scholarly journals The sps Genes Encode an Original Legionaminic Acid Pathway Required for Crust Assembly in Bacillus subtilis

mBio ◽  
2020 ◽  
Vol 11 (4) ◽  
Author(s):  
Thomas Dubois ◽  
Frederic Krzewinski ◽  
Nao Yamakawa ◽  
Christelle Lemy ◽  
Audrey Hamiot ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Food Industry ◽  
Safety Concern ◽  
Superficial Layer ◽  
Biosynthesis Pathway ◽  
Spore Surface ◽  
Human Pathogens ◽  
Adhesion Properties ◽  
Content Type ◽  
Nonulosonic Acid

ABSTRACT The crust is the outermost spore layer of most Bacillus strains devoid of an exosporium. This outermost layer, composed of both proteins and carbohydrates, plays a major role in the adhesion and spreading of spores into the environment. Recent studies have identified several crust proteins and have provided insights about their organization at the spore surface. However, although carbohydrates are known to participate in adhesion, little is known about their composition, structure, and localization. In this study, we showed that the spore surface of Bacillus subtilis is covered with legionaminic acid (Leg), a nine-carbon backbone nonulosonic acid known to decorate the flagellin of the human pathogens Helicobacter pylori and Campylobacter jejuni. We demonstrated that the spsC, spsD, spsE, spsG, and spsM genes of Bacillus subtilis are required for Leg biosynthesis during sporulation, while the spsF gene is required for Leg transfer from the mother cell to the surface of the forespore. We also characterized the activity of SpsM and highlighted an original Leg biosynthesis pathway in B. subtilis. Finally, we demonstrated that Leg is required for the assembly of the crust around the spores, and we showed that in the absence of Leg, spores were more adherent to stainless steel probably because of their reduced hydrophilicity and charge. IMPORTANCE Bacillus species are a major economic and food safety concern of the food industry because of their food spoilage-causing capability and persistence. Their persistence is mainly due to their ability to form highly resistant spores adhering to the surfaces of industrial equipment. Spores of the Bacillus subtilis group are surrounded by the crust, a superficial layer which plays a key role in their adhesion properties. However, knowledge of the composition and structure of this layer remains incomplete. Here, for the first time, we identified a nonulosonic acid (Leg) at the surfaces of bacterial spores (B. subtilis). We uncovered a novel Leg biosynthesis pathway, and we demonstrated that Leg is required for proper crust assembly. This work contributes to the description of the structure and composition of Bacillus spores which has been under way for decades, and it provides keys to understanding the importance of carbohydrates in Bacillus adhesion and persistence in the food industry.

scholarly journals Spore Heat Activation Requirements and Germination Responses Correlate with Sequences of Germinant Receptors and with the Presence of a Specific spoVA2mob Operon in Foodborne Strains of Bacillus subtilis

2017 ◽  
Vol 83 (7) ◽  
Author(s):  
Antonina O. Krawczyk ◽  
Anne de Jong ◽  
Jimmy Omony ◽  
Siger Holsappel ◽  
Marjon H. J. Wells-Bennik ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Heat Resistance ◽  
Spore Germination ◽  
Food Industry ◽  
Heat Treatments ◽  
Phenotypic Traits ◽  
Content Type ◽  
Heat Resistant ◽  
Heat Activation ◽  
High Level

ABSTRACT Spore heat resistance, germination, and outgrowth are problematic bacterial properties compromising food safety and quality. Large interstrain variation in these properties makes prediction and control of spore behavior challenging. High-level heat resistance and slow germination of spores of some natural Bacillus subtilis isolates, encountered in foods, have been attributed to the occurrence of the spoVA 2mob operon carried on the Tn1546 transposon. In this study, we further investigate the correlation between the presence of this operon in high-level-heat-resistant spores and their germination efficiencies before and after exposure to various sublethal heat treatments (heat activation, or HA), which are known to significantly improve spore responses to nutrient germinants. We show that high-level-heat-resistant spores harboring spoVA 2mob required higher HA temperatures for efficient germination than spores lacking spoVA 2mob. The optimal spore HA requirements additionally depended on the nutrients used to trigger germination, l-alanine (l-Ala), or a mixture of l-asparagine, d-glucose, d-fructose, and K+ (AGFK). The distinct HA requirements of these two spore germination pathways are likely related to differences in properties of specific germinant receptors. Moreover, spores that germinated inefficiently in AGFK contained specific changes in sequences of the GerB and GerK germinant receptors, which are involved in this germination response. In contrast, no relation was found between transcription levels of main germination genes and spore germination phenotypes. The findings presented in this study have great implications for practices in the food industry, where heat treatments are commonly used to inactivate pathogenic and spoilage microbes, including bacterial spore formers. IMPORTANCE This study describes a strong variation in spore germination capacities and requirements for a heat activation treatment, i.e., an exposure to sublethal heat that increases spore responsiveness to nutrient germination triggers, among 17 strains of B. subtilis, including 9 isolates from spoiled food products. Spores of industrial foodborne isolates exhibited, on average, less efficient and slower germination responses and required more severe heat activation than spores from other sources. High heat activation requirements and inefficient, slow germination correlated with elevated resistance of spores to heat and with specific genetic features, indicating a common genetic basis of these three phenotypic traits. Clearly, interstrain variation and numerous factors that shape spore germination behavior challenge standardization of methods to recover highly heat-resistant spores from the environment and have an impact on the efficacy of preservation techniques used by the food industry to control spores.

scholarly journals A SpoIID Homolog Cleaves Glycan Strands at the Chlamydial Division Septum

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Nicolas Jacquier ◽  
Akhilesh K. Yadav ◽  
Trestan Pillonel ◽  
Patrick H. Viollier ◽  
Felipe Cava ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Intracellular Bacteria ◽  
Human Pathogens ◽  
Content Type ◽  
Obligate Intracellular ◽  
Peptidoglycan Biosynthesis ◽  
Peptidoglycan Synthesis ◽  
Lytic Transglycosylase ◽  
Obligate Intracellular Bacteria

ABSTRACT Chlamydiales species are obligate intracellular bacteria lacking a classical peptidoglycan sacculus but relying on peptidoglycan synthesis for cytokinesis. While septal peptidoglycan biosynthesis seems to be regulated by MreB actin and its membrane anchor RodZ rather than FtsZ tubulin in Chlamydiales, the mechanism of peptidoglycan remodeling is poorly understood. An amidase conserved in Chlamydiales is able to cleave peptide stems in peptidoglycan, but it is not clear how peptidoglycan glycan strands are cleaved since no classical lytic transglycosylase is encoded in chlamydial genomes. However, a protein containing a SpoIID domain, known to possess transglycosylase activity in Bacillus subtilis, is conserved in Chlamydiales. We show here that the SpoIID homologue of the Chlamydia-related pathogen Waddlia chondrophila is a septal peptidoglycan-binding protein. Moreover, we demonstrate that SpoIID acts as a lytic transglycosylase on peptidoglycan and as a muramidase on denuded glycan strands in vitro. As SpoIID-like proteins are widespread in nonsporulating bacteria, SpoIID might commonly be a septal peptidoglycan remodeling protein in bacteria, including obligate intracellular pathogens, and thus might represent a promising drug target. IMPORTANCE Chlamydiales species are obligate intracellular bacteria and important human pathogens that have a minimal division machinery lacking the proteins that are essential for bacterial division in other species, such as FtsZ. Chlamydial division requires synthesis of peptidoglycan, which forms a ring at the division septum and is rapidly turned over. However, little is known of peptidoglycan degradation, because many peptidoglycan-degrading enzymes are not encoded by chlamydial genomes. Here we show that an homologue of SpoIID, a peptidoglycan-degrading enzyme involved in sporulation of bacteria such as Bacillus subtilis, is expressed in Chlamydiales, localizes at the division septum, and degrades peptidoglycan in vitro, indicating that SpoIID is not only involved in sporulation but also likely implicated in division of some bacteria.

scholarly journals Role Played by Exosporium Glycoproteins in the Surface Properties of Bacillus cereus Spores and in Their Adhesion to Stainless Steel

2011 ◽  
Vol 77 (14) ◽  
pp. 4905-4911 ◽  
Author(s):  
Yannick Lequette ◽  
Estelle Garénaux ◽  
Grégoire Tauveron ◽  
Sylvain Dumez ◽  
Stéphane Perchat ◽  
...  
Keyword(s):  
Stainless Steel ◽  
Bacillus Cereus ◽  
Surface Properties ◽  
Isoelectric Point ◽  
Significant Role ◽  
Distal Part ◽  
Spore Surface ◽  
Adhesion Properties ◽  
Content Type

ABSTRACTBacillus cereusspores are surrounded by a loose-fitting layer called the exosporium, whose distal part is mainly formed from glycoproteins. The role played by the exosporium glycoproteins ofB. cereusATCC 14579 (BclA and ExsH) was investigated by considering hydrophobicity and charge, as well as the properties of spore adhesion to stainless steel. The absence of BclA increased both the isoelectric point (IEP) and hydrophobicity of whole spores while simultaneously reducing the interaction between spores and stainless steel. However, neither the hydrophobicity nor the charge associated with BclA could explain the differences in the adhesion properties. Conversely, ExsH, another exosporium glycoprotein, did not play a significant role in spore surface properties. The monosaccharide analysis ofB. cereusATCC 14579 showed different glycosylation patterns on ExsH and BclA. Moreover, two specific glycosyl residues, namely, 2-O-methyl-rhamnose (2-Me-Rha) and 2,4-O-methyl-rhamnose (2,4-Me-Rha), were attached to BclA, in addition to the glycosyl residues already reported inB. anthracis.

scholarly journals ThespsGene Products Affect the Germination, Hydrophobicity, and Protein Adsorption of Bacillus subtilis Spores

2014 ◽  
Vol 80 (23) ◽  
pp. 7293-7302 ◽  
Author(s):  
Giuseppina Cangiano ◽  
Teja Sirec ◽  
Cristina Panarella ◽  
Rachele Isticato ◽  
Loredana Baccigalupi ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Experimental Validation ◽  
Surface Display ◽  
Deep Understanding ◽  
Transcriptional Analysis ◽  
Functional Study ◽  
Spore Surface ◽  
Wild Type ◽  
Content Type ◽  
Hydrophobic Force

ABSTRACTThe multilayered surface of theBacillus subtilisspore is composed of proteins and glycans. While over 70 different proteins have been identified as surface components, carbohydrates associated with the spore surface have not been characterized in detail yet. Bioinformatic data suggest that the 11 products of thespsoperon are involved in the synthesis of polysaccharides present on the spore surface, but an experimental validation is available only for the four distal genes of the operon. Here, we report a transcriptional analysis of thespsoperon and a functional study performed by constructing and analyzing two null mutants lacking either all or only the promoter-proximal gene of the operon. Our results show that bothspsmutant spores apparently have normal coat and crust but have a small germination defect and are more hydrophobic than wild-type spores. We also show that spores lacking all Sps proteins are highly adhesive and form extensive clumps. In addition,spsmutant spores have an increased efficiency in adsorbing a heterologous enzyme, suggesting that hydrophobic force is a major determinant of spore adsorption and indicating that a deep understanding of the surface properties of the spore is essential for its full development as a surface display platform.

scholarly journals Expansion of the Spore Surface Polysaccharide Layer inBacillus subtilisby Deletion of Genes Encoding Glycosyltransferases and Glucose Modification Enzymes

2019 ◽  
Vol 201 (19) ◽  
Author(s):  
Bentley Shuster ◽  
Mark Khemmani ◽  
Yusei Nakaya ◽  
Gudrun Holland ◽  
Keito Iwamoto ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Bacillus Subtilis ◽  
Surface Properties ◽  
Antimicrobial Agents ◽  
Gene Clusters ◽  
Surface Hydrophobicity ◽  
Ruthenium Red ◽  
Spore Surface ◽  
Content Type ◽  
India Ink

ABSTRACTPolysaccharides (PS) decorate the surface of dormant endospores (spores). In the model organism for sporulation,Bacillus subtilis, the composition of the spore PS is not known in detail. Here, we have assessed how PS synthesis enzymes produced during the late stages of sporulation affect spore surface properties. Using four methods, bacterial adhesion to hydrocarbons (BATH) assays, India ink staining, transmission electron microscopy (TEM) with ruthenium red staining, and scanning electron microscopy (SEM), we characterized the contributions of four sporulation gene clusters,spsABCDEFGHIJKL,yfnHGF-yfnED,ytdA-ytcABC, andcgeAB-cgeCDE, on the morphology and properties of the crust, the outermost spore layer. Our results show that all mutations in thespsoperon result in the production of spores that are more hydrophobic and lack a visible crust, presumably because of reduced PS deposition, while mutations incgeDand theyfnH–Dcluster noticeably expand the PS layer. In addition,yfnH–Dmutant spores exhibit a crust with an unusual weblike morphology. The hydrophobic phenotype fromspsmutant spores was partially rescued by a second mutation inactivating any gene in theyfnHGFoperon. WhilespsI, yfnH, andytdAare paralogous genes, all encoding glucose-1-phosphate nucleotidyltransferases, each paralog appears to contribute in a distinct manner to the spore PS. Our data are consistent with the possibility that each gene cluster is responsible for the production of its own respective deoxyhexose. In summary, we found that disruptions to the PS layer modify spore surface hydrophobicity and that there are multiple saccharide synthesis pathways involved in spore surface properties.IMPORTANCEMany bacteria are characterized by their ability to form highly resistant spores. The dormant spore state allows these species to survive even the harshest treatments with antimicrobial agents. Spore surface properties are particularly relevant because they influence spore dispersal in various habitats from natural to human-made environments. The spore surface inBacillus subtilis(crust) is composed of a combination of proteins and polysaccharides. By inactivating the enzymes responsible for the synthesis of spore polysaccharides, we can assess how spore surface properties such as hydrophobicity are modulated by the addition of specific carbohydrates. Our findings indicate that several sporulation gene clusters are responsible for the assembly and allocation of surface polysaccharides. Similar mechanisms could be modulating the dispersal of infectious spore-forming bacteria.

scholarly journals Bacillus subtilis-Mediated Protection from Citrobacter rodentium-Associated Enteric Disease RequiresespHand Functional Flagella

2011 ◽  
Vol 80 (2) ◽  
pp. 710-719 ◽  
Author(s):  
Sara E. Jones ◽  
Katherine L. Knight
Keyword(s):  
Bacillus Subtilis ◽  
Pathogenic Bacteria ◽  
Cell Loss ◽  
Enteric Infection ◽  
Citrobacter Rodentium ◽  
Human Pathogens ◽  
Wild Type ◽  
Content Type ◽  
Beneficial Microbes ◽  
Pathogen Colonization

ABSTRACTCommensals limit disease caused by invading pathogens; however, the mechanisms and genes utilized by beneficial microbes to inhibit pathogenesis are poorly understood. The attaching and effacing mouse pathogenCitrobacter rodentiumassociates intimately with the intestinal epithelium, and infections result in acute colitis.C. rodentiumis used to model the human pathogens enterohemorrhagicEscherichia coliand enteropathogenicE. coli. To confirm thatBacillus subtilis, a spore-forming bacterium found in the gut of mammals, could reduceC. rodentium-associated disease, mice received wild-typeB. subtilisspores and 24 h later were infected by oral gavage with pathogenicC. rodentium. Disease was assessed by determining the extent of colonic epithelial hyperplasia, goblet cell loss, diarrhea, and pathogen colonization. Mice that received wild-typeB. subtilisprior to enteric infection were protected from disease even thoughC. rodentiumcolonization was not inhibited. In contrast,espHandhagmutants, defective in exopolysaccharides and flagellum production, respectively, did not protect mice fromC. rodentium-associated disease. AmotABmutant also failed to protect mice from disease, suggesting thatB. subtilis-mediated protection requires functional flagella. By expanding our current mechanistic knowledge of bacterial protection, we can better utilize beneficial microbes to prevent intestinal disease caused by pathogenic bacteria, ultimately reducing human disease. Our data demonstrate that wild-typeB. subtilisreduced disease caused byC. rodentiuminfection through a mechanism that requiredespHand functional flagella.

scholarly journals Novel Aggregation Properties of Candida albicans Secreted Aspartyl Proteinase Sap6 Mediate Virulence in Oral Candidiasis

2015 ◽  
Vol 83 (7) ◽  
pp. 2614-2626 ◽  
Author(s):  
Rohitashw Kumar ◽  
Darpan Saraswat ◽  
Swetha Tati ◽  
Mira Edgerton
Keyword(s):  
Candida Albicans ◽  
Epithelial Cells ◽  
Oral Candidiasis ◽  
Oral Microbiome ◽  
Proteinase Activity ◽  
Adhesion Properties ◽  
Content Type ◽  
Oral Epithelial Cells ◽  
Oral Epithelial ◽  
Cell Cell

Candida albicans, a commensal fungus of the oral microbiome, causes oral candidiasis in humans with localized or systemic immune deficiencies. Secreted aspartic proteinases (Saps) are a family of 10 related proteases and are virulence factors due to their proteolytic activity, as well as their roles in adherence and colonization of host tissues. We found that mice infected sublingually withC. albicanscells overexpressing Sap6 (SAP6OE and a Δsap8strain) had thicker fungal plaques and more severe oral infection, while infection with the Δsap6strain was attenuated. These hypervirulent strains had highly aggregative colony structurein vitroand higher secreted proteinase activity; however, the levels of proteinase activity ofC. albicansSaps did not uniformly match their abilities to damage cultured oral epithelial cells (SCC-15 cells). Hyphal induction in cells overexpressing Sap6 (SAP6OE and Δsap8cells) resulted in formation of large cell-cell aggregates. These aggregates could be produced in germinated wild-type cells by addition of native or heat-inactivated Sap6. Sap6 bound only to germinated cells and increasedC. albicansadhesion to oral epithelial cells. The adhesion properties of Sap6 were lost upon deletion of its integrin-binding motif (RGD) and could be inhibited by addition of RGD peptide or anti-integrin antibodies. Thus, Sap6 (but not Sap5) has an alternative novel function in cell-cell aggregation, independent of its proteinase activity, to promote infection and virulence in oral candidiasis.

scholarly journals Interdependent YpsA- and YfhS-Mediated Cell Division and Cell Size Phenotypes in Bacillus subtilis

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Robert S. Brzozowski ◽  
Brooke R. Tomlinson ◽  
Michael D. Sacco ◽  
Judy J. Chen ◽  
Anika N. Ali ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Cell Division ◽  
Cell Size ◽  
Unknown Function ◽  
Suppressor Mutation ◽  
Gram Positive ◽  
Content Type ◽  
Suppressor Mutations ◽  
Extragenic Suppressor ◽  
Cell Division Inhibition

ABSTRACT Although many bacterial cell division factors have been uncovered over the years, evidence from recent studies points to the existence of yet-to-be-discovered factors involved in cell division regulation. Thus, it is important to identify factors and conditions that regulate cell division to obtain a better understanding of this fundamental biological process. We recently reported that in the Gram-positive organisms Bacillus subtilis and Staphylococcus aureus, increased production of YpsA resulted in cell division inhibition. In this study, we isolated spontaneous suppressor mutations to uncover critical residues of YpsA and the pathways through which YpsA may exert its function. Using this technique, we were able to isolate four unique intragenic suppressor mutations in ypsA (E55D, P79L, R111P, and G132E) that rendered the mutated YpsA nontoxic upon overproduction. We also isolated an extragenic suppressor mutation in yfhS, a gene that encodes a protein of unknown function. Subsequent analysis confirmed that cells lacking yfhS were unable to undergo filamentation in response to YpsA overproduction. We also serendipitously discovered that YfhS may play a role in cell size regulation. Finally, we provide evidence showing a mechanistic link between YpsA and YfhS. IMPORTANCE Bacillus subtilis is a rod-shaped Gram-positive model organism. The factors fundamental to the maintenance of cell shape and cell division are of major interest. We show that increased expression of ypsA results in cell division inhibition and impairment of colony formation on solid medium. Colonies that do arise possess compensatory suppressor mutations. We have isolated multiple intragenic (within ypsA) mutants and an extragenic suppressor mutant. Further analysis of the extragenic suppressor mutation led to a protein of unknown function, YfhS, which appears to play a role in regulating cell size. In addition to confirming that the cell division phenotype associated with YpsA is disrupted in a yfhS-null strain, we also discovered that the cell size phenotype of the yfhS knockout mutant is abolished in a strain that also lacks ypsA. This highlights a potential mechanistic link between these two proteins; however, the underlying molecular mechanism remains to be elucidated.

scholarly journals Potentially Human-Pathogenic Escherichia coli O26 in Norwegian Sheep Flocks

2011 ◽  
Vol 77 (14) ◽  
pp. 4949-4958 ◽  
Author(s):  
C. Sekse ◽  
M. Sunde ◽  
B.-A. Lindstedt ◽  
P. Hopp ◽  
T. Bruheim ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Variable Number Tandem Repeat ◽  
Variable Number ◽  
Human Pathogens ◽  
Content Type ◽  
Representative Sampling ◽  
E Coli ◽  
Pathogenic Escherichia Coli ◽  
Close Relationship ◽  
Large Survey

ABSTRACTA national survey ofEscherichia coliO26 in Norwegian sheep flocks was conducted, using fecal samples to determine the prevalence. In total, 491 flocks were tested, andE. coliO26 was detected in 17.9% of the flocks. One hundred forty-twoE. coliO26 isolates were examined for flagellar antigens (H typing) and four virulence genes, includingstxandeae, to identify possible Shiga toxin-producingE. coli(STEC) and enteropathogenicE. coli(EPEC). Most isolates (129 out of 142) were identified asE. coliO26:H11. They possessedeaeand may have potential as human pathogens, although only a small fraction were identified as STEC O26:H11, giving a prevalence in sheep flocks of only 0.8%. Correspondingly, the sheep flock prevalence of atypical EPEC (aEPEC) O26:H11 was surprisingly high (15.9%). The genetic relationship between theE. coliO26:H11 isolates was investigated by pulsed-field gel electrophoresis (PFGE) and multilocus variable number tandem repeat analysis (MLVA), identifying 63 distinct PFGE profiles and 22 MLVA profiles. Although the MLVA protocol was less discriminatory than PFGE and a few cases of disagreement were observed, comparison by partition mapping showed an overall good accordance between the two methods. A close relationship between a few isolates of aEPEC O26:H11 and STEC O26:H11 was identified, but all theE. coliO26:H11 isolates should be considered potentially pathogenic to humans. The present study consisted of a representative sampling of sheep flocks from all parts of Norway. This is the first large survey of sheep flocks focusing onE. coliO26 in general, including results of STEC, aEPEC, and nonpathogenic isolates.

scholarly journals Streptomycin-Induced Expression in Bacillus subtilis of YtnP, a Lactonase-Homologous Protein That Inhibits Development and Streptomycin Production in Streptomyces griseus

2011 ◽  
Vol 78 (2) ◽  
pp. 599-603 ◽  
Author(s):  
Johannes Schneider ◽  
Ana Yepes ◽  
Juan C. Garcia-Betancur ◽  
Isa Westedt ◽  
Benjamin Mielich ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Signaling Pathway ◽  
Streptomyces Griseus ◽  
Aerial Mycelium ◽  
Positive Bacterium ◽  
Gram Positive ◽  
Induced Expression ◽  
Content Type ◽  
Homologous Protein

ABSTRACTBacillus subtilisinduces expression of the geneytnPin the presence of the antimicrobial streptomycin, produced by the Gram-positive bacteriumStreptomyces griseus.ytnPencodes a lactonase-homologous protein that is able to inhibit the signaling pathway required for the streptomycin production and development of aerial mycelium inS. griseus.

Sign in / Sign up

Export Citation Format

Share Document