Paracandidimonas lactea sp. nov., a urea-utilizing bacterium isolated from landfill

A urea-utilizing bacterium, designated Q2-2 T, was isolated from landfill. Cells of strain Q2-2 T were Gram stain-negative, aerobic, short-rod bacteria. Strain Q2-2 T was observed to grow at a temperature range of 15–37℃ (optimum 30 ℃), a pH range of 5.5–9.5 (optimum pH 8.0) and 0–4% (w/v) NaCl (optimum 1%). The major respiratory quinone was Q-8, and the major polar lipids were diphosphatidyl glycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine, and phosphatidyl glycerol. Based on the 16S rRNA gene sequence, strain Q2-2 T had the highest similarity with Paracandidimonas caeni 24 T (98.0%), followed by Pusillimonas soli MJ07T (97.5%), Parapusillimonas granuli Ch07T (97.2%), Pusillimonas ginsengisoli DCY25T (97.1%) and Paracandidimonas soli IMT-305 T (96.4%). The ANI values between strain Q2-2 T and the above related type strains were 71.02%, 73.52%, 74.32%, 74.59% and 72.29%, respectively. The DNA G + C content of strain Q2-2 T was 61.1%. Therefore, strain Q2-2 T represents a novel species of the genus Paracandidimonas, for which the name Paracandidimonas lactea sp. nov. (type strain Q2-2 T = CGMCC 1.19179 T = JCM 34906 T) is proposed.


Introduction
Paracandidimonas is one of the members of the order Burkholderiales and the family Alcaligenaceae. At present, there are only two species reported in the genus of Paracandidimonas, including Paracandidimonas soli IMT-305 T from soil in Alabama (Kämpfer et al. 2017) and Paracandidimonas caeni 24 T from the sludge from a pesticide manufacturing plant (Li et al. 2018). Both species have not been reported as having urease activity.
A bacterial strain utilizing urea, named Q2-2 T , was recently isolated from landfill, which appears to have different properties from the known species of Paracandidimonas. Therefore, this strain was identified by polyphasic taxonomic approaches. Based on the results, it is proposed herein that strain Q2-2 T represents a novel species of the genus Paracandidimonas.

Isolation and culture conditions
Soils were sampled from a landfill site in Hangzhou, Zhejiang Province. The soils (10 g) were diluted with sterile water, and then placed in a shaker at 30 ℃ and 200 rpm for 2 d. After mixing, the samples were spread on R2A plates and incubated at 30 ℃ for 2 d. A translucent milky white colony was isolated and purified which was named Q2-2 T . To preserve it for the subsequent identification experiments,

Genomic characteristics and phylogenetic analysis
The bacterial universal primer pair 27F/1492R was used for the amplification of the 16S rRNA gene of the genome, according to the established procedure (Weisburg et al. 1991). The PCR product was sequenced by Sangon Biotech (Shanghai) Co., Ltd, and the resulting sequence was compared with the closely related sequence of the reference organism using the EzBioCloud server (http:// www. ezbio cloud. net) (Yoon et al. 2017). Phylogenetic trees were constructed using the calculation formulas of the neighbourjoining (Saitou and Nei 1987), least squares (Fitch and Margoliash 1967), maximum likelihood (Joseph and Felsenstein 1981) and minimum-evolution (Fitch 1971) methods with the MEGA X program package (Sudhir et al. 2018). The G + C content of strain Q2-2 T was analysed with the RAST server based on the complete genome sequence (Aziz et al. 2008). The ANI values were computed through the Orthologous Average Nucleotide Identity Tool (OAT) (Chun et al. 2015), and the digital DNA-DNA hybridization (dDDH) values were calculated by Genome-to-Genome Distance Calculator (http:// ggdc. dsmz. de/) (Meier-Kolthoff et al. 2013). To support and demonstrate the 16S rRNA phylogenetic trees of strain Q2-2 T , multi-locus sequence analysis (MLSA) based on housekeeping genes (gyrB, hisS, recA, rpoB) was performed with related strains (Glaeser and Kämpfer 2015).

Morphology, physiology and biochemical analysis
After growth on R2A plates at 30 ℃ for 48 h, the colonies were 1.0-2.0 mm in diameter with smooth surface, circular, milky white and convex. Cells of strain Q2-2 T were Gram negative, aerobic, immobile, short rods of 0.2-0.36 µm width and 0.9-1.8 µm length with no flagella (Fig. S1).The diverse characteristics between strain Q2-2 T and related type strains are shown in Table 1. All negative traits from commercial kits are listed in the Table S1, and the utilization of carbon sources (Biolog GNIII) are listed in the Table S2.

Chemotaxonomic characterization
The cellular fatty acid profiles of strain Q2-2 T are shown in Table S3. There were some comparable qualitative and Table 1 Differential phenotypic characteristics between strain Q2-2 T and its closest related type strains Strains: 1. Strain Q2-2 T ; 2. Paracandidimonas caeni 24 T (Li Y et al. 2018); 3. Pusillimonas soli MJ07 T (Lee et al. 2010); 4. Parapusillimonas granuli Ch07 T (Kim et al. 2010); 5. Pusillimonas ginsengisoli DCY25 T (Srinivasan et al. 2010); 6. Paracandidimonas soli IMT-305 T (Kämpfer et.al. 2017). + , Positive; − , negative; w, weakly positive; ND: no data. All data are from the present study except where indicated otherwise  . 1 The Neighbour-joining tree of strain Q2-2 T and its related type genera constructed based on the 16S rRNA gene sequences, using the Kimura two-parameter model. Bootstrap values were based on 1000 replicates. The filled circles represent the same topological structures in the ML tree. Scale bar indicates 0.010 substitutions per nucleotide position quantitative characteristics of strain Q2-2 T with the interrelated type strains. The major fatty acids of strain Q2-2 T were C 16: 0 (33.95%), C 17:0 cyclo (11.42%) and C 18: 0 (0.83%), which were the same as those of Paracandidimonas caeni 24 T . Compared with Paracandidimonas soli IMT-305 T , the main unsaturated fatty acids of strain Q2-2 T had high consistency. The major fatty acid C 14: 0 was found in strain Q2-2 T , but not in the related strains with the exception of Pusillimonas ginsengisoli DCY25 T . The respiratory quinone of strain Q2-2 T was Q-8. The major polar lipids of strain Q2-2 T were DPG, PE, PME and PG, which were similar to those of the reference strains. According to the polyphasic evidence described above, it is concluded that strain Q2-2 T represents a novel species of the genus Paracandidimonas in the family Alcaligenaceae, for which the name Paracandidimonas lactea sp. nov. is proposed.
The GenBank/EMBL/DDBJ accession numbers of the 16S rRNA gene sequence and complete genome sequence of strain Q2-2 T are MZ520859 and JAJJOZ000000000, respectively.