
Plasmids, bacterial strains, and growth conditions. We modified a Tn5containing pRL27 vector (28) to include an FseI restriction site inside one of the transposon ends in order to increase the annealing temperature and stringency of the targeting oligonucleotide in the modified TraDIS (transposon-directed insertion site sequencing) method (6) we used to identify transposon insertion sites for this study. The resulting pRL27F plasmid was initially electroporated into Escherichia coli EC100D-pirϩ cells (Epicentre), reisolated, and electroporated into E. coli BW20767 cells (40) for conjugation with the host species. Liquid cultures of E. coli strains were grown aerobically at 37°C with shaking at 200 rpm in LB medium with 50 g/ml kanamycin (Kan) for maintenance and 150 g/ml for selection. Aeromonas veronii Hm21R was isolated previously (41) and provided by Joerg Graf. Vibrio sp. strain ZWU0020 was isolated from adult zebrafish intestinal contents and provided by John F. Rawls. A spontaneous rifampin (Rif)-resistant strain was isolated from a wild-type (WT) culture of Vibrio by plating on 100 g/ml Rif. Liquid cultures of Aeromonas veronii Hm21 and Vibrio sp. strain ZWU0020 were grown aerobically at 30°C with shaking at 200 rpm in LB medium with 100 g/ml rifampin.
Targeted deletion of cheA in Aeromonas and Vibrio strains. Targeted deletion of cheA in Aeromonas was accomplished by allelic exchange. A targeting construct consisting of a kanamycin resistance cassette with 5= and 3= flanking homologous regions of the cheA gene was assembled by overlap extension and cloned into the pDMS197 (42) suicide plasmid, which contains a tetracycline resistance gene and the sacB1 counterselection marker. The resulting allelic exchange plasmid was transformed into the donor E. coli strain BW20767 for conjugation with Aeromonas. Conjugations were incubated at 37°C on LB agar for~5 h before being picked and plated onto LB plates containing rifampin and 10% sucrose to select for Aeromonas mutants that had undergone complete allelic exchange. Verification of tetracycline sensitivity and sequencing of the targeted locus were done to confirm Aeromonas ⌬cheA mutants. Similar methods were used in the engineering of the Vibrio ⌬cheA2 mutant derivative with slight modification. The targeting construct containing a kanamycin resistance gene flanked by Ͼ500 bp of sequence homologous to the 5= and 3= regions flanking cheA2 was cloned into the alternative suicide vector pGP704sac28 (43). This vector carries a gene that encodes resistance to ampicillin along with the sacB gene, which allowed more-efficient screening of merodiploids for complete allelic exchange events. Chemotaxis defects were verified for the mutants of both strains by stabbing 1 l of an overnight culture into soft-agar swim plates (TSA [tryptic soy agar] with 0.2% agar) (see Fig. S2 in the supplemental material) and incubating Aeromonas at 30°C for 5 h and Vibrio at room temperature overnight.
Derivation, inoculation, and sequencing of transposon insertion site libraries. The E. coli BW20767 strain harboring the Tn5-containing pRL27F plasmid was conjugated in multiple replicates with the host strain (Vibrio or Aeromonas) overnight at 30°C by spotting mixtures of the two strains on LB agar. Conjugated bacteria were picked with sterile loops and resuspended in liquid LB medium, and multiple dilutions were plated on LB agar with Kan (150 g/ml) and Rif (100 g/ml). Control conjugations of only one strain (host or donor) were included to ensure that antibiotic selection was effective. Selective plates were incubated overnight at 30°C, and multiple plates with good separation of colonies were scraped and resuspended in 3 ml of LB broth. The mix of transposon-tagged bacteria was then split into three 50-ml cultures in order to reduce stochastic loss of transposon-associated genomic insertion sites (TAGs) and grown with Kan and Rif. These cultures represented an initial negative selection against insertions in essential genes. The replicate cultures were combined, and a portion was used to inoculate germ-free fish and extract DNA for sequencing libraries of the input inoculum. Fifty embryos from a single wild-type (WT) parental (strain AB/Tü) cross were derived germ free as previously described (10) and raised in 50 ml of sterile embryo medium in tissue culture flasks. Multiple fish among three replicate flasks were analyzed for monoassociation conditions, while multiple fish from a single flask were analyzed per each diassociation condition. All bacterial inoculations were given at a density of 10 5 CFU ml Ϫ1 in the surrounding fish water. Intestines were dissected and homogenized in 200 l sterile embryo medium with a pellet pestle motor (catalog no. 749540-0000; Kimble-Chase) and pestle (see Text S1 in the supplemental material) at 7 days postfertilization (dpf) and briefly cultured to an optical density at 600 nm (OD 600 ) of less than 1.0 in 1 ml of LB broth with Kan in order to increase the ratio of bacterial to zebrafish DNA used as the template in subsequent PCRs. Cultures were then pelleted, and genomic DNA was extracted using Qiagen DNeasy columns and the protocol for Gram-negative bacteria. Details on the preparation of Illumina sequencing libraries from genomic DNA for identification of transposon insertion sites are provided in Text S1, and the oligonucleotide sequences used are provided in Table S2.
Sequence read filtering, mapping, and identification of differentially abundant TAGs. Raw Illumina reads were first quality filtered using the fastx_toolkit by end trimming reads at a quality score of less than 25 and initially discarding reads shorter than 60 bases, then reads with an overall average quality of less than 20 over 90% of their bases were discarded. Quality sequences were then filtered based on the presence of the target sequence and outside end (OE) sequence from the transposon to verify that the reads came from a sequence adjacent to a Tn5 insertion site. After trimming of transposon sequences, filtering out adapter sequence contamination and sequences shorter than 20 bases, the remaining genomic sequence of each read was mapped to the draft genomes using Bowtie 2 (44). For additional quality control, we filtered out alignments that had poor (Ͻ10) mapping quality scores because we found that at this cutoff, Ͼ96% of these alignments were derived from less than 10 reads, and we suspected that they may have been misalignments due to the draft status of our genome assemblies and sequencing error. The majority of alignments were of very high quality despite the draft status of the genomes (see Fig. S3 in the supplemental material). Alignment files were imported into an SQL database and queried to group reads from the same TAG and retrieve read counts. A unique identifier for each TAG was then assigned, and tables of abundances in each sample were imported into R for further analysis. For identification of differentially abundant TAGs, we used the R Bioconductor package edgeR (45), and estimates of common dispersion within each intestinal output and inoculum input sample comparison to identify significant changes in their relative levels (after Benjamini-Hochberg multiple testing correction).
TAG richness of populations, binomial sampling simulations, and statistical analysis. Tables of counts of TAGs by sample are identical in structure to a community matrix of species by sites. We made use of the R package Vegan to rarefy each experiment's table of counts to the minimum number of TAGs detected in each set of experiments and then determine the total TAG richness in each sample. Binomial sampling simulations were carried out within R using rarefied tables and the binomial distribution set of functions to generate binomial probabilities and random deviates for simulated distributions as described above. To test the significance of differences in COG category representation shown in Fig. 3A, we employed a binomial test using the relative abundance of each COG category annotation in the input inoculum TAG set as the probability of detecting a given COG category and then applied the Benjamini-Hochberg multiple hypothesis testing correction.
Light sheet microscopy. Light sheet microscopy methods, fluorescent bacterial strains, and fluorescent intensity measurements were used as previously described (13). Briefly, germ-free fish were inoculated with 10 5 CFU/ml of a given labeled strain at 5 dpf, followed by inoculation with the second labeled strain at 6 dpf. Fish were rinsed in sterile media and anesthetized prior to mounting and imaging in low-melting-point agarose beginning 24 h after inoculation with the second strain.News coverage of the coronavirus crisis has been credible and trustworthy. No news organization has published any reports whose accuracy could be questioned. On the contrary, news media organizations have proactively debunked coronavirus-related misinformation that has circulated on social media platforms (Heikkilä 2020).
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