# Jennifer C. Flanagan^1,*, Andrew Stump ^1,*, Alexandra Alexiev^1, Jenna M. Lang^1, Jonathan A. Eisen^1,2,#, David A. Coil^11 University of California Davis Genome Center, Davis, CA, USA.2 University of California Davis Department of Evolution and Ecology, Department of Medical Microbiology and Immunology, Davis, California, USA* These authors contributed equally to this work.# Corresponding author: jaeisen@ucdavis.edu

Abstract

Abstract

A novel, Gram-negative, non-spore-forming, pleomorphic yellow-orange bacterial strain was isolated from a stadium seat. Strain Coronado(T) falls within the Erythrobacteraceae family based on 16S rRNA phylogenetic analysis, but is both phylogenetically and physiologically distinct from existing genera in the family. A phylogenetic tree inferred from 16S rRNA gene sequences shows a highly supported clade containing Coronado(T), Porphyrobacter, Erythromicrobium, and Erythrobacter. While this strain has Q-10 as the predominant respiratory lipoquinone, as do other members of the family, the fatty acid profile of this strain is distinct. Coronado(T) contains predominatly C18:1ω7cis and C16:0, a high percentage of the latter not being observed in any other Erythrobacteraceae. This strain is catalase-positive and oxidase-negative, the latter of which is unusual for the other genera present in the same clade. Coronado(T) can grow from 4-28°C, at NaCl concentrations 0.1-1.5%, and at pH 6.0-8.0. On the basis of phenotypic and phylogenetic data presented in this study, strain Coronado(T) represents a novel species in a new genus in the family Erythrobacteraceae for which the name Kirrobacter mercurialis gen. nov., sp. nov. is proposed; the type strain is Coronado(T) (=DSMZ 29971, =LMG 28700).

INTRODUCTION

A 2005 phylogenetic analysis of the Alphaproteobacteria class led to the creation of a new family, Erythrobacteraceae, to house the genera Erythrobacter, Porphyrobacter and Erythromicrobium (Lee 2005). These genera were later joined by Altererythrobacter (Kwon 2007) and Croceicoccus (Xu 2009), the latter work also emended the description of the family. Members of the Erythrobacteraceae family are Gram-negative, aerobic bacteria that contain cartenoids, usually appearing pink, orange or yellow. They do not form spores, are chemo-organotrophic, and are most often associated with aquatic environments.

In this study, strain Coronado(T) was isolated from a stadium seat at Niedermeyer Field, Coronado High School in Coronado, California, USA as part of a nationwide Citizen Science project (Project MERCCURI - www.spacemicrobes.org.) One goal of Project MERCCURI was to collect bacterial isolates to be used for an experiment aboard the International Space Station (ISS). A cotton swab was used to swab the surface of a stadium seat, then colonies were isolated via plating onto agar plates. Almost full length (primers 27F and 1391R) 16S rRNA sequencing showed 99% identity to a few uncultured sequences at NCBI, but no more than 95% identity to the nearest cultured representative (Porphyrobacter donghaensis)(Yoon 2004). Uncultured isolates with high identity (=>99%) were found in studies of deep ocean sediment and the human skin microbiome.

Phylogenetic and biochemical characteristics show that this isolate belongs to a new genus and species within the Erythrobacteraceae family. However, phylogenetic analysis of the entire family would suggest that a major taxonomic revision of the entire group is required, as has been suggested by others (e.g. (Rainey 2003)) Here we describe the genotypic, morphologic, and biochemical characteristics of strain Coronado(T), which represents a novel genus and species. We propose the name of Kirrobacter mercurialis gen. nov., sp. nov.

METHODS

Cells were initially grown on plates containing either Reasoner's 2A agar (R2A), or lysogeny broth agar (LB). LB was made with 10g tryptone, 10g NaCl, and 5g yeast extract per liter. A clear preference for LB was observed and so was used for all subsequent experiments. Salt tolerance was measured in liquid media (25°C) from 0% to 20% w/v NaCl. pH tolerance was measured in liquid media (25°C) from pH 3.4 to pH 8.0. Temperature preference was measured in liquid culture across the range 4°C to 30°C. Growth in microgravity was measured aboard the International Space Station (ISS).

Cell morphology, motility, and presence/absence of flagella were examined by light microscopy (Zeiss Axio Lab.A1) and transmission electron microscopy (TEM). Cell cultures in either exponential or stationary phase were prepared for TEM by the UC Davis Electron Microscopy lab as follows. 400 mesh copper grids with formvar/carbon support film (Ted Pella, Inc., Redding, Ca.) were placed on dental wax. A 10μl drop of fixed or unfixed sample was placed onto the grid and left in a dust-free environment for 10 min. Then excess was wicked off with filter paper. A 10μl drop of 1% PTA pH 5.8 (phosphotungstic acid) or 1% ammonium molybdate in DDH2O was added to the grid and wicked off immediately. Grids were allowed to air-dry completely before viewing in a Philips CM120 (FEI/Philips Inc, Hillsborough, Or.) electron microscope at 80KV.

Catalase and oxidase activity, as well as the hydrolysis of starch and casein, were assayed by standard methods. Carbon source utilization was assayed using the Phenotypic MicroArray(TM) services offered by Biolog, Inc. following standard procedures for gram-negative bacteria. Colonies were grown on blood agar at room temperature and suspended in IF-0a inoculating fluid (Biolog) to a density of 42% transmittance. The cell suspension was diluted 1:6 in IF-0a plus 1x Dye H (Biolog) and a carbon source utilization MicroPlate (PM1; Biolog) was inoculated with 100μl per well. The PM1 microplate was incubated at 23°C and read by the OmniLog instrument every 15 minutes for 96 hours. Duplicate sets of OmniLog data were converted to average read value and a threshold of 78 was required in both replicates for a positive call.

Respiratory quinones, polar lips, and fatty acids

Cells were grown in 1L of LB at 23°C for large-scale biomass production, then centrifuged and lyopholized. Analysis of respiratory quinones/polar lipids and fatty acids were carried out by the Identification Service, DSMZ, Braunschweig. Germany. Details are available on their website (http://www.dsmz.de/services/services-microorganisms/identification.html).

16S rDNA and Genome Sequencing

Genomic DNA was extracted using a Wizard Genomic DNA Purification Kit (Promega). A nearly full-length 16S rRNA gene sequence was amplified using the 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1391R (5'-GACGGGCGGTGTGTRCA-3') "universal" primers. Sanger sequencing was provided by the College of Biological Science UC-DNA Sequencing Facility (UC Davis). This DNA was also used for Illumina sequencing of the draft genome as described elsewhere (Coil and Eisen, submitted). The genome sequence was annotated using the RAST server (Aziz 2008) (Overbeek 2013).

A phylogentic tree was inferred using all type strains from the Erythrobacteraceae family. The 1482bp 16S rDNA sequence was obtained from the genome assembly in RAST, and uploaded to the Ribosomal Database Project (RDP) (Cole 2013). RDP was used to build an alignment including every type strain within the Erythrobacteraceae family, including strain Coronado(T). Because the taxon names exported with this alignment contained special characters that were not compatible with phylogenetic reconstruction software, a custom script was used to remove or replace those characters with underscores. A description of and link to this script can be found in (Dunitz 2015). FastTree (Price 2009) was used with default settings to build an unrooted phylogeny. Dendroscope 3 (Huson 2012) was used to view and edit the phylogenetic tree in order to 1) root the tree with a clade that is sister to a well-supported clade containing strain Coronado(T), 2) prune the tree to remove taxa outside the clade containg the outgroup, and 3) "clean up" the tree.

RESULTS AND DISCUSSION

Morphological, physiological, and biochemical characteristics

Cells were non-motile and not observed to form spores or possess flagella, though over 30 flagella or flagella-associated genes are present in the genome. In contrast, the genome for a non-motile close relative (P. cryptus) does not contain any flagellar genes so it is possible that strain Coronado(T) is motile under specific conditions. Unlike most members of the Erythrobacteraceae family, strain Coronado(T) is oxidase-negative. This strain is catalase-positive, and unable to hydrolze casein or starch.

Cells were oval or rod shaped and ranged in length from 1.2um to 2.2um with an average of 1.6um (Figure 1). Cell width ranged from 0.6um to 1um with an average of 0.8um.

Growth was only observed under aerobic conditions, from 4°C to 28°C, with optimal growth around 25°C. Low levels of growth were observed at pH 6.0 up to pH 8.0, maximum growth occurred around neutral pH. Salt was required for growth, and the strain could not grow at >1.5% NaCl, optimal growth was at 0.5% NaCl. No statistically significant difference in growth was observed between earth and microgravity aboard the International Space Station (ISS).