Journal of Dairy Science and Biotechnology

Korean Society of Dairy Science and Biotechnology (한국낙농식품응용생물학회)
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Complete Genome Sequence of Chryseobacterium mulctrae KACC 21234T : A Potential Proteolytic and Lipolytic Bacteria Isolated from Bovine Raw Milk

  • Elnar, Arxel G. (Department of Animal Science and Technology, Chung-Ang University) ;
  • Kim, Geun-Bae (Department of Animal Science and Technology, Chung-Ang University)
  • Received : 2022.06.24
  • Accepted : 2022.06.27
  • Published : 2022.06.30
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Abstract

Chryseobacterium mulctrae KACC 21234T is a novel species isolated from raw bovine milk. Psychrotrophic bacteria are considered contaminants and are hypothesized to originate from the environment. In this investigation, the C. mulctrae KACC 21234T genome was determined to be 4,868,651 bp long and assembled into four contigs with a G+C ratio of 33.8%. In silico genomic analyses revealed the presence of genes encoding proteases (endopeptidase Clp, oligopeptidase b, carboxypeptidase) and lipases (phospholipase A(2), phospholipase C, acylglycerol lipase) that can catalyze the degradation of the proteins and lipids in milk, causing its quality to deteriorate. Additionally, antimicrobial resistance and putative bacteriocin genes were detected, potentially intensifying the pathogenicity of the strain. The genomic evidence presented highlights the need for improved screening protocols to minimize the potential contamination of milk by proteolytic and lipolytic psychrotrophic bacteria.

Keywords

The genus Chryseobacterium is composed of Gram-negative, aerobic, non-fermentative, psychrotrophic bacilli with characteristic yellow pigmentation. The genus is considered a part of the normal environmental microflora and has been associated with human infections [1,2]. Recently, several novel species have been isolated from raw milk samples, including C. mulctrae KACC 21234T [3-6]. And although Chryseobacterium is not part of the normal microflora of milk, contamination can occur during the milking process (i.e., from cow udder, human handling, and immediate environment) or transport and processing [7]. Psychrotrophs can take advantage of the high nutritional content of milk and grow at low temperatures (7℃ and below). The activity of these microorganisms may lead to different types of milk spoilage – souring, gas production, proteolysis, ropiness, change in milk fat, flavor defect, and color defect. In addition, some contaminating microorganisms are potentially pathogenic [8].

The presence of lipolytic and proteolytic microorganisms in milk may lead to a variety of defects and shortened shelf-life through the production of extracellular enzymes that can hydrolyze proteins (b-casein and as-casein) and triglycerides, ultimately causing spoilage of the product. Proteases, particularly plasmin, are linked with gelation of UHT sterilized milk, development of bitterness in milk, and reduction in yield of soft cheese while the action of lipases affects the flavor profile of dairy products [8]. Moreover, the extracellular enzymes synthesized by psychrotrophic bacteria are heat stable, withstanding pasteurization conditions (72℃ for 15 sec) and even ultrahigh temperature (UHT, 132℃ for 2 sec) processes, presenting a challenge in securing the quality and safety of milk and dairy products [8,9].

C. mulctrae KACC 21234T was isolated based on its proteolytic activity on skim milk agar plate (SMA; 5% skim milk), incubated at 10℃ for 10 days. Strain KACC 21234T was routinely cultured in tryptic soy agar (TSA, BD Difco, USA) at 30℃ [6]. The genomic DNA was extracted using QIAamp PowerFecal DNA kit (Qiagen, Germany) and sent to ChunLab (Korea) for sequencing using PacBio RSII Single Molecule Real-Time (SMRT) platform with 20 kb SMRTbellTM template library. De novo assembly of the PacBio reads was performed using the PacBio SMRT analysis software ver. 2.3.0.

Genome annotation was perdformed using the Rapid Annotation using Subsystem Technology (RAST) using default parameters [10]. Transfer RNAs and ribosomal RNAs were identified using tRNAscan-SE ver. 1.3.1 [11] and INFERNAL ver. 1.1.3 software using the Rfam 12.0 database [12], respectively (Fig. 1, 2). The genome features of C. mulctrae KACC 21234T are listed in Table 1.

OGGGB6_2022_v40n2_86_f0001.png 이미지

Fig. 1. Circular genome map of Chryseobacterium mulctrae KACC 21234T. Circles represent the following characteristics from the outermost circle to the center: (1) contig information, (2) coding sequences on forward strand, (3) coding sequences on reverse strand, (4) transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs), (5) GC skew, and (6) GC ratio. G, guanine; C, cytosine.

OGGGB6_2022_v40n2_86_f0002.png 이미지

Fig. 2. Subsystem category distribution by (A) KEGG annotation and (B) Cluster of Orthologous Groups. KEGG, Kyoto Encyclopedia of Genes and Genomes.

Table 1. Genome features of Chryseobacterium mulctrae KACC 21234T

OGGGB6_2022_v40n2_86_t0001.png 이미지

G, guanine; C, cytosine.

Functional genome annotation revealed several genes encoding for proteases and lipases (Table 2). Several studies have reported that members of Chryseobacterium showed greater spoilage ability than Pseudomonas spp. based on its proteolytic and lipolytic activity [7,13]. Additionally, although less frequently reported, the production of phospholipases (CS110_01857 and CS110_02528) was associated with sweet curdling and bitter cream in milk due to the aggregation of fat globules. C. mulctrae KACC 21234T also has genes for b-galactosidase (CS110_00343), which may cause unwanted hydrolysis of β-galactosidic bonds in lactose.

Table 2. Predicted proteases and lipases from Chryseobacterium mulctrae KACC 21234T genome

OGGGB6_2022_v40n2_86_t0002.png 이미지

Xaa-Cya, cysteic acid residue; Xaa-Asp, aspartic acid residue.

Antimicrobial resistance genes were also detected using the Resistance Gene Identifier with The Comprehensive Antibiotic Resistance Database (https://card.mcmaster.ca/analyze/rgi). Specifically, CPS-1, adeF, and qacG, which confers resistance to carbapenem, fluoroquinolone and tetracycline, and antiseptics, respectively, were identified. Furthermore, two open reading frames (ORF) encoding a putative bacteriocin (Linocin M18 and Carocin D) were identified via BAGEL4 (http://bagel4.molgenrug.nl/). However, there were no immunity and transport proteins associated with the bacteriocin genes.

The in-silico analyses of C. mulctrae KACC 21234T genome revealed the presence of various proteolytic and lipolytic enzymes, bacteriocins, and antimicrobial resistance genes which highlights the risks involved in microbial contamination of milk. Thus, it is imperative to develop effective screening methods for the detection of contaminating microorganisms and their enzymes to improve the quality and safety of milk and related products.

Nucleotide Sequence Accession Number

The Whole Genome Shogun project has been deposited at GenBank under the accession number VAJL00000000. The version described in this paper has the accession number VAJL01000000, consisting of sequences VAJL01000001 – VAJL01000004.

Conflict of Interest

The authors declare no potential conflict of interest.

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