Identification and Characterization of aarF, a Locus Required for Production of Ubiquinone in Providencia stuartii and Escherichia coli and for Expression of 2′-N-Acetyltransferase in P. stuartii

Providencia stuartii contains a chromosomal 2′-N-acetyltransferase [AAC(2′)-Ia] involved in the O acetylation of peptidoglycan. The AAC(2′)-Ia enzyme is also capable of acetylating and inactivating certain aminoglycosides and confers high-level resistance to these antibiotics when overexpressed. We report the identification of a locus in P. stuartii, designated aarF, that is required for the expression of AAC(2′)-Ia. Northern (RNA) analysis demonstrated that aac(2′)-Ia mRNA levels were dramatically decreased in a P. stuartii strain carrying an aarF::Cm disruption. The aarF::Cm disruption also resulted in a deficiency in the respiratory cofactor ubiquinone. The aarF locus encoded a protein that had a predicted molecular mass of 62,559 Da and that exhibited extensive amino acid similarity to the products of two adjacent open reading frames of unknown function (YigQ and YigR), located at 86 min on the Escherichia coli chromosome. An E. coli yigR::Kan mutant was also deficient in ubiquinone content. Complementation studies demonstrated that the aarF and the E. coli yigQR loci were functionally equivalent. The aarF or yigQR genes were unable to complement ubiD and ubiE mutations that are also present at 86 min on the E. coli chromosome. This result indicates that aarF (yigQR) represents a novel locus for ubiquinone production and reveals a previously unreported connection between ubiquinone biosynthesis and the regulation of gene expression.     

Comparison of the functions of glutathionylspermidine synthetase/amidase from E. coli and its predicted homologues YgiC and YjfC

Protein function prediction is very important in establishing the roles of various proteins in bacteria; however, some proteins in the E. coli genome have their function assigned based on low percent sequence homology that does not provide reliable assignments. We have made an attempt to verify the prediction that E. coli genes ygiC and yjfC encode proteins with the same function as glutathionylspermidine synthetase/amidase (GspSA). GspSA is a bifunctional enzyme that catalyzes the ATP-dependent formation and hydrolysis of glutathionylspermidine (G-Sp), a conjugate of glutathione (GSH) and spermidine. YgiC and YjfC proteins show 51% identity between themselves and 28% identity to the synthetase domain of the GspSA enzyme. YgiC and YjfC proteins were expressed and purified, and the properties of GspSA, YgiC, and YjfC were compared. In contrast to GspSA, proteins YgiC and YjfC did not bind to G-Sp immobilized on the affinity matrix. We demonstrated that all three proteins (GspSA, YgiC and YjfC) catalyze the hydrolysis of ATP; however, YgiC and YjfC cannot synthesize G-Sp, GSH, or GSH intermediates. gsp, ygiC, and yjfC genes were eliminated from the E. coli genome to test the ability of mutant strains to synthesize G-Sp conjugate. E. coli cells deficient in GspSA do not produce G-Sp while synthesis of the conjugate is not affected in ΔygiC and ΔyjfC mutants. All together our results indicate that YgiC and YjfC are not glutathionylspermidine synthetases as predicted from the amino acid sequence analysis.     

pH dependent effects of sodium ions on dextransucrase activity in Streptococcus mutans

Dextransuccrase (E.C 2.4.1.5) is a key enzyme in S. mutans for the metabolism of sucrose which helps in the adherence and accumulation of bacteria on tooth surface leading to the formation of dental caries. Dextransuccrase resembles in its catalytic properties with the brush boarder sucrase and exhibits pH dependent inhibitory and stimulatory effects in response to Na⁺. In this communication we studied the effect of monovalent cations on the activity of dextransuccrase from S. mutans. The percentage inhibition of dextransuccrase was 65% at 0.5 mM NaCl which enhanced to 90% at 20 mM sodium concentration. However there was no effect on dextransucrase activity in presence of other monovalent cations (Rb⁺, Cs⁺, and K⁺) tested. Enzyme activity was enhanced 20–24% in acidic pH but was strongly inhibited (59–89%) around neutral and alkaline pH by 0.5–2.0 mM sodium chloride. Upon dialysis, 86% of enzyme activity was restored to control values. There was no effect of 2 mM NaCl on glucosyltransferase activity of the enzyme. Kinetic studies revealed that enzyme showed biphasic effects in response to Na⁺ ions. At acidic pH the enzyme exhibited mixed type of activation affecting both Vmax and Km, while in alkaline pH, the enzyme showed V- type effect reducing Vmax by 74% without affecting Km. The effects of sodium ions on dextransuccrase activity were specific, thus it can be useful to block its catalytic activity, and reducing the cariogenic potential of S. mutans.     

Characterization of aarA, a pleiotrophic negative regulator of the 2'-N-acetyltransferase in Providencia stuartii.

We have utilized transposon mutagenesis to obtain insertional mutations in Providencia stuartii that activate the chromosomal aac(2')-la gene. Two closely linked mini-Tn5Cm insertions were obtained in a locus designated aarA, and a single insertion was obtained in a separate locus, aarC. Nucleotide sequence analysis, complementation studies, and localization of the sites of mini-Tn5Cm insertion have allowed the identification of the aarA coding region. The deduced AarA protein had a molecular mass of 31,086 kDa and displayed characteristics of an integral membrane protein. A strain deleted for the aarA gene by allelic exchange showed at least a fourfold increase in the accumulation of aac(2')-la mRNA and an eightfold increase in aminoglycoside resistance. Mutations in aarA were pleiotrophic and also resulted in loss of pigmentation and a deficiency in cell separation during division.     

Diaphragm adaptations in patients with COPD

Inspiratory muscle weakness in patients with COPD is of major clinical relevance. For instance, maximum inspiratory pressure generation is an independent determinant of survival in severe COPD. Traditionally, inspiratory muscle weakness has been ascribed to hyperinflation-induced diaphragm shortening. However, more recently, invasive evaluation of diaphragm contractile function, structure, and biochemistry demonstrated that cellular and molecular alterations occur, of which several can be considered pathologic of nature. Whereas the fiber type shift towards oxidative type I fibers in COPD diaphragm is regarded beneficial, rendering the overloaded diaphragm more resistant to fatigue, the reduction of diaphragm fiber force generation in vitro likely contributes to diaphragm weakness. The reduced diaphragm force generation at single fiber level is associated with loss of myosin content in these fibers. Moreover, the diaphragm in COPD is exposed to oxidative stress and sarcomeric injury. This review postulates that the oxidative stress and sarcomeric injury activate proteolytic machinery, leading to contractile protein wasting and, consequently, loss of force generating capacity of diaphragm fibers in patients with COPD. Interestingly, several of these presumed pathologic alterations are already present early in the course of the disease (GOLD I/II), although these patients appear not limited in their daily life activities. Treatment of diaphragm dysfunction in COPD is complex since its etiology is unclear, but recent findings indicate the ubiquitin-proteasome pathway as a prime target to attenuate diaphragm wasting in COPD.