SUMMARY The panoply of microorganisms and other species present in our environment influence human health and disease, especially in cities, but have not been profiled with metagenomics at a city-wide scale. We sequenced DNA from surfaces across the entire New York City (NYC) subway system, the Gowanus Canal, and public parks. Nearly half of the DNA (48%) does not match any known organism; identified organisms spanned 1,688 bacterial, viral, archaeal, and eukaryotic taxa, which were enriched for harmless genera associated with skin (e.g., Acinetobacter). Predicted ancestry of human DNA left on subway surfaces can recapitulate U.S. Census demographic data, and bacterial signatures can reveal a station’s history, such as marine-associated bacteria in a hurricane-flooded station. Some evidence of pathogens was found (Bacillus anthracis), but a lack of reported cases in NYC suggests that the pathogens represent a normal, urban microbiome. This baseline metagenomic map of NYC could help long-term disease surveillance, bioterrorism threat mitigation, and health management in the built environment of cities.

Figure 3B has been corrected to show the general coverage of the Yersinia pestis pMT1 plasmid, but not the murine toxin gene (yMT). The initial claim of ''.consistent 203 coverage across the murine toxin gene.'' was erroneously based on looking at gene annotation coordinates from different reference sequences. No reads mapped to the yMT gene when updated annotations were used. The Summary, Results, and Discussion sections have been revised to remove and clarify misleading and speculative text about pathogenic organisms. We now state that although all our metagenomic analysis tools identified reads with similarity to B. anthracis and Y. pestis sequences, there is minimal coverage to the backbone genome of these organisms, and there is no strong evidence to suggest these organisms are in fact present, and no evidence of pathogenicity. The figure and the text have been corrected online and in the print version.

Intraerythrocytic malaria parasites reside within a parasitophorous vacuole membrane (PVM) that closely overlays the parasite plasma membrane. Although the PVM is the site of several transport activities essential to parasite survival, the basis for organisation of this membrane system is unknown. Here, we performed proximity labeling at the PVM with BioID2, which highlighted a group of single‐pass integral membrane proteins that constitute a major component of the PVM proteome but whose function remains unclear. We investigated EXP1, the longest known member of this group, by adapting a CRISPR/Cpf1 genome editing system to install the TetR–DOZI‐aptamers system for conditional translational control. Importantly, although EXP1 was required for intraerythrocytic development, a previously reported in vitro glutathione S‐transferase activity could not account for this essential EXP1 function in vivo. EXP1 knockdown was accompanied by profound changes in vacuole ultrastructure, including apparent increased separation of the PVM from the parasite plasma membrane and formation of abnormal membrane structures. Furthermore, although activity of the Plasmodium translocon of exported proteins was not impacted by depletion of EXP1, the distribution of the translocon pore‐forming protein EXP2 but not the HSP101 unfoldase was substantially altered. Collectively, our results reveal a novel PVM defect that indicates a critical role for EXP1 in maintaining proper organisation of EXP2 within the PVM.

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word count: 250 22 23 24 Importance word count: 149 25 26 Abstract 27Intraerythrocytic malaria parasites reside within a parasitophorous vacuole membrane (PVM) 28 that closely overlays the parasite plasma membrane (PPM) and constitutes the barrier between 29 parasite and host compartments. The PVM is the site of several essential transport activities but 30 the basis for organization of this membrane system is unknown. We utilized the second-31 generation promiscuous biotin ligase BioID2 fused to EXP2 or HSP101 to probe the content of 32 the PVM, identifying known and novel candidate PVM proteins. Among the best represented 33 hits were members of a group of single-pass integral membrane proteins that constitute a major 34 component of the PVM proteome but whose function remains unclear. We investigated the 35 function of EXP1, the longest known member of this group, by adapting a CRISPR/Cpf1 36 genome editing system to install the TetR-DOZI-aptamers system for conditional translational 37 control. EXP1 knockdown was essential for intraerythrocytic development and accompanied by 38 profound changes in vacuole ultrastructure, including increased separation of the PVM and 39 PPM and formation of abnormal membrane structures in the enlarged vacuole lumen. While 40 previous in vitro studies indicated EXP1 possesses glutathione S-transferase activity, a mutant 41 version of EXP1 lacking a residue important for this activity in vitro still provides substantial 42 rescue of endogenous exp1 knockdown in vivo. Intriguingly, while activity of the Plasmodium 43 translocon of exported proteins was not impacted by depletion of EXP1, the distribution of the 44 translocon pore-forming protein EXP2 was substantially altered. Collectively, our results reveal 45 a novel PVM defect that indicates a critical role for EXP1 in maintaining proper PVM 46 organization. 47 48 Importance 49 Like other obligate intracellular apicomplexans, blood-stage malaria parasites reside within a 50 membrane-bound compartment inside the erythrocyte known as the parasitophorous vacuole. 51Although the vacuole is the site of several transport activities essential to parasite survival, little 52 is known about its organization. To explore vacuole biology, we adopted recently developed 53 proteomic (BioID2) and genetic (CRISPR/Cpf1) tools for use in Plasmodium falciparum, which 54 allowed us to query the function of the prototypical vacuole membrane protein EXP1. 55Knockdown of EXP1 showed that a previously reported glutathione S-transferase activity cannot 56 fully account for the essential function(s) of EXP1 and revealed a novel role for this protein in 57 maintaining normal vacuole morphology and PVM protein arrangement. Our results provide new 58 insight into vacuole organization and illustrate the power of BioID2 and Cpf1 (which utilizes a T-59 rich PAM uniquely suited to the P. falciparum genome) for proximity protein identification and 60 genome editing in P. falciparum. 61 62 65principal barrier between the parasite and its host cell (1). Durin...