Ethanol tolerance, yield of melanin, swarming motility and growth are correlated with the expression levels of aiiA gene in Bacillus thuringiensis

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Abstract

To investigate the role of aiiA gene in Bacillus thuringiensis, B. thuringiensis strain BMB171 was selected and three derivatives were constructed, such that (1) aiiA was knocked out (aiiA-KO), (2) aiiA-KO was reversed (aiiA-Rev) and (3) the expression of aiiA was elevated (aiiA-Elevated). First, we found that mutation of aiiA increased the ethanol tolerance in B. thuringiensis. Omitting ethanol from the medium resulted in a decreased generation time for aiiA-Elevated. In contrast, aiiA-KO had the highest ethanol tolerance when the medium contained 2 or 4% ethanol. Second, we found that more melanin was produced at 37 °C when aiiA expression was elevated. Third, we found inhibition of swarming motility in the aiiA-Elevated group. Finally, there was delayed sporulation in Luria–Bertani, ICPM, casein and ammonium sulfate medium in the aiiA-Elevated group. The conclusion was that ethanol tolerance, yield of melanin, swarming motility and growth are correlated with the expression levels of aiiA gene in B. thuringiensis.

Introduction

Three genetically related members of the Bacillus cereus group, Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis, have been important in medical and agricultural studies [13], [14]. B. anthracis, the causative agent of anthrax, is a virulent mammalian pathogen that often causes animal and human mortality. B. cereus is frequently isolated as a contaminant of various foods and can occasionally be an opportunistic human pathogen. B. thuringiensis is distinguished from B. cereus and B. anthracis by production of parasporal crystals in association with spore formation and by its pathogenicity to many species of insects.

The B. cereus group contains a common aiiA gene coding for N-acyl homoserine lactonase. When the complete B. cereus group genome sequence was scanned, the aiiA homologue gene was also found in B. cereus ATCC14579 genome from 3409487 to 3410239 (AE016877), in B. cereus ATCC10987 from 3223999 to 3224751 (AE017194), in B. anthracis str. Sterne from 3226177 to 3226929 (AE017225), in B. anthracis str. A2012 from 3689755 to 3690507 (AAAC01000001), in B. anthracis str. ‘Ames Ancestor’ from 3225611 to 3226363 (AE017334) and in B. thuringiensis serovar konkukian str. 97-27 from 3298004 to 3298756 (AE017355). The aiiA gene was first reported in Bacillus sp. 240B1 where DNA sequencing indicated that the N-acyl homoserine lactonase contained the “HXDHHD” zinc-binding motif [9]. This enzyme inactivates the N-acyl homoserine lactone (AHL) signal molecular by hydrolyzing its lactone bond, which in turn disrupts AHL based cell-to-cell communication [8]. Many bacterial isolates have been reported to produce similar AHL lactonase in B. thuringiensis, B. cereus and Bacillus mycoides [7], [16].

The role of aiiA in B. cereus group is unclear. It has been suggested that insecticidal B. thuringiensis strains might compete with gram-negative bacteria in natural ecosystems by autoinducer-degrading activity [7], [16]. However, some facts contradict this hypothesis. First, the ecological environment and nutrition sources of the B. cereus group are not same as that of gram-negative bacteria, such as Erwinia carotovora. That is, E. carotovora specializes in degrading plant-derived nutrition while the B. cereus group prefers protein and amino acid substrates [3], [14]. Thus, competition seems unlikely. Second, many bacteria that do not contain the aiiA gene can also survive in the same ecological environment as E. carotovora. Additionally, sequence alignment showed that AHL lactonase contained a zinc-binding motif that was conservative in several groups of metallohydrolases. However, AHL lactonase still had AHL-degrading activity even if no zinc was present [25]. Thus, we hypothesize that AHL lactonase may have a self-regulating function in the B. cereus group. Therefore, we mutated, reverse mutated and over-expressed the aiiA gene to assay the function of the B. thuringiensis AHL lactonase.

Section snippets

Bacterial strains and plasmids

The bacterial strains and plasmids used in this study are listed in Table 1. B. thuringiensis acrystalliferous BMB171 was used for the initial strain (aiiA-Normal). Escherichia coli strains were grown at 37 °C in Luria–Bertani (LB) medium. Agrobacterium tumefaciens strains were grown at 28 °C in AB medium [5]. B. thuringiensis stains were grown at 28, 37 or 42 °C in different experiments. The other medium used in this study were ICPM medium (0.3–1% tryptone, 0.5% glucose, 0.1% CaCO3, 0.05% MgSO4,

AiiA protein regulates ethanol tolerance of B. thuringiensis

When we inoculated aiiA-Normal, aiiA-KO, aiiA-Rev and aiiA-Elevated in LB medium containing 0, 2, 4, 6 and 10% ethanol, the rate of growth of the aiiA mutations had an inverse relationship with the ethanol level (Fig. 3). Omitting ethanol from the medium resulted in a decreased generation time for aiiA-Elevated (Fig. 3A). The difference between aiiA-Elevated and aiiA-KO was statistically significant (P < 0.05). When the LB medium contained 2% or 4% ethanol, aiiA-KO had the best ethanol tolerance,

Discussion

AHL lactonase, first reported as AiiA protein, quenches the AHL signal molecular in pathogens [9]. Our data suggested some novel functions of AHL lactonase, as compared to other studies [7], [16]. That is, elevated expression of aiiA reduces ethanol tolerance, increases melanin production, inhibits swarming and delays sporulation of B. thuringiensis. All of the data in this study suggest that elevated expression of aiiA may facilitate the entry and exit of substances around a cell. However, the

Acknowledgements

This work was supported by grants from Chinese 973 Plan (2003CB114201), National Natural Science of Foundation of China (30270053), Chinese National 863 Plan (2003AA223081), and Key Research Project of Ministry of Education of China. We are grateful to Stephen C. Winans (Cornell University, Ithaca, New York), who kindly offer the strains A. tumefaciens WCF47 and E. coli DH5α (pJZ365).

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