
==== Front
Curr Microbiol
Curr Microbiol
Current Microbiology
0343-8651
1432-0991
Springer US New York

39153035
3835
10.1007/s00284-024-03835-1
Original Paper
A Homolog of the Histidine Kinase RetS Controls the Synthesis of Alginates, PHB, Alkylresorcinols, and Motility in Azotobacter vinelandii
Rosales-Cruz Araceli
Reyes-Nicolau Jimena
Minto-González Eduardo
Meneses-Carbajal Alan
Mondragón-Albarrán Claudia
López-Pliego Liliana
http://orcid.org/0000-0002-4565-8514
Castañeda Miguel miguel.castaneda@correo.buap.mx

https://ror.org/03p2z7827 grid.411659.e 0000 0001 2112 2750 Centro de Investigaciones en Ciencias Microbiológicas, Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla, IC-11 Ciudad Universitaria Puebla, Apdo. Postal 1622, C. P. 72000 Puebla, Pue Mexico
17 8 2024
17 8 2024
2024
81 10 31128 5 2024
8 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by/4.0/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
The two-component system GacS/A and the posttranscriptional control system Rsm constitute a genetic regulation pathway in Gammaproteobacteria; in some species of Pseudomonas, this pathway is part of a multikinase network (MKN) that regulates the activity of the Rsm system. In this network, the activity of GacS is controlled by other kinases. One of the most studied MKNs is the MKN-GacS of Pseudomonas aeruginosa, where GacS is controlled by the kinases RetS and LadS; RetS decreases the kinase activity of GacS, whereas LadS stimulates the activity of the central kinase GacS. Outside of the Pseudomonas genus, the network has been studied only in Azotobacter vinelandii. In this work, we report the study of the RetS kinase of A. vinelandii; as expected, the phenotypes affected in gacS mutants, such as production of alginates, polyhydroxybutyrate, and alkylresorcinols and swimming motility, were also affected in retS mutants. Interestingly, our data indicated that RetS in A. vinelandii acts as a positive regulator of GacA activity. Consistent with this finding, mutation in retS also negatively affected the expression of small regulatory RNAs belonging to the Rsm family. We also confirmed the interaction of RetS with GacS, as well as with the phosphotransfer protein HptB.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00284-024-03835-1.

http://dx.doi.org/10.13039/501100015029 Vicerrectoría de Investigación y Estudios de Posgrado, Benemérita Universidad Autónoma de Puebla 100301900-VIEP2022 Castañeda Miguel issue-copyright-statement© Springer Science+Business Media, LLC, part of Springer Nature 2024
==== Body
pmcIntroduction

In bacteria, signal transduction is generally performed by proteins belonging to signaling systems known as two-component systems (TCSs). TCSs are composed of a receptor protein (histidine kinase, HK) that, upon receiving a signal, autophosphorylates a histidine residue in the transmitter domain (H1) and activates, through transphosphorylation, the second element of the system known as the response regulator (RR). RR is phosphorylated on an aspartate residue of its receptor domain, also known as REC or D2 [1]. The HK-RR regulatory pair paradigm has recently been questioned because of the existence of systems with two or more HKs that control the phosphorylation status of an RR but where only one HK phosphorylates the RR. The accessory HK (or HK) is proposed to stimulate or block the kinase activity of the main HK. These signal transduction systems are known as multikinase networks (MKNs), and their study has become a novel area of research in physiology and signaling in prokaryotes [2]. GacS/A is a common TCS in Gammaproteobacteria and is considered a global regulatory system of secondary metabolism [3]. The regulation exercised by GacS/A uses the posttranscriptional control system Rsm (Csr) as an intermediary, which consists of one or more proteins and two or more small regulatory RNAs (sRNAs) [3, 4]. The protein(s) of the Rsm (Csr) system, encoded by the rsmA (csrA) gene, bind to its mRNA targets and, in most cases, block their translation, and promote their degradation, although they can act via other mechanisms [5]. The sRNAs-Rsm counteracts the functions of RsmA, and GacA positively controls its transcription [4]. The TCS GacS/A in Pseudomonas aeruginosa is related to other HKs. It forms a MKN in which GacA activity is negatively controlled by RetS and positively controlled by the HK LadS [2, 6]. Thus, GacS, RetS, and LadS constitute the core of the GacS-MKN that controls the activation of GacA, which in turn regulates the transcription of rsmZ and rsmY, the genes that encode the sRNAs of the Rsm system [2, 7]. Another HK, PA1611, has been reported to block the activity of RetS on GacS [8]. GacS is an unorthodox HK. In addition to the transmitter or H1 domain, this type of kinase has two extra phosphorylation domains: a receiver domain (D1) and a domain-designated H2 or Hpt. Recently, a new pseudoreceptor domain (referred to as the ND) was identified in GacS. This domain is situated between the transmitter and receiver domains and is essential for autokinase activity [9]. In these kinases, phosphate is transferred sequentially from the H1 domain to D1 and from D1 to H2, which transfers the phosphate to the D2 domain of the RR [3]. RetS, LadS, and PA1611 are hybrid HKs; these kinases are similar to unorthodox HKs but do not have an H2 (Hpt) domain [8, 10]. Thus, only GacS can phosphorylate GacA, while the hybrid HKs of this MKN stimulate GacS phosphorylation or promote its dephosphorylation [2, 6, 8].

On the other hand, PA1611 phosphorylates a soluble protein that carries an Hpt domain-designated HptB, which, in turn, indirectly activates the expression of the sRNA RsmY. In addition to PA1611, the kinases ErcS’ and SagS can also phosphorylate HptB, and together with PA1611, form part of the HptB branch of GacS-MKN [8, 10]. In Pseudomonas fluorescens, Pseudomonas protegens, Pseudomonas putida, and Pseudomonas syringae, there are orthologs of these kinases that constitute the core of the GacS-MKN, but their roles in GacS-MKN have not been characterized [11–13]. Outside of the genus Pseudomonas, only in Azotobacter vinelandii has this MKN been studied [14].

A. vinelandii is a nitrogen-fixing soil bacterium that forms cysts resistant to desiccation and produces three metabolites of biotechnological interest: alginates, polyhydroxybutyrate (PHB), and alkylresorcinols (ARs) [15–17]. Alginates are a family of polymers composed of monomers of guluronic acid and mannuronic acid joined by links β (1–4). In industry, alginates are used as additives for viscosifying, stabilizing, emulsifying, and gelling aqueous solutions [18]. PHB is a polyester consisting of β-hydroxybutyrate monomers belonging to the polyhydroxyalkanoate (PHA) family. PHAs are generally synthesized and stored as a source of carbon and energy. PHAs are interesting compounds since they can form plastics with properties similar to those of polypropylene and polyethylene with the advantage of being biodegradable [19]. ARs are long-chain phenolic lipids. The ARs synthesized by A. vinelandii are 5-n-eneicosylresorcinol, 5-n-tricosylresorcinol, and their galactosidase derivatives, which, together with alkylpyrones, replace the phospholipids in the membrane during encystment and are part of the cyst covering [17]. ARs have potential uses as immunomodulators and anticancer and antimicrobial agents [20]. GacS/A regulates alginates, polyhydroxybutyrate (PHB), and ARs through the posttranscriptional control system Rsm, which specifically controls the transcription of the sRNAs RsmZ1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, and RsmY [21–25]. Previously, in A. vinelandii, the presence of a retS homolog was reported and shown to interact with GacS, although it has not been thoroughly characterized [14]. Interestingly, this bacterium does not have a ladS homolog but possesses another HK (designated HrgS) functionally related to GacS; thus, in A. vinelandii, unlike that reported in P. aeruginosa, the core of GacS-MKN could be integrated by GacS, HrgS, and RetS [14]. This work reports the phenotypic characterization of the A. vinelandii retS homolog and its probable function in the GacS-MKN.

Materials and Methods

Microbiological Procedures

The bacterial strains used are listed in Table S1. A. vinelandii strains were grown at 30 °C in Burk’s nitrogen-free medium with salts [26] supplemented with 20 g/L sucrose (BS). Escherichia coli strain DH5-α was grown on Luria–Bertani (LB) medium at 37 °C. The antibiotic concentrations used (in μg/mL) for A. vinelandii and E. coli were as follows: tetracycline (Tc), 40 and 20; kanamycin (Km), 4 and 20; gentamicin (Gm), 1.5 and 10; streptomycin (Sm) 2 and 20; ampicillin (Ap), 0 and 100; nalidixic acid (Nal), 10 and 10. A. vinelandii transformation and conjugation were carried out as previously described [27].

Nucleic Acid Procedures

DNA isolation and cloning procedures were carried out as described previously [28]. DreamTaq polymerase and Fusion High FidelityDNA polymerase (Thermo Fisher Scientific) were used for PCR amplification. The A. vinelandii DJ [29] genome sequence was used to design the oligonucleotides for PCR amplification.

Generation of A. vinelandii retS Mutants

A 1581-bp DNA fragment containing retS was amplified by PCR from A. vinelandii E [30] chromosomal DNA with the primers FRetSZ-BH1 and RRetS-S1. The oligonucleotides used were designed from the DJ strain genome sequence (Table S1). This fragment was subsequently cloned and inserted into the pGEMT-Easy vector, and the resulting plasmid was designated pGEMretS1.5. This plasmid was used to determine the nucleotide sequence of the retS locus of strain E, which showed 100% identity with the corresponding sequences of the DJ strain. To construct the A. vinelandii retS mutant, the plasmid pGEMretS1.5 was cleaved at the single ClaI site located within the sequence corresponding to the transmitter domain of this HK. A 1200-bp ClaI fragment carrying a Km resistance cassette obtained from pBSL98 [31] was subsequently ligated into this plasmid; the resulting plasmids were designated pGEMretS::KmNP and pGEMretS::KmP. In the first plasmid, the resistance cassette was inserted in the same sense orientation as that of the retS gene; in the second plasmid, the Km cassette was inserted in the opposite sense orientation to that of the retS gene. In A. vinelandii, the insertion of resistance cassettes into genes with the same orientation as the direction of transcription produces nonpolar mutations, which allow transcription of the downstream genes in the same operon; otherwise, insertions in the opposite sense generate polar mutations [32]. Later, competent cells of the wild-type strain E were transformed with the plasmids pGEMretS::KmNP and pGEMretS::KmP that were previously linearized with ScaI to ensure allelic exchange by double reciprocal recombination events. Km-resistant transformants were selected, and the corresponding mutations and the absence of wild-type retS alleles were confirmed by PCR analysis and subsequent sequencing (data not shown). The resulting mutants were designated EretSNP and EretSP.

Plasmid pGEMretS::KmNP was used as described above to generate mutations in strains that carried a transcriptional fusion of the genes rsmZ1 (EPrsmZ1-gusA), rsmZ2 (EPrsmZ2-gusA), rsmZ6 (EPrsmZ6-gusA), and rsmY (EPrsmY-gusA) [24, 25]. The resulting strains were designated EPrsmZ1-gusAretS, EPrsmZ2-gusAretS, EPrsmZ6-gusAretS, and EPrsmY-gusAretS.

Construction of retS gacS Double Mutants

To generate the double mutant EretSgacS, the corresponding single mutant retS was conjugated with the plasmid pSUPgacS::Sm, which carried a nonpolar mutation generated by the insertion of a streptomycin cassette in gacS. pSUPgacS::Sm was constructed by subcloning the insert of pMC7 [33] into pSUP202 [34]. Afterward, Sm-resistant transconjugants were selected, and the success of the double recombination was verified by sequencing and PCR analysis with the primers WTS2D and WTS2R (Table S1) (data not shown).

Complementation Analysis of the EretS Mutant

To carry out genetic complementation analysis of the mutant EretS, a 3069-bp DNA fragment was amplified with the primers ER1RR_RetS_Fw and RRetS-S1 (Table S1) and subsequently cloned and inserted into pGEMT-Easy, generating the plasmid pGEMretSwt. The sequence inserted into pGEMretSwt was subcloned and inserted into pUMATc [35]; pUMATc is an integrative suicide vector that promotes the integration of the cloned DNA into the melA locus of the A. vinelandii chromosome. Transformants resistant to Tc were isolated and confirmed by PCR analysis to carry the retS locus inserted into the melA gene. PCR was performed with the ER1RR_RetS_Fw and Hsp70_RTIRv primers (Table S1) (data not shown). The recombinant strain was designated EretS/melA::retS.

Two-Hybrid LexA Assay

This assay, which was carried out in E. coli, allows the interaction between two proteins to be studied. LexA is a repressor that acts as a dimer. An interaction domain is required to form the dimer, which is removed and replaced by the domains of the proteins to be tested. If this occurs, a dimer is formed that represses the expression of lacZ located on the chromosome of the reporter strain of the system. To carry out the LexA two-hybrid assay [36] between GacS and RetS, the plasmids pSR659GacS and pSR658RetS were used. Similarly, these plasmids, along with pSR658RetS and pSR659HptB, were used to determine the RetS and HptB interaction. The plasmid pSR659HptB was constructed for this study by cloning a PCR fragment corresponding to the coding region of hptB; this fragment was amplified with the primers HptBTH SacIFw and HptBTHKpnIRv (Table S1). The plasmid pSR659GacS combined with pSR658RetS was cotransformed into the E. coli strain SU202, and the effects of the protein interactions were visualized on MacConkey-lactose indicator plates. A similar assay was performed for RetS and HptB with the plasmids pSR658RetS and pSR559HptB.

Motility Assays

To perform the motility assays, the bacterial strains were grown on BS medium at 30 °C until they reached the exponential phase (24 h). Samples of the cells (1X105 CFU) were then transferred to BS plates containing 0.15% agar for swimming tests. These plates were incubated at 30 °C for 24 h.

Analytical Methods

Protein content was determined by the Lowry method [37]. Alginate production was determined as previously described [38]. ARs synthesis was measured as reported previously [39]. The PHB content of the bacteria was assayed by the method of Law and Slepecky [40], and β-galactosidase activities were determined as previously reported [41]. All the measurements were performed in triplicate. Glucuronidase activity was measured as previously reported [42]. One U corresponds to 1 nmol of O-nitrophenyl-β-d-glucuronide hydrolyzed per min per µg of protein.

The sequence accession numbers of the A. vinelandii strains used in this work were as follows: DJ strain, GenBank CP001157; E (AEIV) strain, GenBank CP092752.

Results

Search for Putative retS Homologs in A. vinelandii

We initiated our investigation of the A. vinelandii retS gene with Avin_6870, a putative gene annotated as a retS homolog in the genome sequence of the DJ strain. The DJ strain is a nonmucoid type strain of A. vinelandii [29]. This gene has a 48% identity with retS of P. aeruginosa, while the protein it encodes has a 57% identity with its P. aeruginosa counterpart. The prediction of the protein domains revealed the characteristic architecture of RetS homologs, which feature two receiver domains and an unusual 7TMR-DISMED2 input domain in addition to the transmitter domain [6]. The transmitter domain (H1) of the RetS homolog of A. vinelandii has a high identity (69%) with its P. aeruginosa counterpart (Fig. S4). These unique features of RetS homologs are crucial for understanding the protein’s function and potential implications (Fig. S1).

Previously, a 3.5 kb fragment containing retS and its regulatory region from the wild-type mucoid strain E (also named AEIV) was amplified and sequenced. We decided to work with this strain because it produces alginate, and much of our work has focused on studying the production of this polymer. The sequence was found to be practically identical to its counterpart in the DJ strain, with a remarkable 99% identity. Recently, the genome of strain E (AEIV) was released in the GenBank database. The sequence of the retS locus reported in GenBank was 100% identical to that obtained in this work. In A. vinelandii, a gene that encodes a heat shock protein belonging to the Hsp70 family is located 100 bp downstream of retS. Due to the proximity between these genes, mutations in retS could affect hsp70.

RetS Regulates the Synthesis of Alginates in Strain E

By allelic exchange and the use of previously described mutagenic plasmids (see “Materials and Methods” section for details), we generated EretS-polar and EretS-nonpolar mutants (EretSP and EretSNP, respectively). To test the polar and nonpolar nature of the insertions, we performed RT-PCR amplification of a region downstream of the insertions (Fig. S2b); lines 3 and 4 correspond to the samples of the polar and nonpolar mutants, respectively. The absence of amplification in line 3 proved the polarity of the insertion in the EretSP mutant. The amplification shown in line 4 corresponds to the nonpolar mutant (EretSNP), which shows that the insertion did not affect the integrity of the retS mRNA, this is consistent with the published findings regarding these types of mutations [32]. Furthermore, to rule out a polarity effect on the expression of the hsp70 gene, we performed a transcriptional analysis of hsp70 by RT-PCR. The results are shown in Fig. S2c, which shows that the expression of hsp70 was not affected in the EretSP (line 3) and EretSNP (line 4) mutants.

The EretS mutants presented a diminished mucoid phenotype; another relevant phenotypic characteristic was irregular and discontinuous growth on agar (Fig. 1a). In A. vinelandii, the mucoid phenotype is directly related to alginate synthesis; this finding was corroborated by the quantification of alginate production in the EretS mutants [43]. Polar and nonpolar mutants had similar effects on alginate production (Fig. 1b), on the other hand, introducing the wild-type allele of retS into the EretSP mutant restored alginate synthesis. The polarity analysis previously described, together with the complementation of the EretSP mutant with the wild-type gene (Fig. 1b), revealed that the effect observed in the synthesis of alginates was only due to the absence of retS. On the basis of these results, we decided to continue characterizing retS in the polar and nonpolar mutants.Fig. 1 Regulatory effect of AvAEIV_000681 (retS) on alginate synthesis. a Mucoid phenotypes of wild-type strain E and its derivate with an AvAEIV_000681 (retS) mutation. b Alginate production in A. vinelandii retS mutants. All the measurements were done 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. Significant differences were analyzed by Anova test. Statistical significance is indicated. P < 0.1234; ns (not significant), *P < 0.03232; **P < 0.021; ***P < 0.0002; ****P < 0.0001

RetS Also Controls Other Phenotypes Related to HK GacS

In Pseudomonas species, RetS acts as a negative regulator; accordingly, the production of alginates in the EretS mutant should increase; however, Fig. 1b shows that the opposite was true. These data suggested that, in A. vinelandii, RetS acts as a positive regulator. To verify the regulatory character of RetS, we measured the production of other GacS-controlled metabolites in A. vinelandii, such as PHB and ARs [22, 23].

Our experiments also revealed a significant reduction in PHB synthesis as a consequence of the retS mutation. The data in Fig. 2a highlight the crucial role of RetS in this process.Fig. 2 Effect of the retS mutations on ARs synthesis, PHB accumulation, and swimming motility. a PHB production in E WT strain and its mutant derivatives retS. PHB content was determined in cells grown for 48 h in PY liquid medium supplemented with 2% sucrose. b ARs synthesis in wild-type strain E and its mutant derivatives retS. The ARs measurements were done in cells grown for 120 h in Burk’s minimal media amended with n-butanol (0.2%) as a carbon source. In both graphs, the bars represent the statistical media of three measurements and their standard deviation. Significant differences were analyzed by ANOVA test. Statistical significance is indicated. P < 0.1234; ns (not significant), *P < 0.03232; **P < 0.021; ***P < 0.0002; ****P < 0.0001. c Colonies of A. vinelandii E (wild type), and its derivatives mutants retS, Fast Blue B stain makes the ARs production visible with a reddish color. The A. vinelandii strains grew on Burk media amended with 2% of Butanol (BBOH medium) for 120 h of incubation. d Quantitative motility assay of A. vinelandii wild-type strain E and its derivatives retS mutants. The cells were cultivated over BS plates amended with 0.15% agar to test swimming motility. The motility halo was visualized and measured after 24 h of incubation. The bars represent the statistical media of three measurements and their standard deviation. Significant differences were analyzed by Anova test. Statistical significance is indicated. P < 0.1234; ns (not significant), *P < 0.03232; **P < 0.021; ***P < 0.0002; ****P < 0.0001. e Swimming motility phenotypes of A. vinelandii wild-type strain E and its derivatives retS mutants

The effect of the retS mutation on ARs synthesis was evident upon fast blue staining, as shown in Fig. 2c. The absence of retS hindered ARs synthesis, a finding that was subsequently confirmed quantitatively (Fig. 2b).

The GacS-Rsm pathway is also involved in controlling swimming motility in A. vinelandii. Therefore, we investigated whether mutation of retS could indirectly affect motility. The reduced motility in retS mutants (shown in Fig. 2d, e) strongly suggests that this histidine kinase, along with GacS, was involved in flagellum biosynthesis on the basis of the function reported for GacS in A. vinelandii [44].

RetS is Involved in the Transcriptional Control of Genes Encoding sRNAs of the Rsm Family

The phenotypes related to GacS that were affected in the EretS mutant suggest that as in Pseudomonas spp., Rsm-sRNAs could be a regulatory target of RetS [6]. To prove this, the retS mutation was transferred into strains carrying gusA transcriptional fusions of genes encoding some of the A. vinelandii Rsm-sRNAs. A. vinelandii has eight sRNAs belonging to the Rsm family, with seven of the RsmZ (RsmZ1-7) subfamily and one of the RsmY subfamily [24, 25]. After ruling out the polarity mutation effect, the experiments were carried out only with strains derived from the EretSNP mutant because similar effects were observed for the EretSP and EretSNP mutants.

Figure 3 shows the transcription of four of the eight Rsm-sRNAs, rsmZ1, rsmZ2, rsmZ6, and rsmY; in all the cases, the retS mutation diminished the expression of the rsm-sRNAs genes.Fig. 3 Effect of the retS mutation on the activity of the PrsmZ1-gusA, PrsmZ2-gusA, PrsmZ6-gusA, and PrsmY-gusA transcriptional fusions. Promotor activity of the transcriptional fusions PrsmZ1-gusA, PrsmZ1-gusA, and PrsmZ1-gusA in strains EPrsmZ1-gusA, EPrsmZ1-gusA and EPrsmZ1-gusA, and their derivative mutants retS. 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. The bars represent the statistical media of three measurements and their standard deviation. Significant differences were analyzed by t test. Statistical significance is indicated. P < 0.1234; ns (not significant), *P < 0.03232; **P < 0.021; ***P < 0.0002; ****P < 0.0001

GacS is Required for the RetS Function

The HK GacS of A. vinelandii has a DHp subdomain and a HAMP domain that are highly conserved with their counterparts in GacS of P. aeruginosa. Both domains possess up to 70% identity (Fig. S3), suggesting that functional relationships similar to those of its P. aeruginosa homolog could be established.

The data presented, thus, far strongly suggest a functional relationship between GacS and AvAEIV_000681 (RetS). To test this hypothesis, we created a double mutant, EgacSretS, and conducted phenotypic hierarchy studies alongside the single mutants retS and gacS. While both the retS and gacS single mutants presented reduced alginate production, they presented different phenotypes. In semisolid media, the gacS mutant displayed the growth of colonies that were rough and dark (Fig. 4a), whereas in liquid media, it formed flocs (Fig. 4b). Conversely, retS mutants produced clear colonies in semisolid media and did not flocculate in liquid medium (Fig. 4a). In the EgacSretS double mutant, the gacS phenotype was dominant (Fig. 4a), confirming the genetic relationship between the two kinases. Additionally, the dominance of the gacS phenotype indicated the greater genetic hierarchy of gacS over retS.Fig. 4 Phenotypes of single mutants EretS and EgacS and double mutant EretSgacS. a Mucoid phenotypes of the A. vinelandii strain E (wild type), EgacS, EretS, and the double mutant EgacSretS growth on Burks Sucrose (BS medium) semisolid medium at 30 °C for 48 h. b Flocculation phenotype of the strains EgacS, EretS, and the double mutant EgacSretS growth on BS liquid medium at 30 °C, 2000 rpm for 48 h. c Staining of ARs produced by A. vinelandii strains E (wild type) EgacS, EretS, and the double mutant EgacSretS growth on Burk Butanol (BBOH medium) for 120 h of incubation

Previously, a null ARs production phenotype was reported in gacS and gacA mutants [23], which contrasts with the partial phenotype of the EretSP mutant. Interestingly, the partial ARs production phenotype in the EretSP mutant became a null ARs production phenotype through mutation of the gacS gene. As in the previous case, in the EretSgacS double mutant, the phenotype of the gacS mutation prevailed (Fig. 4c).

RetS Interacts with HptB

In a previous study in A. vinelandii, the physical interaction between RetS and GacS was reported [14], whereas in P. aeruginosa, RetS has also been reported to interact with the HptB protein; it has been proposed that HptB can phosphorylate RetS [10]. To establish whether this also occurs in A. vinelandii, a LexA two-hybrid assay was carried out. To perform the assay, we cloned DNA fragments corresponding to the cytoplasmic domains of the gacS and hptB genes into LexA expression vectors. The absence of a color change in the MacConkey medium confirmed the interaction between RetS and HptB (Fig. 5a). The interaction assay between RetS and GacS was repeated as a positive control, confirming the interaction between RetS and GacS. Figure 5b shows the quantitative results of the assays.Fig. 5 Determination of the interaction of RetS with GacS and HptB, established by LexA Two-hybrid assays. a Plate Two-Hybrid assay performed with RetS, GacS, and HptB. b Quantitative Two-Hybrid assay carried out with RetS, GacS, and HptB. The LexA dimerization domain was removed and replaced with GacS, RetS, and HptB proteins. 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. The bars represent the statistical media of three measurements and their standard deviation. Significant differences were analyzed by t test. Statistical significance is indicated. P < 0.1234; ns (not significant), *P < 0.03232; **P < 0.021; ***P < 0.0002; ****P < 0.0001

Discussion

The study of MKNs in bacterial signaling is an emerging area of research that has improved the understanding of how bacteria sense and respond to their environment [2]. The P. aeruginosa GacS network is one of the most studied MKNs; bioinformatic searches suggest the existence of this MKN in many bacteria of the Pseudomonas genus [8], but it has been partially characterized only in Pseudomonas protegens (formerly Pseudomonas fluorescens) [11] and Pseudomonas syringae [45]. GacS forms the core of the GacS-MKN as the central kinase, whereas RetS and LadS are its regulatory kinases [2]. In P. aeruginosa, LadS promotes alternative GacS phosphorylation, increasing GacA phosphorylation. To carry out GacS phosphorylation, LadS requires an REC domain [8]. Interestingly, in P. syringae, LadS also acts as a positive regulator; however, it does not contain the REC domain, suggesting a distinct regulatory mechanism [45].

In P. aeruginosa, RetS acts as a negative regulator, forming heterodimers with GacS. There are three mechanisms through which RetS blocks the function of GacS: inhibition of GacS autophosphorylation, removal of the phosphate from the phosphorylated GacS, and dephosphorylation of the REC domain of phosphorylated GacS [46]. Forming the GacS heterodimer requires the DHp subdomain (a subdomain of the transmitter domain) and an additional HAMP domain, which GacS possesses just above the transmitting domain. The DHp subdomains of both proteins interact with each other, and the GacS HAMP domain contacts the RetS transmitter domain in a region distinct from the DHp subdomain [9]. The high identity and domain conservation between A. vinelandii and P. aeruginosa RetS and GacS are in agreement with the interaction results. Interestingly, and counter to what was expected, the retS mutation diminished alginate production. In A. vinelandii, GacS positively controlled alginate production. Thus, mutations in gacS also decreased alginate synthesis. The regulatory effect of RetS on alginate synthesis was likely due to its interaction with GacS; therefore, a mutation in retS was expected to affect other GacS phenotypes. For that, we tested other phenotypes regulated by GacS, and the results were consistent with the positive regulatory character of RetS found for the synthesis of alginates, thus, ruling out the exceptional situation described above. The positive regulatory effect of RetS is not common; it has only been reported in the control of swimming, swarming, and surfactant production in the strain Pf5 of P. protegens [47]. Interestingly, in the same strain, the production of the antifungal compound 2,4-diacetyl phloroglucinol is negatively regulated by RetS [48]. This last case correlates with the regulatory mechanism originally described for RetS. Overall, previously reported data [47, 49] suggest the existence of alternative mechanisms through which RetS performs its regulatory functions.

The high similarity of GacS and RetS of A. vinelandii with those of its homologs from P. aeruginosa and the results obtained from the two-hybrid assay suggest that the formation of the GacS-RetS heterodimer, which would prevent the transphosphorylation of GacS, is highly probable. It is widely documented that GacS homologs are not phosphorylated by a cis mechanism [48]. Thus, the heterodimer turns off autokinase activity. The transmitter domain of RetS homologs has structural alterations that turn off its kinase activity; the high conservation of the primary and predicted tertiary structures of the RetS transmitter domain of A. vinelandii also indicates that it could not have kinase activity. In A. vinelandii, the phenotypes of the retS mutation strongly suggest that the RetS-GacS interaction did not promote the activation of GacS and therefore the activation of GacA.

One possible explanation for the phenotypes of the retS mutant in A. vinelandii could be the potential inhibition of the phosphatase activity of GacS by dimerization with RetS. In E. coli, UvrY, the GacA homolog, is sometimes phosphorylated by acetyl phosphate, independent of BarA (GacS homolog) [50]. BarA has both kinase and phosphatase activity and, under specific conditions, dephosphorylates UvrY by acting as a homodimer. If a similar process occurs in the GacS/A system of A. vinelandii, RetS interference with the ability of GacS to form homodimers could reduce the phosphatase activity of GacS, favoring the phosphorylated state of GacA. Therefore, under specific conditions, the absence of RetS shifts the kinase/phosphatase balance of GacS toward phosphatase activity, deactivating GacA and negatively impacting its regulatory targets.

HptB can interact with RetS; in this study, we prove that this interaction also occurred in A. vinelandii. In P. aeruginosa, HptB can phosphorylate RetS; however, how this phosphorylation impacts RetS function is unclear. In A. vinelandii, HptB likely transfers its phosphate to RetS; in turn, phosphorylated RetS could transfer the phosphate to GacS through a similar mechanism to that described for the phosphorylation of GacS by LadS [8]. If this occurred in A. vinelandii, the positive regulatory phenotype of RetS could be explained. In this sense, determining the role that the two REC domains could play in the transfer of the phosphate group by HptB would be interesting.

The verification of the proposed hypotheses would be a very interesting subject of study for subsequent studies, which would improve the knowledge of the MKN-GacS. Although the presence of MKN-GacS is presumed in numerous species of the genus Pseudomonas, it has been studied in only a few species. The MKN of P. aeruginosa has become the study paradigm of this signaling system; the MKNs of P. protegens and P. syringae, although they have been less studied, show conserved aspects and other variables that suggest that the network is flexible and could have unique features in each bacterium. Outside of the Pseudomonas genus, the MKN has been studied only in A. vinelandii, where interesting variants have been found; there is no homolog for ladS, and a hybrid kinase (HrgS) related to GacS has not been reported in Pseudomonas species [14]. The positive regulation of GacS/A-related phenotypes by RetS shown in this study is another unique feature of the system that opens a new line of investigation.

Conclusion

In A. vinelandii, RetS positively regulates the synthesis of alginates, PHB, and ARs and motility.

Supplementary Information

Below is the link to the electronic supplementary material.Supplementary file1 (DOCX 20 KB)

Fig. S1 Predicted domains of histidine kinase RetS homologs from A. vinelandii and P. aeruginosa. The analysis was carried out using the SMART protein domain annotation resource (http://smart.embl-heidelberg.de/) (DOCX 1224 KB)

Fig. S2 RT-PCR analysis of retS and hsp70 genes in A. vinelandii EretS mutants. A) Genetic map of retS::Km and hsp70 in A. vinelandii retS mutants. Arrows indicate the orfs, an inverted triangle shows the insertion of the resistance cassette, the primer PCR pairs are indicated, and the amplicons generated are shown as squares. B) Agarose gel electrophoresis of RetS RT-PCR products of lanes: 1) GeneRuler 100bp DNA Ladder (Thermo-Scientific), 2) Strain E (WT), 3) EretSP, 4) EretSNP, 5) Negative control (PCR without template), 6) Positive control (PCR using genomic DNA as template). C) Agarose gel electrophoresis of Hsp70 RT-PCR products of lanes: 1) GeneRuler 100bp DNA Ladder (Thermo Scientific), 2) Strain E (WT), 3) EretSP, 4) EretSNP, 5) Negative control (PCR without template), 6) Positive control (PCR using genomic DNA as template) (DOCX 1160 KB)

Fig. S3 Alignment of the A. vinelandii and P. aeruginosa GacS proteins. Predicted domains are indicated by boxes: blue box, HAMP domain; black box, Transmitter domain (H1); red box, DHp subdomain; green box, receiver domain (D1); orange box, Hpt domain (H2) (DOCX 1325 KB)

Fig. S4 Alignment of the A. vinelandii and P. aeruginosa RetS proteins. The predicted transmitter domain (H1) is indicated by the black box (DOCX 1544 KB)

Acknowledgements

This work was supported by VIEP-BUAP, grant 100301900-VIEP2022 and grant 00093-VIEP2023. A. Rosales-Cruz and C. Mondragón-Albarrán thanks CONAHCyT for M.Sc. scholarships.

Author Contributions

Conceived and designed the experiments: MC and LLP. Performed the experiments: ARC, JRN, EMG, AMC, and CMA. Analyzed the data: MC, ARC, and LLP. Wrote the paper: MC, LLP, and ARC.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
==== Refs
References

1. Gao R Stock AM Biological insights from structures of two-component proteins Annu Rev Microbiol 2009 63 133 154 10.1146/annurev.micro.091208.073214 19575571
Gao R, Stock AM (2009) Biological insights from structures of two-component proteins. Annu Rev Microbiol 63:133–154. 10.1146/annurev.micro.091208.07321419575571
2. Francis VI Porter SL Multikinase networks: two-component signaling networks integrating multiple stimuli Annu Rev Microbiol 2019 73 199 223 10.1146/annurev-micro-020518-115846 31112439
Francis VI, Porter SL (2019) Multikinase networks: two-component signaling networks integrating multiple stimuli. Annu Rev Microbiol 73:199–223. 10.1146/annurev-micro-020518-11584631112439
3. Lapouge K Schubert M Allain FH Gac/Rsm signal transduction pathway of gamma-proteobacteria: from RNA recognition to regulation of social behaviour Mol Microbiol 2008 67 241 253 10.1111/j.1365-2958.2007.06042.x 18047567
Lapouge K, Schubert M, Allain FH et al (2008) Gac/Rsm signal transduction pathway of gamma-proteobacteria: from RNA recognition to regulation of social behaviour. Mol Microbiol 67:241–253. 10.1111/j.1365-2958.2007.06042.x18047567
4. Romeo T Vakulskas CA Babitzke P Post-transcriptional regulation on a global scale: form and function of Csr/Rsm systems Environ Microbiol 2013 15 2 313 324 10.1111/j.1462-2920.2012.02794.x 22672726
Romeo T, Vakulskas CA, Babitzke P (2013) Post-transcriptional regulation on a global scale: form and function of Csr/Rsm systems. Environ Microbiol 15(2):313–324. 10.1111/j.1462-2920.2012.02794.x22672726
5. Pourciau C Lai YJ Gorelik M Babitzke P Romeo T Diverse mechanisms and circuitry for global regulation by the RNA-Binding protein CsrA Front Microbiol 2020 27 11 601352 10.3389/fmicb.2020.601352
Pourciau C, Lai YJ, Gorelik M, Babitzke P, Romeo T (2020) Diverse mechanisms and circuitry for global regulation by the RNA-Binding protein CsrA. Front Microbiol 27(11):601352. 10.3389/fmicb.2020.601352
6. Ventre I Goodman AL Vallet-Gely I Multiple sensors control reciprocal expression of Pseudomonas aeruginosa regulatory RNA and virulence genes Proc Natl Acad Sci U S A 2006 103 1 171 176 10.1073/pnas.0507407103 16373506
Ventre I, Goodman AL, Vallet-Gely I et al (2006) Multiple sensors control reciprocal expression of Pseudomonas aeruginosa regulatory RNA and virulence genes. Proc Natl Acad Sci U S A 103(1):171–176. 10.1073/pnas.050740710316373506
7. Pusic P Sonnleitner E Bläsi U Specific and global RNA regulators in Pseudomonas aeruginosa Int J Mol Sci 2021 22 16 8632 10.3390/ijms22168632 34445336
Pusic P, Sonnleitner E, Bläsi U (2021) Specific and global RNA regulators in Pseudomonas aeruginosa. Int J Mol Sci 22(16):8632. 10.3390/ijms2216863234445336
8. Chambonnier G Roux L Redelberger D The hybrid histidine kinase LadS forms a multicomponent signal transduction system with the GacS/GacA two-component system in Pseudomonas aeruginosa PLoS Genet 2016 12 5 e1006032 10.1371/journal.pgen.1006032 27176226
Chambonnier G, Roux L, Redelberger D et al (2016) The hybrid histidine kinase LadS forms a multicomponent signal transduction system with the GacS/GacA two-component system in Pseudomonas aeruginosa. PLoS Genet 12(5):e1006032. 10.1371/journal.pgen.100603227176226
9. Fadel F Bassim V Francis VI Porter SL Insights into the atypical autokinase activity of the Pseudomonas aeruginosa GacS histidine kinase and its interaction with RetS Structure 2022 30 9 1285 1297.e5 10.1016/j.str.2022.06.002 35767996
Fadel F, Bassim V, Francis VI, Porter SL et al (2022) Insights into the atypical autokinase activity of the Pseudomonas aeruginosa GacS histidine kinase and its interaction with RetS. Structure 30(9):1285-1297.e5. 10.1016/j.str.2022.06.00235767996
10. Hsu JL Chen HC Peng HL Characterization of the histidine-containing phosphotransfer protein B-mediated multistep phosphorelay system in Pseudomonas aeruginosa PAO1 J Biol Chem 2008 283 9933 9944 10.1074/jbc.M708836200 18256026
Hsu JL, Chen HC, Peng HL et al (2008) Characterization of the histidine-containing phosphotransfer protein B-mediated multistep phosphorelay system in Pseudomonas aeruginosa PAO1. J Biol Chem 283:9933–9944. 10.1074/jbc.M70883620018256026
11. Humair B González N Mossialos D Temperature-responsive sensing regulates biocontrol factor expression in Pseudomonas fluorescens CHA0 ISME J 2009 3 955 965 10.1038/ismej.2009.42 19421236
Humair B, González N, Mossialos D et al (2009) Temperature-responsive sensing regulates biocontrol factor expression in Pseudomonas fluorescens CHA0. ISME J 3:955–965. 10.1038/ismej.2009.4219421236
12. Yu X Lund SP Greenwald JW Transcriptional analysis of the global regulatory networks active in Pseudomonas syringae during leaf colonization mBio 2014 5 5 e01683-14 10.1128/mBio.01683-14 25182327
Yu X, Lund SP, Greenwald JW et al (2014) Transcriptional analysis of the global regulatory networks active in Pseudomonas syringae during leaf colonization. mBio 5(5):e01683-14. 10.1128/mBio.01683-1425182327
13. Bernal P Civantos C Pacheco-Sánchez D Quesada JM Filloux A Llamas MA Transcriptional organization and regulation of the Pseudomonas putida K1 type VI secretion system gene cluster Microbiology 2023 169 1 001295 10.1099/mic.0.001295 36748579
Bernal P, Civantos C, Pacheco-Sánchez D, Quesada JM, Filloux A, Llamas MA (2023) Transcriptional organization and regulation of the Pseudomonas putida K1 type VI secretion system gene cluster. Microbiology 169(1):001295. 10.1099/mic.0.00129536748579
14. López-Pliego L González-Acocal V García-González DL HrgS (Avin_34990), a novel histidine-kinase related to GacS, regulates alginate synthesis in Azotobacter vinelandii FEMS Microbiol Lett 2022 369 1 fnac024 10.1093/femsle/fnac024 35266527
López-Pliego L, González-Acocal V, García-González DL et al (2022) HrgS (Avin_34990), a novel histidine-kinase related to GacS, regulates alginate synthesis in Azotobacter vinelandii. FEMS Microbiol Lett 369(1):fnac024. 10.1093/femsle/fnac02435266527
15. Galindo E Peña C Núñez C Molecular and bioengineering strategies to improve alginate and polydydroxyalkanoate production by Azotobacter vinelandii Microb Cell Fac 2007 6 7 10.1186/1475-2859-6-7
Galindo E, Peña C, Núñez C et al (2007) Molecular and bioengineering strategies to improve alginate and polydydroxyalkanoate production by Azotobacter vinelandii. Microb Cell Fac 6:7. 10.1186/1475-2859-6-7
16. Noar JD Bruno-Bárcena JM Azotobacter vinelandii: the source of 100 years of discoveries and many more to come Microbiology 2018 164 4 421 436 10.1099/mic.0.000643 29533747
Noar JD, Bruno-Bárcena JM (2018) Azotobacter vinelandii: the source of 100 years of discoveries and many more to come. Microbiology 164(4):421–436. 10.1099/mic.0.00064329533747
17. Segura D Vite O Romero Y Isolation and characterization of Azotobacter vinelandii mutants impaired in alkylresorcinol synthesis: alkylresorcinols are not essential for cyst desiccation resistance J Bacteriol 2009 191 9 3142 3148 10.1128/JB.01575-08 19270099
Segura D, Vite O, Romero Y et al (2009) Isolation and characterization of Azotobacter vinelandii mutants impaired in alkylresorcinol synthesis: alkylresorcinols are not essential for cyst desiccation resistance. J Bacteriol 191(9):3142–3148. 10.1128/JB.01575-0819270099
18. Urtuvia V Maturana N Acevedo F Bacterial alginate production: an overview of its biosynthesis and potential industrial production World J Microbiol Biotechnol 2017 33 198 10.1007/s11274-017-2363-x 28988302
Urtuvia V, Maturana N, Acevedo F et al (2017) Bacterial alginate production: an overview of its biosynthesis and potential industrial production. World J Microbiol Biotechnol 33:198. 10.1007/s11274-017-2363-x28988302
19. Peña C Castillo T García A Millán M Segura D Biotechnological strategies to improve production of microbial poly-(3-hydroxybutyrate): a review of recent research work Microb Biotechnol 2014 7 4 278 293 10.1111/1751-7915.12129 24898500
Peña C, Castillo T, García A, Millán M, Segura D (2014) Biotechnological strategies to improve production of microbial poly-(3-hydroxybutyrate): a review of recent research work. Microb Biotechnol 7(4):278–293. 10.1111/1751-7915.1212924898500
20. Zabolotneva AA Shatova OP Sadova AA Shestopalov AV Roumiantsev SA An Overview of alkylresorcinols biological properties and effects J Nutr Metab 2022 10.1155/2022/4667607 35036005
Zabolotneva AA, Shatova OP, Sadova AA, Shestopalov AV, Roumiantsev SA (2022) An Overview of alkylresorcinols biological properties and effects. J Nutr Metab. 10.1155/2022/466760735036005
21. Núñez C López-Pliego L Ahumada-Manuel CL Castañeda M Genetic regulation of alginate production in Azotobacter vinelandii a bacterium of biotechnological interest: a mini-review Front Microbiol 2022 13 845473 10.3389/fmicb.2022.845473 35401471
Núñez C, López-Pliego L, Ahumada-Manuel CL, Castañeda M (2022) Genetic regulation of alginate production in Azotobacter vinelandii a bacterium of biotechnological interest: a mini-review. Front Microbiol 13:845473. 10.3389/fmicb.2022.84547335401471
22. Hernandez-Eligio A Moreno S Castellanos M RsmA post-transcriptionally controls PhbR expression and polyhydroxybutyrate biosynthesis in Azotobacter vinelandii Microbiology 2012 158 1956 1963 10.1099/mic.0.059329-0
Hernandez-Eligio A, Moreno S, Castellanos M et al (2012) RsmA post-transcriptionally controls PhbR expression and polyhydroxybutyrate biosynthesis in Azotobacter vinelandii. Microbiology 158:1956–1963. 10.1099/mic.0.059329-0
23. Romero Y Guzmán J Moreno S The GacS/A-RsmA signal transduction pathway controls the synthesis of alkylresorcinol lipids that replace membrane phospholipids during encystment of Azotobacter vinelandii SW136 PLoS ONE 2016 11 4 e0153266 10.1371/journal.pone.0153266 27055016
Romero Y, Guzmán J, Moreno S et al (2016) The GacS/A-RsmA signal transduction pathway controls the synthesis of alkylresorcinol lipids that replace membrane phospholipids during encystment of Azotobacter vinelandii SW136. PLoS ONE 11(4):e0153266. 10.1371/journal.pone.015326627055016
24. López-Pliego L Núñez C García-Ramírez L Transcriptional study of the RsmZ-sRNAs and their relationship to the biosynthesis of alginate and alkylresorcinols in Azotobacter vinelandii Mol Biotechnol 2018 60 9 670 680 10.1007/s12033-018-0102-7 29987520
López-Pliego L, Núñez C, García-Ramírez L et al (2018) Transcriptional study of the RsmZ-sRNAs and their relationship to the biosynthesis of alginate and alkylresorcinols in Azotobacter vinelandii. Mol Biotechnol 60(9):670–680. 10.1007/s12033-018-0102-729987520
25. López-Pliego L Mena-Muñoz G Terán-Melo JL Study of the sRNA RsmY involved in the genetic regulation of the synthesis of alginate and alkyl resorcinols in Azotobacter vinelandii Arch Microbiol 2020 202 3 579 589 10.1007/s00203-019-01769-y 31741014
López-Pliego L, Mena-Muñoz G, Terán-Melo JL et al (2020) Study of the sRNA RsmY involved in the genetic regulation of the synthesis of alginate and alkyl resorcinols in Azotobacter vinelandii. Arch Microbiol 202(3):579–589. 10.1007/s00203-019-01769-y31741014
26. Kennedy C Gamal R Humphrey R The nifH, nifM, and nifN genes of Azotobacter vinelandii: characterization by Tn5 mutagenesis and isolation from pLARF1 gene banks Mol Gen Genet 1986 205 318 325 10.1128/JB.01575-08
Kennedy C, Gamal R, Humphrey R et al (1986) The nifH, nifM, and nifN genes of Azotobacter vinelandii: characterization by Tn5 mutagenesis and isolation from pLARF1 gene banks. Mol Gen Genet 205:318–325. 10.1128/JB.01575-08
27. Ahumada-Manuel CL Guzman J Peña C The signaling protein MucG negatively affects the production and the molecular mass of alginate in Azotobacter vinelandii Appl Microbiol Biotechnol 2017 101 4 1521 1534 10.1007/s00253-016-7931-8 27796435
Ahumada-Manuel CL, Guzman J, Peña C, et al (2017) The signaling protein MucG negatively affects the production and the molecular mass of alginate in Azotobacter vinelandii. Appl Microbiol Biotechnol 101(4):1521–1534. 10.1007/s00253-016-7931-827796435
28. Sambrook J Fritsch EF Maniatis T Molecular Cloning: A Laboratory Manual 1989 Cold Spring Harbor Cold Spring Harbor Laboratory
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor
29. Setubal JC dos Santos P Goldman BS Genome sequence of Azotobacter vinelandii, an obligate aerobe specialized to support diverse anaerobic metabolic processes J Bacteriol 2009 191 14 4534 4545 10.1128/JB.00504-09 19429624
Setubal JC, dos Santos P, Goldman BS et al (2009) Genome sequence of Azotobacter vinelandii, an obligate aerobe specialized to support diverse anaerobic metabolic processes. J Bacteriol 191(14):4534–4545. 10.1128/JB.00504-0919429624
30. Larsen B Haug A Biosynthesis of alginate. 1. Composition and structure of alginate produced by Azotobacter vinelandii (Lipman) Carbohydr Res 1971 17 2 287 296 10.1016/s0008-6215(00)82536-7 5150891
Larsen B, Haug A (1971) Biosynthesis of alginate. 1. Composition and structure of alginate produced by Azotobacter vinelandii (Lipman). Carbohydr Res 17(2):287–296. 10.1016/s0008-6215(00)82536-75150891
31. Alexeyev MF Shokolenko IN Croughan TP Improved antibiotic-resistance gene cassettes and omega elements for Escherichia coli vector construction and in vitro deletion/insertion mutagenesis Gene 1995 160 1 63 67 10.1016/0378-1119(95)00108-i 7628718
Alexeyev MF, Shokolenko IN, Croughan TP (1995) Improved antibiotic-resistance gene cassettes and omega elements for Escherichia coli vector construction and in vitro deletion/insertion mutagenesis. Gene 160(1):63–67. 10.1016/0378-1119(95)00108-i7628718
32. Mouncey NJ Mitchenall LA Pau RN Mutational analysis of genes of the mod locus involved in molybdenum transport, homeostasis, and processing in Azotobacter vinelandii J Bacteriol 1995 177 18 5294 5302 10.1128/jb.177.18.5294-5302.1995 7665518
Mouncey NJ, Mitchenall LA, Pau RN (1995) Mutational analysis of genes of the mod locus involved in molybdenum transport, homeostasis, and processing in Azotobacter vinelandii. J Bacteriol 177(18):5294–5302. 10.1128/jb.177.18.5294-5302.19957665518
33. Castañeda M Guzmán J Moreno S Espín G The GacS sensor kinase regulates alginate and poly-beta-hydroxybutyrate production in Azotobacter vinelandii J Bacteriol 2000 182 9 2624 2628 10.1128/JB.182.9.2624-2628.2000 10762268
Castañeda M, Guzmán J, Moreno S, Espín G (2000) The GacS sensor kinase regulates alginate and poly-beta-hydroxybutyrate production in Azotobacter vinelandii. J Bacteriol 182(9):2624–2628. 10.1128/JB.182.9.2624-2628.200010762268
34. Simon R Priefer U Pühler A A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria Nat Biotechnol 1983 1 784 791 10.1038/nbt1183-784
Simon R, Priefer U, Pühler A (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Nat Biotechnol 1:784–791. 10.1038/nbt1183-784
35. Cocotl-Yañez M Moreno S Encarnación S A small heat shock protein (Hsp20) regulated by RpoS is essential for cyst desiccation resistance in Azotobacter vinelandii Microbiology 2014 160 479 487 10.1099/mic.0.073353-0 24385478
Cocotl-Yañez M, Moreno S, Encarnación S et al (2014) A small heat shock protein (Hsp20) regulated by RpoS is essential for cyst desiccation resistance in Azotobacter vinelandii. Microbiology 160:479–487. 10.1099/mic.0.073353-024385478
36. Daines DA Granger-Schnarr M Dimitrova M Use of LexA-based system to identify protein–protein interactions in vivo Methods Enzymol 2002 358 153 161 10.1016/s0076-6879(02)58087-3 12474385
Daines DA, Granger-Schnarr M, Dimitrova M et al (2002) Use of LexA-based system to identify protein–protein interactions in vivo. Methods Enzymol 358:153–161. 10.1016/s0076-6879(02)58087-312474385
37. Lowry OH Rosebrough NJ Farr AL Protein measurement with the Folin phenol reagent J Biol Chem 1951 193 265 275 10.1016/S0021-9258(19)52451-6 14907713
Lowry OH, Rosebrough NJ, Farr AL et al (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–27514907713
38. Blumenkrantz N Asboe-Hansen G New method for quantitative determination of uronic acids Anal Biochem 1973 54 484 489 10.1016/0003-2697(73)90377-1 4269305
Blumenkrantz N, Asboe-Hansen G (1973) New method for quantitative determination of uronic acids. Anal Biochem 54:484–489. 10.1016/0003-2697(73)90377-14269305
39. Tłuścik F Kazubek A Mejbaum-Katzenellenbogen W Alkylresorcinols in rye (Secale cereale L.) grains. VI. Colorimetric micromethod for the determination of alkylresorcinols with the use of diazonium salt, Fast Blue B Acta Soc Bot Pol 1981 50 645 651 10.5586/asbp.1981.086
Tłuścik F, Kazubek A, Mejbaum-Katzenellenbogen W (1981) Alkylresorcinols in rye (Secale cereale L.) grains. VI. Colorimetric micromethod for the determination of alkylresorcinols with the use of diazonium salt, Fast Blue B. Acta Soc Bot Pol 50:645–651. 10.5586/asbp.1981.086
40. Law JH Slepecky RA Assay of poly-beta-hydroxybutyric acid J Bacteriol 1961 82 1 33 36 10.1128/jb.82.1.33-36.1961 13759651
Law JH, Slepecky RA (1961) Assay of poly-beta-hydroxybutyric acid. J Bacteriol 82(1):33–36. 10.1128/jb.82.1.33-36.196113759651
41. Miller JH Experiments in molecular genetics 1972 Cold Spring Harbor Cold Spring Harbor Laboratory Press 431 435
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp 431–435
42. Wilson KJ Sessitsch A Corbo JC beta-Glucuronidase (GUS) transposons for ecological and genetic studies of rhizobia and other Gram-negative bacteria Microbiology 1995 141 1691 1705 10.1099/13500872-141-7-1691 7551037
Wilson KJ, Sessitsch A, Corbo JC et al (1995) beta-Glucuronidase (GUS) transposons for ecological and genetic studies of rhizobia and other Gram-negative bacteria. Microbiology 141:1691–1705. 10.1099/13500872-141-7-16917551037
43. Mejía-Ruíz H Moreno S Guzmán J Isolation and characterization of an Azotobacter vinelandii algK mutant FEMS Microbiol Lett 1997 156 1 101 106 10.1111/j.1574-6968.1997.tb12712.x 9368366
Mejía-Ruíz H, Moreno S, Guzmán J et al (1997) Isolation and characterization of an Azotobacter vinelandii algK mutant. FEMS Microbiol Lett 156(1):101–106. 10.1111/j.1574-6968.1997.tb12712.x9368366
44. López-Pliego L Lara-Flores N Molina-Romero D The GacS/A-Rsm pathway positively regulates motility and flagella synthesis in Azotobacter vinelandii Curr Microbiol 2021 79 1 17 10.1007/s00284-021-02695-3 34905080
López-Pliego L, Lara-Flores N, Molina-Romero D et al (2021) The GacS/A-Rsm pathway positively regulates motility and flagella synthesis in Azotobacter vinelandii. Curr Microbiol 79(1):17. 10.1007/s00284-021-02695-334905080
45. Records AR Gross DC Sensor kinases RetS and LadS regulate Pseudomonas syringae type VI secretion and virulence factors J Bacteriol 2010 192 14 3584 3596 10.1128/JB.00114-10 20472799
Records AR, Gross DC (2010) Sensor kinases RetS and LadS regulate Pseudomonas syringae type VI secretion and virulence factors. J Bacteriol 192(14):3584–3596. 10.1128/JB.00114-1020472799
46. Francis VI Waters EM Finton-James SE Multiple communication mechanisms between sensor kinases are crucial for virulence in Pseudomonas aeruginosa Nat Commun 2018 9 1 2219 10.1038/s41467-018-04640-8 29880803
Francis VI, Waters EM, Finton-James SE et al (2018) Multiple communication mechanisms between sensor kinases are crucial for virulence in Pseudomonas aeruginosa. Nat Commun 9(1):2219. 10.1038/s41467-018-04640-829880803
47. Ueda A Ogasawara S Horiuchi K Identification of the genes controlling biofilm formation in the plant commensal Pseudomonas protegens Pf-5 Arch Microbiol 2020 202 9 2453 2459 10.1007/s00203-020-01966-0 32607723
Ueda A, Ogasawara S, Horiuchi K (2020) Identification of the genes controlling biofilm formation in the plant commensal Pseudomonas protegens Pf-5. Arch Microbiol 202(9):2453–2459. 10.1007/s00203-020-01966-032607723
48. Ryan Kaler KM Nix JC Schubot FD RetS inhibits Pseudomonas aeruginosa biofilm formation by disrupting the canonical histidine kinase dimerization interface of GacS J Biol Chem 2021 297 4 101193 10.1016/j.jbc.2021.101193 34529974
Ryan Kaler KM, Nix JC, Schubot FD (2021) RetS inhibits Pseudomonas aeruginosa biofilm formation by disrupting the canonical histidine kinase dimerization interface of GacS. J Biol Chem 297(4):101193. 10.1016/j.jbc.2021.10119334529974
49. Jing X Cui Q Li X Engineering Pseudomonas protegens Pf-5 to improve its antifungal activity and nitrogen fixation Microb Biotechnol 2020 13 1 118 133 10.1111/1751-7915.13335 30461205
Jing X, Cui Q, Li X et al (2020) Engineering Pseudomonas protegens Pf-5 to improve its antifungal activity and nitrogen fixation. Microb Biotechnol 13(1):118–133. 10.1111/1751-7915.1333530461205
50. Camacho MI Alvarez AF Chavez RG Effects of the global regulator CsrA on the BarA/UvrY two-component signaling system J Bacteriol 2015 197 983 991 10.1128/JB.02325-14 25535275
Camacho MI, Alvarez AF, Chavez RG et al (2015) Effects of the global regulator CsrA on the BarA/UvrY two-component signaling system. J Bacteriol 197:983–991. 10.1128/JB.02325-1425535275
