Two LysR-type transcriptional regulators, BenM and CatM, control benzoate consumption by the soil bacterium Acinetobacter baylyi ADP1. These homologs play overlapping roles in the expression of multiple genes. This study focuses on the benABCDE operon, which initiates benzoate catabolism. At this locus, BenM and CatM each activate transcription in response to the catabolite cis,cis-muconate. BenM, but not CatM, additionally responds to benzoate as an effector. Regulation by CatM alone is insufficient for growth on benzoate as the sole carbon source. However, three point mutations independently increased CatM-activated benA transcription and enabled growth on benzoate without BenM. Two mutations generate variants with one amino acid change in the 303-residue CatM, CatM(V158M) and CatM(R156H). These substitutions affected regulation of benA differently than that of catB, another CatM-regulated gene involved in benzoate catabolism. In relation to CatM, CatM(V158M) increased cis,cis-muconate-dependent transcription of benA but decreased that of catB. CatM(R156H) increased effector-independent expression of catB compared to CatM. In contrast, cis,cis-muconate was required with CatM(R156H) to activate unusually high benA expression. Thus, induction by cis,cis-muconate depends on both the sequence of CatM and the promoter. A point mutation at position ؊40 of the benA promoter enhanced CatM-activated gene expression and altered regulation by CatM(R156H). BenM and CatM bound to the same locations on ben region DNA. The frequency with which spontaneous mutations allow CatM to substitute for BenM might predict that one regulator would be sufficient for controlling benzoate consumption. This prediction is discussed in light of current and previous studies of the BenM-CatM regulon.
Transcription of both chromosomal and extrachromosomally introduced nifS was regulated (up-expressed) by oxygen or by supplemental iron conditions. This up-expression was not observed in a fur mutant strain background or when an iron chelator was added. Iron-bound Fur (but not apo-Fur) recognized the nifS promoter, and Fur bound significantly farther upstream (؊155 bp to ؊190 bp and ؊210 to ؊240 bp) in the promoter than documented Helicobacter pylori Fur binding regions. This binding was stronger than Fur recognition of the flgE or napA promoter and includes a Fur recognition sequence common to the H. pylori pfr and sodB upstream areas. Studies of Fur-regulated genes in H. pylori have indicated that apo-Fur acts as a repressor, but our results demonstrate that iron-bound Fur activates (nifS) transcription.The human gastric pathogen Helicobacter pylori is well adapted for colonizing a unique niche, the stomach mucosa (3). In the mucosa it is subject to severe oxidative stress from the oxidative burst of the host immune system, which results in production of a variety of reactive oxygen intermediates (ROI) that damage macromolecules of the bacterium (21). Due to the Fenton reaction, the ROI are especially a problem in the presence of excess free intracellular iron (7). Considering the persistent nature of H. pylori, it is not surprising that the pathogen has a repertoire of enzymes involved in detoxification of the oxidative agents or in repairing the oxidized macromolecules in the cell (1,17,18,22,24).Although combating oxidative stress is a key to H. pylori survival, we know little about regulation of expression of the specific genes involved. To aid in understanding the overall oxygen stress-modulated gene expression in H. pylori, we compared the global gene expression of H. pylori grown at 2% versus 12% oxygen by using a microarray approach. Preliminary results from these studies showed an up-expression of expected genes, such as thioredoxin reductase, thioredoxin, superoxide dismutase (SOD), and thiol peroxidase, all enzymes known to be directly involved in combating oxidative stress. In addition, among the other highly up-regulated genes was one encoding the Fe-S cluster synthesis protein NifS. We observed an approximately fivefold up-regulation of the nifSnifU operon (hp0220 and hp0221) after a 2-h shift from 2% to 12% O 2 (Abstr. 105th Gen. Meet. Am. Soc. Microbiol., abstr. K-063, 2005). NifS belongs to the crucial IscS family of proteins, which is involved in Fe-S cluster formation (12). The NifS proteins provide sulfur donation via an L-cysteine desulfurase activity. The cluster maturation proteins are usually considered to be essential "housekeeping" proteins, as nearly all organisms have multiple proteins that require Fe-S clusters for their function. Thus, inactivation of the H. pylori nifS-nifU operon (hp0220 and hp0221) was previously shown to be lethal to the bacterium (12, 13).In this study we performed promoter-reporter gene fusions to determine the regulation of the nifS-nifU operon under condit...
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