Applied and Environmental Microbiology-2021-Michels-AEM.00200-21.full

Title: Amino acid analog induces stress response in marine Synechococcus 6 93407, USA 7 2Proteome Exploration Laboratory, Beckman Institute, Division of Biology and Biological AEM Accept

Title: Amino acid analog induces stress response in marine Synechococcus

6

93407, USA

7

2Proteome Exploration Laboratory, Beckman Institute, Division of Biology and Biological

AEM Accepted Manuscript Posted Online 14 May 2021

Appl Environ Microbiol doi:10.1128/AEM.00200-21

Copyright © 2021 American Society for Microbiology All Rights Reserved.

analysis of Synechococcus sp cells grown with HPG revealed a physiological shift in nitrogen

catalyzes the esterification of Met with its tRNA, methionyl-tRNA synthetase, has low

level, no changes to protein expression or degradation were observed due to NCAA additions

tested (up to 100 µM; Figure 1A, B) However, for Synechococcus sp., HPG concentrations as

and intact when grown in the presence of HPG (Figures 2,3,4) A bright signal in the green

(fluorescence signal of the azide-containing CR-110 fluorophore) was visually brighter for both

also significantly upregulated in HPG treatments Many antenna proteins were upregulated in

two conditions (Table S5, Figure S2) These proteins predominantly indicate that when a cell is

cultures revealed an influence of HPG on cyanobacterial cell physiology (Figure 8) Therefore,

Overall, these culture-based experiments revealed that phytoplankton exhibit varied

would lead to increased products and affect metabolic pathways, or whether the

documented as part of the core general stress response in cyanobacteria (41) Chaperonin

phycobilisome in HPG treated cells This upregulation could also indicate attempts to increase

F-type ATPase, allowing energy to be temporarily stored and easily accessed by many enzymes

(21) Populations of marine cyanobacteria are especially influenced by nutrient availability (53,

exhibit a reduction in growth These findings highlight the importance of coupling HPG

equally wide range of study systems (7, 20, 65-67) and demonstrates potentially less toxicity and

The three organisms were grown to exponential phase according to the conditions

exponential phase in sterile L1 medium minus silica When culture density reached OD450 ~0.4

resuspended in fresh L1 medium, either replete or lacking nitrate according to the treatment and

camera (Zeiss Axiocam 506 mono) Peak channel excitation and emissions wavelength/bandpass

densities in each image: for Synechococcus sp., 500 - 1000 ROIs were selected per image (~5

were quenched with 1 L 5% hydroxylamine for 15 minutes at room temperature, combined,

and an AGC target of 50k in Centroid mode The total cycle time was set at 3 second

(http://proteomecentral.proteomexchange.org) via the PRIDE partner repository (79) with the

9 Kiick KL, Saxon E, Tirrell D A, Bertozzi CR 2002 Incorporation of azides into recombinant

16 Leizeaga A, Estrany M, Forn I, Sebastián M 2017 Using Click-Chemistry for Visualizing in

Insights into adaptation to a coastal environment Proc Natl Acad Sci U.S.A 103:13555–

32 Michelou VK, Cottrell MT, Kirchman DL 2007 Light-stimulated bacterial production and

40 Klotz A, Reinhold E, Doello S, Forchhammer K 2015 Nitrogen Starvation Acclimation in

49 Latifi A, Ruiz M, Zhang C 2009 Oxidative stress in cyanobacteria FEMS Microbiol Revs

57 Azam F, Malfatti F 2007 Microbial structuring of marine ecosystems Nat Rev Microbiol

67 Chakrabarti S, Liehl P, Buchon N, Lemaitre B 2012 Infection-induced host translational

77 Mann HB, Whitney DR 1947 On a Test of Whether one of Two Random Variables is

pigments Right: merged image of all three channels The exposure times were standardized

samples from nitrate replete (+HPG), nitrate limited (+HPG) and control (-HPG) treatments Dye

on May 14, 2021 at CALIFORNIA INST OF TECHNOLOGY

DAPI CR-110 autofluorescence Pigment Merged

DAPI CR-110 autofluorescence Pigment Merged

on May 14, 2021 at CALIFORNIA INST OF TECHNOLOGY

on May 14, 2021 at CALIFORNIA INST OF TECHNOLOGY

DAPI CR-110 autofluorescence Pigment Merged

on May 14, 2021 at CALIFORNIA INST OF TECHNOLOGY

Table 1 Exponential growth and L-homopropargylglycine (HPG), concentration ranges tested

Table 2 Experimental set up conditions to test physiological response of Synechococcus

sp to L-homopropargylglycine (HPG) in nitrate replete and nitrate limited conditions

Replete centrifuge, wash & resuspend 10 µM Replete

Nitrate-limited centrifuge, wash & resuspend 10 µM Limited