Application NoteApplication Note

High-throughput Isolation and Cultivation of Slow Growing Bacteria from Soil

Isolation of Slow Growing Bacteria from SoilThe soil used in this study was collected from an urban community garden in Berkeley, CA (U.S.). 1 g of soil was suspended in 9 mL of 1x Phosphate Buffered Saline (PBS) inside a 15 mL conical tube and vortexed for 30 seconds at 3000 rpm. The tube was inverted 3 times and allowed to sit upright for 2 minutes. After larger soil particles settled to the bottom of the tube, the supernatant was collected. The supernatant was diluted in R2A liquid medium containing 100 µM resazurin and loaded onto arrays at a concentration corresponding to a fill rate of fewer than 0.3 cells per nano-well. The loaded and sealed arrays were incubated in the dark at room temperature for up to 18 days before analysis using the Prospector instrument.The Prospector instrument captures fluorescence images of the array with red (635 nm), green (532 nm), and blue (488 nm) excitation. A shift from red to green fluorescence of the resazurin redox marker in a nano-well indicates bacterial growth. The Prospector software was used to select and generate a target list of putative growth-positive wells for transfer into a 96-well plate by means of a robotic probe using the red/green fluorescence ratio. For more detailed information please refer to the GALT Application Note on Isolation and Cultivation of Bacterial from a Soil Sample1.In order to isolate slow-growing microbes two criteria were followed: A) transferring of cells from putative growth-positive nano-wells into 96-well plates from arrays that had been incubated for 6 to 18 days and B) selection of isolates from those 96-well plates that appeared positive from day 6 onwards. All isolates included in this study met both criteria except for 59 which were selected on the array prior to day

6, but took more than 6 days to appear growth positive on the 96-well plate. A total of 254 isolates were obtained, and their taxonomic identity was assigned using 16S rRNA sequencing. Figure 1 demonstrates the shift in red and green fluorescence signals of selected nano-wells containing slow growing microbes from 24h to 11 days post loading and shows the location of these wells on the Prospector arraysDetailed information of the time requirements for sample preparation, array loading and transferring to 96-well plates followed by bacterial identification can be found in the GALT Application Note on Isolation and Cultivation of Bacterial from a Soil Sample1. For this experiment, minor steps were modified: A) the day positive wells were transferred from arrays to 96-well plates was extended to between 6 and 18 days and B) selection of positive isolates from 96-well plates was extended to those that showed growth from day 6 onwards.

Identification of Slow Growing Bacteria Isolates from Soil A portion of the 16S rRNA gene was amplified using the universal primers (27F and 1391R) and sequenced using Sanger sequencing technology with 27F. The 16S rRNA sequences were identified at the genus or higher taxonomic level using the SILVA database. Figure 2 shows the taxonomy and relative abundance of the slow-growing microbe collection from soil. The 254 isolate collection represented common soil phyla that comprises most of soil diversity: Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes and even the rarely cultivable Acidobacteria 2-7. Among known slow growing bacteria, the collection contained representative isolates from the following genera.

The GALT Prospector™ is a system for high-throughput isolation and cultivation of microbial species from complex environmental samples. The system cultivates and images microbial colonies on a proprietary microfluidic array chip containing over 6000 nano-wells for microbial cultivation. A bench-top system automates the isolation and cultivation workflow through software-driven imaging, selection and transferring of single isolates into standard multi-well plates for downstream analysis. When a diluted sample is loaded into the array, individual cells are captured in the nano-wells in isolation from other cells. This eliminates competition between slow and fast-growing microbes and enables identification and isolation of slow growers within complex samples. Here, we describe successful isolation and cultivation of taxonomically diverse slow-growing soil microbes using the GALT Prospector.

Figure 1: Green versus red fluorescence signals over time. Arrays in this study were imaged with the Prospector instrument at 24 hours, 48 hours, 7 days, and 11 days post loading (from top to bottom). Data were analyzed and visualized using the software package R56. All points in the left-side panel represent the green vs red fluorescence values for all 6109 wells on the array. Points highlighted in blue represent a select example of slow growing microbes included in this application note. Green amplitude values, indicative of microbial growth, are shown in the corresponding array heatmaps in the right-side panel. Wells highlighted in gray show the position of the wells containing slow growing bacteria on the array.

Red Amplitude

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Green Amplitude

Bradyrhizobium19.3% of isolates correspond to the well-known slow growing genus Bradyrhizobium 8, 9. This proteobacteria genus are soil bacteria10 and is highly diverse, comprising 58 species to date11. A key characteristic of this genus is the ability to establish a nitrogen-fixing symbiosis with legumes that increases nitrogen availability to plants, making them independent of exogenous nitrogen fertilization12. For example, several Bradyrhizobium species have been shown to establish a symbiosis with soybean, highlighting the genus’s important role in sustainable agriculture8, 13-18. Bradyrhizobium are also known to perform a wide range of other biochemical functions such as photosynthesis, denitrification and aromatic compound degradation19-22. They are also able to cope with stresses such as heavy metals, extreme pH, and antibiotics23-25.

Mycobacterium21.7% of isolates correspond to the genus Mycobacterium. Although this genus, within Actinobacteria, contains slow as well as rapidly growing bacteria26 many species are characterized by slow growth rates27. The genus Mycobacterium is primarily known for its obligate parasitic human pathogenic species such as M. tuberculosis and M. leprae responsible for causing the important human diseases tuberculosis and leprosy, respectively28, 29. The genus however is extremely diverse, comprising 209 species11, and it can be separated into two

broad categories: tuberculosis-causing mycobacteria and non-tuberculous mycobacteria (NTM)30, 31. NTM are usually highly abundant in environments such as soil and water32, 33 and many NTM species are saprophytes or non-pathogenic to humans and animals34. Although mycobacteria represent a significant portion of the environmental flora, little work has been done in deciphering the role of the so-called environmental mycobacteria (EM) in the environment.

AcidobacteriaWe isolated two Acidobacteria in this study: Acidobacteria GP1 and Edaphobacter aggregans. Acidobacteria represent a highly diverse phylum found in a wide range of habitats as shown by the 16S rRNA gene-based sequencing approaches and environmental shotgun metagenomic studies2, 35-45. In soil, Acidobacteria can comprise up to 50% of 16S rRNA gene sequences in clone libraries2, 38, 46, 47 yet, little is known regarding their physiology or ecology due to the difficulties in cultivating the majority of the members of this phyla and to the poor representation of Acidobacteria in bacterial culture collections45, 48-51. Currently, the phylum is divided into 26 subdivisions (GP)52, however not all subdivisions have cultured representatives. The abundance of Acidobacteria is usually correlated with soil pH as shown by 16S rRNA gene clone libraries and pyrosequencing53. Members of subdivisions (GP) 1, 3, 4 and 6 are most often detected in soil environments2, 53. Thus, the majority of isolates cultivated

Figure 2: Taxonomy of the slow growing bacteria isolate

collection. R visualization [56] of the relative abundance of the 254-isolate collection labeled by genus and colored by phylum.

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to date belong to the subdivision GP154. Both isolates recovered using the Prospector system in this study correspond to subdivision 1 (GP1). One of the isolates obtained correlates taxonomically to Edaphobacter aggregans. E. aggregans was originally isolated from a protorendzina in a deciduous forest near Würzburg, northern Bavaria, Germany55. The type species E. aggregans Wbg-1T is able to grow in a pH range between 4.0-7.0 and has a slow growth, visible colonies on agar can be seen after 2-3 weeks of incubation55. Among other known slow growing bacteria isolated, the collection contains many representative isolates from the Proteobacteria genus Methylobacterium57 (17.7%). Methylotrophs are unique in that they can use one-carbon compounds like methane or methanol58. The collection also includes isolates from the genera Sphingomonas59, these are often isolated from soil and have been demonstrated to have potential to aid in bioremediation of soils60. As well as isolates from the genera Rhodanobacter61, including Nocardia62 and Nocardiodes63, 64 from the Actinomycetales order. While microbial function cannot be inferred solely from a taxonomic assignment, the Prospector system is a powerful tool to isolate slow growing microbes and can be combined with downstream molecular profiling techniques like whole genome sequencing to investigate the potential function of slow growing bacteria.

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