The gut microbiome harbors a 'silent reservoir' of antibiotic resistance (AR) genes that is thought to contribute to the emergence of multidrug-resistant pathogens through horizontal gene transfer (HGT). To counteract the spread of AR, it is paramount to know which organisms harbor mobile AR genes and which organisms engage in HGT. Despite methods that characterize the overall abundance of AR genes in the gut, technological limitations of short-read sequencing have precluded linking bacterial taxa to specific mobile genetic elements (MGEs) encoding AR genes. Here, we apply Hi-C, a high-throughput, cultureindependent method, to surveil the bacterial carriage of MGEs. We compare two healthy individuals with seven neutropenic patients undergoing hematopoietic stem cell transplantation, who receive multiple courses of antibiotics, and are acutely vulnerable to the threat of multidrug-resistant infections. We find distinct networks of HGT across individuals, though AR and mobile genes are associated with more diverse taxa within the neutropenic patients than the healthy subjects. Our data further suggest that HGT occurs frequently over a several-week period in both cohorts. Whereas most efforts to understand the spread of AR genes have focused on pathogenic species, our findings shed light on the role of the human gut microbiome in this process.

The presence of tRNA genes in bacteriophages has been explained on the basis of codon usage (tRNA genes are retained in the phage genome if they correspond to codons more common in the phage than in its host) or amino acid usage (independent of codon, the amino acid corresponding to the retained tRNA gene is more common in the phage genome than in the bacterial host). The existence of a large database of sequenced mycobacteriophages, isolated on the common host Mycobacterium smegmatis, allows us to test the above hypotheses as well as explore other hypotheses for the presence of tRNA genes. Our analyses suggest that amino acid rather than codon usage better explains the presence of tRNA genes in mycobacteriophages. However, closely related phages that differ in the presence of tRNA genes in their genomes are capable of lysing the common bacterial host and do not differ in codon or amino acid usage. This suggests that the benefits of having tRNA genes may be associated with either growth in the host or the ability to infect more hosts (i.e., host range) rather than simply infecting a particular host.

Promoter-proximal pausing of RNA polymerase II (Pol II) is a key regulatory step during transcription. To understand the evolution and function of pausing, we analyzed transcription in 20 organisms across the tree of life. Unicellular eukaryotes have a slow acceleration of Pol II near transcription start sites that matured into a longer and more focused pause in metazoans. Increased pause residence time coincides with the evolution of new subunits in the NELF and 7SK complexes. In mammals, depletion of NELF reverts a focal pause to a proto-paused-like state driven in part by DNA sequence. Loss of this focal pause compromises transcriptional activation for a set of heat shock genes. Overall, we discovered how pausing evolved and increased regulatory complexity in metazoans.

12The gut microbiome harbors a 'silent reservoir' of antibiotic resistance (AR) genes that is thought to 13 contribute to the emergence of multidrug-resistant pathogens through the process of horizontal gene 14 transfer (HGT). To counteract the spread of AR genes, it is paramount to know which organisms harbor 15 mobile AR genes and with which organisms they engage in HGT. Despite methods to characterize the 16 bulk presence 1 , abundance 2 and function 3 of AR genes in the gut, technological limitations of short-read 17sequencing have precluded linking bacterial taxa to specific mobile genetic elements (MGEs) and their 18 concomitant AR genes. Here, we apply and evaluate a high-throughput, culture-independent method for 19surveilling the bacterial carriage of MGEs, based on bacterial Hi-C protocols. We compare two healthy 20individuals with a cohort of seven neutropenic patients undergoing hematopoietic stem cell 21 transplantation, who receive multiple courses of antibiotics throughout their prolonged hospitalizations, 22and are thus acutely vulnerable to the threat of multidrug-resistant infections 4 . We find that the networks 23 of HGT are surprisingly distinct between individuals, yet AR and mobile genes are more dispersed across 24 taxa within the neutropenic patients than the healthy subjects. Our data further suggest that HGT is 25occurring throughout the course of treatment in the microbiomes of neutropenic patients and within the 26 guts of healthy individuals over a similar timeframe. Whereas most efforts to understand the spread of AR 27genes have focused on pathogenic species, our findings shed light on the role of the human gut 28 microbiome in this process. 29 preferentially recruited to contigs that are longer and more abundant, but to a lesser degree than expected, 81reducing potential bias in our dataset toward highly abundant organisms (Extended Data Figure 4). We 82 binned contigs using several tools (Maxbin 22 , MetaBat and Concoct), and applied a binning aggregation 83 strategy, DAS Tool 23 , to obtain a set of draft genomic assemblies. As misassembly can resemble HGT, we 84removed assemblies with greater than 10% contamination, as determined by CheckM, resulting in 85 taxonomically coherent assemblies (Extended Data Figure 5), albeit a greater number of unbinned contigs 86(24.6% of the total) (Extended Data Table 3). We then apply conservative criteria to link mobile and 87 mobile AR-containing contigs with the genomic draft assemblies, considering an MGE part of a genome 88 assembly only if it is directly linked to it by at least two uniquely-mapped Hi-C read-pairs. As MGEs are 89 known to recombine, this mitigates the potential for falsely linking contigs that merely share common 90 mobile genes. However, this also potentially reduces our ability for overall detection, especially for larger 91MGEs, since mobile contigs are often fragmented in metagenomic assemblies 24 . Nevertheless, we 92restricted our analysis to those AR-organism and MGE-organism linkages derived from high-confidence ...

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