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Vol. 15, No. 2, pp. 126-135

International

Journal

of

Nematology

December, 2005

Protein changes in the nematode symbiotic bacterium Photorhabdus luminescens during in vitro serial culture

Cheng Bai*, David I. Shapiro-Ilan*, Yi Wang** , Randy Gaugler**, Elizabeth A. Cowles***, Shu-Xia Yi**** *USDA-ARS, SAA, Southeast Fruit and Tree Nut Research Laboratory, 21 Dunbar Road, Byron, GA 31008, USA. E-mail: [email protected] **Department of Entomology, Rutgers University, New Brunswick, NJ 08901, USA ***Department of Biology, Eastern Connecticut State University, Willimantic, CT 06226, USA ****Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, WI 53226, USA

Abstract. Heterorhabditid nematodes kill their arthropod hosts with the aid of the symbiotic bacteria, Photorhabdus spp. Stability of the bacteria in serial culture is essential for maintaining virulence and success in nematode mass production. Photorhabdus bacteria exist as two variant forms, phase I and phase II; the former is generally more desirable for culture purposes. We investigated changes in protein electrophoretic profiles during 21 in vitro culture cycles of P. luminescens strain HB-GA. Substantial changes were observed in protein assays after 21 culture cycles; the protein alterations were observed in both variant forms of the bacteria (phase I and phase II): the level of change appeared to increase as the number of cultural cycles increased. A search on protein sequence similarity revealed that the majority of protein changes in P. luminescens were related to biologically active proteins including a paralyzed flagella protein, an insecticidal toxin, and an insecticidal toxin complex protein TccCl, catalyzing enzymes including two proteases (that also contribute to insect death), an alkaline phosphatase, an allene oxide cyclase-like enzyme (earlyresponse to dehydration), a Holliday junction DNA helicase, and membrane proteins including an outer membrane fimbrial usher protein. The observed protein variations indicate that genetic changes during in vitro serial culture could affect traits critical to bacterial virulence and survival capacity. Keywords. Entomopathogenic nematode, Heterorhabditis, Photorhabdus luminescens, protein change, serial culture, symbiotic bacterium.

dictate the virulence of the nematodes and their success in mass production.

INTRODUCTION

M

otile gram-negative bacterium, Photorhabdus luminescens (Thomas and Poinar), is a symbiont of the entomopathogenic nematode, Heterorhabditis bacteriophora Poinar (Boemare, 2002; Poinar, 1990). The two species are mutualistically associated with each other. When the bacterium is not inside an arthropod host it is held inside the intestine of the dauer stage nematode; the bacterium does not persist in the soil environment without the nematode (Poinar, 1990). Thus, the bacterium relies on the nematode for entry into the host. The bacterium provides benefits to the nematode by being the primary agent responsible for killing the host, and producing antibiotics to prevent secondary invaders from establishing (Poinar, 1990). The fitness and quality of these symbiotic bacteria can

Photorhabdus bacteria exhibit phase variation (Akhurst, 1980; 1982) Relative to the primary cell type (or phase I), various traits such as antibiotic production are diminished or absent in the secondary (or phase II) form (Akhurst and Boemare, 1990). Thus, the primary cell type is more conducive to nematode growth and reproduction as it is the only cell type retained by the dauer infective juvenile, and therefore phase shift to the secondary cell type during mass in vitro production can be detrimental (Ehlers et ai, 1990). After organisms are serially cultured in the laboratory, certain beneficial traits can be altered or lost due to genetic processes such as inadvertent selection or drift (Hopper et al, 1993; Hoy, 1985; Roush, 1990). Trait deterioration in the 126

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entomopathogenic nematode-bacterium complex has been observed (Bai et al., 2005; Shapiro et al., 1996; Wang and Grewal, 2002). Characterization of the genetic basis for the observed phenotypic changes in the nematode-bacteria complex is fundamental to understanding and overcoming the problem. In laboratory and mass in vitro culture of the nematode-bacterium complex, the bacteria are passed through numerous serial culture cycles (Ehlers, 2001; Shapiro-Ilan and Gaugler, 2002) resulting in countless generations which undoubtedly allow for genetic change. However, trait changes in these symbiotic bacteria have not been investigated separately from the nematodes. In this study, our objective was to analyze bacterial protein changes during in vitro serial culture.

without regard to whether phase shift to secondary form had occurred. In this regime, a very high percentage (> 80%) of cultures had already shifted to phase II by the fifth culture cycle (Yi Wang et al., 2005, unpublished data). In the second regime, only primary phase colonies, as indicated from plating on NBTA, were selected for subsequent culture cycles. Thus, the second regime included an extra "selection step" on solid media in between each of the liquid culture cycles; these selection steps on NBTA were not counted toward the 21 cycles. By running the two regimes in parallel, we were able to qualitatively compare protein changes during in vitro culture when phase shift is left unchecked (first regime) with changes occurring when the primary cell type is maintained throughout the process. Whole strain bacterial proteins from cell pellets were prepared according to methods described by Bai et al. (2002). Briefly, the bacteria from two plates for each culture cycle were suspended in 1.5 ml of cold 2X PBS and centrifuged for 10 min at 4,000 g; and this procedure was repeated. The cell pellets were re-suspended in cold PBS containing 10 mM dithiothreitol and homogenized in a 7-ml tissue grinder. The cell extracts were centrifuged twice for 20 min at 20,000 g and the protein supernatant after centrifugation was used for further analysis (Bai et al., 2002). Protein concentration was determined with the BioRad Protein Assay Kit, using bovine serum albumin as the standard (Bio-Rad; Hercules, CA). Protein samples were suspended in Laemmli sample buffer (Bio-Rad; 1:1, v/v) and boiled for 5 min. The protein samples were loaded on large 10-20% gradient sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (160 x 160 x 1.5 mm) to separate the various proteins. The migration distance on the SDS-PAGE gel indicates the size of protein subunits. The protein subunit molecular masses were estimated with Precision Plus Protein Standards (Bio-Rad;. Hercules, CA).

MATERIALS AND METHODS Bacteria strains Photorhabdus luminescens (strain HB-GA) was analyzed for protein changes during serial culture. Monoxenic cultures of the bacteria were initiated from the haemolymph of H. bacteriophora infected insects (greater waxmoth larvae, Galleria mellonella (L.)) according to procedures described by Dunphy and Webster (1989). The nematodes were originally collected from soil in Georgia, USA by baiting with G. mellonella (Kaya and Stock, 1997). We chose the HB-GA strain of P. luminescens for this study rather than an established or previously studied strain which was already in culture, so that we could observe protein changes starting from the time of initial isolation. Analysis of whole bacterial protein A foundation population of P. luminescens from the original isolate was grown in TSY, i.e., tryptic soy broth with yeast extract (per litre: 40 g tryptic soy broth + 5 g yeast extract [Sigma -Aldrich, Inc. St. Louis, MO]) and stored at -80°C in 20% glycerol (Popiel et al., 1989). A portion of bacteria from the foundation population was serially cultured in TSY according to procedures described in Popiel et al. (1989). Approximately 109 cells were added to 50 ml of TSY in a 250 ml Erlenmeyer flask, which was incubated for 48 hours at 25°C and 200 rpm (Strauch et al, 1994). The bacteria were cultured in this manner (in vitro liquid culture in shaker flasks) for an additional 20 cycles. Following each culture cycle the bacteria were plated onto NBTA to determine cell type (phase I or phase II) (Kaya and Stock, 1997). After liquid culture cycles 1, 5, 10, 15, and 20 the bacteria were cultured on nutrient agar (NA) (BectonDickson and Co., Sparks, MD) for 48 hours at 25°C. Proteins were prepared from each of these additional liquid culture cycles on NA, i.e., protein analysis was conducted after 2, 6, 11, 16, and 21 total cycles, which will hereafter be referred to as C2, C6, Cll, C16 and C21.

Amino acid sequence analysis In this study, only the protein subunits showing changes during serial culture were processed for sequencing. The NH2-terminal sequences were analyzed by automated Edman degration method. The protein bands were transferred onto PVDF membranes (Bio-Rad). After staining with Coomassie Brilliant Blue R-250 solution for 3 min, protein bands of interest were cut out and used for sequencing. For NH2-terminal amino acid sequencing, about 200 pmol of the peptide was run on a Procise cLC sequencer model 492 (Applied Biosystems; Foster City, CA) pulsed with on-line analysis on a built-in separation system. BLAST (Latched et al., 1997) and FASTA Sequence Comparisons (Pearson and Lipman, 1988) were used to search for sequence homologies.

RESULTS SDS-PAGE analyses resulted in numerous (approximately fifty) distinguishable bands in bacterial protein subunits (Fig. 1). Pronounced changes in bacterial

Two culture cycle regimes were performed. In one regime, the bacteria were cultured through the 20 cycles 127

Protein changes in a nematode's symbiont: Bai et al.

Fig. 1A. Large SDS-polyacrelymide gradient gel of Photorhabdus luminescens HB-GA strain bacterial proteins with phase shift (A) and only phase I bacterial protein (B) during in vitro serial cultures. Each lane loaded 40 jig of total protein C2 to C21 indicates serial cultures from cycles 2 to.21 on TSY: the bacteria were grown on TSY from CI to C21 in addition to interval growth on NBTA between the cycles for selection of primary and secondary colonies. Numbers at the left column of the fieure indicate molecular mass in kDa.

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Fig. IB. Comparative sequence homology between known proteins and NH2-terminal sequences of Photorhabdus luminescens protein subunits which changed during serial cultures. The NH2-terminal sequences of P. luminescens proteins are listed along with results of BLAST and FASTA searches. Identical residues are indicated with colons (:), and similar residues are indicated with plus (+). Band numbers 1 to 10 are illustrated in Fig. 1. C2, CI6 and C21 means serial cultures at cycles 2, 16 and 21 on TSY, respectively: the bacteria were grown on TSY from CI to C21, in addition to interval growth on NBTA between the cycles for selection of primary and secondary colonies.

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48- kDa protein (#7), for which no sequence result obtained because the NH2-terminal was blocked. FASTA search showed that significant hits were found for protein subunits #1, 2, 3, and 8. Among these protein subunits, only the 15kDa protein (#6) and the 13- kDa protein (#9) from P. luminescens (HB-GA) shared 61% identity in a 21 amino acid overlap (Fig. 2). Table 1 summarizes the level of protein change in P. luminescens (HB-GA) during serial culture.

protein banding patterns were detected between C2 and C21. Changes in bacterial proteins when phase shift was not controlled More than six changes in bacterial protein banding patterns (#1-6) were observed during serial culture. A band with 19- kDa (#1) was unstable over time and an additional band of 16- kDa (#2) appeared at C16. The thickness of two major bands with 45- (#3) and 33- kDa (#4) increased steadily as the cultural cycles increased from C2 to C16. Two bands of 30- (#5) and 15- kDa (#6) disappeared between C2 to C6 and between C6 to CI 1, respectively (Fig. 1A).

DISCUSSION We detected substantial change in protein molecules during in vitro serial culture of the nematode symbiotic bacterium P. luminescens strain HB-GA. The number and quality of changes that occurred when phase shift was controlled (i.e, primary phase was maintained) differed from changes when phase shift was not controlled. We expect that the extent of change during serial culture may also vary among different strains of P. luminescens.

Whole bacterial proteins when phase shift was controlled (kept at phase I) Four marked changes (#7-10) in bacterial protein bands were observed. A band with 48- kDa (#7) appeared at CI 1, and increased afterwards becoming more intense at CI6 and C21. A very fine band with 33- kDa (#8) appeared at C6 and CI 1, and then became very thick at CI6 and C21. Two bands of 13- and 12- kDa (#9 and 10, respectively) varied in quantity during serial culture (Fig. IB), with the 12-kDa band increasing from CI 1 to C21.

The genome sequencing of P. luminescens strain TT01 has been completed; it contains 5,688,987 base pairs long and 4,893 predicted protein-coding genes (Duchaud et al, 2003). They encode a large number of adhesions, toxins, haemolysins, proteases and lipases, as well as antibiotics. The proteins likely play a role in the elimination of competitors, host colonization, invasion and bioconversion of the insect cadaver (Duchaud et al., 2003). We found that the proteins which changed in P. luminescens during serial culture can be classified into three groups based on their possible functional properties, i.e., biologically active proteins, catalyzing enzymes; and membrane proteins.

Analysis of protein sequences and similarities The NH2-terminal sequences and possible identifications of 10 protein subunits that showed obvious changes in serial cultures were analyzed (Fig. 2). BLAST search analyses indicated that the range for the identity of partial sequence similarities between the separated proteins and known proteins was from 62 to 100%. The exception was the 130

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Protein changes in a nematode's symbiont: Bai et al.

International Journal of Nematology Vol. 15, No, 2, 2005

Insecticidal toxicity in P. luminescens is associated with high molecular weight protein complexes (Bowen et al., 1998). During in vitro serial culture we detected changes in several proteins that appear to be associated with bacterial toxicity or other aspects of pathogenicity. Most pertinent were the alterations in proteins that may be associated with specific insecticidal toxins, i.e. the toxin complex protein TccCl (Bowen et al., 1998) from P. luminescens subsp. laumondii TTOl (Duchaud et al., 2003), and a toxin from the plague bacterium, Yersinsa pestis (Parkhill et al., 2001). Possibly associated with pathogenicity is a protein we detected that is homologous to a paralyzed flagella protein, which may be important to invasion and distribution within the host (Tomb et al., 1997).

would have been detected had we also run 2-D gels. However, these experiments provide important clues as to variations in bacterial cells during in vitro culture. Clearly, further characterization of protein and genomic changes during culture of entomopathogenic nematodes and their bacterial symbionts is needed. Nonetheless, we were able to detect a number of protein subunits which changed during subculturing using the methodology described, and we found high levels of homology between some of those proteins and described sequences. Based on sequence data it appears that the protein variations we observed have direct impact on fitness of the bacteria, e.g., in virulence and survival. Indeed, recent studies investigating phenotypic changes during in vitro culture revealed fluctuations in P. luminescens virulence and reproduction (Yi Wang et al., 2005, unpublished data).

The enzymatic activities in P. luminescens have been identified as general lipases, phospholipases, proteases, and DNases (Forst and Nealson, 1996). Enzyme levels that changed in our study during in vitro culture included two proteases (proteins 6 and 9), an alkaline phosphatase (APASE, #3), an allene oxide cyclase-like enzyme (#8), and a Holliday junction DNA helicase (#10). Photorhabdus luminescens is known to produce FTsH proteases or FTsH endopeptidases. Two of the putative proteases (proteins 6, and 9) may contribute to insect death (Duchaud et al., 2003; Ffrench-Constant et al., 2003). They also are related to a hypothetical protein which is a member of the PF/ ATPases family associated with various cellular activities (Theologis et al., 2000). APASE is involved in the dephosphorylation of various substrates. The NH2-terminal sequence of the APASE with 45- kDa detected in our study is 82% identical to the APASE from P. luminescens subsp. laumondii TTOl (Duchaud et al., 2003). Allene oxide cyclase (#8) might play a role in early-responsive to dehydration (according to BLAST results). Typically, allene oxide cyclase is present in plants. The P. luminescens protein subunit #8 is 100% identical to allene oxide cyclase (by both BLAST and FASTA analyses with an 8 amino acids overlap). Thus, this could be a novel record for presence of an allene oxide cyclase-like enzyme in bacteria.

Characterization of specific elements such as proteins and genes related to trait change during serial culture is an important first step to monitoring and overcoming the problem. Once this characterization is complete, the information can be used for designing probes to screen P. luminescens strains for the presence of genes that are susceptible to change and to study whether the protein synthesis is controlled at the transcriptional or translational level. Furthermore, the probes could be used as a basis in molecular cloning to introduce biologically active genes that stabilize or enhance the insecticidal efficacy of the nematode-bacterium complex. We expect that many of the genes that are susceptible to change in P. luminescens to be common among other bacteria species; thus, development of probes and clones specifically for P. luminescens are likely to have wide utility in other taxa. Acknowledgements. The authors thank Kathy Halat of USDA-ARS, SAA, for technical assistance, and Andy Nyczepir and Brian Kunkel for comments on an earlier draft of this manuscript. This research was supported in part by USDA-NRI grant 0201974.

LITERATURE CITED

The membrane protein that changed during in vitro culture is a hypothetical outer membrane fimbrial usher protein (#4) that shares a common sequence piece with a hypothetical protein from P. luminescens subsp. laumondii TTOl (Duchaud et al, 2003). This protein could be involved in the export and assembly of fimbrial subunits across the outer membrane [results from Search of ecce (The E. coli Cell Envelope Protein Data Collection) files, 2001]. Fimbrial proteins facilitate bacterial adherence to various substrates. Any change of the cell membrane proteins would likely influence ion channel activity.

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