The GacS/A-Rsm Pathway Positively Regulates Motility and Flagella Synthesis in Azotobacter vinelandii.

Azotobacter vinelandii is a motile bacterium that possesses an unusual pattern of peritrichous flagellation for members of the Pseudomonadaceae family. Unlike what has been reported for Pseudomonas spp. FleQ is not the master regulator of motility in A. vinelandii, this role is performed by FlhDC. Other factors involved in the regulation of motility are AlgU (σE) and CydR which act as negative regulators. In some members of the Enterobacteriaceae and Pseudomonadaceae families, the GacS/A-Rsm pathway is another important factor regulating motility. In the present study, the involvement of the GacS/A-Rsm pathway in regulating the motility of A. vinelandii was explored; we found that contrary to what has been reported for most of the strains studied of Pseudomonas species, GacS/A, through the Rsm system, positively controlled swimming motility. We show that the target of this regulation is the synthesis of flagella, which most likely occurs in an FlhDC-independent manner.

Study of the sRNA RsmY involved in the genetic regulation of the synthesis of alginate and alkyl resorcinols in Azotobacter vinelandii.

Azotobacter vineladii is a Gram-negative bacterium that produces alginate and poly-hydroxybutyrate (PHB), two polymers of biotechnological interest. This bacterium has the ability to form desiccation-resistant cysts. In the cyst the membrane phospholipids are replaced with a family of phenolic lipids called alkylresorcinols (ARs). The alginate, PHB, and ARs are controlled by the GacS/A two-component system and the small regulatory RNA (sRNA) RsmZ1, belonging to the Rsm (Csr) regulatory system. The Rsm (Csr) systems usually possess two or more sRNAs, in this regard A. vinelandii is the bacterium with the highest number of rsm-sRNAs. Originally, the presence of two sRNAs of the RsmY family (RsmY1 and RsmY2) was reported, but in a subsequent work it was suggested that they conformed to a single sRNA. In this work we provide genetic evidence confirming that rsmY1 and rsmY2 constitute a single gene. Also, it was established that rsmY mutation decreased alginate and ARs production, but did not affect the PHB synthesis. Transcriptional studies showed that rsmY has its higher expression during the stationary growth phase, and in the absence of RsmZ1, rsmY increases its transcription. Interestingly, rsmY expression was influenced by the carbon source, but its expression did not correlate with alginate production.

Two-component system CbrA/CbrB controls alginate production in Azotobacter vinelandii.

Azotobacter vinelandii, belonging to the Pseudomonadaceae family, is a free-living bacterium that has been considered to be a good source for the production of bacterial polymers such as alginate. In A. vinelandii the synthesis of this polymer is regulated by the Gac/Rsm post-transcriptional regulatory system, in which the RsmA protein binds to the mRNA of the biosynthetic algD gene, inhibiting translation. In several Pseudomonas spp. the two-component system CbrA/CbrB has been described to control a variety of metabolic and behavioural traits needed for adaptation to changing environmental conditions. In this work, we show that the A. vinelandii CbrA/CbrB two-component system negatively affects alginate synthesis, a function that has not been described in Pseudomonas aeruginosa or any other Pseudomonas species. CbrA/CbrB was found to control the expression of some alginate biosynthetic genes, mainly algD translation. In agreement with this result, the CbrA/CbrB system was necessary for optimal rsmA expression levels. CbrA/CbrB was also required for maximum accumulation of the sigma factor RpoS. This last effect could explain the positive effect of CbrA/CbrB on rsmA expression, as we also showed that one of the promoters driving rsmA transcription was RpoS-dependent. However, although inactivation of rpoS increased alginate production by almost 100 %, a cbrA mutation increased the synthesis of this polymer by up to 500 %, implying the existence of additional CbrA/CbrB regulatory pathways for the control of alginate production. The control exerted by CbrA/CbrB on the expression of the RsmA protein indicates the central role of this system in regulating carbon metabolism in A. vinelandii.

The Role of the ncRNA RgsA in the Oxidative Stress Response and Biofilm Formation in Azotobacter vinelandii.

Azotobacter vinelandii is a soil bacterium that forms desiccation-resistant cysts, and the exopolysaccharide alginate is essential for this process. A. vinelandii also produces alginate under vegetative growth conditions, and this production has biotechnological significance. Poly-ß-hydroxybutyrate (PHB) is another polymer synthetized by A. vinelandii that is of biotechnological interest. The GacS/A two-component signal transduction system plays an important role in regulating alginate production, PHB synthesis, and encystment. GacS/A in turn controls other important regulators such as RpoS and the ncRNAs that belong to the Rsm family. In A. vinelandii, RpoS is necessary for resisting oxidative stress as a result of its control over the expression of the catalase Kat1. In this work, we characterized a new ncRNA in A. vinelandii that is homologous to the P16/RsgA reported in Pseudomonas. We found that the expression of rgsA is regulated by GacA and RpoS and that it was essential for oxidative stress resistance. However, the activity of the catalase Kat1 is unaffected in rgsA mutants. Unlike those reported in Pseudomonas, RgsA in A. vinelandii regulates biofilm formation but not polymer synthesis or the encystment process.

A small heat-shock protein (Hsp20) regulated by RpoS is essential for cyst desiccation resistance in Azotobacter vinelandii.

In Azotobacter vinelandii, a cyst-forming bacterium, the alternative sigma factor RpoS is essential to the formation of cysts resistant to desiccation and to synthesis of the cyst-specific lipids, alkylresorcinols. In this study, we carried out a proteome analysis of vegetative cells and cysts of A. vinelandii strain AEIV and its rpoS mutant derivative AErpoS. This analysis allowed us to identify a small heat-shock protein, Hsp20, as one of the most abundant proteins of cysts regulated by RpoS. Inactivation of hsp20 did not affect the synthesis of alkylresorcinols or the formation of cysts with WT morphology; however, the cysts formed by the hsp20 mutant strain were unable to resist desiccation. We also demonstrated that expression of hsp20 from an RpoS-independent promoter in the AErpoS mutant strain is not enough to restore the phenotype of resistance to desiccation. These results indicate that Hsp20 is essential for the resistance to desiccation of A. vinelandii cysts, probably by preventing the aggregation of proteins caused by the lack of water. To our knowledge, this is the first report of a small heat-shock protein that is essential for desiccation resistance in bacteria.

Genetic Regulation of Alginate Production in Azotobacter vinelandii a Bacterium of Biotechnological Interest: A Mini-Review.

Alginates are a family of polymers composed of guluronate and mannuronate monomers joined by ß (1-4) links. The different types of alginates have variations in their monomer content and molecular weight, which determine the rheological properties and their applications. In industry, alginates are commonly used as additives capable of viscosifying, stabilizing, emulsifying, and gelling aqueous solutions. Recently, additional specialized biomedical uses have been reported for this polymer. Currently, the production of alginates is based on the harvesting of seaweeds; however, the composition and structure of the extracts are highly variable. The production of alginates for specialized applications requires a precise composition of monomers and molecular weight, which could be achieved using bacterial production systems such as those based on Azotobacter vinelandii, a free-living, non-pathogenic bacterium. In this mini-review, we analyze the latest advances in the regulation of alginate synthesis in this model.

Corrigendum: Genetic Regulation of Alginate Production in Azotobacter vinelandii a Bacterium of Biotechnological Interest: A Mini-Review.

[This corrects the article DOI: 10.3389/fmicb.2022.845473.].

HrgS (Avin_34990), a novel histidine-kinase related to GacS, regulates alginate synthesis in Azotobacter vinelandii.

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 histidine-kinase (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.

Transcriptional Study of the RsmZ-sRNAs and Their Relationship to the Biosynthesis of Alginate and Alkylresorcinols in Azotobacter vinelandii.

The GacS/A system in Azotobacter vinelandii regulates alginate and alkylresorcinols production through RsmZ1, a small regulatory RNA (sRNA) that releases the translational repression of the algD and arpR mRNAs caused by the RsmA protein. In the Pseudomonadaceae family, the Rsm-sRNAs are grouped into three families: RsmX, RmsY and RsmZ. Besides RsmZ1, A. vinelandii has six other isoforms belonging to the RsmZ family and another one to the RsmY. Environmental signals controlling rsmsRNAs genes in A. vinelandii are unknown. In this work, we present a transcriptional study of the A. vinelandii rsmZ1-7-sRNAs genes, whose transcriptional profiles showed a differential expression pattern, but all of them exhibited their maximal expression at the stationary growth phase. Furthermore, we found that succinate promoted higher expression levels of all the rsmZ1-7 genes compared to glycolytic carbon sources. Single mutants of the rsmZ-sRNAs family were constructed and their impact on alginate production was assessed. We did not observe correlation between the alginate phenotype of each rsmZ-sRNA mutant and the expression level of the corresponding sRNA, which suggests the existence of additional factors affecting their impact on alginate production. Similar results were found in the regulation exerted by the RsmZ-sRNAs on alkylresorcinol synthesis.

BLAST-XYPlot Viewer: A Tool for Performing BLAST in Whole-Genome Sequenced Bacteria/Archaea and Visualize Whole Results Simultaneously.

One of the most commonly used tools to compare protein or DNA sequences against databases is BLAST. We introduce a web tool that allows the performance of BLAST-searches of protein/DNA sequences in whole-genome sequenced bacteria/archaea, and displays a large amount of BLAST-results simultaneously. The circular bacterial replicons are projected as horizontal lines with fixed length of 360, representing the degrees of a circle. A coordinate system is created with length of the replicon along the x-axis and the number of replicon used on the y-axis. When a query sequence matches with a gene/protein of a particular replicon, the BLAST-results are depicted as an "x,y" position in a specially adapted plot. This tool allows the visualization of the results from the whole data to a particular gene/protein in real time with low computational resources.

Characterization of antimicrobial resistance mechanisms in carbapenem-resistant Pseudomonas aeruginosa carrying IMP variants recovered from a Mexican Hospital.

PURPOSE: Pseudomonas aeruginosa infections in hospitals constitute an important problem due to the increasing multidrug resistance (MDR) and carbapenems resistance. The knowledge of resistance mechanisms in Pseudomonas strains is an important issue for an adequate antimicrobial treatment. Therefore, the objective was to investigate other antimicrobial resistance mechanisms in MDR P. aeruginosa strains carrying bla IMP, make a partial plasmids characterization, and determine if modifications in oprD gene affect the expression of the OprD protein. METHODOLOGY: Susceptibility testing was performed by Kirby Baüer and by Minimum Inhibitory Concentration (presence/absence of efflux pump inhibitor); molecular typing by Pulsed-field gel electrophoresis (PFGE), resistance genotyping and integrons by PCR and sequencing; OprD expression by Western blot; plasmid characterization by MOB Typing Technique, molecular size by PFGE-S1; and bla IMP location by Southern blot. RESULTS: Among the 59 studied P. aeruginosa isolates, 41 multidrug resistance and carbapenems resistance isolates were detected and classified in 38 different PFGE patterns. Thirteen strains carried bla IMP; 16 bla GES and four carried both genes. This study centered on the 17 strains har-boring bla IMP. New variants of ß-lactamases were identified (bla GES-32, bla IMP-56, bla IMP-62) inside of new arrangements of class 1 integrons. The presence of bla IMP gene was detected in two plasmids in the same strain. The participation of the OprD protein and efflux pumps in the resistance to carbapenems and quinolones is shown. No expression of the porin OprD due to stop codon or IS in the gene was found. CONCLUSIONS: This study shows the participation of different resistance mechanisms, which are reflected in the levels of MIC to carbapenems. This is the first report of the presence of three new variants of ß-lactamases inside of new arrangements of class 1 integrons, as well as the presence of two plasmids carrying bla IMP in the same P. aeruginosa strain isolated in a Mexican hospital.

Extended-spectrum ß-lactamase-producing Escherichia coli isolated from healthy humans in Mexico, including subclone ST131-B2-O25:H4-H30-Rx.

OBJECTIVES: The resistance mechanisms, molecular type and plasmid content of cefotaxime-resistant Escherichia coli isolated from faecal samples of healthy volunteers in Puebla, Mexico, were characterised. METHODS AND RESULTS: Cefotaxime-resistant E. coli were recovered from 11 (18%) of 60 healthy volunteers. The isolates (one per sample) were characterised as multidrug-resistant and phenotypically extended-spectrum ß-lactamase (ESBL)-producing strains. Genes encoding resistance to ß-lactams (blaCTX-M-15, blaCTX-M-14a, blaCTX-M-14b, blaOXA-1, blaTEM-1b), quinolones [aac(6')-Ib-cr, qnrB19], aminoglycosides [aac(3')-II] and tetracycline [tet(A), tet(B)] were detected among the 11 ESBL-producing E. coli by PCR and sequencing, as well as gene cassette arrays in class 1 (dfrA17-aadA5) and class 2 (dfrA1-sat2-aadA1) integrons. Seven pulsotypes were identified by XbaI PFGE and the strains were distributed into phylogroups (number of isolates) A (2), B2 (4) and D (5). Seven sequence types were identified, four of them novel (ST5060, ST5079, ST5080 and ST5081), associated with phylogroups A-D. Transfer of a 140-kb IncFIA plasmid carrying the blaCTX-M-15 gene was evidenced in the ST5060 strain. Four CTX-M-15-producing E. coli strains of phylogroup B2 belonged to the ST131 complex, and IncFIB plasmids of 130kb and 155kb were detected in two of them. Multiple plasmid addiction systems were also found. Serotyping and fimH subtyping of ST131-B2 strains identified the ST131-B2-O25:H4-H30-Rx subclone. Additionally, this subclone and CTX-M-14-producing isolates were detected among residents living in the same household, suggesting clonal dissemination. CONCLUSIONS: This study reports the detection of E. coli ST131-B2-O25:H4-H30-Rx subclone in healthy humans in Mexico, highlighting its dissemination in the community setting.

Detection and Molecular Characterization of Escherichia coli Strains Producers of Extended-Spectrum and CMY-2 Type Beta-Lactamases, Isolated from Turtles in Mexico.

Multidrug-resistant bacteria are a growing problem in different environments and hosts, but scarce information exists about their prevalence in reptiles. The aim of this study was to analyze the resistance mechanisms, molecular typing, and plasmid content of cefotaxime-resistant (CTX(R)) Escherichia coli isolates recovered from cloacal samples of 71 turtles sheltered in a herpetarium in Mexico. CTX(R)-E. coli were recovered in 11 of 71 samples (15.5%), and one isolate/sample was characterized. Extended-spectrum ß-lactamase (ESBL)-producing E. coli isolates were detected in four samples (5.6%): two strains carried the blaCTX-M-2 gene (phylogroup D and ST2732) and two contained the blaCTX-M-15 gene (phylogroup B1 and lineages ST58 and ST156). The blaCMY-2 gene was detected by PCR in E. coli isolates of eight samples (9.8%) (one of them also carried blaCTX-M-2); these isolates were distributed into phylogroups A (n = 1), B1 (n = 6), and D (n = 1) and typed as ST155, ST156, ST2329, and ST2732. Plasmid-mediated quinolone resistance (PMQR) genes were detected in five isolates [aac(6')Ib-cr, qnrA, qnrB19, and oqxB]. From three to five replicon plasmids were detected among the strains, being IncFIB, IncI1, IncFrep, and IncK the most prevalent. ESBL or pAmpC genes were transferred by conjugation in four strains, and the blaCTX-M-15 and blaCMY-2 genes were localized in IncFIB or IncI1 plasmids by Southern blot hybridization assays. Class 1 and/or class 2 integrons were detected in eight strains with six different structures of gene cassette arrays. Nine pulsed-field gel electrophoresis patterns were found among the 11 studied strains. To our knowledge, this is the first detection of ESBL, CMY-2, PMQR, and mobile determinants of antimicrobial resistance in E. coli of turtle origin, highlighting the potential dissemination of multidrug-resistant bacteria from these animals to other environments and hosts, including humans.