Residue Leu-641 of Acetyl-CoA synthetase is critical for the acetylation of residue Lys-609 by the Protein acetyltransferase enzyme of Salmonella enterica

Posttranslational regulation of protein function by acetylation is present throughout nature. Regulation of protein function by Sir2 protein (sirtuin) deacetylases is conserved in all domains of life. In the prokaryote Salmonella enterica, the metabolic enzyme acetyl-coenzyme A synthetase (Acs) is regulated by a Sir2-dependent protein acetylation/deacetylation system (SDPADS). The recent identification of the acetyltransferase enzyme responsible for the acetylation of Acs defined the SDPADS in prokaryotes. This report identifies one residue in Acs, Leu-641, which is critical for the acetylation of Acs by the protein acetyltransferase enzyme. In vivo and in vitro evidence shows that mutations at Leu-641 prevent the acetylation of Acs by protein acetyltransferase, maintain the Acs enzyme in its active state, and bypass the need for sirtuin deacetylase activity during growth on acetate.     

Vitreoscilla hemoglobin promoter is not responsive to nitrosative and oxidative stress in Escherichia coli

The Vitreoscilla hemoglobin gene (vhb) is expressed under oxygen-limited conditions via an FNR-dependent mechanism. Furthermore, cAMP-CRP has been implicated in its regulation. Recently, VHb protein has been reported to protect a heterologous host from nitrosative stress. In this study we analyzed the regulation of the Vitreoscilla hemoglobin promoter (Pvhb) in Escherichia coli under nitrosative and oxidative stress conditions. Our results show unambiguously that expression of neither VHb nor chloramphenicol acetyltransferase under the control of Pvhb is induced under the experimental conditions used. Thus, a clear discrepancy between in vivo function, i.e. protection against nitrosative stress, and regulation of gene expression is obvious. The regulation of Pvhb reported here is in clear contrast to the expression pattern of flavohemoglobins from various microorganisms, which are generally induced by nitrosative stress. However, the length of Pvhb is only 146 bp and therefore, we cannot rule out that additional regulatory sequences may be located in the upstream region of Pvhb.     

Structure and substrate binding properties of cobB, a Sir2 homolog protein deacetylase from Escherichia coli

Sirtuins are NAD+-dependent protein deacetylase enzymes that are broadly conserved from bacteria to human, and have been implicated to play important roles in gene regulation, metabolism and longevity. cobB is a bacterial sirtuin that deacetylates acetyl-CoA synthetase (Acs) at an active site lysine to stimulate its enzymatic activity. Here, we report the structure of cobB bound to an acetyl-lysine containing non-cognate histone H4 substrate. A comparison with the previously reported archaeal and eukaryotic sirtuin structures reveals the greatest variability in a small zinc-binding domain implicated to play a particularly important role in substrate-specific binding by the sirtuin proteins. Comparison of the cobB/histone H4 complex with other sirtuin proteins in complex with acetyl-lysine containing substrates, further suggests that contacts to the acetyl-lysine side-chain and beta-sheet interactions with residues directly C-terminal to the acetyl-lysine represent conserved features of sirtuin-substrate recognition. Isothermal titration calorimetry studies were used to compare the affinity of cobB for a variety of cognate and non-cognate acetyl-lysine-bearing peptides revealing an exothermic reaction with relatively little discrimination between substrates. In contrast, similar studies employing intact acetylated Acs protein as a substrate reveal a binding reaction that is endothermic, suggesting that cobB recognition of substrate involves a burial of hydrophobic surface and/or structural rearrangement involving substrate regions distal to the acetyl-lysine-binding site. Together, these studies suggest that substrate-specific binding by sirtuin proteins involves contributions from the zinc-binding domain of the enzyme and substrate regions distal to the acetyl-lysine-binding site.     

Restored DNA-binding of the cAMP-CRP activator complex reestablishes negative regulation by the CytR repressor in the deoP2 promoter in Escherichia coli.

Summary We have investigated in vivo the coupling between CytR regulation of the deoP2 promoter in Escherichia coli and the DNA-binding specificity of the cAMP-CRP (cAMP receptor protein) complex in order to obtain a more detailed picture of the role played by cAMP-CRP in CytR regulation. By introducing CRP proteins that exhibit an altered DNA binding specificity into a strain containing a mutant deoP2 promoter in which cAMP-CRP activation was decreased and CytR regulation completely abolished, we show that CytR regulation of this promoter can be reestablished by restored the DNA binding of the cAMP-CRP complex. Hence, CytR regulation of deoP2 can be modulated by simply varying DNA binding of cAMP-CRP. These data confirm the crucial role played by the cAMP-CRP activator complex in CytR regulation of the deoP2 promoter.     

Bacillus subtilis rRNA promoters are growth rate regulated in Escherichia coli.

rRNA promoters from the rrnB locus of Bacillus subtilis and from the rrnB locus of Escherichia coli were fused to the gene for chloramphenicol acetyltransferase (CAT). The level of expression of CAT in E. coli showed growth rate dependence when the CAT gene was linked to either E. coli or B. subtilis tandem promoters. The downstream promoter of the tandem Bacillus pair showed growth rate regulation, while the upstream promoter did not, whereas for the E. coli tandem promoters, only the upstream promoter was growth rate regulated.