Biomedical subjects

Peter L Graumann

Publications and source records attributed to Peter L Graumann.

At least 19 recordsLinked to original sources

PubMed2006

Real time fluorescent resonance energy transfer visualization of ferric pyoverdine uptake in Pseudomonas aeruginosa. A role for ferrous iron.

To acquire iron, Pseudomonas aeruginosa secretes a major fluorescent siderophore, pyoverdine (PvdI), that chelates iron and shuttles it into the cells via the specific outer membrane transporter, FpvAI. We took advantage of the fluorescence properties of PvdI and its metal chelates as well as the efficient FRET between donor tryptophans in FpvAI and PvdI to follow the fate of the siderophore during iron uptake. Our findings with PvdI-Ga and PvdI-Cr uptake indicate that iron reduction is required for the dissociation of PvdI-Fe, that a ligand exchange for iron occurs, and that this dissociation occurs in the periplasm. We also observed a delay between PvdI-Fe dissociation and the rebinding of PvdI to FpvAI, underlining the kinetic independence of metal release and siderophore recycling. Meanwhile, PvdI is not modified but recycled to the medium, still competent for iron chelation and transport. Finally, in vivo fluorescence microscopy revealed patches of PvdI, suggesting that uptake occurs via macromolecular assemblies on the cell surface.

Biological Transport↗

PubMed2006

Dynamic localization and interaction with other Bacillus subtilis actin-like proteins are important for the function of MreB.

Bacterial actin-like proteins play a key role in cell morphology and in chromosome segregation. Many bacteria, like Bacillus subtilis, contain three genes encoding actin-like proteins, called mreB, mbl and mreBH in B. subtilis. We show that MreB and Mbl colocalize extensively within live cells, and that all three B. subtilis actin paralogues interact with each other underneath the cell membrane. A mutation in the phosphate 2 motif of MreB had a dominant negative effect on cell morphology and on chromosome segregation. Expression of this mutant allele of MreB interfered with the dynamic localization of Mbl. These experiments show that the interaction between MreB and Mbl has physiological significance. An mreB deletion strain can grow under special media conditions, however, depletion of Mbl in this mutant background abolished growth, indicating that actin paralogues can partially complement each other. The membrane protein MreC was found to interact with Mbl, but not with MreB, revealing a clear distinction between the function of the two paralogues. The phosphate 2 mutant MreB protein allowed for filament formation of mutant or wild-type MreB, but abolished the dynamic reorganization of the filaments. The latter mutation led to a strong reduction, but not complete loss, of function of MreB, both in terms of chromosome segregation and of cell morphology. Our work shows that that the dynamic localization of MreB is essential for the proper activity of the actin-like protein and that the interactions between MreB paralogues have important physiological significance.

Actins↗

PubMed2006

Bacillus subtilis SbcC protein plays an important role in DNA inter-strand cross-link repair.

BACKGROUND: Several distinct pathways for the repair of damaged DNA exist in all cells. DNA modifications are repaired by base excision or nucleotide excision repair, while DNA double strand breaks (DSBs) can be repaired through direct joining of broken ends (non homologous end joining, NHEJ) or through recombination with the non broken sister chromosome (homologous recombination, HR). Rad50 protein plays an important role in repair of DNA damage in eukaryotic cells, and forms a complex with the Mre11 nuclease. The prokaryotic ortholog of Rad50, SbcC, also forms a complex with a nuclease, SbcD, in Escherichia coli, and has been implicated in the removal of hairpin structures that can arise during DNA replication. Ku protein is a component of the NHEJ pathway in pro- and eukaryotic cells. RESULTS: A deletion of the sbcC gene rendered Bacillus subtilis cells sensitive to DNA damage caused by Mitomycin C (MMC) or by gamma irradiation. The deletion of the sbcC gene in a recN mutant background increased the sensitivity of the single recN mutant strain. SbcC was also non-epistatic with AddAB (analog of Escherichia coli RecBCD), but epistatic with RecA. A deletion of the ykoV gene encoding the B. subtilis Ku protein in a sbcC mutant strain did not resulted in an increase in sensitivity towards MMC and gamma irradiation, but exacerbated the phenotype of a recN or a recA mutant strain. In exponentially growing cells, SbcC-GFP was present throughout the cells, or as a central focus in rare cases. Upon induction of DNA damage, SbcC formed 1, rarely 2, foci on the nucleoids. Different to RecN protein, which forms repair centers at any location on the nucleoids, SbcC foci mostly co-localized with the DNA polymerase complex. In contrast to this, AddA-GFP or AddB-GFP did not form detectable foci upon addition of MMC. CONCLUSION: Our experiments show that SbcC plays an important role in the repair of DNA inter-strand cross-links (induced by MMC), most likely through HR, and suggest that NHEJ via Ku serves as a backup DNA repair system. The cell biological experiments show that SbcC functions in close proximity to the replication machinery, suggesting that SbcC may act on stalled or collapsed replication forks. Our results show that different patterns of localization exist for DNA repair proteins, and that the B. subtilis SMC proteins RecN and SbcC play distinct roles in the repair of DNA damage.

Bacillus subtilis↗

PubMed2006

Different genetic programmes within identical bacteria under identical conditions: the phenomenon of bistability greatly modifies our view on bacterial populations.

Two different behavioural or developmental patters can operate within a single bacterial population in which all of the cells are exposed to the same environmental conditions. Investigations of single cells using green fluorescent protein reveal that a bacterial population can be composed of two distinct fractions in different physiological states, and that cells can even switch between states. Such behaviour, termed 'bistability', can occur even in exponentially growing populations, in which cells have always been regarded as behaving almost identically and having the same pattern of gene expression. In this issue of Molecular Microbiology, Dubnau and Losick review four examples of such bistable populations found in two different bacterial species, and explain why this behaviour makes sense. These investigations have established the new concept of genetically identical cells that behave differently, which will profoundly change how we view bacterial populations.

Bacillus subtilis↗

PubMed2006

Cold-induced putative DEAD box RNA helicases CshA and CshB are essential for cold adaptation and interact with cold shock protein B in Bacillus subtilis.

The nucleic acid binding cold shock proteins (CSPs) and the cold-induced DEAD box RNA helicases have been proposed separately to act as RNA chaperones, but no experimental evidence has been reported on a direct cooperation. To investigate the possible interaction of the putative RNA helicases CshA and CshB and the CSPs from Bacillus subtilis during cold shock, we performed genetic as well as fluorescence resonance energy transfer (FRET) experiments. Both cshA and cshB genes could be deleted only in the presence of a cshB copy in trans, showing that the presence of one csh gene is essential for viability. The combined gene deletion of cshB and cspD resulted in a cold-sensitive phenotype that was not observed for either helicase or csp single mutants. In addition to the colocalization of the putative helicases CshA and CshB with CspB and the ribosomes in areas surrounding the nucleoid, we detected a strong FRET interaction in vivo between CshB and CspB that depended on active transcription. In contrast, a FRET interaction was not observed for CshB and the ribosomal protein L1. Therefore, we propose a model in which the putative cold-induced helicases and the CSPs work in conjunction to rescue misfolded mRNA molecules and maintain proper initiation of translation at low temperatures in B. subtilis.

Adaptation, Physiological↗

PubMed2006

Recruitment of Bacillus subtilis RecN to DNA double-strand breaks in the absence of DNA end processing.

The recognition and processing of double-strand breaks (DSBs) to a 3' single-stranded DNA (ssDNA) overhang structure in Bacillus subtilis is poorly understood. Mutations in addA and addB or null mutations in recJ (DeltarecJ), recQ (DeltarecQ), or recS (DeltarecS) genes, when present in otherwise-Rec+ cells, render cells moderately sensitive to the killing action of different DNA-damaging agents. Inactivation of a RecQ-like helicase (DeltarecQ or DeltarecS) in addAB cells showed an additive effect; however, when DeltarecJ was combined with addAB, a strong synergistic effect was observed with a survival rate similar to that of DeltarecA cells. RecF was nonepistatic with RecJ or AddAB. After induction of DSBs, RecN-yellow fluorescent protein (YFP) foci were formed in addAB DeltarecJ cells. AddAB and RecJ were required for the formation of a single RecN focus, because in their absence multiple RecN-YFP foci accumulated within the cells. Green fluorescent protein-RecA failed to form filamentous structures (termed threads) in addAB DeltarecJ cells. We propose that RecN is one of the first recombination proteins detected as a discrete focus in live cells in response to DSBs and that either AddAB or RecQ(S)-RecJ are required for the generation of a duplex with a 3'-ssDNA tail needed for filament formation of RecA.

3' Flanking Region↗

PubMed2006

Differential and dynamic localization of topoisomerases in Bacillus subtilis.

Visualization of topoisomerases in live Bacillus subtilis cells showed that Topo I, Topo IV, and DNA gyrase differentially localize on the nucleoids but are absent at cytosolic spaces surrounding the nucleoids, suggesting that these topoisomerases interact with many regions of the chromosome. While both subunits of Topo IV were uniformly distributed throughout the nucleoids, Topo I and gyrase formed discrete accumulations, or foci, on the nucleoids in a large fraction of the cells, which showed highly dynamic movements. Three-dimensional time lapse microscopy showed that gyrase foci accumulate and dissipate within a 1-min time scale, revealing dynamic assembly and disassembly of subcellular topoisomerase centers. Gyrase centers frequently colocalized with the central DNA replication machinery, suggesting a major role for gyrase at the replication fork, while Topo I foci were frequently close to or colocalized with the structural maintenance of chromosomes (SMC) chromosome segregation complex. The findings suggest that different areas of supercoiling exist on the B. subtilis nucleoids, which are highly dynamic, with a high degree of positive supercoiling attracting gyrase to the replication machinery and areas of negative supercoiling at the bipolar SMC condensation centers recruiting Topo I.

Artificial Gene Fusion↗

PubMed2005

Dynamic formation of RecA filaments at DNA double strand break repair centers in live cells.

We show that RecN protein is recruited to a defined DNA double strand break (DSB) in Bacillus subtilis cells at an early time point during repair. Because RecO and RecF are successively recruited to DSBs, it is now clear that dynamic DSB repair centers (RCs) exist in prokaryotes. RecA protein was also recruited to RCs and formed highly dynamic filamentous structures, which we term threads, across the nucleoids. Formation of RecA threads commenced approximately 30 min after the induction of DSBs, after RecN recruitment to RCs, and disassembled after 2 h. Time-lapse microscopy showed that the threads rapidly changed in length, shape, and orientation within minutes and can extend at 1.02 microm/min. The formation of RecA threads was abolished in recJ addAB mutant cells but not in each of the single mutants, suggesting that RecA filaments can be initiated via two pathways. Contrary to proteins forming RCs, DNA polymerase I did not form foci but was present throughout the nucleoids (even after induction of DSBs or after UV irradiation), suggesting that it continuously scans the chromosome for DNA lesions.

Bacillus subtilis↗

PubMed2005

Intracellular protein and DNA dynamics in competent Bacillus subtilis cells.

We have found that two DNA repair/recombination proteins localize differentially to the cell poles in competent Bacillus subtilis cells. RecA protein colocalized with competence protein ComGA, and its polar localization largely depended on ComGA and ComK activity, while RecN oscillated between the poles in a minute time frame, independent of any competence factor. Oscillation of RecN arrested upon addition of external DNA, suggesting that an interaction with incoming single-stranded (ss) DNA favors the localization of RecN at the pole containing the competence machinery. In agreement with this model, purified RecN protein showed ATP-dependent binding to ssDNA. Addition of DNA resulted in the formation of RecA threads emanating from the competence machinery. Our data show that in competent bacteria there exists a specifically positioned and dynamic ssDNA binding apparatus that accepts ssDNA taken up through the polar competence machinery and processes ssDNA for recombination with chromosomal DNA via extended RecA filaments.

Bacillus subtilis↗

PubMed2005

The RuvAB branch migration translocase and RecU Holliday junction resolvase are required for double-stranded DNA break repair in Bacillus subtilis.

In models of Escherichia coli recombination and DNA repair, the RuvABC complex directs the branch migration and resolution of Holliday junction DNA. To probe the validity of the E. coli paradigm, we examined the impact of mutations in DeltaruvAB and DeltarecU (a ruvC functional analog) on DNA repair. Under standard transformation conditions we failed to construct DeltaruvAB DeltarecG, DeltarecU DeltaruvAB, DeltarecU DeltarecG, or DeltarecU DeltarecJ strains. However, DeltaruvAB could be combined with addAB (recBCD), recF, recH, DeltarecS, DeltarecQ, and DeltarecJ mutations. The DeltaruvAB and DeltarecU mutations rendered cells extremely sensitive to DNA-damaging agents, although less sensitive than a DeltarecA strain. When damaged cells were analyzed, we found that RecU was recruited to defined double-stranded DNA breaks (DSBs) and colocalized with RecN. RecU localized to these centers at a later time point during DSB repair, and formation was dependent on RuvAB. In addition, expression of RecU in an E. coli ruvC mutant restored full resistance to UV light only when the ruvAB genes were present. The results demonstrate that, as with E. coli RuvABC, RuvAB targets RecU to recombination intermediates and that all three proteins are required for repair of DSBs arising from lesions in chromosomal DNA.

Bacillus subtilis↗

PubMed2005

Dynamic assembly, localization and proteolysis of the Bacillus subtilis SMC complex.

BACKGROUND: SMC proteins are key components of several protein complexes that perform vital tasks in different chromosome dynamics. Bacterial SMC forms a complex with ScpA and ScpB that is essential for chromosome arrangement and segregation. The complex localizes to discrete centres on the nucleoids that during most of the time of the cell cycle localize in a bipolar manner. The complex binds to DNA and condenses DNA in an as yet unknown manner. RESULTS: We show that in vitro, ScpA and ScpB form different complexes with each other, among which the level of the putative 2 ScpA/4 ScpB complex showed a pronounced decrease in level upon addition of SMC protein. Different mutations of the ATPase-binding pocket of SMC reduced, but did not abolish interaction of mutant SMC with ScpA and ScpB. The loss of SMC ATPase activity led to a loss of function in vivo, and abolished proper localization of the SMC complex. The formation of bipolar SMC centres was also lost after repression of gyrase activity, and was abnormal during inhibition of replication, resulting in single central clusters. Resumption of replication quickly re-established bipolar SMC centres, showing that proper localization depends on ongoing replication. We also found that the SMC protein is subject to induced proteolysis, most strikingly as cells enter stationary phase, which is partly achieved by ClpX and LonA proteases. Atomic force microscopy revealed the existence of high order rosette-like SMC structures in vitro, which might explain the formation of the SMC centres in vivo. CONCLUSION: Our data suggest that a ScpA/ScpB sub-complex is directly recruited into the SMC complex. This process does not require SMC ATPase activity, which, however, appears to facilitate loading of ScpA and ScpB. Thus, the activity of SMC could be regulated through binding and release of ScpA and ScpB, which has been shown to affect SMC ATPase activity. The proper bipolar localization of the SMC complex depends on a variety of physiological aspects: ongoing replication, ATPase activity and chromosome supercoiling. Because the cellular concentration of SMC protein is also regulated at the posttranscriptional level, the activity of SMC is apparently regulated at multiple levels.

Adenosine Triphosphatases↗

PubMed2005

Bacillus subtilis actin-like protein MreB influences the positioning of the replication machinery and requires membrane proteins MreC/D and other actin-like proteins for proper localization.

BACKGROUND: Bacterial actin-like proteins have been shown to perform essential functions in several aspects of cellular physiology. They affect cell growth, cell shape, chromosome segregation and polar localization of proteins, and localize as helical filaments underneath the cell membrane. Bacillus subtilis MreB and Mbl have been shown to perform dynamic motor like movements within cells, extending along helical tracks in a time scale of few seconds. RESULTS: In this work, we show that Bacillus subtilis MreB has a dual role, both in the formation of rod cell shape, and in chromosome segregation, however, its function in cell shape is distinct from that of MreC. Additionally, MreB is important for the localization of the replication machinery to the cell centre, which becomes aberrant soon after depletion of MreB. 3D image reconstructions suggest that frequently, MreB filaments consist of several discontinuous helical filaments with varying length. The localization of MreB was abnormal in cells with decondensed chromosomes, as well as during depletion of Mbl, MreBH and of the MreC/MreD proteins, which we show localize to the cell membrane. Thus, proper positioning of MreB filaments depends on and is affected by a variety of factors in the cell. CONCLUSION: Our data provide genetic and cytological links between MreB and the membrane, as well as with other actin like proteins, and further supports the connection of MreB with the chromosome. The functional dependence on MreB of the localization of the replication machinery suggests that the replisome is not anchored at the cell centre, but is positioned in a dynamic manner.

Actins↗

PubMed2005

Genetic interaction of the SMC complex with topoisomerase IV in Bacillus subtilis.

The role of topoisomerase IV (Topo IV) and of the structural maintenance of chromosomes (SMC) complex in chromosome compaction and in global protein synthesis was investigated. Lowering of the levels of Topo IV led to chromosome decondensation, while overproduction induced chromosome hyper-compaction, showing that Topo IV has an influence on the compaction of the whole chromosome, in a manner similar to that of the SMC protein, though different in mechanism. Increased synthesis of Topo IV in smc-deleted cells partially rescued the growth and condensation defect of the deletion, but not the segregation defect, revealing that the two systems interact at a genetic level. Two-dimensional gel investigations showed that global protein synthesis is highly aberrant in smc-deleted cells, and, to a different extent, also in cells lacking ScpA or ScpB, which form the SMC complex together with SMC protein. Overproduction of Topo IV partially rescued the defect in protein synthesis in smc mutant cells, indicating that Topo IV can restore the loss of negative supercoiling caused by the absence of SMC protein, but does not fully rescue the segregation defect. The data also show that the SMC protein has a dual function, in chromosome supercoiling and in active segregation.

Bacillus subtilis↗

PubMed2004

Dynamic movement of actin-like proteins within bacterial cells.

Actin proteins are present in pro- and eukaryotes, and have been shown to perform motor-like functions in eukaryotic cells in a variety of processes. Bacterial actin homologues are essential for cell viability and have been implicated in the formation of rod cell shape, as well as in segregation of plasmids and whole chromosomes. We have generated functional green fluorescent protein fusions of all three Bacillus subtilis actin-like proteins (MreB, Mbl and MreBH), and show that all three proteins form helical filaments underneath the cell membrane, the pattern of which is distinct for each protein. Time-lapse microscopy showed that the filaments are highly dynamic structures. A number of separate filaments of MreB and Mbl continuously move through the cell along helical tracks underneath the cell membrane. The speed of extension of the growing end of filaments is within the range of known actin polymerization (0.1 microm/s), generating a potential poleward or centreward pushing velocity at 0.24 microm/min for MreB or Mbl, respectively. During nutritional downshift and a block in topoisomerase IV activity, the filaments rapidly disintegrated, showing that movement occurs only in growing cells. Contrary to Mbl and MreBH filaments, MreB filaments were generally absent in cells lacking DNA, providing a further distinction between the three orthologues.

Actins↗

PubMed2004

An intracellular actin motor in bacteria?

Actin performs structural as well as motor-like functions in eukaryotic cells. Orthologues of actin have also been identified in bacteria, where they perform an essential function during cell growth. Bacterial actins are implicated in the maintenance of rod-shaped cell morphology, and appear to form a cytoskeletal structure, localising as helical filaments underneath the cell membrane. Recently, a plasmid-borne actin orthologue has been shown to perform a mitotic-like function during segregation of a plasmid, and chromosomally encoded actin proteins were found to play an important role in chromosome segregation. Based on the findings that actin filaments are dynamic structures in two bacterial species, we propose that actins perform motor functions rather than a purely structural role in bacteria. We suggest that an intracellular motor exists in bacteria that could be derived from an ancestral actin motor that was present in cells early in evolution.

Actins↗

PubMed2004

Cytoskeletal elements in bacteria.

It has become clear recently that bacteria contain all of the cytoskeletal elements that are found in eukaryotic cells, demonstrating that the cytoskeleton has not been a eukaryotic invention, but evolved early in evolution. Several proteins that are involved in cell division, cell structure and DNA partitioning have been found to form highly dynamic ring structures or helical filaments underneath the cell membrane or throughout the length of the cell. These exciting findings indicate that several highly dynamic processes occur within prokaryotic cells, during growth or differentiation, that are vital for a wide range of cellular tasks.

Bacteria↗

PubMed2004

Visualization of DNA double-strand break repair in live bacteria reveals dynamic recruitment of Bacillus subtilis RecF, RecO and RecN proteins to distinct sites on the nucleoids.

We have found that SMC-like RecN protein, RecF and RecO proteins that are involved in DNA recombination play an important role in DNA double-strand break (DSB) repair in Bacillus subtilis. Upon induction of DNA DSBs, RecN, RecO and RecF localized as a discrete focus on the nucleoids in a majority of cells, whereas two or three foci were rarely observed. RecN, RecO and RecF co-localized to the induced foci, with RecN localizing first, while RecO localized later, followed by RecF. Thus, three repair proteins were differentially recruited to distinct sites on the nucleoids, potentially constituting active DSB repair centres (RCs). RecF did not form regular foci in the absence of RecN and failed to form any foci in recO cells, demonstrating a central role for RecN and RecO in initializing the formation of RCs. RecN/O/F foci were detected in recA, recG or recU mutant cells, indicating that the proteins act upstream of proteins involved in synapsis or post-synapsis. In the absence of exogenous DNA damage, RCs were rare, but they accumulated in recA and recU cells, suggesting that DSBs occur frequently in the absence of RecA or RecU. The results suggest a model in which RecN that forms multimers in solution and high-molecular-weight complexes in cells containing DSBs initiates the formation of RCs that mediate DSB repair with the homologous sister chromosome, which presents a novel concept for DSB repair in prokaryotes.

Bacillus subtilis↗

PubMed2003

Actin-like proteins MreB and Mbl from Bacillus subtilis are required for bipolar positioning of replication origins.

Actin-like proteins MreB and Mbl are required for proper cell shape and for viability in B. subtilis and form dynamic helical filaments underneath the cell membrane. We have found that depletion of MreB and Mbl proteins leads to a rapid defect in chromosome segregation before a defect in cell shape becomes detectable. Under these conditions, the SMC chromosome segregation complex that is essential for proper chromosome arrangement and segregation loses its specific subcellular localization, and replication origins fail to localize in a regular bipolar manner as in wild type cells. Time-lapse microscopy showed that during depletion of MreB, origin regions can move towards the same cell pole, showing that bipolar orientation of origin separation is lost. Contrarily, depletion of three other cell shape determinants, MreC, MreD, or MreBH (the third B. subtilis actin homolog) had no effect on chromosome segregation but varying effects on cell morphology. Depletion of MreC and MreD resulted in formation of round cells, while depletion of MreBH led to formation of vibrio-shaped cells. The data show that actin proteins Mbl and MreB are required for proper chromosome segregation and that Mre proteins affect different aspects in cell shape.

Actins↗