Morphogenetic surfactants and their role in the formation of aerial hyphae in Streptomyces coelicolor

Withstanding environmental adversity and seeking optimal conditions for reproduction are basic requirements for the survival of all organisms. Filamentous bacteria of the genus Streptomyces produce a remarkable cell type called the aerial hyphae that is central to its ability to meet both of these challenges. Recent advances have brought about a major shift in our understanding of the cell surface proteins that play important roles in the generation of these cells. Here we review our current understanding of one of these groups of proteins, the morphogenetic surfactants, with emphasis on the SapB protein of Streptomyces coelicolor.     

Replisome localization in vegetative and aerial hyphae of Streptomyces coelicolor

Using a functional fusion of DnaN to enhanced green fluorescent protein, we examined the subcellular localization of the replisome machinery in the vegetative mycelium and aerial mycelium of the multinucleoid organism Streptomyces coelicolor. Chromosome replication took place in many compartments of both types of hypha, with the apical compartments of the aerial mycelium exhibiting the highest replication activity. Within a single compartment, the number of "current" ongoing DNA replications was lower than the expected chromosome number, and the appearance of fluorescent foci was often heterogeneous, indicating that this process is asynchronous within compartments and that only selected chromosomes undergo replication.     

Assembly of the cell division protein FtsZ into ladder-like structures in the aerial hyphae of Streptomyces coelicolor

In the filamentous bacterium Streptomyces coelicolor , the cell division protein FtsZ is required for the conversion of multinucleoidal aerial hyphae into chains of uninucleoidal spores, although it is not essential for viability. Using immunofluorescence microscopy, we have shown that FtsZ assembles into long, regularly spaced, ladder-like arrays in developing aerial hyphae, with an average spacing of about 1.3 μm. Within individual hyphae, ladder formation was relatively synchronous and extended for distances over 100 μm. These ladders were present only transiently, decreasing in intensity as chromosomes separated into distinct nucleoids and disappearing upon the completion of septum formation. Evidence from the overall intensity of immunofluorescence staining suggested that ladder formation was regulated in part at the level of the accumulation and degradation of FtsZ within individual aerial hyphae. Finally, FtsZ ladder formation was under developmental control in that long arrays of FtsZ rings could not be detected in certain so-called white mutants ( whiG , whiH and whiB ), which are blocked in spore formation. The assembly of FtsZ into ladders represents the earliest known molecular manifestation of the process of spore formation, and its discovery provides insight into the role of whi genes in the conversion of aerial hyphae into chains of spores. We have also described a novel use of a cell wall-staining technique to visualize apical tip growth in vegetatively growing hyphae.     

Interactions between Bacillus subtilis early spore coat morphogenetic proteins

When challenged by stresses such as starvation, the soil bacterium Bacillus subtilis produces an endospore surrounded by a proteinaceous coat composed of >70 proteins that are organized into three main layers: an amorphous undercoat, lightly staining lamellar inner coat and electron-dense outer coat. This coat protects the spore against a variety of chemicals or lysozyme. Mutual interactions of the coat's building blocks are responsible for the formation of this structurally complex and extraordinarily resistant shell. However, the assembly process of spore coat proteins is still poorly understood. In the present work, the main focus is on the three spore coat morphogenetic proteins: SpoIVA, SpoVID and SafA. Direct interaction between SpoIVA and SpoVID proteins was observed using a yeast two-hybrid assay and verified by coexpression experiment followed by Western blot analysis. Coexpression experiments also confirmed previous findings that SpoVID and SafA directly interact, and revealed a novel interaction between SpoIVA and SafA. Moreover, gel filtration analysis revealed that both SpoIVA and SpoVID proteins form large oligomers.     

Characteristics of Streptomyces coelicolor A3(2) aerial spore rodlet mosaic

Cytochemical analysis of Streptomyces coelicolor (A3(2) indicated that the aerial growth rodlet mosaic is a polysaccharide. Statistical analysis of frequency distributions of individual rodlet lengths from control and ether-reoriented spore mosaics indicated that the rodlet fibrillar image is the result of individual particulates, rather than evaginations in a continuous sheet of material. A model of the mature sport envelope was developed from freeze-etch-replicated, thin-sectioned, and critical point dried S. coelicolor A3(2) mature spores. The rodlet mosaic was situated between the outer spore wall and an external granuloma matrix. Mixture spore envelope layers from the inner surface to the external surface are plasma membrane, inner spore wall, outer spore wall, rodlet mosaic, an undefined granular matrix, and the sheath. The granular matrix had an uneven thickness and much of the matrix was frequently absent from the interspore spaces of mature spore chains. Streptomyces coelicolor A3(2) mosaic rodlets were isolated by acetic acid refluxing, then ethanol precipitation. Complete acid hydrolysis of rodlets released on sugar which cochromatographed with D-glucosamine-HCl and released acetic acid at 139% of the expected level. Cell associated rodlet mosaics and isolated mosaic rodlets were hydrolyzed with chitinase. Infrared spectra of isolated rodlets were similar to crab chitin spectra.     

SapB and the chaplins: connections between morphogenetic proteins in Streptomyces coelicolor

Morphogenesis in the streptomycetes features the differentiation of substrate-associated vegetative hyphae into upwardly growing aerial filaments. This transition requires the activity of bld genes and the secretion of biosurfactants that reduce the surface tension at the colony-air interface enabling the emergence of nascent aerial hyphae. Streptomyces coelicolor produces two classes of surface-active molecules, SapB and the chaplins. While both molecules are important for aerial development, nothing is known about the functional redundancy or interaction of these surfactants apart from the observation that aerial hyphae formation can proceed via one of two pathways: a SapB-dependent pathway when cells are grown on rich medium and a SapB-independent pathway on poorly utilized carbon sources such as mannitol. We used mutant analysis to show that while the chaplins are important, but not required, for development on rich medium, they are essential for differentiation on MS (soy flour mannitol) medium, and the corresponding developmental defects could be suppressed by the presence of SapB. Furthermore, the chaplins are produced by conditional bld mutants during aerial hyphae formation when grown on the permissive medium, MS, suggesting that the previously uncharacterized SapB-independent pathway is chaplin dependent. In contrast, a bld mutant blocked in aerial morphogenesis on all media makes neither SapB nor chaplins. Finally, we show that a constructed null mutant that lacks all chaplin and SapB biosynthetic genes fails to differentiate in any growth condition. We propose that the biosurfactant activities of both SapB and the chaplins are essential for normal aerial hyphae formation on rich medium, while chaplin biosynthesis and secretion alone drives aerial morphogenesis on MS medium.     

Role of acid metabolism in Streptomyces coelicolor morphological differentiation and antibiotic biosynthesis

Studies of citrate synthase (CitA) were carried out to investigate its role in morphological development and biosynthesis of antibiotics in Streptomyces coelicolor. Purification of CitA, the major vegetative enzyme activity, allowed characterization of its kinetic properties. The apparent K(m) values of CitA for acetyl coenzyme A (acetyl-CoA) (32 microM) and oxaloacetate (17 microM) were similar to those of citrate synthases from other gram-positive bacteria and eukaryotes. CitA was not strongly inhibited by various allosteric feedback inhibitors (NAD(+), NADH, ATP, ADP, isocitrate, or alpha-ketoglutarate). The corresponding gene (citA) was cloned and sequenced, allowing construction of a citA mutant (BZ2). BZ2 was a glutamate auxotroph, indicating that citA encoded the major citrate synthase allowing flow of acetyl-CoA into the tricarboxylic acid (TCA) cycle. Interruption of aerobic TCA cycle-based metabolism resulted in acidification of the medium and defects in morphological differentiation and antibiotic biosynthesis. These developmental defects of the citA mutant were in part due to a glucose-dependent medium acidification that was also exhibited by some other bald mutants. Unlike other acidogenic bald strains, citA and bldJ mutants were able to produce aerial mycelia and pigments when the medium was buffered sufficiently to maintain neutrality. Extracellular complementation studies suggested that citA defines a new stage of the Streptomyces developmental cascade.     

Role of phosphopantetheinyl transferase genes in antibiotic production by Streptomyces coelicolor

The phosphopantetheinyl transferase genes SCO5883 (redU) and SCO6673 were disrupted in Streptomyces coelicolor. The redU mutants did not synthesize undecylprodigiosin, while SCO6673 mutants failed to produce calcium-dependent antibiotic. Neither gene was essential for actinorhodin production or morphological development in S. coelicolor, although their mutation could influence these processes.     

Cloning and analysis of a gene cluster from Streptomyces coelicolor that causes accelerated aerial mycelium formation in Streptomyces lividans.

We describe the cloning and analysis of two overlapping DNA fragments from Streptomyces coelicolor that cause aerial mycelium to appear more rapidly than usual when introduced into Streptomyces lividans on a low-copy-number plasmid vector. Colonies of S. lividans TK64 harboring either clone produce visible aerial mycelia after only 48 h of growth, rather than the usual 72 to 96 h. From deletion and sequence analysis, this rapid aerial mycelium (Ram) phenotype appears to be due to a cluster of three genes that we have designated ramA, ramB, and ramR. Both ramA and ramB potentially encode 65-kDa proteins with homology to ATP-dependent membrane-translocating proteins. A chromosomal ramB disruption mutant of S. lividans was found to be severely defective in aerial mycelium formation. ramR could encode a 21-kDa protein with significant homology to the UhpA subset of bacterial two-component response regulator proteins. The overall organization and potential proteins encoded by the cloned DNA suggest that this is the S. coelicolor homolog of the amf gene cluster that has been shown to be important for aerial mycelium formation in Streptomyces griseus. However, despite the fact that the two regions probably have identical functions, there is relatively poor homology between the two gene clusters at the DNA sequence level.     

Early stages in Bacillus subtilis transformation: association between homologous DNA and surface structures.

The addition of ethylenediaminetetraacetate to competent cultures of Bacillus subtilis irreversibly inhibited the transformability as well as the cellular binding of DNA. Our results show that the inhibition of DNA binding by ethylenediaminetetraacetate in whole cells, protoplasts, and membrane vesicles is mainly due to a permanent alteration of the DNA receptors. Transformation absolutely requires free magnesium ions, whereas DNA binding is a magnesium-independent step. In contrast to ethylenediaminetetraacetate, the absence of Mg2+ does not irreversibly affect the capacity of the competent cells to be transformed DNA-binding receptors located at the cell surface remain associated with the plasma membrane after protoplasting and after isolation of membrane vesicles. A Mg2+-dependent endonucleolytic activity associated with the membrane appears to be responsible for the lower levels of binding by protoplasts in the presence of this ion.     

Bacillus subtilis antibiotics: structures, syntheses and specific functions

The endospore-forming rhizobacterium Bacillus subtilis- the model system for Gram-positive organisms, is able to produce more than two dozen antibiotics with an amazing variety of structures. The produced anti-microbial active compounds include predominantly peptides that are either ribosomally synthesized and post-translationally modified (lantibiotics and lantibiotic-like peptides) or non-ribosomally generated, as well as a couple of non-peptidic compounds such as polyketides, an aminosugar, and a phospholipid. Here I summarize the structures of all known B. subtilis antibiotics, their biochemistry and genetic analysis of their biosyntheses. An updated summary of well-studied antibiotic regulation pathways is given. Furthermore, current findings are resumed that show roles for distinct B. subtilis antibiotics beyond the "pure" anti-microbial action: Non-ribosomally produced lipopeptides are involved in biofilm and swarming development, lantibiotics function as pheromones in quorum-sensing, and a "killing factor" effectuates programmed cell death in sister cells. A discussion of how these antibiotics may contribute to the survival of B. subtilis in its natural environment is given.     

Dynamic structures of Bacillus subtilis RecN-DNA complexes

Genetic and cytological evidences suggest that Bacillus subtilis RecN acts prior to and after end-processing of DNA double-strand ends via homologous recombination, appears to participate in the assembly of a DNA repair centre and interacts with incoming single-stranded (ss) DNA during natural transformation. We have determined the architecture of RecN–ssDNA complexes by atomic force microscopy (AFM). ATP induces changes in the architecture of the RecN–ssDNA complexes and stimulates inter-complex assembly, thereby increasing the local concentration of DNA ends. The large CII and CIII complexes formed are insensitive to SsbA (counterpart of Escherichia coli SSB or eukaryotic RPA protein) addition, but RecA induces dislodging of RecN from the overhangs of duplex DNA molecules. Reciprocally, in the presence of RecN, RecA does not form large RecA–DNA networks. Based on these results, we hypothesize that in the presence of ATP, RecN tethers the 3′-ssDNA ends, and facilitates the access of RecA to the high local concentration of DNA ends. Then, the resulting RecA nucleoprotein filaments, on different ssDNA segments, might promote the simultaneous genome-wide homology search.     

Interactions between charged polypeptides and nonionic surfactants

The influence of molecular characteristics on the mutual interaction between peptides and nonionic surfactants has been investigated by studying the effects of surfactants on amphiphilic, random copolymers of alpha-L-amino acids containing lysine residues as the hydrophilic parts. The hydrophobic residues were either phenylalanine or tyrosine. The peptide-surfactant interactions were studied by means of circular dichroism spectroscopy and binding isotherms, as well as by 1D and 2D NMR. The binding of surfactant to the peptides was found to be a cooperative process, appearing at surfactant concentrations just below the critical micellar concentration. However, a certain degree of peptide hydrophobicity is necessary to obtain an interaction with nonionic surfactant. When this prerequisite is fulfilled, the peptide mainly interacts with self-assembled, micelle-like surfactant aggregates formed onto the peptide chain. Therefore, the peptide-surfactant complex is best described in terms of a necklace model, with the peptide interacting primarily with the palisade region of the micelles via its hydrophobic side chains. The interaction yields an increased amount of alpha-helix conformation in the peptide. Surfactants that combine small headgroups with a propensity to form small, nearly spherical micelles were shown to give the largest increase in alpha-helix content.