Highlights d Cities possess a consistent ''core'' set of non-human microbes d Urban microbiomes echo important features of cities and city-life d Antimicrobial resistance genes are widespread in cities d Cities contain many novel bacterial and viral species

Humans have co-evolved with a dense community of microbial symbionts that inhabit the lower intestine. In the colon, secreted mucus creates a physical barrier that separates these microbes from the intestinal epithelium. Some gut bacteria are able to utilize mucin glycoproteins, the main mucus component, as a nutrient source. However, it remains unclear which bacterial enzymes initiate the degradation of the highly complex O-glycans found in mucins. In the colon, these glycans are heavily sulfated, but the specific sulfatases that are active on colonic mucins have not been identified. Here, we show that sulfatases are essential to the utilization of colonic mucin O-glycans by the human gut symbiont Bacteroides thetaiotaomicron. We have characterized the activity of 12 different sulfatases encoded by this species, showing that these enzymes collectively are active on all of the known sulfate linkages in colonic O-glycans. Crystal structures of 3 enzymes provide mechanistic insight into the molecular basis of substrate-specificity.Unexpectedly, we found that a single sulfatase is essential for utilization of sulfated Oglycans in vitro and also plays a major role in vivo. Our results provide insight into the mechanisms of mucin degradation by gut bacteria, an important process for both normal microbial gut colonization and diseases such as inflammatory bowel disease (IBD).Sulfatase activity is likely to be a keystone step in bacterial mucin degradation and inhibition of these enzymes may therefore represent a viable therapeutic path for treatment of IBD and other diseases.

A variety of cell surface structures dictate interactions between bacteria and their environment, including their viruses (bacteriophages). Members of the human gut Bacteroidetes characteristically produce several phase-variable capsular polysaccharides (CPS), but their contributions to bacteriophage interactions are unknown. To begin to understand how CPS impact Bacteroides -phage interactions, we isolated 71 B. thetaiotaomicron -infecting bacteriophages from two locations in the United States. Using B. thetaiotaomicron strains that express defined subsets of CPS, we show that CPS dictates host tropism for these phages and that expression of non-permissive CPS variants are selected under phage predation, enabling survival. In the absence of CPS, B. thetaiotaomicron escapes bacteriophage predation by altering expression of 8 distinct phase-variable lipoproteins. When constitutively expressed, one of these lipoproteins promotes resistance to multiple bacteriophages. Our results reveal important roles for Bacteroides CPS and other cell surface structures that allow these bacteria to persist under bacteriophage predation and hold important implications for using bacteriophages therapeutically to target gut symbionts.

SummaryHumans have co-evolved with a dense community of microbial symbionts that inhabit the lower intestine. In the colon, secreted mucus creates a physical barrier that separates these microbes from the intestinal epithelium. Some gut bacteria are able to utilize mucin glycoproteins, the main mucus component, as a nutrient source. However, it remains unclear which bacterial enzymes initiate the degradation of the highly complex O-glycans found in mucins. In the colon, these glycans are heavily sulfated, but the specific sulfatases that are active on colonic mucins have not been identified. Here, we show that sulfatases are essential to the utilization of colonic mucin O-glycans by the human gut symbiont Bacteroides thetaiotaomicron. We have characterized the activity of 12 different sulfatases encoded by this species, showing that these enzymes collectively are active on all of the known sulfate linkages in colonic O-glycans. Crystal structures of 3 enzymes provide mechanistic insight into the molecular basis of substrate-specificity. Unexpectedly, we found that a single sulfatase is essential for utilization of sulfated O-glycans in vitro and also plays a major role in vivo. Our results provide insight into the mechanisms of mucin degradation by gut bacteria, an important process for both normal microbial gut colonization and diseases such as inflammatory bowel disease (IBD). Sulfatase activity is likely to be a keystone step in bacterial mucin degradation and inhibition of these enzymes may therefore represent a viable therapeutic path for treatment of IBD and other diseases.

21A variety of cell surface structures, including capsular polysaccharides (CPS), dictate 22interactions between bacteria and their environment including their viruses (bacteriophages). 23Members of the prominent human gut Bacteroidetes characteristically produce several phase-24 variable CPS, but their contributions to bacteriophage interactions are unknown. We used 25 engineered strains of the human symbiont Bacteroides thetaiotaomicron, which differ only in the 26 CPS they express, to isolate bacteriophages from two locations in the United States. Testing each 27 of 71 bacteriophages against a panel of strains that express wild-type phase-variable CPS, one of 28 eight different single CPS, or no CPS at all, revealed that each phage infects only a subset of 29 otherwise isogenic strains. Deletion of infection-permissive CPS from B. thetaiotaomicron was 30 sufficient to abolish infection for several individual bacteriophages, while infection of wild-type 31 B. thetaiotaomicron with either of two different bacteriophages rapidly selected for expression of 32 non-permissive CPS. Surprisingly, acapsular B. thetaiotaomicron also escapes complete killing 33 by these bacteriophages, but surviving bacteria exhibit increased expression of 8 distinct phase-34 variable lipoproteins. When constitutively expressed, one of these lipoproteins promotes 35 resistance to multiple bacteriophages. Finally, both wild-type and acapsular B. thetaiotaomicron 36 were able to separately co-exist with one bacteriophage for over two months in the mouse gut, 37suggesting that phase-variation promotes resistance but also generates sufficient numbers of 38 susceptible revertants to allow bacteriophage persistence. Our results reveal important roles for 39Bacteroides CPS and other cell surface structures that allow these bacteria to persist despite 40 bacteriophage predation and hold important implications for using bacteriophages therapeutically 41 to target gut symbionts. 42 43Bacteroides thetaiotaomicron and Bacteroides fragilis each encode 8 different CPS 14,15 and there 61 is broad genetic diversity of cps loci among different strains within these species (e.g., 47 62 different cps biosynthetic loci were identified in just 14 strains of B. thetaiotaomicron) 8 . In 63Bacteroides, CPS structures appear to surround the entire bacterial cell 16,17 and the cps 64 biosynthetic loci that encode these surface coatings are often under the control of phase variable 65 promoters 8,15,18 . In conjunction with other regulatory mechanisms, phase variable CPS 66 4 expression generates phenotypic heterogeneity within an otherwise isogenic population that may 67 facilitate survival in the face of diverse disturbances 8,15,19,20 . 68 Bacterial viruses or bacteriophages (herein, phages), like the bacteria on which they prey, 69 vary greatly across individual gut microbiomes and are even responsive to host dietary changes 70 and disease states [21][22][23][24][25] . Compared to gut bacteria, far less is understood about the phages of the 71 gut microbi...