A quorum-sensing signaling system essential for genetic competence in Streptococcus mutans is involved in biofilm formation

In a previous study, a quorum-sensing signaling system essential for genetic competence in Streptococcus mutans was identified, characterized, and found to function optimally in biofilms (Li et al., J. Bacteriol. 183:897-908, 2001). Here, we demonstrate that this system also plays a role in the ability of S. mutans to initiate biofilm formation. To test this hypothesis, S. mutans wild-type strain NG8 and its knockout mutants defective in comC, comD, comE, and comX, as well as a comCDE deletion mutant, were assayed for their ability to initiate biofilm formation. The spatial distribution and architecture of the biofilms were examined by scanning electron microscopy and confocal scanning laser microscopy. The results showed that inactivation of any of the individual genes under study resulted in the formation of an abnormal biofilm. The comC mutant, unable to produce or secrete a competence-stimulating peptide (CSP), formed biofilms with altered architecture, whereas the comD and comE mutants, which were defective in sensing and responding to the CSP, formed biofilms with reduced biomass. Exogenous addition of the CSP and complementation with a plasmid containing the wild-type comC gene into the cultures restored the wild-type biofilm architecture of comC mutants but showed no effect on the comD, comE, or comX mutant biofilms. The fact that biofilms formed by comC mutants differed from the comD, comE, and comX mutant biofilms suggested that multiple signal transduction pathways were affected by CSP. Addition of synthetic CSP into the culture medium or introduction of the wild-type comC gene on a shuttle vector into the comCDE deletion mutant partially restored the wild-type biofilm architecture and further supported this idea. We conclude that the quorum-sensing signaling system essential for genetic competence in S. mutans is important for the formation of biofilms by this gram-positive organism.     

Transcriptional regulation of post-aggregation genes in Dictyostelium by a feed-forward loop involving GBF and LagC

Expression profiles of developmental genes in Dictyostelium were determined on microarrays during development of wild type cells and mutant cells lacking either the DNA binding protein GBF or the signaling protein LagC. We found that the mutant strains developed in suspension with added cAMP expressed the pulse-induced and early adenylyl cyclase (ACA)-dependent genes, but not the later ACA-dependent, post-aggregation genes. Since expression of lagC itself is dependent on GBF, expression of the post-aggregation genes might be controlled only by signaling from LagC. However, expression of lagC in a GBF-independent manner in a gbfA- null strain did not result in expression of the post-aggregation genes. Since GBF is necessary for accumulation of LagC and both the DNA binding protein and the LagC signal transduction pathway are necessary for expression of post-aggregation genes, GBF and LagC form a feed-forward loop. Such network architecture is a common motif in diverse organisms and can act as a filter for noisy inputs. Breaking the feed-forward loop by expressing lagC in a GBF-independent manner in a gbfA+ strain does not significantly affect the patterns of gene expression for cells developed in suspension with added cAMP, but results in a significant delay at the mound stage and asynchronous development on solid supports. This feed-forward loop can integrate temporal information with morphological signals to ensure that post-aggregation genes are only expressed after cell contacts have been made.     

Novel mutations that alter the regulation of sporulation in Bacillus subtilis. Evidence that phosphorylation of regulatory protein SpoOA controls the initiation of sporulation

Sporulation in Bacillus subtilis is a complex developmental process that occurs in response to nutrient deprivation. To identify components of the mechanism that allows cells to monitor their nutritional status and to understand how this sensory information is transduced into a signal to activate specific sporulation genes, we have isolated mutants that are able to sporulate efficiently under nutritional conditions that strongly inhibit sporulation in wild-type bacteria, a phenotype we refer to as Coi (control of initiation). Four coi mutations were found to be within the coding sequence of spoOA, a gene in which null mutations prevent the initiation of sporulation and a gene whose product shares a domain of homology with phosphorylation-activated proteins that play signal transduction roles in bacteria. All four of the spoOA mutations were within this conserved domain and in close proximity to the presumptive phosphoacceptor site. The wild-type and one of the mutant SpoOA proteins were purified and shown to be competent to accept phosphoryl groups from a phosphohistidine within a bacterial signal transduction kinase (CheA). The mutant SpoOA protein exhibited enhanced phosphoacceptor activity compared with the wild-type. This property of the mutant protein, together with additional genetic information, supports a model for regulation of sporulation initiation by control of the phosphorylation level of SpoOA.     

Induction of beta-lactamase influences the course of development in Myxococcus xanthus.

Myxococcus xanthus is a gram-negative bacterium that develops in response to starvation on a solid surface. The cells assemble into multicellular aggregates in which they differentiate from rod-shaped cells into spherical, environmentally resistant spores. Previously, we have shown that the induction of beta-lactamase is associated with starvation-independent sporulation in liquid culture (K. A. O'Connor and D. R. Zusman, Mol. Microbiol. 24:839-850, 1997). In this paper, we show that the chromosomally encoded beta-lactamase of M. xanthus is autogenously induced during development. The specific activity of the enzyme begins to increase during aggregation, before spores are detectable. The addition of inducers of beta-lactamase in M. xanthus, such as ampicillin, D-cycloserine, and phosphomycin, accelerates the onset of aggregation and sporulation in developing populations of cells. In addition, the exogenous induction of beta-lactamase allows M. xanthus to fruit on media containing concentrations of nutrients that are normally too high to support development. We propose that the induction of beta-lactamase is an integral step in the development of M. xanthus and that this induction is likely to play a role in aggregation and in the restructuring of peptidoglycan which occurs during the differentiation of spores. In support of this hypothesis, we show that exogenous induction of beta-lactamase can rescue aggregation and sporulation of certain mutants. Fruiting body spores from a rescued mutant are indistinguishable from wild-type fruiting body spores when examined by transmission electron microscopy. These results show that the signal transduction pathway leading to the induction of beta-lactamase plays an important role in aggregation and sporulation in M. xanthus.     

The Aspergillus FlbA RGS domain protein antagonizes G protein signaling to block proliferation and allow development.

flbA encodes an Aspergillus nidulans RGS (regulator of G protein signaling) domain protein that is required for control of mycelial proliferation and activation of asexual sporulation. We identified a dominant mutation in a second gene, fadA, that resulted in a very similar phenotype to flbA loss-of-function mutants. Analysis of fadA showed that it encodes the alpha-subunit of a heterotrimeric G protein, and the dominant phenotype resulted from conversion of glycine 42 to arginine (fadA(G42R)). This mutation is predicted to result in a loss of intrinsic GTPase activity leading to constitutive signaling, indicating that activation of this pathway leads to proliferation and blocks sporulation. By contrast, a fadA deletion and a fadA dominant-interfering mutation (fadA(G203R)) resulted in reduced growth without impairing sporulation. In fact, the fadA(G203R) mutant was a hyperactive asexual sporulator and produced elaborate sporulation structures, called conidiophores, under environmental conditions that blocked wild-type sporulation. Both the fadA(G203R) and the fadA deletion mutations suppressed the flbA mutant phenotype as predicted if the primary role of FlbA in sporulation is in blocking activation of FadA signaling. Because overexpression of flbA could not suppress the fadA(G42R) mutant phenotype, we propose that FlbA's role in modulating the FadA proliferation signal is dependent upon the intrinsic GTPase activity of wild-type FadA.     

Identification of major sporulation proteins of Myxococcus xanthus using a proteomic approach

Myxococcus xanthus is a soil-dwelling, gram-negative bacterium that during nutrient deprivation is capable of undergoing morphogenesis from a vegetative rod to a spherical, stress-resistant spore inside a domed-shaped, multicellular fruiting body. To identify proteins required for building stress-resistant M. xanthus spores, we compared the proteome of liquid-grown vegetative cells with the proteome of mature fruiting body spores. Two proteins, protein S and protein S1, were differentially expressed in spores, as has been reported previously. In addition, we identified three previously uncharacterized proteins that are differentially expressed in spores and that exhibit no homology to known proteins. The genes encoding these three novel major spore proteins (mspA, mspB, and mspC) were inactivated by insertion mutagenesis, and the development of the resulting mutant strains was characterized. All three mutants were capable of aggregating, but for two of the strains the resulting fruiting bodies remained flattened mounds of cells. The most pronounced structural defect of spores produced by all three mutants was an altered cortex layer. We found that mspA and mspB mutant spores were more sensitive specifically to heat and sodium dodecyl sulfate than wild-type spores, while mspC mutant spores were more sensitive to all stress treatments examined. Hence, the products of mspA, mspB, and mspC play significant roles in morphogenesis of M. xanthus spores and in the ability of spores to survive environmental stress.     

Gene expression in Bacillus subtilis surface biofilms with and without sporulation and the importance of yveR for biofilm maintenance

Five independent DNA microarray experiments were used to study the gene expression profile of a 5-day Bacillus subtilis air-liquid interface biofilm relative to planktonic cells. Both wild-type B. subtilis and its sporulation mutant (DeltaspoIIGB::erm) were investigated to discern the important biofilm genes (in the presence and absence of sporulation). The microarray results indicated that suspension cells were encountering anaerobic conditions, and the air-liquid interface biofilm was metabolically active. For the statistically significant differential expression (P < 0.05), there were 342 genes induced and 248 genes repressed in the wild-type biofilm, whereas 371 genes were induced and 128 genes were repressed in the sporulation mutant biofilm. The microarray results were confirmed with RNA dot blotting. A small portion of cells (1.5%) in the wild-type biofilm formed spores and sporulation genes were highly expressed. In the biofilm formed by the sporulation mutant, competence genes (comGA, srfAA, srfAB, srfAD, and comS) were induced which indicate a role for quorum sensing (bacterial gene expression controlled by sensing their population) in biofilms. There were 53 genes consistently induced in the biofilms of both the wild-type strain and its spoIIGB mutant-those genes have functions for transport, metabolism, antibiotic production-and 26 genes with unknown functions. Besides the large number of genes with known functions induced in the biofilm (121 genes in the wild-type biofilm and 185 genes in the sporulation mutant biofilm), some genes with unknown functions were also induced (221 genes in the wild-type biofilm and 186 genes in the sporulation mutant biofilm), such as the yve operon which appears to be involved in polysaccharide synthesis and the ybc operon which inhibits the growth of competitors for nutrients. A knockout mutant of yveR was constructed, and the mutant showed major defects in biofilm maintenance. Both the wild-type strain and its sporulation mutant formed normal biofilms, suggesting complete sporulation is not necessary for biofilm formation. The expression profiles of these two strains share more repressed genes than induced genes, suggesting that the biofilm cells repress similar pathways in response to starvation and high cell density.     

Cardiolipin domains in Bacillus subtilis marburg membranes

Recently, use of the cardiolipin (CL)-specific fluorescent dye 10-N-nonyl-acridine orange (NAO) revealed CL-rich domains in the Escherichia coli membrane (E. Mileykovskaya and W. Dowhan, J. Bacteriol. 182: 1172-1175, 2000). Staining of Bacillus subtilis cells with NAO showed that there were green fluorescence domains in the septal regions and at the poles. These fluorescence domains were scarcely detectable in exponentially growing cells of the clsA-disrupted mutant lacking detectable CL. In sporulating cells with a wild-type lipid composition, fluorescence domains were observed in the polar septa and on the engulfment and forespore membranes. Both in the clsA-disrupted mutant and in a mutant with disruptions in all three of the paralogous genes (clsA, ywjE, and ywiE) for CL synthase, these domains did not vanish but appeared later, after sporulation initiation. A red shift in the fluorescence due to stacking of two dye molecules and the lipid composition suggested that a small amount of CL was present in sporulating cells of the mutants. Mass spectrometry analyses revealed the presence of CL in these mutant cells. At a later stage during sporulation of the mutants the frequency of heat-resistant cells that could form colonies after heat treatment was lower. The frequency of sporulation of these cells at 24 h after sporulation initiation was 30 to 50% of the frequency of the wild type. These results indicate that CL-rich domains are present in the polar septal membrane and in the engulfment and forespore membranes during the sporulation phase even in a B. subtilis mutant with disruptions in all three paralogous genes, as well as in the membranes of the medial septa and at the poles during the exponential growth phase of wild-type cells. The results further suggest that the CL-rich domains in the polar septal membrane and engulfment and forespore membranes are involved in sporulation.     

The membrane glycoprotein gp150 is encoded by the lagC gene and mediates cell-cell adhesion by heterophilic binding during Dictyostelium development

gp150 is a membrane glycoprotein which has been implicated in cell-cell adhesion in the postaggregation stages of Dictyostelium development. An analysis of its tryptic peptides by mass spectrometry has identified gp150 as the product of the lagC gene, which was previously shown to play a role in morphogenesis and cell-type specification. Antibodies raised against the GST-LagC fusion protein specifically recognized gp150 in wild-type cells and showed that it is missing in lagC-null cells. Immunolocalization studies have confirmed its enrichment in cell-cell contact regions. In mutant cells that lack the aggregation stage-specific cell adhesion molecule gp80, gp150 is expressed precociously. Moreover, these cells acquire EDTA-resistant cell-cell binding during aggregation, suggesting a role for gp150 in this process. Cells in which the genes encoding gp80 and gp150 are both inactivated do not acquire EDTA-resistant cell adhesion during aggregation. Strains transformed with an actin 15::lagC construct express gp150 precociously, but do not show EDTA-resistant adhesion during early development. However, vegetative cells expressing gp150 can be recruited into aggregates of 16-h lagC-null cells. These results, together with those obtained with the cell-to-substratum binding assay, indicate that gp150 mediates cell-cell adhesion via heterophilic interactions with another component that accumulates during the aggregation stage.