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10 pages, 796 KiB

Open AccessArticle

Characterization by MALDI-TOF MS and 16S rRNA Gene Sequencing of Aerobic Endospore-Forming Bacteria Isolated from Pharmaceutical Facility in Rio de Janeiro, Brazil

by Nathalia Gonçalves Santos Caldeira, Maria Luiza Soares de Souza, Rebeca Vitória da Silva Lage de Miranda, Luciana Veloso da Costa, Stephen James Forsythe, Viviane Zahner and Marcelo Luiz Lima Brandão

Microorganisms 2024, 12(4), 724; https://doi.org/10.3390/microorganisms12040724 - 03 Apr 2024

Viewed by 495

Abstract

Bacillus and related genera are among the most important contaminants in the pharmaceutical production environment, and the identification of these microorganisms at the species level assists in the investigation of sources of contamination and in preventive and corrective decision making. The aim of [...] Read more.

Bacillus and related genera are among the most important contaminants in the pharmaceutical production environment, and the identification of these microorganisms at the species level assists in the investigation of sources of contamination and in preventive and corrective decision making. The aim of this study was to evaluate three methodologies for the characterization of endospore-forming aerobic bacterial strains isolated from a pharmaceutical unit in Rio de Janeiro, Brazil. MALDI-TOF MS was performed using MALDI Biotyper^® and VITEK^® MS RUO systems, and complete 16S rRNA gene sequencing was performed using the Sanger methodology. The results showed the prevalence of the genera Bacillus (n = 9; 36.0%), Priestia (n = 5; 20.0%), and Paenibacillus (n = 4; 16.0%). Three (20.0%) strains showed <98.7% of DNA sequencing similarity on the EzBioCloud Database, indicating possible new species. In addition, the reclassification of Bacillus pseudoflexus to the genus Priestia as Priestia pseudoflexus sp. nov. is proposed. In conclusion, 16S rRNA and MALDI TOF/MS were not sufficient to identify all strains at the species level, and complementary analyses were necessary. Full article

(This article belongs to the Special Issue Assembly, Structure, and Germination of Bacterial Spores)

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12 pages, 2288 KiB

Open AccessArticle

Impact of Protein Aggregates on Sporulation and Germination of Bacillus subtilis

by Julien Mortier, Alexander Cambré, Sina Schack, Graham Christie and Abram Aertsen

Microorganisms 2023, 11(9), 2365; https://doi.org/10.3390/microorganisms11092365 - 21 Sep 2023

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Abstract

In order to improve our general understanding of protein aggregate (PA) management and impact in bacteria, different model systems and processes need to be investigated. As such, we developed an inducible synthetic PA model system to investigate PA dynamics in the Gram-positive model [...] Read more.

In order to improve our general understanding of protein aggregate (PA) management and impact in bacteria, different model systems and processes need to be investigated. As such, we developed an inducible synthetic PA model system to investigate PA dynamics in the Gram-positive model organism Bacillus subtilis. This confirmed previous observations that PA segregation in this organism seems to follow the Escherichia coli paradigm of nucleoid occlusion governing polar localization and asymmetric segregation during vegetative growth. However, our findings also revealed that PAs can readily persist throughout the entire sporulation process after encapsulation in the forespore during sporulation. Moreover, no deleterious effects of PA presence on sporulation, germination and spore survival against heat or UV stress could be observed. Our findings therefore indicate that the sporulation process is remarkably robust against perturbations by PAs and misfolded proteins. Full article

(This article belongs to the Special Issue Assembly, Structure, and Germination of Bacterial Spores)

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15 pages, 3161 KiB

Open AccessArticle

Assessing the Feasibility of Employing a Combination of a Bacteriophage-Derived Endolysin and Spore Germinants to Treat Relapsing Clostridioides difficile Infection

by Khalid Alyahya and Les Baillie

Microorganisms 2023, 11(7), 1651; https://doi.org/10.3390/microorganisms11071651 - 24 Jun 2023

Cited by 1 | Viewed by 1336

Abstract

Clostridioides difficile is a Gram-positive, anaerobic, spore-forming bacillus and is a major cause of healthcare-associated infections. Whereas the vegetative form of the pathogen is susceptible to treatment with antibiotics, its ability to persist in the gut as antibiotic-resistant spores means that reinfection can [...] Read more.

Clostridioides difficile is a Gram-positive, anaerobic, spore-forming bacillus and is a major cause of healthcare-associated infections. Whereas the vegetative form of the pathogen is susceptible to treatment with antibiotics, its ability to persist in the gut as antibiotic-resistant spores means that reinfection can occur in cases were the individual fails to re-establish a protective microflora. Bacteriophages and their lysins are currently being explored as treatment options due to their specificity, which minimizes the disruption to the other members of the gut microflora that are protective. The feasibility of employing recombinant endolysins to target the vegetative form of C. difficile has been demonstrated in animal models. In this study, we cloned and expressed the enzyme active domain of LysCD6356 and confirmed its ability to lyse the vegetative forms of a diverse range of clinical isolates of C. difficile, which included members of the hypervirulent 027 ribotype. Lytic activity was adversely affected by calcium, which is naturally found in the gut and is released from the spore upon germination. Our results suggests that a strategy in which the triggering of spore germination is separated in time from the application of the lysin could be developed as a strategy to reduce the risk of relapsing C. difficile infections. Full article

(This article belongs to the Special Issue Assembly, Structure, and Germination of Bacterial Spores)

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13 pages, 8130 KiB

Open AccessArticle

Structural Analysis of Bacillus subtilis Sigma Factors

by Katherine M. Collins, Nicola J. Evans, James H. Torpey, Jonathon M. Harris, Bethany A. Haynes, Amy H. Camp and Rivka L. Isaacson

Microorganisms 2023, 11(4), 1077; https://doi.org/10.3390/microorganisms11041077 - 20 Apr 2023

Cited by 2 | Viewed by 1967

Abstract

Bacteria use an array of sigma factors to regulate gene expression during different stages of their life cycles. Full-length, atomic-level structures of sigma factors have been challenging to obtain experimentally as a result of their many regions of intrinsic disorder. AlphaFold has now [...] Read more.

Bacteria use an array of sigma factors to regulate gene expression during different stages of their life cycles. Full-length, atomic-level structures of sigma factors have been challenging to obtain experimentally as a result of their many regions of intrinsic disorder. AlphaFold has now supplied plausible full-length models for most sigma factors. Here we discuss the current understanding of the structures and functions of sigma factors in the model organism, Bacillus subtilis, and present an X-ray crystal structure of a region of B. subtilis SigE, a sigma factor that plays a critical role in the developmental process of spore formation. Full article

(This article belongs to the Special Issue Assembly, Structure, and Germination of Bacterial Spores)

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11 pages, 1115 KiB

Open AccessArticle

Divalent Cation Signaling in Clostridium perfringens Spore Germination

by Roua Almatrafi, Saeed Banawas and Mahfuzur R. Sarker

Microorganisms 2023, 11(3), 591; https://doi.org/10.3390/microorganisms11030591 - 26 Feb 2023

Cited by 1 | Viewed by 1671

Abstract

Spore germination plays an essential role in the pathogenesis of Clostridium perfringens-associated food poisoning. Germination is initiated when bacterial spores sense various stimuli, including chemicals and enzymes. A previous study showed that dipicolinic acid (DPA) chelated with calcium (Ca-DPA) significantly stimulated spore [...] Read more.

Spore germination plays an essential role in the pathogenesis of Clostridium perfringens-associated food poisoning. Germination is initiated when bacterial spores sense various stimuli, including chemicals and enzymes. A previous study showed that dipicolinic acid (DPA) chelated with calcium (Ca-DPA) significantly stimulated spore germination in C. perfringens. However, whether Ca²⁺ or DPA alone can induce germination is unknown. Therefore, we aimed to evaluate the possible roles of Ca²⁺ and other divalent cations present in the spore core, such as Mn²⁺ and Mg²⁺, in C. perfringens spore germination. Our study demonstrated that (i) Ca-DPA, but not DPA alone, induced C. perfringens spore germination, suggesting that Ca²⁺ might play a signaling role; (ii) all tested calcium salts induced spore germination, indicating that Ca²⁺ is critical for germination; (iii) the spore-specific divalent cations Mn²⁺ and Mg²⁺, but not Zn²⁺, induced spore germination, suggesting that spore core-specific divalent cations are involved in C. perfringens spore germination; and (iv) endogenous Ca²⁺ and Mg²⁺ are not required for induction of C. perfringens spore germination, whereas exogenous and partly endogenous Mn²⁺ are required. Collectively, our results suggest that exogenous spore core-specific divalent cation signals are more important than endogenous signals for the induction of spore germination. Full article

(This article belongs to the Special Issue Assembly, Structure, and Germination of Bacterial Spores)

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11 pages, 998 KiB

Open AccessArticle

Role of Bacillus subtilis Spore Core Water Content and pH in the Accumulation and Utilization of Spores’ Large 3-Phosphoglyceric Acid Depot, and the Crucial Role of This Depot in Generating ATP Early during Spore Germination

by George Korza, Michelle Goulet, Angela DeMarco, James Wicander and Peter Setlow

Microorganisms 2023, 11(1), 195; https://doi.org/10.3390/microorganisms11010195 - 12 Jan 2023

Cited by 4 | Viewed by 2253

Abstract

The development of Bacillus spore cores involves the accumulation of 3-phosphoglycerate (3PGA) during sporulation, following core acidification to ~6.4, and before decreases in core water content occur due to Ca-dipicolinc acid (CaDPA) uptake. This core acidification inhibits phosphoglycerate mutase (PGM) at pH 6.4, [...] Read more.

The development of Bacillus spore cores involves the accumulation of 3-phosphoglycerate (3PGA) during sporulation, following core acidification to ~6.4, and before decreases in core water content occur due to Ca-dipicolinc acid (CaDPA) uptake. This core acidification inhibits phosphoglycerate mutase (PGM) at pH 6.4, allowing 3PGA accumulation, although PGM is active at pH 7.4. Spores’ 3PGA is stable for months at 4 °C and weeks at 37 °C. However, in wild-type spore germination, increases in core pH to 7.5–8 and in core water content upon CaDPA release and cortex peptidoglycan hydrolysis allow for rapid 3PGA catabolism, generating ATP; indeed, the earliest ATP generated following germination is from 3PGA catabolism. The current work found no 3PGA in those Bacillus subtilis spores that do not accumulate CaDPA during sporulation and have a core pH of ~7.4. The ATP production in the germination of 3PGA-less spores in a poor medium was minimal, and the germinated spores were >99% dead. However, the 3PGA-replete spores that germinated in the poor medium accumulated >30 times more ATP, and >70% of the germinated spores were found to be alive. These findings indicate why 3PGA accumulation during sporulation (and utilization during germination) in all the Firmicute spores studied can be crucial for spore revival due to the generation of essential ATP. The latter finding further suggests that targeting PGM activity during germination could be a novel way to minimize the damaging effects of spores. Full article

(This article belongs to the Special Issue Assembly, Structure, and Germination of Bacterial Spores)

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12 pages, 2242 KiB

Open AccessArticle

Genomic versus Plasmid-Borne Expression of Germinant Receptor Proteins in Bacillus cereus Strain 14579

by Yan Wang, Peter Setlow and Stanley Brul

Microorganisms 2022, 10(9), 1774; https://doi.org/10.3390/microorganisms10091774 - 02 Sep 2022

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Abstract

Germinant receptors (GRs) are proteins in the spore-forming bacteria of Bacillus species that are crucial in triggering spore germination by sensing nutrients in the spores’ environment. In the Gram-positive bacterium Bacillus cereus strain ATCC 14579, the GerR GR initiates germination with L-alanine. While [...] Read more.

Germinant receptors (GRs) are proteins in the spore-forming bacteria of Bacillus species that are crucial in triggering spore germination by sensing nutrients in the spores’ environment. In the Gram-positive bacterium Bacillus cereus strain ATCC 14579, the GerR GR initiates germination with L-alanine. While we have expressed GerR subunits fused to reporter proteins from genes under control of their native promoter on plasmids in this B. cereus strain, here we sought increased flexibility in this work by studying genome integration and plasmid-borne inducible high level (over) expression. However, construction of chromosomal integrants to visualize and localize the GerR B subunit fused to fluorescent reporter protein SGFP2 was not successful in this B. cereus strain using constructs with either shorter (~600 bp) or longer (~1200 bp) regions of homology to the gerR operon. This failure was in contrast to successful IPTG-inducible expression of GerRB-SGFP2 from plasmid pDG148 in vegetative cells and dormant spores, as fluorescent GerRB-SGFP2 foci were present in vegetative cells and the protein was detected by Western blot analysis. In dormant spores, the fluorescence intensity with IPTG-inducible expression from pDG148-gerRB-SGFP2 was significantly higher than in wild type spores. However, the full length GerRB-SGFP2 protein was not detected in spores using Western blots. Clearly, there are still challenges in the construction of B. cereus strains harboring fluorescent reporter proteins in which tagged proteins are encoded by genes incorporated in the chromosome or on extrachromosomal expression plasmids. Full article

(This article belongs to the Special Issue Assembly, Structure, and Germination of Bacterial Spores)

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12 pages, 3053 KiB

Open AccessArticle

Changes in the Spore Proteome of Bacillus cereus in Response to Introduction of Plasmids

by Xiaowei Gao, Bhagyashree N. Swarge, Winfried Roseboom, Yan Wang, Henk L. Dekker, Peter Setlow, Stanley Brul and Gertjan Kramer

Microorganisms 2022, 10(9), 1695; https://doi.org/10.3390/microorganisms10091695 - 24 Aug 2022

Viewed by 1514

Abstract

Fluorescent fusion proteins were expressed in Bacillus cereus to visualize the germinosome by introducing a plasmid that carries fluorescent fusion proteins of germinant receptor GerR subunits or germinosome scaffold protein GerD. The effects of plasmid insertion and recombinant protein expression on the spore [...] Read more.

Fluorescent fusion proteins were expressed in Bacillus cereus to visualize the germinosome by introducing a plasmid that carries fluorescent fusion proteins of germinant receptor GerR subunits or germinosome scaffold protein GerD. The effects of plasmid insertion and recombinant protein expression on the spore proteome were investigated. Proteomic analysis showed that overexpression of the target proteins had negligible effects on the spore proteome. However, plasmid-bearing spores displayed dramatic abundance changes in spore proteins involved in signaling and metabolism. Our findings indicate that the introduction of a plasmid alone alters the spore protein composition dramatically, with 993 proteins significantly down-regulated and 415 proteins significantly up-regulated among 3323 identified proteins. This shows that empty vector controls are more appropriate to compare proteome changes due to plasmid-encoded genes than is the wild-type strain, when using plasmid-based genetic tools. Therefore, researchers should keep in mind that molecular cloning techniques can alter more than their intended targets in a biological system, and interpret results with this in mind. Full article

(This article belongs to the Special Issue Assembly, Structure, and Germination of Bacterial Spores)

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