Complete genome sequence of Chitinophaga pinensis type strain (UQM 2034^T)

The most similar 16S rRNA gene sequences from cultivated strains that are stored in GenBank originate from isolates belonging to different species of the genus Chitinophaga: C. sancti, C. filiformis and C. ginsengisoli with 96-97% sequence similarity; all of them were isolated from soil samples. In metagenomic surveys of environmental samples only 16S rRNA genes with sequence similarity values below 92% to C. pinensis were detected, indicating that members of this species are not abundant in the so far genomically screened habitats (status July 2009).

Figure 1 shows the phylogenetic neighborhood of C. pinensis UQM 2034^T in a 16S rRNA based tree. The sequences of the six copies of the 16S rRNA gene in the genome differ by up to five nucleotides, and differ by up to 21 nucleotides (1.4%) from the previously published sequence generated from ACM 2034 (AF78775). Most likely this discrepancy is caused by sequencing errors in the publicly available C. pinensis 16S sequence.

Cells of C. pinensis stain Gram-negative and form long, filamentous, flexible rods with rounded ends (Table 1). They occur singly and measure 0.5-0.8 by 40 µm in the mature gliding stage. Figure 2 shows cells of C. pinensis during cell division before separation. Upon aging, they transform into spherical bodies without the production of fruiting bodies. Myxospores with a diameter of 0.8-0.9 µm are formed. On charcoal yeast extract agar (CYEA) a yellowish pigment is produced. The temperature for growth ranges from 12-37°C with an optimum of 24°C. The optimal pH for growth is 7, but growth is possible in a wide pH range from 4 to 10. NaCl concentrations of 0-1.5% (w/v) are tolerated [1].

Strain UQM 2034^T produces acid from glucose, lactose, and sucrose. Chitin, casein and gelatin are hydrolyzed, whereas according to Sangkhobol and Skerman (1981) cellulose, starch, alginate, and agar are not hydrolyzed [1]. Nitrate is not reduced to nitrite. C. pinensis UQM 2034^T produces urease and is catalase and oxidase positive [1,3]. UQM 2034^T is susceptible to tetracycline, streptomycin, and chloramphenicol and resistant to neomycin, kanamycin, penicillin G, and erythromycin [1]. C. pinensis UQM 2034^T is able to lyse Staphylococcus aureus cells but not cells of Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis [1].

The genome is 9,127,347 bp long and comprises one main circular chromosome with a 45.2% GC content (Table 3 and Figure 3). Of the 7,397 genes predicted, 7,302 were protein coding genes, and 95 RNAs. In addition, 110 pseudogenes were also identified. The majority of the protein-coding genes (62.4%) were assigned with a putative function while those remaining were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.

The predominant characteristic feature of C. pinensis is the ability to degrade chitin, a β-1,4-glycosidic linked homopolymer of N-acetyl-D-glucosamine and one of the most abundant polysaccharides in nature. It is a component of fungal cell walls and of arthropod exoskeletons. Chitin is degraded by chitinases (EC 3.2.1.14); endochitinases randomly cleave within the chitin molecule and exochitinases hydrolyze diacetylchitobiose from the end of a chitin chain. Diacetylchitobiose is further degraded to N-acetylglucosamine by the action of N-acetylglucosaminidases (EC 3.2.1.52).

These glycosidic bond hydrolyzing enzymes were grouped in glycoside hydrolase (GH) families based on amino acid sequence similarities (Web Site) [22]. For the C. pinensis genome 169 glycoside hydrolases belonging to 49 different GH families are predicted; 18 of the predicted glycoside hydrolases belong to GH family 43 which contains xylosidases, xylanases, arabinanases, arabinofuranosidases and galactosidases.

Because of the chitin degrading ability of C. pinensis a great number of chitinases was expected to be encoded in the genome. According to the CAZY-database, exochitinases and endochitinases belong to GH families 18, 19 and 48. As estimated, there were several glycoside hydrolases predicted, which may be involved in chitin degradation; five members of GH family 18 (Cpin_2184, Cpin_2186, Cpin_2580, Cpin_3805, Cpin_3919) and three members of GH family 19 (Cpin_5850, Cpin_5553, Cpin_5898). The comparison of the amino acid sequence from these chitinase candidates to the databank BlastP indicated no homologs according to the whole length of the proteins. However, similarities to the known GH family domains were observed.

The search for N-acetylglucosaminidases (EC 3.2.1.52) in the genome of C. pinensis revealed gene Cpin_3944 which encodes a protein with a GH family 20 domain. The predicted GH family 20 domain resembles the well characterized catalytic domain of a N-acetylglucosaminidase from Serratia marcescens [23]. Further N-acetylhexoaminidases of C. pinensis are encoded by the genes Cpin_1798, Cpin_4994 and Cpin_1915.

A second way to degrade chitin was described by Davis and Eveleigh in 1984 [24]. First, the chitin molecule is deacetyliated by deacetylases (EC 3.5.1.41), afterwards chitobiose is released from chitosan by the action of chitosanases (EC 3.2.1.132), finally chitobiose is hydrolyzed by glucosaminidases (EC 3.2.1.52) and glucosamine molecules are released.

One putative chitin deacetylase is encoded in the genome of C. pinensis. The deduced amino acid sequence of Cpin_6813 shows a GH family 19 domain and a C-terminal deacetylase domain. Chitosanases that are responsible for the hydrolysis of chitosan are mainly found in GH family 46 but also occur in GH families 5 and 18. In C. pinensis, no GH family 46 members were observed, but the presence of nine GHs belonging to family 5 and five members of GH family 18 are predicted. One of these glycoside hydrolases might have a chitosanase function. It remains unclear which pathway C. pinensis uses for the degradation of chitin and whether the predicted functions of the proteins match the real functions.