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 0 -UTR, far upstream of the ribosome-binding site (RBS). OmrA/B downregulate plasmidborne 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 0 -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.

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. 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.TimmuskPaenibacillus polymyxa (previously Bacillus polymyxa) is one of many plant growth-promoting rhizobacteria (PGPR) and is known to have a broad host plant range. It has been isolated from the rhizospheres of wheat and barley (30), white clover, perennial ryegrass, crested wheatgrass (19), lodgepole pine (18), Douglas fir (43), green bean (37), and garlic (24). In addition to several P. polymyxa antagonistic effects reported previously, we have shown that P. polymyxa antagonizes oomycetic pathogens (49). Due to its broad host range, its ability to form endospores, and its ability to produce different kinds of antibiotics, P. polymyxa is a potential commercially useful biocontrol agent. So far, most studies on the biocontrol activity of P. polymyxa have concentrated on the production of different antibiotic substances. Although biocontrol in general has been used for decades, its application has not been very consistent, perhaps due in part to an incomplete understanding of the biological control system (14, 15). Plant roots are not passive targets for soil organisms. Therefore, in addition to understanding the agent itself and its interaction with the pathogen, knowledge of the interaction of the biological control agent with the plant root is required. Although the plant growthpromoting activity of P. polymyxa has been the subject of numerous studies (references 2, 3, and 53 and references therein), the pattern of colonization of this bacterium on host plants has not been studied in detail previously.We previously reported that a natural isolate of P. polymyxa induces drought tolerance and antagonizes pathogens in Arabidopsis thaliana. These effects were observed with a gnotobiotic system and with soil (48, 49). These studies indicated that, aside from the beneficial effects observed, inoculation of A. thaliana by P. polymyxa (in the absence of biotic or abiotic stress) resulted in a ...

SummaryMreB 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.

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.

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 FtsZenhanced 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.A prokaryotic homologue of tubulin, FtsZ, is the main cell division protein in bacteria, many archaea, and eukaryotic organelles. The first recognizable step of bacterial cell division is the assembly of FtsZ into a ring-like structure, the Z ring, at the future division site (2,11,35,38,44). The Z ring is associated with the cytoplasmic membrane and drives cytokinesis. This process involves several other division proteins, which require FtsZ for their recruitment to the division site and direct the synthesis of the septal peptidoglycan. These proteins have been identified in Escherichia coli and Bacillus subtilis, but the subsets of known division proteins in different bacterial species vary considerably (11, 38). The Z ring determines the division plane in bacteria (11), and the temporal and spatial regulation of Z-ring formation is remarkably precise. The Z ring is highly dynamic, and the assembly and behavior of the ring appears to be strongly influenced by the balance between polymerization and depolymerization of FtsZ (43,48). In E. coli and B. subtilis, the intracellular FtsZ concentration is higher than the critical concentration for polymerization. Thus, factors that inhibit ectopic polymerization are needed. They include MinC, which prevents Z-ring assembly at the cell poles (23), and EzrA, which has a negative impact on polymerization along most of the cell (19, 31). In addition, the nucleoid inhibits Z-ring assembly (50, 51, 58), and SulA is an inhibitor of FtsZ asse...