Isolation and characterization of a heavy metal- and antibiotic-tolerant novel bacterial strain from a contaminated culture plate of Chlamydomonas reinhardtii, a green micro-alga.

Growth media and cultures

Chlamydomonas wild type strain 4A+ (CC-4051 4A+ mt+) was cultured in the lab on TAP agar (Chlamydomonas Resource Center) under dim light (15-20 μmol m^-2s^-1) at 22°C. TAP medium recipes are described in Mitra et al., 2020.⁹ 4A+ liquid TAP cultures were grown on a shaker under low light (70-80 μmol m^-2s^-1) for 3 days for aeration as described in Mitra et al, 2020.^8,9 Clip185 stock was maintained in the lab under dim light (15-30 μmol m^-2s^-1) at 22°C on Lysogeny Broth (LB) agar medium (Cold Spring Harbor Protocols). Experiment-specific temperature and light intensities can be found in the result section of this article under specific experiment descriptions. Clip185 liquid cultures were grown in LB on a shaker at 30°C-37°C for aeration. Light from 1-2 cool white fluorescent lights were used in experiments. A LI-250A light meter (LI-COR, Inc., Lincoln, NE) was used to measure light intensities.

Testing antibiotic sensitivity of Clip185

We tested the following antibiotics: penicillin; neomycin, chloramphenicol, polymyxin B and vancomycin. Antibiotics were purchased from Sigma-Aldrich (St. Louis, MO). Two different amounts (50 μg and 100 μg) of each of these five antibiotics were tested using the Kirby-Bauer (KB) disc diffusion antibiotic susceptibility tests as described in the American Society for Microbiology (ASM) protocol with one modification: we used TAP agar medium instead of Mueller-Hinton agar medium as stated in the ASM. KB tests of 4A+ and Clip185 were performed on TAP-agar plates as described in Mitra et al., 2020.^9,28,29 A 12-hours old LB liquid culture of Clip185 was used for plating on the antibiotic-containing TAP plates. Antibiotic plates were incubated at 22°C. Clip185 and Chlamydomonas plates were imaged after 7 days of incubation. Diameters of zones of inhibitions were measured in Microsoft PowerPoint by importing free Google ruler available on Chrome. Means and standard deviations were calculated using Microsoft Excel. Microsoft Excels’ t-Test: Paired Two Sample for Means tool in the analysis ToolPak was used for statistical analyses of the data, using three biological replicates per experiment (each with three internal replicates). To test the efficacy of vancomycin as a potent antibiotic in eradicating Clip185 contamination on Chlamydomonas TAP plates, Clip185 and 4A+ strains were jointly streaked on TAP media plates containing 50 μg/mL of vancomycin. Streaked TAP + vancomycin plates were imaged after incubation at 22°C for 2.5 weeks. We chose 2.5-weeks incubation period over 7 days in this vancomycin efficacy confirmation experiment to make sure that the selected vancomycin amount is bactericidal to Clip185 and not bacteriostatic.

Clip185 growth analyses

Growth at different temperatures: Clip185 was streaked on LB agar and TAP agar prepared at our laboratory and media plates were incubated at 22°C and 37°C. LB-agar and TAP agar culture plates were imaged after 3 days and 7 days of growth, respectively. Clip185 growth on LB medium plates was monitored for 3 days and that on TAP medium plates was monitored over 7 days because we observed that Clip185 grows slowly in TAP medium compared to that in the LB medium. Three biological replicates per medium per temperature (each with three technical replicates) were used in the experiment.

Growth assays on Tryptic Soy Blood Agar (TSBA): Clip185 and an Acidovorax sp.⁹ were streaked on TSBA plates, purchased from Carolina Biological (Burlington, NC). We used a gamma-hemolytic strain of Acidovorax as a control in this experiment as it does not hemolyze red blood cells. Plates were incubated at 37°C and were imaged after 3 days of growth. Hemolysis classifications were assigned according to the information stated in the ASM protocol. Three biological replicates per strain (each with three technical replicates) were used in the experiment.

Growth assays on Mannitol Salt Agar (MSA): MSA plates purchased from Carolina Biological (Burlington, NC). Clip185 and Staphylococcus aureus (a mannitol fermenter which produces acid, used here as positive control) were streaked on MSA plates, and the plates were incubated at 22°C. The Clip185 MSA plate was imaged after 3-6 days of growth. After 3 days of growth, the S. aureus plate was imaged. Three biological replicates per strain (each with three technical replicates) were used in the experiment.

Growth assays on Tris-Phosphate (TP)-phenol red-sugar-agar medium: TP is the TAP medium without acetate (https://doi.org/10.17504/protocols.io.bgzujx6w).⁹ Clip185 was streaked on TP-phenol red-agar medium (pH of 7.2) containing three different sugars as stated under the result section, and the plates were incubated at 22°C. Culture plates were imaged after 7 days of growth. Results were interpreted as described in Mitra et al., 2020.⁹ Three biological replicates per growth medium (each with three technical replicates) were used in the experiment.

Comparative growth assays on Potato Dextrose Agar (PDA) and Mueller-Hinton-Agar (MHA): Clip185 was streaked on PDA and MHA plates and incubated at 30°C for three days. The plates were imaged after three days of growth. The PDA and MHA plates were purchased from Carolina Biological (Burlington, NC). Three biological replicates per growth medium (each with three technical replicates) were used in the experiment.

Testing Light-regulation of decaprenoxanthin production: Clip185, Corynebacterium glutamicum strain ATCC13032 and Kocuria rhizophila ATCC 9341 strain were streaked on two LB media plates per strain. For each strain, one plate was kept under dim light (20-25 μmol m^-2s^-1) and the other was kept in the dark at 22°C for five days. Culture plates were imaged after 5 days of growth. Three biological replicates per strain for each light condition (each with three technical replicates) were used in the experiment.

Growth assays on LB media containing heavy metals: LB media containing the following concentrations of metal salts were prepared: a) 0.5 mM, 1 mM and 2 mM of cadmium chloride; b) 0.5 mM and 1 mM of cobalt (Co²⁺) chloride; c) 0.5 mM, 1 mM and 2 mM zinc sulfate heptahydrate; d) 0.5 mM and 1 mM of cupric sulfate pentahydrate; e) 1 mM, 2 mM and 4 mM of nickel (Ni²⁺) bromide and; f) 2 mM, 4 mM, 6 mM and 10 mM of chromium (Cr⁶⁺) standard solution. Cobalt chloride and cadmium chloride salts were purchased from Strem Chemicals (Newburyport, MA). Nickel bromide salt was purchased from Acros organics (Fair Lawn, NJ). Cupric sulfate pentahydrate salt and the chromium (Cr⁶⁺) standard solution were purchased from Fisher Scientific (Thermo Fisher Scientific, Waltham, MA). A sterile wooden applicator was lightly tapped over Clip185 growth on a 2-3 days old Clip185 LB-agar plate to collect approximately the same amounts of cells for each experiment. This wooden applicator was used for streaking cells on the respective LB metal plates. The LB metal plates were imaged after a specific period of growth labeled on the figures and also stated in figure legends (Figures 14-19). As a control, Clip185 was streaked on a LB plate and the plate was imaged after 8 days and 28 days of growth. All plates were incubated under 15-20 μmol m^-2s^-1 light intensity. Three biological replicates per metal concentration were used in this experiment. Each biological replicate had three technical replicates.

Gram staining, oxidase test and starch hydrolysis test

Gram staining: Gram staining of Clip185 was performed using commercial Gram stain reagents (VWR, Radnor, PA). Gram-stained Clip185 cells were imaged under an oil immersion lens using a Samsung Galaxy S5 camera and a cell phone adapter for the microscope eyepiece. Three biological replicates (each with three technical replicates) were used in the experiment.

Cytochrome c oxidase test: Oxidase test was performed as described in Mitra et al., 2020^8,9 using Difco DrySlide Oxidase Disposable Slide purchased from Carolina Biological (Burlington, NC). Sphingobium yanoikuyae was used as an oxidase-positive control in this experiment. Three biological replicates per strain (each with three technical replicates) were used in the experiment.

Starch hydrolysis test: Starch hydrolysis test was performed as described in Mitra et al., 2020^8,9 using commercial Mueller-Hinton-agar (MHA) medium plates (Carolina Biological, Burlington, NC). Clip185 and Bacillus subtilis were streaked on MHA medium plates and incubated for 3 days at 30°C. After 3 days of growth, starch hydrolysis tests were performed. MHA plates were imaged before and after the starch hydrolysis tests. Three biological replicates per strain (each with three technical replicates) were used in the experiment.

Decaprenoxanthin extractions and spectrophotometric analyses

Actinobacteria like Corynebacterium glutamicum ATCC 13032 and Kocuria rhizophila ATCC 9341 are known producers of the C₅₀ carotenoid decaprenoxanthin. Hence, we selected these two bacterial strains as controls for in the decaprenoxanthin spectrophotometric analyses experiment. Clip185, C. glutamicum and K. rhizophila strains were grown under a light intensity of 15-20 μmol m^-2s^-1. Cells of these three strains were treated with 5 mL of 100% methanol for carotenoid extraction. Pigment extraction was carried out in the dark for 6 hours at 22°C. Extracted pigments were processed as described in Mitra et al., 2020.⁸ Spectrophotometric wavelength (400 nm – 600 nm) scan analyses of extracted pigments were performed using a Beckman Coulter DU 730 Life science UV/Vis spectrophotometer (Brea, CA). Three maximum absorption peaks of decaprenoxanthin were monitored at 413 nm, 437 nm and 467 nm, as described for decaprenoxanthin and its glucosides in the literature.^30–32 C. glutamicum and K. rhizophila were used as positive controls in spectrophotometric analyses. Three biological replicates per strain were used in this experiment. Each biological replicate had three internal replicates.

Clip185 genomic DNA isolation and PCR amplification of the partial 16S rRNA gene

Isolation of Clip185 genomic DNA and DNA concentration and purity checking of the isolated genomic DNA were performed as described in Mitra et al., 2020.^8,9 Forward and reverse 16S rRNA PCR primer sequences can be found in Mitra et al., 2020^8,9 and in Klindworth et al., 2013.³³ PCR was performed as described in Mitra et al., 2020^8,9 with an extension time of 1 minute.

Gel extraction of the Clip185 16S rRNA amplicon and DNA sequencing

The PCR-amplified partial 16S rRNA Clip185 genomic product (product size approximately 445 bp) was purified from the agarose gel and cloned in pCR4-TOPO TA vector as described in Mitra et al., 2020.^8,9 One clone containing the Clip185 partial 16S rRNA gene was sequenced at the UC Berkeley DNA Sequencing Facility. Analyses of DNA sequences were performed using the Chromas Lite (Technelysium) and BLAST programs.

Imaging

A Samsung Galaxy S5 cell phone camera was used for imaging all culture plates. Image cropping and adjustments were made using the Photos app in Windows 10 and Adobe Photoshop version 22.3. Visualization and imaging of ethidium bromide -stained DNA gels were performed using a Bio-Rad Molecular Imager Gel Doc XR+ (Bio-Rad, Hercules, CA).

Isolation and purification of a yellow-pigmented bacterial strain

We isolated a yellow-pigmented bacterium from a contaminated TAP media plate of the Chlamydomonas strain, CLiP strain LMJ.RY0402.18514 (Figure 1A). We purified the bacterium from the contaminated Chlamydomonas culture plate using the streak plate method. We picked twenty-one single colonies from Clip185 streaked-LB plate and transferred them to a fresh LB agar plate using a numbered grid (Underlying data³⁴). Colony # 37 was selected for further studies (Underlying data³⁴). We maintained colony # 37 stock on LB agar at 22°C under 15-25 μmol m^-2s^-1 light intensity (Figure 1B). We named this bacterium Clip185 after the Chlamydomonas Library Project (CLiP) and the first three numerical digits in the second part of the name of the Chlamydomonas CLiP strain LMJ.RY0402.185141 it contaminated. We have deposited the Clip185 strain to the ARS Culture Collection (NRRL Accession number: B-65609).

Figure 1. Isolation of the bacterial strain Clip185 from a contaminated Chlamydomonas TAP-agar plate.

(A) Tris-Acetate-Phosphate (TAP)-agar medium plate showing bacterial contamination of a Chlamydomonas strain at room temperature (22°C). (B) LB-agar medium plate of purified Clip185 strain. Culture plate shown in (B) was imaged after 5 days of growth at 22°C under 20-25 μmol m^-2s^-1 light intensity.

Clip185 antibiotic susceptibility tests

We monitored relative antibiotic-sensitivities of Clip185 and Chlamydomonas 4A+ wild type strain using KB disc diffusion tests as described in Mitra et al. 2020.^8,9 In our experiments we used two different amounts (50 μg and 100 μg) of penicillin, chloramphenicol, neomycin, polymyxin B and vancomycin. The mean diameters of zones of growth inhibitions for each antibiotic amount with respective standard deviations are shown in Table 1. For detailed statistical analyses of the data from three biological replicates (each of which had three internal replicates) please refer to our underlying data.³⁵

Clip185 and Chlamydomonas were both resistant to penicillin and chloramphenicol for both amounts (50 μg and 100 μg) as no zones of growth inhibitions were observed (Table 1; Underlying data³⁵). Chlamydomonas had a larger diameter of zone of growth inhibition for 50 μg polymyxin B (420.15 IU) than Clip185 (Table 1, Underlying data³⁵). Both Chlamydomonas and Clip185 had a larger diameter of zone of growth inhibition for the 100 μg (840.3 IU) polymyxin B compared to that for 50 μg polymyxin B (Table 1, Underlying data³⁵). P-values from the 1-tailed and 2-tailed hypothesis tests for sensitivity to 100 μg dose of polymyxin B were 3% and 7%, respectively. It is known that if the diameter of the zone of growth inhibition is more than 12 mm with the application of 300 IU of polymyxin B in a KB test, then a bacterium is sensitive to polymyxin B.³⁶ We used polymyxin B amounts that were 1.4-fold to 2.8-fold higher than what is used for KB tests, and growth inhibition zones of Chlamydomonas and Clip185 were less than 12 mm (Table 1). Thus, based on the diameters of the zones of growth inhibitions, both Chlamydomonas and Clip185 are resistant to polymyxin B (Table 1, Underlying data³⁵).

Chlamydomonas had a slightly smaller diameter for zone of growth inhibition for 50 μg (38.75 IU) neomycin than Clip185 (Table 1). P value from the 1-tailed hypothesis test was statistically significant (3%) but the P value from the 2-tailed hypothesis test was not statistically significant for 50 μg neomycin (Underlying data³⁵). Clip185 had a larger dimeter of zone of growth inhibition for 100 μg neomycin (77.5 IU) than Chlamydomonas and, the data was statistically significant (Table 1, Underlying data³⁵). It is known that if the diameter of the growth inhibition zone is more than 16 mm, then a bacterium is sensitive to 30 μg neomycin in a KB test. We used neomycin amounts that were 1.7-fold to 3.3-fold higher than what is used for KB tests (Table 1), and growth inhibition zones of Chlamydomonas and Clip185 were less than 16 mm. Hence both Chlamydomonas and Clip185 are resistant to neomycin (Table 1, Underlying data³⁵). Clip185 was highly sensitive to both 50 μg (50.35 IU) and 100 μg (100.7 IU) vancomycin while Chlamydomonas was resistant to vancomycin as it did not show any zones of growth inhibitions (Table 1, Underlying data³⁵). In summary, our KB test results show that vancomycin is the best drug to minimize Clip185 contamination on TAP agar.

Next, we streaked Chlamydomonas and bacterium Clip185 together on TAP agar plates containing 50 μg of vancomycin per mL of the TAP medium and incubated the plates at room temperature for a period of 2.5 weeks (Figure 2). Clip185 did not grow on the TAP plate containing 50 μg/mL of vancomycin but Chlamydomonas did (Figure 2) after 2.5 weeks. Hence vancomycin at a concentration of 50 μg/mL in the TAP medium is bactericidal to Clip185 on Chlamydomonas culture plates without hindering the algal growth.

Figure 2. Testing the efficacy of Vancomycin in eliminating Clip185 contamination.

Chlamydomonas wild type 4A+ and Clip185 strains were streaked on TAP-agar plate containing 50 μg of Vancomycin/mL of medium. TAP-agar antibiotic plate was incubated at room temperature (22°C) for 2.5 weeks before it was imaged.

Gram staining

Gram staining revealed that Clip185 is a gram-positive small bacillus. Cells were small rods, and often two rods were joined to each other (Figure 3).

Figure 3. Gram-stained Clip185 under 100× magnification.

Clip185 cells from a three days-old LB-agar medium culture plate were used for gram-staining. Gram-stained cells were visualized under an oil immersion lens of a bright-field microscope and imaged with a smart phone camera.

Clip185 growth analyses on TAP- and LB-agar medium plates at different temperatures

Clip185 grew well on LB at 22°C as well at 37°C on LB-agar (Figure 4A-B). It grew slowly on TAP-agar at 22°C and 37°C (Figure 4C-D) as evident from the comparison of 3-days growth on the LB plates and 7-days growth on the TAP plates. Hence, Clip185 is a mesophile.

Figure 4. Clip185 growth on TAP- and LB-agar medium at different temperatures.

(A) Growth on LB-agar medium plate at room temperature (22°C). (B) Growth on LB-agar medium plate at 37°C. (C) Growth on TAP-agar medium plate at 22°C. (D) Growth on TAP-agar medium plate at 37°C. LB-agar culture plates were imaged after 3 days of growth. TAP-agar culture plates were imaged after 7 days of growth.

Testing for Clip185’s ability to lyse red blood cells

Clip185 could partially lyse red blood cells when grown on tryptic soy blood agar plates for three days at 30°C, as evident from the greenish discoloration around the Clip185 growth (Figure 5A).

Figure 5. Clip185 is gamma hemolytic.

(A) 72 hours-growth of Clip185 on tryptic soy-blood agar plate. (B) 72 hours-growth of a gamma-hemolytic Acidovorax sp. on tryptic soy-blood agar plate. Media plates were incubated at 37°C.

We used a gamma-hemolytic strain of Acidovorax as a control in this experiment (Figure 5B) which cannot hemolyze red blood cells. As expected, there was no trace of hemolysis around the growth of Acidovorax⁹ on the tryptic soy agar plate (Figure 5B).

Testing for presence/absence of cytochrome c oxidase in Clip185

We conducted oxidase test on Clip185 and a gram-negative bacterium Sphingobium yanoikuyae. Our oxidase test results showed that S. yanoikuyae is oxidase-positive (Figure 6; left) and Clip185 is oxidase-negative (Figure 6; right). Results were interpreted as stated under the method section of this article.^8,9 Hence Clip185 does not produce cytochrome c oxidase, an enzyme involved in the electron transport chain in bacterial respiration.

Figure 6. Clip185 is oxidase-negative.

Cells of Sphingobium yanoikuyae (on the left) and Clip185 (on the right) streaked on a disposable slide containing a film coated with oxidase reagent tetramethyl-p-phenylenediamine dihydrochloride (TMPD). Image of the slide was taken after 10 seconds of the application of the cells on the slide.

Testing Clip185’s ability to utilize different sugars as carbon sources and ferment them

We did not want to use LB medium to test the ability of Clip185 to utilize other sugars as carbon source, because excess amino acids present in the nutrient-rich LB medium are converted to glucose via gluconeogenesis. TP agar medium is the TAP medium minus acetate. Hence TP medium lacks a carbon source.^8,9 We supplemented TP medium (pH 7.2) separately with three different sugars (glucose, sucrose and lactose) and the pH indicator, phenol red. (Figure 7). We grew Clip185 on these neutral TP + sugar plates. Figure 7A, C and E represent control TP + 1% glucose, TP + 1% sucrose and TP + 1% lactose plates (without Clip185), respectively. These control plates show the light reddish color of phenol red when the pH is 7.2. Clip185 grew on TP + 1% glucose and TP +1% sucrose plates and fermented the respective sugars (Figure 7B; Figure 7D) but it did not grow on TP + 1% lactose plate (Figure 7F). Fermentation of glucose and sucrose produced acids which decreased the pH in the TP medium. With the decrease in pH, phenol red’s light red color changed to a yellow color (Figure 7B; Figure 7D).

Figure 7. Growth of Clip185 on Tris Phosphate (TP) agar medium supplemented with different sugars.

(A) Control TP + 1% glucose agar medium plate with phenol red as a pH indicator. (B) Clip185 on TP +1% glucose agar medium plate with phenol red as a pH indicator. (C) Control TP +1% sucrose agar medium plate with phenol red as a pH indicator. (D) Clip185 growth on TP +1% sucrose agar medium plate with phenol red as a pH indicator. (E) Control TP +1% lactose agar medium plate with phenol red as a pH indicator. (F) Clip185 growth on TP +1% lactose agar medium plate with phenol red as a pH indicator. Culture plates were imaged after 7 days of growth at room temperature.

Clip185’s ability to grow on Mannitol Salt Agar (MSA) and ferment mannitol

Clip185 grew on MSA (Figure 8A-B) and fermented mannitol in MSA to acid slowly compared to the fast mannitol fermentation displayed by the gram-positive bacterium. Staphylococcus aureus (Figure 8C). Acid production decreased the pH of MSA. With the decrease in pH, phenol red’s light red color changed to a yellow color (Figure 8).

Figure 8. Clip185 growth on Mannitol Salt Agar (MSA) medium.

(A) 3 days-growth of Clip185 on MSA plate. (B) 6 days-growth of Clip185 on MSA plate. (C) 3 days-growth of Staphylococcus aureus on MSA plate. Media plates were incubated at 30°C.

Starch hydrolysis test

Starch has two forms: linear amylose and branched amylopectin. The large sizes of amylose and amylopectin prevent these chemicals from being transported across the bacterial cell wall. Bacteria secrete out the α-amylase and oligo-1,6-glucosidase enzymes to degrade starch. These enzymes degrade extracellular starch into glucose/dextran which can be utilized by bacteria as carbon sources. A starch hydrolysis test is used to identify bacteria that can utilize extracellular starch as a carbon/energy source. Traditionally, potato dextrose agar (PDA) is used for starch hydrolysis tests. Clip185 did not grow well on PDA (Underlying data³⁷). It grew well on Mueller-Hinton Agar (MHA), which is a rich medium used for growing bacteria that have special nutritional requirements (fastidious bacteria) (Underlying data³⁷). Hence, we chose MHA, which contains 0.15 % starch, to test Clip185’s ability to hydrolyze starch.

We used Bacillus subtilis, which is known to hydrolyze starch, as a positive control in the experiment. We grew both Clip185 and B. subtilis on MHA for 3 days and imaged the plates (Figure 9A; Figure 9C). Gram iodine was then added to the both Clip185 and B. subtilis plates. Iodine reacted with the starch in MHA to produce a light blue color. Both Clip185 (Figure 9B) and B. subtilis (Figure 9D) tested positive in the starch hydrolysis test as there is a visible clear zone indicated by the black arrows, surrounding the growth of each of these two strains on the MHA due to the hydrolysis of starch.

Figure 9. Clip185 can hydrolyze starch.

(A) 3 days -growth of Clip185 at 30°C on Mueller-Hinton medium which contains 0.15% starch. (B) Clip185 Mueller-Hinton plate shown in (A) was treated with Gram iodine. (C) 3 days-growth of Bacillus subtilis at 30°C on Mueller-Hinton medium which contains 0.15% starch. (D) B. subtilis Mueller-Hinton plate shown in (C) treated with Gram iodine. Black arrows indicate the clear starch-hydrolyzed zones around the bacterial growth.

Partial 16S rRNA gene sequencing of Clip185

The 16S rRNA gene has nine hypervariable (V1-V9) and nine conserved regions (C1-C9).^38–40 We used the Escherichia coli 16S rRNA gene which is 1541 bp long (Figure 10A), as a reference in the 16S rRNA gene schematics in Figure 10. A total of 11 nucleotides (788-798) located within the C4 conserved region are completely conserved in bacteria.⁴¹ The 11 bp super-conserved region is represented by the black box within the C4 region in the schematics (Figure 10). The forward and reverse 16S rRNA gene-specific PCR primers are represented by black arrows in the 16S rRNA gene schematics (Figure 10A). A 445 bp amplicon was generated upon PCR amplification of the partial 16S rRNA gene of Clip185 (Underlying data⁴²). This 445 bp amplicon was cloned. A clone harboring the Clip185 amplicon was then sequenced to determine the taxonomic identity of Clip185.

Figure 10. PCR amplification of the partial 16S rRNA gene of Clip185 and NCBI-BLAST analyses.

(A) A schematic diagram showing the nine conserved and hypervariable regions in the 16S rRNA gene. The interspersed conserved regions (C1– C9) are shown in dark gray, and the hypervariable regions (V1–V9) are depicted in white. The black box within the C4 region represents 11 nucleotides (788-798 base pairs) that are invariant in bacteria.^8,9 Thick black arrows denote 16S rRNA gene-specific forward and reverse PCR primers in the C2 region and in the C4 region, respectively. The gene diagram is based on the 16S rRNA gene sequence of E. coli. See also Underlying data.⁴² (B) A schematic diagram showing the nucleotide changes in Clip185 in the 16S rRNA region spanning the C2 and C4 regions in comparison to the best NCBI- BLAST hit in 2019 (score of 800; E-value 0 and percent identity of 99.10%): Microbacterium binotii strain PK1-12M 16S ribosomal RNA gene, partial sequence (GenBank Accession #: MN428150.1). (C) A schematic diagram showing the nucleotide changes in Clip185 in the 16S rRNA region spanning the C2 and C4 regions in comparison to the best NCBI- BLAST hit in 2021 (score of 806; E-value 0 and percent identity of 99.33%): Microbacterium sp. strain MDP6 16S ribosomal RNA gene, partial sequence (GenBank Accession #: MK128451.1). Black nucleotides show the native nucleotides in the BLAST hits Microbacterium binotii strain PK1-12M and Microbacterium sp. strain MDP6 that were substituted by the depicted red nucleotides in Clip185 16S rRNA gene sequence. The black bold numbers within the parenthesis beside the nucleotides show the specific nucleotide positions where the nucleotide changes have occurred. Nucleotide positions shown in the figures have been assigned according to that of the 16S rRNA gene sequence of E. coli.

In 2019, NCBI-nucleotide BLAST analyses detected Microbacterium binotii strain PK1-12M (GenBank Accession #: MN428150.1) as the nearest match to Clip185 based on the partial 16S ribosomal RNA gene sequence, with a score of 800, an E-value of 0 and percent identity of 99.10%. Four nucleotide substitutions were identified in Clip185’s 16S rRNA partial gene sequence relative to that in Microbacterium binotii strain PK1-12M (Figure 10B). In the C4 region, one transition and one transversion were detected (Figure 10B). We found one transition in the C2 region and one transversion in the 11 bp super-conserved sub-region within C4 region (Figure 10B). We deposited this partial 16S rRNA gene sequence of Clip185 in NCBI GenBank with the description: Microbacterium binotii strain PK1-12M variant 16S ribosomal RNA gene, partial sequence (Accession number: MN633284.1). In early 2021, BLAST analyses detected a new match: Microbacterium sp. strain MDP6 (Accession #: MK128451.1) with a score of 806, E-value of 0 and percent identity of 99.33%) (NCBI last accessed 8-26-21, Figure 10C). We detected three nucleotide changes in the 16S rRNA gene of Clip185 with respect to this new hit: one transition in the C2 region, one transversion in the 11 bp super-conserved sub-region within C4 region and one deletion in the C4 region (Figure 10C). Analyses of available genome sequences of Microbacterium sp. on NCBI revealed that whole genome sequences for Microbacterium binotii strain PK1-12M and Microbacterium sp. strain MDP6 are not available (NCBI data last accessed on 8-26-2021).

Spectrophotometric analyses of pigments synthesized by Clip185

Carotenoids are synthesized from the precursor isopentenyl pyrophosphate and its isomer dimethylallyl pyrophosphate in the terpenoid biosynthetic pathway¹⁶ (Figure 11). Two molecules of geranylgeranyl diphosphate condense to form phytoene (Figure 11). Lycopene (a red C₄₀ carotenoid) is formed by the four-step enzymatic desaturation of phytoene (Figure 11). Lycopene is diprenylated and hydroxylated to form C₅₀ carotenoids^16,17 (Figure 11). To date, three different C₅₀ carotenoid biosynthetic pathways are known in the literature: 1) the ε-cyclic C₅₀ carotenoid decaprenoxanthin biosynthetic pathway in Corynebacterium glutamicum;^16,43,44 2) the β-cyclic C₅₀ carotenoid C.p.450 biosynthetic pathway in Dietzia sp. CQ4⁴⁵ and; 3) the γ-cyclic C50 carotenoid sarcinaxanthin biosynthetic pathway in Micrococcus luteus NCTC2665.¹⁸ Microbacterium belongs to the gram-positive order of Actinomycetales. Many members of Actinomycetales produce the C₅₀ carotenoid, decaprenoxanthin.¹⁶

Figure 11. A schematic representation of the C₅₀ biosynthesis pathways in bacteria.

C₄₀ carotenoid lycopene is diprenylated to form an acyclic C₅₀ carotenoid, flavuxanthin. Flavuxanthin undergoes cyclization in three different ways to form three types of natural C₅₀ carotenoids in bacteria. Substrates and intermediate products names are abbreviated and are shown in black. Genes encoding enzymes catalyzing specific reactions/steps in the pathways are shown in blue. Abbreviations: IPP, isopentenyl pyrophosphate; DMPP, dimethylallyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farensyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate; C.p. 496, 2,2′-Bis-(3-methylbut-2-enyl)-3,4,3′,4′-tetradehydro-1,2,1′,2′-tetrahydro-ψ, ψ-carotene-1, 1-diol; C.p.450, 2,2′-Bis-(4-hydroxy-3- methybut-2-enyl)-β, β-carotene. Superscripted numbers: 1 denotes lbtC in the lbtBC fused gene; 2 denotes lbtA and lbtB in the lbtBC fused gene; 3 denotes Micrococcus luteus gene; 4 denotes Corynebacterium glutamicum genes and 5 denotes Dietzia sp. CQ4 genes.

Since our 16S rRNA partial gene sequencing showed that Clip185 is a Microbacterium sp. we extracted the yellow pigment from Clip185 (Figure 12A; Figure 12C) and performed spectrophotometric analyses to determine whether Clip185 produces decaprenoxanthin (Figure 12). As actinobacteria C. glutamicum and Kocuria rhizophila are known to produce decaprenoxanthin,^16,30 we used extracted pigments from these two bacterial strains (Figure 12A; Figure 12C) as positive controls in the spectrophotometric assays (Figure 12).

Figure 12. Spectrophotometric analyses of extracted pigments of Clip185.

(A) Falcon tubes containing methanol-extracted pigments of Corynebacterium glutamicum (X) and Clip185 (Y). (B) Absorption spectra of extracted pigments C. glutamicum (X, dark blue spectrum) and Clip185 (Y, pale blue spectrum). (C) Falcon tubes containing methanol-extracted pigments of Kocuria rhizophila (Z) and Clip185 (Y). (D) Absorption spectra of extracted pigments K. rhizophila (Z, dark blue spectrum) and Clip185 (Y, pale blue spectrum). Three major absorption peaks of decaprenoxanthin are labeled.

We found that the C. glutamicum (X spectrum in Figure 12B) and K. rhizophila (Z spectrum in Figure 12D) pigment absorption spectra exhibited three major absorption peaks that are representative of decaprenoxanthin absorption at 413 nm, 437 nm and 467 nm as described in the literature.^30–32 These three absorption peaks from C. glutamicum absorption spectrum X and K. rhizophila absorption spectrum Z very nicely overlaid against the three absorption peaks from the Clip185 absorption spectrum (Y spectrum in Figure 12B and Figure 12D). This result strongly suggests the presence of decaprenoxanthin in Clip185. HPLC and/or LC/MS analyses could confirm the presence of decaprenoxanthin in Clip185. However, we do not have access to such analytical tools at our institution.

Light-regulation of decaprenoxanthin production in Clip185

It is known that in non-phototrophic bacteria, biosynthesis of C₅₀ carotenoids can be modulated by growth conditions (e.g. presence/absence of light).^15,46,47 We studied the production of decaprenoxanthin in Clip185, C. glutamicum and K. rhizophila under dim light (20-25 μmol m^-2s^-1). In dark conditions, Clip185 appears cream color with a light yellowish tinge (Figure 13A, left). Decaprenoxanthin production in Clip185 was induced under light (Figure 13A, right). Decaprenoxanthin production is strongly induced under light in C. glutamicum as stated in published literature¹⁹ in contrast to that in Clip185 (Figure 13A; Figure 13B). K. rhizophila produces decaprenoxanthin constitutively irrespective of light conditions (Figure 13C).

Figure 13. Decaprenoxanthin production under light and dark on LB media.

(A) Decaprenoxanthin production in Clip185 under darkness and light. (B) Decaprenoxanthin production in C. glutamicum under darkness and light. (C) Decaprenoxanthin production in K. rhizophila under darkness and light. Bacterial culture plates were exposed to darkness or light for 5 days at 22°C and then imaged. Light intensity was 20-25 μmol m^-2s^-1.

Growth of Clip185 on LB media containing toxic concentrations of heavy metals

Since Microbacterium sp. has been isolated from heavy metal-contaminated environments,¹⁵ we decided to test whether Clip185 could tolerate toxic concentrations (ranging from 0.5 mM - 6 mM) of six different heavy metals: cadmium, cobalt (Co²⁺), zinc, copper (Cu²⁺), nickel (Ni²⁺) and chromium [Cr⁶⁺] (Figures 14-19). Clip185 can grow well with yellow pigmentation on 0.5 mM of cadmium plate (Figure 14A) but grows slowly on 1 mM cadmium plate and retains pigment production (Figure 14B). Clip185 slowly grows on 2 mM cadmium plate, but over time stops growing and is white in color (Figure 14C; Figure 14D). Growth of Clip185 for 8 days and 28 days on LB plate are shown for comparisons (Figure 14E). Clip185 grew well with yellow pigmentation on 0.5 mM cobalt plate (Figure 15A) but failed to grow on 1 mM cobalt plate (Figure 15B). Clip185 grew well with yellow pigmentation on 0.5 mM and 1 mM zinc plates (Figure 16A-B) but grew very slowly on 2 mM zinc plate (Figure 16C). Clip185 grew well with yellow pigmentation on 0.5 mM copper plate (Figure 17A) but failed to grow on 1 mM copper plate (Figure 17B). Clip185 grew well with yellow pigmentation on 1 mM and 2 mM nickel plates (Figure 18A-B) but failed to grow on 4 mM nickel plate (Figure 18C). Clip185 could grow well with yellow pigmentation on 2 mM and 4 mM chromium plate (Figure 19A-D) but grew extremely slowly with yellow pigmentation on 6 mM chromium plate (Fig 19E-H). Clip185 failed to grow on 10 mM of chromium plate after 90 days of incubation (Underlying data⁴⁸).

Figure 14. Growth of Clip185 on LB media containing Cadmium.

(A) 8 days-growth of Clip185 on LB media containing 0.5 mM Cadmium. (B) 8 days (left) and 28 days (right)-growth of Clip185 on LB media containing 1 mM Cadmium. (C) 8 days-growth of Clip185 on LB media containing 2 mM Cadmium. (D) 45 days-growth of Clip185 on LB media containing 2 mM Cadmium. (E) 8 days (left) and 28 days (right)-growth of Clip185 on LB media (control). Number of days of growth are labeled on the figures. Light intensity was 10-15 μmol m^-2s^-1.

Figure 15. Growth of Clip185 on LB media containing Cobalt.

(A) 8 days-growth of Clip185 on LB media containing 0.5 mM Cobalt. (B) 8 days (left) and 28 days (right)-growth of Clip185 on LB media containing 1 mM Cobalt. Number of days of growth are labeled on the figures. Light intensity was 10-15 μmol m^-2s^-1.

Figure 16. Growth of Clip185 on LB media containing Zinc.

(A) 8 days-growth of Clip185 on LB media containing 0.5 mM Zinc. (B) 8 days-growth of Clip185 on LB media containing 1 mM Zinc. (C) 8 days (left) and 28 days (right)-growth of Clip185 on LB media containing 2 mM Zinc. Number of days of growth are labeled on the figures. Light intensity was 10-15 μmol m^-2s^-1. Light intensity was 10-15 μmol m^-2s^-1.

Figure 17. Growth of Clip185 on LB media containing Copper.

(A) 8 days-growth of Clip185 on LB media containing 0.5 mM Copper. (B) 8 days (left) and 28 days (right)-growth of Clip185 on LB media containing 1 mM Copper. Number of days of growth are labeled on the figures. Light intensity was 10-15 μmol m^-2s^-1.

Figure 18. Growth of Clip185 on LB media containing Nickel.

(A) 8 days-growth of Clip185 on LB media containing 1 mM Nickel. (B) 8 days-growth of Clip185 on LB media containing 2 mM Nickel. (C) 8 days (left) and 28 days (right)-growth of Clip185 on LB media containing 4 mM Nickel. Number of days of growth are labeled on the figures. Light intensity was 10-15 μmol m^-2s^-1.

Figure 19. Growth of Clip185 on LB media containing hexavalent Chromium (Cr⁶⁺).

(A) 8 days-growth of Clip185 on LB media containing 2 mM Cr⁶⁺. (B) 28 days-growth of Clip185 on LB media containing 2 mM Cr⁶⁺. (C) 8 days-growth of Clip185 on LB media containing 4 mM Cr⁶⁺. (D) 28 days-growth of Clip185 on LB media containing 4 mM Cr⁶⁺. (E) 8 days-growth of Clip185 on LB media containing 6 mM Cr⁶⁺. (F) 28 days-growth of Clip185 on LB media containing 6 mM Cr⁶⁺. (G) 67 days-growth of Clip185 on LB media containing 6 mM Cr⁶⁺. (H) 90 days-growth of Clip185 on LB media containing 6 mM Cr⁶⁺. Number of days of growth are labeled on the figures. Light intensity was 10-15 μmol m^-2s^-1. See also Underlying data.⁴⁸

Underlying data

NRRL ARS Culture Collection: Clip185/Microbacterium binotii strain PK1-12M variant; Accession number: B-65609.

NCBI GenBank: Microbacterium binotii strain PK1-12M variant; 16S ribosomal RNA gene, partial sequence, Accession number MN633284.1: https://www.ncbi.nlm.nih.gov/nuccore/MN633284.1.

Figshare: Antibiotic sensitivity data of the bacterial strain Clip185/Microbacterium binotii strain PK1-12M variant (Accession number: MN633284.1) and Chlamydomonas reinhardtii strain 4A+ from disc diffusion antibiotic susceptibility tests, https://doi.org/10.6084/m9.figshare.14668914.³⁵

This project contains the following underlying data:

Figshare: Image of a LB plate with single colonies of Clip185/Microbacterium binotii strain PK1-12M variant (Accession number: MN633284.1), https://doi.org/10.6084/m9.figshare.14669214.³⁴

This project contains single colonies of Clip185 Clip185/Microbacterium binotii strain PK1-12M variant (Accession number: MN633284.1) on a LB plate.

Figshare: Images of Clip185/Microbacterium binotii strain PK1-12M variant (Accession number: MN633284.1) growth on Potato Dextrose Agar (PDA) and Mueller-Hinton Agar (MHA), https://doi.org/10.6084/m9.figshare.14669397.³⁷

This project contains an image that shows the growth of Clip185 on Potato Dextrose Agar (PDA) and Mueller-Hinton Agar (MHA).

Figshare: Image of the DNA agarose gel showing the electrophoretic analysis of PCR products obtained using the 16S rRNA gene-specific primers on Clip185/Microbacterium binotii strain PK1-12M variant (Accession number: MN633284.1) genomic DNA, https://doi.org/10.6084/m9.figshare.14669400.⁴²

This project contains one DNA agarose gel image that shows DNA agarose gel electrophoresis analysis of PCR products obtained using the 16S rRNA gene-specific primers on Clip185/Microbacterium binotii strain PK1-12M variant (Accession number: MN633284.1) genomic DNA.

Figshare: Image of growth of Clip185/Microbacterium binotii strain PK1-12M variant (Accession number: MN633284.1) on a LB plate containing 10 mM hexavalent Chromium (Cr⁶⁺), https://doi.org/10.6084/m9.figshare.14669421.⁴⁸

This project contains one image of a 10 mM hexavalent Chromium (Cr⁶⁺) containing LB media plate on which Clip185/Microbacterium binotii strain PK1-12M variant (Accession number: MN633284.1) was streaked and incubated for 90 days for growth analysis.

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).