Two antisense RNAs target the transcriptional regulator CsgD to inhibit curli synthesis.

Escherichia coli produces proteinaceous surface structures called curli that are involved in adhesion and biofilm formation. CsgD is the transcriptional activator of curli genes. We show here that csgD expression is, in part, controlled post-transcriptionally by two redundant small RNAs (sRNAs), OmrA and OmrB. Their overexpression results in curli deficiency, in accordance with the inhibition of chromosomally encoded, FLAG-tagged CsgD. Downregulation of csgD occurs by a direct antisense interaction within the csgD 5'-UTR, far upstream of the ribosome-binding site (RBS). OmrA/B downregulate plasmid-borne csgD-gfp fusions in vivo, and inhibit CsgD translation in vitro. The RNA chaperone Hfq is required for normal csgD mRNA and OmrA/B levels in the cell, and enhances sRNA-dependent inhibition of csgD translation in vitro. Translational inhibition involves two phylogenetically conserved secondary structure modules that are supported by chemical and enzymatic probing. The 5'-most element is necessary and sufficient for regulation, the one downstream comprises the RBS and affects translational efficiency. OmrA/B are two antisense RNAs that regulate a transcription factor to alter a morphotype and group behaviour.

Non-sporulating ftsZ mutants in Streptomyces coelicolor reveal amino acid residues critical for FtsZ polymerization dynamics.

During sporulation of Streptomyces coelicolor, the cytokinetic protein FtsZ is assembled into dozens of regularly spaced Z rings, which orchestrate the division of aerial hyphae into spores. We have previously found that a missense allele of ftsZ, ftsZ17(Spo), primarily affects sporulation septation rather than formation of cross-walls in vegetative mycelium. To clarify what aspect of FtsZ function is compromised in such non-sporulating mutants, we here use a genetic strategy to identify new ftsZ(Spo) alleles and describe how some of the mutations affect the biochemical properties of FtsZ. We have established a system for purification of recombinant untagged S. coelicolor FtsZ, and shown that it assembles dynamically into single protofilaments, displays a critical concentration indicative of cooperative assembly and has a rate of GTP hydrolysis that is substantially higher than that of the closely related Mycobacterium tuberculosis FtsZ. Of the nine isolated ftsZ(Spo) mutations, four affect the interface between the two main subdomains of FtsZ that is implicated in the assembly-induced conformational changes thought to mediate the GTP/GDP-driven cooperative assembly of FtsZ. We find that all these four mutations affect the polymerization behaviour of FtsZ in vitro. In addition, at least one ftsZ(Spo) mutation at the longitudinal contact surface between subunits in protofilaments strongly affects formation of polymers in vitro. We conclude that the assembly of Z rings during sporulation of S. coelicolor is highly sensitive to disturbances of FtsZ polymerization and therefore constitutes an excellent system for analysis of the elusive properties of FtsZ that mediate its characteristic polymerization dynamics.

Hfq and Hfq-dependent small RNAs are major contributors to multicellular development in Salmonella enterica serovar Typhimurium.

The RNA chaperone Hfq and its associated small RNAs (sRNAs) regulate a variety of phenotypes in bacteria. In this work, we show that Hfq is a master regulator of biofilm formation in Salmonella enterica serovar Typhimurium. Hfq and two Hfq-dependent sRNAs (ArcZ and SdsR) are required for rdar morphotype expression in S. typhimurium. Hfq controls rdar biofilm formation through the major biofilm regulator CsgD. While csgD mRNA steady-state levels are altered in a sdsR mutant, ArcZ seems to work mainly at the post-transcriptional level. Overexpression of ArcZ complemented rdar morphotype formation of an hfq mutant under plate-grown conditions. Although ArcZ activates rpoS expression, its effect on csgD expression is mainly independent of RpoS. ArcZ does not only regulate rdar morphotype expression, but also the transition between sessility and motility and the timing of type 1 fimbriae vs. curli fimbriae surface-attachment at ambient temperature. Consequently, ArcZ is a major regulator of rdar biofilm development.

Bistable expression of CsgD in biofilm development of Salmonella enterica serovar typhimurium.

Bacterial persistence in the environment and in the infected host is often aided by the formation of exopolymer-enclosed communities known as biofilms. Heterogeneous gene expression takes place in microcompartments formed within the complex biofilm structure. This study describes cell differentiation within an isogenic bacterial cell population based on the example of biofilm formation by Salmonella enterica serovar Typhimurium. We analyzed the expression of the major biofilm regulator CsgD at the single-cell level with a chromosomal CsgD-green fluorescent protein (GFP) translational fusion. In individual cells, CsgD-GFP expression is mostly found in the cytoplasm. Quantitative expression analysis and results from three different models of S. Typhimurium biofilms demonstrated that CsgD is expressed in a bistable manner during biofilm development. CsgD expression is, however, monomodal when CsgD is expressed in larger amounts due to a promoter mutation or elevated levels of the secondary signaling molecule c-di-GMP. High levels of CsgD-GFP are associated with cellular aggregation in all three biofilm models. Furthermore, the subpopulation of cells expressing large amounts of CsgD is engaged in cellulose production during red, dry, and rough (rdar) morphotype development and in microcolony formation under conditions of continuous flow. Consequently, bistability at the level of CsgD expression leads to a corresponding pattern of task distribution in S. Typhimurium biofilms.

Influence of CrgA on assembly of the cell division protein FtsZ during development of Streptomyces coelicolor.

The product of the crgA gene of Streptomyces coelicolor represents a novel family of small proteins. A single orthologous gene is located close to the origin of replication of all fully sequenced actinomycete genomes and borders a conserved gene cluster implicated in cell growth and division. In S. coelicolor, CrgA is important for coordinating growth and cell division in sporogenic hyphae. In this study, we demonstrate that CrgA is an integral membrane protein whose peak expression is coordinated with the onset of development of aerial hyphae. The protein localizes to discrete foci away from growing hyphal tips. Upon overexpression, CrgA localizes to apical syncytial cells of aerial hyphae and inhibits the formation of productive cytokinetic rings of the bacterial tubulin homolog FtsZ, leading to proteolytic turnover of this major cell division determinant. In the absence of known prokaryotic cell division inhibitors in actinomycetes, CrgA may have an important conserved function influencing Z-ring formation in these bacteria.

MreB of Streptomyces coelicolor is not essential for vegetative growth but is required for the integrity of aerial hyphae and spores.

MreB forms a cytoskeleton in many rod-shaped bacteria which is involved in cell shape determination and chromosome segregation. PCR-based and Southern analysis of various actinomycetes, supported by analysis of genome sequences, revealed mreB homologues only in genera that form an aerial mycelium and sporulate. We analysed MreB in one such organism, Streptomyces coelicolor. Ectopic overexpression of mreB impaired growth, and caused swellings and lysis of hyphae. A null mutant with apparently normal vegetative growth was generated. However, aerial hyphae of this mutant were swelling and lysing; spores doubled their volume and lost their characteristic resistance to stress conditions. Loss of cell wall consistency was observed in MreB-depleted spores by transmission electron microscopy. An MreB-EGFP fusion was constructed to localize MreB in the mycelium. No clearly localized signal was seen in vegetative mycelium. However, strong fluorescence was observed at the septa of sporulating aerial hyphae, then as bipolar foci in young spores, and finally in a ring- or shell-like pattern inside the spores. Immunogold electron microscopy using MreB-specific antibodies revealed that MreB is located immediately underneath the internal spore wall. Thus, MreB is not essential for vegetative growth of S. coelicolor, but exerts its function in the formation of environmentally stable spores, and appears to primarily influence the assembly of the spore cell wall.

Dynamics of FtsZ assembly during sporulation in Streptomyces coelicolor A3(2).

FtsZ, the bacterial tubulin homologue, is the main player in at least two distinct processes of cell division during the development of Streptomyces coelicolor A3(2). It forms cytokinetic rings and is required for the formation of both the widely spaced hyphal cross walls in the substrate mycelium and the specialized septation that converts sporogenic aerial hyphae into spores. The latter developmentally controlled septation involves the coordinated assembly of large numbers of FtsZ rings in each sporulating hyphal cell. We used an FtsZ-enhanced green fluorescent protein (EGFP) translational fusion to visualize the progression of FtsZ ring assembly in vivo during sporulation of aerial hyphae. This revealed that the regular placement of multiple FtsZ rings and initiation of cytokinesis was preceded by a protracted phase during which spiral-shaped FtsZ intermediates were detected along the length of the aerial hyphal cell. Time course experiments indicated that they were remodeled and gradually replaced by regularly spaced FtsZ rings. Such spiral-shaped filaments could also be detected with immunofluorescence microscopy using an antiserum against FtsZ. Based on our observations, we propose a model for the progression of Z-ring assembly during sporulation of S. coelicolor. Furthermore, mutants lacking the developmental regulatory genes whiA, whiB, whiG, whiH, and whiI were investigated. They failed in up-regulation of the expression of FtsZ-EGFP in aerial hyphae, which is consistent with the known effects of these genes on ftsZ transcription.

Paenibacillus polymyxa invades plant roots and forms biofilms.

Paenibacillus polymyxa is a plant growth-promoting rhizobacterium with a broad host range, but so far the use of this organism as a biocontrol agent has not been very efficient. In previous work we showed that this bacterium protects Arabidopsis thaliana against pathogens and abiotic stress (S. Timmusk and E. G. H. Wagner, Mol. Plant-Microbe Interact. 12:951-959, 1999; S. Timmusk, P. van West, N. A. R. Gow, and E. G. H. Wagner, p. 1-28, in Mechanism of action of the plant growth promoting bacterium Paenibacillus polymyxa, 2003). Here, we studied colonization of plant roots by a natural isolate of P. polymyxa which had been tagged with a plasmid-borne gfp gene. Fluorescence microscopy and electron scanning microscopy indicated that the bacteria colonized predominantly the root tip, where they formed biofilms. Accumulation of bacteria was observed in the intercellular spaces outside the vascular cylinder. Systemic spreading did not occur, as indicated by the absence of bacteria in aerial tissues. Studies were performed in both a gnotobiotic system and a soil system. The fact that similar observations were made in both systems suggests that colonization by this bacterium can be studied in a more defined system. Problems associated with green fluorescent protein tagging of natural isolates and deleterious effects of the plant growth-promoting bacteria are discussed.

A missense mutation in ftsZ differentially affects vegetative and developmentally controlled cell division in Streptomyces coelicolor A3(2).

Streptomyces coelicolor A3(2) undergoes at least two kinds of cell division: vegetative septation leading to cross-walls in the substrate mycelium; and developmentally regulated sporulation septation in aerial hyphae. By isolation and characterization of a non-sporulating ftsZ mutant, we demonstrate a difference between the two types of septation. The ftsZ17(Spo) allele gave rise to a classical white phenotype. The mutant grew as well as the parent on plates, and formed apparently normal hyphal cross-walls, although with a small reduction in frequency. In contrast, sporulation septation was almost completely abolished, resulting in a phenotype reminiscent of whiH and ftsZdelta2p mutants. The ftsZ17(Spo) allele was partially dominant and had no detectable effect on the cellular FtsZ content. As judged from both immunofluorescence microscopy of FtsZ and translational fusion of ftsZ to egfp, the mutation prevented correct temporal and spatial assembly of Z rings in sporulating hyphae. Homology modelling of S. coelicolor FtsZ indicated that the mutation, an A249T change in the C-terminal domain, would be expected to alter the protein on the lateral face of FtsZ protofilaments. The results suggest that cytokinesis may be developmentally controlled at the level of Z-ring assembly during sporulation of S. coelicolor A3(2).