Putative Response Regulator Research Articles

Molecular analysis of the regulation of nisin immunity.

The genetic determinants controlling immunity to nisin are coordinately regulated, along with biosynthesis genes, in response to an environmental signal, nisin or a nisin analogue. The nisR gene product, the putative response regulator of nisin biosynthesis, was found to be a vital component of this induction mechanism. This protein forms part of a two-component regulatory system which controls the expression of genes involved in nisin immunity and biosynthesis. Analysis of the structural requirements of the external signal, using nisin fragments and engineered nisin variants, indicated that the 12 amino-terminal residues of the molecule are a minimum requirement for induction, with an intact ring A being an essential component. Changes throughout the molecule also affected its induction capacity. The production of certain variant nisins by engineered lactococcal strains is reduced in parallel with the strains' immunity to nisin. This can be attributed to inefficient induction by the variant molecule. Treating growing cultures with nisin restored full immunity and maximized the yields of nisin variants by the producer strains.

Cloning and nucleotide sequence of regA, a putative response regulator gene ofRhodobacter sphaeroides

A 0.9 kb DNA fragment carrying the Rhodobacter capsulatus regA gene, which encodes an oxygen-dependent, positively-acting response regulator of photosynthetic gene expression, was used as a probe in Southern hybridisation experiments to determine whether a similar gene occurs in R. sphaeroides. A strongly hybridising DNA fragment isolated from a R. sphaeroides plasmid gene bank was isolated, sequenced and found to contain an open reading frame which exhibits 75% identity with the R. capsulatus regA gene. The deduced amino acid sequence of 184 residues shows 81% identity and 89% similarity with the R. capsulatus RegA protein, and significant similarities with other response regulators of the two component sensor-regulator type. Introduction of the R. sphaeroides gene into a R. capsulatus regA mutant, which exhibits abnormally low levels of membrane-bound photosynthetic complexes, resulted in a 22-33-fold increase in these complexes to approximately 62-65% of wild-type levels. This is the first study to identify a putative response regulator in R. sphaeroides and to complement a regulatory mutation in R. capsulatus with a gene from another species. Further studies of associated genes may identify the different mechanisms by which the regulation of photosynthesis complex formation occurs in response to environmental stimuli in R. sphaeroides and R. capsulatus.

Cloning and nucleotide sequence of regA, a putative response regulator gene of Rhodobacter sphaeroides

A 0.9 kb DNA fragment carrying the Rhodobacter capsulatus regA gene, which encodes an oxygen-dependent, positively-acting response regulator of photosynthetic gene expression, was used as a probe in Southern hybridisation experiments to determine whether a similar gene occurs in R. sphaeroides. A strongly hybridising DNA fragment isolated from a R. sphaeroides plasmid gene bank was isolated, sequenced and found to contain an open reading frame which exhibits 75% identity with the R. capsulatus regA gene. The deduced amino acid sequence of 184 residues shows 81% identity and 89% similarity with the R. capsulatus RegA protein, and significant similarities with other response regulators of the two component sensor-regulator type. Introduction of the R. sphaeroides gene into a R. capsulatus regA mutant, which exhibits abnormally low levels of membrane-bound photosynthetic complexes, resulted in a 22–33-fold increase in these complexes to approximately 62–65% of wild-type levels. This is the first study to identify a putative response regulator in R. sphaeroides and to complement a regulatory mutation in R. capsulatus with a gene from another species. Further studies of associated genes may identify the different mechanisms by which the regulation of photosynthesis complex formation occurs in response to environmental stimuli in R. sphaeroides and R. capsulatus.

PrrA, a putative response regulator involved in oxygen regulation of photosynthesis gene expression in Rhodobacter sphaeroides

A new locus, prrA, involved in the regulation of photosynthesis gene expression in response to oxygen, has been identified in Rhodobacter sphaeroides. Inactivation of prrA results in the absence of photosynthetic spectral complexes. The prrA gene product has strong homology to response regulators associated with signal transduction in other prokaryotes. When prrA is present in multiple copies, cells produce light-harvesting complexes under aerobic growth conditions, suggesting that prrA affects photosynthesis gene expression positively in response to oxygen deprivation. Analysis of the expression of puc::lacZ fusions in wild-type and PrrA- cells revealed a substantial decrease in LacZ expression in the absence of prrA under all conditions of growth, especially when cells were grown anaerobically in the dark in the presence of dimethyl sulfoxide. Northern (RNA) and slot blot hybridizations confirmed the beta-galactoside results for puc and revealed additional positive regulation of puf, puhA, and cycA by PrrA. The effect of truncated PrrA on photosynthesis gene expression in the presence of low oxygen levels can be explained by assuming that PrrA may be effective as a multimer. PrrA was found to act on the downstream regulatory sequences (J. K. Lee and S. Kaplan, J. Bacteriol. 174:1146-1157, 1992) of the puc operon regulatory region. Finally, two spontaneous prrA mutations that abolish prrA function by changing amino acids in the amino-terminal domain of the protein were isolated.

A novel response-regulator is able to suppress the nodulation defect of a Bradyrhizobium japonicum nodW mutant

The two-component regulatory system Nod-VW of Bradyrhizobium japonicum is essential for the nodulation of the legume host plants Vigna radiata, V. unguiculata and Macroptilium atropurpureum. The NodV protein shares homology with the sensor-kinases, whereas the NodW protein is a member of the response-regulator class. We report here the identification of a new B. japonicum DNA region that is able to suppress the phenotypic defect of a nodW mutant, provided that this region is expressed from a foreign promoter. The minimal complementing region, which itself is not essential for nodulation in a nodW+ background, consists of one gene designated nwsB (nodW-suppressor). The deduced amino acid sequence of the nwsB gene product shows a high degree of homology to NodW. The nws B gene is preceded by a long open reading frame, nwsA, whose putative product appears to be a sensor-kinase. Downstream of nwsB, an open reading frame encoding a second putative response-regulator was identified. Interspecies hybridization revealed the presence of nwsAB-like DNA also in other Bradyrhizobium strains. Using nwsB'-'lacZ fusions, the nwsB gene was found to be expressed rather weakly in B. japonicum. This low level of expression is obviously not sufficient to compensate for a nodW- defect, whereas strong overexpression of nwsB is a condition that leads to suppression of the nodW- mutation.

Phosphorylation and dephosphorylation catalyzed in vitro by purified components of the nitrate sensing system, NarX and NarL.

The regulation of specific gene expression by nitrate in Escherichia coli is mediated by the NarX/NarQ-NarL system. Based on sequence homologies with a family of two-component regulatory systems in bacteria, NarL has been identified as a putative response regulator while NarX and NarQ were proposed to be alternative membrane-associated sensors that activate NarL in the presence of nitrate. To investigate the interaction of NarX and NarL in vitro, both proteins were purified from overproducing strains. Purified NarX was rapidly labeled when incubated with [gamma-32P] ATP but not with [alpha-32P]ATP in a reaction that required Mg2+ but was unaffected by nitrate. Incubation of the labeled NarX with purified NarL resulted in the transient phosphorylation of NarL. Both the phosphorylation and dephosphorylation of NarL required Mg2+, and neither reaction was affected by the presence of nitrate. NarL-phosphate, stabilized by the addition of EDTA, ran as a monomer on gel filtration. Dephosphorylation of the isolated NarL-phosphate required the addition of both Mg2+ and the NarX protein. The relative stabilities of the phosphorylated forms of the two proteins at different pH values were consistent with the proposal that, in analogy to other related two-component regulatory systems, NarX and NarL were phosphorylated on specific histidine and aspartate residues, respectively.