A Universal Eluent System for Method Scouting and Separation of Biotherapeutic Proteins by Ion-Exchange, Size-Exclusion, and Hydrophobic Interaction Chromatography.

Characterization and quality control of biotherapeutic proteins commonly require the application of several orthogonal separation techniques in order to establish product identity and purity. Many of the techniques used rely on a buffered aqueous mobile phase system to maintain the native conformation of the protein and its variants. Optimal pH, buffer substance(s), and chromatography methods vary with each protein of interest and result in tedious method development for each new drug product. Linear controlled pH gradient systems from pH 5.6 to pH 10.2 has been shown to provide a global method for the separation of charge variants of monoclonal antibodies. This can be realized using two balanced zwitterionic buffer blends. The pH linearity of the resulting system, with a cation ion exchange column in place, can generate any pH value in this accessible pH range. This study expands the scope of this buffer system and demonstrates its application in conjunction with a quaternary HPLC pump for several analytical techniques: the pH optimization of salt gradient-based anion and cation exchange during method development, as well as performing pH gradient elution. In addition, the same universal buffers are used for hydrophobic interaction and size exclusion chromatography. This eluent system omits the need to prepare different buffers for each method and flushing of the HPLC system between method changes. The implementation of this concept is further demonstrated to allow an automated method scouting approach and selection of different methods that requires minimal manual intervention.

Site-specific glycan-conjugated NISTmAb antibody drug conjugate mimetics: synthesis, characterization, and utility.

Antibody drug conjugates (ADCs) represent a rapidly growing modality for the treatment of numerous oncology indications. The complexity of analytical characterization method development is increased due to the potential for synthetic intermediates and process-related impurities. In addition, the cytotoxicity of such materials provides an additional challenge with regard to handling products and/or sharing materials with analytical collaborators and/or vendors for technology development. Herein, we have utilized a site-specific chemoenzymatic glycoconjugation strategy for preparing ADC mimetics composed of the NIST monoclonal antibody (NISTmAb) conjugated to non-cytotoxic payloads representing both small molecules and peptides. The materials were exhaustively characterized with high-resolution mass spectrometry-based approaches to demonstrate the utility of each analytical method for confirming the conjugation fidelity as well as deep characterization of low-abundance synthetic intermediates and impurities arising from payload raw material heterogeneity. These materials therefore represent a widely available test metric to develop novel ADC analytical methods as well as a platform to discuss best practices for extensive characterization.

Potential transceptor AtNRT1.13 modulates shoot architecture and flowering time in a nitrate-dependent manner.

Compared with root development regulated by external nutrients, less is known about how internal nutrients are monitored to control plasticity of shoot development. In this study, we characterize an Arabidopsis thaliana transceptor, NRT1.13 (NPF4.4), of the NRT1/PTR/NPF family. Different from most NRT1 transporters, NRT1.13 does not have the conserved proline residue between transmembrane domains 10 and 11; an essential residue for nitrate transport activity in CHL1/NRT1.1/NPF6.3. As expected, when expressed in oocytes, NRT1.13 showed no nitrate transport activity. However, when Ser 487 at the corresponding position was converted back to proline, NRT1.13 S487P regained nitrate uptake activity, suggesting that wild-type NRT1.13 cannot transport nitrate but can bind it. Subcellular localization and ß-glucuronidase reporter analyses indicated that NRT1.13 is a plasma membrane protein expressed at the parenchyma cells next to xylem in the petioles and the stem nodes. When plants were grown with a normal concentration of nitrate, nrt1.13 showed no severe growth phenotype. However, when grown under low-nitrate conditions, nrt1.13 showed delayed flowering, increased node number, retarded branch outgrowth, and reduced lateral nitrate allocation to nodes. Our results suggest that NRT1.13 is required for low-nitrate acclimation and that internal nitrate is monitored near the xylem by NRT1.13 to regulate shoot architecture and flowering time.

New Insights into the Chromatography Mechanisms of Ion-Exchange Charge Variant Analysis: Dispelling Myths and Providing Guidance for Robust Method Optimization.

Charge variant analysis is a widely used analytical tool in characterization of monoclonal antibodies (mAbs). It depicts the heterogeneity of charge variant forms, some of which may differ by only minor modifications of a single amino acid. The analysis ensures product consistency with no unwanted changes to the protein. With increasing numbers of new mAb drug products emerging in the market, the need for a robust charge variant analysis has intensified. The charge variant profiles often display partially resolved peaks on shoulders of larger peaks. This puts considerably more pressure on the robustness of the method to maintain the suboptimum selectivity. New products and techniques have emerged to address these requirements, in addition to the pre-existing older methods that may not have been optimized correctly in the past. This has led to some confusion as to the best approach and strategies in optimization of charge variant analysis. We show studies from several different approaches using on-line pH monitoring to check the performance characteristics of the methods. This has led to new insights on the interactions between the protein, column, and buffer constituents. We dispel some inaccurate assumptions about the different ion-exchange elution mechanisms and suggest ways to develop high-throughput methods that remain robust and of high resolution. Streamlined automatable method development tools are presented that will result in more efficient method optimization. The mechanisms behind poor chromatography design have provided an alternative explanation behind some methods failing when in the QC laboratories.

In-depth analyses of native N-linked glycans facilitated by high-performance anion exchange chromatography-pulsed amperometric detection coupled to mass spectrometry.

Characterization of glycans present on glycoproteins has become of increasing importance due to their biological implications, such as protein folding, immunogenicity, cell-cell adhesion, clearance, receptor interactions, etc. In this study, the resolving power of high-performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) was applied to glycan separations and coupled to mass spectrometry to characterize native glycans released from different glycoproteins. A new, rapid workflow generates glycans from 200 µg of glycoprotein supporting reliable and reproducible annotation by mass spectrometry (MS). With the relatively high flow rate of HPAE-PAD, post-column splitting diverted 60% of the flow to a novel desalter, then to the mass spectrometer. The delay between PAD and MS detectors is consistent, and salt removal after the column supports MS. HPAE resolves sialylated (charged) glycans and their linkage and positional isomers very well; separations of neutral glycans are sufficient for highly reproducible glycoprofiling. Data-dependent MS2 in negative mode provides highly informative, mostly C- and Z-type glycosidic and cross-ring fragments, making software-assisted and manual annotation reliable. Fractionation of glycans followed by exoglycosidase digestion confirms MS-based annotations. Combining the isomer resolution of HPAE with MS2 permitted thorough N-glycan annotation and led to characterization of 17 new structures from glycoproteins with challenging glycan profiles.

Serum metabolomics strategy for understanding pharmacological effects of ShenQi pill acting on kidney yang deficiency syndrome.

Kidney yang deficiency syndrome, a diagnostic pattern in Chinese medicine, is similar with clinical features of the glucocorticoid withdrawal syndrome. The aim of this present study was to explore low molecular mass differentiating metabolites between control group and model group of kidney yang deficiency rats induced with corticosterone as well as the therapeutic effect of Shen Qi Pill, a classic traditional Chinese medicine formula for treating Kidney yang deficiency syndrome in China. This study utilized ultra-performance liquid chromatography coupled with electrospray ionization synapt quadrupole time-of-flight high definition mass spectrometry (UPLC/ESI-SYNAPT-QTOF-HDMS) to identify the underlying biomarkers for clarifying mechanism of Shen Qi Pill in treating Kidney yang deficiency syndrome based on metabolite profiling of the serum samples and in conjunction with multivariate and pathway analysis. Meanwhile, blood biochemistry assay and histopathology were examined to identify specific changes in the model group rats. Distinct changes in the pattern of metabolites were observed by UPLC-HDMS. The changes in metabolic profiling were restored to their baseline values after treatment with Shen Qi Pill according to the combined with a principal component analysis (PCA) score plots. Altogether, the current metabolomics approach based on UPLC-HDMS and orthogonal projection to latent structures discriminate analysis (OPLS-DA) demonstrated 27 ions (18 in the negative mode, 9 in the positive mode, 17 ions restored by Shen Qi Pill). These results indicated that effectiveness of Shen Qi Pill in Kidney yang deficiency syndrome rats induced a substantial change in the metabolic profiles by regulating the biomarkers and adjusting the metabolic disorder. It suggested that the metabolomics approach was a powerful approach for elucidation of pathologic changes of Chinese medicine syndrome and action mechanisms of traditional Chinese medicine.

Integration of nitrogen and potassium signaling.

Sensing and responding to soil nutrient fluctuations are vital for the survival of higher plants. Over the past few years, great progress has been made in our understanding of nitrogen and potassium signaling. Key components of the signaling pathways including sensors, kinases, miRNA, ubiquitin ligases, and transcriptional factors. These components mediate the transcriptional responses, root-architecture changes, and uptake-activity modulation induced by nitrate, ammonium, and potassium in the soil solution. Integration of these responses allows plants to compete for limited nutrients and to survive under nutrient deficiency or toxic nutrient excess. A future challenge is to extend the present fragmented sets of data to a comprehensive signaling network. Then, such knowledge and the accompanying molecular tools can be applied to improve the efficiency of nutrient utilization in crops.

Consistency of a two clinical site sample collection: a proteomics study.

PURPOSE: We investigated the ability to perform a clinical proteomic study using samples collected at different times from two independent clinical sites. EXPERIMENTAL DESIGN: Label-free 2-D-LC-MS proteomic analysis was used to differentially quantify tens of thousands of peptides from human plasma. We have asked whether samples collected from two sites, when analyzed by this type of peptide profiling, reproducibly contain detectable peptide markers that are differentially expressed in the plasma of disease (advanced renal cancer) patients relative to healthy normals. RESULTS: We have demonstrated that plasma proteins enriched in disease patients are indeed detected reproducibly in both clinical collections. Regression analysis, unsupervised hierarchical clustering and PCA detected no systematic bias in the data related to site of sample collection and processing. Using a genetic algorithm, support vector machine classification method, we were able to correctly classify disease samples at 88% sensitivity and 94% specificity using the second site as an independent validation set. CONCLUSIONS AND CLINICAL RELEVANCE: We conclude that multiple site collection, when analyzed by label-free 2-D-LC-MS, generates data that are sufficiently reproducible to guide reliable biomarker discovery.

The Arabidopsis nitrate transporter NRT1.7, expressed in phloem, is responsible for source-to-sink remobilization of nitrate.

Several quantitative trait locus analyses have suggested that grain yield and nitrogen use efficiency are well correlated with nitrate storage capacity and efficient remobilization. This study of the Arabidopsis thaliana nitrate transporter NRT1.7 provides new insights into nitrate remobilization. Immunoblots, quantitative RT-PCR, beta-glucuronidase reporter analysis, and immunolocalization indicated that NRT1.7 is expressed in the phloem of the leaf minor vein and that its expression levels increase coincidentally with the source strength of the leaf. In nrt1.7 mutants, more nitrate was present in the older leaves, less (15)NO(3)(-) spotted on old leaves was remobilized into N-demanding tissues, and less nitrate was detected in the phloem exudates of old leaves. These data indicate that NRT1.7 is responsible for phloem loading of nitrate in the source leaf to allow nitrate transport out of older leaves and into younger leaves. Interestingly, nrt1.7 mutants showed growth retardation when external nitrogen was depleted. We conclude that (1) nitrate itself, in addition to organic forms of nitrogen, is remobilized, (2) nitrate remobilization is important to sustain vigorous growth during nitrogen deficiency, and (3) source-to-sink remobilization of nitrate is mediated by phloem.

CHL1 functions as a nitrate sensor in plants.

Ions serve as essential nutrients in higher plants and can also act as signaling molecules. Little is known about how plants sense changes in soil nutrient concentrations. Previous studies showed that T101-phosphorylated CHL1 is a high-affinity nitrate transporter, whereas T101-dephosphorylated CHL1 is a low-affinity transporter. In this study, analysis of an uptake- and sensing-decoupled mutant showed that the nitrate transporter CHL1 functions as a nitrate sensor. Primary nitrate responses in CHL1T101D and CHLT101A transgenic plants showed that phosphorylated and dephosphorylated CHL1 lead to a low- and high-level response, respectively. In vitro and in vivo studies showed that, in response to low nitrate concentrations, protein kinase CIPK23 can phosphorylate T101 of CHL1 to maintain a low-level primary response. Thus, CHL1 uses dual-affinity binding and a phosphorylation switch to sense a wide range of nitrate concentrations in the soil, thereby functioning as an ion sensor in higher plants. For a video summary of this article, see the PaperFlick file with the Supplemental Data available online.

Mutation of the Arabidopsis NRT1.5 nitrate transporter causes defective root-to-shoot nitrate transport.

Little is known about the molecular and regulatory mechanisms of long-distance nitrate transport in higher plants. NRT1.5 is one of the 53 Arabidopsis thaliana nitrate transporter NRT1 (Peptide Transporter PTR) genes, of which two members, NRT1.1 (CHL1 for Chlorate resistant 1) and NRT1.2, have been shown to be involved in nitrate uptake. Functional analysis of cRNA-injected Xenopus laevis oocytes showed that NRT1.5 is a low-affinity, pH-dependent bidirectional nitrate transporter. Subcellular localization in plant protoplasts and in planta promoter-beta-glucuronidase analysis, as well as in situ hybridization, showed that NRT1.5 is located in the plasma membrane and is expressed in root pericycle cells close to the xylem. Knockdown or knockout mutations of NRT1.5 reduced the amount of nitrate transported from the root to the shoot, suggesting that NRT1.5 participates in root xylem loading of nitrate. However, root-to-shoot nitrate transport was not completely eliminated in the NRT1.5 knockout mutant, and reduction of NRT1.5 in the nrt1.1 background did not affect root-to-shoot nitrate transport. These data suggest that, in addition to that involving NRT1.5, another mechanism is responsible for xylem loading of nitrate. Further analyses of the nrt1.5 mutants revealed a regulatory loop between nitrate and potassium at the xylem transport step.

Quantification of intermediate-abundance proteins in serum by multiple reaction monitoring mass spectrometry in a single-quadrupole ion trap.

A method is presented to quantify intermediate-abundance proteins in human serum using a single-quadrupole linear ion trap mass spectrometer-in contrast, for example, to a triple-quadrupole mass spectrometer. Stable-isotope-labeled (tryptic) peptides are spiked into digested protein samples as internal standards, aligned with the traditional isotope dilution approach. As a proof-of-concept experiment, four proteins of intermediate abundance were selected, coagulation factor V, adiponectin, C-reactive protein (CRP), and thyroxine binding globulin. Stable-isotope-labeled peptides were synthesized with one tryptic sequence from each of these proteins. The normal human serum concentration ranges of these proteins are from 1 to 30 microg/mL (or 20 to 650 pmol/mL). These labeled peptides and their endogenous counterparts were analyzed by LC-MS/MS using multiple reaction monitoring, a multiplexed form of the selected reaction monitoring technique. For these experiments, only one chromatographic dimension (on-line reversed-phase capillary column) was used. Improved limits of detection will result with multidimensional chromatographic methods utilizing more material per sample. Standard curves of the spiked calibrants were generated with concentrations ranging from 3 to 700 pmol/mL using both neat solutions and peptides spiked into the complex matrix of digested serum protein solution where ion suppression effects and interferences are common. Endogenous protein concentrations were determined by comparing MS/MS peak areas of the endogenous peptides to the isotopically labeled internal calibrants. The derived concentrations from a normal human serum pool (neglecting loss of material during sample processing) were 9.2, 110, 120, and 246 pmol/mL for coagulation factor V, adiponectin, CRP, and thyroxine binding globulin, respectively. These concentrations generally agree with the reported normal ranges for these proteins. As a measure of analytical reproducibility of this single-quadrupole assay, the coefficients of variance based on 12 repeated measurements for each of the endogenous tryptic peptides were 17.0, 25.4, 24.2, and 14.0% for coagulation factor V, adiponectin, CRP, and thyroxine binding globulin, respectively.

Reducing protein concentration range of biological samples using solid-phase ligand libraries.

The discovery of specific polypeptides of diagnostic relevance from a biological liquid is complicated by the overall vast number and the large concentration range of all polypeptides/proteins in the sample. Depletion or fractionation methodologies have been used for selectively removing abundant proteins; however, they failed to significantly enrich trace proteins. Here we expand upon a new method that allows the reduction of the protein concentration range within a complex mixture, like neat serum, through the simultaneous dilution of high abundance proteins and the concentration of low abundance ones in a single, simple step. This methodology utilizes solid-phase ligand libraries of large diversity. With a controlled sample-to-ligand ratio it is possible to modulate the relative concentration of proteins such that a large number of peptides or proteins that are normally not detectable by classical analytical methods become, easily detectable. Application of this method for reducing the dynamic range of unfractionated serum is specifically described along with treatment of other biological extracts. Analytical surface enhanced laser desorption/ionization mass spectrometry (SELDI-MS) technology and mono- and two-dimensional electrophoresis (1-DE and 2-DE) demonstrate the increase in the number of proteins detected. Examples linking this approach with additional fractionation methods demonstrate a further increase in the number of detectable species using either the so-called "top down" or "bottom up" approaches for proteomics analysis. By enabling the detection of a greater proportion of polypeptides/proteins within a sample, this method may contribute significantly towards the discovery of new biomarkers of diagnostic relevance.

Reduction of the concentration difference of proteins in biological liquids using a library of combinatorial ligands.

The discovery of polypeptides and proteins with relevance to a particular biological state is complicated by their vast number and concentration range in most biological mixtures. Depletion methodologies are frequently used to remove the most abundant species; however, this removal not only fails significantly to enrich trace proteins, it may also nonspecifically deplete them due to their interactions with the removed high-abundance proteins. Here we report a simple-to-use methodology that reduces the protein concentration range of a complex mixture like whole serum through the simultaneous dilution of high-abundance proteins and the concentration of low-abundance proteins. This methodology utilizes solid-phase ligand libraries of immense diversity, generated by "split, couple, recombine" combinatorial chemistry, that are used for affinity-based binding to the proteins of a given mixture. With a controlled sample-to-ligand ratio it is possible to modulate the relative concentration of proteins such that many peptides or proteins that are undetectable by classical analytical methods become easily accessible. The reduction in the dynamic range of unfractionated serum is specifically described along with treatment of other proteomes such as extracts from Escherichia coli, chicken egg white and cell culture supernatant. Mono- and bi-dimensional electrophoresis (1-DE and 2-DE respectively) and surface-enhanced laser desorption/ionization-mass spectrometry (SELDI-TOF-MS) technology demonstrate the reduction in protein concentration range. Combining this approach with additional fractionation methods further increased the number of detectable species.

Classification of cancer types by measuring variants of host response proteins using SELDI serum assays.

Protein expression profiling has been increasingly used to discover and characterize biomarkers that can be used for diagnostic, prognostic or therapeutic purposes. Most proteomic studies published to date have identified relatively abundant host response proteins as candidate biomarkers, which are often dismissed because of an apparent lack of specificity. We demonstrate that 2 host response proteins previously identified as candidate markers for early stage ovarian cancer, transthyretin and inter-alpha trypsin inhibitor heavy chain 4 (ITIH4), are posttranslationally modified. These modifications include proteolytic truncation, cysteinylation and glutathionylation. Assays using Surface Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry (SELDI-TOF-MS) may provide a means to confer specificity to these proteins because of their ability to detect and quantitate multiple posttranslationally modified forms of these proteins in a single assay. Quantitative measurements of these modifications using chromatographic and antibody-based ProteinChip array assays reveal that these posttranslational modifications occur to different extents in different cancers and that multivariate analysis permits the derivation of algorithms to improve the classification of these cancers. We have termed this process host response protein amplification cascade (HRPAC), since the process of synthesis, posttranslational modification and metabolism of host response proteins amplifies the signal of potentially low-abundant biologically active disease markers such as enzymes.

A mannosyl transferase required for lipopolysaccharide inner core assembly in Rhizobium leguminosarum. Purification, substrate specificity, and expression in Salmonella waaC mutants.

The lipopolysaccharide (LPS) core domain of Gram-negative bacteria plays an important role in outer membrane stability and host interactions. Little is known about the biochemical properties of the glycosyltransferases that assemble the LPS core. We now report the purification and characterization of the Rhizobium leguminosarum mannosyl transferase LpcC, which adds a mannose unit to the inner 3-deoxy-d-manno-octulosonic acid (Kdo) moiety of the LPS precursor, Kdo(2)-lipid IV(A). LpcC containing an N-terminal His(6) tag was assayed using GDP-mannose as the donor and Kdo(2)-[4'-(32)P]lipid IV(A) as the acceptor and was purified to near homogeneity. Sequencing of the N terminus confirmed that the purified enzyme is the lpcC gene product. Mild acid hydrolysis of the glycolipid generated in vitro by pure LpcC showed that the mannosylation occurs on the inner Kdo residue of Kdo(2)-[4'-(32)P]lipid IV(A). A lipid acceptor substrate containing two Kdo moieties is required by LpcC, since no activity is seen with lipid IV(A) or Kdo-lipid IV(A). The purified enzyme can use GDP-mannose or, to a lesser extent, ADP-mannose (both of which have the alpha-anomeric configuration) for the glycosylation of Kdo(2)-[4'-(32)P]lipid IV(A). Little or no activity is seen with ADP-glucose, UDP-glucose, UDP-GlcNAc, or UDP-galactose. A Salmonella typhimurium waaC mutant, which lacks the enzyme for incorporating the inner l-glycero-d-manno-heptose moiety of LPS, regains LPS with O-antigen when complemented with lpcC. An Escherichia coli heptose-less waaC-waaF deletion mutant expressing the R. leguminosarum lpcC gene likewise generates a hybrid LPS species consisting of Kdo(2)-lipid A plus a single mannose residue. Our results demonstrate that heterologous lpcC expression can be used to modify the structure of the Salmonella and E. coli LPS cores in living cells.

An outer membrane enzyme that generates the 2-amino-2-deoxy-gluconate moiety of Rhizobium leguminosarum lipid A.

The structures of Rhizobium leguminosarum and Rhizobium etli lipid A are distinct from those found in other Gram-negative bacteria. Whereas the more typical Escherichia coli lipid A is a hexa-acylated disaccharide of glucosamine that is phosphorylated at positions 1 and 4', R. etli and R. leguminosarum lipid A consists of a mixture of structurally related species (designated A-E) that lack phosphate. A conserved distal unit, comprised of a diacylated glucosamine moiety with galacturonic acid residue at position 4' and a secondary 27-hydroxyoctacosanoyl (27-OH-C28) as part of a 2' acyloxyacyl moiety, is present in all five components. The proximal end is heterogeneous, differing in the number and lengths of acyl chains and in the identity of the sugar itself. A proximal glucosamine unit is present in B and C, but an unusual 2-amino-2-deoxy-gluconate moiety is found in D-1 and E. We now demonstrate that membranes of R. leguminosarum and R. etli can convert B to D-1 in a reaction that requires added detergent and is inhibited by EDTA. Membranes of Sinorhizobium meliloti and E. coli lack this activity. Mass spectrometry demonstrates that B is oxidized in vitro to a substance that is 16 atomic mass units larger, consistent with the formation of D-1. The oxidation of the lipid A proximal unit is also demonstrated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry in the positive and negative modes using the model substrate, 1-dephospho-lipid IV(A). With this material, an additional intermediate (or by product) is detected that is tentatively identified as a lactone derivative of 1-dephospho-lipid IV(A). The enzyme, presumed to be an oxidase, is located exclusively in the outer membrane of R. leguminosarum as judged by sucrose gradient analysis. To our knowledge, an oxidase associated with the outer membranes of Gram-negative bacteria has not been reported previously.

An Escherichia coli mutant lacking the cold shock-induced palmitoleoyltransferase of lipid A biosynthesis: absence of unsaturated acyl chains and antibiotic hypersensitivity at 12 degrees C.

An acyltransferase induced by cold shock in Escherichia coli, designated LpxP, incorporates a palmitoleoyl moiety into nascent lipid A in place of the secondary laurate chain normally added by LpxL(HtrB) (Carty, S. M., Sreekumar, K. R., and Raetz, C. R. H. (1999) J. Biol. Chem. 274, 9677-9685). To determine whether the palmitoleoyl residue alters the properties of the outer membrane and imparts physiological benefits at low growth temperatures, we constructed a chromosomal insertion mutation in lpxP, the structural gene for the transferase. Membranes from the lpxP mutant MKV11 grown at 12 degrees C lacked the cold-induced palmitoleoyltransferase present in membranes of cold-shocked wild type cells but retained normal levels of the constitutive lauroyltransferase encoded by lpxL. When examined by mass spectrometry, about two-thirds of the lipid A molecules isolated from wild type E. coli grown at 12 degrees C contained palmitoleate in place of laurate, whereas the lipid A of cold-adapted MKV11 contained only laurate in amounts comparable with those seen in wild type cells grown at 30 degrees C or above. To probe the integrity of the outer membrane, MKV11 and an isogenic wild type strain were grown at 30 or 12 degrees C and then tested for their susceptibility to antibiotics. MKV11 exhibited a 10-fold increase in sensitivity to rifampicin and vancomycin at 12 degrees C compared with wild type cells but showed identical resistance when grown at 30 degrees C. We suggest that the palmitoleoyltransferase may confer a selective advantage upon E. coli cells growing at lower temperatures by making the outer membrane a more effective barrier to harmful chemicals.