Requirement offlhAfor Swarming Differentiation, Flagellin Export, and Secretion of Virulence-Associated Proteins inBacillus thuringiensis

Abstract

Bacillus thuringiensis is being used worldwide as a biopesticide, although increasing evidence suggests that it is emerging as an opportunistic human pathogen. While phospholipases, hemolysins, and enterotoxins are claimed to be responsible for B. thuringiensis virulence, there is no direct evidence to indicate that the flagellum-driven motility plays a role in parasite-host interactions. This report describes the characterization of a mini-Tn10 mutant of B. thuringiensis that is defective in flagellum filament assembly and in swimming and swarming motility as well as in the production of hemolysin BL and phosphatidylcholine-preferring phospholipase C. The mutant strain was determined to carry the transposon insertion in flhA, a flagellar class II gene encoding a protein of the flagellar type III export apparatus. Interestingly, the flhA mutant of B. thuringiensis synthesized flagellin but was impaired in flagellin export. Moreover, a protein similar to the anti-sigma factor FlgM that acts in regulating flagellar class III gene transcription was not detectable in B. thuringiensis, thus suggesting that the flagellar gene expression hierarchy of B. thuringiensis differs from that described for Bacillus subtilis. The flhA mutant of B. thuringiensis was also defective in the secretion of hemolysin BL and phosphatidylcholine-preferring phospholipase C, although both of these virulence factors were synthesized by the mutant. Since complementation of the mutant with a plasmid harboring the flhA gene restored swimming and swarming motility as well as secretion of toxins, the overall results indicate that motility and virulence in B. thuringiensis may be coordinately regulated by flhA, which appears to play a crucial role in the export of flagellar as well as nonflagellar proteins.Bacillus thuringiensis is a gram-positive, rod-shaped, sporeforming bacterium that is motile by peritrichous flagella. During sporulation this organism produces a variety of ␦-endotoxins (Cry proteins) that are primarily responsible for a highly specific insecticidal activity (53). For this reason, B. thuringiensis is being used presently as a biological insecticide, accounting for more than 90% of the biopesticides employed worldwide (37).Despite a long record of being safe, there is increasing public concern about the potential pathogenicity of B. thuringiensis to humans. This is due to the sporadic but sometimes severe infections caused by this organism, which include diarrheal food poisoning (34), corneal ulcer (52), cellulitis (57), and burn (13) as well as war wound (28) infections. Moreover, the pathogenicity of B. thuringiensis has been demonstrated in an animal model of infection by administering spores to mice via nasal inoculation (27,50). Similar to Bacillus cereus, B. thuringiensis produces a variety of virulence factors, which include phosphatidylcholine-preferring phospholipase C (PC-PLC) and phosphatidylinositol-specific phospholipase C (38, 40), hemolysins, and enterotoxins (1,18,21). Heierson et al. (24) isolated an avirulent...

Characterization of a broad-host-range flagellum-dependent phage that mediates high-efficiency generalized transduction in, and between, Serratia and Pantoea

“…The flhA mutant, which is deficient in flagellum export (Ghelardi et al, 2002), had the dual phenotype of phage resistance and non-motility, as predicted. This mutant was chosen for genetic complementation tests because, by comparison with gene organization in other genomes, the gene is predicted not to be part of a large operon.…”

Genome-wide Expression Analyses of Campylobacter jejuni NCTC11168 Reveals Coordinate Regulation of Motility and Virulence by flhA

“…These included fliA and rpoN, which encode 28 and 54 , respectively; flgR, which encodes a 54 activator (14); and flhA and flhB, which have been implicated in the regulation of flagellar genes in campylobacter and in other organisms (26,27,31). PCR products of flagellar regulators generated from V1 and V26 were sequenced in both directions, including at least 200 nt of the upstream region.…”

Quorum signal molecules as biosurfactants affecting swarming in Rhizobium etli

“…This process and other forms of surface motility (reviewed in ref. 9) are found in many bacterial genera (11)(12)(13)(14)(15). Swarmer cells are often hyperflagellated, elongated, and multinucleated.…”