The effect of the pH of the sporulation environment on the heat resistance ofClostridium perfringens spores

The inactivation ofClostridium perfringens NCTC 8239 spores at 95° and 105° C, as determined by colony formation on an agar base containing lysozyme (BASE + lysozyme), was influenced by the initial pH of the sporulation medium. In the pH range of 7.0–8.5, established by the addition of each of several biological buffers or carbonate buffer to Duncan-Strong (DS) medium, increased pH resulted in formation of spores with greater resistance to inactivation at elevated temperatures. An increase of pH from 8.5 to 9.0 resulted in increased resistance of spores formed in DS-carbonate but not DS-TAPS (N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid) medium. Resistance to spore injury, as determined by reduced recovery on BASE compared with BASE + lysozyme, was not increased for spores formed in media with higher pH's. As the pH of the medium increased, cell growth and number of spores formed were decreased, but the percentage of sporulation was apparently not affected.     

Intracellular nitrite accumulation: The cause of growth inhibition of Microcystis aeruginosa exposure to high nitrite level

The aim of the present study is to test the role of intracellular nitrite in external nitrite suppressing algal growth. We examined the growth of Microcystis aeruginosa at different nitrite levels under high nitrate conditions and without nitrate conditions. There were higher intracellular nitrite and lower P_m^chla, R_d ^chla, α^chl, maximum cell density and specific growth rate in high nitrate group than nitrate absence group at 5 mg NO₂^?‐N L^?1. At 10 and 15 mg NO₂^?‐N L^?1, P_m^chla, R_d ^chla, α^chl, maximum cell densities and specific growth rates in the high nitrate group became higher than those of the nitrate absence group, while a lower intracellular nitrite in the high nitrate group than nitrate absence group was observed. In addition, the intracellular nitrite and the growth of M. aeruginosa in the high nitrate group did not change from 5 to 10 mg NO₂^?‐N L^?1. In the nitrite uptake experiment, with nitrite concentration increasing from 5 to 15 mg NO₂^?‐N L^?1, maximum nitrite uptake rate of alga increased, and half‐saturation constant of alga decreased. These results indicate that external nitrite inhibited algal growth through stimulating intracellular nitrite rise, which resulted from overexpression of nitrite transporter.     

Effect of glucose on sporulation and extracellular amylase production byClostridium perfringens type A in a defined medium

The effect of glucose and other sugars on sporulation and extracellular amylase production byClostridium perfringens NCTC 8679 type A in a defined medium was studied. Cells grown in the presence of glucose and mannose yielded the highest levels of amylase activity, while disaccharides such as lactose, maltose, and sucrose resulted in moderate amylase production. Little amylase activity was detected in the medium in the presence of ribose or galactose. The concentration of each sugar resulting in highest amylase production was between 6 and 10mm except for fructose (25mm). Levels of heat-resistant spores decreased as sugar concentrations increased. The addition of even small amounts of glucose to the medium before exponential growth suppressed sporulation but maximized amylase activity. The addition of glucose after the initiation of sporulation did not inhibit spore formation. However, its addition to 3-h amylase-producing cells did inhibit subsequent sporulation but promoted the continued excretion of amylase. The different response to glucose between sporulating cells and amylase-producing cells suggests that the mechanisms of catabolite repression of extracellular amylase production and sporulation are distinct in this strain ofC. perfringens.     

Analysis of the germination of individual Clostridium perfringens spores and its heterogeneity

Aims: To analyse the germination and its heterogeneity of individual spores of Clostridium perfringens. Methods and Results: Germination of individual wild‐type Cl. perfringens spores was followed by monitoring Ca‐dipicolinic acid (CaDPA) release and by differential interference contrast (DIC) microscopy. Following the addition of KCl that acts via germinant receptors (GRs), there was a long variable lag period (T_lag) with slow release of c. 25% of CaDPA, then rapid release of remaining CaDPA in c. 2 min (ΔT_release) and a parallel decrease in DIC image intensity, and a final decrease of c. 25% in DIC image intensity during spore cortex hydrolysis. Spores lacking the essential cortex‐lytic enzyme (CLE) (sleC spores) exhibited the same features during GR‐dependent germination, but with longer average T_lag values, and no decrease in DIC image intensity because of cortex hydrolysis after full CaDPA release. The T_lag of wild‐type spores in KCl germination was increased significantly by lower germinant concentrations and suboptimal heat activation. Wild‐type and sleC spores had identical average T_lag and ΔT_release values in dodecylamine germination that does not utilize GRs. Conclusions: Most of these results were essentially identical to those reported for the germination of individual spores of Bacillus species. However, individual sleC Cl. perfringens spores germinated inefficiently with either KCl or exogenous CaDPA, in contrast to CLE‐deficient Bacillus spores, indicating that germination of these species’ spores is not completely identical. Significance and Impact of the Study: This work provides information on the kinetic germination and its heterogeneity of individual spores of Cl. perfringens.     

A respiratory nitrate reductase active exclusively in resting spores of the obligate aerobe Streptomyces coelicolor A3(2)

The Gram‐positive aerobe Streptomyces coelicolor undergoes a complex life cycle including growth as vegetative hyphae and the production of aerial hyphae and spores. Little is known about how spores retain viability in the presence of oxygen; however, nothing is known about this process during anaerobiosis. Here, we demonstrate that one of the three respiratory nitrate reductases, Nar‐1, synthesized by S. coelicolor is functional exclusively in spores. A tight coupling between nitrite production and the activity of the cytoplasmically oriented Nar‐1 enzyme was demonstrated. No exogenous electron donor was required to drive nitrate reduction, which indicates that spore storage compounds are used as electron donors. Oxygen reversibly inhibited nitrate reduction by spores but not by spore extracts, suggesting that nitrate transport might be the target of oxygen inhibition. Nar‐1 activity required no de novo protein synthesis indicating that Nar‐1 is synthesized during sporulation and remains in a latently active state throughout the lifetime of the spore. Remarkably, the rates of oxygen and of nitrate reduction by wetted spores were comparable. Together, these findings suggest that S. coelicolor spores have the potential to maintain a membrane potential using nitrate as an alternative electron acceptor.     

β-nitropropionic acid and nitrite in relation to nitrate formation by Aspergillus flavus

Summary -nitropropionic acid (BNP) was converted to nitrate in media inoculated with A. flavus spores or with replacement cultures of mycelium pregrown in glucose-peptone medium. Conversion by replacement cultures was rapid: 8–30% in 2 days; influenced by pH: most rapid at pH 3.5; and extensive: as much as 80% BNP nitrogen appeared as nitrate after 14 days. Nitrite was detectable in BNP replacement cultures at low levels or not at all, and nitrate was formed in BNP replacement media with or without glucose. Nitrite was not oxidized in growing cultures inoculated with spores, but replacement cultures oxidized over 50% of added nitrite to nitrate in 8 days. No nitrite or nitrate appeared in replacement systems with pyruvic oxime, oxalacetic acid oxime, acetoxime, ketoglutaric acid oxime, or hydroxylamine.Of the three non-nitrifying mutants of A. flavus obtained, all formed nitrate from BNP in replacement but only one oxidized nitrite to nitrate. No accumulation of free or bound hydroxylamine or of nitrite could be detected in the mutants. BNP was detected by qualitative test in cultures of the wild type but not the mutants. Evidence indicates that the pathway in A. flavus is BNPNO₃ ^- rather than BNPNO₂ ^-NO₃ ^-.     

Genome-Wide Transcriptional Profiling of Clostridium perfringens SM101 during Sporulation Extends the Core of Putative Sporulation Genes and Genes Determining Spore Properties and Germination Characteristics

The formation of bacterial spores is a highly regulated process and the ultimate properties of the spores are determined during sporulation and subsequent maturation. A wide variety of genes that are expressed during sporulation determine spore properties such as resistance to heat and other adverse environmental conditions, dormancy and germination responses. In this study we characterized the sporulation phases of C. perfringens enterotoxic strain SM101 based on morphological characteristics, biomass accumulation (OD₆₀₀), the total viable counts of cells plus spores, the viable count of heat resistant spores alone, the pH of the supernatant, enterotoxin production and dipicolinic acid accumulation. Subsequently, whole-genome expression profiling during key phases of the sporulation process was performed using DNA microarrays, and genes were clustered based on their time-course expression profiles during sporulation. The majority of previously characterized C. perfringens germination genes showed upregulated expression profiles in time during sporulation and belonged to two main clusters of genes. These clusters with up-regulated genes contained a large number of C. perfringens genes which are homologs of Bacillus genes with roles in sporulation and germination; this study therefore suggests that those homologs are functional in C. perfringens. A comprehensive homology search revealed that approximately half of the upregulated genes in the two clusters are conserved within a broad range of sporeforming Firmicutes. Another 30% of upregulated genes in the two clusters were found only in Clostridium species, while the remaining 20% appeared to be specific for C. perfringens. These newly identified genes may add to the repertoire of genes with roles in sporulation and determining spore properties including germination behavior. Their exact roles remain to be elucidated in future studies.     

Phospholipid fatty acid and lipopolysaccharide fatty acid signature lipids in methane-utilizing bacteria

The effect of human bile juice and bile salts (sodium cholate, sodium taurocholate, sodium glycochenodeoxycholate and sodium chenodeoxycholate) on growth, sporulation and enterotoxin production by enterotoxin-positive and enterotoxin-negative strains of Clostridium perfringens was determined. Each bile salt inhibited growth to a different degree. A mixture of bile salts completely inhibited the growth of enterotoxin-positive strains of this organism. Human bile juice completely inhibited the growth of all the strains at a dilution of 1:320. A distinct stimulatory effect of the bile salts on sporulation was observed in the case of C. perfringens strains NCTC 8239 and NCTC 8679. The salts also increased enterotoxin concentrations in the cell extracts of the enterotoxin-positive strains tested. No effect on enterotoxin production was detected when an enterotoxin-negative strain was examined.     

Characterization of two putative fibronectin-binding proteins of Clostridium perfringens

Clostridium perfringens is a Gram-positive anaerobic pathogen that causes gas gangrene and food poisoning in humans and animals. Genomic analysis of C. perfringens strain 13 revealed that this bacterium contains two genes (CPE0737 and CPE1847) that encode putative fibronectin (Fn)-binding proteins (Fbps). These genes, named fbpA and fbpB, were found to be constitutively expressed in all three strains (13, NCTC8237, CPN50) of C. perfringens, isolated from gas gangrene of human, that were tested. Both fbpA and fbpB were cloned and His-tagged, recombinant FbpA (rFbpA) and recombinant FbpB (rFbpB) were purified by Ni²⁺-Sepharose column chromatography from transformed Escherichia coli. These recombinant Fbps were shown to bind to Fn, purified from human serum, in a ligand blotting assay. Additionally, Fn bound to these rFbps in a dose-dependent manner in an enzyme-linked immunosorbent assay. Furthermore, it was found that pre-incubation of Fn with either rFbpA or rFbpB inhibited the binding of Fn to C. perfringens cells.     

ISOLATION AND PARTIAL CHARACTERIZATION OF NITRATE ASSIMILATION MUTANTS OF DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE)1

Fifteen nitrate assimilation-deficient mutants of the euryhaline green alga, Dunaliella tertiolecta Butcher were selected by their chlorate resistance. Ten mutants, unable to grow on NO₃^? but able to grow on NO₂^?, had no detectable nitrate reductase activity. Five mutants, unable to grow on either NO₃^? or NO₂^?, had depressed levels of both nitrate and nitrite reductase. A method for assaying methyl viologen-nitrate reductase in the presence of nitrite reductase is described.     

Nitrogen and sulphur requirements of Colletotrichum inamdarii Lal

Summary Nitrogen and sulphur requirements ofColletotrichum inamdarii Lal isolated from the leaves ofCarissa carandas L. have been studied. DL-serine, L-asparagine and L-phenylalanine have been found to be of good nitrogen source followed by potassium nitrate, calcium nitrate, magnesium nitrate, DL-alanine, ammonium nitrate, glutamic acid, ammonium sulphate, DL-valine, aspartic acid, ammonium chloride, ammonium hydrogencarbonate, L-histidine and potassium nitrite. There was no growth in the absence of nitrogen.Sporulation was excellent on calcium nitrate and sodium nitrate, Very good on DL-serine, potassium nitrate, and magnesium nitrate. Good on L-asparagine, L-phenylalanine and ammonium oxalate. Fair on DL-alanine, DL-leucine, ammonium sulphate, DL-valine, ammonium chloride and L-histidine whereas poor on glutamic acid, aspartic acid, ammonium tartarate and ammonium nitrate. Few spores were observed on ammonium hydrogencarbonate but potassium nitrite did not show any sporulation.Amongst the sulphur compounds sodium bisulphate gave the best growth and good sporulation, followed by sodium thiosulphate, magnesium sulphate, ammonium sulphate and potassium sulphate. Thiourea gave negligible growth whereas it failed to grow on zinc sulphate and potassium persulphate.     

Effects of non-steady-state iron limitation on nitrogen assimilatory enzymes in the marine diatom thalassiosira weissflogii (BACILLARIOPHYCEAE)

Since the recognition of iron‐limited high nitrate (or nutrient) low chlorophyll (HNLC) regions of the ocean, low iron availability has been hypothesized to limit the assimilation of nitrate by diatoms. To determine the influence of non‐steady‐state iron availability on nitrogen assimilatory enzymes, cultures of Thalassiosira weissflogii (Grunow) Fryxell et Hasle were grown under iron‐limited and iron‐replete conditions using artificial seawater medium. Iron‐limited cultures suffered from decreased efficiency of PSII as indicated by the DCMU‐induced variable fluorescence signal (F_v/F_m). Under iron‐replete conditions, in vitro nitrate reductase (NR) activity was rate limiting to nitrogen assimilation and in vitro nitrite reductase (NiR) activity was 50‐fold higher. Under iron limitation, cultures excreted up to 100 fmol NO₂^?·cell^?1·d^?1 (about 10% of incorporated N) and NiR activities declined by 50‐fold while internal NO₂^? pools remained relatively constant. Activities of both NR and NiR remained in excess of nitrogen incorporation rates throughout iron‐limited growth. One possible explanation is that the supply of photosynthetically derived reductant to NiR may be responsible for the limitation of nitrogen assimilation at the NO₂^? reduction step. Urease activity showed no response to iron limitation. Carbon:nitrogen ratios were equivalent in both iron conditions, indicating that, relative to carbon, nitrogen was assimilated at similar rates whether iron was limiting growth or not. We hypothesize that, diatoms in HNLC regions are not deficient in their ability to assimilate nitrate when they are iron limited. Rather, it appears that diatoms are limited in their ability to process photons within the photosynthetic electron transport chain which results in nitrite reduction becoming the rate‐limiting step in nitrogenassimilation.     

Effects of Mn levels on resistance of Bacillus megaterium spores to heat, radiation and hydrogen peroxide

Aims: To determine the effects of Mn levels in Bacillus megaterium sporulation and spores on spore resistance. Methods and Results: Bacillus megaterium was sporulated with no added MnCl₂ and up to 1 mmol l^?1 MnCl₂. The resultant spores were purified and loosely bound Mn removed, and spore Mn levels were found to vary c. 100‐fold. The Mn level had no effect on spore γ‐radiation resistance, but B. megaterium spores with elevated Mn levels had higher resistance to UVC radiation (as did Bacillus subtilis spores), wet and dry heat and H₂O₂. However, levels of dipicolinic acid and the DNA‐protective α/β‐type small, acid‐soluble spore proteins were the same in spores with high and low Mn levels. Conclusions: Mn levels either in sporulation or in spores are important factors in determining levels of B. megaterium spore resistance to many agents, with the exception of γ‐radiation. Significance and Impact of the Study: The Mn level in sporulation is an important factor to consider when resistance properties of B. megaterium spores are examined, and will influence the UV resistance of B. subtilis spores, some of which are used as biological dosimeters.     

Stimulation of the onset of sporulation ofClostridium perfringens type A by netropsin and distamycin

The basic peptide antibiotics, netropsin and distamycin, previously shown to inhibit sporulation ofBacillus subtilis, stimulated low levels of sporulation ofClostridium perfringens strain NCTC 8798 at concentrations of 1.0 and 0.1 g/ml respectively. Most sporulating cells produced in the presence of the antibiotics were defective. These were blocked at Stage III of sporulation, and many possessed forespores exterior to the sporangium. The same antibiotics could also inhibit the caffeine-induced stimulation of sporulation of this strain.     

AbrB and Spo0E Control the Proper Timing of Sporulation in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

We have shown previously that Spo0AP-dependent sinIR operon expression was substantially down-regulated in abrB null mutant backgrounds. In this report, we show that loss of function mutations in abrB also cause phosphorelay gene expression to be down regulated. abrB null mutations caused diminished vegetative growth-associated sporulation and resulted in a significant reduction in sporulation frequencies at T₂₄. These mutants, however, sporulated at wild-type levels at T₄₈, indicating that sporulation timing was affected. The rvtA11 mutation in spo0A, a deletion mutation in spo0E, and a null mutation in hpr (scoC) rescued sporulation and Spo0AP-dependent gene expression in an abrB mutant background. These data indicate that AbrB and Spo0E may comprise a checkpoint system that regulates the progression of sporulation, allowing exploration of alternate cell states prior to the irrevocable commitment to sporulation.