Biomedical subjects

Klas Flärdh

Publications and source records attributed to Klas Flärdh.

9 recordsLinked to original sources

PubMed2006

Signal sequence directs localized secretion of bacterial surface proteins.

All living cells require specific mechanisms that target proteins to the cell surface. In eukaryotes, the first part of this process involves recognition in the endoplasmic reticulum of amino-terminal signal sequences and translocation through Sec translocons, whereas subsequent targeting to different surface locations is promoted by internal sorting signals. In bacteria, N-terminal signal sequences promote translocation across the cytoplasmic membrane, which surrounds the entire cell, but some proteins are nevertheless secreted in one part of the cell by poorly understood mechanisms. Here we analyse localized secretion in the Gram-positive pathogen Streptococcus pyogenes, and show that the signal sequences of two surface proteins, M protein and protein F (PrtF), direct secretion to different subcellular regions. The signal sequence of M protein promotes secretion at the division septum, whereas that of PrtF preferentially promotes secretion at the old pole. Our work therefore shows that a signal sequence may contain information that directs the secretion of a protein to one subcellular region, in addition to its classical role in promoting secretion. This finding identifies a new level of complexity in protein translocation and emphasizes the potential of bacterial systems for the analysis of fundamental cell-biological problems.

Adhesins, Bacterial↗

PubMed2006

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.

Bacterial Proteins↗

PubMed2006

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.

Amino Acid Sequence↗

PubMed2005

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.

Bacterial Proteins↗

PubMed2003

Growth polarity and cell division in Streptomyces.

Streptomycetes are mycelial bacteria that resemble filamentous fungi in their apical growth, branching, and morphogenetic development. One inroad into the largely unknown mechanisms underlying this prokaryotic growth polarity is provided by Streptomyces DivIVA, a protein localized at hyphal tips and involved in tip extension. Another aspect is a proposed migration of nucleoids. During sporulation, the modes of growth and cell division are reorganised. This involves dynamic assembly of FtsZ into a multitude of cytokinetic rings. Controlled by developmental regulators and intriguingly coordinated with chromosome segregation, this leads to spores with a single chromosome each. Genome sequences have shed new light on these aspects and reinforced the role of Streptomyces in bacterial cell biology.

Chromosomes, Bacterial↗

PubMed2003

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).

Alleles↗

PubMed2003

Essential role of DivIVA in polar growth and morphogenesis in Streptomyces coelicolor A3(2).

Streptomycetes grow by cell wall extension at hyphal tips. The molecular basis for such polar growth in prokaryotes is largely unknown. It is reported here that DivIVASC, the Streptomyces coelicolor homologue of the Bacillus subtilis protein DivIVA, is essential and directly involved in hyphal tip growth and morphogenesis. A DivIVASC-EGFP hybrid was distinctively localized to hyphal tips and lateral branches. Reduction of divIVASC expression to about 10% of the normal level produced a phenotype strikingly similar to that of many tip growth mutants in fungi, including irregular curly hyphae and apical branching. Overexpression of the gene dramatically perturbed determination of cell shape at the growing tips. Furthermore, staining of nascent peptidoglycan with a fluorescent vancomycin conjugate revealed that induction of overexpression in normal hyphae disturbed tip growth, and gave rise to several new sites of cell wall assembly, effectively causing hyperbranching. The results show that DivIVASC is a novel bacterial morphogene, and it is localized at or very close to the apical sites of peptidoglycan assembly in Streptomyces hyphae.

Amino Acid Sequence↗

PubMed2002

Antisense RNAs everywhere?

In recent years, systematic searches of both prokaryote and eukaryote genomes have identified a staggering number of small RNAs, the biological functions of which remain unknown. Small RNA-based regulators are well known from bacterial plasmids. They act on target RNAs by sequence complementarity; that is, they are antisense RNAs. Recent findings suggest that many of the novel orphan RNAs encoded by bacterial and eukaryotic chromosomes might also belong to a ubiquitous, heterogeneous class of antisense regulators of gene expression.

Base Sequence↗

PubMed1999

Association of early sporulation genes with suggested developmental decision points in Streptomyces coelicolor A3(2).

Cytological analysis of a series of Streptomyces coelicolor A3(2) mutants with disruptions of early sporulation (whi, for white aerial mycelium) genes in an isogenic background has provided new information about the role of whiH, and confirmed and extended previous knowledge about whiG, whiA and whiB. The characteristic straight aerial hyphae of whiG mutants contained normally spaced vegetative-like septa, while mutants in whiA or whiB had abnormally long and coiled aerial hyphae almost devoid of septation. whiG, whiA and whiB were all absolutely required for sporulation septation, and for all visible signs of nucleoid condensation and partitioning and other changes associated with later stages of sporulation. On the other hand, whiH appeared to enhance low basal levels of these processes. Thus, whiH mutant aerial hyphae were divided into loosely coiled fragments of variable sizes by what appeared to be a few sporulation septa. These fragments showed some spore-like characteristics and contained condensed and aberrantly partitioned nucleoids. whiG, whiA and whiB were epistatic to whiH on the criterion that they prevented such fragments from forming in double mutants. These spore-like features and the synthesis of clearly detectable levels of the whiE-directed grey spore pigment were not due to any residual activity of previously studied whiH alleles since they were retained by a constructed whiH null mutant. A model is presented that explains the mutant phenotypes by proposing two early developmental decision points involved in commitment to sporulation septation, one requiring whiG and the other requiring whiA and whiB.

Alleles↗