A Tale of Transmission: Aeromonas veronii Activity withinLeech-Exuded Mucus

Brittany M. Ott,a Andrew M. Dacks,a Kenneth J. Ryan,b Rita V. M. Rioa

Department of Biology, West Virginia University, Morgantown, West Virginia, USAa; Department of Statistics, West Virginia University, Morgantown, West Virginia, USAb

Transmission, critical to the establishment and persistence of host-associated microbiotas, also exposes symbionts to new envi-ronmental conditions. With horizontal transmission, these different conditions represent major lifestyle shifts. Yet genome-wide analyses of how microbes adjust their transcriptomes toward these dramatic shifts remain understudied. Here, we providea comprehensive and comparative analysis of the global transcriptional profiles of a symbiont as it shifts between lifestyles dur-ing transmission. The gammaproteobacterium Aeromonas veronii is transmitted from the gut of the medicinal leech to otherhosts via host mucosal castings, yet A. veronii can also transition from mucosal habitancy to a free-living lifestyle. These threelifestyles are characterized by distinct physiological constraints and consequently lifestyle-specific changes in the expression ofstress-response genes. Mucus-bound A. veronii had the greatest expression in terms of both the number of loci and levels oftranscription of stress-response mechanisms. However, these bacteria are still capable of proliferating within the mucus, sug-gesting the availability of nutrients within this environment. We found that A. veronii alters transcription of loci in a syntheticpathway that obtains and incorporates N-acetylglucosamine (NAG; a major component of mucus) into the bacterial cell wall,enabling proliferation. Our results demonstrate that symbionts undergo dramatic local adaptation, demonstrated by widespreadtranscriptional changes, throughout the process of transmission that allows them to thrive while they encounter new environ-ments which further shape their ecology and evolution.

Mutualistic bacteria provide numerous advantages to their eu-karyotic hosts, such as the provisioning of essential nutrients

(1, 2), protection from pathogens (3, 4), and immunologicalpriming (5). These mutualists also serve as some of the best exam-ples of the extended phenotype (6) by providing opportunities forrapid adaptation and resilience toward dynamic ecological condi-tions, including broadening the host dietary range via enzymaticactivities (reviewed in references 7 and 8), detoxification mecha-nisms (reviewed in references 9 and 10), or enhancing tolerance toenvironmental stressors such as fluctuating temperatures (11). Al-though the persistence of these relations over evolutionary time iscrucial for securing adaptive potential, little is known about localadaptation and the activity of symbionts within a transmissionsetting.

Mixed-mode transmission (MMT), integrating componentsof both vertical and horizontal microbial acquisition, is the pre-dominant mechanism for obtaining microbial symbionts by ahost (reviewed in reference 12). A recently described model ofMMT involves the acquisition of the heterotrophic Gammapro-teobacteria Aeromonas veronii, a predominant member, along withthe Bacteroidetes Mucinivorans hirudinis (13, 14), of the Europeanmedicinal leech (Hirudo verbana) digestive tract microbiota. Spe-cifically, A. veronii is found within the leech crop (13), which is thelargest component of the digestive tract, where the blood meal isstored over a period of weeks to months (15). These A. veroniisymbionts are known to utilize both type II and type III secretionsystems (16, 17) during initial leech host colonization. Specifi-cally, the type II secretion system enables A. veronii establishmentthrough the export of hemolysin (16), while the type III secretionsystem prevents A. veronii from being phagocytized by macro-phage-like cells (18), demonstrating the use of traditionally rec-ognized virulence mechanisms to establish a beneficial associa-tion. Two other Aeromonas species are associated with distinctmedicinal leech host species: Hirudo medicinalis harbors Aeromo-

nas hydrophila (19), while Hirudo orientalis has been shown tohouse both A. veronii and Aeromonas jandaei (20).

In the H. verbana symbiosis, acquisition of A. veronii incorpo-rates vertical transmission via the leech cocoon (21). The cocoonis secreted from glandular cells of the parental mouth and containsapproximately 3 to 36 fertilized eggs (15). These cocoons are wa-tertight and yolk filled, providing nutrients for albumenotrophiclarvae to develop into nonfeeding juveniles approximately mid-way into embryogenesis. Imperfect vertical transmission (i.e.,only a subset of juveniles are infected with A. veronii upon cocoonemergence) is complemented by environmental acquisitionthrough regularly secreted mucus that conspecific leeches activelyseek in their environment (22). This mucus is believed to consistof glucosaminoglycans (23) though little else is known about itscomposition. Although A. veronii is a minor microbial constituentof these mucosal secretions, it has a high prevalence, being foundin all wild-type mucosal samples (22, 24). Within the mucus, A.veronii is capable of surviving and even proliferating at a tempothat synchronizes with the host shedding frequency (22). The hor-izontal transmission of A. veronii likely enables additional life-styles, including that as a waterborne human pathogen (reviewed

Received 19 January 2016 Accepted 16 February 2016

Accepted manuscript posted online 19 February 2016

Citation Ott BM, Dacks AM, Ryan KJ, Rio RVM. 2016. A tale of transmission:Aeromonas veronii activity within leech-exuded mucus. Appl Environ Microbiol82:2644 –2655. doi:10.1128/AEM.00185-16.

Editor: H. L. Drake, University of Bayreuth

Address correspondence to Rita V. M. Rio, rita.rio@mail.wvu.edu.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.00185-16.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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in reference 25), free-living in H2O (26), and that as a zebrafishsymbiont (27). Each lifestyle represents a major shift on the phys-iological constraints exerted upon A. veronii by a variety of eco-logical factors, and thus adjustments to gene expression must oc-cur for persistence under these different conditions.

Shifting niches results in changes in several environmental fac-tors, including ambient temperatures (28) and nutrient resources(reviewed in reference 29), with significant impacts on the “tran-scriptional tuning” of organisms, necessitating a balance betweenseveral responses, including growth and stress (reviewed in refer-ence 29). Stress is a known selective force (30) for either preexist-ing or novel mutations within the population (30). Several factorslikely make the transition from gut to mucosal occupancy stressfulfor A. veronii, including alterations in nutrient availability (i.e.,blood to glucosaminoglycan environment), abrupt shifts in atmo-spheric conditions (i.e., anaerobic to aerobic, temperature, etc.),and the likely rise in competition due to increased communitydiversity (24). Notably, A. veronii is capable of not only remainingviable after such a sudden lifestyle shift but also replicating to adensity high enough for transmission to another leech individual(22), indicating the availability of nutrients to support populationgrowth. However, the extent to which A. veronii gene expressionshifts in response to these dramatic environmental changes is un-known.

In this study, we addressed three major questions. First, howdoes A. veronii respond to a lifestyle shift from a mutualistic to afree-living state? Second, how is A. veronii capable of surviving inthe mucosal environment that likely involves numerous stressors?Finally, what nutrient sources enable A. veronii to proliferatewithin shed mucus? To address these questions, high-throughputIllumina-based deep sequencing was used to characterize the A.veronii transcriptome within leech mucosal secretions. Differen-tial analyses of high-throughput sequencing of RNA transcripts(RNA-Seq) were used to compare A. veronii transcript abun-dances within leech mucosal secretions to those in the leech diges-tive tract and within monoculture (31).

In addition to these transcriptome analyses, lectin staining identi-fied N-acetylglucosamine (NAG; a major glycan element of pepti-doglycan) as an abundant component of shed mucus and the bacte-rial cell membrane. We describe the expression profile of lociinvolved in the import and processing of NAG that would facilitate itsincorporation into peptidoglycan, thereby enabling A. veronii pro-liferation within this niche. While the antimicrobial properties ofmucosal secretions have received strong interest (32, 33), thisstudy focuses on the activity of a microbe within a transmissionsubstrate. Here, we describe the significance of this “in-between”state as a selective milieu for microbial ecology and evolution.

MATERIALS AND METHODSLeech husbandry. H. verbana leeches were obtained from Leeches USA(Westbury, NY, USA) and maintained in sterilized, Leech Strength InstantOcean (IO) H2O (0.004%) in the Department of Biology at West VirginiaUniversity (WVU) at 15°C. Leeches were maintained on defibrinated bovineblood (Hemostat, CA) through an artificial feeding system.

Mucosal sampling. Tanks housing leeches were cleaned to remove allmucus, and fresh Leech Strength IO was added (designated day 0). Mu-cosal samples collected the following day were considered 1-day samplesand were aged to 3 days at 15°C within sterile Leech Strength IO H2O.

RNA extraction and library preparation. Total RNA was extractedfrom three pooled 3-day wild-type leech mucus samples using a Master-Pure RNA purification kit (Epicentre, Madison, WI) according to themanufacturer’s protocol for fluid samples. DNA was removed fromthe RNA samples using a Turbo DNA-free kit (Ambion, Austin, TX) usingthe rigorous DNase treatment option. DNA removal was confirmedthrough Aeromonas-specific primers AvgyrB-F and AvgyrB-R with theamplification settings described in Table 1 and an RNA template lacking areverse-transcription step.

Total RNA was quantified using a NanoDrop 2000 instrument(Thermo Scientific, Waltham, MA), and integrity was examined using anAgilent 2100 Bioanalyzer (Agilent, Santa Clara, CA). The pooled-mucusRNA sample with the highest integrity was then processed with a Ribo-Zero magnetic kit for Gram-negative bacteria (Epicentre, Madison, WI)according to the manufacturer’s individual washing protocol. The result-ing mRNA-enriched RNA was then purified using an RNeasy MinElute

TABLE 1 Primer list and amplification settings

Primer target/group and namea Sequence (5=–3=) Amplicon size (bp) Amplification conditionsb

A. veronii gfp (gfp199) 199 30 s at 55°C; 30 s at 72°C*gfp199-F GCA GAT TGG CGA CAG CAC GTgfp199-R CAA TGT TGT GGC GAA TTT TG

General eubacterial primers (16S rRNA) 1,400 30 s at 50°C; 1.5 min at 72°C27F= AGA GTT TGA TCM TGG CTC AG1492R= TAC GGY TAC CTT GTT ACG ACT T

A. veronii gyrase B (gyrB) 514 30 s at 53°C; 30 s at 72°CAVgyrB-F GCA GAT TGG CGA CAG CACAVgyrB-R GCA CCT TGA CGG AGA TAA CG

Phosphoglucosamine mutase (glmM) 488 30 s at 52°C; 30 s at 72°C*AvglmM-F CAG ATT ATG TGG CCG GAG TTAvglmM-R ATG AAT CTG GAT GGG GTG AA

UDP-N-acetylglucosamine 1-carboxyvinyltransferase (murA) 500 30 s at 52°C; 30 s at 72°C*AvmurA-F TAG CCA CGG TCG ATG TGA TAAvmurB-R ATC GAA ACC GGT ACC TTC CT

UDP-N-acetylmurate dehydrogenase (murB) 507 30 s at 53°C; 30 s at 72°C*AvmurB-F CAG GTA CCA CCG GAT TAT TGAvmurB-R AGG GGA TTA CCG CTG AAG AT

a F, forward; R, reverse.b Each amplification reaction was initiated with a 3-min denaturation step at 95°C. Asterisks indicate that 40 cycles were used when NAG-related gene expression in vitro wasanalyzed as a loading control.

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Cleanup kit (Qiagen, Valencia, CA). Approximately 250 ng of the en-riched mRNA was ethanol precipitated and reeluted in Elute, Prime, Frag-ment mix. The eluted mRNA was then processed using a TruSeq RNASample Prep kit, version 2 (Illumina, San Diego, CA), by the WVUGenomics Core Facility, and the libraries were sequenced using an Illu-mina MiSeq instrument (Illumina, San Diego, CA) (2 by 250 bp) at theWVU Genomics Core Facility.

Transcriptome analyses. Previously published transcriptomes (31)from the A. veronii monoculture (in vitro mid-log cultures at 30°C) andleech crop (leech host was maintained at room temperature for 42 h post-feeding prior to dissection) were used for comparative purposes in ouranalyses (Table 2). It is important to note that while paired-end reads anda higher starting quantity of RNA (i.e., 250 ng for mucus compared with100 ng for gut and culture, due to A. veronii being a minor constituent ofexuded mucus) were used for the generation of the mucosal RNA-Seqlibrary, the preparation, sequencing, and bioinformatics analyses of theselibraries were identical for all three environments. In support of this, it hasbeen demonstrated that interlaboratory variation in transcriptome pro-filing due to technical variation is minimal, given proper standardizationas stated above (34).

The cDNA reads from all three libraries were assembled de novo intocontiguous sequences (contigs) using Trinity (35), with those specific toA. veronii identified by mapping to the A. veronii strain HM21 genome(36) using blastn (37). Bowtie 2 (38), using default parameters, and cus-tom perl scripts (Niel Infante, personal communication) were utilized todetermine the number of reads corresponding to each contig. The func-tional annotation of contigs was accomplished using the NCBI nonredun-dant (nr) database. The resulting BLAST files were organized and visual-ized using MEGAN5 (39), which incorporates the Kyoto Encyclopedia ofGenes and Genomes (KEGG) and SEED databases. To enable analysis of

differential gene expression (31) and to minimize systematic bias (40),gene expression values (EV), which normalize transcripts based on genelength (41), were determined using the following formula: number ofreads mapped/[length of gene � (total number of reads/1,000,000)],where gene length is in kb.

Gene ontology (GO) terms for each A. veronii-specific contig weredetermined using Blast2GO, which was followed by enrichment analysisusing Fisher’s exact test (42) with a false-discovery rate (FDR) correction.Differences in EV between KEGG functional categories (i.e., cellular pro-cesses [CP], environmental information processing [EIP], genetic infor-mation processing [GIP], and metabolism [Met]) within each environ-ment (i.e., mucus, gut, or monoculture) were also analyzed by firsttransforming values to log(EV � 1) and testing for statistical significanceusing a one-way analysis of variance (ANOVA) with SAS JMP (version 10)(43). Graphs were built using JMP Graph Builder. Fold change was deter-mined as the ratio of EV within the mucosal environment to the value witheither the culture or gut. Significance was based on a P value of �0.001,with a Bonferroni correction for multiple comparisons.

Lectin staining and immunocytochemistry. Lectin staining was usedto visualize the distribution of N-acetylglucosamine (NAG), predicted tobe the major mucopolysaccharide component of leech mucus (23), andmannose, which was not predicted to be present as mannose is not acomponent of mucopolysaccharides, and was thus used as a specificitycontrol. A GFP-expressing Aeromonas strain (HM21S::Tn7gfp; J. Graf lab,University of Connecticut) was orally administered to leeches to replicatea natural shedding scenario in the mucosal secretions (22). Subsequently,GFP immunocytochemistry was used to visualize Aeromonas localizationwithin the mucus. Mucus castings (i.e., secreted host mucus) were placedin a 24-well culture plate and fixed using 4% paraformaldehyde, followedby dehydration using an ethanol series (i.e., incubation in 100% ethanolfor 5 min three times, followed by a 30-min evaporation step). To ensurelectin binding specificity, a subset of mucosal casts were inoculated withmannose in vitro. Slides were then blocked in blocking solution (1% bo-vine serum albumen [BSA]– 0.3% Triton X-100 in phosphate-bufferedsaline [PBS]) for 1 h, followed by an overnight incubation at room tem-perature (RT) with 20 �g/ml of rhodamine-labeled succinylated wheatgerm agglutinin (WGA) (RL-1022S; Vector Laboratories, Burlingame,CA) and fluorescein-labeled concanavalin A (mannose, FL-1001; VectorLaboratories, Burlingame, CA) in blocking solution. Slides were washedin a PBS buffer solution series, blocked for 1 h (see above), followed byanother overnight incubation at RT with a 1:1,000 dilution of rabbit anti-GFP primary antibody (A11122; Life Technologies) in PBSAT (1% so-dium azide, 2% BSA). Slides were subsequently washed in a PBS buffersolution series and blocked again for 1 h, which was followed by anotherovernight incubation at RT using a 1:1,000 dilution of goat anti-rabbit 633secondary antibody (A21070; Life Technologies) in PBSAT. Followingovernight incubation, slides were washed in a PBS buffer solution series,followed by washes in 40%, 60%, and 80% glycerol. Samples were thenmounted using a hard-set mounting medium containing 4=,6=-di-amidino-2-phenylindole (DAPI) (H-1500; Vector Laboratories, Burlin-game, CA). A no-lectin sample was used as a negative control. Mucosalcasts supplemented with mannose exhibited a high signal in the targetedwavelength, while those without mannose showed little to no signal (datanot shown), indicating that the lectin-based dyes do not bind mucus non-specifically. Slides were visualized using an Olympus Fluoview FV1000confocal microscope equipped with argon and green HeNe lasers andappropriate filters and an Olympus AX70 fluorescence microscope. Im-ages were examined and adjusted to appropriate contrast and brightnessusing Olympus Fluoview software.

Reverse transcription-PCR (RT-PCR) of NAG-related genes undervarious NAG concentrations. As the mucosal environment was hypoth-esized to consist of N-acetylglucosamine (NAG) (23), we further exam-ined whether the RNA-Seq library contained loci involved in the break-down of this carbohydrate. While our transcriptome revealed theexpression of a complete pathway for both the import and metabolism of

TABLE 2 Next-generation sequencing and de novo assembly statistics

Parameter Value for the parameter

Illumina MiSeq data (raw)No. of sequenced bases (Gbp) 1.064No. of paired end reads 9,356,767Avg length of read (bp) 208 � 40

Assembly data (mucus metatranscriptome)No. of reads assembled 8,140,387Total transcriptome size (bp) 77,274,024No. of contigs generated 128,964Avg contig size (bp) 599 � 755N25 contig length (bp) 1,893N50 contig length (bp) 797N75 contig length (bp) 382

Aeromonas-specific data by environmentMucus

No. of reads assembled 301,818Total transcriptome size (bp) 848,739No. of contigs assembled 626Avg contig size (bp) 1,355 � 54

Culturea

No. of reads assembled 918,475Total transcriptome size (bp) 402,746No. of contigs assembled 960Avg contig size (bp) 420 � 17

Guta

No. of reads assembled 4,898,329Total transcriptome size (bp) 81,208No. of contigs assembled 200Avg contig size (bp) 406 � 23

a Raw data obtained from Bomar and Graf (31).

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NAG (KEGG, map00520 and map00550), we chose to validate A. veroniiexpression of this pathway by further examining three loci (i.e., glmM[phosphoglucosamine mutase], an intermediate gene involved in theaminosugar metabolism pathway for NAG processing; murA [UDP-N-acetylglucosamine 1-carboxyvinyltransferase] and murB [UDP-N-acetyl-murate dehydrogenase], the first and second loci involved in thepeptidoglycan synthesis pathway, respectively) under various NAG con-centrations. Primers were generated and verified for species specificityusing Primer BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/)and through PCR under the settings listed in Table 1.

An artificial in vitro culture assay was used as a model for analyzing theexpression within NAG-enriched environments, such as leech mucus. AsLB broth contains N-acetylglucosamine, A. veronii was cultured in mini-mal medium made with M9 salts (44) in which the sole carbon source wasglucose. Overnight cultures were generated with the GFP-expressing A.veronii strain HM21S::Tn7gfp with streptomycin (Str; 100 �g/ml) andkanamycin (Km; 100 �g/ml) and grown at 30°C. To ensure that livingcells were not cannibalizing dead cell debris and salvaging the NAG rem-nants for incorporation into peptidoglycan, live cells were selected using aBD FACSAria (BD Biosciences, San Jose CA) at the WVU Flow CytometryCore Facility. First, overnight cultures were incubated with propidiumiodide, which stains only dead cells, and then cells with GFP expressionand no propidium iodide staining (representing live cells) were separated.These aliquots were run a second time through the flow cytometer toensure that samples were clear of dead cell debris. Subsequently, culturesconsisting of �5,000,000 live cells were allowed to recover and grow at30°C, with shaking at 225 rpm, in minimal medium supplemented withStr and Km and containing no NAG (S-9002; Vector Laboratories, Bur-lingame, CA), a low concentration of NAG (90 nmol/ml), or a high con-centration of NAG (360 nmol/ml) (45) for 42 h, which signified entry intomid-log phase, as verified through readings of the optical density at 600nm (OD600). Cells were then centrifuged at 4,000 � g for 5 min at 4°C andresuspended in RNAlater (Life Technologies, Carlsbad, CA) and placed at�20°C until further processing.

RNA was isolated using the TRIzol protocol (Invitrogen, Carlsbad,CA) and verified free of DNA contamination through PCR amplificationwith an RNA-only template. First-strand cDNA synthesis was performedwith 500 ng of RNA, a 2 mM primer cocktail of AVgfp199-R, AVglmM-R,AVMmurA-R, and AVmurB-R (Table 1), and Superscript II reverse trans-criptase (Life Technologies). Second-strand synthesis was then performedwith 2 �l of cDNA template and complementary 5= end gene primers withthe settings described in Table 1. Amplicons were then analyzed by aga-rose gel electrophoresis and visualized with Kodak one-dimensional im-age analysis software. The expression level of A. veronii gfp199 was used asa loading control.

Nucleotide sequence accession number. Data from this study weredeposited in the NCBI Sequence Read Archive under accession numberSRP067591.

RESULTSA. veronii undergoes transcriptional tuning within differentlifestyles. To determine changes in A. veronii activity within leechmucosal secretions compared with activity in the leech crop andmonoculture environments, we analyzed an RNA-Seq library of apooled 3-day-old mucus sample (n � 3). This time point corre-sponds with a peak in A. veronii density within mucus followinghost shedding (22). In total, approximately 9.3 million paired-endreads were generated, with the average length of each read being208 � 40 bp (Table 2). Overall read quality was high for the sampleset, based on FASTQC analysis. Using the Trinity software pack-age (35), approximately 8 million reads were assembled de novointo 128,964 contigs. Single-end read A. veronii libraries generatedwithin the crop and culture environments (31) were processed

using the same pipeline and used for comparative analyses withthe mucus sequences.

A total of 626 contigs (constituting 301,818 reads), averaging1,355 � 54 bp, matched A. veronii sequences within the mucus.Similarly, 200 contigs generated for the leech crop and 960 contigsfor culture were mapped as stated above, with average sizes of406 � 23 bp and 420 � 17 bp, respectively. To validate that ourassembly matched previously reported results (31) regarding gutand monoculture environments, we searched for and found allgenes reported in the study referenced, indicating consistencyacross our analyses. Further, the expression values (EV) of canon-ical housekeeping loci (e.g., rpoD, rpoB, and infA) were similaracross the three libraries, further validating RNA-Seq normaliza-tion and the lack of systematic bias (see Fig. 2E).

If a gene was transcribed in multiple environments, it tended tobe more highly expressed in the mucosal environment (Fig. 1Aand B). Specifically, 70 and 18 loci were more highly expressedwithin exuded mucus than in the monoculture and gut, respec-tively. In comparison, 18 and 3 loci were more highly expressedwithin culture (Fig. 1A) and gut (Fig. 1B), respectively, than inmucus. All A. veronii-specific contigs were then analyzed usingBlast2GO (42), which determined functional classification andassigned gene ontology (GO) terms to each contig. All A. veroniiexpressed genes that were enriched in at least one environment fellinto 1 of 31 general GO terms (Fig. 1C). We focused on thosesignificantly enriched within the mucosal environment (i.e., 12out 31) (Fig. 1C, indicated with red boxes). While many GO termsdescribe general biological processes (e.g., cellular activity, cellbinding, and transcription), an enrichment of reads correspond-ing to genes involved in oxidoreductase activity, iron/sulfur bind-ing, hydrolase activity, heat shock response, amino acid metabo-lism and biosynthesis, and membrane transport is indicative of theenvironmental resources and pressures encountered by A. veroniiwithin mucus.

Mucosal secretions represent a stressful environment. To en-sure evolutionary persistence, microbes must be able to adapt toenvironmental changes. As a previous study compared differencesin A. veronii gene expression levels between the leech host cropand within monoculture (31), we used these data sets to identifyloci that may be necessary for the transition and survival of A.veronii during horizontal transmission, specifically, the shiftingfrom localization in the host digestive tract to exuded mucus (22).To elucidate the impact of lifestyle shifts on A. veronii transcrip-tional activity, the EV of transcribed genes within all three envi-ronments were determined. The A. veronii genes that were solelyexpressed within a given environment (for gut, 53; culture, 300;mucus, 105) were identified and categorized based on KEGG class(Fig. 2A). First, using a one-sample test for a proportion, we de-termined that in the culture environment there was a higher like-lihood of finding a uniquely expressed gene in the 1:10 EV binthan in any other bin (P 0.001), a finding primarily driven by ahigh diversity of genes at low EV within monoculture. Using Pear-son’s chi-square test for independence, we also determined thatthe bin distribution of uniquely expressed genes depends on theenvironment (P 0.001). Additionally, the mean EV was signif-icantly (P 0.001) higher in the gut than in mucus or culture foruniquely expressed metabolism-related genes, with the value inmucus also being higher than that in culture. For environmentalinformation processing (EIP), the mean expression level ofuniquely expressed loci was also significantly (P 0.001) higher in

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the gut, with values in mucus and culture not differing from eachother. Analyses regarding the genetic information processing(GIP) and other (P 0.001) categories followed a pattern similarto that of EIP. However, no uniquely expressed A. veronii lociinvolved in cellular processes (CP) were identified within the gutenvironment. As such, Student’s t test determined that the meanexpression of CP-related genes that were uniquely expressed wassignificantly (P 0.001) higher in the mucus than in the culture.Finally, when we examined if there were significant differences inmean EV values between KEGG classes for uniquely expressedgenes within specific environments, we observed no significantdifferences. Interestingly, higher mean expression levels of CP-

related genes within the mucosal environment relative to those ofEIP, Met, and the other classes were primarily driven by a singlegene, catalase (katE) (Fig. 2E), although this lacked statistical sig-nificance (one-way ANOVA, P � 0.0170).

While identifying genes exclusively expressed within each en-vironment elucidates what processes may be unique to that habi-tat, this alone does not provide a comprehensive understanding ofthe lifestyle shift experienced by A. veronii. Therefore, we contin-ued with analyses that incorporated all genes expressed withineach of the three environments. As we were mainly interested ingenes within the four main KEGG classes, we compared the EVwithin these classes to see which groups contained the highest

FIG 1 A. veronii expression based on RNA-Seq analyses. (A) Relative expression for each shared contig, based on expression value (EV), within mucus andculture environments. (B) Relative expression for each shared contig, based on EV, comparing the mucus and gut environments. In panels A and B greenindicates contigs with higher expression in the mucus, while red indicates contigs with higher expression in the compared environment (i.e., culture for panel A;gut for panel B). (C) Gene ontology (GO) terms differentially expressed as determined by enrichment analysis using Fisher’s exact test. GO terms boxed in redrepresent categories that are significantly overexpressed in the mucus compared with expression in other environments (Fisher’s exact test, P 0.0031).

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expression levels within given environments. In every environ-ment, GIP contained the highest levels of expression (Fig. 2C).The majority of GIP genes overexpressed (i.e., genes that had anEV of 1,000) within the culture and gut environments were as-sociated with housekeeping functions such as transcription andtranslation (e.g., large-subunit ribosomal proteins and RNA poly-merases). In contrast, a number of GIP genes associated withstress responses were overexpressed within mucus (Fig. 2D andTable 3). Additionally, an overexpressed stress-related gene, glu-tathione peroxidase (Fig. 2E and Table 3), binning to metabolism,was also identified within mucus. Interestingly, upregulation ofglutathione peroxidase indicates that A. veronii is challenged byoxidative stress in the mucus as this enzyme uses lipids to reducefree H2O2 into H2O (46). In support of the oxidative stress en-countered by A. veronii within mucus, cysteine desulfurase (Fig.2E) was also overexpressed. Cysteine desulfurase catalyzes the for-mation of sulfur and alanine from cysteine (47). The sulfur fromthis reaction can then form Fe-S clusters (47), which are fre-quently used in oxidative stress response.

While glutathione peroxidase and cysteine desulfurase are as-sociated with oxidative stress, genes associated with other forms ofstress, including temperature and osmotic stress, were also over-expressed. For example, the ATP-dependent helicases DeaD andRhlE (Fig. 2D and E) are known as DEAD box RNA helicases andare involved in various cell processes at lower temperatures, suchas ribosome biogenesis (48), RNA degradation (48), and transla-tion initiation (reviewed in reference 49). Additionally, the chap-erone DnaK assists cells with protein folding after a heat shockevent (50), indicating that this is associated with thermotolerance,while the chaperone GroEL is useful for the cell to adapt to os-motic stressors and protein misfolds (50).

Beyond managing the multiple levels of stress, A. veronii mustalso be able to use nutrients within the host-secreted mucus forsurvival and proliferation. The overexpression of the isocitratelyase, aceA (Fig. 2E), suggests the presence of glycans in the mu-

cosal environment (51). Isocitrate lyase is a key enzyme in theconversion of isocitrate to succinate, a critical component of thetricarboxylic acid (TCA) cycle, and glyoxylate, a substrate in-volved in numerous metabolic pathways including the produc-tion of malate. The expression of malate dehydrogenase (mdh)within mucus confirms the presence of malate and its metabolisminto oxaloacetate, the latter of which can feed back into the TCAcycle or a number of other pathways. Interestingly, isocitrate lyaseis also active within the leech gut (51), which not only indicates asimilarity in available nutrients but also reveals a crossover of A.veronii genetic function and some redundancy between the gutand mucosal environments.

Mucus consists of N-acetylglucosamine that may enable A.veronii proliferation. In addition to investigating activity relatedto lifestyle adaptation, we also wanted to determine what nutrientsources may enable A. veronii proliferation within the stressfulenvironment of mucus. As shed mucus is thought to consist ofglucosaminoglycans (23), we searched for genes involved in themetabolism of this substance within the A. veronii mucosal RNA-Seq library. Although mining for genes related to the degradationof extracellular NAG polymers did not result in identification ofsuch enzymes (e.g., chitinases), the A. veronii transcriptome didcontain reads corresponding to loci involved in both the import ofextracellular N-acetylglucosamine (NAG; a major component ofglycosaminoglycans) (Fig. 3A) and processing of NAG toward thegeneration of N-acetylmuramic acid (MurNAc). NAG and Mur-NAc are significant components of the glycan backbone of pepti-doglycan (i.e., the bacterial cell wall), which is necessary for cellu-lar proliferation. As such, we hypothesized that A. veroniiscavenges NAG for a nutrient source from the surrounding mu-cosal environment for use in peptidoglycan synthesis.

In order for A. veronii to scavenge environmental NAG, we firstconfirmed that mucus contains this monosaccharide. Mucosal se-cretions were examined for the presence of NAG using a succiny-lated wheat germ agglutinin (WGA-S) probe (52) (Fig. 3B). This

FIG 2 Comparison of the A. veronii transcriptome within three habitats. (A) Proportion of A. veronii genes expressed exclusively within exuded mucus (M),gut (G), or monoculture (C). Genes are categorized based on expression value (EV) in 10-fold increments as well as KEGG class. The “other” category containsgenes that are not classified by KEGG into the four main functional classes. (B) A. veronii genes, binned into KEGG functional categories, expressed onlywithin mucosal casts. Circle diameter corresponds to relative transcript abundance. (C) A one-way ANOVA was performed to test for significantdifferences in mean EV among categories containing all expressed genes [plotted on a log(EV � 1) scale] within each environment. Letters indicatestatistically significant differences in expression levels between KEGG categories. (D) Mucosal secretions represent a stressful environment. Genes overexpressedwithin mucus (EV of 1,000) indicate the presence of oxidative, temperature, and osmotic stressors. (E) Histogram showing the distribution of log(EV � 1) for everygene captured within the RNA-Seq libraries. Gene position is based on alphabetical order. Sunbursts are identified as follows: red, ATP-dependent RNA helicasedeaD transcript; blue, ATP-dependent RNA helicase rhlE; green, molecular chaperone dnaK; brown, the chaperonin groEL; orange, cysteine desulfurase (CYD);yellow, catalase katE; and purple, glutathione peroxidase (GLP). The Xs are identified as follows: black, isocitrate lyase; red, rpoB; blue, infA; and purple, rpoD.The colors of the individual histogram bars correspond to KEGG functional categories, as indicated on the x axis.

TABLE 3 Stress-related genes overexpressed in the mucus

Function KEGG class KEGG accession no.

Expression value in:Fold change relativetoa:

Mucus Gut Culture Gut Culture

Molecular chaperone DnaK GIP K04043 5,365.789,863 0 254.5522701 � 21.07932434Chaperonin GroEL GIP K04077 3,153.165,011 0 119.1977161 � 26.45323344ATP-dependent RNA helicase DeaD GIP K05592 1,055.642,763 0 175.7359135 � 6.006983672ATP-dependent RNA helicase RhlE GIP K11927 9,360.359,259 0 31.08526087 � 301.1188904Glutathione peroxidase Met K00432 1,379.377,862 0 0 � �Cysteine desulfurase Met, GIP K04487 5,039.419,963 0 185.4926594 � 27.16775951a Expression in mucus relative to gut or culture.

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staining demonstrates that mucus consists of an abundantamount of NAG, and it can be used to visualize the structure ofexuded mucus (Fig. 3B, frame i). To determine that A. veronii iswithin the vicinity of NAG for import, a GFP-expressing A. veroniistrain was orally administered to leeches to ensure natural shed-ding into the mucus (22), and immunocytochemistry was used tovisualize the GFP. Bacterial rods can be seen throughout the mu-cus (Fig. 3B, frame ii) with small clusters of GFP-expressing fluo-rescent rods (Fig. 3B, frame iii; see also Movie S1 in the supple-mental material), consistent with the low density of A. veroniiwithin the mucosal microbiota (24).

To determine if A. veronii will alter its transcriptional profile inresponse to nutrient availability, key genes involved in the import

and processing of NAG (Fig. 3A) were examined using semiquan-titative RT-PCR within an in vitro culture system as a model forenvironments containing various concentrations of NAG. Tocontrol for glucose as the sole carbon source, A. veronii cultureswere grown in minimal medium (44), and to reduce the likelihoodthat A. veronii was acquiring NAG from cell wall debris by canni-balizing dead cells, cultures underwent flow cytometry for theisolation of live cells (Fig. 3C, P columns). The recovery mediumwas then supplemented with low or high levels of NAG. An addi-tional set of cultures was supplemented with similar NAG concen-trations; however, these cultures did not undergo flow cytometry(Fig. 3C, N columns).

Figure 3C shows that glmM, murA, and murB had increased ex-

FIG 3 Mucus consists of N-acetylglucosamine that may enable A. veronii proliferation. (A) Metabolic pathway depicting the influx of NAG and its conversion toN-acetylmuramic acid, both of which compose the glycan portion of peptidoglycan. Loci depicted in red were chosen for expression analyses (panels C and D).(B) Lectin staining and immunocytochemistry images of mucosal secretions. Mucosal secretions were stained using WGA-S (60� oil objective) (frame i). Theboxed area indicates a cluster of GFP-expressing A. veronii cells. Frame ii shows only WGA-S staining within the boxed area of frame i while frame iii depicts onlythe immunostaining of GFP protein. Arrowheads indicate examples of bacterial cells (frame ii) with their corresponding GFP signal (frame iii). Scale bar, 10 �m.(C) RT-PCR gel electrophoresis images showing the expression of key NAG-related genes in samples that did not undergo flow cytometry (N) and in samplespost-flow cytometry (P). Cultures were grown in three environments: control (no NAG), low NAG, or high NAG, as indicated. (D) The full names of the genesshown in panel A and analyzed in panel C.

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pression levels when cultures were incubated with increasing concen-trations of NAG. Interestingly, control samples (i.e., no NAG presentin the medium) that were not subjected to flow cytometry to removedead cell debris had a high level of expression for all genes examinedrelative to levels in their flow cytometry counterparts (Fig. 3C). Thus,A. veronii can use both the surrounding mucosal environment andthe cellular remnants of other microbial community members as asource of NAG. These results indicate that in a NAG-enrichedenvironment, these genes are turned on to process NAG into Mur-NAc, likely for peptidoglycan synthesis.

DISCUSSION

In this study, we identify the transcriptional responses that enablethe transition between different microbial lifestyles, in this casefrom a mutualistic to a free-living state. Additionally, we demon-strate that the A. veronii symbiont is capable of not only survivingin a stressful environment but also using host exuded mucus andthe surrounding microbiota for nutritional resources, which iscrucial for proliferation.

The leech mucosal transcriptome consists of 129,964 contigs, 626of which were identified as A. veronii specific. The low proportionof A. veronii transcripts within the total transcriptome (i.e., �1%of total size) corresponds with the relatively low abundance of A.veronii within shed mucus, as characterized by previous Illuminadeep sequencing of the microbiota composition (24). Compari-sons with previous studies of A. veronii in other distinct habitats,specifically the leech digestive tract and monoculture (31), fur-thered our understanding of how this microbe adapts to a majorlifestyle shift, i.e., from a digestive tract symbiont to being shedinto exuded mucus. Bacteria cope with environment shifts viaseveral mechanisms, including lateral gene transfer (LGT) andelevated mutation rates coupled with high effective populationsizes (53, 54); however, less is known concerning adaptive tran-scriptional tuning. Our results demonstrate that as symbiontsshift between lifestyles, they undergo widespread alterations ingene expression, here termed transcriptional tuning (reviewed inreference 29).

The expression of loci either unique to A. veronii living in themucus or shared with the gut and/or monoculture is indicative ofthe activities and conditions within each environment. The ma-jority of genes found to be expressed in all three environments(Fig. 1A and B) are most highly expressed within the mucosalenvironment. This indicates that while certain functions are re-dundant within the culture and gut, these roles are amplifiedwithin mucus, likely due to alterations in nutrient resources andincreased competition from the diverse surrounding microbiota(24). Interestingly, A. veronii in culture express a higher quantityof unique genes at lower EV. While this may be due to the presenceof an environment rich in precursors, it may also suggest that A.veronii, under low stress (i.e., rich monoculture) and residing inan ideal environment, can afford to exert energy on auxiliary geneproducts, such as adenylosuccinate synthase, haloacid dehydroge-nase, and chorismate synthase. In contrast, these products may beavailable to mucosal-bound A. veronii from the surrounding mi-crobial community in a form of nutrient cross-feeding (reviewedin reference 55) or to gut-bound A. veronii from the host (51) orthe secondary leech gut symbiont, M. hirudinis (14, 51).

Conversely, while genes uniquely expressed within the gut en-vironment are low in number, the EV of these genes tend to bemuch larger than those of genes expressed in the other environ-

ments (Fig. 2A, 1,000 to 10,000 EV bin). While the majority ofthese genes are involved in housekeeping functions (e.g., ribo-somal proteins), genes related to phage shock were also found tobe highly expressed, confirming results found in the original guttranscriptome study (31). Phage shock genes are important to thegut environment as a response to osmotic stress, high tempera-tures, stationary growth phase, and filamentous phage infection(56). As phage shock genes were not expressed within the mucosalenvironment, we can conclude that A. veronii does not requiresuch a response and likely does not encounter some of these stres-sors or that deeper sequencing is necessary within the mucosalenvironment to identify such loci.

While residing in mucus, A. veronii does not spend energy on anumber of costly syntheses, as in culture, or use phage shock geneproducts to respond to its surrounding environments, as withinthe leech gut. Instead, A. veronii shifts its energy balance in favor ofmembrane transport (Fig. 1C), likely indicating that the sur-rounding microbial community and/or composition of the mu-cus provides necessary metabolites. Additionally, a significant in-crease in expression related to cellular processes, mainly driven bycatalase activity, as well as the enrichment of genes involved iniron/sulfur binding and responses to temperature fluctuations,denotes a stressful environment. Generally, microbes respond tostress either by acclimating (e.g., transferring limited resourcesfrom growth to survival pathways [57]), becoming dormant (re-viewed in reference 58), or dying. In mucus-bound A. veronii, anumber (30%) of overexpressed genes (i.e., with a correspondingEV of 1,000) are related to stress response, indicating that A.veronii adapts to the environmental transition by shifting re-sources, at least partly, to survival pathways.

While mucosal secretions of aquatic animals are known to har-bor high levels of antimicrobial peptides (32, 33), the overexpres-sion of genes related to the processing of these compounds was notobserved in the A. veronii mucosal transcriptome. However, fluc-tuating temperature may be a stressful condition encountered byA. veronii during transition from the host to the mucus. A numberof genes enriched and overexpressed in the mucosal environmentwere related to heat shock, such as dnaK (50), as well as functionswithin colder environments, such as deaD and rhlE (48, 59; re-viewed in reference 49). This indicates that A. veronii is likelyexperiencing fluctuations in temperature not observed in thegut or monoculture environments, which is a reasonable con-clusion as the monoculture was maintained at a steady 30°C(31). Interestingly, this also indicates that the leech gut envi-ronment is more thermally stable when the host is maintainedat room temperature (31) than free-floating mucus maintainedat 15°C (reflective of a pond’s base within the geographic rangeof the H. verbana host).

In addition to its involvement in temperature stress, the DnaKchaperone is also known for its involvement in oxidative stress.The presence of the latter stressor is supported by a significant biastoward a single, overexpressed gene, catalase, consistent with ahigh abundance of H2O2 (60). Catalase may protect against theoxidative damage produced by reactive oxygen species (ROS),driving the increased need for chaperones in the mucosal environ-ment. Interestingly, catalase was also observed within the coralmucus metagenome (61) and is known to provide Vibrio fischeri, amutualist of sepiolid squid, protection from ROS within host-secreted mucus (reviewed in reference 62), indicating that muco-sal secretions of other aquatic organisms are also characterized by

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high levels of oxidative stress. Additionally, the enrichment ofgenes related to iron/sulfur-dependent enzymes by A. veronii inleech mucus, supported by the overexpression of cysteine desul-furase, indicates the catabolism of sulfur and alanine from cys-teine (47), followed by the formation of Fe-S clusters (47) whichcan respond to oxidative stress. Finally, the overexpression of glu-tathione peroxidase, which uses lipids to reduce hydrogen perox-ide to water (46), further supports the presence of oxidative stresswithin the mucosal environment.

Additionally, a shift in osmolarity within the mucosal environ-ment, relative to that of the gut or culture, is supported by higherexpression of genes related to osmotic stress (e.g., groEL [50])within mucus. The chaperonin groEL is also well known for itsincreased expression in the obligate symbionts of many inverte-brates, such as aphids (63), tsetse (64), and weevils (65), comparedwith levels in nonobligate symbionts, such as the opportunisticpathogen P. aeruginosa (63). This high expression compensatesfor lower protein stability resulting from an AT bias typically se-lected for in many endosymbionts (66). However, as A. veronii isable to maintain a larger genomic inventory, similar to that of ageneralist, and lacks an AT-biased genome (36), the overexpres-sion of groEL is likely due to a variety of other stressors (e.g.,oxidative and temperature stress) (Fig. 2D) that may impact pro-tein folding in the mucosal environment.

Even within the presence of such stressors, A. veronii is capableof survival and proliferation (22), indicating that there are suffi-cient nutrients within the mucus for growth. While the enrich-ment of amino acid metabolism and biosynthesis genes in themucus supports protein synthesis, we decided to focus on carbonsources in the mucus as heterotrophic bacteria rely on externalsources of organic carbon for growth. The overexpression ofisocitrate lyase aceA transcripts suggests not only a source ofnutrition but also an overlap of metabolic function between thegut and mucus (51). As expression of isocitrate lyase within thegut suggests that A. veronii uses acetate or other fatty acids ascarbon and energy sources (51), it is likely that similar com-pounds are also available in the mucus. Interestingly, the ace-tate and short-chain fatty acids in the gut were generated byMucinivorans hirudinis from host mucin glycans (51). This in-dicates that M. hirudinis, which also survives and proliferateswithin mucus (24), may be playing a parallel role in both envi-ronments. Alternatively, one of the many additional mucosalmicrobiota members may provide this service. In either case,these results indicate a redundancy that exists between the gutand mucosal environments.

While little is known regarding the available nutrients withinmucus, we determined that these castings contain the glucosami-noglycan N-acetylglucosamine (NAG) (Fig. 3B), which is a majorcomponent of peptidoglycan (67). As the WGA-S stain used todetermine the mucosal composition detects both monomers andpolymers of NAG (68, 69), long chains of which are also known aschitin, we could not ascertain which was available for A. veroniiuptake. Within the mucosal environment, the enrichment of hy-drolases, a category that contains some enzymes related to chitinmetabolism (i.e., chitinases), indicates that smaller NAG poly-mers may be freed from chitin. While none of these A. veroniihydrolases were identified as chitinases, further investigation intothe mucosal microbial community metatranscriptome revealedthat major community members, such as Curvibacter (24), doproduce endochitinases (B. M. Ott and R. V. M. Rio, unpub-

lished data). Additionally, the most abundant microbial com-munity member with mucus, Pedobacter (24), expresses an al-pha-N-acetylglucosaminidase (Ott and Rio, unpublished),which cleaves off individual NAG monomers from longer poly-mer chains, indicating the availability of free monosaccharideswithin the environment.

The A. veronii mucosal transcriptome did contain transcriptsencompassing the complete pathway involved in the import ofextracellular NAG. Notably, NAG can be imported into A. veroniithrough the use of a phosphotransferase (PTS) system, the N-acetylglucosamine-specific enzyme (NagE), which transportsmonomers across the cell membrane (reviewed in reference 70).Subsequently, this NAG can be incorporated into the productionof N-acetylmuramic acid (MurNAc). Additionally, A. veroniiforms small clusters within the vicinity of NAG. The small num-bers of fluorescent cells confirm the low relative abundance of A.veronii in the mucosal microbiota (24) yet also highlight the effi-ciency of mucus as a transmission platform (22). We also foundthat A. veronii increases the expression of several peptidoglycansynthesis genes in response to high NAG concentrations, confirm-ing that A. veronii may alter nutritional consumption with chang-ing environmental conditions. When the expression of NAG-re-lated genes within in vitro culture assays was examined, the abilityof A. veronii to utilize the NAG of dead cells, through mechanismssuch as cannibalism or obtaining cellular remnants resulting fromenzymatic activity within the mucus, was also observed. This isinteresting as in other NAG-limited environments, A. veronii maybe capable of scavenging other dead community members to pro-mote its proliferation.

In conclusion, these RNA-Seq analyses indicate that trans-mission substrates, such as mucosal castings which underlie thetransmission of many host-associated microbiotas (71, 72; re-viewed in reference 73), expose symbionts to a variety of novelstressors that impact the evolution of the microbiota. Mostorganisms experience different selective pressures during theirlife spans, with horizontally transmitted microbes, in particu-lar, experiencing dramatic shifts in their exposure to a widevariety of biotic and abiotic factors. In particular, stress-relatedgenes are upregulated for the shift from a sheltered existencewithin a host to an environmentally exposed transmission sub-strate, addressing ecological pressures while permitting the ac-quisition of nutrients. These results provide insight into howmucosally derived bacterial symbionts of other aquatic ani-mals, such as corals and the Hawaiian bobtail squid, may copewith the stress involved in horizontal transmission, notably thefluctuating conditions associated with environmental shifts.Our results emphasize the significance of lifestyle shifts withintransmission settings toward shaping microbial evolution.

ACKNOWLEDGMENTS

We thank the Graf lab at the University of Connecticut for providing theGFP-expressing A. veronii strain, Niel Infante of the WVU Genomics Corefor consultation regarding bioinformatic techniques and providing perlscripts, Kristyn Lizbinski for assistance with confocal microscopy, Ste-phen DiFazio for comments on the manuscript, and Kathleen Brundage atthe Flow Cytometry and Single Cell Core Facility.

We gratefully acknowledge funding support from WV Research Cor-poration PSCoR and NSF IOS-1025274 (R.V.M.R.).

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FUNDING INFORMATIONThis work, including the efforts of Rita Vitorino Moreira Rio, was fundedby WV Research Corporation (PSCoR). This work, including the effortsof Rita Vitorino Moreira Rio, was funded by National Science Foundation(NSF) (IOS-1025274).

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