A prokaryotic condensin/cohesin-like complex can actively compact chromosomes from a single position on the nucleoid and binds to DNA as a ring-like structure.

We show that Bacillus subtilis SMC (structural maintenance of chromosome protein) localizes to discrete foci in a cell cycle-dependent manner. Early in the cell cycle, SMC moves from the middle of the cell toward opposite cell poles in a rapid and dynamic manner and appears to interact with different regions on the chromosomes during the cell cycle. SMC colocalizes with its interacting partners, ScpA and ScpB, and the specific localization of SMC depends on both Scp proteins, showing that all three components of the SMC complex are required for proper localization. Cytological and biochemical experiments showed that dimeric ScpB stabilized the binding of ScpA to the SMC head domains. Purified SMC showed nonspecific binding to double-stranded DNA, independent of Scp proteins or ATP, and was retained on DNA after binding to closed DNA but not to linear DNA. The SMC head domains and hinge region did not show strong DNA binding activity, suggesting that the coiled-coil regions in SMC mediate an association with DNA and that SMC binds to DNA as a ring-like structure. The overproduction of SMC resulted in global chromosome compaction, while SMC was largely retained in bipolar foci, suggesting that the SMC complex forms condensation centers that actively affect global chromosome compaction from a defined position on the nucleoid.

Localization of cold shock proteins to cytosolic spaces surrounding nucleoids in Bacillus subtilis depends on active transcription.

Using immunofluorescence microscopy and a fusion of a cold shock protein (CSP), CspB, to green fluorescent protein (GFP), we showed that in growing cells Bacillus subtilis CSPs specifically localize to cytosolic regions surrounding the nucleoid. The subcellular localization of CSPs is influenced by the structure of the nucleoid. Decondensed chromosomes in smc mutant cells reduced the sizes of the regions in which CSPs localized, while cold shock-induced chromosome compaction was accompanied by an expansion of the space in which CSPs were present. As a control, histone-like protein HBsu localized to the nucleoids, while beta-galactosidase and GFP were detectable throughout the cell. After inhibition of translation, CspB-GFP was still present around the nucleoids in a manner similar to that in cold-shocked cells. However, in stationary-phase cells and after inhibition of transcription, CspB was distributed throughout the cell, indicating that specific localization of CspB depends on active transcription and is not due to simple exclusion from the nucleoid. Furthermore, we observed that nucleoids are more condensed and frequently abnormal in cspB cspC and cspB cspD double-mutant cells. This suggests that the function of CSPs affects chromosome structure, probably through coupling of transcription to translation, which is thought to decondense nucleoids. In addition, we found that cspB cspD and cspB cspC double mutants are defective in sporulation, with a block at or before stage 0. Interestingly, CspB and CspC are depleted from the forespore compartment but not from the mother cell. In toto, our findings suggest that CSPs localize to zones of newly synthesized RNA, coupling transcription with initiation of translation.

Specific polar localization of ribosomes in Bacillus subtilis depends on active transcription.

The large subunit of ribosomes in Bacillus subtilis was tagged by generation of a fusion of ribosomal protein L1 to blue fluorescent protein (BFP). The fusion was fully active and localized around the nucleoids, predominantly close to the cell poles, in growing cells. However, in stationary phase cells, and in growing cells treated with rifampicin, L1-BFP was distributed throughout the cells, in contrast to cells treated with chloramphenicol, in which ribosomes still localized around nucleoids. These data show that specific localization of ribosomes is not due to nucleoid exclusion, but is a dynamic process due to active synthesis of RNA. Dual labelling of ribosomes and cold shock proteins (CSPs) tagged with green fluorescent protein (GFP) revealed colocalization of both protein classes. CSPs are implicated in coupling of transcription with translation and may bridge the spatial separation of ribosomes and nucleoid-associated RNA polymerase.

Coupling of asymmetric division to polar placement of replication origin regions in Bacillus subtilis.

Entry into sporulation in Bacillus subtilis is characterized by the formation of a polar septum, which asymmetrically divides the developing cell into forespore (the smaller cell) and mother cell compartments, and by migration of replication origin regions to extreme opposite poles of the cell. Here we show that polar septation is closely correlated with movement of replication origins to the extreme poles of the cell. Replication origin regions were visualized by the use of a cassette of tandem copies of lacO that had been inserted in the chromosome near the origin of replication and decorated with green fluorescent protein-LacI. The results showed that extreme polar placement of replication origin regions is not under sporulation control and occurred in stationary phase under conditions under which entry into sporulation was prevented. On the other hand, the formation of a polar septum, which is under sporulation control, was almost invariably associated with the presence of a replication origin region in the forespore. Moreover, cells in which the polar placement of origin regions was perturbed by deletion of the gene (smc) for the structural maintenance of chromosomes (SMC) protein were impaired in polar division. A small proportion ( approximately 1%) of the mutant cells were able to undergo asymmetric division, but the forespore compartment of these exceptional cells was generally observed to contain a replication origin region. Immunofluorescence microscopy experiments indicated that the block in polar division caused by the absence of SMC occurred at or prior to the step of bipolar Z-ring formation by the cell division protein FtsZ. A model is discussed in which polar division is under the dual control of sporulation and an event associated with the placement of a replication origin at the cell pole.

SMC proteins in bacteria: condensation motors for chromosome segregation?

SMC proteins are a ubiquitous protein family, present in almost all organisms so far analysed except for a few bacteria. They function in chromosome condensation, segregation, cohesion, and DNA recombination repair in eukaryotes, and can introduce positive writhe into DNA in vitro. SMC proteins and the structurally homologous MukB protein are unusual ATPases that form antiparallel dimers, with long coiled coil segments separating globular ends capable of binding DNA. Recently, SMC proteins have been shown to be essential for chromosome condensation, segregation and cell cycle progression in bacteria. Identification of a suppressor mutation for MukB in topoisomerase I in Escherichia coli suggests that SMC proteins are involved in negative DNA supercoiling in vivo, and by this means organize and compact chromosomes. A model is discussed in which bacterial SMC proteins act after an initial separation of replicated chromosome origins into the future daughter cell, separating sister chromatids by condensing replicated DNA strands within both cell halves. This would be analogous to a pulling of DNA strands into opposite cell halves by a condensation mechanism exerted at two specialised subregions in the cell.

Bacillus subtilis SMC is required for proper arrangement of the chromosome and for efficient segregation of replication termini but not for bipolar movement of newly duplicated origin regions.

SMC protein is required for chromosome condensation and for the faithful segregation of daughter chromosomes in Bacillus subtilis. The visualization of specific sites on the chromosome showed that newly duplicated origin regions in growing cells of an smc mutant were able to segregate from each other but that the location of origin regions was frequently aberrant. In contrast, the segregation of replication termini was impaired in smc mutant cells. This analysis was extended to germinating spores of an smc mutant. The results showed that during germination, newly duplicated origins, but not termini, were able to separate from each other in the absence of SMC. Also, DAPI (4',6'-diamidino-2-phenylindole) staining revealed that chromosomes in germinating spores were able to undergo partial or complete replication but that the daughter chromosomes were blocked at a late stage in the segregation process. These findings were confirmed by time-lapse microscopy, which showed that after duplication in growing cells the origin regions underwent rapid movement toward opposite poles of the cell in the absence of SMC. This indicates that SMC is not a required component of the mitotic motor that initially drives origins apart after their duplication. It is also concluded that SMC is needed to maintain the proper layout of the chromosome in the cell and that it functions in the cell cycle after origin separation but prior to complete segregation or replication of daughter chromosomes. It is proposed here that chromosome segregation takes place in at least two steps: an SMC-independent step in which origins move apart and a subsequent SMC-dependent step in which newly duplicated chromosomes condense and are thereby drawn apart.

The family of cold shock proteins of Bacillus subtilis. Stability and dynamics in vitro and in vivo.

Bacillus subtilis possesses three homologous small cold shock proteins (CSPs; CspB, CspC, CspD, sequence identity >72%). They share a similar beta-sheet structure, as shown by circular dichroism, and have a very low conformational stability, with CspC being the least stable. Similar to CspB, CspC and CspD unfold and refold extremely fast in a N <==> U two-state reaction with average lifetimes of only 100-150 ms for the native state and 1-6 ms for the unfolded states at 25 degreesC. As a consequence of their low stability and low kinetic protection against unfolding, all three cold shock proteins are rapidly degraded by proteases in vitro. Analysis of the CSP stabilities in vivo by pulse-chase experiments revealed that CspB and CspD are stable during logarithmic growth at 37 degreesC as well as after cold shock. The cellular half-life of CspC is shortened at 37 degreesC, but under cold shock conditions CspC becomes stable. The proteolytic susceptibility of the CSPs in vitro was strongly reduced in the presence of a nucleic acid ligand, suggesting that the observed stabilization of CSPs in vivo is mediated by binding to their substrate mRNA at 37 degreesC and, in particular, under cold shock conditions.

Cold shock proteins CspB and CspC are major stationary-phase-induced proteins in Bacillus subtilis.

Shortly after the transition from exponential growth to stationary phase, the pattern of protein synthesis in Bacillus subtilis changes markedly. Among the most profoundly induced proteins are two homologous small acidic proteins, CspB and CspC, which are also major cold-shock-induced proteins. The third cold shock protein (CSP) in B. subtilis, CspD, is not induced following entry into stationary phase. Deletion of both cspB and cspC genes has been previously shown to lead to lysis of cells during stationary phase. These findings reveal that CSPs in B. subtilis are induced under several stress conditions, and that an increase in the synthesis of CspB and CspC is needed for efficient adaptation to stationary phase. Enhanced synthesis of CspB occurs through a combination of transcriptional and post-transcriptional activation, indicating a mechanism similar to that mediating cold shock induction of CSPs. Induction of CSPs in bacteria may be triggered by a common signal, the inactivation of ribosomes, occurring under both cold shock and stationary-phase conditions.

Cold shock response in Bacillus subtilis.

Following a rapid decrease in temperature, the physiology of Bacillus subtilis cells changes profoundly. Cold shock adaptation has been monitored at the level of membrane composition, adjustment in DNA topology, and change in cytosolic protein synthesis/composition. Some major players in these processes (cold-stress induced proteins and cold acclimatization proteins, CIPs and CAPs) have been identified and mechanisms in cold shock acclimatization begin to emerge; however, important questions regarding their cellular function still need to be answered.

Chromosome arrangement within a bacterium.

BACKGROUND: The contour length of the circular chromosome of bacteria is greater than a millimeter but must be accommodated within a cell that is only a few micrometers in length. Bacteria do not have nucleosomes and little is known about the arrangement of the chromosome inside a prokaryotic cell. RESULTS: We have investigated the arrangement of chromosomal DNA within the bacterium Bacillus subtilis by using fluorescence microscopy to visualize two sites on the chromosome simultaneously in the same cell. Indirect immunofluorescence with antibodies against the chromosome partition protein Spo0J were used to visualize the replication origin region of the chromosome. Green fluorescent protein fused to the lactose operon repressor Lacl was used to decorate tandem copies of the lactose operon operator lacO. A cassette of tandem operators was separately inserted into the chromosome near the origin (359 degrees), near the replication terminus (181 degrees), or at two points in between (90 degrees and 270 degrees). The results show that the layout of the chromosome is dynamic but is principally arranged with the origin and terminus maximally apart and the quarter points of the chromosome in between. CONCLUSIONS: The use of cytological methods to visualize two chromosomal sites in the same cell has provided a glimpse of the arrangement of a bacterial chromosome. We conclude that, to a first approximation, the folding of the bacterial chromosome is consistent with, and may preserve, the linear order of genes on the DNA.

A superfamily of proteins that contain the cold-shock domain.

Members of a family of cold-shock proteins (CSPs) are found throughout the eubacterial domain and appear to function as RNA-chaperones. They have been implicated in various cellular processes, including adaptation to low temperatures, cellular growth, nutrient stress and stationary phase. The discovery of a domain--the cold-shock domain--that shows strikingly high homology and similar RNA-binding properties to CSPs in a growing number of eukaryotic nucleic-acid-binding proteins suggests that these proteins have an ancient origin.

Subcellular localization of Bacillus subtilis SMC, a protein involved in chromosome condensation and segregation.

We have investigated the subcellular localization of the SMC protein in the gram-positive bacterium Bacillus subtilis. Recent work has shown that SMC is required for chromosome condensation and faithful chromosome segregation during the B. subtilis cell cycle. Using antibodies against SMC and fluorescence microscopy, we have shown that SMC is associated with the chromosome but is also present in discrete foci near the poles of the cell. DNase treatment of permeabilized cells disrupted the association of SMC with the chromosome but not with the polar foci. The use of a truncated smc gene demonstrated that the C-terminal domain of the protein is required for chromosomal binding but not for the formation of polar foci. Regular arrays of SMC-containing foci were still present between nucleoids along the length of aseptate filaments generated by depleting cells of the cell division protein FtsZ, indicating that the formation of polar foci does not require the formation of septal structures. In slowly growing cells, which have only one or two chromosomes, SMC foci were principally observed early in the cell cycle, prior to or coincident with chromosome segregation. Cell cycle-dependent release of stored SMC from polar foci may mediate segregation by condensation of chromosomes.

Use of time-lapse microscopy to visualize rapid movement of the replication origin region of the chromosome during the cell cycle in Bacillus subtilis.

We describe the use of time-lapse fluorescence microscopy to visualize the movement of the DNA replication origin and terminus regions on the Bacillus subtilis chromosome during the course of the cell cycle. The origin and terminus regions were tagged with a cassette of tandem lac operator repeats and visualized through the use of a fusion of the green fluorescent protein to the LacI repressor. We have discovered that origin regions abruptly move apart towards the cell poles during a brief interval of the cell cycle. This movement was also seen in the absence of cell wall growth and in the absence of the product of the parB homologue spo0J. The origin regions moved apart an average distance of 1.4 microm in an 11 min period of abrupt movement, representing an average velocity of 0.17 microm min(-1), and reaching a maximum velocity of greater than 0.27 microm min(-1). The terminus region also exhibited a striking pattern of movement but not as far or a rapid as the origin region. These results provide evidence for a mitotic-like motor that is responsible for segregation of the origin regions of the chromosomes.

Surface-exposed phenylalanines in the RNP1/RNP2 motif stabilize the cold-shock protein CspB from Bacillus subtilis.

In the cold-shock protein CspB from Bacillus subtilis three exposed Phe residues (F15, F17, and F27) are essential for its function in binding to single-stranded nucleic acids. Usually, the hydrophobic Phe side chains are buried in folded proteins. We asked here whether the exposition of the essential Phe residues could be a cause for the very low conformational stability of CspB. Urea-induced and heat-induced equilibrium unfolding transitions were measured for three mutants of CspB, where Phe 15, Phe 17, and Phe 27 were individually replaced by alanine. Unexpectedly, all three mutations strongly destabilized CspB. The aromatic side chains of Phe 15, Phe 17, and Phe 27 in the active site are thus important for both binding to nucleic acids and conformational stability. There is no compromise between function and stability in the active site. Model calculations indicate that, although they are partially exposed to solvent, all three Phe residues nevertheless lose accessible surface upon folding, and this should favor the native state. A different result is obtained with the F38A variant. Phe 38 is hyperexposed in native CspB, and its substitution by Ala is in fact stabilizing.

A family of cold shock proteins in Bacillus subtilis is essential for cellular growth and for efficient protein synthesis at optimal and low temperatures.

Like other bacteria, Bacillus subtilis possesses a family of homologous small acidic proteins (CspB, CspC and CspD, identity >70%) that are strongly induced in response to cold shock. We show that deletion of cspC or cspD genes did not result in a detectable phenotype; in contrast, csp double mutants exhibited severe reduction in cellular growth at 15 degrees C as well as at 37 degrees C, including impairment of survival during the stationary phase. Two-dimensional gel analysis showed that protein synthesis was deregulated in csp double mutants and that the loss of one or two CSPs led to an increase in the synthesis of the remaining CSP(s) at 37 degrees C and after cold shock, suggesting that CSPs down-regulate production of members from this protein family. A cspB/C/D triple mutant (64BCDbt) could only be generated in the presence of cspB in trans on a plasmid that was not lost, in spite of lack of antibiotic pressure, indicating that a minimum of one csp gene is essential for viability of B. subtilis. After cold shock, synthesis of CspB in 64BCDbt was drastically lower than in wild-type cells accompanied by cessation in growth and strong reduction in general protein synthesis. As CspB, CspC and CspD are shown to bind to RNA in a co-operative and interactive manner, CSPs are suggested to function as RNA chaperones facilitating the initiation of translation under optimal and low temperatures.

Effects of heterologous expression of CspB, the major cold shock protein of Bacillus subtillis, on protein synthesis in Escherichia coli.

The major cold shock protein of Bacillus subtilis, CspB, has been shown to affect the level of several cold-induced proteins in B. subtilis after cold shock. Here we show that the expression of CspB in Escherichia coli at 37 degrees C - conditions where the cold shock proteins CspA and CspB of E. coli are not present - resulted in a marked decrease in cellular growth rate and had a profound influence on the pattern of protein synthesis, as revealed by two-dimensional gel electrophoresis. This involves both decreases and increases in the rates of synthesis of specific proteins. Specifically, CspB induction resulted in enhanced beta-galactosidase activity expressed from a transcriptional hns-lacZ fusion. This increase reflects the induction of hns transcription and H-NS synthesis after cold shock, which has been demonstrated to be dependent on CspA in vitro. In contrast, expression of a mutant form of CspB (CspBF15A) that is unable to bind to ssDNA in vitro had no effect on growth rate, pattern of protein synthesis or beta-galactosidase activity. Our data demonstrate a strong influence of CspB on protein synthesis in E. coli and suggest a similar function for CspA in E. coli to that of CspB in B. subtilis.

Some like it cold: response of microorganisms to cold shock.

Bacteria respond to an abrupt decrease in temperature with a specific response, in which cold-induced proteins (CIPs) are transiently expressed at a higher level. Employing two-dimensional gel electrophoresis, several CIPs have been identified. In spite of this, the overall function of the cold shock response is unclear. Recently, the main attention has focused on a group of conserved cold shock proteins (CSPs) that have been shown to have the highest induction after cold shock and to play a major regulatory role in the physiology of adaptation to low temperatures. CSPs, of which Escherichia coli, Bacillus subtilis, and B. cereus possess a family comprising at least 3-7 proteins, are small acidic proteins that share over 45% of sequence identity. Recent evidence suggests that members of this wide-spread protein family can function both at the transcriptional and translational level in vitro. However, the exact mode of action has yet to be established. In addition, post-transcriptional regulation seems to play a major role in the induction of CSPs, a process in which the ribosome may be involved. This is in accordance with a model in which the ribosome has been proposed to be the sensor of temperature in bacteria.

Cold shock stress-induced proteins in Bacillus subtilis.

Bacteria respond to a decrease in temperature with the induction of proteins that are classified as cold-induced proteins (CIPs). Using two-dimensional gel electrophoresis, we analyzed the cold shock response in Bacillus subtilis. After a shift from 37 to 15 degrees C the synthesis of a majority of proteins was repressed; in contrast, 37 proteins were synthesized at rates higher than preshift rates. One hour after cold shock, the induction of CIPs decreased, and after 2 h, general protein synthesis resumed. The identified main CIPs were excised from two-dimensional gels and were subjected to microsequencing. Three small acidic proteins that showed the highest relative induction after cold shock were highly homologous and belonged to a protein family of which one member, the major cold shock protein, CspB, has previously been characterized. Two-dimensional gel analyses of a cspB null mutant revealed that CspB affects the level of induction of several CIPs. Other identified CIPs function at various levels of cellular physiology, such as chemotaxis (CheY), sugar uptake (Hpr), translation (ribosomal proteins S6 and L7/L12), protein folding (PPiB), and general metabolism (CysK, Ilvc, Gap, and triosephosphate isomerase).

A case of convergent evolution of nucleic acid binding modules.

Divergent evolution can explain how many proteins containing structurally similar domains, which perform a variety of related functions, have evolved from a relatively small number of modules or protein domains. However, it cannot explain how protein domains with similar, but distinguishable, functions and similar, but distinguishable, structures have evolved. Examples of this are the RNA-binding protein containing the RNA-binding domain (RBD), and a newly established protein group, the cold-shock domain (CSD) protein family. Both protein domains contain conserved RNP motifs on similar single-stranded nucleic acid-binding surfaces. Apart from the RNP motifs, which have a similar function, the two families show little similarity in topology or amino acid sequence. This can be considered an interesting example of convergent evolution at the molecular level. Previously, a beta-sheet surface was found to interact with RNA in non-homologous proteins from yeast, phage and man, revealing that this mode of RNA binding may be a widely recurring theme.

Mutational analysis of the putative nucleic acid-binding surface of the cold-shock domain, CspB, revealed an essential role of aromatic and basic residues in binding of single-stranded DNA containing the Y-box motif.

The major cold-shock protein of Bacillus subtilis, CspB, is a member of a protein family widespread among prokaryotes and eukaryotes that share the highly conserved cold-shock domain (CSD). The CSD domain is involved in transcriptional and translational regulation and was shown to bind the Y-box motif, a cis-element that contains the core sequence ATTGG, with high affinity. The three-dimensional structure of CspB, a prototype of this protein family, revealed that this hydrophilic CSD domain creates a surface rich in aromatic and basic amino acids that may act as the nucleic acid-binding site. We have analysed the potential role of conserved aromatic and basic residues in nucleic acid binding by site-directed mutagenesis. In gel retardation and ultraviolet cross-linking experiments, the ability of CspB mutants to bind single-stranded oligonucleotides (ssDNA) that contain the Y-box motif was investigated. Single substitutions of three highly conserved phenylalanine residues (Phe-15, Phe-17, Phe-27) by alanine and substitution of one histidine (His-29) by glutamine, all located within the putative RNA-binding sites RNP-1 and RNP-2, abolished the nucleic acid-binding activity of CspB. Conservative substitutions of Phe-15 to tyrosine (F15Y) showed a small increase in binding affinity, whereas separate replacement of Phe-17 and Phe-27 by tyrosine caused a reduction in binding activity. These and other substitutions including the conserved basic residues Lys-7, Lys-13 and Arg-56 as well as the aromatic residues Trp-8 and Phe-30 strongly suggest that CspB uses the side-chains of these amino acids for specific interaction with nucleic acids. Ultraviolet cross-linking experiments for CspB mutants with ssDNA supported the idea of specific CspB/nucleic acid interaction and indicated an essential role for the aromatic and basic residues in this binding. In addition, two-dimensional nuclear magnetic resonance studies with F17A, K13Q, F15Y and F27Y revealed that the mutants have the same overall structure as the wild-type CspB protein.