iTRAQ analysis reveals the effect of gabD and sucA gene knockouts on lysine metabolism and crystal protein formation in Bacillus thuringiensis

Lysine metabolism plays an important role in the formation of the insecticidal crystal proteins of Bacillus thuringiensis (Bt). The genes lam, gabD and sucA encode three key enzymes of the lysine metabolic pathway in Bt4.0718. The lam gene mainly affects the cell growth at stable period, negligibly affected sporulation and insecticidal crystal protein (ICP) production. While, the deletion mutant strains of the gabD and sucA genes showed that the growth, sporulation and crystal protein formation were inhibited, cells became slender, and insecticidal activity was significantly reduced. iTRAQ proteomics and qRT-PCR used to analyse the differentially expressed protein (DEP) between the two mutant strains and the wild type strain. The functions of DEPs were visualized and statistically classified, which affect bacterial growth and metabolism by regulating biological metabolism pathways: the major carbon metabolism pathways, amino acid metabolism, oxidative phosphorylation pathways, nucleic acid metabolism, fatty acid synthesis and peptidoglycan synthesis. The gabD and sucA genes in lysine metabolic pathway are closely related to the sporulation and crystal proteins formation. The effects of DEPs and functional genes on basic cellular metabolic pathways were studied to provide new strategies for the construction of highly virulent insecticidal strains, the targeted transformation of functional genes.     

Quorum sensing intervened bacterial signaling: Pursuit of its cognizance and repression

Bacteria communicate within a system by means of a density dependent mechanism known as quorum sensing which regulate the metabolic and behavioral activities of a bacterial community. This sort of interaction occurs through a dialect of chemical signals called as autoinducers synthesized by bacteria. Bacterial quorum sensing occurs through various complex pathways depending upon specious diversity. Therefore the cognizance of quorum sensing mechanism will enable the regulation and thereby constrain bacterial communication. Inhibition strategies of quorum sensing are collectively called as quorum quenching; through which bacteria are incapacitated of its interaction with each other. Many virulence mechanism such as sporulation, biofilm formation, toxin production can be blocked by quorum quenching. Usually quorum quenching mechanisms can be broadly classified into enzymatic methods and non-enzymatic methods. Substantial understanding of bacterial communication and its inhibition enhances the development of novel antibacterial therapeutic drugs. In this review we have discussed the types and mechanisms of quorum sensing and various methods to inhibit and regulate density dependent bacterial communication.     

Poly(3‐hydroxybutyrate) fuels the tricarboxylic acid cycle and de novo lipid biosynthesis during Bacillus anthracis sporulation

Numerous bacteria accumulate poly(3‐hydroxybutyrate) (PHB) as an intracellular reservoir of carbon and energy in response to imbalanced nutritional conditions. In Bacillus spp., where PHB biosynthesis precedes the formation of the dormant cell type called the spore (sporulation), the direct link between PHB accumulation and efficiency of sporulation was observed in multiple studies. Although the idea of PHB as an intracellular carbon and energy source fueling sporulation was proposed several decades ago, the mechanisms underlying PHB contribution to sporulation have not been defined. Here, we demonstrate that PHB deficiency impairs Bacillus anthracis sporulation through diminishing the energy status of the cells and by reducing carbon flux into the tricarboxylic acid (TCA) cycle and de novo lipid biosynthesis. Consequently, this metabolic imbalance decreased biosynthesis of the critical components required for spore integrity and resistance, such as dipicolinic acid (DPA) and the spore's inner membrane. Supplementation of the PHB deficient mutant with exogenous fatty acids overcame these sporulation defects, highlighting the importance of the TCA cycle and lipid biosynthesis during sporulation. Combined, the results of this work reveal the molecular mechanisms of PHB contribution to B. anthracis sporulation and provide valuable insight into the metabolic requirements for this developmental process in Bacillus species.     

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.     

GK4, a G‐protein‐coupled receptor with a phosphatidylinositol phosphate kinase domain in Phytophthora infestans,is involved in sporangia development and virulence

For dispersal and host infection plant pathogens largely depend on asexual spores. Pathogenesis and sporulation are complex processes that are governed by cellular signalling networks including G‐protein and phospholipid signalling. Oomycetes possess a family of novel proteins called GPCR‐PIPKs (GKs) that are composed of a seven‐transmembrane spanning (7‐TM) domain fused to a phosphatidylinositol phosphate kinase (PIPK) domain. Based on this domain structure GKs are anticipated to link G‐protein and phospholipid signal pathways; however, their functions are currently unknown. Expression analyses of the 12 GK genes in Phytophthora infestans and their orthologues in Phytophthora sojae, revealed differential expression during asexual development. PiGK1 and PiGK4 were fused to monomeric red fluorescent protein (mRFP) and ectopically expressed in P. infestans. In growing hyphae different subcellular distribution patterns were observed indicating that these two GKs act independently during development. We focused on the functional analyses of PiGK4. Its localization suggested involvement in cell differentiation and elongation and its 7‐TM domain showed a canonical GPCR membrane topology. Silencing of GK4 and overexpression of full‐length and truncated constructs in P. infestans revealed that PiGK4 is not only involved in spore germination and hyphal elongation but also in sporangia cleavage and infection.     

Effect of dilution rate, cellobiose and ammonium availabilities on Clostridium cellulolyticum sporulation

The nutritional and physiological factors affecting sporulation of Clostridium cellulolyticum were studied using steady-state continuous cultures grown in both complex and synthetic media. Under cellobiose limitation, the probability that cells will sporulate appears to be directly related to the growth rate. In complex medium, the highest percentage of sporulation was 20% at a dilution rate of 0.015 h⁻¹ whereas in synthetic medium it was 10% at 0.035 h⁻¹. In both media, when the dilution rate was either higher or lower the percentage of sporulation decreased by between 2% and 4%. At low dilution rates, endospore formation was repressed under cellobiose-sufficient concentrations, suggesting catabolite repression by cellobiose. Furthermore, the concentration of ammonium was important in determining the percentage of sporulation, as ammonium limitation induced extensive sporulation at low growth rates even in an excess of cellobiose. The sporulation process is not triggered when cells are cellobiose-exhausted both in complex and synthetic media. These data suggest that, in C. cellulolyticum, an exogenous supply of carbon is required throughout the sporulation process. In the experimental conditions used in this work, no relationship between glycogen accumulation or glycogen mobilization and endospore formation was detected in C. cellulolyticum. Received: 15 April 1999 / Received revision: 15 June 1999 / Accepted: 22 June 1999     

Effect of septum-initiation mutations on sporulation and competent cell formation in Bacillus subtilis

Summary Sporulation and competent cell formation have been studied in four Bacillus subtilis strains, carrying septum-initiation mutations of different loci, div-31, div-341, div-12 and div-355 which exhibit filamentous growth at 45° C. The div-31 mutant was found to be defective in competence development at 30°–40°C whereas the div-12 mutant was affected only slightly. The div-341 and div-355 mutants showed lower competence, particularly at the higher temperatures. The four div mutant strains all showed poor sporulation at higher temperatures compared to the wild-type strain. We propose that some of the initial steps of septation are involved both in sporulation (possibly in forespore septum formation) and in competent cell formation and that these two processes share certain common features distinct from those in vegetative cell division.     

The tetrameric pheromone module SteC‐MkkB‐MpkB‐SteD regulates asexual sporulation,sclerotia formation and aflatoxin production in Aspergillus flavus

For eukaryotes like fungi to regulate biological responses to environmental stimuli, various signalling cascades are utilized, like the highly conserved mitogen‐activated protein kinase (MAPK) pathways. In the model fungus Aspergillus nidulans, a MAPK pathway known as the pheromone module regulates development and the production of secondary metabolites (SMs). This pathway consists five proteins, the three kinases SteC, MkkB and MpkB, the adaptor SteD and the scaffold HamE. In this study, homologs of these five pheromone module proteins have been identified in the plant and human pathogenic fungus Aspergillus flavus. We have shown that a tetrameric complex consisting of the three kinases and the SteD adaptor is assembled in this species. It was observed that this complex assembles in the cytoplasm and that MpkB translocates into the nucleus. Deletion of steC, mkkB, mpkB or steD results in abolishment of both asexual sporulation and sclerotia production. This complex is required for the positive regulation of aflatoxin production and negative regulation of various SMs, including leporin B and cyclopiazonic acid (CPA), likely via MpkB interactions in the nucleus. These data highlight the conservation of the pheromone module in Aspergillus species, signifying the importance of this pathway in regulating fungal development and secondary metabolism.     

Branched-chain fatty acids: the case for a novel form of cell-cell signalling during Myxococcus xanthus development

The esg locus is required for the formation of muiti-cellular fruiting bodies and spores by the developmental bacterium Myxococcus xanthus Studies have suggested that esg mutants are defective in the production of an essential signal (E-signal) used in cell-cell communication and that E-signalling is required for the expression of many developmental genes. Recently we have determined that the esg locus encodes components of a branched-chain keto acid dehydrogenase. a multienzyme complex involved in branched-chain amino acid metabolism in many bacteria and higher organisms. During vegetative growth in M. xanthus. this enzyme complex appears to participate in the production of the branched-chain fatty acids found in this organism. M. xanthus fatty acids (including the branched-chain fatty acids) have been observed to have a variety of effects on developing cells. These effects include; (i) the lysis of M. xanthus cells (autocide activity), (ii) acceleration of the rate of sporulation and (iii) rescue of sporulation by certain development-defective mutants. These and other results suggest a model in which the branched-chain fatty acids. Synthesized during growth, are released from cellular phospholipid by a developmentally regulated phospholipase during fruiting-body formation. This model proposes that one or more of the branched-chain fatty acids that are released constitutes the E-signal which must be transmitted between cells to complete M. xanthus development.     

The MAP1 family of microtubule-associated proteins

MAP1-family proteins are classical microtubule-associated proteins (MAPs) that bind along the microtubule lattice. The founding members, MAP1A and MAP1B, are predominantly expressed in neurons, where they are thought to be important in the formation and development of axons and dendrites. Mammalian genomes usually contain three family members, MAP1A, MAP1B and a shorter, more recently identified gene called MAP1S. By contrast, only one family member, Futsch, is found in Drosophila. After their initial expression, the MAP1A and MAP1B polypeptides are cleaved into light and heavy chains, which are then assembled into mature complexes together with the separately encoded light chain 3 subunit (LC3). Both MAP1A and MAP1B are well known for their microtubule-stabilizing activity, but MAP1 proteins can also interact with other cellular components, including filamentous actin and signaling proteins. Furthermore, the activity of MAP1A and MAP1B is controlled by upstream signaling mechanisms, including the MAP kinase and glycogen synthase kinase-3 β pathways.     

Mutants of the fission yeast Schizosaccharomyces pombe which alter the shift between cell proliferation and sporulation

Summary Mutants of S. pombe have been isolated which undergo conjugation and sporulation in rich medium, conditions which are normally inhibitory for these processes. Two of these mutants are also able to sporulate from the haploid state in the absence of heterozygosity at the mating type locus. These recessive mutants define a single nuclear gene called ran1 which is unlinked to mating type. It is proposed that the ran1 gene codes for an inhibitor in the control of the initiation of conjugation and sporulation. In wild type cells the inhibitory effect is released by nutritional starvation and heterozygosity at the mating type locus. This allows the cells to proceed to sporulation. The ran1 mutants are unusual in that they attempt to undergo a reductional meiotic division from the haploid state. They are also genetically unstable and generate extragenic suppressors at high frequency.     

Changes in patterns of alkaline serine protease and bacilysin formation caused by common effectors of sporulation inBacillus subtilis 168

Bacilysin biosynthesis and alkaline serine protease production inBacillus subtilis 168 were monitored and compared in batch cultures when various effectors of sporulation were added at different stages of growth in a medium containing sucrose and glutamate. Depending on the time of addition, glucose affected sporulation and serine protease formation to the same extent, but had no effect on bacilysin production. Ammonium andl-alanine additions suppressed all three processes. Casamino acids severely interfered with bacilysin formation and sporulation, but not with protease formation. Decoyinine, a well-known inducer of sporulation, induced protease formation as well, but did not affect bacilysin biosynthesis. The extent of the observed effects depended largely on the time of metabolite additions. The results are discussed with reference to a possible coregulation of sporulation and the formation of bacilysin and alkaline serine protease inB. subtilis.