Description and charactrization of the Amazonian entomopathogenic bacterium Photorhabdus luminescens MN7

Many isolates of the genus Photorhabdus have been reported around the world. Here we describe the first Brazilian Photorhabdus isolate, found in association with the entomopathogenic nematode Heterorhabditis baujardi LPP7, from the Amazonian forest in Monte Negro (RO, Brazil). The new isolate can be grouped with the Hb-Hm clade of P. luminescens subsp. luminescens, close to the new subspecies P. luminescens subsp. sonorensis. P. luminescens MN7 has several characteristics expected of variant form I cells, such as the presence of intracellular crystals, secretion of hydrolytic enzymes (lipases and proteases) and bioluminescence. Although H. baujardi LPP7 is not prolific when compared to H. bacteriophora HP88, P. luminescens MN7 is clearly pathogenic and probably secretes the same toxins as P. luminescens subsp. luminescens W14, when fed to larvae of the greater wax moth Galleria mellonella. This behavior is different from what is found in Photorhabdus luminescens subsp. laumondii HP88, which was used as a control in our experiments, and P. l. subsp. laumondii TT01. Besides the toxin secretion, P. luminescens MN7 secretes proteolytic polypeptides that have molecular masses different from those found in P. l. subsp. laumondii TT01. Finally, the crude extract from spent culture medium was shown to contain 3,5-dihydroxy-4-isopropyl-cis-stilbene and 1,3,8-trihydroxy-9,10-anthraquinone as the major compounds, similarly to other Photorhabdus luminescens strains.


Introduction
Bacteriological assays and antibiotic analysis  This kit allowed the simultaneous assay of glucose fermentation; H 2 S production 161 (desulfhydrase activity); CO 2 production from glucose fermentation; L-tryptophan 162 deaminase activity; urease activity; motility; tryptophan hydrolase activity; and lysine 163 decarboxilase activity. After an overnight incubation at 28°C, results were read as 164 recommended by the manufacturer. Antibiotic resistance was tested using antibiotic-  performed by growing ten Galleria mellonella larvae at 28°C in the different pollen 236 mixtures plus 3.6 g of bee wax in a Petri dish. Every other day the larvae were 237 individually weighed and larval growth was expressed as relative weight gain (RWG) 238 [21], calculated as follows: RWG = 1 + [(sample mean -control mean)/control mean]. 239 Growth of the larvae was followed for 8 days in the dark. Initial mean weight of the   (Figs 1a and 1b). These cells present prominent 250 inclusions that probably correspond to crystals of polypeptides CipA and CipB, whose 251 function is unknown but are necessary for the growth of the nematode [48]. When P. 252 luminescens MN7 is cultivated in LB-pyruvate, starting from 24h after the inoculum, 253 crystals are easily detected within the cells by differential interference contrast (DIC) 254 microscopy. Negative staining and electron microscopy of LB-pyruvate grown bacteria 255 clearly show the presence of peritrichous flagella (Fig 1c) suggesting that MN7 is   Table) and includes DNA sequences from 40  Table). Complete results are shown in S8-S11 Figs The tree 274 constructed with five concatenated gene sequences puts MN7 in a basal position of the 275 Hb-Hm clade (that includes strains MX4A, Caborca, and CH35) (Fig 2a-b). On the . We could detect the activity of two different secreted 290 enzymes in P. luminescens MN7: a lipase, using a direct method on LB-agar (Fig 3a), 291 and a protease, using an electrophoretic method (Fig 3b). These findings confirm that 292 our isolate contains mainly variant form I cells. Also, the lipase activity (measured by 293 the diameter of the precipitation zone around the bacterial colony) of P. luminescens 294 MN7 is more intense than TT01 colonies (Fig 3a). After 18h of bacterial growth, 295 protease activity was more intense in MN7 than in TT01 culture supernatants (Fig 3b).  P. luminescens MN7 is unable to grow in minimal medium with NH 4 Cl as the only 309 nitrogen source but is capable of glucose fermentation and CO 2 production. MN7 cells 310 are motile and do not produce lysine decarboxylase, tryptophanase and desulfhydrase.

311
Tryptophan hydrolase and urease activities were not detected in the biochemical tests 312 (Table 1). MN7 is unable to grow in LB-agar at temperatures higher than 32°C.

314
Antibiotic resistance 315 316 We tested the MN7 isolate against a set of multiple antibiotics using a classical 317 agar-diffusion assay (Fig 4). MN7 showed resistance to ampicillin (10 μg/disc), partial 318 resistance to cefalotin (30 μg/disc) and gentamicin (10 μg/disc) and susceptibility to the 319 other antibiotics tested.   of LPP7-infected G. mellonella larvae is weaker (Fig 5c) than larvae infected with HP88 329 (Fig 5d). When LB-agar plated with the isolated bacteria were analyzed in the same way 330 we could observe that MN7 colonies (Fig 5g) emit much less light than TT01 cells (Fig   331   5h). that significantly decreased larval weight gain after eight days. (Fig 6a). The toxin is 344 heat-sensitive, since heating the MN7 spent medium at 70°C for 10 min was enough to 345 abolish its effect on the RWG (Fig 6a). The toxin present in the spent medium of MN7 346 or cells of HP88 has a gradual effect on the decrease of the RWG of G. mellonella 347 larvae as observed in the time curve (Fig 6b).  The results presented here show that the MN7 strain is closely related to a 370 recently described subspecies which has been classified as P. luminescens subsp.  Table. Peaks 4 and 5, corresponding to stilbene and anthraquinone, have retention times of 27.4 and 31.0 min respectively (see S13 Fig). (b) Growth inhibition of Staphylococcus aureus by preparative TLC-purified stilbene. Filter paper discs impregnated with 10 or 20 µg of purified stilbene (S) were applied over a continuous lawn of Staphylococcus aureus culture in LB agar. Control discs (C) consisted of filter paper discs impregnated with methanol and dried.
sequences [12], but with 68% support values. In the last six years, this clade was classifying MN7 within the Hb-Hm clade (Fig 2) corresponds to PrtS, a poorly known protease with a broad spectrum of substrates [60].

480
These protease activities of P. luminescens MN7 show more intense bands than P. 481 luminescens TT01 (see Fig 3b).   Table). The tree was constructed using  Table). The