Evaluating the occurrence and possible roles for an intermediate-filament homolog in the dimorphic prosthecate bacteria Jake Bailey Department of Earth Sciences University of Southern California Abstract Isolates of eight prosthechate alpha-proteobacteria (Caulobacter halobacteroides CM13a, unnamed Hyphomicrobium sp., Caulobacter leidya, Hyphomonas MHS-1, Maricaulis washingtonensis, Maricaulis maris CM11, Woodsholea maritime CM243, and an unnamed marine Caulobacter, strain CM261) were screened for the presence of the bacterial intermediate-filament homolog, crescentin. The protein was not detectable by immunofluorescence techniques in any of the isolates, with the exception of Woodsholea maritime, where it exhibited a diffuse cytoplasmic occurrence in fully-extended prosthecate cells, a narrow mid-cell band localization in swarmer cells, and an extended helical localization in intermediate-sized prosthecate cells. These results suggest that crescentin is not widely utilized among the prosthecate bacteria, and in the single case outside the freshwater Caulobacters, in W. maritime, localization hints at an alternative role for this cytoskeletal protein. Preliminary results indicated that C. crescentus mutants lacking crescentin suffer no detectable fitness loss are further supported here by growth, motility and chemotaxis assays that show little difference between the laboratory performance of the C. crescentus wild type and creS- deficient mutant. The limited occurrence of crescentin amongst the prosthecate bacteria, and the absence of deleterious phenotypic effects of the creS mutant, add further to the mystery of crescentin’s ecophysiological role, and generally illustrates our poor-understanding of the selective advantage(s) that might be provided by a vibroidal cell morphology. Introduction In eukaryotic cells, the cytoskeleton serves as an active support structure for processes such as cell division, maintenance of cell shape, motility, and intracellular transport. The recent discovery of cytoskeletal protein homologs in bacteria, such as mreB, mbl (mreB- like), crescentin, and ftsZ, has greatly increased our understanding of cell structure and function in bacteria and archaea (Carballido-López, 2006; Jones et al., 2001; Kruse et al., 2005). Bacterial cytoskeletal proteins are now known to play a pivotal roll in cell shape, cell division, and cell polarity in bacteria (Klint et al., 2007; Møller-Jensen & Löwe, 2005). A bacterial homolog to the intermediate filaments, known as crescentin, was recently identified in the prosthecate bacterium Caulobacter crecsentus (Ausmees et al., 2003). Transposon insertions in the crescentin gene resulted in mutants that formed straight cells, rather than the vibroidal morphology characteristic of C. crescentus. Preliminary unpublished results indicate similar laboratory growth of the wild type and the mutant that lacks crescentin, prompting questions about the selective advantage of a protein that confers a vibroidal cell shape (Ausmees et al., 2003). Previous authors suggested that the selective advantage of crescentin may only be obvious under natural conditions (e.g., oligotrophic settings). Here I use immunofluorescence techniques to investigate the occurrence of crescentin among eight prosthecate bacteria that are closely-related to C. crescentus and that inhabit a wide range of environmental conditions, in hopes of gaining insight into crescentin’s phylogenetic and environmental occurrence, in the hopes that these data might offer clues about crescentin’s ecophysiological role. Additionally, I compare the growth, motility, and chemotactic response of the C. crescentus wild type with the crescentus CresC:Tn 5 mutant using various laboratory experiments. Methods Bacterial strains and growth conditions: Each of the bacterial strains used in this study are listed in Table 1. Caulobacter crescentus CB15N, CJW763, and C. leidyia were grown in PYE or minimal media at 30oC with shaking. All marine strains were grown on CPS complex media at 25oC with shaking. Marine strains were grown on Caulobacter complex media. Hyphomicrobium sp. was isolated using methanol as the carbon source under denitrifying conditions as described by (Sperl & Hoare, 1971). Biomass for immunofluorescence was collected 1) during mid-exponential phase; or 2) during stationary phase. Both conditions were examined to monitor crescentin expression in different morphotypes. Media compositions: PyE: 0.2% Peptone, 0.1% Yeast Extract, 0.02% MgSO4 • 7H2O. PyCM: 0.25% peptone, 0.05% yeast extract, 1mM CaCl2, 1 mM MgSO4, 1% Bacto agar, prepared in milli-Q water. CPS Complex Medium: 0.05% casamini acids, 0.05 % Bacto peptone, 80% seawater. MG2 Minimal media: 20X M2 Salts (17.4 g Na2HPO4, 10.6 g KH2PO4, 10.0 g NH4Cl) dissolved in 1L of Milli-Q water was autoclaved. 50 ml of salt solution, was added to 938ml of sterile water, followed by the sequential addition of 0.5 ml of 1M MgSO4, 10ml of 20% glucose, 1 ml of 10mM FeSO4/10mM EDTA, and 0.5 ml of CaCl2. Fe-deficient minimal media was made using the same recipe, but adding only 1 mM FeSO4/1mM EDTA. Similarly, P-deficient minimal media contained 1X phosphate salts. Immunofluorescence assays: Immunofluorescence detection using anti-crescentin polyclonal antibodies was performed using methods similar to those described by (Pogliano et al., 1995). Cells grown under the growth conditions provided in Table 1 were fixed using 12.5% formaldehyde. After 4x centrifuge washes with PBS, cells were re-suspended in 75 μl of GTE. 25 of 0.01 mg/ml lysozyme diluted in GTE was added to the cell suspension and ~20 μl was spotted on poly-l lysine-coated, 8-well glass slides. After a 10-minute incubation, the lysozyme/cell solution was aspirated. 2% filtered BSA in PBS was then used to block the cells for 15 minutes in a humid chamber. Anti- crescentin antibodies diluted 1:40 in 2% BSA were added and allowed to incubate for 1 hr. at 25oC. Following aspiration and 10X washing with 2%BSA in PBS, FITC-labeled goat anti-rabbit secondary antibodies (diluted 1:200) were applied and allowed to incubate in the dark for 1 hr. at 25oC. Following 10X washes with PBS, 5 μl of 2 μM FM 4-64 (Invitrogen) was added to Citfluor (Citifluor Ltd., London) anti-fade mounting solution and applied to each well before adding a cover slip and imaging. Images were acquired using a Hsm Axiocam attached to a Zeiss M1 Imager microscope. Image processing was performed using Axiovision Rel 4.6 and Photoshop CS2 software. All isolates were evaluated in triplicate. Each immunofluorescence 8-well slide included C. crescentus as a positive control and the CresC:Tn 5 mutant as a negative control. Motility assays: 0.3% agar swarm plates were inoculated with five microliters of saturated culture (either CB15 or the creS::Tn5 mutant) normalized to the same optical density (OD600) similar to the assay employed by (Pierrce et al., 2006). In cases where cultures were maintained on peptone yeast extract, and later transferred to experiments on minimal media, all cultures were transferred and grown overnight on minimal media before experiment inoculation. All plates were incubated and kept at room temperature in sealed humid containers for 4.5 days prior to swarm halo measurement. Chemotaxis assays: 10 μm glass capillary tubes containing PyE media were suspended in the wells of a 96-well culture plate. Each well contained freshwater base, and was inoculated with 5 μl of exponential phase culture (either CB15 or the creS::Tn5 mutant). Cell recruitment into the capillaries was compared after 12 hours of exposure, by expelling the capillary fluid onto a Hausser counting chamber slide and counting cells in nine randomly-selected 1 sq. mm. grids. Phylogenetic tree construction: 16s rDNA sequences for all organisms except for Hyphomicrobium sp. and Caulobacter strain CM261were downloaded from GenBank and the Ribosomal Database Project (RDP) and aligned using the alignment tools provided on the RDP II website (Cole, 2007). Organisms selected to fill out the tree were selected based on the groupings of prosthecate -Proteobacteria that resulted from the analyses of (Sly et al., 1999). A 16S rDNA phylogenetic tree was constructed using the RDP Weighbor-weighted, neighbor-joining tree building algorithm. Results Evaluating crescentin’s occurrence using immunofluorescence microscopy: In each slide examined, C. crescentus wild type was used as a positive control and the CresC:Tn 5 mutant as a negative control. Positive controls exhibited consistent localization of the anti-cres antibodies to the concave portion of the cell (Fig. 1 a-c). Negative controls consistently showed no, or very low, levels of background binding, and no localization of anti-cres antibodies (Fig. 1d). No consistent anti-crescentin antibody binding or localization was observed in Caulobacter halobacteroides CM13a, unnamed Hyphomicrobium sp., Caulobacter leidya, Hyphomonas MHS-1, Maricaulis washingtonensis, Maricaulis maris CM11, Woodsholea maritime CM243, and marine Caulobacter strain CM261 (Fig 1 e-h). Binding of anti-crescentin was observed in all morphological variants of Woodsholea maritime, but with a different localization observed in each cell morphotype (Fig.1 i-l). The phylogenetic distribution of organisms screened for crescentin in this study, along with the results, are presented in Figure 2. Chemotaxis assays: A similar chemotactic response, as measured by the relative numbers of bacteria in each of six replicate capillary tubes after 12 hours, was observed for both