Bacterial filament formation, a defense mechanism against flagellate grazing, is growth rate controlled in bacteria of different phyla

Abstract

A facultatively filamentous bacterium was isolated from eutrophic lake water and was identified as Flectobacillus sp. strain MWH38 (a member of the Cytophaga-Flavobacterium-Bacteroides phylum) by comparative 16S rRNA gene sequence analysis. Filament formation by Flectobacillus sp. strain MWH38 and filament formation by Flectobacillus major, the closest known relative of strain MWH38, were studied in chemostat cultures under grazing pressure by the bacterivorous flagellate Ochromonas sp. strain DS and without predation at several growth rates. The results clearly demonstrated that filament formation by the two flectobacilli is growth rate controlled and thus independent of the presence of a predator. However, flagellate grazing positively influenced bacterial growth rates by decreasing bacterial biomass and thus indirectly stimulated filament formation. The results of investigations of cell elongation and filament formation by Comamonas acidovorans PX54 (a member of the beta subclass of the class Proteobacteria) supported the recent proposal that in this species the mechanism of filament formation is growth rate controlled. The finding that the grazing defense mechanism consisting of filament formation is growth rate controlled in the flectobacilli investigated and C. acidovorans PX54 (i.e., in bacteria belonging to divergent evolutionary phyla) may indicate that this mechanism is a phylogenetically widely distributed defense strategy against grazing.

FIG. 1

Dendrogram showing the estimated phylogenetic position of Flectobacillus sp. strain MWH38 within the Cytophaga-Flavobacterium-Bacteroides evolutionary lineage. The dendrogram was generated by using the FITCH algorithm of the PHYLIP package (8) and evolutionary distances (16) calculated from 16S rRNA (and rRNA gene) sequence dissimilarities. The positions of the genera of the Cytophaga-Flavobacterium-Bacteroides evolutionary lineage were calculated from the sequences of the type strains of the type species of the genera.

FIG. 2

Results of the predation chemostat experiment performed with Flectobacillus sp. strain MWH38 and the bacterivorous flagellate Ochromonas sp. strain DS (introduced on day 13). (A) Influence of flagellate grazing on bacterial numbers. (B) Influence of the predator on the mean length of bacteria (including single cells and filaments) and the percentage of long filaments (length, >10 μm). d, days.

FIG. 3

Results of chemostat experiment performed with Flectobacillus sp. strain MWH38, showing the influence of growth rate on the morphology of the strain. Initially, bacteria were grown in both reactors at a growth rate of 0.5 day⁻¹. On day 13, the growth rate of the bacteria in reactor 1 was increased to 1.0 day⁻¹, and 9 days later the growth rate of the bacteria in reactor 2 was increased to 2.0 day⁻¹. (A) Influence of growth rate on the percentage of long filaments (length, >10 μm) in the population. (B) Influence of growth rate on the mean bacterial length (including single cells and filaments). d, days.

FIG. 4

Photomicrographs of Flectobacillus sp. strain MWH38 populations grown in chemostats with and without predation and at different growth rates. (A and B) Flectobacillus sp. strain MWH38 from the predation experiment before (A) and after (B) inoculation with the flagellate. Before the start of predation, the bacteria were cultured at a growth rate of 0.5 day⁻¹. (C and D) Flectobacillus sp. strain MWH38 from the growth rate chemostat experiment at growth rates of 0.5 day⁻¹ (C) and 2.0 day⁻¹ (D). Flagellates were not included in this experiment.

FIG. 5

Cell size distributions of chemostat-grown Flectobacillus sp. strain MWH38, F. major, and C. acidovorans PX54 with and without flagellate predation and at different growth rates in flagellate-free culture. Most of the distributions shown were based on three to eight chemostat samples; the exceptions were the distributions shown in panels K (two samples) and L (one sample). Data for the latter two distributions were obtained from a chemostat experiment described by Hahn and Höfle (15). This experiment was carried out by using the same experimental conditions as those used for the chemostat experiments performed in this study. Note that the graph in panel F represents only the last 5 days of the flagellate-controlled phase of the grazing experiment (Fig. 7), while the graph in panel H represents the first 4 days after a steady state was established in the growth rate experiment (Fig. 8). d, day.

FIG. 6

Photomicrographs of F. major cells grown in chemostats with and without predation and at different growth rates. (A through C) F. major from the predation experiment before inoculation with the flagellate (A) and 4 days (B) and 15 days (C) after inoculation with the flagellate. Before the start of predation, the bacteria were cultured at a growth rate of 0.5 day⁻¹. Three Ochromonas sp. strain DS cells are shown in panel B. (D and E) F. major from the growth rate chemostat experiment at growth rates of 0.5 day⁻¹ (D) and 2.0 day⁻¹ (E). Flagellates were not included in this experiment.

FIG. 7

Results of the predation chemostat experiment performed to determine the influence of grazing by the bacterivorous flagellate Ochromonas sp. strain DS on the morphology of F. major. Bacteria were grown in two parallel reactors, reactor 1 (solid symbols) and reactor 2 (open symbols), at the same growth rate, 0.5 day⁻¹. Both reactors were inoculated on day 11 with the flagellate, and on day 31 flagellates were eliminated from both reactors by treatment with specific inhibitors (cycloheximide and colchicine). In the period between introduction and elimination of flagellates, the predators established populations (data not shown) comparable to the populations in other chemostat experiments. (A) Influence of flagellate grazing on bacterial numbers. (B) Influence of flagellate grazing on the percentage of long filaments (length, >10 μm) in the population. (C) Influence of flagellate grazing on the mean length of bacteria (including single cells and filaments). d, days.

FIG. 8

Influence of growth rate on the cell morphology of F. major in two parallel chemostat reactors, reactor 1 (solid symbols) and reactor 2 (open symbols). (A) Changes in bacterial cell number and percentage of long filaments (length, >10 μm) in the population with changes in growth rate. (B) Influence of growth rate on the mean length of bacteria (including single cells and filaments). d, days.

FIG. 9

Size class distribution of C. acidovorans PX54 cells cultured in chemostats at different growth rates (0.5, 1.0, 2.0, 3.0, 3.5, 4.0, and 4.5 day⁻¹) and in batch culture at the maximum growth rate (bar on the right). The same medium with the same substrate concentration was used for chemostat and batch cultures. The bacterial populations were divided into size classes with different sensitivities to grazing by bacterivorous flagellates (28). Because no C. acidovorans PX54 cells were smaller than 0.4 μm, all cells smaller than 1.2 μm were considered edible bacteria. In chemostat cultures the percentage of short filaments (length, 5 to 10 μm) increased with growth rate from 0% (growth rate, 0.5 day⁻¹) to 1% (growth rate, 4.5 day⁻¹) and was thus much smaller than the percentage observed in samples obtained from the exponential stage of batch cultures. d, day.

FIG. 10

Influence of growth rate on the length of Flectobacillus sp. strain MWH38, F. major, and C. acidovorans PX54 cells and filaments in flagellate-free chemostat cultures. C. acidovorans PX54 never formed chainlike filaments, and only a small percentage (1 to 3%) of F. major cells formed chainlike filaments consisting of two or three cells. High percentages of Flectobacillus sp. strain MWH38 cells formed chainlike filaments at high growth rates (Fig. 3 and 4), and the data for this species distinguish between the influence of growth rate on cell length (L_c) (○) and the influence of growth rate on the total length of bacteria (single cells and filaments (L_T) (•). The curves are the result of a regression analysis (see text). In addition, the mean lengths of C. acidovorans PX54 cells observed in the exponential phase of batch cultures are shown. d, day.