Stenotrophomonas nematodicola sp. nov., a novel intestinal 1 lifespan-prolonging bacterium for Caenorhabditis elegans 2 that assists in host resistance to Bacillus nematocida 3 colonization

19 Background: The gut microbiota of Caenorhabditis elegans , a tiny worm that feeds on 20 bacteria, is significantly dominated by the bacteria upon which it feeds. These bacteria 21 may not only interfere with the intestinal flora of C. elegans but also assist in resisting 22 pathogen infection. Understanding the interactions between the microbiota of C. 23 elegans and pathogens will shed light on how to achieve biological control of 24 agricultural pests. 25 Results: The lifespan of Caenorhabditis elegans fed on strain CPCC 101271 T was 26 extended by approximately 40% compared with that of worms fed on Escherichia coli 27 OP50. In addition, the colonization of C. elegans by the pathogenic bacterium Bacillus 28 nematocida B16 was inhibited when it was pre-fed with strain CPCC 101271 T . Based 29 on a polyphasic taxonomy study including genotypic, chemotaxonomic and phenotypic 30 characteristics, we propose that strain CPCC 101271 T represents a novel bacterial 31 species with the name Stenotrophomonas nematodicola sp. nov. and CPCC 101271 T as 32 the type strain. Metagenomic sequence analysis of the intestinal microbiota of C. 33 elegans fed with strain CPCC 101271 T and then infected with B16 revealed that pre- 34 feeding with CPCC 101271 T improved the diversity of intestinal bacteria, while the 35 community structure varied significantly together with the fluctuation of 36 Stenotrophomonas spp. and Bacillus spp. abundance during competition between strain 37 CPCC 101271 T and B16. 38 Conclusions: The nematode microbiota strain CPCC 101271 T assisted in its host 39 resistance to the pathogen Bacillus nematocida colonization, so as to act as an intestinal 40 life span-prolonging for C. elegans. The genotypic and phenotypic properties of strain 41 CPCC 101271 T supported to the proposal of strain CPCC 101271 T as a novel species 42 of the genus Stenotrophomonas.

The worm Caenorhabditis elegans is susceptible to many of the pathogens that infect 58 plant parasitic nematodes (Sinha et al., 2012). Like most pathogens that infect C. 59 elegans, pathogenic bacteria colonize the digestive tract and ultimately kill the 60 nematode. In contrast, most bacteria such as Escherichia coli and Bacillus subtilis are 61 usually not toxic to C. elegans (Garsin et al., 2001). Thus, C. elegans has proven to be  However, in nature the nematode C. elegans is a 'microbivore' because of its ability to 67 consume various types of bacteria. To some extent, the gut microbiota of nematodes 68 may be dominated by the bacteria that they feed on; these bacteria may shape the 69 microbiota community structure, regulate metabolism and even alter the lifespan of the  In our previous study, we found that the bacterial pathogen strain B. nematocida B16 75 4 killed C. elegans nematodes by employing a "Trojan horse" mechanism (Niu et al.,76 2010). We have isolated several bacteria inside worms from various origins including 77 soil and rotten fruit. Some bacteria, like Phytobacter sp. SCO41, showed inhibitory 78 effects on pathogenic bacterium B16 . To explore the relationships 79 between microbiota and pathogens of nematodes in depth, we combined metageomic 80 sequencing analysis and culture-dependent methods to collect evidence. As a result of 81 this analysis, we found that strain CPCC 101271 T , originally isolated from the intestinal 82 lumen of C. elegans in nature, acts as a component of beneficial microbiota for C. 83 elegans by extending the lifespan of the host, as well inhibiting the colonization of the 84 host by B. nematocida B16, an opportunistic pathogen, which was previously proposed 85 as a candidate biological control agent for nematodes (Huang et al., 2005). 86 Here, we report the results of a taxonomic study of strain CPCC 101271 T , which we

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The isolation and identification of strain CPCC 101271 T , which represents a novel      To investigate whether strain CPCC 101271 T has colonization-resistance activity 183 against B. nematocida B16, we first performed an in vitro bacteriostatic activity test.

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The results showed that strain CPCC 101271 T could not inhibit B. nematocida B16 but worms not pre-fed with CPCC 101271 T scored as "full". In contrast, only 10% of the 205 animals pre-fed with CPCC 101271 T were scored as "full". Moreover, compared with 206 worms fed only B16, the percentage of worms fed with both B16 and CPCC 101271 T 207 that had undetectable B16 colonization was much higher at 48 h (10% vs. 80%) and at 208 10 72 h (0% vs. 70%). The worms pre-fed with E. coli showed little difference compared 209 with those in the no pre-feeding group. The ability of B16 to colonize the nematodes 210 pre-fed with JCM 13333 T was stronger than its ability to colonize those pre-fed with 211 CPCC 101271 T , but a little weaker than its ability to colonize the negative controls pre-212 fed with E. coli OP50. Differences between the abilities of B16 to colonize the 213 nematodes pre-fed with CPCC 101271 T and pre-fed with OP50 were notable when we       In contrast, worms in nature are exposed to complex microbial communities.

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Understanding the worm's natural microbiome is essential to help explain their realistic   We should explore substantial evidence to confirm the above inference in the following 402 studies. Understanding this inference mechanism can help quickly inhibit the growth of   Gram-staining reaction was performed according to Magee et al. (1975). Cell the neighbor-joining method (Saitou & Nei, 1987) with Knuc values (Kimura 1980(Kimura & 496 1983) and complete deletion gaps. Phylogenetic trees were also constructed using and 497 the maximum-parsimony (Kluge & Farris, 1969) and maximum-likelihood (Felsenstein,   Colonization capability assay 519 Colonization capability was assayed using approximate fifty 1-day-old adult 520 hermaphrodite worms were placed on each plate at 25 °C following the procedures Transfer' experiments, the worms were transferred by hair and repeated washed using 523 sterilized NaCl solution (0.85 %, w/v). Three nematode treatment groups were set up.

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In the first group of nematodes pre-fed with CPCC 101271 T then infected by B16g, the 525 worms were transferred onto LB plates containing a low concentration (10 6 cells/mL) 526 of CPCC 101271 T and co-cultivated for 4 h. The worms were then removed from the 527 plates, washed twice, transferred to plates containing B16g and co-cultivated for 72 h.

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In the second group of nematodes pre-fed with JCM13333 T then infected by B16g, the 529 worms were first seeded on an LB agar plate containing JCM13333 T (10 6 cells/mL) and 530 cultivated for 4 h, and then the worms were transferred to plates containing B16g and 531 cultivated for 72 h. In the third group, the worms were first fed on the same 532 concentration of OP50 for 4 h, then transferred to B16g plates and cultivated for 72 h.

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The control group of nematodes without being pre-fed with bacteria were directly 534 seeded on blank medium and then cultivated for 4 h before being transferred to B16g 535 plates, which were also defined as B16 direct infection group. The colonization process The tested nematodes were divided into five groups as follows: (I) CW00h group, 551 which was pre-fed with CPCC 101271 T for 4 h; (II-V) CW04h, CW08h, CW12h and 552 CW16h groups, which were separately co-cultivated with B16 for 4 h, 8 h, 12 h and 16 553 h, respectively, after being pre-fed with CPCC 101271 T for 4 h.

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The worms were collected and then washed and surface sterilized as described above.