High quality permanent draft genome sequence of Chryseobacterium bovis DSM 19482^T, isolated from raw cow milk

Abstract

Introduction

Chryseobacterium bovis DSM 19482 ^T (=LMG 24227 ^T; CIP 110170 ^T), was isolated by Hantsis-Zacharov and Halpern [1] from raw cow milk when psychrotolerant bacterial communities in raw milk, and their proteolytic and lipolytic traits, were studied. This study revealed that 5% out of the culturable psychrotolerant bacterial communities belonged to the genus Chryseobacterium. Chryseobacterium bovis proliferates at low temperatures and produce heat-stable proteolytic and lipolytic enzymes which remain active after the milk pasteurization process. This may be a limiting factor in maintaining the flavor quality of fluid milk and its products [1]. Strain C. bovis H9^T DSM 19482 ^T was isolated in April 2004 from a modern farm equipped with automated milking facilities in northern Israel [2]. Three novel psychrotolerant Chryseobacterium species were isolated and identified from raw milk in the same study [1]: C. bovis, C. haifense and C. oranimense [2–4]. The genus Chryseobacterium [5] is a member of the family Flavobacteriaceae and currently consists of about 100 species with Chryseobacterium gleum as the type species. Species belonging to this genus exist in diverse environments such as milk, water, sludge, soil, animals, insects, plants and human samples [2, 6].

Here we describe a summary classification and a set of the features of the species C. bovis, together with the permanent draft genome sequence description and annotation of the type strain (DSM 19482 ^T).

Organism information

Classification and features

C. bovis strain DSM 19482 ^T shares typical characteristics of Chryseobacterium such as Gram-negative staining, occurrence as chemoheterotrophic rods and positive catalase and oxidase reactions. The strain contains flexirubin-type pigments, which are also typical for Chryseobacterium [2] (Table 1). The phylogenetic tree based on the 16S rRNA, also supports the fact that strain DSM 19482 ^T belongs to Chryseobacterium genus (Fig. 1).

Table 1

Classification and general features of Chryseobacterium bovis DSM 19482^T according to the MIGS recommendations [25], published by the Genome Standards Consortium [26] and the Names for Life database [27]

MIGS ID	Property	Term	Evidence codea
	Current classification	Domain Bacteria	TAS [28]
		Phylum Bacteroidetes	TAS [29]
		Class Flavobacteriia	TAS [30]
		Order Flavobacteriales	TAS [31]
		Family Flavobacteriaceae	TAS [32]
		Genus Chryseobacterium	TAS [5]
		Species Chryseobacterium bovis	TAS [2]
		Type strain DSM 19482T	TAS [2]
	Gram stain	Negative	TAS [2]
	Cell shape	Rod	TAS [2]
	Motility	Non-motile	TAS [2]
	Sporulation	Non-sporulating	IDS
	Temperature range	7–37 °C	TAS [2]
	Optimum Temperature	30–32 °C	TAS [2]
	pH range, Optimum	5.0–9.8; 6.5–8.5	NAS
	Carbon source	Glucose, lactose, Maltose	TAS [2]
MIGS-6	Habitat	Cow milk	TAS [2]
MIGS-6.3	Salinity, Optimum	0–2.5%; 0–1.75%	NAS
MIGS-22	Oxygen requirement	Facultative anaerobe	TAS [2]
MIGS-15	Biotic relationship	Unknown	TAS [2]
MIGS-14	Pathogenicity	Unknown	TAS [2]
MIGS-4	Geographic location	Northern Israel	TAS [2]
MIGS-5	Sample collection	2004	TAS [2]
MIGS-4.1	Latitude	32.635149	NAS
MIGS-4.2	Longitude	35.362050	NAS
MIGS-4.4	Altitude	Not reported	-

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^aEvidence codes - IDA Inferred from Direct Assay, TAS Traceable Author Statement (ie, a direct report exists in the literature), NAS Non-traceable Author Statement (ie, not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). Evidence codes are from the Gene Ontology project [33]

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Fig. 1

Phylogenetic tree highlighting the position of Chryseobacterium bovis relative to type species within the genus Chryseobacterium. Maximum likelihood (ML) tree inferred under the GTR + CAT model and rooted with Elizabethkingia. The branches are scaled in terms of the expected number of substitutions per site. The numbers above the branches are support values when larger than 60% from ML (left) and maximum parsimony (MP, right) bootstrapping. Phylogenies were inferred by the GGDC web server [34] available at (http://ggdc.dsmz.de) using the DSMZ phylogenomics pipeline [35] adapted to single genes. A multiple sequence alignment was created with MUSCLE [36]. ML and MP trees were inferred from the alignment with RAxML [37] and TNT [38], respectively. For ML, rapid bootstrapping in conjunction with the autoMRE bootstopping criterion [39] and subsequent search for the best tree was used; for MP, 1000 bootstrapping replicates were used in conjunction with tree-bisection-and-reconnection branch swapping and ten random sequence addition replicates

Cells of C. bovis strain DSM 19482 ^T are non-motile rods, measuring 0.5–0.9 μm in width and 1.1–2.3 μm in length (Fig. 2). After 48 h incubation on standard plate-count agar (SPC) at 30 °C in the dark, colonies are circular with entire edges, opaque, smooth and cream-colored. When light is provided during growth, colonies are yellow-colored because of the production of carotenoid-type pigments (absorbance peaks at 454 and 481 nm). They also contain small amounts of flexirubin-type pigments [2–4].

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Fig. 2

Electron micrograph of negatively stained cells of Chryseobacterium bovis strain DSM 19482^T. Cells are nonflagellated rods. Bar, 2 μm

Growth is observed under anaerobic conditions on SPC agar containing 0.1% (w/v) potassium nitrate but not on SPC agar with the addition of 0.5% glucose (indicating that glucose is not fermented) [2]. The strain grows at 7–37 °C (optimum, 30–32 °C), with 0–2.5% NaCl (optimum, 0–1.75%) and at pH 5.0–9.8 (optimum, pH 6.5–8.5) (Table 1). C. bovis does not grow on MacConkey or cetrimide agar. Casein, aesculin and tributyrin are hydrolysed. Glucose, mannose, maltose, arabinose, mannitol, N-acetylglucosamine, gluconate and adipic and malic acids are assimilated. Acid is produced from D-glucose, maltose, D-lactose and D-mannose. Acetoin is produced; gelatin is hydrolyzed; H₂S and indole are not produced; urea is not hydrolyzed; citrate is not utilized; and arginine dihydrolase, lysine and ornithine decarboxylases and tryptophan deaminase activities are absent. Alkaline and acid phosphatases, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, naphthol-AS-BI-phosphohydrolase, α-glucosidase, ß-galactosidase and cystine arylamidase activities are present [2].

Chemotaxonomic data

The major fatty acids of the type strains are: iso-C_15:0; antesio-C_15:0 and iso-C_17:0 3OH. Some strains in this species also possess iso-C_17:0 ω9c as a major fatty acid [2].

Genome sequencing information

Genome properties

The assembly of the draft genome sequence consists of 96 scaffolds amounting to 3,346,045 bp, and the G + C content is 38.19% (Table 3). Of the 3341 genes predicted, 3236 were protein-coding genes, and 105 RNAs. The majority of the protein-coding genes (69.95%) were assigned a putative function while the remaining ones were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.

Insights from the genome sequence

C. bovis DSM 19482 ^T showed the ability to hydrolyze casein and tributyrin [2] and these traits can also be observed in its genome. The following protease genes were detected: Membrane-associated serine protease, rhomboid family; ATP-dependent Clp protease ATP-binding subunit ClpB; Do/DeqQ family serine protease; ATP-dependent Clp protease ATP-binding subunit ClpX and transglutaminase-like enzyme, putative cysteine protease; ATP-dependent Lon protease (Lon functions in the cytosol) and cell division protease FtsH. The lipolytic properties of C. bovis DSM 19482 ^T are evident from the presence of the following genes: phospholipase/carboxylesterase; esterase/lipase superfamily enzyme and GDSL-like lipase/acylhydrolase.

C. bovis DSM 19482 ^T is producing carotenoid-type pigments under light conditions. Indeed, genes which are part of the carotenoid biosynthesis are found in its genome: phytoene desaturase (lycopene-forming), phytoene desaturase (neurosporene-forming), phytoene desaturase (zeta-carotene-forming), all-trans-zeta-carotene desaturase and beta-carotene 3-hydroxylase.

C. bovis DSM 19482 ^T was able to grow under anaerobic conditions when nitrate was provided. This ability is supported by the presence of the following genes: MFS transporter, NNP family, nitrate/nitrite transporter (two genes) and assimilatory nitrate reductase catalytic subunit.

Gliding motility properties are reflected by the presence of the genes that are exclusive to the Bacteroidetes phylum such as gliding motility-associated lipoprotein GldK and gliding motility-associated lipoprotein GldH. Another gene that supports the motility feature is the chemotaxis protein MotB gene.

Among the genes found in C. bovis DSM 19482 ^T genome are genes for resistance to different components. For example a gene for multidrug resistance protein, MATE family. Members of the Multi-Antimicrobial Extrusion (MATE) family function as drug/sodium antiporters. These proteins mediate resistance to a wide range of cationic dyes, fluroquinolones, aminoglycosides and other structurally diverse antibodies and drugs. These proteins are predicted to have twelve alpha-helical transmembrane regions. The Strain DSM 19482 ^T genome, also possesses a gene for cobalt-zinc-cadmium resistance protein CzcA. CzcA has a low cation-transport activity for cobalt and is essential for the expression of cobalt, zinc and cadmium resistance. Another gene found in the genome is a tellurite resistance protein TerC. TerC has been implicated in resistance to tellurium, and may be involved in efflux of tellurium ions. The quaternary ammonium compound-resistance protein SugE gene that is found in C. bovis DSM 19482 ^T genome encodes an efflux pump which confers resistance to cetylpyridinium, cetyldimethylethyl ammonium and cetrimide cations.

Resistance to antibiotics is revealed by the following genes: glycopeptide antibiotics resistance protein (plays a role in resistance to glycopeptide antibiotics such as vancomycin); MFS transporter, DHA1 family; tetracycline resistance protein gene; and Fusaric acid resistance protein-like gene, which is involved in the resistance (detoxification) of the fungal toxin Fusaric acid.

A gene for putative auto-transporter adhesin head GIN domain demonstrates the function of cell adhesion. Two genes indicate the possibility of C. bovis DSM 19482 ^T to produce a capsule, capsular exopolysaccharide family protein and polysaccharide export outer membrane protein.

Conclusions

In the current study we characterized the genome of C. bovis strain DSM 19482 ^T that was isolated from raw cow milk [2]. C. bovis is a psychrotolerant bacterium which can grow at 7 °C, although its optimal growth temperature is higher (30–32 °C). After milk collection, the milk is kept in cold storage, and psychrotolerants dominate the bacterial flora. These bacteria possess extracellular enzymes, mainly proteases and lipases which contribute to the spoilage of dairy products, as their enzymes can resist pasteurization [1]. The C. bovis DSM 19482 ^T genome demonstrates that indeed, this genome encodes proteases and lipases which may play a role in milk products spoilage.

C. bovis strain DSM 19482 ^T produces a carotenoid pigment, a feature that was also observed for C. haifense [3], but not for other species in this genus. This trait could be used for the commercial production of carotene.

C. bovis DSM 19482 ^T genome demonstrated the strains' potential to produce a multidrug-resistance protein, resistance to cobalt, zinc, cadmium, tellurite, cetylpyridinium, cetyldimethylethyl ammonium and cetrimide cations as well as resistance to glycopeptide antibiotics, tetracycline and resistance to the fungal toxin fusaric acid. The whole-genome sequence of C. oranimense G311, a strain that was isolated from a cystic fibrosis patient, also demonstrated multi-drug resistance [24]. Indication for a capsule-forming ability was apparent in both C. bovis DSM 19482 ^T and C. oranimense G311. Sharma et al. [24] suggested that the resistance of C. oranimense G311 to colistin maybe due to the production of capsular polysaccharides.

Acknowledgements

Abbreviations

References