Early studies on mutants that overexpressed the AAC(2 )-Ia enzyme indicated that they possessed altered cell morphology, forming small rounded cells. To further address the role of AAC(2' )-Ia, a null allele was created by introducing a frameshift mutation into the aac(2')-Ia coding region by allelic replacement . The loss of aac(2)-Ia resulted in cells with a slightly elongated phenotype . Furthermore, the staining properties of aac(2 )-Ia mutant cells with uranyl acetate was altered, relative to wild-type cells. The basis for this phenotype is unknown; however, it suggests changes in the surface properties of cells. These data suggested a possible role for AAC(2 )-Ia that is related to the cell envelope. Work done by Payie and Clarke has revealed that AAC(2')-Ia functions as a peptidoglycan O-acetyltransferase . The O-acetylation of peptidoglycan is a modification that regulates the activity of autolytic enzymes involved in peptidoglycan breakdown and turnover [80,81]. The altered cell morphology seen in cells with changes in aac(2')-Ia expression may be due to the changes in the activity of autolytic enzymes. The AAC(2')-Ia enzyme is capable of obtaining acetate from peptidoglycan, N-acetylglucosamine, and acetyl-coenzymeA . Interestingly, the AAC(2' )-Ia enzyme is released by osmotic shock and may be located in the periplasm. Since acetyl-CoA is not located within the periplasm, the use of this substrate as a source of acetate would require a mechanism for transfer into the periplasm. The mechanism for such a transfer is unknown in P. stuartii.
Studies on the regulation of aac(2)-Ia have been conducted using lacZreporter gene fusions to the aac(2)-Ia promoter region. Early studies demonstrated that aac(2')-Ia transcription was not inducible by sub-inhibitory amounts of aminoglycoside antibiotics . Using these fusions, two approaches have been used to identify gene products that act in trans to regulate aac(2')-Ia. The first approach involved selecting spontaneous gentamicin resistant mutants of a P. stuartii strain harboring an aac(2)-lacZ fusion on a low-copy plasmid. One mechanism for the increased gentamicin resistance of these mutants would be increased expression of the chromosomal aac(2')-Ia gene. During these isolations, the predominant class of mutants were darker blue in the presence of X-gal indicating increased transcription from the aac(2')-Ia promoter region on the plasmid. A second approach to identify regulatory mutants involved isolating transposon insertions (mini-Tn5Cm) that activated the aac(2')-lacZ fusion. Insertions that resulted in aac(2')-lacZ activation were then tested for increased expression of the chromosomal aac(2')-Ia gene. Using both of these strategies, genes designated aar (aminoglycoside acetyltransferase regulator) have been identified. The surprising number of regulatory genes that have been identified suggests the importance of modifying aac(2')-Ia expression in response to various environmental conditions. This would allow cells to fine-tune the levels of peptidoglycan acetylation and regulate autolysis.
The aar genes are grouped into two classes. The first class of genes act pheno-typically as negative regulatory genes since loss of function mutations increase aac(2')-Ia expression. The second class of regulatory genes are those that act in a positive manner and are required for normal levels of aac(2')-Ia expression.
The aarA gene encodes a very hydrophobic polypeptide of 31.1 kDa in size . The AarA protein contains at least two possible transmembrane domains, suggesting that it is an integral membrane protein. The AarA protein has been shown to be a member of the Rhomboid family of intramembrane serine proteases that are widely distributed in prokaryotes and eukaryotes [84,85]. The AarA protein is required for the production or activity of an extracellular pheromone signal, AR-factor, that acts to reduce aac(2')-Ia expression. The aarA gene was identified as a mini-Tn5Cm insertion that increased gentamicin resistance levels eight-fold above wild-type. The aarA mutants increase aac(2')-Ia transcription 3- to 10-fold depending on the growth phase of cells. Null mutations in aarA are highly pleiotropic and additional pheno-types include loss of production of a diffusible yellow pigment and a cell chaining phenotype that is most prominent in cells at mid-log phase.
The aarB3 mutation originally designated aar3  results in a 10- to 12-fold increase in aac(2')-Ia transcription. In the aarB3 background, the levels of aminoglycoside resistance are increased 128-fold above wild-type, suggesting that this mutation further increases aminoglycoside resistance in a manner independent of aac(2')-Ia expression. The aarB3 also results in altered cell morphology and a slow growth phenotype. The identity of the aarB gene has not been determined.
The aarC gene encodes a homolog of gcpE, a protein widely distributed in bacteria and required for isoprenoid biosynthesis. A missense allele, aarCl, resulted in a number of pleiotropic phenotypes including slow growth, altered cell morphology, and increased aac(2')-Ia expression at high cell density . The biochemical function of AarC remains to be determined.
The aarD was identified by a mini-Tn5Cm insertion that resulted in a five-fold activation of an aac(2')-lacZ fusion and a three-fold increase in the levels of aac(2')-Ia mRNA accumulation . In addition, a 32-fold increase in aminoglycoside resistance was observed in aarD mutants, relative to wild-type P. stuartii. The aarD locus encodes two polypeptides that are homologs of the E. coli CydD and CydC proteins [87-89]. The CydD and CydC proteins act in a heterodimeric ABC transporter complex required for formation of a functional cytochrome d oxidase complex [90-93]. P. stuartii aarD mutants exhibit phenotypic characteristics consistent with a defect in the cytochrome d oxidase including hyper-susceptibility to the respiratory inhibitors Zn2+ and toluidine blue .
The increased aac(2')-Ia expression observed in the aarD1 background contributes minimally to the overall increase in gentamicin resistance since introduction of the aarD1 mutation into an aac(2')-Ia mutant strain also results in a 32-fold increase in gentamicin resistance. Previous studies have demonstrated that uptake of aminoglycosides is dependent on the presence of a functional electron transport system [94-96]. Since electron transport is defective in the aarD1 background , it is probable that a decrease in aminoglycoside uptake accounts for the high level of resistance observed in aarD mutants. However, the mechanism that contributes to increased aac(2')-Ia transcription is unknown. A direct role for aarD in the regulation of aac(2')-Ia is unlikely, since ABC transporters are not known to function as transcriptional regulators . A regulatory protein may couple changes in the redox state of the membrane to aac(2')-Ia expression (see below) . Mutations in aarD are predicted to alter the redox state of the membrane and thus indirectly affect aac(2')-Ia expression.
The aarG gene encodes a protein with similarity to sensor kinases of the two-component family with the strongest identity to PhoQ (57%). Immediately upstream of aarG is an open reading frame designated aarR, which encoded a protein with 75% amino acid identity to PhoP, a response regulator [99,100]. The regulatory phenotypes associated with the aarG1 mutation may result from a failure to phos-phorylate the putative response regulator AarR, which functions as a repressor of aarP, and possibly aac(2')-Ia.
A recessive mutation (aarG1) results in an 18-fold increase in the expression of P-galactosidase from an aac(2')-lacZ fusion . Direct measurements of RNA from the chromosomal copy of aac(2')-Ia have confirmed this increase occurs at the level of RNA accumulation. Taken together, these results demonstrate that loss of aarG results in increased aac(2')-Ia transcription. The aarG1 allele also results in enhanced expression of aarP, encoding a transcriptional activator of aac(2')-Ia (see below) . Genetic experiments have shown that in an aarG1, aarP double mutant, the expression of aac(2')-Ia is significantly reduced over that seen in the aarG1 background. However, the levels of aac(2')-Ia in this double mutant are still significantly higher than in a strain with only an aarP mutation. Therefore, the aarG1 mutation increases aac(2')-Ia expression by both aarP-dependent and -independent mechanisms.
The aarGI allele confers a Mar phenotype to P. stuartii, resulting in increased resistance to tetracycline, chloramphenicol, and fluoroquinolones. This Mar phenotype in the aarGI background is partially due to overexpression of aarP, which is known to confer a Mar phenotype in both P. stuartii and E. coli (see below). However, an aarP-independent mechanism also accounts for increased levels of intrinsic resistance in the aarGI background. This mechanism could involve increased expression of a second activator with a target specificity similar to that of AarP.
Positive Regulators of aac(2')-Ia aarE
The aarE gene is ubiA, which encodes an octaprenyltransferase required for the second step of ubiquinone biosynthesis . Although the aarE mutations increase aminoglycoside resistance, accumulation of aac(2')-Ia mRNA is significantly reduced in the aarEI background. The loss of ubiquinone function is predicted to decrease the uptake of aminoglycosides, which accounts for the high-level aminoglycoside resistance. The decreased aac(2')-Ia mRNA accumulation may reflect a requirement for ubiquinone, either directly or indirectly in a regulatory process involved in aac(2')-Ia mRNA expression.
The aarF locus of P. stuartii acts as a positive regulator of aac(2 )-Ia expression with the level of aac(2')-Ia mRNA decreased in an aarF null mutant . Despite the lack of aac(2')-Ia expression, aarF null mutants exhibit a 256-fold increase in gentamicin resistance over the wild-type strain. P. stuartii aarF null mutants also exhibit severe growth defects under aerobic growth conditions and have been found to lack detectable quantities of the respiratory cofactor ubiquinone. The aarF gene is the ubiB homolog of P. stuartii, and heterologous complementation studies demonstrated that these genes were functionally equivalent .
The high-level gentamicin resistance observed in the aarF(ubiB) mutants is likely associated with decreased accumulation of the drug resulting from the absence of aerobic electron transport. It seems unlikely that aarF is directly involved in the regulation of aac(2')-Ia. It has been proposed that a reduced form of ubiquinone acts as an effector molecule in an uncharacterized regulatory pathway that activates the expression of aac(2 )-Ia . In ubiquinone-deficient aarF mutant strains, this regulatory cascade would be disrupted, resulting in decreased aac(2 )-Ia expression (see below).
The aarP gene was originally isolated from a multi-copy library of P. stuartii chromosomal DNA based on the ability to activate aac(2 )-Ia expression in trans . The presence of aarP in multiple copies led to an eight-fold increase in aac(2')-Ia mRNA accumulation. Studies utilizing an aac(2')-lacZ transcriptional fusion demonstrate that this increase results from an activation of aac(2 )-Ia transcription. Chromosomal disruption of the aarP locus resulted in a five-fold reduction in aac(2 )-Ia mRNA levels and eliminated the induction of aac(2 )-Ia expression normally observed during logarithmic growth . Expression of aarP has been shown to be increased in the aarB, aarC, and aarG mutants, demonstrating that aarP contributes to the overexpression of aac(2')-Ia in these mutant backgrounds [82,86,99].
The aarP gene encodes a 16 kDa protein that contains a putative DNA binding helix-turn-helix motif and belongs to the AraC/XylS family of transcriptional activators [101,104]. The AarP protein exhibits extensive homology with the E. coli MarA and SoxS proteins that were discussed above. AarP exhibits high homology to MarA and SoxS in the helix-turn-helix domain and was found to activate targets of both MarA and SoxS in vivo . The purified AarP protein binds to a wild-type aac(2')-Ia promoter fragment in electrophoretic mobility shift assays .
Expression of aarP appears to be governed by a mechanism that differs from those controlling MarA and SoxS expression. Unlike the MarA and SoxS proteins, which are located in operons containing a gene that regulates their expression, the aarP message appears to be monocistronic. Expression of aarP is not elevated in the presence of SAL, a potent inducer of MarA. Recent studies of aarP expression have revealed that the AarP message accumulates as cell density increases . At least three aar genes (aarB, aarC, and aarG) are involved in aarP regulation [82,86,99]. In addition, we have recently identified a role for the stationary phase starvation protein SspA as an activator of aarP . The SspA protein is a global regulator that is proposed to interact with RNA polymerase during starvation and redirect new gene expression [107,108].
Role of Quorum Sensing in aac(2')-Ia Regulation
The regulation of aac(2')-Ia expression is mediated by cell-to-cell signaling . The accumulation of aac(2')-Ia mRNA exhibits two levels of growth phase dependent expression. First, as cells approach mid-log phase, a significant increase is observed relative to cells at early-log phase. This increase at mid-log phase is the result of increased aarP expression. Second, as cells approach stationary phase, the levels of aac(2')-Ia mRNA are decreased to levels that are at least 20-fold lower than those at mid-log phase. This decrease at high density is mediated by the accumulation of an extracellular factor (AR-factor) . The growth of P. stuartii cells in spent (conditioned) media from stationary phase cultures resulted in the premature repression of aac(2')-Ia in cells at mid-log phase. The ability to produce AR-factor is dependent on the AarA protein described previously.
In summary, the large number of genes that influence aac(2')-Ia regulation suggest that the expression of aac(2')-Ia and the subsequent O-acetylation of pepti-doglycan must be tightly controlled in P. stuartii. The AAC(2')-Ia enzyme represents a minor O-acetyltransferase in P. stuartii . The physiological function of AAC(2')-Ia may be to "fine-tune" the levels of peptidoglycan O-acetylation in response to different environmental conditions or phases of growth. For example, in cells at mid-log phase, there is a burst of aac(2')-Ia expression that may be required for peptidoglycan turnover in rapidly growing cells. As cells increase in density and approach stationary phase, the accumulation of AR-factor leads to decreased aac(2')-Ia expression at stationary phase. This may reflect a requirement for lower peptido-glycan turnover at stationary phase. The additional levels of aac(2')-Ia regulation, namely, the role of ubiquinone and/or electron transport, are understood in less detail. The simplest model, proposed earlier, is that aac(2')-Ia expression is also coupled to electron transport via regulatory protein(s) that sense the redox status of the cell. The AarG/AarR two-component system may have a role in this process. At the present time, interplay among the aar genes, electron transport, and quorum sensing in controlling aac(2)-Ia expression is being investigated. The mechanisms identified may serve as a model for the regulation of other chromosomally encoded acetyl-transferases. In addition, the identification of physiological roles for the other intrinsic acetyltransferases will allow us to better predict how the modification of intrinsic genes can lead to antibiotic resistance.
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