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| Spotlight Selection | Bacteriology | Full-Length Text
Genome editing in ubiquitous freshwater Actinobacteria
Nachiketa Bairagi,1 Jessica L. Keer,2 Jordan C. Heydt,3 Julia A. Maresca1
AUTHOR AFFILIATIONS See aliation list on p. 11.
ABSTRACT Development of genome-editing tools in diverse microbial species is an
important step both in understanding the roles of those microbes in dierent environ
ments, and in engineering microbes for a variety of applications. Freshwater-specic
clades of Actinobacteria are ubiquitous and abundant in surface freshwaters world
wide. Here, we show that Rhodoluna lacicola and Aurantimicrobium photophilum, which
represent widespread clades of freshwater Actinobacteria, are naturally transformable.
We also show that gene inactivation via double homologous recombination and
replacement of the target gene with antibiotic selection markers can be used in both
strains, making them convenient and broadly accessible model organisms for freshwater
systems. We further show that in both strains, the predicted phytoene synthase is the
only phytoene synthase, and its inactivation prevents the synthesis of all pigments.
The tools developed here enable targeted modication of the genomes of some of
the most abundant microbes in freshwater communities. These genome-editing tools
will enable hypothesis testing about the genetics and (eco)physiology of freshwater
Actinobacteria and broaden the available model systems for engineering freshwater
microbial communities.
IMPORTANCE To advance bioproduction or bioremediation in large, unsupervised
environmental systems such as ponds, wastewater lagoons, or groundwater systems,
it will be necessary to develop diverse genetically amenable microbial model organisms.
Although we already genetically modify a few key species, tools for engineering more
microbial taxa, with dierent natural phenotypes, will enable us to genetically engineer
multispecies consortia or even complex communities. Developing genetic tools for
modifying freshwater bacteria is particularly important, as wastewater, production ponds
or raceways, and contaminated surface water are all freshwater systems where micro
bial communities are already deployed to do work, and the outputs could potentially
be enhanced by genetic modications. Here, we demonstrate that common tools for
genome editing can be used to inactivate specic genes in two representatives of a very
widespread, environmentally relevant group of Actinobacteria. These Actinobacteria are
found in almost all tested surface freshwater environments, where they co-occur with
primary producers, and genome-editing tools in these species are thus a step on the way
to engineering microbial consortia in freshwater environments.
KEYWORDS genome editing, transformation, selectable markers, freshwater,
Actinobacteria, carotenoids
Diversication of genetically amenable bacterial systems is critically important for
advancing bioproduction, bioprospecting, and biodegradation. The workhorse
model organisms such as Escherichia coli or Saccharomyces cerevisiae are relatively easily
modied, but starting with an organism that already has all or some of the desired
metabolic capabilities or environmental tolerances would mean that fewer genomic
modications are required (1–3). To have a library of genetically tractable organisms that
November 2024 Volume 90 Issue 11 10.1128/aem.00865-24 1
Editor Arpita Bose, Washington University in St.
Louis, St. Louis, Missouri, USA
Address correspondence to Julia A. Maresca,
jamaresc@esf.edu.
The authors declare no conict of interest.
See the funding table on p. 11.
Received 1 May 2024
Accepted 3 September 2024
Published 16 October 2024
Copyright © 2024 Bairagi et al. This is an open-access
article distributed under the terms of the Creative
Commons Attribution 4.0 International license.
can operate in the full range of environmental conditions, we need a broad phylogenetic
and phenotypic range of microbes that can be genetically engineered.
In freshwater environments, freshwater-specic clades of low-GC Actinobacteria
comprise up to 60% of the bacteria in surface waters and likely mediate much of the
heterotrophic conversion of dissolved organic carbon to CO2 and buried biomass (4–
16). Globally, metagenomic analyses in combination with geochemical measurements
have indicated that freshwater Microbacteriaceae play key roles in carbon, nitrogen,
phosphorus, and sulfur cycling in lakes and ponds, from coastal lagoons in South
America to bogs and ponds in the American Midwest, to alpine lakes in Europe and
Asia (7, 8, 16, 17). Genome analysis of these freshwater strains indicates that they form
coherent clades within the Microbacteriaceae family of Actinobacteria and have quite
similar, very small, genomes (<2 Mbp) (18–24). Despite their limited genomic resources,
they are keystone species in freshwater metabolic networks (18, 25–29), and may also be
useful bioindicators of trophic status (30).
Because these clades are ubiquitous and abundant, understanding their genetics,
metabolism, and ecophysiology is fundamental to understanding freshwater biogeo
chemistry. However, no genome-editing tools yet exist for any of the freshwater-spe
cic clades—the closest relatives in which genes can be inactivated or heterologously
expressed are phytopathogenic Clavibacter spp. (31–33), which are high-GC species with
larger genomes (34). Investigation into the functions of specic genes in freshwater
Actinobacterial clades has thus relied on heterologous expression in model organisms
such as E. coli until now (35, 36).
A broad understanding of how and why freshwater Actinobacteria dominate in
diverse freshwater environments will require systems-level tools for genome editing
and analysis. Therefore, our goal was to develop tools for targeted gene inactivation in
Rhodoluna (R.) lacicola strain MWH-Ta8, representative of the Luna-1 clade of freshwa
ter Actinobacteria (12, 19), and Aurantimicrobium (A.) photophilum strain MWH-Mo1,
representative of the Luna2/acIII clade (20). Isolates from these clades tend to be brightly
colored due to carotenoid pigments (17). The crtB gene, encoding the rst committed
step in carotenoid biosynthesis, was chosen for these proof-of-principle experiments
because of the ubiquity of carotenoids in these strains (17, 35), the lack of existing
biochemical data about pigment biosynthesis in freshwater Actinobacteria (37) and
because we previously isolated a spontaneous mutant in the crtB gene of R. lacicola.
Inactivation of this gene via a point mutation that introduces an early stop codon caused
a loss of pigmentation but had no eect on growth (38). We show that R. lacicola can take
up DNA via natural transformation with either linear and plasmid DNA or via electropo
ration with plasmid DNA and that A. photophilum can take up linear DNA via natural
transformation. Targeted gene inactivation is possible in both strains using antibiotic
selection for double homologous recombination, enabling the characterization of the
roles of specic genes and pathways in the ecophysiology of this group of keystone
organisms in freshwater environments.
MATERIALS AND METHODS
Strains and growth conditions
Genome-editing tools were developed for two cultures: Rhodoluna lacicola MWH-Ta8
(19) and Aurantimicrobium photophilum MWH-Mo1 (20). The R. lacicola culture is
available at the DSMZ culture collection under strain no. 23834; its genome sequence
is available at NCBI under accession no. GCA_000699505.1. The A. photophilum culture is
available at the DSMZ under strain no. DSM 107758 and its genome sequence is available
at NCBI under accession no. GCA_003194085.1. Both strains were grown aerobically
either in NSY medium [per liter: 10 mL 100× inorganic basal medium with 1 g L−1 each
nutrient broth, soytone, and yeast extract (12)] supplemented with vitamin B12 (1 mg
mL−1) and sodium thiosulfate (50 mM), or in minimal media composed of, per liter,
10 mL 100× inorganic basal medium (12) with 1 g NaCl, 0.4 g MgCl2 × 6H2O, 0.1 g
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CaCl2 × 2H2O, 0.2 g KH2PO4, 0.5 g KCl, 1 g NH4Cl, and 50 µM thiosulfate. The pH was
adjusted to 8.0, and after autoclaving, the medium was supplemented with (per liter)
40 mg L-asparagine, 40 mg L-cysteine, 1 mL 1,000 × 8-vitamin mix, 1 µg L−1 vitamin B12,
and 0.1% (vol/vol) D-fructose. The 8-vitamin mix was composed of 100 mg mL−1 each
thiamine-HCl, D-Calcium pantothenate, folic acid, nicotinic acid, 4-aminobenzoic acid,
pyridoxine-HCl, lipoic acid, and biotin.
Solid NSY and minimal medium had the same compositions, solidied with 15 g
L−1 agar. For R. lacicola, the medium was supplemented with ampicillin (20 µg mL−1),
chloramphenicol (20 µg mL−1), kanamycin (30 µg mL−1), or tetracycline (10 µg mL−1),
as appropriate. For A. photophilum, the medium was supplemented with ampicillin (75
µg mL−1). Cells in liquid culture were grown at 30°C with shaking (~150 rpm); cells on
solid medium were incubated at room temperature (~26°C). When applicable, growth in
liquid culture was quantied by measuring optical density (OD) at 600 nm using a Fisher
Scientic BioMate 3S UV-Vis spectrophotometer.
For plasmid propagation, E. coli strain NEB 5-alpha (New England Biolabs, catalog #
C2987H) was grown on liquid or solid LB medium supplemented with ampicillin (100
µg mL−1), chloramphenicol (34 µg mL−1), kanamycin (30 µg mL−1), or tetracycline (15 µg
mL−1), as appropriate.
Antibiotic sensitivity assays
R. lacicola was grown in liquid NSY medium until late exponential phase, then harvested
by centrifugation (5,000 rpm, 20 min) and resuspended in NSY medium. Cell suspensions
were spread on solid NSY medium and sensitivity to the antibiotics ampicillin, chloram
phenicol, kanamycin, streptomycin, and tetracycline was assessed by agar disk diusion
assays. Solutions of antibiotics (20 µg mL−1 or 200 µg mL−1) were applied to 6 mm paper
disks (BBL item no. 231039) and air-dried. The dried disks were then placed on the R.
lacicola lawns and the plates were incubated until growth was visible. The presence of a
zone of no growth around the plates indicated sensitivity.
A. photophilum was grown in liquid NSY medium until late exponential phase, then
harvested by centrifugation (5,000 rpm, 20 min) and resuspended in NSY medium to
an optical density at 600 nm (OD600 nm) of 0.05. A series of 10-fold dilutions (10−1 to
10−4) were prepared. Then 1 µL of each dilution was placed on solid NSY medium with
dierent concentrations of antibiotics and allowed to sink into the medium without
spreading. All trials were done in triplicate. The plates were incubated at 29°C for 5 days,
then growth was evaluated.
Gene inactivation constructs
To inactivate crtB using double homologous recombination, linear constructs
were synthesized using double-joint PCR (39). First, ~500 bp regions upstream
and downstream of crtB (locus tag rhola_00010860) were amplied from R.
lacicola genomic DNA using KOD-ONE polymerase master mix (Sigma Aldrich,
item no. KMM-101NV) and primers Ta8_crtB_US_Fp/ Ta8_crtB_US_Rp_TcR and
Ta8_crtB_DS_Fp_TcR/Ta8_crtB_DS_Rv, respectively (Table S1); the reverse primer for the
upstream region and the forward primer for the downstream region also included 20–24
nucleotides homologous to the selectable marker to be used. The selectable markers
were amplied using primers that were the reverse/complement of the upstream reverse
and downstream forward primers from the anking regions. A second round of PCR
was then done using the products of the rst three reactions as primers (39). Then,
the product of the second reaction was used as the template for PCR using primers
Ta8_crtB_US_Fp and Ta8_crtB_DS_Rv to amplify the full-length construct. The antibiotic
resistance gene was inserted in-frame in the crtB locus because we have observed that
crtB is expressed throughout the cell cycle, though expression levels vary (40).
The same approach was used to produce an inactivation construct to replace crtB
(AURMO_01714) with bla (a beta-lactamase providing ampicillin resistance, amplied
from pUC19) in A. photophilum. Primer sequences can be found in Table S2.
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For inactivation of cryB (rhola_00013030), a knockout construct was ligated into
pUC19. 500 bp regions upstream and downstream of rhola_00013030 in the R. lacicola
genome and the tetR gene from plasmid pEX18-Tc were PCR-amplied, and then joined
using double-joint PCR (Table S1). This product and pUC19 were then digested with
KpnI and SalI (New England Biolabs, catalog nos. R0142 and R0138, respectively). The
plasmid backbone was dephosphorylated and the two fragments were ligated with T4
DNA ligase (ThermoFisher, catalog no. EL0011) and the resulting plasmid was trans
formed into chemically competent E. coli strain NEB 5-alpha (New England Biolabs,
catalog #C2987H) for plasmid propagation. The resulting plasmid encoded the tetR gene
inserted in-frame in the rhola_00013030 locus. Plasmids were extracted and puried from
a 2 mL E. coli culture using the GeneJet plasmid miniprep kit (Thermo Fisher, catalog
#K0502).
Transformation procedure
Natural transformation of R. lacicola
For natural transformation, R. lacicola cells (~10 mL) were grown in NSY on the benchtop
(exposed to normal day/night light cycles) for 10 days, until the OD600 nm was ~0.1,
diluted to an OD600 nm of ~0.01 and grown 24 h in the dark. They were then harvested
by centrifugation for 10 min at 4,700 × g , washed two times in NSY media, then
resuspended in 500 μL NSY. Linear constructs or plasmid DNA (~500 ng) were mixed
with the cells and the solution was incubated without selection at room temperature
overnight in the dark. The cells were transferred to solid selective NSY and incubated
at 28°C until colonies were visible (~2 weeks), then individual colonies were restreaked
on solid selective NSY once more. Putative mutants were screened after two rounds of
colony growth on selective media.
Electroporation of R. lacicola
To test the ecacy of electroporation as a transformation method, a plasmid con
struct for inactivation of the gene encoding a putative CryB-type cryptochrome
(rhola_00013030) in R. lacicola was made. To prepare R. lacicola for electroporation, cells
(~50 mL) were grown in the dark in NSY amended with vitamin B12 (1 mg mL−1) and
thiosulfate (50 mM) for 24 h. Then glycine was added to a nal concentration of 1% and
the cells were incubated one more hour, then harvested by centrifugation for 10 min
at 4,700 × g and 4°C. The supernatant was discarded and the cells were washed twice
in 10% ice-cold glycerol, then resuspended in 200 µL 10% glycerol, quick-frozen in a
dry-ice ethanol bath, and stored at −80°C until use. Cells were thawed on ice for 10 min,
then plasmid DNA (~500 ng) was mixed with cells (50 µL). The mixtures were kept on
ice for 5 min and then transferred to an electroporation cuvette with a 1 mm gap. Cells
were electroporated at 1.7 kV (4.8 ms) using an Eppendorf Eporator. Cells were then
resuspended in 950 µL room temperature NSY media and incubated without selection
at room temperature for 3 h with shaking. The cells were then transferred to solid NSY
amended with tetracycline and incubated at 28°C until colonies were visible. Individual
colonies were then selected and restreaked on solid selective NSY.
Natural transformation of A. photophilum
For natural transformation, A. photophilum cells (~100 mL) were grown in the dark in NSY
for 24 h, until the OD600 nm was ~0.08, then harvested by centrifugation for 10 min at
4,700 × g, washed two times in NSY media, and resuspended in 500 μL NSY. Linear DNA
(~500 ng) was mixed with the cells and the solution was incubated without selection at
room temperature for 6 h in the dark, then 6 h in the light. The cells were transferred
to solid selective NSY and incubated at 28°C until colonies were visible (~10 days),
then individual colonies were restreaked on solid selective NSY. Putative mutants were
screened after two rounds of colony growth on selective media.
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Conrmation of insertions
For initial conrmation that double homologous recombination had occurred, replacing
crtB with dierent selectable markers, individual transformant colonies were picked
and resuspended in 50 uL water. 1–2 uL of this solution was used as the template for
colony PCR. Primers complementary to the R. lacicola genome outside the upstream
and downstream regions in the constructs were used to amplify the region of insertion.
Amplicons from mutant strains were compared to amplicons from wild-type R. lacicola
to conrm that the size corresponded to the expected size if crtB had been replaced
with the correct marker gene. PCR across the region could not be used to conrm the
replacement of crtB with bla in A. photophilum since the crtB and bla genes are very
similar in size. In addition to PCR-amplication of the whole crtB region, PCR with one
primer complementary to the region upstream of crtB and one complementary to the
inserted beta-lactamase gene (bla) was used; this primer pair should generate a product
of 1,475 bp in the mutant strain. Additionally, the bla gene was amplied from both the
mutant strain and the plasmid construct.
For inactivation of the predicted CryB-type cryptochrome in R. lacicola, tetracycline-
resistant colonies were obtained after one round of selection. However, the tetR cassette
is similar in size to rhola_00013030, so PCR across the region was inconclusive. Instead,
after a subsequent round of growth on non-selective medium, nested PCR using rst
a pair of primers complementary to the region of chromosomal DNA outside the
construct, followed by amplication of tetR and a fragment of the wild-type gene from
that product, was used to demonstrate both that tetR had been integrated into the
chromosome at the appropriate place and that no wild-type copy of the gene remained
in the cells.
Pigment analysis
Wild-type and ΔcrtB cultures (100 mL) of R. lacicola and A. photophilum were grown
in NSY medium as described above. Cells were harvested by centrifugation at 5,000
× g for 15 min and resuspended in 0.4 mL high-performance liquid chromatography
(HPLC)-grade acetone: methanol (7:2 vol/vol). Cells were sonicated on ice (50% duty
cycle, 1 s on/o pulses) using a Fisher Scientic probe sonicator (Sonic Dismembrator
model 120, probe model CL-18). The lysate was centrifuged at 12,000 × g for 2 min
to remove cell debris, and the supernatant was ltered through a 0.2 µm polytetrauoro-
ethylene syringe lter (Thermo Scientic) into glass vials. Pigment extracts were then
transferred to quartz cuvettes and absorption spectra from 350 to 600 nm were recorded
on a Thermo Scientic BioMate 3S UV/visible spectrophotometer.
RESULTS
Antibiotic sensitivity of freshwater actinobacterial strains
The sensitivity of R. lacicola to the antibiotics ampicillin, chloramphenicol, kanamycin,
streptomycin, and tetracycline was assessed by an agar disk diusion assay. R. lacicola
is not sensitive to streptomycin, but is sensitive to chloramphenicol, kanamycin, and
tetracycline at 20 µg mL−1, as well as to ampicillin at 200 µg mL−1 (Table 1). In contrast,
A. photophilum was resistant to chloramphenicol and kanamycin at all concentrations
tested, and sensitive only to ampicillin (Table 2).
Transformation and homologous recombination
Inactivation of crtB via double homologous recombination
For a proof of principle experiment, the crtB gene encoding phytoene synthase,
the rst committed step in carotenoid biosynthesis, was targeted for inactivation. A
linear construct was made in which the gene encoding tetracycline resistance (tetR
from pEX18-Tc) was placed in-frame between the regions immediately upstream and
downstream of the crtB gene from the R. lacicola genome (Fig. 1A). R. lacicola was
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transformed using natural transformation, and tetracycline-resistant transformants were
white rather than pink in color, demonstrating that pigment biosynthesis was success
fully abolished in those strains. PCR on white colonies subsequently conrmed the
insertion of the selectable marker in each case (Fig. 1B). No PCR products corresponding
to the wild-type genotype were detected, suggesting that the mutation was completely
segregated.
The crtB gene in A. photophilum was inactivated using the same approach, with the
bla gene inserted between the homology arms upstream and downstream of crtB (Fig.
1C). PCR reactions using white colonies as templates again conrmed the insertion of bla
in the correct location (Fig. 1D).
Conrmation of loss of crtB activity
Wild-type R. lacicola synthesizes pink carotenoids and wild-type A. photophilum
synthesizes yellow carotenoids, which have not yet been characterized (19, 20, 38). The
rst committed step in carotenoid biosynthesis is the synthesis of phytoene from two
molecules of geranylgeranyl diphosphate by CrtB, the phytoene synthase (41), so the
loss of crtB should result in loss of pigmentation. The crtB mutants of both strains are
colorless. The absorption spectra of the pigments extracted from wild-type cells have
maxima between 460 and 530 nm, consistent with mixtures of C40 and C50 carotenoids
(Fig. 2). These peaks are absent from the absorption spectra of the ΔcrtB mutants,
indicating that phytoene synthase activity was successfully disrupted.
Other selectable markers tested in R. lacicola
In addition to tetracycline (tetR amplied from plasmid pEX18-TC (42)) for antibiotic
selection, we tested the kanamycin resistance cassette npt-ii from plasmid pEB001 (43),
the chloramphenicol resistance gene cat from pMCL200 (44), and the beta-lactamase bla
encoding ampicillin resistance from pUC19 as selectable markers for double homologous
recombination. In all four cases, recombinant strains were selected and PCR conrmed
that the gene replacement had occurred. However, none of these strains survived either
with or without antibiotic selection for more than two rounds of restreaking.
Electroporation of R. lacicola
To test the ecacy of electroporation as a transformation method, a plasmid construct
was made for the inactivation of rhola_00013030, encoding a putative cryptochrome
TABLE 1 R. lacicola is sensitive to chloramphenicol, kanamycin, and tetracyclinea
Antibiotic 20 µg mL−1 200 µg mL−1
Chloramphenicol S S
Kanamycin S S
Streptomycin R R
Tetracycline S S
aSensitivity was evaluated with disk diusion assays on solid NSY medium. R. lacicola was scored as resistant (“R”) if
cells grew up to the edge of the disk containing the antibiotic, or sensitive (“S”) if there was a clear ring around the
disk.
TABLE 2 A. photophilum is sensitive to ampicillina
Ampicillin Chloramphenicol Kanamycin
Dilution 150 75 25 10 200 100 50 200 100 50
10−1 0 0 0 (+) ++ ++ ++ ++ ++ ++
10−2 0 0 0 (+) ++ ++ ++ ++ ++ ++
10−3 0 0 0 0 ++ ++ ++ ++ ++ ++
10−4 0 0 0 0 0 + + + (+) +
aSensitivity was evaluated by the growth of 1 µL culture spotted onto solid NSY medium amended with antibiotics
at the indicated concentrations. Concentrations are in µg mL−1. “(+)” indicates very little growth; “+” indicates
growth of a few colonies in the droplet area; “++” indicates conuence within the droplet area.
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FIG 1 Inactivation of crtB by natural transformation and double homologous recombination in R. lacicola and A. photophilum, replacing crtB with tetR and
bla, respectively. Primer sequences can be found in Tables S1 and S2. (A) Inactivation construct for R. lacicola. Primers Ta8_crtB_FR_Fw and Ta8_crtB_FR_Rv
were used to evaluate mutants (dashed gray line). (B) PCR conrmation of tetR insertion into the crtB locus. The molecular weight marker used was GeneRuler
1 KB Plus (ThermoFisher, catalog no. SM1331). The size of the amplicon in the putative mutant strain is the same as the amplicon from the plasmid, indicating
the replacement of the 894 bp crtB gene with the 1,191 bp tetR gene. (C) Inactivation construct for A. photophilum. Primers Mo1_crtB_Up_cPCR_Fw and
Mo1_AmpR_Rp were used to evaluate mutants (dashed gray line). (D) PCR conrmation of bla insertion into the crtB locus. The size of the crtB amplicon in the
putative mutant strain is similar in size to the amplicon from the plasmid and wild type, as expected (PCR1). However, amplication using one primer outside
of crtB and one complementary to bla yields a product only in the mutant, indicating replacement of the crtB gene in the mutant strain. Amplication with
bla-specic primers yields identical products from the mutant and plasmid, but no product from wild-type A. photophilum.
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potentially relevant to light capture and utilization in R. lacicola. Tetracycline-resistant
colonies were obtained after one round of selection, and after a subsequent round of
growth on non-selective NSY, PCR using a pair of primers complementary to a region of
chromosomal DNA outside the construct was used to demonstrate insertion of tetR into
the chromosome at the appropriate place (Fig. 3).
DISCUSSION
Actinobacteria are abundant, cosmopolitan, heterotrophic members of freshwater
ecosystems, and they regulate their organic carbon metabolism in response to both
biotic and abiotic parameters. Extensive (meta) genomic analysis of these bacteria
has predicted a variety of genotype-phenotype relationships (18, 24, 28, 40). Here,
we demonstrate that the freshwater Actinobacteria R. lacicola and A. photophilum are
naturally transformable and that gene inactivation via homologous recombination is
feasible in both strains. We hope that the availability of methods for genome editing in
model organisms from two clades within this group will catalyze research that directly
tests cause and eect hypotheses between genetic content and phenotype in freshwater
ecosystems. Additionally, there is broad interest in exploiting microbes and microbial
consortia in freshwater systems to remove contaminants, produce biofuels, or provide
ecosystem services (1, 45–47). The tools provided here make it feasible to modify
the genomes of some of the most abundant microbes in freshwater communities, a
necessary step in engineering synthetic microbial communities in those environments.
Targeted gene inactivation succeeded in R. lacicola using selection with a tetR
(encoding tetracycline resistance) cassette on complex medium amended with
tetracycline. Electroporation can be used to transform R. lacicola. Additionally, our prior
FIG 2 Pigment analysis of crtB mutants. Cells were grown to late stationary phase and pigments were extracted in acetone:methanol (7:2 vol/vol), then
absorption was measured using a UV/Vis spectrophotometer. (A) Wild-type R. lacicola makes pink carotenoid pigments (solid line), but the signal from the
carotenoids disappears when crtB is inactivated (dashed line) since it encodes the rst committed step in carotenoid biosynthesis. (B) Wild-type A. photophilum
makes yellow carotenoid pigments (solid lines), which disappear when crtB is inactivated (dashed line).
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work found that transcripts for a DNA uptake system encoded by comEC are more
abundant in the dark (40), suggesting that both strains might be naturally transformable.
Here, we conrm that if electroporation is not available, both A. photophilum and R.
lacicola can take up DNA during a dark incubation. Both PCR products and plasmid
DNA are stable in the cells for enough time that homologous recombination is feasible.
Additionally, no special equipment is needed for natural transformation, making these
convenient and accessible model organisms.
Direct selection can be used to select for double homologous recombination in both
strains. Because this method requires that the selectable markers be expressed from
the chromosome, this method also demonstrates that the expression of heterologous
genes from the chromosome is feasible. These genome-editing tools can now be used
to investigate the roles of specic genes and combinations of genes in freshwater
Actinobacterial physiology, both by inactivating genes of interest in R. lacicola and A.
photophilum or by expressing genes of interest from other freshwater Actinobacteria in a
phylogenetically related, physiologically relevant host.
We also tested the replacement of crtB in R. lacicola with genes encoding resistance to
ampicillin, chloramphenicol, and kanamycin. Although preliminary data after one round
of selection on solid medium with antibiotics indicated successful gene replacement
via double homologous recombination, the mutant strains did not survive more than
two rounds of restreaking on selective medium. In the case of tetracycline resistance,
the mutants are stable on non-selective medium after one round of selection, and the
same may be true for the other selectable markers. This may not be a strong enough
selection to inactivate genes whose loss would be deleterious to the cell, and might
instead select for merodiploidy (48). It also suggests that until a stable plasmid replicon
FIG 3 Inactivation of cryB by electroporation and double homologous recombination in R. lacicola. (A) Construct for replacement of the cryB gene, encoding a
putative cryptochrome, with the tetracycline resistance gene from plasmid pEX18-Tc. (B) PCR across the insertion site conrms that in the mutant, the product
is ~200 bp smaller than in the wild type (PCR1), that the tetR gene is present in the mutant but not the wild type (PCR2), and that an internal fragment of cryB is
present in the wild type but not the mutant strain (PCR3).
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is found for these strains, genes for heterologous expression will have to be inserted
into the chromosome. We suggest that the crtB locus may be suitable for this, since its
inactivation has no apparent eect on growth, and replacement of crtB has a visually
identiable phenotype.
Carotenoid pigments appear to be universal in freshwater Actinobacteria and
carotenoid biosynthetic pathways have been predicted in several strains based on
genome and metagenome-assembled genome sequences (35, 37, 49, 50). However,
these pathways have not yet been biochemically characterized, with the exception
of two beta-carotene cleavage dioxygenases that produce retinal (35, 37). Here, we
demonstrate that in both strains, inactivation of the predicted phytoene synthase,
crtB, led to the loss of all pigment production. This result conrms that crtB indeed
encodes a phytoene synthase, and indicates that all of the pigments in both R. lacicola
and A. photophilum are carotenoid pigments. This also conrms our prior result that
carotenoid production is not required for viability under ordinary laboratory conditions
(38). Inactivation of any gene or combination of genes in this pathway should therefore
be feasible, enabling follow-up genetic studies of carotenoid biosynthetic pathways in
freshwater species.
Additional follow-up studies investigating fundamental processes, ecological
interactions, and potential applications in freshwater Actinobacteria are now possible.
Targeted gene inactivation and physiological comparisons to wild type can be used to
better understand the role(s) of the actinorhodopsins and heliorhodopsins commonly
identied in freshwater Actinobacteria (22, 36, 37, 50, 51) or the identities of the
genes required for their unique cell shape (12). The very small genomes of freshwater
Actinobacteria appear to change and rearrange rapidly, suggesting a role for horizontal
gene transfer (18): now, the importance of the predicted comEC competence genes
for DNA uptake can be tested. The freshwater Actinobacteria appear to be resistant to
grazing (7, 52, 53), so the contributions of specic membrane components to grazing
resistance can now be investigated, as can the roles of specic transporters in organic
carbon uptake and processing, which may mediate metabolic interactions with other
organisms (18).
As members of a ubiquitous and abundant keystone clade, freshwater Actinobacteria
likely play important roles in supporting other microbes in highly diverse freshwater
environments (54). Targeted gene inactivation or insertion of specic genes into the
chromosome for heterologous expression can now be used to investigate the mech
anisms of survival, adaptation, and interaction that freshwater Actinobacteria use to
thrive in this enormous range of environments, in communities with wide varieties of
other microbes. As we use outdoor systems for the synthesis of high-value products
(47, 55, 56) or removal of contaminants (57, 58), engineering the microbial communities
around the strains with traits of interest may enhance and stabilize the desired activities.
Genome-editing tools for more of these “supporting” species could therefore enable
better bioproduction in mixed communities.
In sum, here we establish R. lacicola and A. photophilum as convenient model
organisms for investigating the genetics and physiology of freshwater Actinobacteria.
Linear and plasmid DNA can be transformed into R. lacicola using either natural
transformation or electroporation. We have developed selectable markers for each
strain, driven by the native crtB promoters, for targeted gene inactivation via double
homologous recombination. We anticipate that other antibiotic-based selections will
soon be usable in this strain, and hope that these tools will be widely used. Although A.
photophilum is sensitive to fewer antibiotics than R. lacicola, it is naturally transformable
with linear DNA, and bla works as a selectable marker for insertional gene inactivation in
this strain. These tools will enable genome engineering in freshwater Actinobacteria for
fundamental genetic and ecophysiological characterization as well as for application in
engineered freshwater microbial communities.
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ACKNOWLEDGMENTS
This work was funded by grant #2118003 from the National Science Foundation Enabling
Discovery through GEnomics (EDGE) program.
The authors gratefully acknowledge Kristin Walz for assistance with the initial
antibiotic sensitivity analyses, Eric Rouviere for early tests of competence in R. lacicola,
Dr. Priscilla Hempel for bioinformatics assistance, Dr. Thomas Hanson (University of
Delaware) for support and lab space, and Dr. Adam Guss (Oak Ridge National Laboratory)
for helpful discussions.
AUTHOR AFFILIATIONS
1Department of Civil and Environmental Engineering, University of Delaware, Newark,
Delaware, USA
2Department of Earth Sciences, University of Delaware, Newark, Delaware, USA
3School of Marine Science and Policy, University of Delaware, Newark, Delaware, USA
PRESENT ADDRESS
Julia A. Maresca, Department of Chemistry, SUNY College of Environmental Science and
Forestry, Syracuse, New York, USA
AUTHOR ORCIDs
Jessica L. Keer http://orcid.org/0000-0002-0302-3588
Julia A. Maresca http://orcid.org/0000-0002-3955-1585
FUNDING
Funder Grant(s) Author(s)
National Science Foundation (NSF) 2118003 Nachiketa Bairagi
Julia A. Maresca
AUTHOR CONTRIBUTIONS
Nachiketa Bairagi, Formal analysis, Investigation, Methodology, Validation, Visualization,
Writing – review and editing | Jessica L. Keer, Investigation, Methodology, Writing
– review and editing | Jordan C. Heydt, Investigation, Methodology, Writing – review
and editing | Julia A. Maresca, Conceptualization, Funding acquisition, Investigation,
Methodology, Project administration, Resources, Supervision, Visualization, Writing –
original draft, Writing – review and editing
ADDITIONAL FILES
The following material is available online.
Supplemental Material
Supplemental tables (AEM00865-24-s0001.docx). Tables S1 and S2.
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