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Phosphorus Deprivation Responses and Phosphonate Utilization in a Thermophilic Synechococcus Sp

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Phosphorus Deprivation Responses and Phosphonate Utilization in a Thermophilic Synechococcus Sp JOURNAL OF BACTERIOLOGY, Dec. 2008, p. 8171–8184 Vol. 190, No. 24 0021-9193/08/$08.00ϩ0 doi:10.1128/JB.01011-08 Copyright © 2008, American Society for Microbiology. All Rights Reserved. Phosphorus Deprivation Responses and Phosphonate Utilization in a Thermophilic Synechococcus sp. from Microbial Matsᰔ† Melissa M. Adams,1,2 María R. Go´mez-García,1 Arthur R. Grossman,1 and Devaki Bhaya1* Carnegie Institution for Science, Department of Plant Biology, Stanford University, Stanford, California 94305,1 and Department of Biology, Stanford University, Stanford, California 943052 Received 22 July 2008/Accepted 3 October 2008 The genomes of two closely related thermophilic cyanobacterial isolates, designated Synechococcus isolate OS-A and Synechococcus isolate OS-B؅, from the microbial mats of Octopus Spring (Yellowstone National Park) have been sequenced. An extensive suite of genes that are controlled by phosphate levels constitute the Downloaded from putative Pho regulon in these cyanobacteria. We examined physiological responses of an axenic OS-B؅ isolate as well as transcript abundances of Pho regulon genes as the cells acclimated to phosphorus-limiting condi- tions. Upon imposition of phosphorus deprivation, OS-B؅ stopped dividing after three to four doublings, and absorbance spectra measurements indicated that the cells had lost most of their phycobiliproteins and chlorophyll a. Alkaline phosphatase activity peaked and remained high after 48 h of phosphorus starvation, and there was an accumulation of transcripts from putative Pho regulon genes. Interestingly, the genome of jb.asm.org Synechococcus isolate OS-B؅ harbors a cluster of phn genes that are not present in OS-A isolates. The proteins encoded by the phn genes function in the transport and metabolism of phosphonates, which could serve as an alternative phosphorus source when exogenous phosphate is low. The phn genes were upregulated within a day of eliminating the source of phosphate from the medium. However, the ability of OS-B؅ to utilize methylphos- at MONTANA STATE UNIV AT BOZEMAN on December 18, 2008 phonate as a sole phosphorus source occurred only after an extensive period of exposure to the substrate. Once acclimated, the cells grew rapidly in fresh medium with methylphosphonate as the only source of phosphorus. The possible implications of these results are discussed with respect to the ecophysiology of the microbial mats. Significant advances have been made in characterizing the erated an axenic culture of OS-BЈ and investigated acclimation diversity and ecophysiology of microorganisms in the alkaline responses to light intensities that they would experience in the hot spring microbial mats of Yellowstone National Park (4, microbial mat (19). It was found that OS-BЈ cultures grew best 53). However, relatively little is known about specific physio- at relatively low light intensities (25 to 200 ␮mol photon mϪ2 logical adaptations and the acclimation responses of microbes sϪ1), despite the high irradiation levels experienced by the cells in the mats with respect to nutrient limitation (1, 40, 42, 43). A in the natural environment (43). In fact, relatively high light unicellular cyanobacterium, Synechococcus sp., is the domi- elicited several acclimation responses, including chlorosis or nant, primary producer in these hot spring microbial mats at cell bleaching, which reflects a reduction in the levels of chlo- temperatures above 50°C and is found exclusively in the top rophyll a (chl a) and phycobiliprotein (PBP). green layer (comprising 1 to 2 mm of the ϳ1.5-cm-thick mat) We are studying the ways in which axenic cultures of ther- (53). Based on denaturing gradient gel electrophoresis analysis mophilic cyanobacteria respond to a variety of defined envi- and the subsequent determination of the 16S rRNA/internal ronmentally relevant conditions in the laboratory. We have transcribed spacer sequence, it appears that Synechococcus sp. initiated such studies by defining the potential gene members ecotypes are delineated along environmental gradients, includ- of the Pho regulon and characterizing accumulation of tran- ing temperature and light (9, 10, 37). For instance, the Syn- scripts from these genes in response to P deprivation. The echococcus OS-A isolate (hereafter OS-A) is abundant in the levels of inorganic phosphate (Pi) in the effluent channel of OS high-temperature region of the mat (58 to 65°C), while the have been determined to be as low as 0.37 ␮M (33), although OS-BЈ isolate is abundant in the lower-temperature regions (50 much higher levels (17 ␮M) have also been reported (4). As a to 60°C). OS indicates that the organisms were isolated from reference, P starvation responses of Escherichia coli are elic- Octopus Spring (9). ited when the extracellular Pi concentrations drops below 4 We have complete genome sequences for Synechococcus ␮M (49, 51). The dominant P source within the hot spring Ј 31 OS-A and OS-B isolates, which have enabled examination of microbial mat is Pi, as determined by P-nuclear magnetic the genetic potential of these organisms to respond to fluctu- resonance analysis of the total P content (M. Adams and B. ating environmental conditions, such as nutrient levels, light Cade-Menun, unpublished data); other P compounds detected intensities, and oxygen tension (3, 42, 43). Recently, we gen- include phosphonates and polyphosphate. Based on the 31P- nuclear magnetic resonance analysis, phosphonates could con- stitute up to 5% of the total P content in the microbial mat, and * Corresponding author. Mailing address: Carnegie Institution for levels may fluctuate within the microbial mat over the diel Science, 260 Panama Street, Stanford, CA 94305. Phone: (650) 325- cycle. However, the P composition of the aqueous environment 1521. Fax: (650) 325-6857. E-mail: [email protected]. † Supplemental material for this article may be found at http://jb surrounding the microbial mat (extracellular) was not investi- .asm.org/. gated. ᰔ Published ahead of print on 17 October 2008. Pi metabolism has been studied extensively in E. coli (46, 8171 8172 ADAMS ET AL. J. BACTERIOL. 49). The levels of over 80 identified proteins increase in re- pounds markedly different from other P sources (32). Al- sponse to P starvation, and many of these proteins are encoded though Phns are found in organisms across phylogenetic by genes that are coregulated as part of the Pho regulon (47, groupings, only prokaryotic microorganisms have, so far, been 49). The multigene transcriptional induction associated with shown to have the capability to cleave the Phn bond. Phns are the Pho regulon is accomplished by a mechanism in which the ubiquitous and have been identified in eukaryotes, bacteria, transcriptional regulator PhoB binds to the Pho box sequence, and archaea as antibiotics and phosphonolipids, although the which is an upstream activation sequence that precedes each exact role of the latter within the lipid bilayer is still not clear operon of the Pho regulon (25, 49). Many of the transcriptional (54). The most common naturally occurring Phn is aminoeth- units that are part of the Pho regulon represent multigenic ylphosphonate (AEPhn), which is present in phosphonolipid operons, such as the multigenic transport operon that encodes head groups as well as in polysaccharide and glycoprotein side the four subunits (PstS, PstC, PstA, and PstB) of the Pst high- groups (54). In the marine environment, Phns can comprise as affinity Pi transport systems (25, 49). The PstSCAB transporter much as 25% of the high-molecular-weight dissolved organic P operon in bacteria often includes the gene encoding PhoU, and serve as a P source for some bacteria (7). which is thought to function as a negative regulator of the Many Phns found in terrestrial and aquatic environments Downloaded from PhoR-PhoB two-component system (31). Genes of the Pho are anthropogenic in origin, as they are components of flame regulon also encode phosphatases (e.g., PhoA), which liberate retardants, plasticizers, pharmaceuticals, and herbicides. Pi from organic sources in the environment, and the enzymes Methylphosphonate is a component of several synthetic com- required for utilization of other P sources, including UGP- pounds, such as the flame extinguisher Pyrol 67, which is com- glycerol phosphate (41). The Pho regulon in Pseudomonas posed of vinylphosphonate and methylphosphonate. Glypho- fluorescens Pf0-1 lacks a PhoA homolog but encodes a phos- sate [N-(phosphonomethyl)glycine] is the active ingredient in jb.asm.org phatase, PhoX, which is predominantly responsible for extra- the widely used agricultural herbicide Roundup (26). There cellular phosphatase activity during P limitation (29). Secretion are potential repercussions of the widespread use of Phns as of PhoX by the twin-arginine transport pathway is a feature herbicides, since some are potentially toxic and have long re- at MONTANA STATE UNIV AT BOZEMAN on December 18, 2008 that distinguishes this family of phosphatases from the PhoA tention times in the environment (20). family (17). In this study, we examined the physiological consequences of The enzymes polyphosphate kinase 1 (PPK1) and exopoly- P deprivation on axenic isolates of the thermophilic cyanobac- phosphatase (PPX) have been shown to be members of the terium isolate Synechococcus OS-BЈ. We used a number of Pho regulon of E. coli K-12 (GenBank accession numbers different methods, including assays for alkaline phosphatase NP_416996 and NP_416997 for PPK1 and PPX, respectively) activity, quantification
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