The effect of bacterial community members on the proteome of the ammonia- 1 oxidizing bacterium Nitrosomonas sp. Is79 2

31 Microorganisms in the environment do not exist as the often-studied pure cultures but as 32 members of complex microbial communities. Characterizing the interactions within microbial 33 communities is essential to understand their function in both natural and engineered 34 environments. In this study we investigated how the presence of a nitrite-oxidizing bacterium 35 (NOB) and heterotrophic bacteria affect the growth and proteome of the chemolithoautotrophic 36 ammonia-oxidizing bacterium (AOB) Nitrosomonas sp. Is79. We investigated Nitrosomonas sp. 37 Is79, in co-culture with Nitrobacter winogradskyi , in co-cultures with selected heterotrophic 38 bacteria and as a member of the nitrifying enrichment culture G5-7 . In batch culture, N. 39 winogradskyi and heterotrophic bacteria had positive effects on the growth of Nitrosomonas sp. 40 Is79. An isobaric tag for a relative quantification (iTRAQ) LC MS/MS proteomics approach was 41 used to investigate the effect of N. winogradskyi and the co-cultured heterotrophic bacteria from 42 G5-7 on the proteome of Nitrosomonas sp. Is79. In co-culture with N. winogradskyi , several 43 Nitrosomonas sp. Is79 oxidative stress response proteins changed in abundance with periplasmic 44 proteins increasing and cytoplasmic proteins decreasing in abundance. In the presence of 45 heterotrophic bacteria, the abundance of proteins directly related to the ammonia oxidation 46 pathway increased, while the abundance of proteins related to amino acid synthesis and 47 metabolism decreased. In summary, the proteome of Nitrosomonas sp. Is79 was differentially 48 influenced by the presence of either N. winogradskyi or heterotrophic bacteria. Together, N. 49 winogradskyi and heterotrophic bacteria reduced the oxidative stress for Nitrosomonas sp. Is79, 50 which resulted in a more efficient metabolism. Aerobic ammonia oxidizing microorganisms play an important role in the global nitrogen cycle, 55 converting ammonia to nitrite. In their natural environment they coexist and interact with nitrite 56 oxidizers, which convert nitrite to nitrate, and with heterotrophic microorganisms. The presence 57 of nitrite oxidizers and heterotrophic bacteria has a positive influence on the growth of the 58 ammonia oxidizers. Here we present a study investigating the effect of nitrite oxidizers and 59 heterotrophic bacteria on the proteome of a selected ammonia oxidizer in a defined culture to 60 elucidate how these two groups improve the performance of the ammonia oxidizer. The results 61 show that the presence of nitrite oxidizers and heterotrophic bacteria reduced stress for the 62 ammonia oxidizers and resulted in a more efficient energy generation. This study contributes to 63 our understanding of microbe-microbe interactions, in particular between ammonia oxidizers and 64 their neighboring microbial community. 65 (c)aa 3 ; ATP Syn, ATP synthase; NADH DH, NADH dehydrogenase; PMF, proton- 807 motive force; P460, Cytochrome P460; GDH1, glycine dehydrogenase subunit 1; 808 GDH2, glycine dehydrogenase subunit2; ThrS, threonine synthase; GlyT, glycine 809 hydroxymethyltransferase; GroEL, 60 kDa chaperonin; GroES 10 kDa chaperonin; 810 TDST, 2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N-succinyltransferase; AADH, Glu/Leu/Phe/Val dehydrogenase; PRT, ATP phosphoribosyltransferase; GluA, 812 Glutamate-1-semialdehyde 2,1-aminomutase; SOD, superoxide dismutase; Rr, 813 rubrerythrin; NirK, nitrite reductase; TPX, thiol peroxidase; Ahp, alkyl hydroperoxide reductase; CcP, di-heme cytochrome c peroxidase; Clp, Clp protease subunit; αGP, α- glucan phosphorylase; PNPT, polyribonucleotide nucleotidyltransferase; CA, cytosol aminopeptidase; CopC, copper resistance protein; ESBP, extracellular solute-binding protein family 1; PSBP, sulfate ABC transporter periplasmic binding protein; HCBR, calcium-binding region; Lipase, lipase class 3; T2, ribonuclease T2.