The metabolism of (R)-3-hydroxybutyrate is regulated by the enhancer-binding protein PA2005 and the alternative sigma factor RpoN in Pseudomonas aeruginosa PAO1

Lundgren, Benjamin R.; Harris, Joshua R.; Sarwar, Zaara; Scheel, Ryan A.; Nomura, Christopher T.

doi:10.1099/mic.0.000163

Cited by 14 publications

(16 citation statements)

References 32 publications

(35 reference statements)

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“…In contrast, the acetate-minimal media used for these specific experiments were not supplemented with gentamicin due to the presence of sulfate in the purchased antibiotic preparation (gentamicin sulfate). Previous studies by our group have shown that the addition of an antibiotic for plasmid selection is not necessary for genetic complementation experiments involving P. aeruginosa (32,35).…”

Section: Methodsmentioning

confidence: 93%

“…The msuE and sfnG PCR products were gel purified and subsequently cloned into pJET1.2 (Thermo Fisher Scientific), yielding the plasmids pZS458 and pKSF01, respectively. The Cy5-labeled msuE and sfnG probes were generated by PCR comprised of the Cy5-labeled primers JRH05.f and JRH05.r, which anneal specifically to pJET1.2 (35), and either pZS458 or pKSF01 as the template. The resulting Cy5-labeled msuE and sfnG PCR products were gel purified and quantified using a NanoDrop spectrophotometer.…”

Section: Methodsmentioning

confidence: 99%

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SfnR2 Regulates Dimethyl Sulfide-Related Utilization in Pseudomonas aeruginosa PAO1

et al. 2019

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Dimethyl sulfide (DMS) is a volatile sulfur compound produced mainly from the degradation of dimethylsulfoniopropionate (DMSP) in marine environments. DMS undergoes oxidation to form dimethyl sulfoxide (DMSO), dimethyl sulfone (DMSO 2 ), and methanesulfonate (MSA), all of which occur in terrestrial environments and are accessible for consumption by various microorganisms. The purpose of the present study was to determine how the enhancer-binding proteins SfnR1 and SfnR2 contribute to the utilization of DMS and its derivatives in Pseudomonas aeruginosa PAO1. First, results from cell growth experiments showed that deletion of either sfnR2 or sfnG, a gene encoding a DMSO 2 -monooxygenase, significantly inhibits the ability of P. aeruginosa PAO1 to use DMSP, DMS, DMSO, and DMSO 2 as sulfur sources. Deletion of the sfnR1 or msuEDC genes, which encode a MSA desulfurization pathway, did not abolish the growth of P. aeruginosa PAO1 on any sulfur compound tested. Second, data collected from ␤-galactosidase assays revealed that the msuEDC-sfnR1 operon and the sfnG gene are induced in response to sulfur limitation or nonpreferred sulfur sources, such as DMSP, DMS, and DMSO, etc. Importantly, SfnR2 (and not SfnR1) is essential for this induction. Expression of sfnR2 is induced under sulfur limitation but independently of SfnR1 or SfnR2. Finally, the results of this study suggest that the main function of SfnR2 is to direct the initial activation of the msuEDC-sfnR1 operon in response to sulfur limitation or nonpreferred sulfur sources. Once expressed, SfnR1 contributes to the expression of msuEDC-sfnR1, sfnG, and other target genes involved in DMS-related metabolism in P. aeruginosa PAO1. IMPORTANCE Dimethyl sulfide (DMS) is an important environmental source of sulfur, carbon, and/or energy for microorganisms. For various bacteria, including Pseudomonas, Xanthomonas, and Azotobacter, DMS utilization is thought to be controlled by the transcriptional regulator SfnR. Adding more complexity, some bacteria, such as Acinetobacter baumannii, Enterobacter cloacae, and Pseudomonas aeruginosa, possess two, nonidentical SfnR proteins. In this study, we demonstrate that SfnR2 and not SfnR1 is the principal regulator of DMS metabolism in P. aeruginosa PAO1. Results suggest that SfnR1 has a supportive but nonessential role in the positive regulation of genes required for DMS utilization. This study not only enhances our understanding of SfnR regulation but, importantly, also provides a framework for addressing gene regulation through dual SfnR proteins in other bacteria.

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Section: Methodsmentioning

confidence: 93%

Section: Methodsmentioning

confidence: 99%

SfnR2 Regulates Dimethyl Sulfide-Related Utilization in Pseudomonas aeruginosa PAO1

et al. 2019

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“…It was originally identified as a sigma factor required for transcription of the genes involved in nitrogen assimilation (Hirschman, Wong, Sei, Keener, & Kustu, ; Reitzer & Schneider, ). However, a great deal of evidence has now accumulated that RpoN also controls many other biological activities in bacteria, ranging from utilization of alternative carbon sources (Lundgren, Harris, Serwar, Scheel, & Nomura, ) and biodegradation of pollutants (Shingler, ) to motility (Dasgupta et al., ; Saldias, Lamothe, Wu, & Valvano, ; Totten, Lara, & Lory, ), and biofilm formation (Saldias et al., ; Thompson, Webb, Rice, & Kjelleberg, ; Wolfe, Millikan, Campbell, & Visick, ). It has been shown that RpoN controls flagellum‐mediated motility in B. cenocepacia , which was found to be essential for biofilm formation in microtiter trays (Saldias et al., ).…”

Section: Discussionmentioning

confidence: 99%

Regulation of Burkholderia cenocepacia biofilm formation by RpoN and the c‐di‐GMP effector BerB

et al. 2017

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Knowledge about the molecular mechanisms that are involved in the regulation of biofilm formation is essential for the development of biofilm‐control measures. It is well established that the nucleotide second messenger cyclic diguanosine monophosphate (c‐di‐GMP) is a positive regulator of biofilm formation in many bacteria, but more knowledge about c‐di‐GMP effectors is needed. We provide evidence that c‐di‐GMP, the alternative sigma factor RpoN (σ54), and the enhancer‐binding protein BerB play a role in biofilm formation of Burkholderia cenocepacia by regulating the production of a biofilm‐stabilizing exopolysaccharide. Our findings suggest that BerB binds c‐di‐GMP, and activates RpoN‐dependent transcription of the berA gene coding for a c‐di‐GMP‐responsive transcriptional regulator. An increased level of the BerA protein in turn induces the production of biofilm‐stabilizing exopolysaccharide in response to high c‐di‐GMP levels. Our findings imply that the production of biofilm exopolysaccharide in B. cenocepacia is regulated through a cascade involving two consecutive transcription events that are both activated by c‐di‐GMP. This type of regulation may allow tight control of the expenditure of cellular resources.

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“…PCR was used to amplify the 5=-regulatory regions adjacent to the open reading frames (ORFs) for exaC, PA4022, and eat. The amplified 5=-regulatory regions (850 bp for exaC, 986 bp for PA4022, 2,716 bp for eat, and 928 bp for truncated eat) were then fused to the ␤-galactosidase (lacZ) ORF of E. coli through PCR (24,27). The PA4022::lacZ, eat:: lacZ, and truncated eat::lacZ fusions were subcloned into the XbaI site of ⌬Plac-pBBR1MCS-5 (24) to yield the plasmids pBRL601, pBRL678, and pBRL597, respectively.…”

Section: Methodsmentioning

confidence: 99%

Ethanolamine Catabolism in Pseudomonas aeruginosa PAO1 Is Regulated by the Enhancer-Binding Protein EatR (PA4021) and the Alternative Sigma Factor RpoN

et al. 2016

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Although genes encoding enzymes and proteins related to ethanolamine catabolism are widely distributed in the genomes of Pseudomonas spp., ethanolamine catabolism has received little attention among this metabolically versatile group of bacteria. In an attempt to shed light on this subject, this study focused on defining the key regulatory factors that govern the expression of the central ethanolamine catabolic pathway in Pseudomonas aeruginosa PAO1. This pathway is encoded by the PA4022-eateutBC operon and consists of a transport protein (Eat), an ethanolamine-ammonia lyase (EutBC), and an acetaldehyde dehydrogenase (PA4022). EutBC is an essential enzyme in ethanolamine catabolism because it hydrolyzes this amino alcohol into ammonia and acetaldehyde. The acetaldehyde intermediate is then converted into acetate in a reaction catalyzed by acetaldehyde dehydrogenase. Using a combination of growth analyses and ␤-galactosidase fusions, the enhancer-binding protein PA4021 and the sigma factor RpoN were shown to be positive regulators of the PA4022-eat-eutBC operon in P. aeruginosa PAO1. PA4021 and RpoN were required for growth on ethanolamine, and both of these regulatory proteins were essential for induction of the PA4022-eat-eutBC operon. Unexpectedly, the results indicate that acetaldehyde (and not ethanolamine) serves as the inducer molecule that is sensed by PA4021 and leads to the transcriptional activation of the PA4022-eat-eutBC operon. Due to its regulatory role in ethanolamine catabolism, PA4021 was given the name EatR. Both EatR and its target genes are conserved in several other Pseudomonas spp., suggesting that these bacteria share a mechanism for regulating ethanolamine catabolism. IMPORTANCEThe results of this study provide a basis for understanding ethanolamine catabolism and its regulation in Pseudomonas aeruginosa PAO1. Interestingly, expression of the ethanolamine-catabolic genes in this bacterium was found to be under the control of a positive-feedback regulatory loop in a manner dependent on the transcriptional regulator PA4021, the sigma factor RpoN, and the metabolite acetaldehyde. Previously characterized regulators of ethanolamine catabolism are known to sense and respond directly to ethanolamine. In contrast, PA4021 (EatR) appears to monitor the intracellular levels of free acetaldehyde and responds through transcriptional activation of the ethanolamine-catabolic genes. This regulatory mechanism is unique and represents an alternative strategy used by bacteria to govern the acquisition of ethanolamine from their surroundings. E thanolamine serves as a source of carbon and nitrogen for a variety of bacteria, including members of the Enterobacteriaceae, Pseudomonadaceae, and Firmicutes (1-5). From studies mostly centered on Salmonella enterica subsp. enterica serovar Typhimurium and Escherichia coli, there is now a basic understanding of the catabolic steps involved in ethanolamine utilization. Extracellular ethanolamine enters the bacterial cell through simple diffusion or carrier-me...

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The metabolism of (R)-3-hydroxybutyrate is regulated by the enhancer-binding protein PA2005 and the alternative sigma factor RpoN in Pseudomonas aeruginosa PAO1

Cited by 14 publications

References 32 publications

SfnR2 Regulates Dimethyl Sulfide-Related Utilization in Pseudomonas aeruginosa PAO1

SfnR2 Regulates Dimethyl Sulfide-Related Utilization in Pseudomonas aeruginosa PAO1

Regulation of Burkholderia cenocepacia biofilm formation by RpoN and the c‐di‐GMP effector BerB

Ethanolamine Catabolism in Pseudomonas aeruginosa PAO1 Is Regulated by the Enhancer-Binding Protein EatR (PA4021) and the Alternative Sigma Factor RpoN

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