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Strains of Bacillus Cereus Vary in the Phenotypic Adaptation of Their Membrane Lipid Composition in Response to Low Water Activity, Reduced Temperature and Growth in Rice Starch

Md Anwarul Haque
Grain Quality and Nutrition Division, Bangladesh Rice Research Institute, Joydebpur, Gazipur-1701, Bangladesh; Department of Agricultural Sciences, Imperial College, London, Wye campus, Ashford, Kent TN25 5AH, UK

Nicholas J. Russell
Department of Agricultural Sciences, Imperial College London, Wye campus, Ashford, Kent TN25 5AH, UK

Abstract/ Executive Summary:

The phenotypic adaptation of membrane lipids in seven strains of the food-poisoning bacterium Bacillus cereus, isolated from Bangladeshi rice, is reported in relation to their ability to grow under conditions of low water activity (aw), reduced temperature and the presence of soluble rice starch. The strains have different membrane phospholipid head-group and fatty acyl compositions, and they display individual differences in their responses to both low aw and reduced temperature. The extent of the increase in anionic membrane lipids in response to low aw varies from strain to strain, is solute specific and in one strain does not occur. Growth is stimulated by the presence of soluble rice starch and results in a large rise in the proportion of diphosphatidylglycerol (DPG) at the expense of phosphatidylglycerol (PG), without any change in the proportion of total anionic phospholipids. Growth at 15 6C compared with 37 6C increases the proportions of DPG and phosphatidylethanolamine at the expense of PG. At the lower temperature, there are changes in phospholipid fatty acyl composition characteristic of those expected to maintain membrane fluidity, including increases in the amount of total branched fatty acids and the anteiso-/iso-branched ratio, and a decrease in the equivalent chain-length, but there are strain differences in how those changes were achieved. In contrast to some other bacilli, there are persistent large increases in the proportions of unsaturated fatty acyl chains in phospholipids during growth at 15 6C.

Keywords: Strains of Bacillus cereus, Phenotypic adaptation, Low water activity, Reduced temperature, Rice starch

Location:

Start Date:

End Date:

Program Area: Quality and Nutrition

Commodity: Rice

Objectives:

It was of interest to investigate the effects of stress due to lowered temperature and aw, as well as the influence of rice starch, on membrane lipid adaptation to understand better how this organism can grow in heated rice as it cools and cause food poisoning.

Methodology:

Bacillus cereus strains. The seven B. cereus strains used in this study were isolated and identified from two high-yielding modern varieties of Bangladeshi rice (BR5 and BRRI Dhan28) (Haque, 2002). The BRRI Dhan28 variety is parboiled, dried and milled prior to retail sale. Preparation of soluble rice-starch medium. Soluble rice starch was prepared by boiling 100 g rice in 1 l sterile water for approximately 17 min (according to cooking time), and the rice was removed by filtration through 12-fold cheesecloth. The filtrate was termed 100 % soluble rice starch and appropriate volumes were added to subtilis minimal medium, prepared according to Clowes & Hayes (1968), to give 70 % (v/v) soluble rice-starch medium; when required, this medium was solidified by the addition of agar (Difco; 1-5 %, w/v, final concn). Extraction of lipid and quantification of phospholipid. Cultures of B. cereus were grown in nutrient broth (Oxoid) at 37 or 15 uC in the presence or absence of NaCl or sucrose to lower aw. Bacteria were harvested in the late-exponential phase by centrifuging at 16 266 gav for 15 min (rav=10 cm) and the cell pellet was washed twice with 01 M phosphate buffer (pH 7-0). To test the effect of rice starch on lipid composition, bacteria were grown in subtilis minimal medium containing 70 % (v/v) soluble rice starch. Total lipids were extracted using the method of Bligh & Dyer (1959) as described by Kates (1986). Phospholipids were separated by two-dimensional thin-layer chromatography and quantified on the basis of their phosphorus content as described by Ntougias & Russell (2001). Fatty acid analysis by capillary GC. Fatty acid methyl esters (FAMEs) were produced from total lipid via transmethylation with 2-5 % (v/v) concentrated sulphuric acid in dry methanol. Hydrogenation of FAMEs was carried out to identify those fatty acids containing unsaturated bonds (Kates, 1986); double bond positions were not determined. The FAMEs were analyzed by capillary GC using a Pye Unicam PU4500 gas chromatograph equipped with a Supelco SP2380 fused silica capillary column (length 30 m, i.d. 0-25 mm and film thickness 0<20 mm) and a Spectraphysics SP420 integrator. The carrier gas was nitrogen with a flow rate of 40 ml min 21. Samples were analyzed isothermally at 160 ⁰C. The injector and detector temperatures were 250 ⁰C. The peaks were identified by comparison of their retention times with those of a standard bacterial FAME mix (Supelco 4-7080) and confirmed by GC-MS when necessary. Statistical analysis. The student’s t-test was used to determine the statistical significance of paired values, with 95 % being taken as the significant limit.

Source of Information: Microbiology (2004), 150, 1397–1404

Article Link (Online Journal): DOI 10.1099/mic.0.26767-0

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

At the lower temperature, there are changes in phospholipid fatty acyl composition characteristic of those expected to maintain membrane fluidity, including increases in the amount of total branched fatty acids and the anteiso-/iso-branched ratio, and a decrease in the equivalent chain-length, but there are strain differences in how those changes were achieved. In contrast to some other bacilli, there are persistent large increases in the proportions of unsaturated fatty acyl chains in phospholipids during growth at 15 ⁰C.

Knowledge Management: Journal