Biosynthesis of UDP-glucuronic acid and UDP-galacturonic acid in Bacillus cereus subsp. cytotoxis NVH 391-98

The food borne pathogen Bacillus cereus produces uronic acid-containing glycans that are secreted in a shielding biofilm environment, and certain alkaliphilic Bacillus deposit uronate-glycan polymers in the cell wall when adapting to alkaline environments. The source of these acidic sugars is unknown and, in the present study, we describe the functional identification of an operon in Bacillus cerues subsp. cytotoxis NVH 391-98 that comprises genes involved in the synthesis of UDP-uronic acids in Bacillus spp. Within the operon, a UDP-glucose 6-dehydrogenase converts UDP-glucose in the presence of NAD(+) to UDP-glucuronic acid and NADH, and a UDP-GlcA 4-epimerase (UGlcAE) converts UDP-glucuronic acid to UDP-galacturonic acid. Interestingly, in vitro, both enzymes can utilize the TDP-sugar forms as well, albeit at lower catalytic efficiency. Unlike most of the very few bacterial 4-epimerases that have been characterized, which are promiscuous, the B. cereus UGlcAE enzyme is very specific and cannot use UDP-glucose, UDP-N-acetylglucosamine, UDP-N-acetylglucosaminuronic acid or UDP-xylose as substrates. Size exclusion chromatography suggests that UGlcAE is active as a monomer, unlike the dimeric form of plant enzymes; the Bacillus UDP-glucose 6-dehydrogenase is also found as a monomer. Phylogenic analysis further suggests that the Bacillus UGlcAE may have evolved separately from other bacterial and plant epimerases. Our results provide insight into the formation and function of uronic acid-containing glycans in the lifecycle of B. cereus and related species containing homologous operons, as well as a basis for determining the importance of these acidic glycans. We also discuss the ability to target UGlcAE as a drug candidate.     

Biosynthesis of a New UDP-sugar,UDP-2-acetamido-2-deoxyxylose,in the Human Pathogen Bacillus cereus Subspecies cytotoxis NVH 391-98

We have identified an operon and characterized the functions of two genes from the severe food-poisoning bacterium, Bacillus cereus subsp. cytotoxis NVH 391-98, that are involved in the synthesis of a unique UDP-sugar, UDP-2-acetamido-2-deoxyxylose (UDP-N-acetyl-xylosamine, UDP-XylNAc). UGlcNAcDH encodes a UDP-N-acetyl-glucosamine 6-dehydrogenase, converting UDP-N-acetylglucosamine (UDP-GlcNAc) to UDP-N-acetyl-glucosaminuronic acid (UDP-GlcNAcA). The second gene in the operon, UXNAcS, encodes a distinct decarboxylase not previously described in the literature, which catalyzes the formation of UDP-XylNAc from UDP-GlcNAcA in the presence of exogenous NAD⁺. UXNAcS is specific and cannot utilize UDP-glucuronic acid and UDP-galacturonic acid as substrates. UXNAcS is active as a dimer with catalytic efficiency of 7 mm⁻¹ s⁻¹. The activity of UXNAcS is completely abolished by NADH but unaffected by UDP-xylose. A real-time NMR-based assay showed unambiguously the dual enzymatic conversions of UDP-GlcNAc to UDP-GlcNAcA and subsequently to UDP-XylNAc. From the analyses of all publicly available sequenced genomes, it appears that UXNAcS is restricted to pathogenic Bacillus species, including Bacillus anthracis and Bacillus thuringiensis. The identification of UXNAcS provides insight into the formation of UDP-XylNAc. Understanding the metabolic pathways involved in the utilization of this amino-sugar may allow the development of drugs to combat and eradicate the disease.     

Biofilm Formation and Cell Surface Properties among Pathogenic and Nonpathogenic Strains of the Bacillus cereus Group

Biofilm formation by 102 Bacillus cereus and B. thuringiensis strains was determined. Strains isolated from soil or involved in digestive tract infections were efficient biofilm formers, whereas strains isolated from other diseases were poor biofilm formers. Cell surface hydrophobicity, the presence of an S layer, and adhesion to epithelial cells were also examined.The Bacillus cereus group includes B. cereus sensu stricto, B. anthracis, and B. thuringiensis, three genetically close pathogenic species. Based on genetic evidence, it has been suggested that they could represent one species (7). B. cereus sensu stricto is itself an opportunistic human pathogen occasionally found to cause various diseases such as endophthalmitis or periodontitis but is more frequently involved in gastrointestinal diseases with diarrheal or emetic syndromes (4, 12). Emetic syndromes result from the presence of cereulide, a heat-stable toxin produced in food before ingestion, whereas diarrheal syndromes require survival of the bacterium in the host digestive tract. B. thuringiensis is an insect pathogen, and B. anthracis causes anthrax, a lethal human disease.The persistent contamination of industrial food processing systems by B. cereus (12) may facilitate its involvement in gastroenteritis. This persistence is due to spores, which may survive pasteurization, heating, and gamma-ray irradiation (9, 13), and to biofilms, which have been shown to be highly resistant to cleaning procedures (18). Biofilms are also suspected to be involved in bacterial pathogenicity, as they may form on host epithelia (15).In this study, we wanted to test whether biofilm formation by species of the B. cereus group could be connected to the pathogenicity of the bacterium. For this purpose, we screened a collection of 102 pathogenic (diarrheal, emetic, and oral diseases) and nonpathogenic strains of B. cereus and B. thuringiensis for their capability to form biofilms. As adhesion to inert or living surfaces is a prerequisite for biofilm formation, we have investigated relationships within our collection of strains between biofilm formation and cell surface hydrophobicity, the presence of an S-layer, or adhesion to epithelial cells.     

The Hemolytic Enterotoxin HBL Is Broadly Distributed among Species of the Bacillus cereus Group

The prevalence of the hemolytic enterotoxin complex HBL was determined in all species of the Bacillus cereus group with the exception of Bacillus anthracis. hblA, encoding the binding subunit B, was detected by PCR and Southern analysis and was confirmed by partial sequencing of 18 strains. The sequences formed two clusters, one including B. cereus and Bacillus thuringiensis strains and the other one consisting of Bacillus mycoides, Bacillus pseudomycoides, and Bacillus weihenstephanensis strains. From eight B. thuringiensis strains, the enterotoxin gene hblA could be amplified. Seven of them also expressed the complete HBL complex as determined with specific antibodies against the L₁, L₂, and B components. Eleven of 16 B. mycoides strains, all 3 B. pseudomyoides strains, 9 of 15 B. weihenstephanensis strains, and 10 of 23 B. cereus strains carried hblA. While HBL was not expressed in the B. pseudomycoides strains, the molecular assays were in accordance with the immunological assays for the majority of the remaining strains. In summary, the hemolytic enterotoxin HBL seems to be broadly distributed among strains of the B. cereus group and relates neither to a certain species nor to a specific environment. The consequences of this finding for food safety considerations need to be evaluated.     

耐冷菌Bacillus cereus SYP-A3-2低温蛋白酶的发酵条件

研究了营养因子及环境条件对中国冰川1号耐冷菌Bacillus cereus SYP-A3-2产低温蛋白酶的影响。结果表明对该菌产酶影响较大的因素分别是温度、酪蛋白浓度和pH,在酪蛋白浓度0．8％、pH7、15℃下摇瓶发酵48h产酶水平可达3,800U／mL。     

The CasKR Two-Component System Is Required for the Growth of Mesophilic and Psychrotolerant Bacillus cereus Strains at Low Temperatures

The different strains of Bacillus cereus can grow at temperatures covering a very diverse range. Some B. cereus strains can grow in chilled food and consequently cause food poisoning. We have identified a new sensor/regulator mechanism involved in low-temperature B. cereus growth. Construction of a mutant of this two-component system enabled us to show that this system, called CasKR, is required for growth at the minimal temperature (T_min). CasKR was also involved in optimal cold growth above T_min and in cell survival below T_min. Microscopic observation showed that CasKR plays a key role in cell shape during cold growth. Introducing the casKR genes in a ΔcasKR mutant restored its ability to grow at T_min. Although it was first identified in the ATCC 14579 model strain, this mechanism has been conserved in most strains of the B. cereus group. We show that the role of CasKR in cold growth is similar in other B. cereus sensu lato strains with different growth temperature ranges, including psychrotolerant strains.     

Discrimination Between Mesophilic and Psychrotolerant Strains in the Bacillus cereus Group Based on the PstI Digestion of the pycA Gene

A simple and rapid assay for the detection of Bacillus weihenstephanensis isolates and other psychrotolerant strains in the Bacillus cereus group was developed. It is based on the presence of a nucleotide substitution at position 795 on the housekeeping pycA gene in all B. weihenstephanensis strains. This mutation creates a PstI recognition site. It is absent in mesophilic strains in the B. cereus group. The pycA gene is amplified by PCR and the amplicons submitted to PstI digestions. In mesophilic strains, a single band of 1,718 bp in length is visualised on an agarose gel. In B. weihenstephanensis strains and in all other psychrotolerant strains from the B. cereus group, the amplicons are cleaved and two bands of 1,175 and 543 bp, respectively, are visualised. This method could be used for the screening of B. cereus collections and for the identification of psychrotolerant and mesophilic isolates from different environments.     

The Novel PRRSV Strain HBap4-2018 with a Unique Recombinant Pattern Is Highly Pathogenic to Piglets

Virologica Sinica - Currently, various porcine reproductive and respiratory syndrome virus (PRRSV) variants emerged worldwide with different genetic characteristics and pathogenicity, increasing...     

Discrimination of Psychrotrophic and Mesophilic Strains of the Bacillus cereus Group by PCR Targeting of Major Cold Shock Protein Genes

Detection of psychrotrophic strains (those able to grow at or below 7°C) of the Bacillus cereus group (Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides) in food products is at present extremely slow with conventional microbiology. This is due to an inability to discriminate these cold-adapted strains from their mesophilic counterparts (those able to grow only above 7°C) by means other than growth at low temperature, which takes 5 to 10 days for detection. Here we report the development of a single PCR assay that, using major cold shock protein-specific primers and appropriate annealing temperatures, is capable of both rapidly identifying bacteria of the B. cereus group and discriminating between psychrotrophic and mesophilic strains. It is intended that this development help to more accurately predict the shelf life of refrigerated pasteurized food and dairy products and to reduce the incidence of food poisoning by psychrotrophic strains of the B. cereus group.     

The ISBsu2 Mobile Element Is Present in a Plasmid of a Soil Strain and in the Chromosomes of Several Other Strains of Bacillus subtilis

Chromosomes of several Bacillus subtilis strains were shown to contain homologs of the ISBsu2 mobile genetic element, which was earlier revealed in a cryptic plasmid of a soil strain of B. subtilis.     

Comparison of Minisatellite Polymorphisms in the Bacillus cereus Complex: a Simple Assay for Large-Scale Screening and Identification of Strains Most Closely Related to Bacillus anthracis

Polymorphism of five tandem repeats that are monomorphic in Bacillus anthracis was investigated in 230 isolates of the B. cereus group and in 5 sequenced B. cereus genomes in search for markers allowing identification of B. cereus and B. thuringiensis strains most closely related to B. anthracis. Using this multiple-locus variable number of tandem repeat analysis (MLVA), a cluster of 30 strains was selected for further characterization. Eventually, six of these were characterized by multilocus sequence type analysis. One of the strains is only six point mutations (of almost 3,000 bp) away from B. anthracis and was also proposed to be closest to B. anthracis by MLVA analysis. However, this strain remains separated from B. anthracis by a number of significant genetic events observed in B. anthracis, including the loss of the hemolysin activity, the presence of four prophages, and the presence of the two virulence plasmids, pXO1 and pXO2. One particular minisatellite marker provides an efficient assay to identify the subset of B. cereus and B. thuringiensis strains closely related to B. anthracis. Based on these results, a very simple assay is proposed that allows the screening of hundreds of strains from the B. cereus complex, with modest equipment and at a low cost, to eventually fill the gap with B. anthracis and better understand the origin and making of this dangerous pathogen.     

Identification of the Critical Role of Tyr-194 in the Catalytic Activity of a Novel N-Acyl-Homoserine Lactonase from Marine Bacillus cereus Strain Y2

Enzymatic disruption of quorum-sensing (QS) pathways in pathogenic organisms is a promising anti-infection therapeutic strategy. AHL-lactonase, a potent tool for biocontrol, can hydrolyze QS signal molecule N-acyl-homoserine lactones (AHLs) into inactive products, thereby blocking the QS systems. A marine bacterial isolate Y2, identified as a Bacillus cereus subsp., was found capable of inactivating AHLs. The aiiA gene encoding the AHL-degrading enzyme from bacterial strain Y2 was cloned and expressed in Escherichia coli. The 28-kDa recombinant Y2-AiiA protein was purified and showed strong AHL-degrading activity. Sequence comparisons of Y2-aiiA with known AHL-lactonases revealed high identities in the deduced amino-acid sequences. Functional determination of a potential catalytic residue Tyr-194 of AHL-lactonases was performed by site-directed mutagenesis. As judged by AHL-degrading bioassay, substitution of Tyr-194 with Ala resulted in a dramatic decrease of activity compared with wild-type (WT) recombinant Y2-AiiA, although the expression level of the mutated Y2-AiiA protein was equivalent to that of WT Y2-AiiA. These results suggested that the conserved residue Tyr-194 is critical for catalytic function of the novel AHL-lactonase.     

The certification of a reference material for the evaluation of methods for the enumeration of Bacillus cereus

A reference material containing Bacillus cereus was certified by the Community Bureau of Reference (BCR) for its number of colony-forming particles (cfp) in 0.1 ml reconstituted capsule solution. To this end, a batch of approximately 15,000 capsules was produced and tested for its homogeneity and stability. The variation in the number of cfp between capsules (homogeneity) was not found to be significantly different from a Poisson distribution. Stability was tested for extended periods at storage temperature (-20 degrees C) and at various higher temperatures up to 37 degrees C for 4 weeks to simulate transport conditions. Only at 37 degrees C did a small but significant decrease in the number of cfp occur. At -20 degrees C, no decrease in the number of cfp was observed over a period of about 4 years. For certification, 12 laboratories determined the number of cfp on two agars: Mannitol Egg-Yolk Polymyxin agar (MEYP, incubated at 30 degrees C) and Polymyxin pyruvate Egg-yolk Bromothymol blue Agar (PEMBA, incubated at 37 degrees C). The certified geometric mean value on MEYP after 24 h of incubation was 53.4 cfp 0.1 ml-1 of the reconstituted capsule solution (95% confidence interval 51.7-55.2) and on PEMBA, 55.0 (95% confidence interval 52.8-57.4). Based on these certified values, user tables were constructed specifying the 95% confidence limits when testing a smaller number of capsules, as would be done in individual laboratories. Based on the information on homogeneity, stability and the certification study, the BCR decided to certify the material as CRM 528.