Control of bacterial motility by environmental factors in polarly flagellated and peritrichous bacteria isolated from Lake Baikal

Abstract

Despite numerous studies on bacterial motility, little is known about the regulation of this process by environmental factors in natural isolates. In this study we investigated the control of bacterial motility in response to environmental parameters in two strains isolated from the natural habitat of Lake Baikal. Morphological characterization, carbon source utilization, fermentation analysis, and sequence comparison of 16S rRNA genes showed that these strains belong to two distinct genera, i.e., Enterobacter and Pseudomonas; they were named strains 22 and Y1000, respectively. Both strains swarmed at 25 degrees C and remained motile at low temperatures (4 degrees C), especially the Pseudomonas strain, which further supports the psychrotrophic characteristics of this strain. In contrast, a strong inhibition of motility was observed at above 30 degrees C and with a high NaCl concentration. The existence of flagellar regulatory proteins FlhDC and FleQ was demonstrated in Enterobacter strain 22 and Pseudomonas strain Y1000, respectively, and environmental conditions reduced the expression of the structural genes potentially located at the first level in the flagellar cascade in both organisms. Finally, as in Enterobacter strain 22, a strong reduction in the transcription of the master regulatory gene fleQ was observed in Pseudomonas strain Y1000 in the presence of novobiocin, a DNA gyrase inhibitor, suggesting a link between DNA supercoiling and motility control by environmental factors. Thus, striking similarities observed in the two organisms suggest that these processes have evolved toward a similar regulatory mechanism in polarly flagellated and laterally flagellated (peritrichous) bacteria.

FIG. 1

Motility assay on semisolid medium plates. 1, Pseudomonas strain Y1000; 2, Enterobacter strain 22. Assays were performed at 25°C (A), at 25°C in the presence of 500 mM NaCl (B), at 37°C (C), or at 4°C (D). Plates were incubated for 13 to 15 h at 25 and 37°C and for 132 h at 4°C. The results are representative of those from three independent experiments.

FIG. 2

(A) Regulatory region of the flhDC master operon in Enterobacter strain 22. Nucleotides are numbered relative to the transcriptional start site (+1), indicated by a broken arrow. The unique CAP-binding site consensus sequence is indicated by a box. The positions of the −10 and −35 sequences are underlined. A putative ribosome-binding site (RBS) is indicated in boldface. Only the residues corresponding to the N- and C-terminal parts of FlhD and FlhC are indicated. The dashed arrow labeled E1 represents the oligonucleotide used in +1 mapping. (B) Regulatory region of the fleQ gene in Pseudomonas strain Y1000. Transcriptional start sites are indicated by broken arrows; the two major transcriptional start sites P1 and P2 are indicated in boldface. The position of a putative ς⁵⁴-binding site is indicated by a box. A putative ribosome-binding site with similarities with corresponding regions in the P. aeruginosa fleQ gene (accession no. L49378) and V. cholerae flrA gene (accession no. AF014113) and close to the consensus sequence of P. aeruginosa 16S rRNA (23) is indicated in boldface. Only the residues corresponding to the N- and C-terminal parts of FleQ are indicated. Dashed arrows labeled Y1 and Y2 represent oligonucleotides used in +1 mapping.

FIG. 3

Identification of the fleQ transcriptional start site and effects of growth conditions on fleQ expression with primer Y1 (A) and with primer Y2 (B) (see Materials and Methods). Primer extension analysis was performed with RNA extracted from Pseudomonas strain Y1000 grown in exponential phase at room temperature (lanes 1); to late logarithmic phase (lanes 2); at 4°C (lanes 3); in the presence of 500 mM NaCl (lanes 5); at 30°C (lanes 6); or in the presence of 100 μM (lanes 7), 200 μM (lanes 8), and 400 μM (lanes 9) novobiocin and from Pseudomonas strain Y1000 carrying plasmid pDIA576 grown in exponential phase at 25°C (lanes 4). As a reference, a DNA sequencing ladder is shown (lanes G, A, T, and C). The sequence is complementary to the strand shown on the right and was obtained with the same primer as that used for primer extension. Major transcription start sites P1 and P2 are indicated by arrows. The mRNA level was quantified with the PDI software PDQuest on a SUN computer system. Quantitative data are indicated at the bottom of each lane and were expressed relative to the standard condition level (lanes 1), which was assigned a value of 100%. -, background level undetectable by quantification procedure; N.D., not determined.