SummaryThe level of penicillin resistance in clinical isolates of Streptococcus pneumoniae depends not only on the reduced affinity of penicillin binding proteins (PBPs) but also on the functioning of enzymes that modify the stem peptide structure of cell wall precursors. We used mariner mutagenesis in search of additional genetic determinants that may further attenuate the level of penicillin resistance in the bacteria. A mariner mutant of the highly penicillinresistant S. pneumoniae strain Pen6 showed reduction of the penicillin minimum inhibitory concentration (MIC) from 6 to 0.75 mg ml -1 . Decrease in penicillin MIC was also observed upon introduction of the mutation (named provisionally adr, for attenuator of drug resistance) into representatives of major epidemic clones of penicillin-resistant pneumococci. Attenuation of resistance levels was specific for b-lactams. The adr mutant has retained unchanged (low affinity) PBPs, unaltered murM gene and unchanged cell wall stem peptide composition, but the mutant became hypersensitive to exogenous lysozyme and complementation experiments showed that both phenotypes -reduced resistance and lysozyme sensitivity -were linked to the defective adr gene. DNA sequence comparison and chemical analysis of the cell wall identified adr as the structural gene of the pneumococcal peptidoglycan O-acetylase.

Two-dimensional liquid chromatography (2D-LC) is increasingly being viewed as a viable tool for solving difficult separation problems, ranging from targeted separations of structurally similar molecules to untargeted separations of highly complex mixtures. In spite of this performance potential, though, many users find method development challenging and most frequently cite the "incompatibility" between the solvent systems used in the first and second dimensions as a major obstacle. This solvent strength related incompatibility can lead to severe peak distortion and loss of resolution and sensitivity in the second dimension. In this paper, we describe a novel approach to address the incompatibility problem, which we refer to as Active Solvent Modulation (ASM). This valve-based approach enables dilution of D effluent with weak solvent prior to transfer to theD column but without the need for additional instrument hardware. ASM is related to the concept we refer to as Fixed Solvent Modulation (FSM), with the important difference being that ASM allows toggling of the diluent stream during each D separation cycle. In this work, we show that ASM eliminates the major drawbacks of FSM including complex elution solvent profiles, baseline disturbances, and slowD re-equilibration and demonstrate improvements in D separation quality using both simple small molecule probes and degradants of heat-treated bovine insulin as case studies. We believe that ASM will significantly ease method development for 2D-LC, providing a path to practical methods that involve both highly complementaryD and D separations and sensitive detection.

The separation of ionized bases by reversed-phase liquid chromatography with alkyl silica columns often leads to severely tailed bands that are highly detrimental. Band shape and its dependence on sample mass are notably different when mobile-phase pH is changed, and this behavior has not been previously explained. Ionized silanols present in the stationary phase have been credited with a role in determining peak shape. In the present study, separations on two different polymer columns were compared with those previously obtained on alkyl silica phases. Because silanols are absent from polymer columns, this comparison enabled us to assess the role of silanols in separations on alkyl silica phases and to offer an explanation of why band shape changes with sample size and mobile-phase pH for both polymer and silica-based phases.

Streptococcus pneumoniae has unusually complex cell wall teichoic acid and lipoteichoic acid, both of which contain a ribitol phosphate moiety. The lic region of the pneumococcal genome contains genes for the uptake and activation of choline, the attachment of phosphorylcholine to teichoic acid precursors, and the transport of these precursors across the cytoplasmic membrane. The role of two other, so far uncharacterized, genes, spr1148 and spr1149, in the lic region was determined. TarJ (spr1148) encodes an NADPH-dependent alcohol dehydrogenase for the synthesis of ribitol 5-phosphate from ribulose 5-phosphate. TarI (spr1149) encodes a cytidylyl transferase for the synthesis of cytidine 5-diphosphate (CDP)-ribitol from ribitol 5-phosphate and cytidine 5-triphosphate. We also present the crystal structure of TarI with and without bound CDP, and the structures present a rationale for the substrate specificity of this key enzyme. No transformants were obtained with insertion plasmids designed to interrupt the tarIJ genes, indicating that their function could be essential for cell growth. CDP-activated ribitol is a precursor for the synthesis of pneumococcal teichoic acids and some of the capsular polysaccharides. Thus, all eight genes in the lic region have a role in teichoic acid synthesis.Teichoic acids are the major cell wall components of most gram-positive bacteria (36); they are made up of anionic polymers of glycerol phosphate or ribitol phosphate, with esterlinked D-alanine or sugar additions. There are two types of teichoic acids: wall teichoic acid (WTA), which is covalently linked via a phosphodiester bond to C-6 of N-acetylmuramic acid in the peptidoglycan layer, and lipoteichoic acid (LTA), which contains a terminal glycolipid that acts as an anchor within the cytoplasmic membrane. A number of important physiological functions have been assigned to teichoic acids, including cation homeostasis; trafficking of ions, nutrients, proteins, and antibiotics; regulation of autolysins; and binding of envelope proteins (36,50). LTA has recently been shown to be essential for cell growth in Staphylococcus aureus (20). Teichoic acids and their structural modifications also play important roles in the interaction of pathogenic bacteria with host organisms. For example, an S. aureus mutant lacking WTA, although showing normal growth in the laboratory, has strongly reduced capability for nasal colonization in a cotton rat model (49). Furthermore, a Streptococcus pneumoniae mutant lacking the choline modification in WTA and LTA shows drastically reduced virulence in different animal models of infection (27).The WTA and LTA of the human pathogen S. pneumoniae (the pneumococcus) are unique among bacterial teichoic acids with respect to several features. First, unlike in most other species, the repeating units of WTA and LTA in the pneumococci have identical chemical structures (18). Second, these repeating units contain the amino alcohol choline, which has been detected rarely in bacteria and is an essential growth fac...

Loop-based multiple heart-cutting (MHC) two-dimensional liquid chromatography (2D-LC) is presented as a solution to quantify target components in complex matrices, such as additives in polymers, at very high chromatographic resolution. The determination of hexabromocyclododecane (HBCD) in polystyrene (PS) is described. One dimensional ((1)D) LC analysis with UV detection did not allow quantitation of the main isomers of HBCD due to peak overlap with polymer components. MHC 2D-LC analysis provided the separation power, accuracy, and repeatability needed for quantitative analysis of the additives of interest. Heart-cuts from peaks of the (1)D-chromatogram or entire regions of interest are sampled into loops, where they remain parked until their sequential reinjection onto the second dimension ((2)D) column. A column set consisting of phenyl ((1)D) and C18 ((2)D) stationary phases gave baseline separation in (2)D between HBCD and PS background. Linearity for spiked polymer samples was achieved over a range of 0.02-1.00 wt % HBCD relative to the amount of polymer. The limit of quantitation was estimated at 0.01 wt % HBCD in PS. A peak area RSD of 0.7% obtained for ten replicates of a real sample demonstrated excellent repeatability of the analysis. MHC 2D-LC is an elegant solution for quantitative analyses of difficult-to-separate samples when conventional (1)D separation fails.

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The present contribution illustrates the utilization of a chiral × chiral two-dimensional liquid chromatography (2DLC) setup with tert-butylcarbamoyl quinine chiral stationary phase (CSP) in the first dimension (D) and tert-butylcarbamoyl quinidine CSP in the second dimension (D) to analyze FMOC-derivatized d and l amino acids from peptide hydrolysates. Hereby, in the D andD chiral separation dimensions factors such as selector and immobilization chemistry of the CSPs, mobile phase, temperature, column hardware dimensions, stationary phase supports, particle type and packing were identical. Orthogonality between D andD CSPs was solely based on their stereochemistry, i.e. their opposite configurations in two chiral centers of the selector molecules, which results in inversion of enantiomer elution orders in the two dimensions. Using Coreshell CSPs for fast chromatography allowed D-flow rates which were 60 times faster than theD-flow rates to enable online comprehensive two-dimensional chromatography (LC × LC). Due to very similar chemoselectivity, yet opposite elution orders of corresponding enantiomers in D andD, characteristic 2D-elution patterns for achiral and chiral components can be generated. Peaks of achiral components and impurities are lined up on the diagonal line in the 2D separation space (contour plot) and thereby removed from the chromatographic space of the target enantiomers avoiding overlaps with potential interferences. Corresponding enantiomers provide cross peaks on the 2D chromatogram. Moreover, enantioselectivity of both single CSPs is combined to result in an enhanced overall 2D enantioselectivity. The concept is illustrated for the therapeutic peptides gramicidin and bacitracin. Since all amino acids give a consistent elution order as FMOC-derivatives, all enantiomers of the same configuration are either above or below the diagonal line allowing straightforward imaging of the configuration of the amino acids in peptides by the 2D chromatogram.

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