The Effect of Different Nutritional Resources on Gram-Negative Bacterial Communities Isolated from Marine Environments

The effect of different nutritional resources on gram-negative bacterial communities isolated from marine environments

Shannon Lynch Microbial Diversity 2018 Mini Project Final Report

Introduction The rise of antibiotic and pesticide resistance in clinical, agricultural, and environmental settings had prompted new ways to combat diseases caused by microbial pathogens across multiple disciplines. In particular, treatment options for many multidrug-resistant gram-negative bacterial human pathogens have become limited and pose an urgent threat to human health (Livermore, 2011). One alternative to antibiotics that has been explored to address the problem is the use of Bdellovibrio as potential biocontrol agents (Kadouri & O’Toole, 2004).

Bdellovibrio are small gram-negative predatory bacteria that invade the periplasm of other gram- negative bacteria in a variety of wet, aerobic environments, digesting and killing them from the inside (Sockett, 2009; Stolp, 1973). In the attack phase of their life cycle, Bdellovibrio cells are tiny (< 0.2µm) and highly mobile (Sockett, 2009). Once they invade prey cells, they grow and divide, causing the prey cell to form a structure called a bdelloplasts, which then lyses to release a new generation of Bdellovibrio (Sockett, 2009). With a broad host range that includes plant, animal, and human pathogens (Rendulic et al., 2004), and the capacity to kill many antibiotic resistant pathogens in vitro without adversely affecting animal health (Negus et al., 2017), Bdellovibrio shows promise as a control measure in clinical settings. However, studies appear to focus on the effects of Bdellovibrio in the presence of a single prey under various conditions (Kadouri & O’Toole, 2004, Pantanella et al., 2017). In predation assays against Escherichia coli, Medina & Kadouri (2009) demonstrated that a nutrient rich environment favored the development of axenic host independent biofilm variants, but these variants also switch to predatory growth when nutrients are depleted.

To understand how microbial community composition changes in the presence of Bdellovibrio in different nutritive environments, the predatory activities of Bdellovibrio must be explored in a multispecies context to better understand its efficacy as a biocontrol agent in applied settings where more complex communities occur. While enriching for bioluminescent bacteria in our first week of the Microbial Diversity Course, we observed signs of Bdellovibrio on a wet mount collected from plaques on a lawn of many different bacteria colonies growing on saltwater complete (SWC) agar media. Bacteria were recovered from sea water collected from Eel Pond on the MBL Village Campus, presenting a unique opportunity to explore changes in a bacterial predator-prey system recovered from a natural environment, and experimentally assess dynamics when nutritional resources are limited or abundant.

Statement of Hypothesis: 1) Abundances of bacteria in nutrient rich media will exhibit a lognormal distribution when growing together in the absence of Bdellovibrio, consistent with community patterns; 2) Relative abundances of bacteria in nutrient poor media may or may not change in the absence of Bdellovibrio, but the overall community load will reduce; 3) In the presence of Bdellovibrio, I expect community composition will either remain unchanged or change in various ways depending on the relative susceptibility of community members to Bdellovibrio and its attack rate in either medium.

Methods

Microcosm Experiment To assess composition changes of a gram-negative bacteria community in the presence of Bdellovibrio when resources are either abundant or limited, I assembled a synthetic community consisting of five marine bacterial strains in either nutrient rich or nutrient poor liquid media with or without Bdellovibrio sp. and determined their relative abundances at the initial and final stage The effect of different nutritional resources on gram-negative bacterial communities isolated from marine environments

Shannon Lynch Microbial Diversity 2018 Mini Project Final Report

of the experiment (Tinitial = 0 h; Tfinal = 48 h). Selected nutrient rich and nutrient poor media included Sea Water Complete (SWC) and Growth Curve Media (GCM) (MBL 2018, Chapter 2), and prey bacteria were isolated from marine environments (Table 1) using previously described protocols (Hansel & Frances, 2006; MBL 2018, Chapter 2). After colony PCR of pure isolates, identities were confirmed through BLASTn searches of sequence data from the 16S rRNA gene in the National Center for Biotechnology Information (NCBI) database.

Table 1. Bacterial strains used in the microcosm experiment. Strain Species Classification L-5 Source 12 Vibrio sp. Vibrionaceae Garbage Beach, Woods Hole, MA 56 Pseudomonas umsongenus Pseudomonadaceae Eel Pond, Woods Hole, MA 122 Vibrio cyclitrophicus Vibrionaceae Eel Pond, Woods Hole, MA 162C2LK2G Tenacibaculum discolor Flavobacteriaceae Woods Hole, MA (Lynn Kee, Microbial Diversity Course 2016) AzwK-3b Roseobacter sp. Rhodobacteraceae Elkhorn Slough, California (Hansel & Francis, 2006; Learman & Hansel, 2014)

Synthetic Community Assembly I grew each prey strain from a single colony in GCM at 30 C with shaking and then inoculated 1:1 ratios of overnight cultures into 50-mL flasks containing 20mL of either SWC or GCM. The initial OD600 in each flask was 0.03 and communities were maintained at 30 C with shaking for the duration of the experiment. I used three replicates for each of the following treatments:

1) prey community in SWC with Bdellovibrio; 2) prey community in SWC without Bdellovibrio; 3) prey community in GCM with Bdellovibrio; 4) prey community in GCM without Bdellovibrio.

For controls, I used one replicate each of SWC and GCM only, and one replicate each of SWC and GCM with Bdellovibrio only.

To generate predator inoculum, I re-suspended five three-week old plaques from spread plates intended for Vibrio enrichments (MBL 2018, Chapter 2) in 2mL SWC and passed the suspension through a 0.2µm filter to remove non-Bdellovibrio bacteria. To the filtrate, I added 50µL of a 100µL concentrated sample from Buzzards Bay (2018.Groups3_4.12.07.2, Table 3) that was processed for virus isolations (MBL 2018, Chapter 18), but instead enriched for Bdellovibrio. I added 150µL of the filtrate-concentrated Bdellovibrio mixture to each Bdellovibrio treatment flask. I used the remaining filtrate mix and part of the remaining concentrated Buzzards Bay sample in a Top Agar Spot Test to confirm the presence of Bdellovibrio and produce additional inoculum (see below).

Unfortunately, I did not recover plaques from the assay, indicating that I did not have viable inoculum at my initial time point. As such, I pursued an enrichment of Bdellovibrio to repeat the experiment with sufficient inoculum, assessed community changes in the present experiment without a predator, and further assessed the effect of different nutrient environments on the growth and survival of two of the five bacterial strains in a competition experiment (see below).

Relative Abundance Measures To assess changes in relative abundances of bacterial strains for each treatment, I sampled 1mL from each flask at Tinitial and Tfinal, and froze each sample at -20 C for further processing. I later extracted the total genomic DNA from each sample using a Maxwell® RSC Cultured Cells DNA Kit and associated protocols (Promega Corporation), quantified extracted DNA using the The effect of different nutritional resources on gram-negative bacterial communities isolated from marine environments

Shannon Lynch Microbial Diversity 2018 Mini Project Final Report

Integrated QuantusTM Fluorometer (Promega Corporation), and amplified 7ng/µL of the DNA from each sample using the GoTaq Green Master Mix and 515F-EMP and 926R-EMP primers for 16S rRNA amplicon sequencing. After amplicon sequencing, I processed fastq files for each sample using the dada2 denoise-paired command in the Qiime2 pipeline, aligned sequences using mafft, and assigned taxonomy using the 13_8 99% release of the Greengenes reference OTUs. Relative abundances of OTUs at taxonomic level 5 were visualized on bar charts for each treatment/time point.

Interspecific Competition Experiment For this experiment, I assessed the effect of nutritional resources on interspecific competition of two species of gram negative bacteria. First, I conducted in vitro antibiotic assays to determine the appropriate concentration of an antibiotic that would effectively eliminate one competitor. I used results from these assays to inform the selection of two strains for the competition assay and to develop a tool to quantify abundances of taxa on spread plates that were growing together in liquid culture.

In Vitro Antibiotic Assays For the first assay, I conducted a disk-diffusion antibiotic sensitivity test to assess the extent to which the five strains in the microcosm experiment were affected by ten different antibiotics (Table 2, Fig. 1). Briefly, I spread plated 100µL of overnight cultures representing each strain and 100 µL of sterile water as a control onto SWC agar plates. I then added 5µL of each antibiotic of an appropriate working concentration (www.adgene.org) to 5mm diameter sterile filter paper disks and immediately placed one disk representing each antibiotic around the perimeter of a corresponding strain plate (Fig.1). Sterile disks without antibiotics were applied to plates as an additional control. After 24 h, I observed that 100µg/mL ampicillin did not inhibit the growth of P. umsongenus (no zone of inhibition), but inhibited T. discolor growth (clear zone of inhibition) (Fig. 1). As such, I decided to utilize these species for the competition assay and further tested ampicillin inhibition at different antibiotic and strain concentrations.

Table 2. Working concentrations of different antibiotics used in the preliminary screening assay. Working Concentration Antibiotic (µg/mL) Ampicillin 100 Carbenicillin 100 Chloramphenicol 35 Gentamicin Sulfate 10 Kanamycin 50 Lysozyme 10 Spectinomycin 50 Streptomycin 100 Tetracycline 10 Vancomycin 100

Figure 1. Disk-diffusion test results of ten antibiotics In the second assay, I determined the optimal on plates containing bacteria species selected for the ampicillin and bacterial concentrations that competition assay. Red arrows indicate the absence would eliminate T. discolor growing in liquid and presence of a zone of inhibition produced by 100 culture with P. umsongenus and produce µg/mL ampicillin on Pseudomonas umsongenus and Tenacibaculum discolor. countable P. umsongenus colony forming units The effect of different nutritional resources on gram-negative bacterial communities isolated from marine environments

Shannon Lynch Microbial Diversity 2018 Mini Project Final Report

(CFUs). I performed a ten-fold serial dilution (1 x 10-7 maximum) of T. discolor, P. umsongenus, and a 1:1 mixture of both in liquid culture (initial OD600= 0.2). I then spot plated 10µL of each dilution suspension on SWC media amended with 100µg/mL, 50µg/mL, or 10µg/mL ampicillin. After 24 h I was able to easily count P. umsongenus CFUs from a 1 x 10-6 diluted mixed culture and thus used this dilution to quantify species abundances in the competition assay.

Interspecific Competition Assay For the competition experiment, I inoculated two replicate flasks each containing SWC or GCM with T. discolor, P. umsongenus, and a 1:1 mixture of both, and quantified their abundance from CFUs on spread plates at the initial and final stage of the experiment (Tfinal= 0 h; Tfinal =14 h). All treatments were maintained in 50-mL flasks as described above, and the initial OD600 for each -6 20-mL suspension was 0.02. At Tinitial and Tfinal, I spread plated 100µL of a 1 x 10 dilution from each flask onto SWC and SWC amended with 100µg/mL ampicillin (SWC-amp) and counted CFUs 15 hours later. Abundance of each species growing together was determined from the difference between CFUs counted on SWC and those counted on SWC-amp, the latter of which eliminated T. discolor colonies. To account for differences in kinetics between each bacterium alone in nutrient rich or nutrient poor media, I additionally measured their OD600 as a proxy for growth every hour for 10 hours and plotted measurements on a semi-log scale.

Bdellovibrio Enrichment

Enrichment Strategies To produce a sufficient inoculum load of attack-phase Bdellovibrio for use in a community microcosm experiment, I adapted published protocols designed to enrich for Bdellovibrio (Hobley, 2005; Jurkevitch, 2006; Lambert & Sockett, 2008) and performed a Top Agar Spot Test on existing samples (MBL 2819, Chapter 18).

Given that wild-type Bdellovibrio are prey-dependent, the general approach involved the use of pure cultures of gram-negative bacteria for prey (Table 3), a sample potentially containing wild-type Bdellovibrio (Table 4), and media that favors Bdellovibrio predation, growth, and reproduction over prey growth (Lambert & Sockett, 2008). For media, I used combinations of SWC and GCM liquid or agar media, sterile sea water (SW; Hobley, 2005), and HEPES buffer amended with CaCl2 (Lambert & Sockett, 2008). The HEPES buffer is specifically used to make prey lysates containing a suspension of prey cells in the buffer that are the predominant nutrient source for Bdellovibrio; the CaCl2 increases the efficiency of prey lysis (Lambert & Sockett, 2008). I enlisted a modular approach using different prey, media, and sample sites following liquid enrichment protocols and a double-layer overlay technique (Rittenberg, 1982).

Table 3. Sample ID, site, and coordinates for each sample collected from marine environments in southern Cape Cod for Bdellovibrio enrichment. Sample ID Site Coordinates Comments 2018.Groups3_4.12.07.2 Buzzards Bay, 9 m depth 41.548060, -70.711886 P. 20 in sample book. Processed by Dr. Maria Bautista to enrich for viruses; unintentionally recovered Bdellovibrio. Stored at 4 C. 2018.SL.05.08.1 Eel Pond 41.526406, -70.671780 2018.SL.05.08.1 Garbage Beach 41.525138, -70.673567

The effect of different nutritional resources on gram-negative bacterial communities isolated from marine environments

Shannon Lynch Microbial Diversity 2018 Mini Project Final Report

Table 4. Prey species utilized for Bdellovibrio enrichments. Strain Species Source Site APA752 Escherichia coli Whetmore et al. (2015) Retrieved from genetics week laboratory activities 12 Vibrio sp. 2018.PSL.18.07.15 Garbage Beach 13 Vibrio harveyi 2018.PSL.18.07.15 Garbage Beach 14 Vibrio harveyi 2018.PSL.18.07.15 Garbage Beach 15 Vibrio harveyi 2018.PSL.18.07.15 Garbage Beach 41 Vibrio cyclitrophicus 2018.SMS.09.07.02 Marine Resource Center unbleached coral 54 Pseudoalteromonas sp. 2018.SMS.09.07.2 Marine Resource Center unbleached coral. Prey source in Bdellovibrio glycerol stock 2018.07.18.MB.1 recovered from sample 2018.Groups3_4.12.07.2.

Top Agar Spot Test To revive and grow Bdellovibrio from a -80◦C frozen glycerol stock (2018.07.12.MB.1), I resuspended a small piece of frozen sample in 30µL SWC and spotted three 10µL aliquots onto 0.7% SWC top agar containing E. coli (adapted from MBL 2018, Chapter 18). This approach was repeated using the remaining inoculum from the initial microcosm experiment.

Liquid Enrichments For liquid enrichments, I mixed 750µL sample, 1mL pure culture prey, and 10mL of media (adapted from Hobley, 2005), or 10mL sample and 40mL media. I used the latter approach because Bdellovibrio was unintentionally isolated from Buzzards Bay using this method (Table 3 & 4). Samples were either passed through a 0.2µm filter or left unfiltered prior to mixing. I incubated suspensions at 30◦C with shaking and checked them daily for turbidity and for Bdellovibrio activity under the microscope at 100x magnification using a Zeiss Imager A2 microscope. I diluted (1x; 1 x 10-4; 1 x 10-6) and plated enrichments showing signs of bdelloplasts and small, highly motile cells on overlay plates with corresponding prey species (Hobley, 2005).

Double Layer Overlay Technique Adapting procedures from Hobley (2005), I added 5mL 0.7% SWC or GCM agar mixed with 200µL overnight prey culture and 100µL of sample onto 1.5% SWC or GCM agar plates. After setting, I placed plates in the 30 C incubator and monitored them daily for zones of clearing.

Microscopy Select enrichments containing cells resembling bdelloplasts were further used for live imaging on a Nikon Eclipse Ti2 inverted microscope system (Nikon Instruments Inc., Melville, NY) to test whether these structures lysed prey cells, begot more Bdellovibrio, and to confirm the presence of bdelloplasts in my enrichments. To prep samples for imaging, I pipetted 1µL of sample onto a 35mm #1.5 glass bottom dish with a 20mm bottom well and covered the sample with a 2% GCM agarose pad. I then placed the dish into a 30 C stage top incubator on the microscope and took snapshots every three seconds for four hours using Nikon NIS-Elements imaging software (Nikon Instruments Inc.).

Results and Discussion

Microcosm Experiment As I did not have enough viable inoculum to assess composition changes in a synthetic gram- negative bacteria community in the presence of Bdellovibrio in different nutritive environments, I instead examined the effect of different nutritional resources on their relative abundances in the absence of a predator. The effect of different nutritional resources on gram-negative bacterial communities isolated from marine environments

Shannon Lynch Microbial Diversity 2018 Mini Project Final Report

For the Low Nutrient treatment at Tinitial, the total genomic DNA in all three replicates amplified during PCR prior to amplicon sequencing; DNA for two of the three replicates amplified in all other treatments. Only one of the two replicates in the High Nutrient treatment at Tfinal came back from sequencing with reads. Dada2 ran on paired end reads produced 29,969 – 73,922 non-chimeric reads across samples. Of the 147 features with assigned taxonomy using the Greengenes reference OTUs, 28 were assigned to Pseudoalteromonadaceae. However, 23 of those features (82%) returned Vibrio spp. with 99 – 100% identity in BLASTn searches. This discrepancy is attributed to annotation errors that have incorrectly assigned the Vibrio spp. to Pseudoalteromonadaceae in Greengenes (Lydon & Lipp 2018). As such, Vibrionaceae dominated all samples, accounting for roughly 67 – 95% by relative abundance (Fig. 2). Unfortunately, abundances of the two strains in Vibrionaceae could not be discerned with these methods. The next most abundant taxon across samples (5 – 33% by relative abundance) was Flavobacteriaceae, which contains the genus Tenacibaculum. Rhodobacteraceae (containing Roseobacter sp.), Pseudomonadaceae (containing Pseudomonas umsongenus) were present in very low relative abundances across all samples (<1%).

Initial Final Initial Final

k__Bacteria;p__Tenericutes;c__Mollicutes;o__Mycoplasmatales;f__Mycoplasmataceae 100% k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Vibrionales;f__Vibrionaceae k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Vibrionales;f__Pseudoalteromonadaceae k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Vibrionales;__ k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Thiotrichales;f__Thiotrichaceae 90% k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Pseudomonadales;f__Pseudomonadaceae k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Oceanospirillales;f__Saccharospirillaceae k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Oceanospirillales;f__Oceanospirillaceae 80% k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Oceanospirillales;f__Halomonadaceae k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Legionellales;f__Francisellaceae k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Enterobacteriales;f__Enterobacteriaceae k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Chromatiales;f__Halothiobacillaceae 70% k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Alteromonadales;f__J115 k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Alteromonadales;f__Colwelliaceae k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Alteromonadales;f__Alteromonadaceae k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Alteromonadales;__ 60% k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;__;__ k__Bacteria;p__Proteobacteria;c__Alphaproteobacteria;o__Sphingomonadales;f__Erythrobacteraceae k__Bacteria;p__Proteobacteria;c__Alphaproteobacteria;o__Rickettsiales;f__Pelagibacteraceae k__Bacteria;p__Proteobacteria;c__Alphaproteobacteria;o__Rhodospirillales;f__Rhodospirillaceae 50% k__Bacteria;p__Proteobacteria;c__Alphaproteobacteria;o__Rhodobacterales;f__Rhodobacteraceae k__Bacteria;p__Proteobacteria;c__Alphaproteobacteria;o__;f__ k__Bacteria;p__Proteobacteria;c__Alphaproteobacteria;__;__ 40% k__Bacteria;p__Planctomycetes;c__Planctomycetia;o__Pirellulales;f__Pirellulaceae k__Bacteria;p__Planctomycetes;c__OM190;o__agg27;f__ k__Bacteria;p__Cyanobacteria;c__Synechococcophycideae;o__Synechococcales;f__Synechococcaceae k__Bacteria;p__Cyanobacteria;c__Oscillatoriophycideae;o__Chroococcales;f__Xenococcaceae 30% k__Bacteria;p__Chlorobi;c__Chlorobia;o__Chlorobiales;f__Chlorobiaceae k__Bacteria;p__Bacteroidetes;c__[Saprospirae];o__[Saprospirales];f__Saprospiraceae k__Bacteria;p__Bacteroidetes;c__[Rhodothermi];o__[Rhodothermales];f__[Balneolaceae] k__Bacteria;p__Bacteroidetes;c__Sphingobacteriia;o__Sphingobacteriales;f__NS11-12 20% k__Bacteria;p__Bacteroidetes;c__Flavobacteriia;o__Flavobacteriales;f__Flavobacteriaceae k__Bacteria;p__Bacteroidetes;c__Flavobacteriia;o__Flavobacteriales;f__Cryomorphaceae k__Bacteria;p__Bacteroidetes;c__Flavobacteriia;o__Flavobacteriales;f__ k__Bacteria;p__Bacteroidetes;c__Cytophagia;o__Cytophagales;f__Flammeovirgaceae 10% k__Bacteria;p__Bacteroidetes;c__Cytophagia;o__Cytophagales;__ k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__ k__Bacteria;p__Bacteroidetes;__;__;__ 0% k__Bacteria;p__Actinobacteria;c__Acidimicrobiia;o__Acidimicrobiales;f__OCS155 k__Bacteria;__;__;__;__ Low Nutrients High Nutrients

Vibrionaceae Flavobacteriaceae (Tenacibaculum sp.) Figure 2. Relative abundances of taxa (level 5) growing together for 48 hours in low and high nutrient liquid media in a microcosm experiment. Annotation errors in Greengenes incorrectly assigned Vibrio spp. in the Vibrionaceae to Pseudoalteromonadaceae.

There are a number of possible explanations as to why taxa were not present in equal ratios in low and high nutrient media at Tinitial. One explanation is that species were introduced at equivalent OD600 under the assumption that equivalent ODs confer equivalent CFUs. As I did not empirically determine the relationship between CFUs and OD600 for the species in my communities, they were likely inoculated into media at unequal ratios. Additionally, there was a lag between setting up the experiment and sampling for amplicon sequencing at Tinitial. This could have been enough time for community changes to occur and may explain the differences in relative abundances at Tinitial between dominant taxa in low and high nutrients.

The effect of different nutritional resources on gram-negative bacterial communities isolated from marine environments

Shannon Lynch Microbial Diversity 2018 Mini Project Final Report

Regardless, the general pattern suggests that species ratios change over time and these changes differ depending on nutritional resources. Whereas the ratio between Vibrionaceae Flavobacteriaceae decreases in low nutrients after 48 hours, the ratio increases when nutrients are abundant. These conclusions are tentative, however, for a number of reasons. First, samples were taken from the center of each flask and may not reflect actual community compositions given that species of Pseudomonas attach to surfaces (Madigan et al., 2018). This may explain why the Pseudomonadaceae is poorly represented in the results. Second, conclusions are based on a single replicate at Tfinal in the high nutrient treatment and few replicates across all treatments. Third, annotation errors in Greengenes may have confounded these results for other taxa.

Interspecific Competition Experiment

Growth Curve Analysis Visual inspection of growth curves for both species in different nutritive environments indicates differences within and among species, but with caveats (Fig. 3). P. umsongenus exhibited an overall greater load when nutrients were abundant and grew at a faster rate than P. umsongenus in a low nutrient 600 environment. Regrettably, I failed to dilute samples when OD absorbance measures reached a 10 maximum of 2.5, which occurred when P. umsongenus growth Log Low High appeared to asymptote in high T. discolor nutrients (6 h). In low and high P. umsongenus nutrients, T. discolor exhibited an overall load that was intermediate between that for P. umsongenus in each environment. However, Time Point sampling from the center of each flask may have led to inaccurate Figure 3. Tenacibaculum discolor and Pseudomonas P. umsongenus growth patterns under umsongenus growth curves over ten hours in low and high nutrient liquid media. limited resources as it is likely that the majority of cells were attached to the glass surface of the flasks. T. discolor did not asymptote after ten hours in high nutrients, but plateaued after seven hours in low nutrients. This trial needs to be repeated over a longer time period to capture growth patterns after ten hours and to understand differences in kinetics between P. umsongenus and T. discolor when resources are abundant. Prior to kinetic analysis between species, it will also be necessary to empirically determine CFU – OD600 equivalents so that species start at equal abundances when comparing growth rates.

The effect of different nutritional resources on gram-negative bacterial communities isolated from marine environments

Shannon Lynch Microbial Diversity 2018 Mini Project Final Report

Competition Assay 0 1 After accounting for differences between Pseudomonas Tenacibaculum ● colonies counted on ● ● ● 10

SWC-amp (P. ● ● umsongenus only) 8 and SWC (both ● species), the 6 number of CFUs on 4 a log scale were on average similar at 2 Tinitial and Tfinal when growing together in 0 Nutrients Both_Pseudomonas Both_Tenacibaculum High ● low and high Low ● nutrient media for ● ● log10 CFU/mL 10 15 h (Fig. 4). ● ● Although not 8 significant, T. discolor appears to 6 be slightly more 4 abundant in low nutrient media, 2 which is consistent ● ● with patterns in the 0 growth curve 0 1 analysis (Fig. 3). Time Point

When growing Figure 4. Cell abundances (log10CFU/mL) for each species growing alone, the overall separately or together in different nutritional resources (T1= 15 h). magnitude of P. umsongenus at Tinitial appears to be lower compared to T. discolor. Contrary to growth curve patterns, P. umsongenus increases in abundance at a faster rate compared to T. discolor and itself in high nutrient media. Caveats aside, these data tentatively suggest that within a 15 h time frame, T. discolor and P. umsongenus can coexist in equal ratios regardless of nutritional resources.

It is possible that species of Vibrio are better competitors given that Pseudomonadaceae was scarcely detected in the microcosm experiment. Further experiments exploring single comparisons of more species with more replicates and sampling over longer time periods will provide a better understanding as to what drives these interactions and likely reveal more distinct differences among treatments.

Finally, this study explores gram-negative marine bacteria interactions in concentrated and dilute complex media. Given that competitive exclusion or stable coexistence could occur in the same microbial community depending on certain ratios of nutrient concentrations (Cherif & Loreau, 2007; Hibbing et al., 2010), future work should be devoted to exploring species interactions in defined media and manipulating essential nutrients. These experiments could be refined using phenotypic profiling of isolates with BiOLOG (MBL 2018, Chapter 11) to quantitatively determine microbial interactions on various carbon, nitrogen, phosphorus and sulfur substrates. The effect of different nutritional resources on gram-negative bacterial communities isolated from marine environments

Shannon Lynch Microbial Diversity 2018 Mini Project Final Report

Bdellovibrio Enrichment Top Agar Spot Test The Bdellovibrio sample retrieved from A B a glycerol stock did not produce plaques in the top agar spot test, nor did the remaining inoculum from the initial microcosm experiment. Part of the inoculum from the microcosm experiment contained concentrated 10 µm 10 µm sample that previously produced Bdellovibrio plaques in a spot test C D (Maria Bautista, personal communication). However, I could not reproduce these results because the sample was initially processed to isolate viruses and stored at 4 C for over one week in the absence of prey. As such, the Bdellovibrio cells were no longer viable at the time I conducted a spot Figure 5. Signs of Bdellovibrio under a wet mount viewed at 100x including small, highly motile cells (a), a rounded bdelloplasts-like test. structure (b), a bdelloplasts-like structure zoomed in to reveal a dark mobile body within (c), and a zoomed in image of a Liquid Enrichments potentially lysed bdelloplasts (d). Ultimately, I was unable to produce enough Bdellovibrio inoculum to repeat the microcosm experiment, but signs of Bdellovibrio were observed in the majority of the 35 flasks I monitored regardless of prey and sample origin (Figs. 5 & 6). These signs included highly motile < 0.2µm cells throughout the planktonic region of a wet mount (Fig. 5a), rounded bdelloplasts-like structures (Fig. 5b) containing a dark, mobile body resembling Bdellovibrio in the process of degrading infected prey cell contents for use in its own growth and replication (Fig. 5c), and structures appearing to be bdelloplasts that had lysed after releasing a new generation of attack-phase Bdellovibrio (Fig. 5d).

Signs of Bdellovibrio in turbid cultures varied depending on the type and volume of media used (Fig. 6). In particular, suspensions passaged into flasks containing HEPES buffer from overnight turbid cultures of samples in SWC became turbid at 30 C with shaking (Fig. 7a), and showed signs of Bdellovibrio under the microscope (Fig. 6). In contrast, flasks containing HEPES inoculated with sample from the start did not (Fig. 6). However, when flasks containing sample and HEPES were amended with prey cells, signs of Bdellovibrio activity in turbid cultures were observed (Figs. 6). More of these flasks also had more attack-phase Bdellovibrio, suggesting that HEPES amended with CaCl2 is a better medium for inoculum production. Not all liquid cultures in SW showed turbidity because two of the suspensions exhibited clearing (Fig. 7b); qualitatively, I also observed a greater degree of Bdellovibrio activity in these suspensions.

To isolate and concentrate the small motile cells, I spun 50mL of the liquid cultures for three minutes at 3,000rcf to pellet down the larger cells in the mixed culture, passed the supernatant through a 0.2µm filter, and then spun the supernatant in 2mL aliquots for 30 minutes at 14,000rcf. However, I did not observe a pellet after spinning down the supernatant, nor did I see highly motile cells under the microscope after re-suspending the non-visible pellet in 100µL SWC, though it is possible that 30 minutes was not long enough to produce a pellet. Furthermore, liquid enrichments did not produce plaques within the time frame of the course when diluted and plated using the double layer overlay technique. The effect of different nutritional resources on gram-negative bacterial communities isolated from marine environments

Shannon Lynch Microbial Diversity 2018 Mini Project Final Report

Turbid Motile Cells Bdelloplast

100

50 of Total Flasks Total of 40 %

0 10mL HEPES 10mL SW 40mL GCM 40mL HEPES PASSAGED FROM 750µL Sample 750µL Sample 10mL Sample 10mL Sample 40mL SWC 1mL Prey 1mL Prey 10mL Sample INTO 40mL HEPES

Figure 6. Percentage of flasks containing different quantities and types of media that exhibited turbidity, signs of small highly motile cells, and bdelloplast-like structures.

Double Layer Overlay Technique After two weeks and two days A B prior to the course ending, plaques were observed on one SWC plate containing a sample from Garbage Beach on a top agar containing V. harveyi (Strain 13; Fig. 8a&b). If not for the course time constraints, clearing may have been observed on other top agar plates over longer incubation periods. Hobley Figure 7. Suspensions exhibiting turbidity (a) and clearing (b). Turbid cultures were passaged into HEPES buffer from overnight liquid (2005) reported the apperance of cultures of sample in SWC. Cleared cultures showed a surfeit of plaques after 2 – 4 days, Bdellovibrio activity when viewed under the microscope at 100x. suggesting that Bdellovibrio activity is highly variable depending on sample conditions. On the last day of the course, I prepared a wet mount from a plaque and observed bdelloplast-like structures and small highly motile cells. Crystal violet staining of the supension revealed the presence of potential Bdellovibrio cells within the round bdelloplast-like structures (Fig. 8c) and small attack-phase cells (Fig. 8d). With no time left, I unfortunately missed a step during the fixation process when preparing a stain of the filtered suspension and lost the cells on the slide. However, I did prepare a glycerol stock from one plaque resuspended in SWC (2018.SCL.05.08.1, strain 199) that could be visualized and used in future studies. The effect of different nutritional resources on gram-negative bacterial communities isolated from marine environments

Shannon Lynch Microbial Diversity 2018 Mini Project Final Report

Microscopy Surprisingly, the round structures I previously observed under the microscope A B that resembled bdelloplasts divided and produced a Vibrio sp. colony during live imaging. Hence, these structures were not bdelloplasts giving rise to a new generation of Bdellovibrio. Species of Vibrio are known to exhibit a similar morphology under stress conditions, in which cells enter a physiologically viable but non-culturable C D (VBNC) state (Ramamurthy et al., 2014). For example, Vibrio cholerae transformed into coccoid cells in an aquatic microcosm after incubation for 60 days at 4°C (Chaiyanan, 2007). These results do not rule out the possibility that other the structures I observed under wet mounts 10 µm 10 µm were bdelloplasts, but the sample we Figure 8. Plaque from a garbage beach sample in SWC top particularly live-imaged captured actively agar containing Vibrio harveyi strain 13 (a & b), bdelloplasts dividing cells of a VBNC Vibrio culture. With at 100x containing dividing Bdellovibrio cells stained with more time and optimization, it could be crystal violet (c), and crystal violet-stained attack-phase possible to capture the lysing activity of a cells (d). bdelloplast with live imaging. In hindsight, however, I would have used TEM or SEM microscopy to visualize these structures at higher magnification prior to live imaging.

Conclusions and Future Work I can tentatively conclude that species ratios change over time and these changes differ depending on nutritional resources. However, dynamics are confounded in the presence of a predator, which has larger implications when using such organisms like Bdellovibrio as therapeutic agents. I can expect a number of different possible outcomes given the complexity of the system in either environment. One potential outcome is that relative abundances in the community do not significantly change and Bdellovibrio sp. has very little impact relative to growth and competition for resources. Another possible outcome is that Bdellovibrio sp. has a strong preference for one species in which case changes in relative abundances will depend on whether the preferred prey species is rare or abundant in the community, and the relative susceptibility of other community members. If all community members are equally susceptible, I would expect that the relative abundances do not change, but the overall abundance of the community would decrease in the presence of Bdellovibrio. The stage is set to explore these dynamics with further optimization and lessons learned herein.

Acknowledgements I would like to thank my funding sources which allowed me to take this course: Microbial Diversity - DOE grant scholarship, the Natural Communities Coalition of Orange County, The California Avocado Commission, and the Department of Environmental Studies at UC Santa Cruz. I also wish to thank Rachel Whitaker and George O’Toole for granting me the opportunity to take this formative course, guiding me through many iterations of my mini project, and setting a precedent for a mutually respectful and supportive academic community. I am especially grateful to Deb Hogan who advised me on the design of my project, Nicki Limoli and Danielle Campbell who mentored me on all aspects of my project, and Sandra Rizk who helped me get The effect of different nutritional resources on gram-negative bacterial communities isolated from marine environments

Shannon Lynch Microbial Diversity 2018 Mini Project Final Report out of the lab by midnight on many occasions! I thank Kurt Hanselmann for devoting his time, energy, and creativity to any question or problem I threw at him – Kurt, you are truly an inspiration! Many thanks to our course coordinator, Gabriela Kovacikova, for her intellectual and emotional support and to the course assistants, Rebecca Wipfler, Dee Sanders, and Jared Livingston – they were truly the backbone of the course. I also want to give a big thank you to all the MD 2018 participants, especially to Sarah Schwenk, Nutriphi (#group4rules), Peggy Lai, and Jake Weissman for providing levity when needed in this wonderful adventure. I can’t thank you all enough! Finally, I wish to thank Gregory Gilbert, my Ph.D. advisor, for supporting me to take this course.

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Shannon Lynch Microbial Diversity 2018 Mini Project Final Report

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