci.​2020.​103730
Malik H, Sur B, Singhal N, Bihari V (2008) Antimicrobial protein
from Streptomyces fulvissimus inhibitory to methicillin resistant
Staphylococcus aureus. Indian J Exp Biol 46:254–257
Matsumoto H, Haniu H, Komori N (2019) Determination of protein
molecular weights on SDS-PAGE. Methods Mol Biol 1855:101–
105. https://​doi.​org/​10.​1007/​978-1-​4939-​8793-1_​10
Miao V, Davies J (2010) Actinobacteria: The good, the bad, and the
ugly. Antonie Van Leeuwenhoek 98:143–150. https://​doi.​org/​10.​
1007/​s10482-​010-​9440-6
Nesvizhskii AI, Keller A, Kolker E, Aebersold R (2003) A statistical
model for identifying proteins by tandem mass spectrometry. Anal
Chem 75:4646–4658. https://​doi.​org/​10.​1021/​ac034​1261
Pidutti P, Federici F, Brandi J et al (2017) Purification and characteri-
zation of ribosomal proteins L27 and L30 having antimicrobial
activity produced by the Lactobacillus salivarius SGL 03. J Appl
Microbiol 124:398–407. https://​doi.​org/​10.​1111/​jam.​13646
Pott AS, Dahl C (1998) Sirohaem sulfite reductase and other proteins
encoded by genes at the dsr locus of Chromatium vinosum are
involved in the oxidation of intracellular sulfur. Microbiology
144:1881–1894. https://​doi.​org/​10.​1099/​00221​287-​144-7-​1881
13
 687   Page 12 of 12
Putsep K, Branden CI, Boman HG, Normark S (1999) Antibacterial
peptide from H. pylori. Nature 398:671–672. https://​doi.​org/​10.​
1038/​19439
Qu B, Ma Z, Yao L et al (2020) Preserved antibacterial activity of
ribosomal protein S15 during evolution. Mol Immunol 127:57–66.
https://​doi.​org/​10.​1016/j.​molimm.​2020.​08.​024
Rodríguez JM (1996) Review: Antimicrobial spectrum, structure,
properties and mode of action of nisin, a bacteriocin produced by
Lactococcus lactis. Food Sci Technol Int 2:61–68. https://d​ oi.o​ rg/​
10.​1177/​10820​13296​00200​202
Seo JK, Kim DG, Oh R et al (2017) Antimicrobial effect of the 60S
ribosomal protein L29 (cgRPL29), purified from the gill of pacific
oyster, Crassostrea gigas. Fish Shellfish Immunol 67:675–683.
https://​doi.​org/​10.​1016/j.​fsi.​2017.​06.​058
Sidders B, Withers M, Kendall SL et al (2007) Quantification of global
transcription patterns in prokaryotes using spotted microarrays.
Genome Biol 8:R265. https://​doi.​org/​10.​1186/​gb-​2007-8-​12-​r265
Sierra JM, Fusté E, Rabanal F et al (2017) An overview of antimicro-
bial peptides and the latest advances in their development. Expert
13
Archives of Microbiology (2022) 204:687
Opin Biol Ther 17:663–676. https://​doi.​org/​10.​1080/​14712​598.​
2017.​13154​02
Yaghoubi A, Khazaei M, Ghazvini K et al (2021) Peptides with dual
antimicrobial-anticancer activity derived from the N-terminal
region of H. pylori ribosomal protein L1 (RpL1). Int J Pept Res
Ther 27:1057–1067. https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 10989-0​ 20-1​ 0150-3
Zhou X, Liao WJ, Liao JM et al (2015) Ribosomal proteins: functions
beyond the ribosome. J Mol Cell Biol 7:92–104. https://​doi.​org/​
10.​1093/​jmcb/​mjv014
Publisher's Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
Springer Nature or its licensor (e.g. a society or other partner) holds
exclusive rights to this article under a publishing agreement with the
author(s) or other rightsholder(s); author self-archiving of the accepted
manuscript version of this article is solely governed by the terms of
such publishing agreement and applicable law.