scholarly journals H2-Independent Growth of the Hydrogenotrophic Methanogen Methanococcus maripaludis

mBio ◽  
2013 ◽  
Vol 4 (2) ◽  
Author(s):  
Kyle C. Costa ◽  
Thomas J. Lie ◽  
Michael A. Jacobs ◽  
John A. Leigh
Keyword(s):  
Carbon Monoxide ◽  
Slow Growth ◽  
Glycolytic Enzyme ◽  
Wild Type ◽  
Methanococcus Maripaludis ◽  
Content Type ◽  
Hydrogenotrophic Methanogen ◽  
Alternative Pathways ◽  
Lyase Activity ◽  
First Time

ABSTRACTHydrogenotrophic methanogenicArchaearequire reduced ferredoxin as an anaplerotic source of electrons for methanogenesis. H2oxidation by the hydrogenase Eha provides these electrons, consistent with an H2requirement for growth. Here we report the identification of alternative pathways of ferredoxin reduction inMethanococcus maripaludisthat operate independently of Eha to stimulate methanogenesis. A suppressor mutation that increased expression of the glycolytic enzyme glyceraldehyde-3-phosphate:ferredoxin oxidoreductase resulted in a strain capable of H2-independent ferredoxin reduction and growth with formate as the sole electron donor. In this background, it was possible to eliminate all seven hydrogenases ofM. maripaludis. Alternatively, carbon monoxide oxidation by carbon monoxide dehydrogenase could also generate reduced ferredoxin that feeds into methanogenesis. In either case, the reduced ferredoxin generated was inefficient at stimulating methanogenesis, resulting in a slow growth phenotype. As methanogenesis is limited by the availability of reduced ferredoxin under these conditions, other electron donors, such as reduced coenzyme F420, should be abundant. Indeed, when F420-reducing hydrogenase was reintroduced into the hydrogenase-free mutant, the equilibrium of H2production via an F420-dependent formate:H2lyase activity shifted markedly toward H2compared to the wild type.IMPORTANCEHydrogenotrophic methanogens are thought to require H2as a substrate for growth and methanogenesis. Here we show alternative pathways in methanogenic metabolism that alleviate this H2requirement and demonstrate, for the first time, a hydrogenotrophic methanogen that is capable of growth in the complete absence of H2. The demonstration of alternative pathways in methanogenic metabolism suggests that this important group of organisms is metabolically more versatile than previously thought.

scholarly journals Identification of Genes Required for Glucan Exopolysaccharide Production in Lactobacillus johnsonii Suggests a Novel Biosynthesis Mechanism

2020 ◽  
Vol 86 (8) ◽  
Author(s):  
Melinda J. Mayer ◽  
Alfonsina D’Amato ◽  
Ian J. Colquhoun ◽  
Gwénaëlle Le Gall ◽  
Arjan Narbad
Keyword(s):  
Slow Growth ◽  
Alternative Mechanism ◽  
Wild Type ◽  
Lactobacillus Johnsonii ◽  
Neighboring Gene ◽  
Amino Acid Homology ◽  
Content Type ◽  
Exopolysaccharide Production ◽  
Growth Phenotype ◽  
Slow Growth Phenotype

ABSTRACT Lactobacillus johnsonii FI9785 makes two capsular exopolysaccharides—a heteropolysaccharide (EPS2) encoded by the eps operon and a branched glucan homopolysaccharide (EPS1). The homopolysaccharide is synthesized in the absence of sucrose, and there are no typical glucansucrase genes in the genome. Quantitative proteomics was used to compare the wild type to a mutant where EPS production was reduced to attempt to identify proteins associated with EPS1 biosynthesis. A putative bactoprenol glycosyltransferase, FI9785_242 (242), was less abundant in the Δeps_cluster mutant strain than in the wild type. Nuclear magnetic resonance (NMR) analysis of isolated EPS showed that deletion of the FI9785_242 gene (242) prevented the accumulation of EPS1, without affecting EPS2 synthesis, while plasmid complementation restored EPS1 production. The deletion of 242 also produced a slow-growth phenotype, which could be rescued by complementation. 242 shows amino acid homology to bactoprenol glycosyltransferase GtrB, involved in O-antigen glycosylation, while in silico analysis of the neighboring gene 241 suggested that it encodes a putative flippase with homology to the GtrA superfamily. Deletion of 241 also prevented production of EPS1 and again caused a slow-growth phenotype, while plasmid complementation reinstated EPS1 synthesis. Both genes are highly conserved in L. johnsonii strains isolated from different environments. These results suggest that there may be a novel mechanism for homopolysaccharide synthesis in the Gram-positive L. johnsonii. IMPORTANCE Exopolysaccharides are key components of the surfaces of their bacterial producers, contributing to protection, microbial and host interactions, and even virulence. They also have significant applications in industry, and understanding their biosynthetic mechanisms may allow improved production of novel and valuable polymers. Four categories of bacterial exopolysaccharide biosynthesis have been described in detail, but novel enzymes and glycosylation mechanisms are still being described. Our findings that a putative bactoprenol glycosyltransferase and flippase are essential to homopolysaccharide biosynthesis in Lactobacillus johnsonii FI9785 indicate that there may be an alternative mechanism of glucan biosynthesis to the glucansucrase pathway. Disturbance of this synthesis leads to a slow-growth phenotype. Further elucidation of this biosynthesis may give insight into exopolysaccharide production and its impact on the bacterial cell.

scholarly journals c-Type Cytochrome Assembly Is a Key Target of Copper Toxicity within the Bacterial Periplasm

mBio ◽  
2015 ◽  
Vol 6 (5) ◽  
Author(s):  
Anne Durand ◽  
Asma Azzouzi ◽  
Marie-Line Bourbon ◽  
Anne-Soisig Steunou ◽  
Sylviane Liotenberg ◽  
...  
Keyword(s):  
Radical Scavenging ◽  
Copper Toxicity ◽  
Chlorophyll Synthesis ◽  
Copper Tolerance ◽  
Wild Type ◽  
Tight Control ◽  
Content Type ◽  
Excess Copper ◽  
Rubrivivax Gelatinosus ◽  
First Time

ABSTRACT In the absence of a tight control of copper entrance into cells, bacteria have evolved different systems to control copper concentration within the cytoplasm and the periplasm. Central to these systems, the Cu+ ATPase CopA plays a major role in copper tolerance and translocates copper from the cytoplasm to the periplasm. The fate of copper in the periplasm varies among species. Copper can be sequestered, oxidized, or released outside the cells. Here we describe the identification of CopI, a periplasmic protein present in many proteobacteria, and show its requirement for copper tolerance in Rubrivivax gelatinosus. The ΔcopI mutant is more susceptible to copper than the Cu+ ATPase copA mutant. CopI is induced by copper, localized in the periplasm and could bind copper. Interestingly, copper affects cytochrome c membrane complexes (cbb3 oxidase and photosystem) in both ΔcopI and copA-null mutants, but the causes are different. In the copA mutant, heme and chlorophyll synthesis are affected, whereas in ΔcopI mutant, the decrease is a consequence of impaired cytochrome c assembly. This impact on c-type cytochromes would contribute also to the copper toxicity in the periplasm of the wild-type cells when they are exposed to high copper concentrations. IMPORTANCE Copper is an essential cation required as a cofactor in enzymes involved in vital processes such as respiration, photosynthesis, free radical scavenging, and pathogenesis. However, copper is highly toxic and has been implicated in disorders in all organisms, including humans, because it can catalyze the production of toxic reactive oxygen species and targets various biosynthesis pathways. Identifying copper targets, provides insights into copper toxicity and homeostatic mechanisms for copper tolerance. In this work, we describe for the first time a direct effect of excess copper on cytochrome c assembly. We show that excess copper specifically affects periplasmic and membrane cytochromes c, thus suggesting that the copper toxicity targets c-type cytochrome biogenesis.

scholarly journals Novel Aminoglycoside-Tolerant Phoenix Colony Variants of Pseudomonas aeruginosa

2020 ◽  
Vol 64 (9) ◽  
Author(s):  
Devin Sindeldecker ◽  
Kelly Moore ◽  
Anthony Li ◽  
Daniel J. Wozniak ◽  
Matthew Anderson ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Calcium Sulfate ◽  
Bacterial Pathogen ◽  
Wild Type ◽  
Antibiotic Resistant ◽  
Content Type ◽  
Persistent Infections ◽  
High Concentrations ◽  
First Time ◽  
Very High

ABSTRACT Pseudomonas aeruginosa is an opportunistic bacterial pathogen and is known to produce biofilms. We previously showed the emergence of colony variants in the presence of tobramycin-loaded calcium sulfate beads. In this study, we characterized the variant colonies, which survived the antibiotic treatment, and identified three distinct phenotypes—classically resistant colonies, viable but nonculturable colonies (VBNC), and phoenix colonies. Phoenix colonies, described here for the first time, grow out of the zone of clearance of antibiotic-loaded beads from lawn biofilms while there are still very high concentrations of antibiotic present, suggesting an antibiotic-resistant phenotype. However, upon subculturing of these isolates, phoenix colonies return to wild-type levels of antibiotic susceptibility. Compared with the wild type, phoenix colonies are morphologically similar aside from a deficiency in green pigmentation. Phoenix colonies do not recapitulate the phenotype of any previously described mechanisms of resistance, tolerance, or persistence and, thus, form a novel group with their own phenotype. Growth under anaerobic conditions suggests that an alternative metabolism could lead to the formation of phoenix colonies. These findings suggest that phoenix colonies could emerge in response to antibiotic therapies and lead to recurrent or persistent infections, particularly within biofilms where microaerobic or anaerobic environments are present.

scholarly journals Deletion ofv-chiAfrom a Baculovirus Reduces Horizontal Transmission in the Field

2013 ◽  
Vol 79 (13) ◽  
pp. 4056-4064 ◽  
Author(s):  
Vincent D'Amico ◽  
James Slavicek ◽  
John D. Podgwaite ◽  
Ralph Webb ◽  
Roger Fuester ◽  
...  
Keyword(s):  
Cell Culture ◽  
Horizontal Transmission ◽  
Laboratory Data ◽  
Disease Outbreaks ◽  
Wild Type ◽  
Content Type ◽  
Transmission Process ◽  
Naturally Occurring ◽  
First Time

ABSTRACTNucleopolyhedroviruses (NPVs) can initiate devastating disease outbreaks in populations of defoliating Lepidoptera, a fact that has been exploited for the purposes of biological control of some pest insects. A key part of the horizontal transmission process of NPVs is the degradation of the larval integument by virus-coded proteins called chitinases, such as V-CHIA produced by thev-chiAgenes. We used recombinant and naturally occurring strains of theLymantria disparNPV (LdMNPV) to test horizontal transmission in the field, release of virus from dead larvae under laboratory conditions, and cell lysis and virus release in cell culture. In the field, strains of LdMNPV lacking functionalv-chiAgenes showed reduced horizontal transmission compared to wild-type or repaired strains. These findings were mirrored by a marked reduction in released virus in laboratory tests and cell culture when the same strains were used to infect larvae or cells. Thus, this study tests the pivotal role of liquefaction and thev-chiAgene in field transmission for the first time and uses complementary laboratory data to provide a likely explanation for our findings.

scholarly journals Minicells as a Damage Disposal Mechanism inEscherichia coli

mSphere ◽  
2018 ◽  
Vol 3 (5) ◽  
Author(s):  
Camilla U. Rang ◽  
Audrey Proenca ◽  
Christen Buetz ◽  
Chao Shi ◽  
Lin Chao
Keyword(s):  
Wild Type ◽  
Chromosomal Dna ◽  
Cell Level ◽  
Content Type ◽  
E Coli ◽  
Survival And Growth ◽  
First Time ◽  
Antibiotic Level ◽  
Type Strains ◽  
Sister Cells

ABSTRACTMany bacteria produce small, spherical minicells that lack chromosomal DNA and therefore are unable to proliferate. Although minicells have been used extensively by researchers as a molecular tool, nothing is known about why bacteria produce them. Here, we show that minicells helpEscherichia colicells to rid themselves of damaged proteins induced by antibiotic stress. By comparing the survival and growth rates of wild-type strains with theE. coliΔminCmutant, which produces excess minicells, we found that the mutant was more resistant to streptomycin. To determine the effects of producing minicells at the single-cell level, we also tracked the growth ofΔminClineages by microscopy. We were able to show that the mutant increased the production of minicells in response to a higher level of the antibiotic. When we compared two sister cells, in which one produced minicells and the other did not, the daughters of the former had a shorter doubling time at this higher antibiotic level. Additionally, we found that minicells were more likely produced at the mother’s old pole, which is known to accumulate more aggregates. More importantly, by using a fluorescent IbpA chaperone to tag damage aggregates, we found that polar aggregates were contained by and ejected with the minicells produced by the mother bacterium. These results demonstrate for the first time the benefit to bacteria for producing minicells.IMPORTANCEBacteria have the ability to produce minicells, or small spherical versions of themselves that lack chromosomal DNA and are unable to replicate. A minicell can constitute as much as 20% of the cell’s volume. Although molecular biology and biotechnology have used minicells as laboratory tools for several decades, it is still puzzling that bacteria should produce such costly but potentially nonfunctional structures. Here, we show that bacteria gain a benefit by producing minicells and using them as a mechanism to eliminate damaged or oxidated proteins. The elimination allows the bacteria to tolerate higher levels of stress, such as increasing levels of streptomycin. If this mechanism extends from streptomycin to other antibiotics, minicell production could be an overlooked pathway that bacteria are using to resist antimicrobials.

scholarly journals GFPuv-Expressing Recombinant Rickettsia typhi: a Useful Tool for the Study of Pathogenesis and CD8+ T Cell Immunology in R. typhi Infection

2017 ◽  
Vol 85 (6) ◽  
Author(s):  
Matthias Hauptmann ◽  
Nicole Burkhardt ◽  
Ulrike Munderloh ◽  
Svenja Kuehl ◽  
Ulricke Richardt ◽  
...  
Keyword(s):  
T Cell ◽  
Genetic Manipulation ◽  
Interleukin 2 ◽  
Scid Mice ◽  
Wild Type ◽  
Rickettsia Typhi ◽  
Content Type ◽  
First Time

ABSTRACT Rickettsia typhi is the causative agent of endemic typhus, a disease with increasing incidence worldwide that can be fatal. Because of its obligate intracellular life style, genetic manipulation of the pathogen is difficult. Nonetheless, in recent years, genetic manipulation tools have been successfully applied to rickettsiae. We describe here for the first time the transformation of R. typhi with the pRAM18dRGA plasmid that originally derives from Rickettsia amblyommatis and encodes the expression of GFPuv (green fluorescent protein with maximal fluorescence when excited by UV light). Transformed R. typhi (R. typhi GFPuv) bacteria are viable, replicate with kinetics similar to those of wild-type R. typhi in cell culture, and stably maintain the plasmid and GFPuv expression under antibiotic treatment in vitro and in vivo during infection of mice. CB17 SCID mice infected with R. typhi GFPuv succumb to the infection with kinetics similar to those for animals infected with wild-type R. typhi and develop comparable pathology and bacterial loads in the organs, demonstrating that the plasmid does not influence pathogenicity. In the spleen and liver of infected CB17 SCID mice, the bacteria are detectable by immunofluorescence microscopy in neutrophils and macrophages by histological staining. Finally, we show for the first time that transformed rickettsiae can be used for the detection of CD8+ T cell responses. GFP-specific restimulation of spleen cells from R. typhi GFPuv-infected BALB/c mice elicits gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin 2 (IL-2) secretion by CD8+ T cells. Thus, R. typhi GFPuv bacteria are a novel, potent tool to study infection with the pathogen in vitro and in vivo and the immune response to these bacteria.

scholarly journals Stability of Proteins Out of Service: the GapB Case of Bacillus subtilis

2017 ◽  
Vol 199 (20) ◽  
Author(s):  
Ulf Gerth ◽  
Eleonora Krieger ◽  
Daniela Zühlke ◽  
Alexander Reder ◽  
Uwe Völker ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Arginine Kinase ◽  
Adaptor Protein ◽  
Glycolytic Enzyme ◽  
Dependent Manner ◽  
Wild Type ◽  
Recognition Mechanism ◽  
Content Type ◽  
In The Wild

ABSTRACT Bacillus subtilis possesses two glyceraldehyde-3-phosphate dehydrogenases with opposite roles, the glycolytic NAD-dependent GapA and the NADP-dependent GapB enzyme, which is exclusively required during gluconeogenesis but not active under conditions promoting glycolysis. We propose that proteins that are no longer needed will be recognized and proteolyzed by Clp proteases and thereby recycled. To test this postulation, we analyzed the stability of the glycolytic enzyme GapA and the gluconeogenetic enzyme GapB in the presence and absence of glucose. It turned out that GapA remained rather stable under both glycolytic and gluconeogenetic conditions. In contrast, the gluconeogenetic enzyme GapB was degraded after a shift from malate to glucose (i.e., from gluconeogenesis to glycolysis), displaying an estimated half-life of approximately 3 h. Comparative in vivo pulse-chase labeling and immunoprecipitation experiments of the wild-type strain and isogenic mutants identified the ATP-dependent ClpCP protease as the enzyme responsible for the degradation of GapB. However, arginine protein phosphorylation, which was recently described as a general tagging mechanism for protein degradation, did not seem to play a role in GapB proteolysis, because GapB was also degraded in a mcsB mutant, lacking arginine kinase, in the same manner as in the wild type. IMPORTANCE GapB, the NADP-dependent glyceraldehyde-3-phosphosphate dehydrogenase, is essential for B. subtilis under gluconeogenetic conditions. However, after a shift to glycolytic conditions, GapB loses its physiological function within the cell and becomes susceptible to degradation, in contrast to GapA, the glycolytic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase, which remains stable under glycolytic and gluconeogenetic conditions. Subsequently, GapB is proteolyzed in a ClpCP-dependent manner. According to our data, the arginine kinase McsB is not involved as adaptor protein in this process. ClpCP appears to be in charge in the removal of inoperable enzymes in B. subtilis, which is a strictly regulated process in which the precise recognition mechanism(s) remains to be identified.

scholarly journals Pyocyanin Stimulates Quorum Sensing-Mediated Tolerance to Oxidative Stress and Increases Persister Cell Populations in Acinetobacter baumannii

2014 ◽  
Vol 82 (8) ◽  
pp. 3417-3425 ◽  
Author(s):  
Nidhi Bhargava ◽  
Prince Sharma ◽  
Neena Capalash
Keyword(s):  
Oxidative Stress ◽  
Quorum Sensing ◽  
Acinetobacter Baumannii ◽  
Homoserine Lactone ◽  
Protective Mechanism ◽  
Wild Type ◽  
Content Type ◽  
Sensing System ◽  
Anti Oxidant ◽  
First Time

ABSTRACTAcinetobacter baumanniiandPseudomonas aeruginosaare nosocomial pathogens with overlapping sites of infection. This work reports that the two can coexist stably in mixed-culture biofilms. In a study intended to improve our understanding of the mechanism of their coexistence, it was found that pyocyanin, produced byP. aeruginosathat generally eliminates competition from other pathogens, led to the generation of reactive oxygen species (ROS) inA. baumanniicells, which in response showed a significant (P≤ 0.05) increase in production of enzymes, specifically, catalase and superoxide dismutase (SOD). This work shows for the first time that the expression of catalase and SOD is under the control of a quorum-sensing system inA. baumannii. In support of this observation, a quorum-sensing mutant ofA. baumannii(abaI::Km) was found to be sensitive to pyocyanin compared to its wild type and showed significantly (P≤ 0.001) lower levels of the antioxidant enzymes, which increased on addition of 5 μMN-(3-hydroxydodecanoyl)-l-homoserine lactone. Likewise, in wild-typeA. baumannii, there was a significant (P< 0.01) decrease in the level of anti-oxidant enzymes in the presence of salicylic acid, a known quencher of quorum sensing. In the presence of amikacin and carbenicillin,A. baumanniiformed 0.07 and 0.02% persister cells, which increased 4- and 3-fold, respectively, in the presence of pyocyanin. These findings show that pyocyanin induces a protective mechanism inA. baumanniiagainst oxidative stress and also increases its persistence against antibiotics which could be of clinical significance in the case of coinfections withA. baumanniiandP. aeruginosa.

scholarly journals The Endospore-Forming Pathogen Bacillus cereus Exploits a Small Colony Variant-Based Diversification Strategy in Response to Aminoglycoside Exposure

mBio ◽  
2015 ◽  
Vol 6 (6) ◽  
Author(s):  
Elrike Frenzel ◽  
Markus Kranzler ◽  
Timo D. Stark ◽  
Thomas Hofmann ◽  
Monika Ehling-Schulz
Keyword(s):  
Antibiotic Resistance ◽  
Bacillus Cereus ◽  
Differential Diagnostics ◽  
Wild Type ◽  
Content Type ◽  
Small Colony Variant ◽  
Enhanced Production ◽  
Small Colony ◽  
First Time ◽  
Slow Growing

ABSTRACTBacillus cereusis among the microorganisms most often isolated from cases of food spoilage and causes gastrointestinal diseases as well as nongastrointestinal infections elicited by the emetic toxin cereulide, enterotoxins, and a panel of tissue-destructive virulence factors. This opportunistic pathogen is increasingly associated with rapidly fatal clinical infections especially linked to neonates and immunocompromised individuals. Fatality results from either the misdiagnosis ofB. cereusas a contaminant of the clinical specimen or from failure of antibiotic therapy. Here we report for the first time that exposure to aminoglycoside antibiotics induces a phenotype switching of emeticB. cereussubpopulations to a slow-growing small colony variant (SCV) state. Along with altered antibiotic resistance, SCVs showed distinct phenotypic and metabolic properties, bearing the risk of antibiotic treatment failure and of clinical misdiagnosis by standard identification tests used in routine diagnostic. The SCV subpopulation is characterized by enhanced production of the toxin cereulide, but it does not secrete tissue-destructive and immune system-affecting enzymes such as sphingomyelinase and phospholipase. SCVs showed significantly prolonged persistence and decreased virulence in theGalleria mellonellamodel for bacterial infections, indicating diversification concerning their ecological lifestyle. Importantly, diversification into coexisting wild-type and SCV subpopulations also emerged during amikacin pressure duringin vivoinfection experiments.IMPORTANCEThis study shows for the first time that pathogenic spore-formingB. cereusstrains are able to switch to a so far unreported slow-growing lifestyle, which differs substantially in terms of developmental, phenotypic, metabolic, and virulence traits from the wild-type populations. This underpins the necessity of molecular-based differential diagnostics and a well-chosen therapeutic treatment strategy in clinical environments to combatB. cereusin a tailored manner. The reported induction of SCV in an endospore-forming human pathogen requires further research to broaden our understanding of a yet unexplored antibiotic resistance mechanism in sporulating bacteria. Our work also raises a general question about the ecological meaning of SCV subpopulation emergence and importance of SCV in sporeformer populations as an alternative route, next to sporulation, to cope with stresses encountered in natural niches, such as soil or host interfaces.

scholarly journals The PA3177 Gene Encodes an Active Diguanylate Cyclase That Contributes to Biofilm Antimicrobial Tolerance but Not Biofilm Formation by Pseudomonas aeruginosa

2018 ◽  
Vol 62 (10) ◽  
Author(s):  
Bandita Poudyal ◽  
Karin Sauer
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Antimicrobial Agents ◽  
Drug Tolerance ◽  
Diguanylate Cyclase ◽  
Wild Type ◽  
Planktonic Cells ◽  
Swarming Motility ◽  
Content Type ◽  
First Time

ABSTRACT A hallmark of biofilms is their heightened resistance to antimicrobial agents. Recent findings suggested a role for bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) in the susceptibility of bacteria to antimicrobial agents; however, no c-di-GMP modulating enzyme(s) contributing to the drug tolerance phenotype of biofilms has been identified. The goal of this study was to determine whether c-di-GMP modulating enzyme(s) specifically contributes to the biofilm drug tolerance of Pseudomonas aeruginosa. Using transcriptome sequencing combined with biofilm susceptibility assays, we identified PA3177 encoding a probable diguanylate cyclase. PA3177 was confirmed to be an active diguanylate cyclase, with overexpression affecting swimming and swarming motility, and inactivation affecting cellular c-di-GMP levels of biofilm but not planktonic cells. Inactivation of PA3177 rendered P. aeruginosa PAO1 biofilms susceptible to tobramycin and hydrogen peroxide. Inactivation of PA3177 also eliminated the recalcitrance of biofilms to killing by tobramycin, with multicopy expression of PA3177 but not PA3177_GGAAF harboring substitutions in the active site, restoring tolerance to wild-type levels. Susceptibility was linked to BrlR, a previously described transcriptional regulator contributing to biofilm tolerance, with inactivation of PA3177 negatively impacting BrlR levels and BrlR-DNA binding. While PA3177 contributed to biofilm drug tolerance, inactivation of PA3177 had no effect on attachment and biofilm formation. Our findings demonstrate for the first time that biofilm drug tolerance by P. aeruginosa is linked to a specific c-di-GMP modulating enzyme, PA3177, with the pool of PA3177-generated c-di-GMP only contributing to biofilm drug tolerance but not to biofilm formation.

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