14 research outputs found
Precise Timing of Transcription by c-di-GMP Coordinates Cell Cycle and Morphogenesis in Caulobacter
Bacteria adapt their growth rate to their metabolic status and environmental conditions by modulating the length of their G1 period. Here we demonstrate that a gradual increase in the concentration of the second messenger c-di-GMP determines precise gene expression during G1/S transition in Caulobacter crescentus . We show that c-di-GMP stimulates the kinase ShkA by binding to its central pseudo-receiver domain, activates the TacA transcription factor, and initiates a G1/S-specific transcription program leading to cell morphogenesis and S-phase entry. Activation of the ShkA-dependent genetic program causes c-di-GMP to reach peak levels, which triggers S-phase entry and promotes proteolysis of ShkA and TacA. Thus, a gradual increase of c-di-GMP results in precise control of ShkA-TacA activity, enabling G1/S-specific gene expression that coordinates cell cycle and morphogenesis
Mechanistic Basis of Branch-Site Selection in Filamentous Bacteria
Many filamentous organisms, such as fungi, grow by tip-extension and by forming new branches behind the tips. A similar growth mode occurs in filamentous bacteria, including the genus Streptomyces, although here our mechanistic understanding has been very limited. The Streptomyces protein DivIVA is a critical determinant of hyphal growth and localizes in foci at hyphal tips and sites of future branch development. However, how such foci form was previously unknown. Here, we show experimentally that DivIVA focus-formation involves a novel mechanism in which new DivIVA foci break off from existing tip-foci, bypassing the need for initial nucleation or de novo branch-site selection. We develop a mathematical model for DivIVA-dependent growth and branching, involving DivIVA focus-formation by tip-focus splitting, focus growth, and the initiation of new branches at a critical focus size. We quantitatively fit our model to the experimentally-measured tip-to-branch and branch-to-branch length distributions. The model predicts a particular bimodal tip-to-branch distribution results from tip-focus splitting, a prediction we confirm experimentally. Our work provides mechanistic understanding of a novel mode of hyphal growth regulation that may be widely employed
Males and Females Contribute Unequally to Offspring Genetic Diversity in the Polygynandrous Mating System of Wild Boar
The maintenance of genetic diversity across generations depends on both the number of reproducing males and females. Variance in reproductive success, multiple paternity and litter size can all affect the relative contributions of male and female parents to genetic variation of progeny. The mating system of the wild boar (Sus scrofa) has been described as polygynous, although evidence of multiple paternity in litters has been found. Using 14 microsatellite markers, we evaluated the contribution of males and females to genetic variation in the next generation in independent wild boar populations from the Iberian Peninsula and Hungary. Genetic contributions of males and females were obtained by distinguishing the paternal and maternal genetic component inherited by the progeny. We found that the paternally inherited genetic component of progeny was more diverse than the maternally inherited component. Simulations showed that this finding might be due to a sampling bias. However, after controlling for the bias by fitting both the genetic diversity in the adult population and the number of reproductive individuals in the models, paternally inherited genotypes remained more diverse than those inherited maternally. Our results suggest new insights into how promiscuous mating systems can help maintain genetic variation
Regulation of apical growth and hyphal branching in Streptomyces
The filamentous bacteria Streptomyces grow by tip extension and through the initiation of new branches, and this apical growth is directed by a polarisome-like complex involving the essential polarity protein DivIVA. New branch sites must be marked de novo and, until recently, there was no understanding of how these new sites are selected. Equally, hyphal branching patterns are affected by environmental conditions, but there was no insight into how polar growth and hyphal branching might be regulated in response to external or internal cues. This review focuses on recent discoveries that reveal the principal mechanism of branch site selection in Streptomyces, and the first mechanism to be identified that regulates polarisome behaviour to modulate polar growth and hyphal branching
Determination of Phosphorylation Sites in the DivIVA Cytoskeletal Protein of Streptomyces coelicolor by Targeted LC-MS/MS
The filamentous bacterium Streptomyces coelicolor modulates polar growth and branching by phosphorylating the cytoskeletal protein DivIVA. Previous MALDI-TOF analysis of DivIVA showed that a large 7.2 kDa tryptic peptide was multiply phosphorylated. To aid localization of the phosphorylation sites, we introduced additional tryptic cleavage sites into DivIVA, and the resulting phosphopeptides were analyzed by LC-MS/MS. Phosphopeptide isomers could be separated chromatographically, but because of overlapping elution and spectrum quality, site assignment by standard software tools was ambiguous. Because fragment ions carrying the phosphate group are essential for confident localization, large numbers of spectra were collected using targeted LC-MS/MS, and a special script was developed for plotting the elution of site-determining fragments from those spectra under the XIC of the parent ions. Where multiple phosphopeptide isomers were present, the elution of the site-determining y-ions perfectly coincided with the elution of the corresponding phosphopeptide isomer. This method represents a useful tool for user inspection of spectra derived from phosphopeptide isomers and significantly increases confidence when defining phosphorylation sites. In this way, we show that DivIVA is phosphorylated in vivo on five sites in the C-terminal part of the protein (T304, S309, S338, S344, and S355). The data have been deposited to the ProteomeXchange Consortium with identifier PXD00009S
Evidence of tip-focus splitting, growth of foci and emergence of branches, in fluorescence-imaged <i>Streptomyces coelicolor</i> expressing <i>divIVA-egfp</i>.
<p>The tip always contains a large DivIVA focus and established tips extend at an approximately constant speed. At about 12 minutes, the DivIVA tip-focus undergoes splitting, leaving behind a new focus (arrow). As the tip continues to extend, the new focus remains in place on the membrane and grows in intensity. After about 42 minutes a new branch is formed at the position of the new focus, with the new focus now sitting at the tip of the new branch. Both the new branch and the original branch now continue to extend in length. Time in hoursâśminutes. Scale bar: .</p
Determination of Phosphorylation Sites in the DivIVA Cytoskeletal Protein of <i>Streptomyces coelicolor</i> by Targeted LCâMS/MS
The
filamentous bacterium <i>Streptomyces coelicolor</i> modulates
polar growth and branching by phosphorylating the cytoskeletal
protein DivIVA. Previous MALDI-TOF analysis of DivIVA showed that
a large 7.2 kDa tryptic peptide was multiply phosphorylated. To aid
localization of the phosphorylation sites, we introduced additional
tryptic cleavage sites into DivIVA, and the resulting phosphopeptides
were analyzed by LCâMS/MS. Phosphopeptide isomers could be
separated chromatographically, but because of overlapping elution
and spectrum quality, site assignment by standard software tools was
ambiguous. Because fragment ions carrying the phosphate group are
essential for confident localization, large numbers of spectra were
collected using targeted LCâMS/MS, and a special script was
developed for plotting the elution of site-determining fragments from
those spectra under the XIC of the parent ions. Where multiple phosphopeptide
isomers were present, the elution of the site-determining y-ions perfectly
coincided with the elution of the corresponding phosphopeptide isomer.
This method represents a useful tool for user inspection of spectra
derived from phosphopeptide isomers and significantly increases confidence
when defining phosphorylation sites. In this way, we show that DivIVA
is phosphorylated in vivo on five sites in the C-terminal part of
the protein (T304, S309, S338, S344, and S355). The data have been
deposited to the ProteomeXchange Consortium with identifier PXD000095
The Ser/Thr protein kinase AfsK regulates polar growth and hyphal branching in the filamentous bacteria Streptomyces.
In cells that exhibit apical growth, mechanisms that regulate cell polarity are crucial for determination of cellular shape and for the adaptation of growth to intrinsic and extrinsic cues. Broadly conserved pathways control cell polarity in eukaryotes, but less is known about polarly growing prokaryotes. An evolutionarily ancient form of apical growth is found in the filamentous bacteria Streptomyces, and is directed by a polarisome-like complex involving the essential protein DivIVA. We report here that this bacterial polarization machinery is regulated by a eukaryotic-type Ser/Thr protein kinase, AfsK, which localizes to hyphal tips and phosphorylates DivIVA. During normal growth, AfsK regulates hyphal branching by modulating branch-site selection and some aspect of the underlying polarisome-splitting mechanism that controls branching of Streptomyces hyphae. Further, AfsK is activated by signals generated by the arrest of cell wall synthesis and directly communicates this to the polarisome by hyperphosphorylating DivIVA. Induction of high levels of DivIVA phosphorylation by using a constitutively active mutant AfsK causes disassembly of apical polarisomes, followed by establishment of multiple hyphal branches elsewhere in the cell, revealing a profound impact of this kinase on growth polarity. The function of AfsK is reminiscent of the phoshorylation of polarity proteins and polarisome components by Ser/Thr protein kinases in eukaryotes