Lactic acid bacteria isolated from rye sourdoughs produce bacteriocin-like inhibitory substances active against Bacillus subtilis and fungi

Aim: To screen five strains of lactic acid bacteria (LAB) isolated from rye sourdoughs for the potential production of antimicrobial substances. Methods and Results: Lactobacillus sakei KTU05‐06, Pediococcus acidilactici KTU05‐7, Pediococcus pentosaceus KTU05‐8, KTU05‐9 and KTU05‐10 isolated from rye sourdoughs were investigated for the production of bacteriocin‐like inhibitory substances (BLIS). The supernatants of analysed LAB inhibited growth of up to 15 out of 25 indicator bacteria strains as well as up to 25 out of 56 LAB strains isolated from rye sourdoughs. Moreover, these five LAB were active against ropes‐producing Bacillus subtilis and the main bread mould spoilage causing fungi –Aspergillus, Fusarium, Mucor and Penicillium. Lactobacillus sakei KTU05‐6 demonstrated the best antibacterial properties and is resistant towards heat treatment even at 100°C for 60 min. Conclusions: The use of LAB‐producing antibacterial substances may be a good choice as a co‐starter culture to ensure the stability of sourdoughs and to avoid the bacterial and fungi spoilage of the end product. Significance and Impact of the Study: The antimicrobial compounds designated as sakacin KTU05‐6, pediocin KTU05‐8 KTU05‐9, KTU05‐10 and AcKTU05‐67 were not identical to any other known BLIS, and this finding leads up to the assumption that they might be the novel.     

Sensitivity ofBacillus subtilis sporulation to methylene blue inhibition

The dye methylene blue was found to inhibit sporulation inBacillus subtilis 168. The compound blocked spore formation at concentrations subinhibitory to vegetative growth while allowing synthesis of serine protease, antibiotic, and certain catabolite-repressed enzymes. The sporulation process was sensitive to the inhibitor through T₆, but germination and outgrowth were not affected by the presence of the compound. The inhibition of sporulation may be related to the ability of the compound to inhibit oxidative phosphorylation.     

Anti-Listeria monocytogenes Bacteriocin-Like Inhibitory Substances From Enterococcus faecium UQ31 Isolated from Artisan Mexican-Style Cheese

Artisan fresh Mexican-style cheeses are commonly made from raw milk that provides not only rich flavors, but also a diversity of associated lactic acid bacteria (LAB) strains. Enterococcus faecium UQ31 was isolated from panela cheese and produced bacteriocin-like inhibitory substances (BLIS) with a strong anti-Listeria activity. A modified pH–mediated adsorption-desorption purification process resulted in (after SDS-PAGE) two bands showing antimicrobial activities, where most of the activity corresponded to the band with an estimated molecular weight of 7.5 kDa. The BLIS produced by E. faecium UQ31 were heat resistant, stable at ambient storage conditions, and active in the pH range 5–9. The BLIS antimicrobial activities were detected during logarithmic growth phase and remained constant until the end of incubation time (19 h). These BLIS showed a wide anti-Listeria monocytogenes spectra. The E. faecium UQ31 strain or their BLIS represent a promising potential as antimicrobial food preservatives.     

Live Cell Imaging of Germination and Outgrowth of Individual Bacillus subtilis Spores; the Effect of Heat Stress Quantitatively Analyzed with SporeTracker

Spore-forming bacteria are a special problem for the food industry as some of them are able to survive preservation processes. Bacillus spp. spores can remain in a dormant, stress resistant state for a long period of time. Vegetative cells are formed by germination of spores followed by a more extended outgrowth phase. Spore germination and outgrowth progression are often very heterogeneous and therefore, predictions of microbial stability of food products are exceedingly difficult. Mechanistic details of the cause of this heterogeneity are necessary. In order to examine spore heterogeneity we made a novel closed air-containing chamber for live imaging. This chamber was used to analyze Bacillus subtilis spore germination, outgrowth, as well as subsequent vegetative growth. Typically, we examined around 90 starting spores/cells for ≥4 hours per experiment. Image analysis with the purposely built program “SporeTracker” allows for automated data processing from germination to outgrowth and vegetative doubling. In order to check the efficiency of the chamber, growth and division of B. subtilis vegetative cells were monitored. The observed generation times of vegetative cells were comparable to those obtained in well-aerated shake flask cultures. The influence of a heat stress of 85°C for 10 min on germination, outgrowth, and subsequent vegetative growth was investigated in detail. Compared to control samples fewer spores germinated (41.1% less) and fewer grew out (48.4% less) after the treatment. The heat treatment had a significant influence on the average time to the start of germination (increased) and the distribution and average of the duration of germination itself (increased). However, the distribution and the mean outgrowth time and the generation time of vegetative cells, emerging from untreated and thermally injured spores, were similar.     

Spatiotemporally regulated proteolysis to dissect the role of vegetative proteins during Bacillus subtilis sporulation: cell‐specific requirement of σH and σA

Sporulation in Bacillus subtilis is a paradigm of bacterial development, which involves the interaction between a larger mother cell and a smaller forespore. The mother cell and the forespore activate different genetic programs, leading to the production of sporulation‐specific proteins. A critical gap in our understanding of sporulation is how vegetative proteins, made before sporulation initiation, contribute to spore formation. Here we present a system, spatiotemporally regulated proteolysis (STRP), which enables the rapid, developmentally regulated degradation of target proteins, thereby providing a suitable method to dissect the cell‐ and developmental stage‐specific role of vegetative proteins. STRP has been used to dissect the role of two major vegetative sigma factors, σ^H and σ^A, during sporulation. The results suggest that σ^H is only required in predivisional cells, where it is essential for sporulation initiation, but that it is dispensable during subsequent steps of spore formation. However, evidence has been provided that σ^A plays different roles in the mother cell, where it replenishes housekeeping functions, and in the forespore, where it plays an unexpected role in promoting spore germination and outgrowth. Altogether, the results demonstrate that STRP has the potential to provide a comprehensive molecular dissection of every stage of sporulation, germination and outgrowth.     

Interaction of Apurinic/Apyrimidinic Endonucleases Nfo and ExoA with the DNA Integrity Scanning Protein DisA in the Processing of Oxidative DNA Damage during Bacillus subtilis Spore Outgrowth

Oxidative stress-induced damage, including 8-oxo-guanine and apurinic/apyrimidinic (AP) DNA lesions, were detected in dormant and outgrowing Bacillus subtilis spores lacking the AP endonucleases Nfo and ExoA. Spores of the Δnfo exoA strain exhibited slightly slowed germination and greatly slowed outgrowth that drastically slowed the spores'' return to vegetative growth. A null mutation in the disA gene, encoding a DNA integrity scanning protein (DisA), suppressed this phenotype, as spores lacking Nfo, ExoA, and DisA exhibited germination and outgrowth kinetics very similar to those of wild-type spores. Overexpression of DisA also restored the slow germination and outgrowth phenotype to nfo exoA disA spores. A disA-lacZ fusion was expressed during sporulation but not in the forespore compartment. However, disA-lacZ was expressed during spore germination/outgrowth, as was a DisA-green fluorescent protein (GFP) fusion protein. Fluorescence microscopy revealed that, as previously shown in sporulating cells, DisA-GFP formed discrete globular foci that colocalized with the nucleoid of germinating and outgrowing spores and remained located primarily in a single cell during early vegetative growth. Finally, the slow-outgrowth phenotype of nfo exoA spores was accompanied by a delay in DNA synthesis to repair AP and 8-oxo-guanine lesions, and these effects were suppressed following disA disruption. We postulate that a DisA-dependent checkpoint arrests DNA replication during B. subtilis spore outgrowth until the germinating spore''s genome is free of damage.     

Characterization of bacteriocin-like inhibitory substances (BLIS) from sourdough lactic acid bacteria and evaluation of their in vitro and in situ activity

AIMS: To identify and characterize bacteriocion-producing lactic acid bacteria (LAB) in sourdoughs and to compare in vitro and in situ bacteriocin activity of sourdough- and nonsourdough LAB. METHODS AND RESULTS: Production of antimicrobial compounds by 437 Lactobacillus strains isolated from 70 sourdoughs was investigated. Five strains (Lactobacillus pentosus 2MF8 and 8CF, Lb. plantarum 4DE and 3DM and Lactobacillus spp. CS1) were found to produce distinct bacteriocin-like inhibitory substances (BLIS). BLIS-producing Lactococcus lactis isolated from raw barley showed a wider inhibitory spectrum than sourdough LAB, but they did not inhibit all strains of the key sourdough bacterium Lb. sanfranciscensis. Antimicrobial production by Lb. pentosus 2MF8 and Lc. lactis M30 was also demonstrated in situ. CONCLUSIONS: BLIS production by sourdough LAB appears to occur at a low frequency, showing limited inhibitory spectrum when compared with BLIS-producing Lc. lactis. Nevertheless, they are active BLIS producers under sourdough and bread-making conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The activity of BLIS has been demonstrated in situ. It may influence the complex sourdough microflora and support the implantation and stability of selected insensitive bacteria, such as Lb. sanfranciscensis, useful to confer good characteristics to the dough.     

Role of the Nfo and ExoA apurinic/apyrimidinic endonucleases in repair of DNA damage during outgrowth of Bacillus subtilis spores

Germination and outgrowth are critical steps for returning Bacillus subtilis spores to life. However, oxidative stress due to full hydration of the spore core during germination and activation of metabolism in spore outgrowth may generate oxidative DNA damage that in many species is processed by apurinic/apyrimidinic (AP) endonucleases. B. subtilis spores possess two AP endonucleases, Nfo and ExoA; the outgrowth of spores lacking both of these enzymes was slowed, and the spores had an elevated mutation frequency, suggesting that these enzymes repair DNA lesions induced by oxidative stress during spore germination and outgrowth. Addition of H₂O₂ also slowed the outgrowth of nfo exoA spores and increased the mutation frequency, and nfo and exoA mutations slowed the outgrowth of spores deficient in either RecA, nucleotide excision repair (NER), or the DNA-protective α/β-type small acid-soluble spore proteins (SASP). These results suggest that α/β-type SASP protect DNA of germinating spores against damage that can be repaired by Nfo and ExoA, which is generated either spontaneously or promoted by addition of H₂O₂. The contribution of RecA and Nfo/ExoA was similar to but greater than that of NER in repair of DNA damage generated during spore germination and outgrowth. However, nfo and exoA mutations increased the spontaneous mutation frequencies of outgrown spores lacking uvrA or recA to about the same extent, suggesting that DNA lesions generated during spore germination and outgrowth are processed by Nfo/ExoA in combination with NER and/or RecA. These results suggest that Nfo/ExoA, RecA, the NER system, and α/β-type SASP all contribute to the repair of and/or protection against oxidative damage of DNA in germinating and outgrowing spores.     

Effects of modification of membrane lipid composition on Bacillus subtilis sporulation and spore properties

Aims: To determine effects of inner membrane lipid composition on Bacillus subtilis sporulation and spore properties. Methods and Results: The absence of genes encoding lipid biosynthetic enzymes had no effect on B. subtilis sporulation, although the expected lipids were absent from spores’ inner membrane. The rate of spore germination with nutrients was decreased c. 50% with mutants that lacked the major cardiolipin (CL) synthase and another enzyme for synthesis of a major phospholipid. Spores lacking the minor CL synthase or an enzyme essential for glycolipid synthesis exhibited 50–150% increases in rates of dodecylamine germination, while spores lacking enzymes for phosphatidylethanolamine (PE), phosphatidylserine (PS) and lysylphosphatidylglycerol (l‐PG) synthesis exhibited a 30–50% decrease. Spore sensitivity to H₂O₂ and tert‐butylhydroperoxide was increased 30–60% in the absence of the major CL synthase, but these spores’ sensitivity to NaOCl or Oxone^? was unaffected. Spores of lipid synthesis mutants were less resistant to wet heat, with spores lacking enzymes for PE, PS or l‐PG synthesis exhibiting a two to threefold decrease and spores of other strains exhibiting a four to 10‐fold decrease. The decrease in spore wet heat resistance correlated with an increase in core water content. Conclusions: Changing the lipid composition of the B. subtilis inner membrane did not affect sporulation, although modest effects on spore germination and wet heat and oxidizing agent sensitivity were observed, especially when multiple lipids were absent. The increases in rates of dodecylamine germination were likely due to increased ability of this compound to interact with the spore’s inner membrane in the absence of some CL and glycolipids. The effects on spore wet heat sensitivity are likely indirect, because they were correlated with changes in core water content. Significance and Impact of the Study: The results of this study provide insight into roles of inner membrane lipids in spore properties.     

Study of Bacillus subtilis Endospores in Soil by Use of a Modified Endospore Stain

The Schaeffer-Fulton endospore stain was modified so that it would stain Bacillus subtilis endospores in soil smears. The modified stain differentiated among dormant spores, spores undergoing activation, and spores which had germinated but had not yet shown outgrowth. These differentiations were seen for spores in soil and for pure spore preparations in the laboratory. This stain was used to show reversible B. subtilis spore activation promoted by an Ensifer adhaerens-like indigenous bacterium in soil and by pure cultures of E. adhaerens added to spores in the laboratory. Under the specific conditions in the laboratory, spore germination did not proceed beyond the activation stage, and relatively little change occurred in the numbers of both E. adhaerens and B. subtilis. This was also true in soil, although some germination with destruction of spores and vegetative cells did occur if the soil had been nutritionally enriched by preincubation with incorporated ground alfalfa.     

Carbohydrate Composition during Germination and Outgrowth of Ascospores of Saccharomyces cerevisiae

Single spores of Saccharomyces cerevisiae were examined to distinguish changes in the synthesis and degradation of intracellular and wall carbohydrates during germination and outgrowth. Intracellular carbohydrate was fractionated into trehalose and glycogen. Trehalose degradation occurred during germination and outgrowth. The intracellular glycogen was degraded during germination and then synthesized during outgrowth. Wall carbohydrate was fractionated into glycogen, glucan and mannan. The wall glycogen and the KOH-soluble glucan were degraded during germination and then synthesized prior to and during outgrowth, respectively. The major component of the KOH-insoluble glucan in the wall is β-1,3-glucan. The glucan and mannan were synthesized during outgrowth.

The study revealed that the development of a vegetative cell from a spore follows rapid decreases in the amounts of trehalose, glycogen and KOH-soluble glucan during germination, and great increases in the amounts of glycogen, β-1,3-glucan and mannan during outgrowth.     

Quantitative Analysis of Polymyxin B Released from Polymyxin B-Treated Dormant Spores of Bacillus subtilis and Relationship between Its Permeability and Inhibitory Effect on Outgrowth

Polymyxin B, one of the cyclic polypeptide antibiotics, binds to the coat of Bacillus subtilis dormant spores and inhibits them from growing after germination. When about 2.8 × 10⁸ cells/ml of polymyxin B-treated dormant spores were incubated in heart infusion broth, 3.6 μg/ml of polymyxin B were released into the liquid medium during germination. Incubation of the same concentration of polymyxin B-treated ones in 100 mM CaCl₂ solution released 4.0 μg/ml of the antibiotic. The effect of various concentrations of polymyxin B on germination, outgrowth and vegetative growth of the dormant spores was investigated; the results showed that concentrations of 4.0 μg/ml and higher of the antibiotic inhibited their outgrowth and vegetative growth after germination. Young vegetative cells were less sensitive to the antibiotic than germinated spores. In addition to these results, immunoelectron microscopy with colloidal gold particles indicated that polymyxin B permeated into the core of the germinated spores and inhibited them from outgrowing.     

The Identification of in Vitro Production of Lectin from Callus Cultures of Korean Mistletoe (Viscum album L. var. coloratum)

The hydrolysis of the bacterial spore peptidoglycan (cortex) is a crucial event in spore germination. It has been suggested that SleC and SleM, which are conserved among clostridia, are to be considered putative cortex-lytic enzymes in Clostridium perfringens. However, little is known about the details of the hydrolytic process by these enzymes during germination, except that SleM functions as a muramidase. Muropeptides derived from SleC-digested decoated spores of a Bacillus subtilis mutant that lacks the enzymes, SleB, YaaH and CwlJ, related to cortex hydrolysis were identified by amino acid analysis and mass spectrometry. The results suggest that SleC is most likely a bifunctional enzyme possessing lytic transglycosylase activity and N-acetylmuramoyl-L-alanine amidase activity confined to cross-linked tetrapeptide-tetrapeptide moieties of the cortex structure. Furthermore, it appears that during germination of Clostridium perfringens spores, SleC causes merely small and local changes in the cortex structure, which are necessary before SleM can function.     

Inhibition of Vancomycin and High-Level Aminoglycoside-Resistant Enterococci Strains and Listeria monocytogenes by Bacteriocin-Like Substance Produced by Enterococcus faecium E86

Three hundred and thirty nine lactic bacteria strains isolated from food samples were screened for antimicrobial activity. Only one strain isolated from meat pie and identified as Enterococcus faecium produced a bacteriocin-like inhibitory substance (BLIS) showing activity against Enterococcus, Leuconostoc, Lactobacillus, Listeria, Corynebacterium and Staphylococcus aureus. The BLIS produced was resistant to acid and alkali treatment and 121oC for 15 min. The addition of BLIS in BHI contaminated with Listeria monocytogenes decreased the contamination in 4.8 log cycles in 24 h. The inhibition of listeria was also obtained in milk. Forty multiresistant enterococci strains were inhibited in the well-diffusion test. Two vancomycin resistant strains tested in liquid with BLIS were also inhibited. The BLIS producer showed no pathogenicity marker.     

The expression of <Emphasis Type="Italic">Bacillus intermedius</Emphasis> glutamyl endopeptidase gene in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> recombinant strains

The gene encoding for B. intermedius glutamyl endopeptidase (gseBi) has previously been cloned and its nucleotide sequence analyzed. In this study, the expression of this gene was explored in protease-deficient strain B. subtilis AJ73 during stationary phase of bacterial growth. We found that catabolite repression usually involved in control of endopeptidase expression during vegetative growth was not efficient at the late stationary phase. Testing of B. intermedius glutamyl endopeptidase gene expression with B. subtilis spo0-mutants revealed slight effect of these mutations on endopeptidase expression. Activity of glutamyl endopeptidase was partly left in B. subtilis ger-mutants. Probably, gseBi expression was not connected with sporulation. This enzyme might be involved in outgrowth of the spore, when germinating endospore converts into the vegetative cell. These data suggest complex regulation of B. intermedius glutamyl endopeptidase gene expression with contribution of several regulatory systems and demonstrate changes in control of enzyme biosynthesis at different stages of growth.     

Chloride dependence of endospore germination in Halobacillus halophilus

The ion requirement for germination and outgrowth of endospores from the moderately halophilic salt marsh bacterium Halobacillus halophilus was studied. Germination and outgrowth of endospores plated onto nutrient broth was dependent on the salt concentration in the artificial seawater used as the source of ions. Maximal germination and outgrowth were observed when double-concentrated artificial seawater was used. Replacement of chloride salts in the artificial seawater by other salts resulted in a complete loss of germination and outgrowth that was restored upon addition of chloride. To analyze the role of chloride more directly and quantitatively, a defined growth medium was used in which the artificial seawater was substituted by a solution of magnesium sulfate and sodium chloride. Spore germination and outgrowth were strictly dependent on the chloride concentration; maximal germination and outgrowth were observed at ≈ 1.3 M Cl^–. Chloride could be substituted by bromide, but not by sulfate or nitrate. Microscopic examinations of single spores clearly showed that germination is the chloride-dependent step. This first report on chloride dependence of spore germination in any endospore-forming bacterium adds another function to chloride in H. halophilus apart from its being essential for the physiology of the vegetative cell. Received: 21 May 1999 / Accepted: 26 July 1999     

RNA synthesis during spore germination in Bacillus subtilis.

Summary We have analyzed the RNA synthesized during spore germination in Bacillus subtilis. Early in germination there is little incorporation of [³H]uridine into RNA. A large increase in incorporation into RNA was found at 45–60 min into germination which was in part due to increases in the specific activity of the UTP pool. When corrected for specific activity changes, the instantaneous rate of RNA synthesis showed a seven to tenfold increase between 30 and 45 min of germination. Polyacrylamide gel electrophoresis studies showed that the RNA synthesized during germination appeared very similar to the RNA made during vegetative growth. DNA-RNA hybridization studies indicated that mRNA and rRNA were synthesized throughout germination. Their relative proportions remained constant and were very similar to the composition of RNA synthesized during vegetative growth.In partial fulfillment of the requirements for the doctoral degree by A.S. in the Department of Microbiology at the New York University School of Medicine     

A conserved ClpP‐like protease involved in spore outgrowth in Bacillus subtilis

Germination and outgrowth of endospores of the Gram‐positive bacterium Bacillus subtilis involves the degradation and conversion to free amino acids of abundant proteins located in the spore core known as small acid‐soluble proteins (SASP). This degradation is mediated primarily by the germination protease Gpr. Here we show that YmfB, a distant homologue of ClpP serine proteases that is highly conserved among endospore‐forming bacteria, contributes to SASP degradation but that its function is normally masked by Gpr. Spores from a ymfB gpr double mutant were more delayed in spore outgrowth and more impaired in SASP degradation than were spores from a gpr single mutant. The activity of YmfB relied on three putative active‐site residues as well as on the product of a small gene ylzJ located immediately downstream of, and overlapping with, ymfB. We propose that YmfB is an orphan ClpP protease that is dedicated to the degradation of a specialized family of small protein substrates.     

Uptake of and Resistance to the Antibiotic Berberine by Individual Dormant,Germinating and Outgrowing Bacillus Spores as Monitored by Laser Tweezers Raman Spectroscopy

Berberine, an alkaloid originally extracted from the plant Coptis chinensis and other herb plants, has been used as a pharmacological substance for many years. The therapeutic effect of berberine has been attributed to its interaction with nucleic acids and blocking cell division. However, levels of berberine entering individual microbial cells minimal for growth inhibition and its effects on bacterial spores have not been determined. In this work the kinetics and levels of berberine accumulation by individual dormant and germinated spores were measured by laser tweezers Raman spectroscopy and differential interference and fluorescence microscopy, and effects of berberine on spore germination and outgrowth and spore and growing cell viability were determined. The major conclusions from this work are that: (1) colony formation from B. subtilis spores was blocked ~ 99% by 25 μg/mL berberine plus 20 μg/mL INF55 (a multidrug resistance pump inhibitor); (2) 200 μg/mL berberine had no effect on B. subtilis spore germination with L-valine, but spore outgrowth was completely blocked; (3) berberine levels accumulated in single spores germinating with ≥ 25 μg/mL berberine were > 10 mg/mL; (4) fluorescence microscopy showed that germinated spores accumulated high-levels of berberine primarily in the spore core, while dormant spores accumulated very low berberine levels primarily in spore coats; and (5) during germination, uptake of berberine began at the time of commitment (T₁) and reached a maximum after the completion of CaDPA release (T_release) and spore cortex lysis (T_lysis).     

Bacillus subtilis Cells Lacking Penicillin-Binding Protein 1 Require Increased Levels of Divalent Cations for Growth

Bacillus subtilis strains lacking penicillin-binding protein 1 (PBP1), encoded by ponA, required greater amounts of Mg²⁺ or Ca²⁺ for vegetative growth or spore outgrowth than the wild-type strain and strains lacking other high-molecular-weight (HMW) PBPs. Growth of ponA cells in a medium low in Mg²⁺ also resulted in greatly increased cell bending compared to wild-type cells or cells lacking other HMW PBPs. The addition of high levels of Mg²⁺ to growth media eliminated these phenotypes of a ponA mutant. In contrast to the effects of divalent cations, NaCl did not restore ponA cell growth in a divalent-cation-deficient medium. Surprisingly, wild-type cells swelled and then lysed during both vegetative growth and spore outgrowth when 500 mM NaCl was included in a divalent-cation-deficient medium. Again, Mg²⁺ addition was sufficient to allow normal vegetative growth and spore outgrowth of both wild-type and ponA cells in a medium with 500 mM NaCl. These studies demonstrate that (i) while HMW PBPs possess largely redundant functions in rich medium, when divalent cations are limiting, PBP1 is required for cell growth and spore outgrowth; and (ii) high levels of NaCl induce cell lysis in media deficient in divalent cations during both vegetative growth and spore outgrowth.