Discovery of a Unique Extracellular Polysaccharide in Members of the Pathogenic Bacillus That Can Co-form with Spores

Abstract

An exopolysaccharide, produced during the late stage of stationary growth phase, was discovered and purified from the culture medium of Bacillus cereus, Bacillus anthracis, and Bacillus thuringiensis when strains were grown in a defined nutrient medium that induces biofilm. Two-dimensional NMR structural characterization of the polysaccharide, named pzX, revealed that it is composed of an unusual three amino-sugar sequence repeat of [-3)XylNAc4OAc(␣1-3)GlcNAcA4OAc(␣1-3)XylNAc(␣1-] n . The sugar residue XylNAc had never been described previously in any glycan structure. The XNAC operon that contains the genes for the assembly of pzX is also unique and so far has been identified only in members of the Bacillus cereus sensu lato group. Microscopic and biochemical analyses indicate that pzX co-forms during sporulation, so that upon the release of the spore to the extracellular milieu it becomes surrounded by pzX. The relative amounts of pzX produced can be manipulated by specific nutrients in the medium, but rich medium appears to suppress pzX formation. pzX has the following unique characteristics: a surfactant property that lowers surface tension, a cell/spore antiaggregant, and an adherence property that increases spores binding to surfaces. pzX in Bacillus could represent a trait shared by many spore-producing microorganisms. It suggests pzX is an active player in spore physiology and may provide new insights to the successful survival of the B. cereus species in natural environments or in the hosts.In 1881, Louis Pasteur developed the first vaccine for anthrax, the devastating disease caused by Bacillus anthracis, a Gram-positive endospore-forming bacterium. Since then, outbreaks of anthrax affecting humans and animals have dramatically decreased (1, 2), although infrequent incidents have been reported (3). However, letters containing B. anthracis spores sent by mail to United States officials a week after the September 11, 2001 attack, led to public fear of infection and the use of its spores as a bioweapon agent (4). B. anthracis, which elicits different disease phenotypes, is closely related in terms of gene content and synteny (5) to other Bacillus species collectively named Bacillus cereus sensu lato (6) or B. cereus group. These highly related bacilli are able to colonize in diverse hosts, including insects and mammals, and they are commonly found in soil, water, and depending on the species, in cadavers, vegetation, and food. In addition to B. anthracis, this group includes B. cereus, recognized as a cause of food-poisoning toxins, and Bacillus thuringiensis that produces insecticidal proteins. In the past 15 years, several B. cereus strains were reported to cause severe anthrax-like disease in humans (7,8) and apes (9). Some of these virulent strains, while retaining B. cereus diagnostic phenotypes, harbor plasmids similar to the toxin and capsule virulence plasmids pXO1 and pXO2 present in B. anthracis (10 -12). Hence, it is no wonder that the need to distinguish these Bacillus strains has led to a mas...

Two genomic regions encoding exopolysaccharide production systems have complementary functions in B. cereus multicellularity and host interaction

“…These polymers can play various structural functions, including coating of the cell wall, capsule formation, spore surface decoration, and providing adhesion or structural support for biofilms; furthermore, they also offer protection against various stresses, provide a storage reservoir for carbon and protect the bacterial cells against host defenses 49,50 . B. cereus has been shown to produce several different polysaccharides, including (i) exopolysaccharides associated with sporulation (genes BC0484-90) 51 , (ii) two spore-decorating sugars (genes BC3358-61) 31 , and (iii) two secondary cell wall polysaccharides (SCWP) (genes unknown) 52 . In this work, we have investigated the roles of two B. cereus genomic regions putatively dedicated to the production of two different polysaccharides, referred to as eps1 and eps2.…”

A four-gene operon in Bacillus cereus produces two rare spore-decorating sugars

“…In this study, we examined sugar composition under different hydrolytic conditions using B. cereus ATCC 14579 cells. In addition, B. cereus was grown in different media because its glycans change as the medium is altered (14). Using this approach, we detected two unknown sugar peaks (labeled B and C in Fig.…”

“…A 1:100 dilution of an overnight BHI culture of B. cereus ATCC 14579 was used to inoculate 50 ml of liquid medium (BHI or Msgg), and the cultures were grown for up to 72 h at 30°C with shaking (200 rpm). Cells were rapidly pelleted by centrifugation (10,000 ϫ g for 1 min at 4°C), resuspended in 0.8 ml of lysozyme solution (14), and incubated at room temperature for 10 min. To each sample 80 l of 10ϫ EB (0.3 M NaOAc, pH 5.2, 50 mM EDTA, 5% sarkosyl, and 1.42 M ␤-mercaptoethanol) was added.…”

“…The resulting alditol-acetate derivatives were analyzed using a GC/EI-MS system (Agilent 7890a/5975c) equipped with an autosampler injector (Agilent 7693). A 1-l sample was injected into the GC column (Equity-1 or DB-5, 30 m ϫ 0.25 mm, 0.25-m film thickness) using split mode (1:50) with an injector inlet setting of 250°C (helium at 3 ml/min), and chromatography was performed as described previously (14). In some cases the GC column used was Rtx-2330 by RESTEK.…”

“…Gene knock-out was achieved by double crossover recombination as described previously (14). Two DNA fragments flanking gene BC3360 and two DNA fragments flanking gene BC3361 were PCR-amplified by specific primer sets ZL479 and ZL480, ZL481 and ZL482, ZL489 and ZL490, and ZL491 and ZL492, respectively (see supplemental Table S2).…”