Nora Ausmees

The Bacterial Cytoskeleton: An Intermediate Filament-Like Function in Cell Shape

Various cell shapes are encountered in the prokaryotic world, but how they are achieved is poorly understood. Intermediate filaments (IFs) of the eukaryotic cytoskeleton play an important role in cell shape in higher organisms. No such filaments have been found in prokaryotes. Here, we describe a bacterial equivalent to IF proteins, named crescentin, whose cytoskeletal function is required for the vibrioid and helical shapes of Caulobacter crescentus. Without crescentin, the cells adopt a straight-rod morphology. Crescentin has characteristic features of IF proteins including the ability to assemble into filaments in vitro without energy or cofactor requirements. In vivo, crescentin forms a helical structure that colocalizes with the inner cell curvatures beneath the cytoplasmic membrane. We propose that IF-like filaments of crescentin assemble into a helical structure, which by applying its geometry to the cell, generates a vibrioid or helical cell shape depending on the length of the cell.

Structural and putative regulatory genes involved in cellulose synthesis in Rhizobium leguminosarum bv. trifolii.

Six genes involved in cellulose synthesis in Rhizobium leguminosarum bv. trifolii were identified using Tn5 mutagenesis. Four of them displayed homology to the previously cloned and sequenced Agrobacterium tumefaciens cellulose genes celA, celB, celC and celE. These genes are organized similarly in R. leguminosarum bv. trifolii. In addition, there were strong indications that two tandemly located genes, celR1 and celR2, probably organized as one operon, are involved in the regulation of cellulose synthesis. The deduced amino acid sequences of these genes displayed a high degree of similarity to the Caulobacter crescentus DivK and PleD proteins that belong to the family of two-component response regulators. This is to our knowledge the first report of genes involved in the regulation of cellulose synthesis. Results from attachment assays and electron microscopic studies indicated that cellulose synthesis in R. leguminosarum bv. trifolii is induced upon close contact with plant roots during the attachment process.

Bacterial cell curvature through mechanical control of cell growth

The cytoskeleton is a key regulator of cell morphogenesis. Crescentin, a bacterial intermediate filament‐like protein, is required for the curved shape of Caulobacter crescentus and localizes to the inner cell curvature. Here, we show that crescentin forms a single filamentous structure that collapses into a helix when detached from the cell membrane, suggesting that it is normally maintained in a stretched configuration. Crescentin causes an elongation rate gradient around the circumference of the sidewall, creating a longitudinal cell length differential and hence curvature. Such curvature can be produced by physical force alone when cells are grown in circular microchambers. Production of crescentin in Escherichia coli is sufficient to generate cell curvature. Our data argue for a model in which physical strain borne by the crescentin structure anisotropically alters the kinetics of cell wall insertion to produce curved growth. Our study suggests that bacteria may use the cytoskeleton for mechanical control of growth to alter morphology.

Functions of the CckA histidine kinase in Caulobacter cell cycle control

The CtrA master transcriptional regulator is a central control element in Caulobacter cell cycle progression and polar morphogenesis. Because of its critical role, CtrA activity is temporally regulated by multiple mechanisms including phosphorylation and ClpXP‐dependent degradation of CtrA. The CckA histidine kinase is known to contribute to CtrA phosphorylation. We show here that genes differentially expressed in a ctrA temperature‐sensitive (ts) mutant are similarly affected in a cckA ts mutant, that the phosphorylation of CckA coincides temporally with CtrA phosphorylation during the cell cycle, and that CckA is essential for viability because it is required for CtrA phosphorylation. Thus, it is the signal transduction pathway mediated by CckA that culminates in CtrA activation, which is temporally regulated and essential for cell cycle progression. CckA also positively regulates CtrA activity by a mechanism that is independent of CtrA phosphorylation. CtrA is more stable in the presence of CckA than it is absence, suggesting that CckA may also be involved, directly or indirectly, in the regulation of CtrA proteolysis.

Intermediate filament‐like proteins in bacteria and a cytoskeletal function in Streptomyces

Actin and tubulin cytoskeletons are conserved and widespread in bacteria. A strikingly intermediate filament (IF)‐like cytoskeleton, composed of crescentin, is also present in Caulobacter crescentus and determines its specific cell shape. However, the broader significance of this finding remained obscure, because crescentin appeared to be unique to Caulobacter. Here we demonstrate that IF‐like function is probably a more widespread phenomenon in bacteria. First, we show that 21 genomes of 26 phylogenetically diverse species encoded uncharacterized proteins with a central segmented coiled coil rod domain, which we regarded as a key structural feature of IF proteins and crescentin. Experimental studies of three in silico predicted candidates from Mycobacterium and other actinomycetes revealed a common IF‐like property to spontaneously assemble into filaments in vitro. Furthermore, the IF‐like protein FilP formed cytoskeletal structures in the model actinomycete Streptomyces coelicolor and was needed for normal growth and morphogenesis. Atomic force microscopy of living cells revealed that the FilP cytoskeleton contributed to mechanical fitness of the hyphae, thus closely resembling the function of metazoan IF. Together, the bioinformatic and experimental data suggest that an IF‐like protein architecture is a versatile design that is generally present in bacteria and utilized to perform diverse cytoskeletal tasks.

Characterization of a new 2,4-dichlorophenoxyacetic acid degrading plasmid pEST4011: physical map and localization of catabolic genes

SUMMARY: Plasmid pEST4011 enables Pseudomonas putida PaW85 to degrade 2,4-dichlorophenoxyacetic acid (2,4-D) and 3-chlorobenzoate (3-CBA). This new 2,4-D degradative plasmid has considerable homology with the regions of pJP4 containing the 2,4-D degradative genes (tfd). Restriction fragment BamHI-B of plasmid pEST4011, which has homology with this region, was cloned into the broad-host-range vector pKT240 and studied in P. putida PaW85. Restriction mapping, hybridization analysis and enzyme assays established the location of the genes for 2,4-D monooxygenase (tfdA), 2,4-dichlorophenol hydroxylase (tfdB), chlorocatechol 1,2-dioxygenase (tfdC) and the tfdR and tfdS regulatory genes on this fragment. Plasmid pEST4012 is a derivative of pEST4011 derived through the spontaneous deletion of a 42 kbp DNA fragment, which results in the loss of the 2,4-D+ and 3-CBA+ phenotype. We present here the physical maps of pEST4011 and pEST4012. In spite of the similarities in functions, the size (70 kbp), order of catabolic genes and restriction pattern of pEST4011 are clearly different from those of pJP4.

The rhizobial adhesion protein RapA1 is involved in adsorption of rhizobia to plant roots but not in nodulation

The effect of the rhizobium adhesion protein RapA1 on Rhizobium leguminosarum bv. trifolii adsorption to Trifolium pratense (red clover) roots was investigated. We altered RapA1 production by cloning its encoding gene under the plac promoter into the stable vector pHC60. After introducing this plasmid in R. leguminosarum bv. trifolii, three to four times more RapA1 was produced, and two to five times higher adsorption to red clover roots was obtained, as compared with results for the empty vector. Enhanced adsorption was also observed on soybean and alfalfa roots, not related to R. leguminosarum cross inoculation groups. Although the presence of 1 mM Ca2+ during rhizobial growth enhanced adsorption, it was unrelated to RapA1 level. Similar effects were obtained when the same plasmid was introduced in Rhizobium etli for its adsorption to bean roots. Although root colonization by the RapA1-overproducing strain was also higher, nodulation was not enhanced. In addition, in vitro biofilm formation was similar to the wild-type both on polar and on hydrophobic surfaces. These results suggest that RapA1 receptors are present in root but not on inert surfaces, and that the function of this protein is related to rhizosphere colonization.

Dynamic gradients of an intermediate filament-like cytoskeleton are recruited by a polarity landmark during apical growth.

Significance Here, we show that FilP, a bacterial cytoskeletal protein related to metazoan intermediate filament (IF) proteins, can self-assemble into a regular network structure. This finding offers a possible explanation for its previously characterized role in cellular rigidity and elasticity and might offer insights into the mechanical role of human IFs. The assembly of FilP cytoskeleton is coupled to the function of the polarisome, a protein complex orchestrating the polar growth characteristic of Streptomyces. These results suggest that apical assembly of a stress-bearing cytoskeleton is a common strategy in tip-growing walled cells, such as filamentous fungi, pollen tubes, and mycelial bacteria. Intermediate filament (IF)-like cytoskeleton emerges as a versatile tool for cellular organization in all kingdoms of life, underscoring the importance of mechanistically understanding its diverse manifestations. We showed previously that, in Streptomyces (a bacterium with a mycelial lifestyle similar to that of filamentous fungi, including extreme cell and growth polarity), the IF protein FilP confers rigidity to the hyphae by an unknown mechanism. Here, we provide a possible explanation for the IF-like function of FilP by demonstrating its ability to self-assemble into a cis-interconnected regular network in vitro and its localization into structures consistent with a cytoskeletal network in vivo. Furthermore, we reveal that a spatially restricted interaction between FilP and DivIVA, the main component of the Streptomyces polarisome complex, leads to formation of apical gradients of FilP in hyphae undergoing active tip extension. We propose that the coupling between the mechanism driving polar growth and the assembly of an IF cytoskeleton provides each new hypha with an additional stress-bearing structure at its tip, where the nascent cell wall is inevitably more flexible and compliant while it is being assembled and matured. Our data suggest that recruitment of cytoskeleton around a cell polarity landmark is a broadly conserved strategy in tip-growing cells.

SmeA, a small membrane protein with multiple functions in Streptomyces sporulation including targeting of a SpoIIIE/FtsK-like protein to cell division septa.

Sporulation in aerial hyphae of Streptomyces coelicolor involves profound changes in regulation of fundamental morphogenetic and cell cycle processes to convert the filamentous and multinucleoid cells to small unigenomic spores. Here, a novel sporulation locus consisting of smeA (encoding a small putative membrane protein) and sffA (encoding a SpoIIIE/FtsK‐family protein) is characterized. Deletion of smeA‐sffA gave rise to pleiotropic effects on spore maturation, and influenced the segregation of chromosomes and placement of septa during sporulation. Both smeA and sffA were expressed specifically in apical cells of sporogenic aerial hyphae simultaneously with or slightly after Z‐ring assembly. The presence of smeA‐like genes in streptomycete chromosomes, plasmids and transposons, often paired with a gene for a SpoIIIE/FtsK‐ or Tra‐like protein, indicates that SmeA and SffA functions might be related to DNA transfer. During spore development SffA accumulated specifically at sporulation septa where it colocalized with FtsK. However, sffA did not show redundancy with ftsK, and SffA function appeared distinct from the DNA translocase activity displayed by FtsK during closure of sporulation septa. The septal localization of SffA was dependent on SmeA, suggesting that SmeA may act as an assembly factor for SffA and possibly other proteins required during spore maturation.

Mutations in the Lipopolysaccharide Biosynthesis Pathway Interfere with Crescentin-Mediated Cell Curvature in Caulobacter crescentus

Bacterial cell morphogenesis requires coordination among multiple cellular systems, including the bacterial cytoskeleton and the cell wall. In the vibrioid bacterium Caulobacter crescentus, the intermediate filament-like protein crescentin forms a cell envelope-associated cytoskeletal structure that controls cell wall growth to generate cell curvature. We undertook a genetic screen to find other cellular components important for cell curvature. Here we report that deletion of a gene (wbqL) involved in the lipopolysaccharide (LPS) biosynthesis pathway abolishes cell curvature. Loss of WbqL function leads to the accumulation of an aberrant O-polysaccharide species and to the release of the S layer in the culture medium. Epistasis and microscopy experiments show that neither S-layer nor O-polysaccharide production is required for curved cell morphology per se but that production of the altered O-polysaccharide species abolishes cell curvature by apparently interfering with the ability of the crescentin structure to associate with the cell envelope. Our data suggest that perturbations in a cellular pathway that is itself fully dispensable for cell curvature can cause a disruption of cell morphogenesis, highlighting the delicate harmony among unrelated cellular systems. Using the wbqL mutant, we also show that the normal assembly and growth properties of the crescentin structure are independent of its association with the cell envelope. However, this envelope association is important for facilitating the local disruption of the stable crescentin structure at the division site during cytokinesis.