Role of GerKB in Germination and Outgrowth of Clostridium perfringens Spores

Previous work indicated that Clostridium perfringens gerKA gerKC spores germinate significantly, suggesting that gerKB also has a role in C. perfringens spore germination. We now find that (i) gerKB was expressed only during sporulation, likely in the forespore; (ii) gerKB spores germinated like wild-type spores with nonnutrient germinants and with high concentrations of nutrients but more slowly with low nutrient concentrations; and (iii) gerKB spores had lower colony-forming efficiency and slower outgrowth than wild-type spores. These results suggest that GerKB plays an auxiliary role in spore germination under some conditions and is required for normal spore viability and outgrowth.Spores of Bacillus and Clostridium species can break dormancy upon sensing a variety of compounds (termed germinants), including amino acids, nutrient mixtures, a 1:1 chelate of Ca²⁺ and pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]), and cationic surfactants such as dodecylamine (20). Nutrient germinants are sensed by their cognate receptors, located in the spore''s inner membrane (6), which are composed of proteins belonging to the GerA family (10, 11). In Bacillus subtilis, three tricistronic operons (gerA, gerB, and gerK) expressed uniquely during sporulation in the developing forespore each encode the three major germinant receptors, with different receptors responding to a different spectrum of nutrient germinants (5, 9, 20). Null mutations in any cistron in a gerA family operon inactivate the function of the respective receptor (9, 11). In contrast, Clostridium perfringens, a gram-positive, spore-forming, anaerobic pathogenic bacterium, has no tricistronic gerA-like operon but only a monocistronic gerAA that is far from a gerK locus. This locus contains a bicistronic gerKA-gerKC operon and a monocistronic gerKB upstream of and in the opposite orientation to gerKA-gerKC (Fig. ​(Fig.1A)1A) (16). GerAA has an auxiliary role in the germination of C. perfringens spores at low germinant concentrations, while GerKA and/or GerKC are required for l-asparagine germination and have partial roles in germination with KCl and a mixture of KCl and l-asparagine (AK) (16). In contrast to the situation with B. subtilis, where germinant receptors play no role in Ca-DPA germination (12, 13), GerKA and/or GerKC is required for Ca-DPA germination (16). The partial requirement for GerKA and/or GerKC in C. perfringens spore germination by KCl and AK suggests that the upstream gene product, GerKB, might also have some role in KCl and AK germination of C. perfringens spores. Therefore, in this study we have investigated the role of GerKB in the germination and outgrowth of C. perfringens spores.Open in a separate windowFIG. 1.Arrangement and expression of gerKB in C. perfringens SM101. (A) The arrangement of the gerK locus in C. perfringens SM101 is shown, and the locations of the primers used to amplify the upstream regions of the gerKB gene and the putative promoters of gerKB and gerKA are indicated. The gerKB promoter was predicted to be within the intergenic regions between gerKB and the gerK operon. (B) GUS specific activities from the gerKB-gusA fusion in strain SM101(pDP84) grown in TGY vegetative (filled squares) and DS sporulation (open squares) media were determined as described in the text. Data represent averages from three independent experiments with the error bars representing standard deviations, and time zero denotes the time of inoculation of cells into either TGY or DS medium.To determine if gerKB is expressed during sporulation, 485 bp upstream of the gerKB coding sequence, including DNA between gerKB and gerKA, was PCR amplified with primer pair CPP389/CPP391, which had SalI and PstI cleavage sites, respectively (see Table S2 in the supplemental material). The PCR fragment was cloned between SalI and PstI cleavage sites in plasmid pMRS127 (17) to create a gerKB-gusA fusion in plasmid pDP84 (see Table S1 in the supplemental material). This plasmid was introduced into C. perfringens SM101 by electroporation (3), and Em^r transformants were selected. The SM101 transformant carrying plasmid pDP84 was grown in TGY vegetative growth medium (3% Trypticase soy, 2% glucose, 1% yeast extract, 0.1% l-cysteine) (7) and in Duncan-Strong (DS) (4) sporulation medium and assayed for β-glucuronidase (GUS) activity as described previously (23). Vegetative cultures of strain SM101 carrying plasmid pMRS127 (empty vector) or pDP84 (gerKB-gusA) exhibited no significant GUS activity, and strain SM101 grown in DS medium also exhibited no significant GUS activity (Fig. ​(Fig.1B1B and data not shown). However, GUS activity was observed in sporulating cultures of SM101(pDP84) (Fig. ​(Fig.1B),1B), indicating that a sporulation-specific promoter is located upstream of gerKB. The expression of the gerKB-gusA fusion began ∼3 h after induction of sporulation and reached a maximum after ∼6 h of sporulation (Fig. ​(Fig.1B).1B). The decrease in GUS activity observed after ∼6 h is consistent with the GerKB-GusA protein being packaged into the dormant spore where it cannot be easily assayed and thus with gerKB being expressed in the forespore compartment of the sporulating cell (8). These results confirm that, as with the gerKA-gerKC operon (16), gerKB is also expressed only during sporulation.To investigate the role of GerKB in C. perfringens spore germination, we constructed a gerKB mutant strain (DPS108) as described previously (14-16). A 2,203-bp DNA fragment carrying 2,080 bp upstream of and 123 bp from the N-terminal coding region of gerKB was PCR amplified using primers CPP369 and CPP367, which had XhoI and BamHI cleavage sites at the 5′ ends of the forward and reverse primers, respectively (see Table S2 in the supplemental material). A 1,329-bp fragment carrying 134 bp from the C-terminal and 1,195 bp downstream of the coding region of gerKB was PCR amplified using primers CPP371 and CPP370, which had BamHI and KpnI cleavage sites at the 5′ ends of the forward and reverse primers, respectively (see Table S2 in the supplemental material). These PCR fragments were cloned into plasmid pCR-XL-TOPO, giving plasmids pDP67 and pDP69, respectively (see Table S1 in the supplemental material). An ∼2.2-kb BamHI-XhoI fragment from pDP67 was cloned into pDP1 (pCR-XL-TOPO carrying an internal fragment of gerAA), giving plasmid pDP68, and an ∼1.4-kb KpnI-BamHI fragment from pDP69 was cloned in pDP68, giving pDP73 (see Table S1 in the supplemental material). The latter plasmid was digested with BamHI, the ends were filled, and an ∼1.3-kb NaeI-SmaI fragment carrying catP from pJIR418 (1) was inserted, giving plasmid pDP74. Finally, an ∼4.8-kb KpnI-XhoI fragment from pDP74 (see Table S1 in the supplemental material) was cloned between the KpnI and SalI sites of pMRS104, giving pDP75, which cannot replicate in C. perfringens. Plasmid pDP75 was introduced into C. perfringens SM101 by electroporation (3), and the gerKB deletion strain DPS108 was isolated as described previously (18). The presence of the gerKB deletion in strain DPS108 was confirmed by PCR and Southern blot analyses (data not shown). Strain DPS108 gave ∼70% sporulating cells in DS sporulation medium, similar to results with the wild-type strain, SM101 (data not shown).Having obtained evidence for successful construction of the gerKB mutant, we compared the germinations of heat-activated (80°C; 10 min) gerKB and wild-type spores as previously described (16). Both the gerKB and wild-type spores germinated identically and nearly completely in 60 min at 40°C in brain heart infusion (BHI) broth as determined by the fall in optical density at 600 nm (OD₆₀₀) of germinating cultures and phase-contrast microscopy (data not shown). This result suggests that GerKB plays no essential role in spore germination in rich medium. The role of GerKB in C. perfringens spore germination was also assessed with individual germinants identified previously (16). Heat-activated wild-type and gerKB spores germinated similarly with high (100 mM) concentrations of KCl, l-asparagine, and AK, all in 25 mM sodium phosphate (pH 7.0), and in 50 mM Ca-DPA adjusted to pH 8.0 with Tris base (Fig. 2A to D). These results were also confirmed by phase-contrast microscopy (data not shown). However, with lower (10 to 20 mM) concentrations of KCl, l-asparagine, and AK, gerKB spore germination was very slightly (Fig. ​(Fig.2A)2A) to significantly (Fig. 2B and C) slower than that of wild-type spores. These results suggest that while GerKB is not essential for germination with high concentrations of KCl, l-asparagine, or AK, it plays a significant role in germination with low l-asparagine and AK concentrations and, further, that GerKB is not required for Ca-DPA germination. This latter finding is similar to the situation with B. subtilis spores where germinant receptors play no role in Ca-DPA germination (19, 20). However, in C. perfringens spores, GerKA and/or GerKC do play a significant role in Ca-DPA germination (16).Open in a separate windowFIG. 2.Germination of spores of C. perfringens strains with various germinants. Heat-activated spores of strains SM101 (wild type) (filled symbols) and DPS108 (gerKB) (open symbols) were incubated at an OD₆₀₀ of 1 at 40°C with high (squares) and low (triangles) germinant concentrations of 100 and 10 mM KCl (A), 100 and 20 mM l-asparagine (B), 100 and 10 mM AK (C), and 50 mM Ca-DPA (D) as described in the text, and at various times the OD₆₀₀ was measured. No significant germination was observed when heat-activated spores of SM101 and DPS108 were incubated for 60 min at 40°C in 25 mM sodium phosphate buffer (pH 7.0) (data not shown). The data shown are averages from duplicate determinations with two different spore preparations, and error bars represent standard deviations.Bacterial spores can also germinate with dodecylamine, a cationic surfactant (19). In B. subtilis spores, dodecylamine induces germination most likely by opening channels composed, at least in part, of SpoVA proteins (22), allowing release of the spores'' Ca-DPA (19). Spores of B. subtilis lacking all three functional germinant receptors release DPA, as do wild-type spores, upon incubation with dodecylamine (19), while C. perfringens spores lacking GerKA-GerKC incubated with dodecylamine release DPA slower than wild-type spores (16). However, when C. perfringens gerKB spores at an OD₆₀₀ of 1.5 were incubated with 1 mM dodecylamine in Tris-HCl (pH 7.4) at 60°C (2, 16), gerKB spores released their DPA slightly faster than wild-type spores (Fig. ​(Fig.3)3) when DPA release was measured as described previously (16). These results suggest that GerKB has no role in dodecylamine germination.Open in a separate windowFIG. 3.Germination of spores of C. perfringens strains with dodecylamine. Spores of strains SM101 (wild type) (filled squares) and DPS108 (gerKB) (open squares) were germinated with dodecylamine, and germination was monitored by measuring DPA release as described in the text. There was no significant DPA release in 60 min by spores incubated similarly but without dodecylamine (data not shown). Error bars represent standard deviations.Previous work (16) found that C. perfringens spores lacking GerKA-GerKC had lower viability than wild-type spores on rich medium plates, and it was thus of interest to determine gerKB spore viability, which was measured as previously described (14, 16). Strikingly, the colony-forming ability of gerKB spores was ∼7-fold lower (P < 0.01) than that of wild-type spores after 24 h on BHI plates (Table ​(Table1),1), and no additional colonies appeared when plates were incubated for up to 3 days (data not shown). The colony-forming ability of spores lacking GerKA and GerKC determined in parallel was ∼12-fold lower than that of wild-type spores (Table ​(Table1).1). Phase-contrast microscopy of C. perfringens spores incubated in BHI broth for 24 h under aerobic conditions to prevent vegetative cell growth indicated that >90% of wild-type spores not only had germinated but had also released the nascent vegetative cell, while >85% of gerKA gerKC and gerKB spores remained as only phase-dark germinated spores with no evidence of nascent cell release (data not shown), as found previously with gerKA gerKC spores (16). The fact that >85% of gerKB spores germinated in BHI medium in 24 h but most of these germinated spores did not progress further in development strongly suggests that GerKB is needed for normal spore outgrowth (and see below) as well as for normal spore germination.

TABLE 1.

Colony formation by spores of C. perfringens strains^a

The LaCEP1 peptide modulates cluster root morphology in Lupinus albus

White lupin cluster roots are specialized brush‐like root structures that are formed in some species under phosphorus (P)‐deficient conditions. They intensely secrete protons and organic acid anions for solubilization and acquisition of sparingly soluble phosphates. Phytohormones and sucrose modulate cluster root number, but the molecular mechanisms of cluster root formation have been elusive. Here, a novel peptide phytohormone was identified that affects cluster root development. It belongs to the C‐TERMINALLY-ENCODED PEPTIDE (CEP) family. Members of that family arrest root growth and modulate branching in model species. LaCEP1 was highly expressed in the pre‐emergence zone of clusters. Over‐expression of the gene encoding the LaCEP1 propeptide resulted in moderate inhibition of cluster root formation. The primary and lateral root lengths of lupin were little affected by the overexpression, but LaCEP1 reduced cluster rootlet and root hair elongation. Addition of a 15‐mer core peptide derived from LaCEP1 similarly altered root morphology and modified cluster activity, suggesting that a core sequence of the propeptide is functionally sufficient. Stable overexpression in Arabidopsis confirmed the LaCEP1 function in root growth inhibition across species. Taken together, the root inhibitory effects of the LaCEP1 phytohormone suggest a role as of a regulatory module involved in cluster root development in white lupin.     

Genetic diversity of Lathyrus sp collected from different geographical regions

Molecular Biology Reports - The demand for grass peas (Lathyrus sativus L.) had increased as high nutritional safe food, but most of the accessions of South Asia and Africa had low grain harvest....     

Systems biological approach to investigate the lack of familial link between Down's Syndrome & Neural Tube Disorders

Systems Biology involves the study of the interactions of biological systems and ultimately their functions. Down''s syndrome (DS) is one of the most common genetic disorders which are caused by complete, or occasionally partial, triplication of chromosome 21, characterized by cognitive and language dysfunction coupled with sensory and neuromotor deficits. Neural Tube Disorders (NTDs) are a group of congenital malformations of the central nervous system and neighboring structures related to defective neural tube closure during the first trimester of pregnancy usually occurring between days 18-29 of gestation. Several studies in the past have provided considerable evidence that abnormal folate and methyl metabolism are associated with onset of DS & NTDs. There is a possible common etiological pathway for both NTDs and Down''s syndrome. But, various research studies over the years have indicated very little evidence for familial link between the two disorders. Our research aimed at the gene expression profiling of microarray datasets pertaining to the two disorders to identify genes whose expression levels are significantly altered in these conditions. The genes which were 1.5 fold unregulated and having a p-value <0.05 were filtered out and gene interaction network were constructed for both NTDs and DS. The top ranked dense clique for both the disorders were recognized and over representation analysis was carried out for each of the constituent genes. The comprehensive manual analysis of these genes yields a hypothetical understanding of the lack of familial link between DS and NTDs. There were no genes involved with folic acid present in the dense cliques. Only – CBL, EGFR genes were commonly present, which makes the allelic variants of these genes – good candidates for future studies regarding the familial link between DS and NTDs.

Abbreviations

NTD - Neural Tube Disorders, DS - Down''s Syndrome, MTHFR - Methylenetetrahydrofolate reductase, MTRR– 5 - methyltetrahydrofolate-homocysteine methyltransferase reductase.     

NHE3 Activity Is Dependent on Direct Phosphoinositide Binding at the N Terminus of Its Intracellular Cytosolic Region

The small intestinal BB Na⁺/H⁺ antiporter NHE3 accounts for the majority of intestinal sodium and water absorption. It is highly regulated with both postprandial inhibition and stimulation sequentially occurring. Phosphatidylinositide 4,5-bisphosphate (PI(4,5)P₂) and phosphatidylinositide 3,4,5-trisphosphate (PI(3,4,5)P₃) binding is involved with regulation of multiple transporters. We tested the hypothesis that phosphoinositides bind NHE3 under basal conditions and are necessary for its acute regulation. His₆ proteins were made from the NHE3 C-terminal region divided into four parts as follows: F1 (amino acids 475–589), F2 (amino acids 590–667), F3 (amino acids 668–747), and F4 (amino acids 748–832) and purified by a nickel column. Mutations were made in the F1 region of NHE3 and cloned in pet30a and pcDNA3.1 vectors. PI(4,5)P₂ and PI(3,4,5)P₃ bound only to the NHE3 F1 fusion protein (amino acids 475–589) on liposomal pulldown assays. Mutations were made in the putative lipid binding region of the F1 domain and studied for alterations in lipid binding and Na⁺/H⁺ exchange as follows: Y501A/R503A/K505A; F509A/R511A/R512A; R511L/R512L; R520/FR527F; and R551L/R552L. Our results indicate the following. 1) The F1 domain of the NHE3 C terminus has phosphoinositide binding regions. 2) Mutations of these regions alter PI(4,5)P₂ and PI(3,4,5)P₃ binding and basal NHE3 activity. 3) The magnitude of serum stimulation of NHE3 correlates with PI(4,5)P₂ and PI(3,4,5)P₃ binding of NHE3. 4) Wortmannin inhibition of PI3K did not correlate with PI(4,5)P₂ or PI(3,4,5)P₃ binding of NHE3. Two functionally distinct phosphoinositide binding regions (Tyr⁵⁰¹–Arg⁵¹² and Arg⁵²⁰–Arg⁵⁵²) are present in the NHE3 F1 domain; both regions are important for serum stimulation, but they display differences in phosphoinositide binding, and the latter but not the former alters NHE3 surface expression.     

The serotonin transporter is an exclusive client of the coat protein complex II (COPII) component SEC24C

The transporters for serotonin (SERT), dopamine, and noradrenaline have a conserved hydrophobic core but divergent N and C termini. The C terminus harbors the binding site for the coat protein complex II (COPII) cargo-binding protein SEC24. Here we explored which SEC24 isoform was required for export of SERT from the endoplasmic reticulum (ER). Three lines of evidence argue that SERT can only exit the ER by recruiting SEC24C: (i) Mass spectrometry showed that a peptide corresponding to the C terminus of SERT recruited SEC24C-containing COPII complexes from mouse brain lysates. (ii) Depletion of individual SEC24 isoforms by siRNAs revealed that SERT was trapped in the ER only if SEC24C was down-regulated, in both, cells that expressed SERT endogenously or after transfection. The combination of all siRNAs was not more effective than that directed against SEC24C. A SERT mutant in which the SEC24C-binding motif ((607)RI(608)) was replaced by alanine was insensitive to down-regulation of SEC24C levels. (iii) Overexpression of a SEC24C variant with a mutation in the candidate cargo-binding motif (SEC24C-D796V/D797N) but not of the corresponding mutant SEC24D-D733V/D734N reduced SERT surface levels. In contrast, noradrenaline and dopamine transporters and the more distantly related GABA transporter 1 relied on SEC24D for ER export. These observations demonstrate that closely related transporters are exclusive client cargo proteins for different SEC24 isoforms. The short promoter polymorphism results in reduced SERT cell surface levels and renders affected individuals more susceptible to depression. By inference, variations in the Sec24C gene may also affect SERT cell surface levels and thus be linked to mood disorders.     

Characterization of Clostridium perfringens spores that lack SpoVA proteins and dipicolinic acid

Spores of Clostridium perfringens possess high heat resistance, and when these spores germinate and return to active growth, they can cause gastrointestinal disease. Work with Bacillus subtilis has shown that the spore's dipicolinic acid (DPA) level can markedly influence both spore germination and resistance and that the proteins encoded by the spoVA operon are essential for DPA uptake by the developing spore during sporulation. We now find that proteins encoded by the spoVA operon are also essential for the uptake of Ca(2+) and DPA into the developing spore during C. perfringens sporulation. Spores of a spoVA mutant had little, if any, Ca(2+) and DPA, and their core water content was approximately twofold higher than that of wild-type spores. These DPA-less spores did not germinate spontaneously, as DPA-less B. subtilis spores do. Indeed, wild-type and spoVA C. perfringens spores germinated similarly with a mixture of l-asparagine and KCl (AK), KCl alone, or a 1:1 chelate of Ca(2+) and DPA (Ca-DPA). However, the viability of C. perfringens spoVA spores was 20-fold lower than the viability of wild-type spores. Decoated wild-type and spoVA spores exhibited little, if any, germination with AK, KCl, or exogenous Ca-DPA, and their colony-forming efficiency was 10(3)- to 10(4)-fold lower than that of intact spores. However, lysozyme treatment rescued these decoated spores. Although the levels of DNA-protective alpha/beta-type, small, acid-soluble spore proteins in spoVA spores were similar to those in wild-type spores, spoVA spores exhibited markedly lower resistance to moist heat, formaldehyde, HCl, hydrogen peroxide, nitrous acid, and UV radiation than wild-type spores did. In sum, these results suggest the following. (i) SpoVA proteins are essential for Ca-DPA uptake by developing spores during C. perfringens sporulation. (ii) SpoVA proteins and Ca-DPA release are not required for C. perfringens spore germination. (iii) A low spore core water content is essential for full resistance of C. perfringens spores to moist heat, UV radiation, and chemicals.