Oligomerization into multimeric complexes is a prerequisite for the chaperone function of almost all α‐crystallin type heat shock proteins (α‐Hsp), but the molecular details of complex assembly are poorly understood. The α‐Hsp proteins from Bradyrhizobium japonicum are suitable bacterial models for structure‐function studies of these ubiquitous stress proteins. They fall into two distinct classes, A and B, display chaperone activity in vitro and form oligomers of ≈ 24 subunits. We constructed 19 derivatives containing truncations or point mutations within the N‐ and C‐terminal regions and analyzed them by gel filtration, citrate synthase assay and coaffinity purification. Truncation of more than the initial few amino acids of the N‐terminal region led to the formation of distinct dimeric to octameric structures devoid of chaperone activity. In the C‐terminal extension, integrity of an isoleucine‐X‐isoleucine (I‐X‐I) motif was imperative for α‐Hsp functionality. This I‐X‐I motif is one of the characteristic consensus motifs of the α‐Hsp family, and here we provide experimental evidence of its structural and functional importance. α‐Hsp proteins lacking the C‐terminal extension were inactive, but still able to form dimers. Here, we demonstrate that the central α‐crystallin domain alone is not sufficient for dimerization. Additional residues at the end of the N‐terminal region were required for the assembly of two subunits.

Induction of the heat shock response in Escherichia coli requires the alternative sigma factor 32 (RpoH). The cellular concentration of 32 is controlled by proteolysis involving FtsH, other proteases, and the DnaKJ chaperone system. To identify individual 32 residues critical for degradation, we used a recently developed bacterial one-hybrid system and screened for stabilized versions of 32 . The five single point mutations that rendered the sigma factor more stable mapped to positions L47, A50, and I54 in region 2.1. Strains expressing the stabilized 32 variants exhibited elevated transcriptional activity, as determined by a groE-lacZ fusion. Structure calculations predicted that the three mutated residues line up on the same face of an ␣-helix in region 2.1, suggesting that they are positioned to interact with proteins of the degradation machinery.

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Lactate Dehydrogenase 1 (Ldh1) is a key enzyme involved in Staphylococcus aureus NO·-resistance. Full ldh1-induction requires the presence of glucose, and mutants lacking the Carbon-Catabolite Protein (CcpA) exhibit decreased ldh1 transcription and diminished Ldh1 activity. The redox-regulator Rex represses ldh1 directly by binding to Rex-sites within the ldh1 promoter (Pldh 1). In the absence of Rex, neither glucose nor CcpA affect ldh1 expression implying that glucose/CcpA-mediated activation requires Rex activity. Rex-mediated repression of ldh1 depends on cellular redox status and is maximal when NADH levels are low. However, compared to WT cells, the ΔccpA mutant exhibited impaired redox balance with relatively high NADH levels, yet ldh1 was still poorly expressed. Furthermore, CcpA did not drastically alter Rex transcript levels, nor did glucose or CcpA affect the expression of other Rex-regulated genes indicating that the glucose/CcpA effect is specific for Pldh 1. A putative catabolite response element (CRE) is located ∼30 bp upstream of the promoter-distal Rex-binding site in Pldh 1. However, CcpA had no affinity for Pldh 1 in vitro and a genomic mutation of CRE upstream of Pldh 1 in S. aureus had no affect on Ldh1 expression in vivo. In contrast to WT, ΔccpA S. aureus preferentially consumes non-glycolytic carbon sources. However when grown in defined medium with glucose as the primary carbon source, ΔccpA mutants express high levels of Ldh1 compared to growth in media devoid of glucose. Thus, the actual consumption of glucose stimulates Ldh1 expression rather than direct CcpA interaction at Pldh 1.