Localization of Cold Shock Proteins to Cytosolic Spaces Surrounding Nucleoids in Bacillus subtilis Depends on Active Transcription

Abstract

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 ␤-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 doublemutant 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.

Coping with the cold: the cold shock response in the Gram-positive soil bacterium Bacillus subtilis

“…This is evident since in contrast to the addition of translation inhibitor chloramphenicol, which results in nucleoid compaction, it was shown that for B. subtilis the addition of RNA polymerase inhibitor rifampicin results in expansion of the nucleoid (Weber et al 2001c). According to Woldringh's model, the inhibition of transcription should lead to a more condensed nucleoid since under such conditions expansion forces mediated by translation of mRNAs encoding membrane-directed proteins are abolished.…”

“…Conversely, a lack of CSPs in csp double mutants leads to nucleoid compaction (Weber et al 2001c) that, according to Woldringh's model, might be a result of compromised protein synthesis in such strains (Graumann et al 1997). Therefore, the function of CSPs, in terms of coupling transcription to translation, is somehow interconnected to nucleoid structure.…”

“…Although the size and shape of B. subtilis cells do not change detectably after cold shock, microscopic analyses have revealed that cold shock results in nucleoid compaction, as is also the case at 37°C upon entry into the stationary growth phase (Weber et al 2001c). Such compaction might be interpreted in terms of classical gyrasemediated increase in negative DNA supercoil, increased amounts or altered activity of other DNA structuring proteins, and/or reduced synthesis of proteins that are directed towards the membrane that is thought to expand the nucleoid as a result of expansion forces mediated by coupling transcription and translation to membrane insertion (Woldringh et al 1995).…”

“…Interestingly, in growing B. subtilis cells, the reported subcellular localization of CSPs and ribosomes to areas surrounding the nucleoid, is dependent on active transcription and consequently on nucleoid structure, since upon addition of rifampicin or entry of cells into the stationary growth phase, CSPs and ribosomes localize throughout the cell (Mascarenhas et al 2001;Weber et al 2001c). Conversely, a lack of CSPs in csp double mutants leads to nucleoid compaction (Weber et al 2001c) that, according to Woldringh's model, might be a result of compromised protein synthesis in such strains (Graumann et al 1997).…”

“…It is also evident that a hierarchy of importance exists, in which CspB appears to be most important, followed by either CspD or CspC depending on optimal or low-temperature growth conditions, respectively. An analysis of a cspB/cspC doubledeletion mutant revealed that removal of these csp genes results in pleiotropic alteration of protein synthesis, cell lysis during entry into the stationary growth phase, alteration of nucleoid structure, and the inability to differentiate to endospores indicating a fundamental yet poorly understood function for CSPs (Graumann et al 1997;Weber et al 2001c). Cell lysis upon entry into the stationary phase might be explained by the observation that both CspB and CspC are stationary-phase induced which, in the double mutant, is no longer possible and shows that in B. subtilis, CSPs are not only inducible after cold shock (Graumann & Marahiel 1999b).…”