aarC, an essential gene involved in density-dependent regulation of the 2'-N-acetyltransferase in Providencia stuartii

Abstract

The 2-N-acetyltransferase [AAC(2)-Ia] in Providencia stuartii has a dual function where it is involved in the acetylation of peptidoglycan and certain aminoglycosides. A search for negative regulators of the aac(2)-Ia gene has resulted in the identification of aarC. A missense allele (aarC1) resulted in an 8.9-fold increase in ␤-galactosidase accumulation from an aac(2)-lacZ transcriptional fusion. Northern blot analysis demonstrated an increase in aac(2)-Ia mRNA accumulation that was specific to cells at high density. In addition, the aarC1 allele also resulted in a substantial increase in the expression of aarP, a transcriptional activator of the aac(2)-Ia gene. The wild-type aarC gene was isolated by complementation and encodes a predicted protein of 365 amino acids with a molecular mass of 39,815 Da. The predicted AarC protein exhibited 88% amino acid homology to the previously identified GcpE protein of Escherichia coli and 86% homology to a gene product from Haemophilus influenzae. The E. coli gcpE gene was able to functionally complement the aarC1 allele in P. stuartii. The aarC1 allele was identified as a T to G transversion that resulted in a valine to glycine substitution at position 136 in the AarC protein.The aarC gene appears to be essential for cell viability as construction of a disrupted copy (aarC::lacZ) was possible only in cells that carried an episomal copy of aarC or gcpE.

GcpE Is Involved in the 2- C -Methyl- d -Erythritol 4-Phosphate Pathway of Isoprenoid Biosynthesis in Escherichia coli

“…However, as gcpE homologues are highly conserved in bacteria lacking aac(2Ј)-Ia such as E. coli and Haemophilus influenzae, the authors concluded that GcpE must additionally carry out essential housekeeping functions. A single point mutation in the aarC gene of P. stuartii resulted in a slow-growth phenotype and altered cell morphology, with the formation of very short rods, many of which were spherical (31). This observation is consistent with the fact that inhibition of the MEP pathway impaires cell wall biosynthesis (37).…”

“…Using the primer pair C plus D, the gcpE gene (1,116 bp) was amplified in the wild-type strain, and no product was obtained in the gcpE mutant strain. In earlier work, the gcpE homologue of Providencia stuartii was described as aarC and identified as a negative regulator of the 2Ј-N-acetyltransferase [Aac(2Ј)-Ia] involved in the acetylation of peptidoglycan and certain aminoglycosides in P. stuartii (31). However, as gcpE homologues are highly conserved in bacteria lacking aac(2Ј)-Ia such as E. coli and Haemophilus influenzae, the authors concluded that GcpE must additionally carry out essential housekeeping functions.…”

Identification and Characterization of the Point Mutation Which Affects the Transcription Level of the Chromosomal 3- N -Acetyltransferase Gene of Streptomyces griseus SS-1198

“…In contrast, the cloning of the wild-type gene with a high-copy-number vector demonstrated that the wild-type gene could confer a low level (50 g/ml) of Km resistance to S. lividans, suggesting that the difference between resistance levels conferred by the wild-type gene and the mutant genes would be due not to derepression in the heterologous host but to a gene dosage effect. However, we cannot rule out the presence of negative regulators, such as aar factors which regulate the expression of the aac(2Ј)-Ia gene in P. stuartii (21)(22)(23)(24). We have currently found that protoplast regeneration treatment is not necessary for obtaining Km-resistant mutants, because we isolated such mutants without protoplasting (unpublished data).…”

The chromosomal 2 -N-acetyltransferase of providencia stuartii physiological functions and genetic regulation

“…The deduced AarC protein is 40 kDa and is highly conserved to a family of proteins that is widespread in bacteria. The E. coli homolog is GcpE and studies in our lab have demonstrated that gcpE is essential for E. coli viability and have also shown that aarC is an essential gene in P. stuartii (28). Furthermore, complementation experiments have shown that aarC and gcpE are functionally equivalent.…”

“…The aarC gene was identified by a mutation (aarC1) which simultaneously activated the chromosomal aac(2')-Ia gene and a plasmid encoded aac(2')-lacZ transcriptional fusion (28). The deduced AarC protein is 40 kDa and is highly conserved to a family of proteins that is widespread in bacteria.…”