FEMS Microbiology Letters, 2022, 369, 1–7
DOI: 10.1093/femsle/fnac024
Advance access publication date: 10 March 2022
Research Letter – Physiology, Biochemistry & Genetics
Liliana López-Pliego, Verónica González-Acocal, Diana Laura García-González, Jimena Itzel Reyes-Nicolau, Zaira Sánchez-Cuapio,
Alan Shared Meneses-Carbajal, Luis Ernesto Fuentes-Ramírez and Miguel Castañeda
*
Centro de Investigaciones en Ciencias Microbiológicas, Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla. IC-11 Ciudad Universitaria Puebla,
Pue., C.P. 72000, México
∗
Corresponding author: Centro de Investigaciones en Ciencias Microbiológicas, Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla. Apdo. Postal
1622, C.P. 72000, Mexico. Tel: 52-222-2295500; Ext: 2527; E-mail: miguel.castaneda@correo.buap.mx
One sentence summary: Regulation of alginate synthesis by the novel histidine-kinase HrgS.
Editor: Geertje van Keulen
Abstract
Azotobacter vinelandii is a soil bacterium that produces alginates, a family of polymers of biotechnological interest. In A. vinelandii, alginate production is controlled by the two-component system GacS/GacA. GacS/GacA, in turn, regulates the Rsm post-transcriptional
regulatory system establishing a cascade that regulates alginate biosynthesis by controlling the expression of the algD biosynthetic
gene. In Pseudomonas aeruginosa, GacS/GacA is influenced by other histidine-kinases constituting a multicomponent signal transduction system. In this study, we explore the presence of GacS-related histidine-kinases in A. vinelandii and discover a novel histidinekinase (Avin_34990, renamed HrgS). This histidin-kinase acts as a negative regulator of alginate synthesis by controlling the transcription of the sRNAs belonging to the Rsm post-transcriptional regulatory system, for which a functional GacS is required.
Keywords: Azotobacter, Alginate, GacS, GacA, RsmZ, algD
Introduction
Alginates are a family of polysaccharides composed of monomers
of mannuronic and glucuronic acids. Alginates are compounds of
biotechnological interest used as stabilizing, thickening, gelling,
or film-forming agents (Galindo et al. 2007). Azotobacter vinelandii
is a GRAS (Generally Recognized As Safe) microorganism that produces copious amounts of alginates under vegetative growth conditions, which makes it a good candidate for the production of bacterial alginates (Urtuvia et al. 2017, Noar and Bruno-Barcena 2018).
In A. vinelandii, alginate synthesis is a highly regulated process; an important point of control is the expression of the algD
gene; this gene encodes the GDP-mannose dehydrogenase (EC
1.1.1.132), a key enzyme in alginate production. This enzyme oxidizes GDP-mannose to produce GDP-mannuronic acid, which is
the direct precursor of alginate synthesis (Galindo et al. 2007).
The Gacs/A two-component signal transduction system controls
the expression of the algD gene through activation of the posttranscriptional control system known as Rsm (Csr) (Manzo et al.
2011, López-Pliego et al. 2020).
The two-component signal transduction systems (TCS) are
composed of a homodimeric transmembrane histidine-kinase
(HK) that senses environmental signals and a cytoplasmic
response regulator (RR) that activates transcription upon phosphorylation by the sensor (Gao and Stock 2009). The TCS GacS/A
is conserved in a wide variety of gammaproteobacteria (Lapouge
et al. 2008). In Pseudomonas aeruginosa, GacS/A controls metabolites and physiological processes related to its pathogenesis
(Goodman et al. 2004), while in Pseudomonas fluorescens it regulates
metabolites involved in biocontrol (Humair et al. 2009). GacS is
an unorthodox HK with three phosphorylation domains (a transmitter or H1 domain, a receiver or D1 domain, and an Hpt or H2
domain) which are sequentially phosphorylated once a suitable
signal activates the protein. Subsequently, the phosphorylated
HK GacS transfers the phosphate group to the GacA response
regulator (Lapouge et al. 2008). Generally, GacA is a transcriptional
regulator that positively regulates the transcription of genes that
encode small RNAs regulators (sRNAs) belonging to Rsm (Csr)
family (Lapouge et al. 2008, Romeo et al. 2013). In the homolog
Rsm systems of Pseudomonas species, the number of Rsm-sRNAs
is variable, finding from two to five sRNAs grouped into three
families: RsmZ, RsmY, and RsmX (Moll et al. 2010). The RNA
binding protein RsmA is the other element of the Rsm control
system, and also, in Pseudomonas species it is common to find
more than one ortholog (Ferreiro et al. 2018, Sobrero and Valverde
2020). RsmA is a post-transcriptional regulator that usually functions as a translational repressor of its target genes, although,
in some cases, it stabilizes its target mRNAs and promotes their
translation. Rsm sRNAs counteract its repressor activity (Lapouge
et al. 2008, Romeo et al. 2013). Unlike Pseudomonas species, A.
vinelandii has only an RsmA ortholog, but possesses an unusually
high number of sRNAs: eight belonging to RmZ (RsmZ1-8) family
and one of the RsmY family, all of them are controlled by GacS/A,
and are involved in the control of alginate synthesis (Manzo et al.
2011, Lopez-Pliego et al. 2018).
In P. aeruginosa, GacS regulates the transcription of rsmZ and
rsmY, taking part in a multicomponent signal transduction sys-
Received: August 15, 2021. Revised: January 31, 2022. Accepted: March 8, 2022
C The Author(s) 2022. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail:
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HrgS (Avin_34990), a novel histidine-kinase related to
GacS, regulates alginate synthesis in Azotobacter
vinelandii
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FEMS Microbiology Letters, 2022, Vol. 369, No. 1
Material and methods
Microbiological procedures
The bacterial strains and plasmids used in this study are listed
in Table S1. Azotobacter vinelandii was grown at 30◦ C in Burk’s
nitrogen-free salts medium (Kennedy et al. 1986) supplemented
with 2% sucrose (BS). Escherichia coli strain DH5α was grown in
Luria-Bertani medium (LB) at 37◦ C. Antibiotic concentrations used
(in micrograms per milliliter) for A. vinelandii and E. coli, respectively, were as follows: tetracycline, 20 and 20; ampicillin (Ap), not
used and 100; nalidixic acid (Nal) and gentamicin (Gm), 1.5 and
10. Azotobacter vinelandii transformation and conjugation were carried out as previously described (Bali et al. 1992).
Nucleic acid procedures
DNA isolation and cloning procedures were carried out as described previously (Sambrook et al. 1989). The DreamTaq polymerase and Fusion High Fidelity DNA polymerase (Thermo Fisher
Scientific) were used for PCR amplifications.
Cloning the A. vinelandii E Avin_34990, retS, and
hptB loci
A 3069-bp DNA fragment containing Avin_34990 was amplified
by PCR from A. vinelandii E chromosomal DNA using the primers
ladScomFw and ladScomRv. The oligonucleotides used were designed from the DJ strain genome sequence. This fragment was
cloned into pCR2.1-TOPO, and the plasmid generated was named
pCRAvin_34990. This plasmid was used to determine the nucleotide sequences of the Avin_34990 locus of the strain E. Similarly, retS and hptB loci of strain E were amplified using the pair
of primers ER1RR_RetS_Fw/RRetS-S1 and hptBFw/hptBRv, respectively, the resulting amplicons of 2930-bp for retS and 1300-bp
for hptB. These amplicons were cloned in pGEMT-Easy Vector and
were sequenced, showing 100% of identity with the corresponding
sequences of DJ strain.
Generation of A. vinelandii Avin_34990 mutants
To construct the A. vinelandii Avin_34990 mutants, a 1027-bp fragment that encodes the cytoplasmic region of A.vin_34990 was amplified by PCR using 34990D and 34990R primers. The amplicon
was cloned in pGEMT-Easy, generating plasmid pGEMAvin_34990.
To create a mutation by insertion in Avin_34990 the plasmid pGEMAvin_34990 was cut at a single XhoI site located in the sequence corresponding to the transmitter domain of this HK. Subsequently, a 800-bp XhoI fragment carrying a Gm resistance cassette (obtained from pBSL141) was ligated to this plasmid. The resulting plasmid was named pGEMAvin_34990::Gm. Later, competent cells of the wild-type strain E were transformed with plasmid
pGEMAvin_34990::Gm, which had been previously linearized with
ScaII to ensure allelic exchange by double reciprocal recombination events. Gm resistant transformants were selected, and the
presence of the corresponding mutations and the absence of wild
type Avin_34990 alleles were confirmed by PCR analysis and subsequent sequencing (data not shown). The resulting mutant was
named EAvin_34990.
The same plasmid was used as described above to generate mutations in strains that carried transcriptional fusion of
the genes, rsmZ1 (EPrsmZ1-gusA), rsmZ2 (EPrsmZ2-gusA), and
rsmY (EPrsmY-gusA), and the translational fusion algD (AEDgusA). The resultant strains were called AED-gusAAvin34990,
EPrsmZ1-gusAAvin_34990, EPrsmZ2-gusAAvin_34990, EPrsmYgusAAvin_34990.
Construction of double mutants Avin_34990 gacS
To generate the double mutants EAvin_34990gacS, EPrsmYgusAAvin_34990gacS, EPrsmZ1-gusAAvin_34990gacS, and EPrsmZ2gusAAvin_34990gacS, the corresponding simple mutants
Avin_34990 were transformed with the ScaI linearized plasmid pMC7, which carried a non-polar mutation generated by
insertion of a streptomycin cassette in gacS. Sm resistant transformants were selected, and the success of double recombination
was verified by sequencing and PCR analysis using the primers
WTS2D y WTS2R (data not shown). Similarly, the EgacS mutant
was generated.
Complementation analysis of EAvin34990
mutant
To carry out the genetic complementation analysis of the mutant
Evin_34990, the 3069-bp DNA fragment cloned in pCRAvin_34990
was sub-cloned into pUMATc, an integrative suicidal vector that
promotes integration of the cloned DNA into the melA locus of the
A. vinelandii chromosome. For this, the insert was released from
pCRTOPOAvin_34990, by HindIII and XbaI double digestion, and
ligated into pUMATc cut with the same enzymes. This fragment
contains the Avin_34990 orf plus 250-bp located upstream of the
open reading frame with its putative regulatory region. The plasmid was called pUMATcAvin34990 and was transformed into the
mutant Eavin_34990. Transformants resistant to Tc were isolated
and confirmed by PCR analysis to carry the Avin34990 locus in-
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tem, also referred to as Multikinase Network (MKN), that involves
at least two additional HKs (Ventre et al. 2006, Chambonnier et al.
2016, Francis and Porter 2019). The MKNs are formed by a central pathway (Core Network) and by one or more lateral pathways
known as branches (Francis and Porter 2019). RetS is one of the
HKs that form the Core GacS Network; this protein is a hybrid HK
with an H1 domain, two D1 domains, and does not possess the
H2 (HptB) domain. RetS lacks its kinase activity but conserves its
phosphatase activity; it has been reported that RetS interacts with
GacS attenuating its kinase activity through several mechanisms
(Francis et al. 2018).
LadS is another hybrid HK member of these GacS-MKN. LadS
possesses a characteristic input domain 7TMR-DISMED2, a classic transmitter domain (H1) linked to a single receiver domain
(D1) but does not have the H2 domain. (Ventre et al. 2006). In P.
aeruginosa, LadS acts as a positive regulator of genes controlled
positively by GacS, establishing a phosphorelay that promotes the
phosphorylation of GacA. When the H1LadS domain is phosphorylated, the phosphate group is transferred to the D1LadS domain
and then to the H2GacS domain (Chambonnier et al. 2016).
This GacS-MKN also has been studied in P. fluorescens. RetS plays
an essential role in the repression at 35◦ C of the biocontrol factors controlled by GacS. LadS positively regulates the expression
of the Rsm-sRNAs, thus impacting the production of biocontrol
factors. It is proposed that this regulatory mechanism is carried
out through GacS (Humair et al. 2009).
The present work started the study of a possible GacS-MKN in
A. vinelandii involved in alginate synthesis. It explored the presence of putative homologs of ladS and found an HK with all the
characteristic domains of the LadS homologs but with a low identity percentage. The data obtained revealed that the HK studied
(Avin_34990) is not a functional homolog of LadS, but they also
show that it is a novel HK that participates in the GacS-MKN in A.
vinelandii.
López-Pliego et al.
| 3
serted into the melA gene. The PCR was done using the 34990D
and 34990R primers (data not shown). The recombinant strain was
named EAvin_34990/melA::Avin_34990.
Avin_34990 overexpression
Two-hybrid LexA assay
To carry out the LexA two-hybrid assay (Daines et al. 2002) among
GacS, Avin_34990 and RetS, a fragment that encoded the cytoplasmic region of GacS was amplified using the primers FgacSZSacI
and RgacSZKpnI and cloned as äbaitä in pSR659, using the restriction sites SacI and KpnI, generating the plasmid pSR659GacS.
The sequences corresponding to the cytoplasmic regions of RetS
and Avin_34990 were amplified by PCR and cloned in pSR658
to generate the prey proteins. The primers used in retS amplification were FretSZBamHI, RretSZSacI, and for Avin_34990 they
were F34990ZBamHI, R34990ZSacI. Both amplicons were cloned
into BamHI and SacI sites of pSR658, generating the plasmids
pSR658RetS, pSR658Avin_34990. The hptB gene was amplified using the FhptBSacI and RhptBKpnI primers and cloned in pSR658
using the restriction sites SacI and KpnI; the constructed plasmid was named pSR658HptB. The plasmid pSR659GacS combined
with pSR658RetS and pSR658Avin_34990 were co-transformed
into E.coli strain SU202, and the effect of the protein interactions
was visualized on MacConkey-lactose indicator plates. A similar
assay was carried out between RetS and Avin_34990 and HptB using the plasmids pSR559HptB and pSR658Avin_34990. For quantitative assays, the beta-galactosidase activity was measured as
indicated in the next paragraph.
Analytical methods
Protein was determined by the Lowry method (Lowry et al. 1951).
Alginate production was determined as previously described (Blumenkrantz and Asboe-Hansen 1973). β-Galactosidase activities
were determined as reported by Miller (1972). All measurements
were done in triplicate. Glucuronidase activity was measured as
reported by Wilson et al. (1995). About 1 U corresponds to 1 nmol
of O-nitrophenyl- ß-D-glucuronide hydrolyzed per min per μg of
protein.
Results
Search for putative LadS homologs in A.
vinelandii
To determine the presence of ladS homologs in the A. vinelandii DJ
genome sequence, we performed a BLASTN search using P. aeruginosa ladS as a query sequence due to the phylogenetic closeness
between P. aeruginosa and A. vinelandii. (Rediers et al. 2004). The
search was unsuccessful, and when a similar search was conducted using BLASTP it found a protein (encoded by Avin_34990)
with 33.76% of identity and 96% of cover .Similar results (33.25%
of identity and 53.45% of similarity) were obtained when aligning with the CLUSTAL algorithm. Although the identity is on
the borderline of significance, the prediction of the domains contained in the protein showed the architecture characteristic of the
Figure 1. Regulatory effect of Avin_34990 on alginate synthesis. (A)
Mucoid phenotypes of wild-type strain E and its derivate with an
Avin_34990 mutation. (B) Alginate production in Avin_34990 mutants,
and effect of Avin_34990 overexpression in the wild-type strain E over
alginate production. (C) Effect of the Avin_34990 mutation on the
activity of the PalgD-gusA translational fusion in strains AED-gusA and
AED-Avin_34990. All the measurements were carried out in cells grown
for 48h in Burk´s minimal media with sucrose. The bars represent the
statistical media of three measurements and their standard deviation.
LadS homologs. LadS, in addition to the transmitter domain, have
a receiver domain and an unusual 7TMR-DISMED2 input domain
(Ventre et al. 2006). Interestingly, when we carried out a BLAST
search of Avin_34990 in P. aeruginosa PAO1 genome sequence, we
found that this HK has high homology with the protein PA3462
(Score of 942, with 56% of identity and 97% of cover). The CLUSTAL
alignment resulted in 55.54% of identity and 78.74% of similarity (Fig. S1). PA3462 is a HK located in a group of orthologs of LadS
in Pseudomonas Genome DB (https://www.pseudomonas.com/orth
ologs/list?id = 110768); however, the function of PA3462 has not
been completely characterized as yet (Kollaran et al. 2019).
Avin_34990 regulates the synthesis of alginates
in the strain E
To start studying A. vin_34990 in the wild type E strain first, we
established the conservation of the Avin_34990 in this strain. The
gene was amplified, cloned, and sequenced, finding that it is practically identical to its counterpart of the strain DJ (99% of identity). Subsequently, by allelic exchange, we generated the mutant
EAvin_34990, which showed a hyper-mucoid phenotype (Fig. 1A).
In A. vinelandii, the mucoid phenotype is directly related to alginate synthesis, this fact was corroborated by quantifying alginate
production in the mutant (Fig. 1B). The complementation carried
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With the aim of over-expressing Avin_34990 we sub-cloned the
insert of the plasmid pCRAvin_34990 into the pBBR1MCS-5 vector. A fragment obtained from pCRAvin_34990 by digestion with
HindIII and XbaI was ligated into pBBR1MCS-5 which had been previously cut with the same enzymes. The resultant plasmid, named
pBBRAvin_34990, was conjugated to the wild-type strain to generate the strain E/pBBR Avin_34990.
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FEMS Microbiology Letters, 2022, Vol. 369, No. 1
out ruled out a polarity effect of the mutation. Consistent with the
negative regulatory character of Avin_34990, its over-expression
diminished alginate production (Fig. 1B).
Upon discovering that Avin_34990 controlled alginate synthesis, the question arose about the ultimate regulatory target among
alginate biosynthetic genes. Since the GacS/A-Rsm pathway regulates the expression of the algD gene at the post-transcriptional
level, we used the translational fusion PalgD-gusA to determine
the effect of the Avin_34990 mutation on the expression of algD.
The results of this experiment answered the question; Fig. 1C
shows that in the mutant AED-gusAAvin_34990, algD expression
is increased threefold, which is evidence that Avin_34990 regulates alginate synthesis negatively through the control it exerts at
the post-transcriptional level on the algD gene.
HK Avin_34990 is involved in the transcriptional
control of genes encoding sRNAs of the Rsm
family
In A. vinelandii, Rsm-sRNAs control alginate biosynthesis, although the impact of the mutation in each one is different. Of the
nine Rsm-sRNAs, RsmZ1, RsmZ2, and RsmY are the ones that have
the most significant influence on alginate synthesis (López-Pliego
et al. 2018, López-Pliego et al. 2020). Therefore, we assessed the
effect of the Avin_34990 mutation in the transcription of rsmZ1,
rsmZ2, and rsmY. Using gusA transcriptional fusions, we found
that rsmZ1, rsmZ2, and rsmY significantly increased their transcription due to the Avin_34990 mutation (Fig. 2A). As can be seen
in the Fig. 2A, the negative effect of the mutation is proportional
in all three cases. These results suggest that the regulator is part
of a regulatory pathway that controls the transcription of these
Rsm-sRNAs in a common way.
GacS is required for the Avin_34990 function
GacS/A-Rsm pathway controls alginate synthesis by positively
regulating the algD gene expression (Manzo et al. 2011, LopezPliego et al. 2020). Avin_34990 also controls the expression of algD
through the Rsm system, but it does so negatively. So it would
be possible that both HKs were related in the regulatory pathway. To test the existence of this relationship a double mutant
EgacSAvin_34990 was generated. The mucoid phenotype of the
double mutant (Fig. 2B), clearly related Avin_34990 with GacS,
subsequently, to verify what had been observed we carried out
quantifications of alginate production in the single mutants EgacS
and EAvin_34990, and in the double mutant EgacSAvin_34990. The
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Figure 2. Alginate synthesis and transcription of the rsmZ1, rsmZ2, and rsmY sRNAs in double mutants Avin_34990 gacS. (A) Promotor activity of the
transcriptional fusions PrsmZ1-gusA, PrsmZ1-gusA, and, PrsmZ1-gusA in strains EPrsmZ1-gusA, EPrsmZ1-gusA, and EPrsmZ1-gusA, and in their derivative
mutants Avin_34990. (B) Mucoid Phenotype in A. vinelandii wild-type strain E and their derivative mutants EgacS, EAvin_34990, and EgacSAvin_34990. (C)
Alginate production in strains E, EAvin_34990, EgacS, and EAvin_34990gacS. (D) β-glucuronidase activity of the PrsmZ1-gusA, PrsmZ1-gusA, and
PrsmZ1-gusA transcriptional fusions that carry the strains E, EAvin_34990, EgacS, and EAvin_hrgSgacS. All the measurements were carried out in cells
grown for 48 h in Burk´s minimal media with sucrose. The bars represent the statistical media of three measurements and their standard deviation.
López-Pliego et al.
| 5
Discussion
Figure 3. Determination of the interaction of GacS with Avin_34990,
RetS, and HptB, established by LexA Two-hybrid assays. (A) Plate
Two-hybrid assay performed with the HKs GacS, Avin_34990, and RetS.
(B) Quantitative Two-hybrid assay carried out with the HKs GacS,
Avin_34990, and RetS. (C) Plate Two-hybrid assay carried out with the HK
GacS and the Hpt containing protein HptB. The LexA dimerization
domain was removed and replaced with GacS, RetS and Avin_34990
proteins (A) or GacS, RetS and HptB proteins (C). Since LexA is an active
repressor only as a dimer, dimerization of the tested proteins could
allow chimeric LexA to bind to its operator site and repress transcription
of the lacZ reporter gene, resulting in a lactose-negative phenotype of
the E. coli reporter strain.
results obtained show that the gacS mutation was epistatic to
the Avin_34990 mutation (Fig. 2C). For its role in alginate regulation and its relationship with GacS we propose re-naming the
Avin_34990 gene as hrgS (Histidine-kinase related to GacS).
Given the above results, it would be possible that the gacS mutation could revert the effect of the Avin_34990 mutation on the
transcription of rsm-sRNAs. This fact was verified by measuring
the transcription of rsmZ, rsmZ2, and rsmY in the double mutant
EgacSAvin_34990 (Fig. 2D).
Histidine Kinase Avin_34990 does not interact
with GacS and HptB
Finally, we explored the possibility that Avin_34990 needs to contact GacS to carry out its function. For this purpose, we assessed
the interaction using the LexA two-hybrid system (Daines et al.
2002). This assay allows genetic analysis to be carried out in Escherichia coli, useful for revealing the formation of heterodimers
The ubiquitous presence of GacS/A among gammaproteobacteria contrasts with the selective presence of HKs LadS and RetS
(Brinkman et al. 2001, Chambonnier et al. 2016). To date, the MKN
composed of GacS, LadS, and RetS has only been found in Pseudomonas, Lysobacter, and Alcanivorax species (Chambonnier et al.
2016, Sobrero and Valverde 2020). The Azotobacter genus belongs
to the Pseudomonadecea family, but there is controversy about
whether species of the genus Azotobacter should be considered
Pseudomonas (Rediers et al. 2004, Gomila et al. 2015). Studies based
on multi-locus sequence typing (MLST) and genomic phylogenetic
analyses showed that the Pseudomonadaceae family included the
genera Azotobacter (Lalucat et al. 2020). An argument against reclassification is based on the disparity of the biological characteristics of the species of the Azotobacter genera and Pseudomonas
(Martínez-Carranza et al. 2019, Lalucat et al. 2020). Furthermore,
analysis of the percentage of conserved protein (POCP) clearly separates A. vinelandii from the Pseudomonas species (Lalucat et al.
2020). The physiological and phenotypical differences between
the two genera could be due to differences in signaling systems.
From this, it was interesting to determine the presence of the
HKs of the MKN related to GacS. Our search led us to find a homolog of the hypothetical protein PA3462 of P. aeruginosa PAO1
(Avin_34990). PA3462 belongs to a group of LadS orthologs; although several studies strongly suggest that this HK is not a functional homolog of LadS. PA3462 regulates phenotypes non-related
with LadS, such as swarming motility and antibiotic resistance
(Kollaran et al. 2019, Kollaran et al. 2019); in addition, even though
both HKs have a DISMED2 sensor domain, only LadS responds to
calcium ( Basu Roy and Sauer 2014).
Avin_34990 (HrgS) is an orphan hybrid HK that does not have an
Hpt domain, so it could not directly activate a response regulator.
Therefore, to achieve it, interaction with Hpt-containing proteins
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between the proteins analyzed. The heterodimer formed by the
LexA hybrid proteins represses the expression of the reporter gene
lacZ located on the chromosome of the reporter strain of the system. We cloned DNA fragments corresponding to the cytoplasmic
domains of gacS and Avin_34990 into LexA expression vectors to
perform the assay. Figure 3A shows the results of the qualitative
assay, the color change in the MacConkey medium evidenced the
absence of interaction between GacS and Avin_ 34990. Figure 3B
shows quantitative confirmation of the previous result. For use as
a positive control, we cloned a DNA fragment corresponding to the
cytoplasmic part of RetS of A. vinelandii. The interaction between
GacS and RetS in P. aeruginosa is widely documented; although the
RetS homolog in A. vinelandii has not yet been characterized, it is
highly conserved regarding RetS of P. aeruginosa (56% of identity).
As expected, a positive interaction was revealed (Fig. 3A).
There are reports of some phosphorylated hybrid HKs, as HrgS,
which can transfer their phosphate to Hpt-containing proteins
and subsequently can activate a response regulator (Hsu et al.
2008). Azotobacter vinelandii only has one Hpt-containing protein
(Avin_34350, homologous to HptB of P. aeruginosa) that could interact with HrgS. For this reason, we assessed by LexA two-hybrid
assays the possible interaction between HptB and HrgS. We cloned
hptB into LexA expression vector pSR659, which, together with
pSR658Avin_34990, was used to carry out the assay. Figure 3C
shows a MacConkey medium plate where the color change indicates no interaction strong enough to be evidenced by this genetic
test. The interaction between GacS and RetS was used as a positive control for the assay.
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FEMS Microbiology Letters, 2022, Vol. 369, No. 1
species do not have more than 38 HKs (http://www.p2cs.org). In
these conditions, in A. vinelandii, the combinatorial use of HKs in
the MKNs could be essential to optimize the signaling processes.
Supplementary data
Supplementary data are available at FEMSLE online.
Acknowledgments
V. González-Acocal thanks CONACyT for M.Sc. scholarships.
Funding
This work was supported by VIEP-BUAP, grant 100301900-VIEP-21.
Conflict of interest statement. The authors declare that they have no
conflict of interest.
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would be necessary; since Avin_34350 (HptB) is the only protein
of this family in A. vinelandii we hypothesized that it could function as an intermediary. The result of the two-hybrid assay ruled
out the above possibility (Fig. 3C). In P aeruginosa, HptB is related to
four hybrid HKs, including RetS (Hsu et al. 2008). Thus, RetS participates in two ways in the GacS-MKN: acting in the pathway where
it directly interacts with GacS (Core GacS Network); and in a pathway related to HptB called HptB branch (Francis and Porter 2019).
The GacS-HptB branch only controls the rsmY transcription (Bordi
et al. 2010), unlike the Core GacS Network (formed by GacS, RetS,
and LadS), which regulates rsmY and rsmZ transcription (Chambonnier et al. 2016). In A. vinelandii HrgS controls the expression
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like Core GacS does, which suggests that HrgS could be part of a
homolog Core GacS Network.
HrgS controls alginate synthesis negatively; among the GacSrelated HKs in Pseudomonas spp, RetS also acts as a negative regulator and requires a strong physical contact with GacS to carry out
its function as phosphatase of GacS (Goodman et al. 2009, Francis et al. 2018). Unlike RetS, the two-hybrid assay results suggest
that HrgS does not physically interact with GacS, which indicates
that HrgS performs a different mechanism than the one proposed
for RetS. It is known that LadS also influence the phosphorylation
state of GacS; although there are no experimental data that evidence a strong contact between LadS and GacS, it is proposed
that at least a transitory and weak contact is established (Chambonnier et al. 2016), a similar fact can occur between GacS and
HrgS, and this could influence the phosphorylation state of GacS.
On the other hand, it is known that some hybrid HKs have the
capacity, under certain conditions, to dephosphorylate response
regulators (Alvarez et al. 2016), so it is possible that GacA is being
dephosphorylated by HrgS. Assuming that the putative RetS of A.
vinelandii had a role similar to its homolog in P. aeruginosa, RetS
and HrgS could negatively control the state of phosphorylation
of GacA, thus controlling the expression levels of the genes that
encode RNAs of the Rsm family.
In addition to HrgS, in the genome sequence of the A. vinelandii
strain DJ, we found another gene (Avin_41190) that encodes an
HK with the characteristic domains of LadS homologs; however, it
also presents a low identity and similarity percentages with LadS
of P. aeruginosa (25.51% and 51.18%, respectively). Likewise, this HK
has a low percentage of identity and similarity with HrgS (26.23%
and 42.52%, respectively). It would be interesting to study this HK
and establish if it is related to this GacS-MKN having a similar
function to LadS in Pseudomonas spp.
In P. aeruginosa, in addition to GacS, RetS, and LadS, the HK
PA1611 also participates in the GacS-MKN; this HK counteracts
the interaction of RetS with GacS, thus favoring the phosphorylated state of GacS (Chambonnier et al. 2016). Contrary to what
was expected in A. vinelandii, there is no homolog of PA1611. Thus,
the hetero-dimerization found between GacS and RetS (Fig. 3A)
suggests that RetS is part of a slightly different GacS-MKN in
A.vinelandii composed of GacS, RetS, and HrgS.
Unlike LadS, which is found in any Pseudomonas species, PA3462
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Pseudomonas resinovorans. Interestingly, homologs to HrgS/PA3462
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and Azotobacter, there are variants in GacS-MKN and this could
partially explain the differences between these genera. Another
aspect to consider is the number of TCS in these genera; the
Pseudomononas species possesses up to 60 HKs while Azotobacter
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