No abstract

Expression of several heat shock operons, mainly coding for small heat shock proteins, is under the control of ROSE (repression of heat shock gene expression) in various rhizobial species. This negatively cis-acting element confers temperature control by preventing expression at physiological temperatures. We provide evidence that ROSE-mediated regulation occurs at the post-transcriptional level. A detailed mutational analysis of ROSE(1)-hspA translationally fused to lacZ revealed that its highly conserved 3'-half is required for repression at normal temperatures (30 degrees C). The mRNA in this region is predicted to form an extended secondary structure that looks very similar in all 15 known ROSE elements. Nucleotides involved in base pairing are strongly conserved, whereas nucleotides in loop regions are more divergent. Base substitutions leading to derepression of the lacZ fusion at 30 degrees C exclusively resided in potential stem structures. Optimised base pairing by elimination of a bulged residue and by introduction of complementary nucleotides in internal loops resulted in ROSE elements that were tightly repressed not only at normal but also at heat shock temperatures. We propose a model in which the temperature-regulated secondary structure of ROSE mRNA influences heat shock gene expression by controlling ribosome access to the ribosome-binding site.

The sigma factor RpoH ( 32 ) is the key regulator of the heat shock response in Escherichia coli. Many structural and functional properties of the sigma factor are poorly understood. To gain further insight into RpoH regions that are either important or dispensable for its cellular activity, we generated a collection of tetrapeptide insertion variants by a recently established in vitro linker insertion mutagenesis technique. Thirty-one distinct insertions were obtained, and their sigma factor activity was analyzed by using a groE-lacZ reporter fusion in an rpoH-negative background. Our study provides a map of permissive sites which tolerate linker insertions and of functionally important regions at which a linker insertion impairs sigma factor activity. Selected linker insertion mutants will be discussed in the light of known sigma factor properties and in relation to a modeled structure of an RpoH fragment containing region 2.The induction of heat shock proteins (Hsps) at elevated temperatures is a universal cellular response. The majority of Hsps are either chaperones or proteases involved in protein quality control (7,42). The evolutionary conservation of Hsps is contrasted by a multitude of regulatory mechanisms established for temperature perception and heat shock gene expression. Numerous positive-and negative-control mechanisms acting at the transcriptional or posttranscriptional level have been described previously (7,13,25,26,31,33,42).In Escherichia coli, expression of more than 30 heat shock genes is under the control of the alternative sigma factor RpoH ( 32 ) (7,42). Its intracellular level is very low during growth at 30°C and increases transiently after temperature upshift (35). The cellular concentration of 32 is tightly controlled at four different levels: transcription and translation of the rpoH gene and activity and stability of the RpoH protein (42). Heat induction of 32 mainly occurs at the posttranscriptional level. An extended secondary structure in the rpoH transcript blocks translation at low temperatures (20,22). Thermal melting of that structure permits ribosome entry followed by translation initiation. Once produced, the fate of 32 is determined by its interaction with a number of other proteins. Under nonstress conditions, the sigma factor is neutralized through an interaction with DnaK and DnaJ (6,36). Sequestration by the chaperones serves two regulatory functions. It inactivates 32 by preventing it from interaction with the RNA polymerase core enzyme and renders it susceptible to FtsH-mediated degradation (36, 38). Accumulation of unfolded proteins under heat stress conditions titrates away the DnaK system, leaving behind free RpoH, which associates with RNA polymerase and in turn initiates transcription of heat shock genes. Accumulation of 32 only occurs in the initial phase of the heat shock response (35). Elevated temperatures introduce a conformational change in 32 which specifically abolishes interaction with DnaK (5). The structurally altered sigma factor is rapidly turned ...

This work addresses the biogenesis of heme-copper terminal oxidases in Bradyrhizobium japonicum, the nitrogen-fixing root nodule symbiont of soybean. B. japonicum has four quinol oxidases and four cytochrome oxidases. The latter include the aa 3 -and cbb 3 -type oxidases. Although both have a Cu B center in subunit I, the subunit II proteins differ in having either a Cu A center (in aa 3 ) or a covalently bound heme c (in cbb 3 ). Two biogenesis factors were genetically studied here, the periplasmically exposed CoxG and ScoI proteins, which are the respective homologs of the mitochondrial copper-trafficking chaperones Cox11 and Sco1 for the formation of the Cu B center in subunit I and the Cu A center in subunit II of cytochrome aa 3 . We could demonstrate copper binding to ScoI in vitro, a process for which the thiols of cysteine residues 74 and 78 in the ScoI polypeptide were shown to be essential. Knock-out mutations in the B. japonicum coxG and scoI genes led to loss of cytochrome aa 3 assembly and activity in the cytoplasmic membrane, whereas the cbb 3 -type cytochrome oxidase apparently remained unaffected. This suggests that subunit I of the cbb 3 -type oxidase obtains its copper cofactor via a different pathway than cytochrome aa 3 . In contrast to the coxG mutation, the scoI mutation caused a decreased symbiotic nitrogen fixation activity. We hypothesize that a periplasmic B. japonicum protein other than any of the identified Cu A proteins depends on ScoI and is required for an effective symbiosis.

Multidrug efflux systems not only cause resistance against antibiotics and toxic compounds but also mediate successful host colonization by certain plant-associated bacteria. The genome of the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum encodes 24 members of the family of resistance/nodulation/cell division (RND) multidrug efflux systems, of which BdeAB is genetically controlled by the RegSR two-component regulatory system. Phylogenetic analysis of the membrane components of these 24 RND-type transporters revealed that BdeB is more closely related to functionally characterized orthologs in other bacteria, including those associated with plants, than to any of the other 23 paralogs in B. japonicum. A mutant with a deletion of the bdeAB genes was more susceptible to inhibition by the aminoglycosides kanamycin and gentamicin than the wild type, and had a strongly decreased symbiotic nitrogen-fixation activity on soybean, but not on the alternative host plants mungbean and cowpea, and only very marginally on siratro. The host-specific role of a multidrug efflux pump is a novel feature in the rhizobia-legume symbioses. Consistent with the RegSR dependency of bdeAB, a B. japonicum regR mutant was found to have a greater sensitivity against the two tested antibiotics and a symbiotic defect that is most pronounced for soybean.