Bacterial size matters:Multiple mechanisms controlling septum cleavage and diplococcus formation are critical for the virulence of the opportunistic pathogen Enterococcus faecalis

Enterococcus faecalis is an opportunistic pathogen frequently isolated in clinical settings. This organism is intrinsically resistant to several clinically relevant antibiotics and can transfer resistance to other pathogens. Although E. faecalis has emerged as a major nosocomial pathogen, the mechanisms underlying the virulence of this organism remain elusive. We studied the regulation of daughter cell separation during growth and explored the impact of this process on pathogenesis. We demonstrate that the activity of the AtlA peptidoglycan hydrolase, an enzyme dedicated to septum cleavage, is controlled by several mechanisms, including glycosylation and recognition of the peptidoglycan substrate. We show that the long cell chains of E. faecalis mutants are more susceptible to phagocytosis and are no longer able to cause lethality in the zebrafish model of infection. Altogether, this work indicates that control of cell separation during division underpins the pathogenesis of E. faecalis infections and represents a novel enterococcal virulence factor. We propose that inhibition of septum cleavage during division represents an attractive therapeutic strategy to control infections

Microscale synthesis and kinetic isotope effect analysis of (4R)-[Ad-C-14,4-H-2] NADPH and (4R)-[Ad-H-3,4-H-2] NADPH

We present a one-pot chemo-enzymatic microscale synthesis of NADPH with two different patterns of isotopic labels: (4R)-[Ad-(14)C,4-(2)H] NADPH and (4R)-[Ad-(3)H,4-(2)H] NADPH. These co-factors are required by an enormous range of enzymes, and isotopically labeled nicotinamides are consequently of significant interest to researchers. In the current procedure, [Ad-(14)C] NAD(+) and [Ad-(3)H] NAD(+) were phosphorylated by NAD(+) kinase to produce [Ad-(14)C] NADP(+) and [Ad-(3)H] NADP(+), respectively. Thermoanaerobium brockii alcohol dehydrogenase was then used to stereospecifically transfer deuterium from C2 of isopropanol to the re face of C4 of NADP(+). After purification by HPLC, NMR analysis indicated the deuterium content at the 4R position is more than 99.7 %. The labeled cofactors were then used to successfully and sensitively measure kinetic isotope effects for R67 dihydrofolate reductase, providing strong evidence for the utility of this synthetic methodology

Triple isotopic labeling and kinetic isotope effects:exposing H-transfer steps in enzymatic systems

Kinetic isotope effect (KIE) studies can provide insight into the mechanism and kinetics of specific chemical steps in complex catalytic cascades. Recent results from hydrogen KIE measurements have examined correlations between enzyme dynamics and catalytic function, leading to a surge of studies in this area. Unfortunately, most enzymatic H-transfer reactions are not rate-limiting and the observed KIEs do not reliably reflect the intrinsic KIEs on the chemical step of interest. Given their importance to understanding the chemical step under study, accurate determination of the intrinsic KIE from the observed data is essential. In 1975, Northrop developed an elegant method to assess intrinsic KIEs from their observed values [Northrop, D. B. (1975) Steady-state analysis of kinetic isotope effects in enzymic reactions, Biochemistry, 14, 2644–2651]. The Northrop method involves KIE measurements using all three hydrogen isotopes, where one of them serves as the reference isotope. This method has been successfully used with different combinations of observed KIEs over the years but criteria for a rational choice of reference isotope have never before been experimentally determined. Here we compare different reference isotopes (and hence distinct experimental designs) using the reduction of dihydrofolate and dihydrobiopterin by two dissimilar enzymes as model reactions. A number of isotopic labeling patterns have been applied to facilitate the comparative study of reference isotopes. The results demonstrate the versatility of the Northrop method, and that such experiments are limited only by synthetic techniques, availability of starting materials, and the experimental error associated with the use of distinct combinations of isotopologues

Nuclear Magnetic Resonance Solution Structure of the Peptidoglycan-Binding SPOR Domain from <i>Escherichia coli</i> DamX: Insights into Septal Localization

SPOR domains are present in thousands of bacterial proteins and probably bind septal peptidoglycan (PG), but the details of the SPOR–PG interaction have yet to be elucidated. Here we characterize the structure and function of the SPOR domain for an <i>Escherichia coli</i> division protein named DamX. Nuclear magnetic resonance revealed the domain comprises a four-stranded antiparallel β-sheet buttressed on one side by two α-helices. A third helix, designated α3, associates with the other face of the β-sheet, but this helix is relatively mobile. Site-directed mutagenesis revealed the face of the β-sheet that interacts with α3 is important for septal localization and binding to PG sacculi. The position and mobility of α3 suggest it might regulate PG binding, but although α3 deletion mutants still localized to the septal ring, they were too unstable to use in a PG binding assay. Finally, to assess the importance of the SPOR domain in DamX function, we constructed and characterized <i>E. coli</i> mutants that produced DamX proteins with SPOR domain point mutations or SPOR domain deletions. These studies revealed the SPOR domain is important for multiple activities associated with DamX: targeting the protein to the division site, conferring full resistance to the bile salt deoxycholate, improving the efficiency of cell division when DamX is produced at normal levels, and inhibiting cell division when DamX is overproduced