Artificial Seawater Media FacilitateCultivating Members of the MicrobialMajority from the Gulf of Mexico

Michael W. Henson, David M. Pitre, Jessica Lee Weckhorst, V. Celeste Lanclos,Austen T. Webber, J. Cameron ThrashDepartment of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana, USA

ABSTRACT High-throughput cultivation studies have been successful at bringingnumerous important marine bacterioplankton lineages into culture, yet these fre-quently utilize natural seawater media that can hamper portability, reproducibility,and downstream characterization efforts. Here we report the results of seven experi-ments with a set of newly developed artificial seawater media and evaluation of cul-tivation success via comparison with community sequencing data from the inocula.Eighty-two new isolates represent highly important marine clades, including SAR116,OM60/NOR5, SAR92, Roseobacter, and SAR11. For many, isolation with an artificialseawater medium is unprecedented, and several organisms are also the first of theirtype from the Gulf of Mexico. Community analysis revealed that many isolates wereamong the 20 most abundant organisms in their source inoculum. This method willexpand the accessibility of bacterioplankton cultivation experiments and improve re-peatability by avoiding normal compositional changes in natural seawater.

IMPORTANCE The difficulty in cultivating many microbial taxa vexes researchersintent on understanding the contributions of these organisms to natural systems,particularly when these organisms are numerically abundant, and many cultivationattempts recover only rare taxa. Efforts to improve this conundrum with marine bac-terioplankton have been successful with natural seawater media, but that approachsuffers from a number of drawbacks and there have been no comparable artificialalternatives created in the laboratory. This work demonstrates that a newly devel-oped suite of artificial seawater media can successfully cultivate many of the mostabundant taxa from seawater samples and many taxa previously only cultivated withnatural seawater media. This methodology therefore significantly simplifies efforts tocultivate bacterioplankton and greatly improves our ability to perform physiologicalcharacterization of cultures postisolation.

KEYWORDS: Gulf of Mexico, SAR11, artificial seawater, coastal microbiology, high-throughput culturing, marine microbiology

The study of microorganisms and their roles in remediation and biogeochemicalcycling requires the observation of microbial communities and genetics in nature

coupled with experimental testing of hypotheses both in situ and in laboratory settings.The latter is best accomplished by cultivation of microorganisms, yet the majority of themicroorganisms observed under a microscope are not readily cultivated (1–3). High-throughput, dilution-to-extinction culturing (HTC) with natural seawater has beenresponsible for the isolation of strains representing numerically abundant clades suchas SAR11 (4, 5), SUP05/Arctic96BD-19 (6), OM43 (7), a small-genome Roseobacter strain(8), and others. HTC benefitted from the oligotrophy theory and the research of DonButton and colleagues (9, 10), which demonstrated that because marine systems arefrequently in a state of nutrient limitation, adaptation by the microbial denizens has led

Received 1 February 2016 Accepted 3 April2016 Published 27 April 2016

Citation Henson MW, Pitre DM, Weckhorst JL,Lanclos VC, Webber AT, Thrash JC. 2016.Artificial seawater media facilitate cultivatingmembers of the microbial majority from theGulf of Mexico. mSphere 1(2):e00028-16.doi:10.1128/mSphere.00028-16.

Editor Steven J. Hallam, University of BritishColumbia

Copyright © 2016 Henson et al. This is anopen-access article distributed under the termsof the Creative Commons Attribution 4.0International license.

Address correspondence to J. Cameron Thrash,thrashc@lsu.edu.

Cultivating the microbial majority with@DrJCthrash @HensonMW_08 #LSUResearch

RESOURCE REPORTApplied and Environmental Science

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to unusual genomics and physiology that must be accounted for in cultivation strat-egies. These oligotrophic microorganisms are generally small, with streamlined ge-nomes that eschew many complicated regulatory systems (11), resulting in slow growthand complicated nutrient requirements, including poor or no growth at high carbonconcentrations (4, 7, 9, 10, 12). Thus, successful HTC experiments have provided anoncompetitive growth environment with naturally occurring compounds at in situconcentrations.

Until now, this has been accomplished predominantly via filtration and autoclavingof natural seawater by using protocols (9, 10, 13) that avoided excessive nutrients foundin “traditional” artificial marine media (14) (see Table 3) and provided complex, butlargely unknown, natural dissolved organic matter (DOM) components. However, inspite of its success, there are many drawbacks to using a natural seawater medium.First, it requires regular access to large volumes (20 liters or more) of seawater, whichcan be a major logistical hurdle for research labs not located near a coastal sourceand/or without vessel access. Second, natural seawater is inherently complex andundefined, thus limiting the amount of physiological characterization that can beaccomplished postisolation (e.g., salinity growth optima or single-carbon substrateutilization). Third, seawater at a given location may experience significant intra- andinterannual chemical fluxes, thereby creating “vintages” from specific sample collec-tions that can prevent reproducible growth or repeated transfers when attempting topassage an organism from one vintage to another. We therefore developed a complexyet defined low-nutrient set of artificial seawater media (ASM) incorporating the theoryof Button and colleagues and published measurements for marine systems, includingthe Gulf of Mexico, that account for variable salinity in coastal ecosystems (12, 15–18)(see Table S1 in the supplemental material; all supplemental material for this article maybe found at https://thethrashlab.com/publications/).

RESULTS AND DISCUSSION

To create the JW1 medium (Table 1; see Table 3), the basic salt mixture was derived byusing values in the medium designed by Kester et al. (19). Concentrations of phosphate,iron, and trace metals were taken from a defined medium for SAR11 (12), with modifiedvitamin concentrations based on data in references 20 and 21. DOM is one of the mostimportant yet least understood components of natural seawater, having varied carbonand nitrogen constituents that can be difficult to distinguish (22). A complex butdefined list of carbon and nitrogen compounds was used to generate our modularDOM mixtures (see Table S1 in the supplemental material), and the total carbon andnitrogen concentrations were based on previously reported Gulf of Mexico data(16–18). Ultimately, our modular recipe allows for customization of the different carbonand nitrogen components, making it easily adaptable to a variety of environments.

Using the media in Table S1 in the supplemental material, we conducted seven HTCexperiments with a combined total of 3,360 distinct cultivation wells according to theprotocol in reference 15 and included community characterization (via 16S rRNA genetag sequencing) of the source water for each experiment. These experiments usedwater collected from six sites along the southern Louisiana coastline (Fig. 1) thatrepresent varied estuarine-marine systems (23). Our goals in sampling these differentsites were to provide varied source inocula and to simultaneously collect coastalnorthern Gulf of Mexico microbial biogeography data. Initial cultivability (10) rangedfrom 0 to 53.1% (Table 2), and 82 of the 231 positive cultures across all of theexperiments were capable of repeated transfer after initial exchange from polytetra-fluoroethylene (PTFE) to polycarbonate flasks and/or did not contain multiple organ-isms. The 82 isolates represent various marine clades of Gammaproteobacteria, Beta-proteobacteria, Alphaproteobacteria, and Actinobacteria (see Table S1 and Fig. S1 to S3in the supplemental material), including five isolates from the Roseobacter clade, 23isolates from the so-called “oligotrophic marine Gammaproteobacteria” (24), two iso-lates from clade OM43, and most notably the first two reported isolates of SAR11 andSAR116 from the Gulf of Mexico (see Table S1). The maximum observed cell yields of

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cultures grown in the JW1 to JW4 media generally ranged between 105 and106 cells·ml�1, as determined by flow cytometry, with growth rates spanning a widerange (see Fig. S4 in the supplemental material).

Phylogenetic inspection shows that the ASM provided for cultivation success similarto that provided for by natural seawater media, as evidenced by the numerous closerelationships to cultivars obtained with natural seawater media (designated with HTCC,HIMB, and IMCC) (see Fig. S1 to S3 and Table S1 in the supplemental material).LSUCC0245 is only the second isolate from SAR11 subclade Va, and LSUCC0261represents only the third isolate from SAR11 subclade IIIa (25, 26). Two clades werenotable for their phylogenetic novelty in the Gammaproteobacteria—those containingLSUCC0096 and LSUCC0101 (see Fig. S1). The clade containing LSUCC0096 was affili-ated with a sole isolate, HIMB30, of the OM252 clade, and therefore, Louisiana StateUniversity Culture Collection (LSUCC) isolates represent a significant expansion ofcultured representatives of this group. Similarly, LSUCC0101-type organisms matchedbest to an only recently recovered isolate, IMCC14953 (27), and this clade likelyrepresents a novel gammaproteobacterial family on the basis of the depth of branchingand best-BLAST identities of 91% to sister clade members (see Fig. S1).

Comparison of isolate sequences with operational taxonomic units (OTUs) fromwhole-community sequencing of the source water demonstrated that our methodfrequently captured some of the most abundant organisms in the system (Fig. 2 and 3;see Table S1 in the supplemental material). For the most successful experiment,

TABLE 1 JW1 medium components

Component Concn

Basic componentsCl 0.54 MNa 0.48 MMg 52 mMS (SO4

2�) 30 mMCa 10 mMK 10 mMBr 0.8 mMB 0.42 mMSr 0.09 mMF 0.055 mMFe 101 nMP (PO4

3�) 51 �MHCO3

� 10 mMTotal inorganic Na 45 �MTotal organic Nb 23 �MTotal organic Cc 71 �M

Trace metalsMn 9 nMZn 1 nMCo 0.5 nMMo 0.3 nMSe 1 nMNi 1 nM

VitaminsB1/thiamine 500 nMB2/riboflavin 0.7 nMB3/niacin 800 nMB5/pantothenate 425 nMB6/pyridoxine 500 nMB7/biotin 4 nMB9/folic acid 4 nMB12 0.7 nMmyo-Inositol 500 nM4-Aminobenzoic acid 60 nM

aAs NO3�, NO2

�, and NH4�.

bAs urea and amino acids.cAs amino acids, carboxylic acids, sugars, and fatty acids.

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designated CJ2, 11 of the 30 isolates cultivated represented 7 of the top 20 OTUs(Fig. 3). Specifically, LSUCC0245/SAR11 subgroup Va was the 6th ranked OTU, whileLSUCC0247/OM60/NOR5 clade was 7th, LSUCC0225 and 0226/SAR116 were 8th, andLSUCC0227 and 0268/OM43 were 10th (Fig. 3). Similar results were obtained in theother experiments, although success was varied (Fig. 2; see Table S1 in the supple-mental material). Often, if members of the dominant taxa were not recovered in aparticular experiment, a representative of that OTU was recovered in a separateexperiment. For example, the SAR11 subclade Va isolate LSUCC0245, represented byOTU10, was recovered only in the CJ2 experiment (Fig. 3), but it nevertheless repre-sented the 3rd, 6th, 8th, and 8th ranks in CJ, JLB, LB, and Tbon source waters,respectively (Fig. 2A, C, E, and F). In an even more extreme example, cultivation wastotally unsuccessful at LB and ARD, yet isolates from other cultivation attempts repre-sented 5 and 3 of the top 25 OTUs, respectively, in the rank abundance curves fromthose experiments (Fig. 2B and E, respectively). While the SAR11 subclade Va organismand some other isolates were cultivated only once, several other closely related taxawere obtained more frequently. For example, 10 isolates of the OM252 clade, repre-sented by OTU55, were cultured from three different experiments. This OTU showedvarious relative abundances (ranks 20 to 540) but regular occurrence, being observed

TABLE 2 Initial cultivability statistics and salinity values for seven HTC experiments

Site Date

No. of wells:

P X Va

%Cultivability Medium

Mediumsalinity

SourcesalinityInoculated Positive

CJ 9/12/14 460 15 0.033 1.27 0.026 2.6 JWAMPFe 34.8 24.6ARD 11/24/14 460 1 0.002 1.5 0.001 0.1 JW1 34.8 1.7JLB 1/9/15 460 61 0.133 1.96 0.073 7.3 JW1 34.8 26FWCb 3/21/15 460 301 0.654 2 0.531 53.1 JW4 5.8 5.4LB 6/9/15 460 0 0 1.8 0 0 JW4 5.8 1.4Tbon 7/24/15 460 41 0.089 1.56 0.06 6 JW3 11.6 14.2CJ2 10/1/15 460 61 0.133 2 0.071 7.1 JW2 23.2 22.2aAccording to V � �ln(1 � p)/X, where p is the number of positive wells divided by the number of inoculated wells and X is the number cellsinoculated per well (10). Percent cultivability � V � 100.

bSample was counted by microscopy and believed to have underestimated the total number of cells, resulting in a higher percent cultivability.

CJ

FWC ARDTBON

LBJLBCJ2

27

28

29

30

31

32

-94 -92 -90Longitude

Latit

ude

FIG 1 Locations of the seven experiments along the coast of the northern Gulf of Mexico (map datacopyright 2016 Google; imagery copyright 2016 TerraMetrics).

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FIG 2 Positions of LSUCC isolates relative to matching OTUs within the top 60 ranks for the first six experiments. OTUs are ordered by decreasing relativeabundance, according to the average of duplicate samples. Experiments are in the following order according to Table 2: CJ (A), ARD (B), JLB (C), FWC (D), LB (E),and Tbon (F). The LSUCC isolates with the best BLAST hits to the representative sequence for a given OTU are in bold. Stars indicate OTUs with cultured representativesfrom other LSUCC experiments. Isolates with matching OTUs with ranks lower than 60 are detailed in Table S1 in the supplemental material.

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LSUCC0218, 0220, 0222, 0258, 0264, 0270, 0272LSUCC0225,

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LSUCC0228,0248, 0255,

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FIG 3 Positions of LSUCC isolates relative to matching OTUs within the top 60 ranks for the most successful experiment,CJ2, which is enlarged to allow for OTU taxonomy labels. OTUs are ordered by decreasing relative abundance, accordingto the average of duplicate samples. LSUCC isolates with best blast hits to the representative sequence for a given OTUare in bold. Stars indicate OTUs with cultured representatives from other LSUCC experiments. Isolates with matching OTUswith ranks lower than 60 are detailed in Table S1 in the supplemental material.

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in four of the seven experiments within the top 35 ranks (see Table S1 in thesupplemental material). Continued cultivation efforts will provide opportunities forbiogeographic analyses of individual isolates, as well as the efficacy and reproducibilityof the cultivability of organisms from abundant clades.

Why some experiments were more successful than others can only be speculatedupon at this time. There are numerous potential reasons why some organisms have notbeen isolated at all or why some organisms can be cultivated under some circum-stances but not others, even though they are present in the starting inocula. It mayhave to do with a dependence on an unsupplied compound or combination ofcompounds, the specific concentration thresholds of compounds, or the requirementfor a better surface than PTFE. There is also evidence that many taxa are dormant andcan be cultivated after stochastic release from dormancy (28). Additionally, our thresh-old of 104 cells·ml�1 for calling a culture positive may prevent us from obtaining certaintaxa that are always at low abundance or those that have not achieved this thresholdin the allotted time.

Beginning in March 2015, we attempted to match salinity with measured valuesobtained with the JW2 to JW4 media by diluting the basic salts and Mg/Ca components(excluding P) (Table 3). Our motivation for making these adaptations was the evidencethat salinity is one of the most important determinants of microbial communitycomposition, if not the most important one, in general (29, 30) and in estuarineenvironments in particular (31–33). The six sites ranged in salinity from 1.4 to 26 ppt(Table 2). The values at CJ, CJ2, and JLB were the most similar to those of marineenvironments (�22 ppt), while Tbon was brackish (14.6 ppt) and ARD, FWC, and LBnearly qualified as freshwater (�6 ppt) (Table 2). We matched salinity as closely aspossible for the FWC, LB, Tbon, and CJ2 experiments (Table 2), but the impact of theseefforts was inconclusive. Although there may be a trend emerging across all of ourexperiments whereby cultivability is correlated with the similarity between in situ andmedium salinities, as of now, there are too few data points to ascertain a significantpattern.

We attribute the success of the ASM to keeping constituent concentrations close toenvironmentally relevant values and providing a complex suite of carbon and nitrogensources while simultaneously keeping the total organic C level low. However, there arestill many taxa that remained uncultivated. Notably, the most dominant members ofthese communities were Actinobacteria, belonging to either the OM1 clade (i.e., “marine

TABLE 3 Comparison of the compositions of natural seawater, marine broth 2216, andJW artificial seawatera

Element SeawaterMarinebroth 2216 JW1 JW2 JW3 JW4

Chlorine 19 19.8 19.2 12.8 6.4 3.2Sodium 10.5 8.8 11 7.4 3.8 2Magnesium 1.35 2.2 1.27 0.85 0.42 0.211Sulfur 885 0.72 0.96 0.64 0.32 0.16Calcium 0.4 0.65 0.41 0.28 0.14 0.07Potassium 0.38 0.32 0.39 0.26 0.13 0.07Bromine 0.65 0.054 0.063 0.042 0.022 0.01Carbon 0.028 4.78 0.0008b 0.0008b 0.0008b 0.0008b

Boron 0.0046 0.0047 0.0046 0.003 0.0015 0.00075Silicon 0.003 0.00085Fluorine 0.0013 0.00109 0.001 0.0009 0.00046 0.00023Nitrogen 0.0005 0.72 0.0096c 0.0096c 0.0096c 0.0096c

Phosphorus 0.0007 0.045 0.0016 0.0016 0.0015 0.0008Iron 0.00001 0.0226 0.000006 0.000006 0.000006 0.000006aAll values are in grams per liter. The data in the seawater and marine broth 2216 columns are fromreference 14.

bC as mixed amino acids, fatty acids, carboxylic acids, and sugars (17, 18).cN as NO3

�, NO2�, NH4

�, urea, and amino acids (17, 18). Differences between JW media and seawater insome element values are related to the geochemical nature of the Gulf of Mexico compared to the Atlanticsubtropical gyre.

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Actinobacteria” [34]) in marine samples (OTU1; Fig. 2A, C, and F and 3) or the hgcI cladein more freshwater samples (OTU2; Fig. 2B, D, and E). Furthermore, it must be remem-bered that because of incubation in the dark, this method excluded the cultivation ofphototrophs, yet some, like Synechococcus, were among the most abundant organismsin some samples (e.g., OTU4; Fig. 2A). Incubation in the light could prove fruitful for theisolation of phototrophs.

We did not attempt to precisely match the concentrations of inorganic nutrients inour media to those at sampled sites during these experiments, but this could also bea key way to improve cultivability. At 5, 2, and 38 �M, the concentrations of ammo-nium, nitrite, and nitrate, respectively, in our ASM are near the measured values at thesites where we sampled for the cultivation experiments; however, these values fluctu-ated (see Table S1 in the supplemental material). The nitrate concentration at site CJwas measured at 0.6 �M, while it was measured at 85 �M at ARD and Tbon (seeTable S1). Our ASM phosphate concentration (51 �M) was based on previous successwith the cultivation of SAR11 (12); however, this was considerably higher than the insitu levels (1 to 3 �M) (Table 2; see Table S1). Measurements of other constituentconcentrations (e.g., DOM components) could help determine what drives the success-ful cultivation of more of the microbial majority. Additionally, we expect the physio-logical and genomic characterization of our isolates to shed additional light on whysome taxa may have been preferentially isolated rather than others. On the basis of ourobservations, future experiments will include additional alterations to the medium totry to obtain additional key members of the microbial consortia.

Nevertheless, while there is always room for improvement, this is the first reportedisolation of SAR11, SAR116, OM43, HIMB11-type members of the Roseobacter clade, andmany other taxa in ASM, which validates the JW medium design and provides a suiteof isolates from the Gulf of Mexico that should prove valuable for linking genomics andphysiology with biogeography. The modularity of our media also facilitates easycustomization for targeted cultivation approaches informed by genomics, metagenom-ics, or marine chemistry. Furthermore, the success of the ASM recipes in the HTCcontext fulfills the goal of providing a portable, reproducible cultivation strategy thatcan be utilized by researchers constrained in their capacity to obtain seawater andthereby provides another valuable tool for the cultivation of important marine clades.

All of the cultures described here are currently archived and available upon requestas part of the LSUCC.

MATERIALS AND METHODSSampling. One liter of surface water was collected at each site (see Table S1 in the supplementalmaterial) in a sterile, acid-washed polycarbonate bottle (Nalgene). Duplicate 120-ml volumes werefiltered serially through 2.7-�m Whatman GF/D and 0.22-�m Sterivex (Millipore) filters, which wereimmediately stored on ice and transferred to �20°C upon return to the lab until DNA extractionswere performed. For biogeochemical measurements, duplicate 50-ml volumes of the 0.22-�m filtratewere collected and placed on ice for measurements of SiO4, PO4

3�, NH4�, NO3

�, and NO2� at the

University of Washington, Marine Chemistry Laboratory (http://www.ocean.washington.edu/story/Marine�Chemistry�Laboratory). Ten milliliters of whole water for cell counts was fixed with 10% (finalvolume) formalin and placed on ice immediately. Temperature, pH, dissolved oxygen, and salinity weremeasured with a handheld YSI 556 multiprobe system.

HTC experiments. The remaining surface water was stored on ice and immediately returned to thelab for use in HTC experiments as described by Thrash et al. (15). Briefly, a subsample was filtered througha 2.7-�m GF/D filter, stained with 1� Sybr green (Lonza), and enumerated with a Guava EasyCyte(Millipore) flow cytometer. For each experiment, a total of 480 dilution-to-extinction cultures wereestablished with five 96-well (2.1 ml per well) PTFE plates (Radleys, Essex, United Kingdom) containingartificial media (see Table S1 in the supplemental material). Each well was inoculated with an estimated1 to 3 cells from the 2.7-�m-filtered water, and plates were stored in the dark at in situ temperatures andmonitored for growth at 2 to 4 weeks, depending on the temperature, and again at 6 weeks. Growth wasmonitored with a Guava EasyCyte (Millipore) flow cytometer, and isolates reaching at least 104 cells·ml�1

were transferred to 50-ml polycarbonate flasks and cryopreserved in liquid N2 with 10% glycerol or 5%(final volume) dimethyl sulfoxide. Normally, all of the isolates reaching this minimum growth weretransferred, with one exception, experiment FWC. In that experiment, cells were counted by microscopy(35), and we believe the total cell concentration in our inoculum was underestimated. The resultantcultivability was �53%, and we therefore subselected only 60 wells for transfer. All transferred isolates,upon reaching at least 105 cells·ml�1, were collected on 25-mm, 0.22-�m polycarbonate filters (Millipore),

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and DNA extractions were performed with the MO BIO PowerWater DNA kit in accordance with themanufacturer’s instructions. Additional cryogenic stocks were also made at this stage to increase thenumber preserved in the LSUCC. Isolate 16S rRNA genes were amplified with recombinant Taq (Invitro-gen) and primers 27F/1492R (S-d-Bact-0008-d-S-20/S-*-Univ-1492-a-A-21) (36) under the following PCRconditions: denaturation at 94°C for 30 s, annealing at 50.8°C for 30 s, elongation at 72°C for 2 min, anda final elongation step at 72°C for 10 min; repeated 35 times. Prior to Sanger sequencing, successful PCRproducts were cleaned with the QIAquick PCR purification kit (Qiagen) to remove any inhibitors and PCRmaterials. Sanger sequencing of amplicons, with both the forward and reverse primers, was completedat the Michigan State RTSF Genomics Core (https://rtsf.natsci.msu.edu/genomics/). Sanger sequenceswere automatically curated by the Finch software (Geospiza Finch Suite distribution v 2.21.0) as providedby the Michigan State RTSF Genomic Core. Forward and reverse sequences were assembled, whensufficient overlap permitted, via the CAP3 web server (http://doua.prabi.fr/software/cap3), after conver-sion of the reverse read to its reverse complement at http://www.bioinformatics.org/sms/rev_comp.html.The purity of isolates was evaluated on the basis of the consistency of the quality scores across the lengthof the read. All chromatograms are available in the supplemental material with the reference to thisreport linked to “Supplementary Information.”

Community iTag analysis. Community DNA was isolated from the Sterivex filters by removing thefilters from their housing under sterile conditions in a biosafety cabinet. Filters were then extracted withthe PowerWater kit as well. DNA was sequenced in the 16S rRNA gene V4 region (515F 806R) withIllumina MiSeq 2 � 250-bp paired-end sequencing at Argonne National Laboratories (37). Raw 16S rRNAgene amplicon data were analyzed with mothur v.1.33.3 (38) by using the Silva v119 database (39).Briefly, 16S rRNA sequences were assembled into contigs and discarded if the contig had any ambiguousbase pairs, possessed repeats of �8 bp, or were �253 bp long. Contigs were aligned with the Silva rRNAv.119 database, checked for chimeras with UCHIME (40), and classified by using the Silva rRNA v.119database. Contigs classifying to chloroplast, eukaryotes, mitochondria, or “unknown” affinities wereremoved from the data, and the remaining contigs were clustered into distinctive OTUs with a 0.03dissimilarity threshold (OTU0.03). Rank abundance curves were arranged by plotting the mean relativeabundance of two biological replicates collected and sequenced separately, with only the top 60 OTUsincluded for clarity. Complete OTU tables are available upon request. Plots were created in R (v. 3.2.1) (41)with the graphing program GGPLOT2 (v. 2.0.0) (42). To determine which OTUs represented LSUCCisolates, sequences from the OTU representative fasta file, provided by mothur with get.Oturep(), wasused to create a database file against which the LSUCC isolate 16S rRNA genes could be used in a BLASTsearch. The commands used are (BLAST v 2.2.26) formatdb -i database.faa -o T -p F and blastall -iqueryfile.faa -d database.faa -p blastn -b 2 -v 2 -o outputfile.txt.

All of the best hits ranked at �98% identity, with the exception of three sequences that had 96 to97% matches to very low-ranking OTUs (LSUCC0175, LSUCC0115, and LSUCC0141). The BLAST results(BOR_bestblasthit), OTU rep file (BOR_16S.rep.fasta), OTU taxonomic assignments (BOR_16S.taxonomy),and OTU table (BOR_OTU.csv) are available in the supplemental material with the reference to this reportlinked to “Supplementary Information.”

Phylogenetic trees. Full-length or, if a contig was unavailable, forward 16S rRNA gene sequencesfrom isolates were compiled with best hits from BLAST to the NR database and sequences of knownrepresentatives of the various marine clades to which isolates were matching (All fasta files areprovided in the supplemental material). Sequences were aligned with MUSCLE (43) and culled withGblocks (44), and phylogeny was inferred with FastTree2 (45). These processes were linked by usinga custom shell script (FT_pipe) that is also available in the supplemental material with the referenceto this report linked to “Supplementary Information.” Visualization was performed with Archaeop-teryx (46).

Nucleotide sequence accession numbers. Community 16S rRNA gene sequence fastq files areavailable at the NCBI Sequence Read Archive under accession numbers SRR3085688 to SRR3085701.Individual isolate sequences have been submitted to NCBI under accession numbers KU382357 toKU382438.

ACKNOWLEDGMENTSThis work was supported by the Department of Biological Sciences at Louisiana StateUniversity and the Louisiana Board of Regents [grant LEQSF(2014 –2017)-RD-A-06]. Thefunders had no role in study design, data collection and interpretation, or the decisionto submit the work for publication.

FUNDING INFORMATIONThis work, including the efforts of J. Cameron Thrash, was funded by Louisiana Boardof Regents (Board of Regents) (LEQSF(2014-17)-RD-A-06).

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Correction for Henson et al., ArtificialSeawater Media Facilitate CultivatingMembers of the Microbial Majority fromthe Gulf of Mexico

Michael W. Henson, David M. Pitre, Jessica Lee Weckhorst, V. Celeste Lanclos,Austen T. Webber, J. Cameron ThrashDepartment of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana, USA

Volume 1, no. 2, doi:10.1128/mSphere.00028-16, 2016. Two additional referencesshould have been cited:

Sosa OA, Gifford SM, Repeta DJ, DeLong EF. 2015. High molecular weightdissolved organic matter enrichment selects for methylotrophs in dilution to extinc-tion cultures. ISME J 9:2725–2739. http://dx.doi.org/10.1038/ismej.2015.68.

Hahnke RL, Bennke CM, Fuchs BM, Mann AJ, Rhiel E, Teeling H, Amann R,Harder J. 2015. Dilution cultivation of marine heterotrophic bacteria abundant aftera spring phytoplankton bloom in the North Sea. Environ Microbiol 17:3515–3526.http://dx.doi.org/10.1111/1462-2920.12479.

Sosa et al. should be cited on PDF page 1, line 8 of the introduction, after “OM43”(together with reference 7) and also on page 2, line 11, after “components.”

Hahnke et al. should be cited on page 1, line 9 of the introduction, after “others” andalso on page 2, line 5, after “concentrations” (together with references 4, 7, 9, 10,and 12).

Published 1 June 2016

Citation Henson MW, Pitre DM, Weckhorst JL,Lanclos VC, Webber AT, Thrash JC. 2016.Correction for Henson et al., Artificial seawatermedia facilitate cultivating members of themicrobial majority from the Gulf of Mexico.mSphere 1(3):e00124-16.doi:10.1128/mSphere.00124-16.

Copyright © 2016 Henson et al. This is anopen-access article distributed under the termsof the Creative Commons Attribution 4.0International license.

Address correspondence to J. Cameron Thrash,joz.ennn@gmail.com.

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Volume 1 Issue 3 e00124-16 msphere.asm.org 1

Second Correction for Henson et al., “Artificial Seawater MediaFacilitate Cultivating Members of the Microbial Majority fromthe Gulf of Mexico”

Michael W. Henson,a David M. Pitre,a Jessica Lee Weckhorst,a V. Celeste Lanclos,a Austen T. Webber,a J. Cameron Thrasha

aDepartment of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana, USA

Volume 1, no. 2, e00028-16, 2016, https://doi.org/10.1128/mSphere.00028-16. Wehave identified two errors in our 2016 mSphere article. The first error occurred in

the reported amino acid composition. In Table S1, we listed cysteine � 2 HCl in ouramino acid mix, but the correct amino acid was cystine � 2 HCl. While this does notaffect the overall rounded organic carbon concentration reported in Table 1, it doesaffect the organic nitrogen concentration calculation in Table 1 and the total nitrogenconcentration reported in Table 3. In addition, we found a second calculation error fororganic nitrogen in Table S1, sheet “C and N mixes,” cell O42. These two errors lead usto make the following corrections.

Page 3 of the PDF, Table 1: the concentration for total organic nitrogen should be17 �M instead of 23 �M.

Page 6 of the PDF, Table 3: total nitrogen should be reported in the JW1 to JW4columns as 0.00087 g/liter instead of 0.0096 g/liter (and the originally reported value of0.0096 g/liter should have been 0.00096 g/liter).

Table S1 is updated on our laboratory website, where it is currently hosted (https://thethrashlab.com/publications/).

While the errors described above do not impact the conclusions made in our article,they do inhibit proper reconstruction of the media we used in our experiments. We verymuch regret the errors.

Published 15 August 2018

Citation Henson MW, Pitre DM, Weckhorst JL,Lanclos VC, Webber AT, Thrash JC. 2018.Second correction for Henson et al., “Artificialseawater media facilitate cultivating membersof the microbial majority from the Gulf ofMexico.” mSphere 3:e00415-18. https://doi.org/10.1128/mSphere.00415-18.

Copyright © 2018 Henson et al. This is anopen-access article distributed under the termsof the Creative Commons Attribution 4.0International license.

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July/August 2018 Volume 3 Issue 4 e00415-18 msphere.asm.org 1