Outer Membrane Defect Research Articles

A hierarchical approach towards identification of novel inhibitors against L, D-transpeptidase YcbB as an anti-bacterial therapeutic target

The bacterial cell wall, being a vital component for cell viability, is regarded as a promising drug target. The L, D-Transpeptidase YcbB enzyme has been implicated for a significant role in cell wall polymers cross linking during typhoid toxin release, β-lactam resistance and outer membrane defect rescue. These observations have been recorded in different bacterial pathogens such as Salmonella Typhimurium, Citrobacter rodentium, and Salmonella typhi. In this work, we have shown structure based virtual screening of diverse natural and synthetic drug libraries against the enzyme and revealed three compounds as LAS_32135590, LAS_34036730 and LAS-51380924. These compounds showed highly stable energies and the findings are very competitive with the control molecule ((1RG or (4 R,5S)-3-({(3S,5S)-5-[(3-carboxyphenyl)carbamoyl]pyrrolidin-3-yl}sulfanyl)-5-[(1S,2R)-1-formyl-2-hydroxypropyl]-4-methyl-4,5-dihydro-1H-pyrrole-2-carboxylic acid or ertapenem)) used. Compared to control (which has binding energy score of −11.63 kcal/mol), the compounds showed better binding energy. The binding energy score of LAS_32135590, LAS_34036730 and LAS-51380924 is −12.63 kcal/mol, −12.22 kcal/mol and −12.10 kcal/mol, respectively. Further, the docked snapshot of the lead compounds and control were investigated for stability under time dependent dynamics environment. All the three leads complex and control system showed significant equilibrium (mean RMSD < 3 Å) both in term of intermolecular docked conformation and binding interactions network. Further validation on the complex’s stability was acquired from the end-state MMPB/GBSA analysis that observed greater contribution from van der Waals forces and electrostatic energy while less contribution was noticed from solvation part. The compounds were also showed good drug-likeness and are non-toxic and non-mutagenic. In short, the compounds can be used in experimental testing’s and might be subjected to structure modification to get better results. Communicated by Ramaswamy H. Sarma

The role of CenKR in the coordination of Rhodobacter sphaeroides cell elongation and division.

Cell elongation and division are essential aspects of the bacterial life cycle that must be coordinated for viability and replication. The impact of misregulation of these processes is not well understood as these systems are often not amenable to traditional genetic manipulation. Recently, we reported on the CenKR two-component system (TCS) in the Gram-negative bacterium Rhodobacter sphaeroides that is genetically tractable, widely conserved in α-proteobacteria, and directly regulates the expression of components crucial for cell elongation and division, including genes encoding subunit of the Tol-Pal complex. In this work, we show that overexpression of cenK results in cell filamentation and chaining. Using cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET), we generated high-resolution two-dimensional (2D) images and three-dimensional (3D) volumes of the cell envelope and division septum of wild-type cells and a cenK overexpression strain finding that these morphological changes stem from defects in outer membrane (OM) and peptidoglycan (PG) constriction. By monitoring the localization of Pal, PG biosynthesis, and the bacterial cytoskeletal proteins MreB and FtsZ, we developed a model for how increased CenKR activity leads to changes in cell elongation and division. This model predicts that increased CenKR activity decreases the mobility of Pal, delaying OM constriction, and ultimately disrupting the midcell positioning of MreB and FtsZ and interfering with the spatial regulation of PG synthesis and remodeling.IMPORTANCEBy coordinating cell elongation and division, bacteria maintain their shape, support critical envelope functions, and orchestrate division. Regulatory and assembly systems have been implicated in these processes in some well-studied Gram-negative bacteria. However, we lack information on these processes and their conservation across the bacterial phylogeny. In R. sphaeroides and other α-proteobacteria, CenKR is an essential two-component system (TCS) that regulates the expression of genes known or predicted to function in cell envelope biosynthesis, elongation, and/or division. Here, we leverage unique features of CenKR to understand how increasing its activity impacts cell elongation/division and use antibiotics to identify how modulating the activity of this TCS leads to changes in cell morphology. Our results provide new insight into how CenKR activity controls the structure and function of the bacterial envelope, the localization of cell elongation and division machinery, and cellular processes in organisms with importance in health, host-microbe interactions, and biotechnology.

Caulobacter lipid A is conditionally dispensable in the absence of fur and in the presence of anionic sphingolipids.

SUMMARYLipid A, the membrane-anchored portion of lipopolysaccharide (LPS), is an essential component of the outer membrane (OM) of nearly all Gram-negative bacteria. Here we identify regulatory and structural factors that together render lipid A nonessential in Caulobacter crescentus. Mutations in the ferric uptake regulator fur allow Caulobacter to survive in the absence of either LpxC, which catalyzes an early step of lipid A synthesis, or CtpA, a tyrosine phosphatase homolog we find is needed for wild-type lipid A structure and abundance. Alterations in Fur-regulated processes, rather than iron status per se, underlie the ability to survive when lipid A synthesis is blocked. Fitness of lipid A-deficient Caulobacter requires an anionic sphingolipid, ceramide phosphoglycerate (CPG), which also mediates sensitivity to the antibiotic colistin. Our results demonstrate that, in an altered regulatory landscape, anionic sphingolipids can support the integrity of a lipid A-deficient OM.

Hyperproduction of PHA copolymers containing high fractions of 4-hydroxybutyrate (4HB) by outer membrane-defected Halomonas bluephagenesis grown in bioreactors.

SummaryBacterial outer membrane (OM) is a self‐protective and permeable barrier, while having many non‐negligible negative effects in industrial biotechnology. Our previous studies revealed enhanced properties of Halomonas bluephagenesis based on positive cellular properties by OM defects. This study further expands the OM defect on membrane compactness by completely deleting two secondary acyltransferases for lipid A modification in H. bluephagenesis, LpxL and LpxM, and found more significant advantages than that of the previous lpxL mutant. Deletions on LpxL and LpxM accelerated poly(3‐hydroxybutyrate) (PHB) production by H. bluephagenesis WZY229, leading to a 37% increase in PHB accumulation and 84‐folds reduced endotoxin production. Enhanced membrane permeability accelerates the diffusion of γ‐butyrolactone, allowing H. bluephagenesis WZY254 derived from H. bluephagenesis WZY229 to produce 82wt% poly(3‐hydroxybutyrate‐co‐23mol%4‐hydroxybutyrate) (P(3HB‐co‐23mol%4HB)) in shake flasks, showing increases of 102% and 307% in P(3HB‐co‐4HB) production and 4HB accumulation, respectively. The 4HB molar fraction in copolymer can be elevated to 32 mol% in the presence of more γ‐butyrolactone. In a 7‐l bioreactor fed‐batch fermentation, H. bluephagenesis WZY254 supported a 84 g l−1 dry cell mass with 81wt% P(3HB‐co‐26mol%4HB), increasing 136% in 4HB molar fraction. This study further demonstrated that OM defects generate a hyperproduction strain for high 4HB containing copolymers.

Structural and Cellular Insights into the l,d-Transpeptidase YcbB as a Therapeutic Target in Citrobacter rodentium, Salmonella Typhimurium, and Salmonella Typhi Infections.

The bacterial cell wall plays a key role in viability and is an important drug target. The cell wall is made of elongated polymers that are cross-linked to one another to form a load-bearing mesh. An alternative cell wall cross-linking mechanism used by the l,d-transpeptidase YcbB has been implicated in the stress-regulated roles of β-lactam resistance, outer membrane defect rescue, and typhoid toxin release. The role for this stress-linked cross-linking in the context of a host infection was unclear. Here, we resolve the crystallographic structures of both Salmonella Typhi YcbB and Citrobacter rodentium YcbB acylated with ertapenem that delineate the conserved structural characteristics of YcbB. In parallel, we show that the general involvement of YcbB in peptidoglycan reinforcement under conditions of bacterial outer envelope stress does not play a significant role in acute infections of mice by C. rodentium and S Typhimurium. Cumulatively, in this work we provide a foundation for the development of novel YcbB-specific antibacterial therapeutics to assist in treatment of increasingly drug-resistant S Typhi infections.

Overexpression of lpxT Gene in Escherichia coli Inhibits Cell Division and Causes Envelope Defects without Changing the Overall Phosphorylation Level of Lipid A.

LpxT is an inner membrane protein that transfers a phosphate group from the essential lipid undecaprenyl pyrophosphate (C-55PP) to the lipid A moiety of lipopolysaccharide, generating a lipid A tris-phosphorylated species. The protein is encoded by the non-essential lpxT gene, which is conserved in distantly related Gram-negative bacteria. In this work, we investigated the phenotypic effect of lpxT ectopic expression from a plasmid in Escherichia coli. We found that lpxT induction inhibited cell division and led to the formation of elongated cells, mostly with absent or altered septa. Moreover, the cells became sensitive to detergents and to hypo-osmotic shock, indicating that they had cell envelope defects. These effects were not due to lipid A hyperphosphorylation or C-55PP sequestering, but most likely to defective lipopolysaccharide transport. Indeed, lpxT overexpression in mutants lacking the L,D-transpeptidase LdtD and LdtE, which protect cells with outer membrane defects from osmotic lysis, caused cell envelope defects. Moreover, we found that pyrophosphorylated lipid A was also produced in a lpxT deletion mutant, indicating that LpxT is not the only protein able to perform such lipid A modification in E. coli.

The Acinetobacter baumannii Mla system and glycerophospholipid transport to the outer membrane.

The outer membrane (OM) of Gram-negative bacteria serves as a selective permeability barrier that allows entry of essential nutrients while excluding toxic compounds, including antibiotics. The OM is asymmetric and contains an outer leaflet of lipopolysaccharides (LPS) or lipooligosaccharides (LOS) and an inner leaflet of glycerophospholipids (GPL). We screened Acinetobacter baumannii transposon mutants and identified a number of mutants with OM defects, including an ABC transporter system homologous to the Mla system in E. coli. We further show that this opportunistic, antibiotic-resistant pathogen uses this multicomponent protein complex and ATP hydrolysis at the inner membrane to promote GPL export to the OM. The broad conservation of the Mla system in Gram-negative bacteria suggests the system may play a conserved role in OM biogenesis. The importance of the Mla system to Acinetobacter baumannii OM integrity and antibiotic sensitivity suggests that its components may serve as new antimicrobial therapeutic targets.

Francisella tularensis D-Ala D-Ala Carboxypeptidase DacD Is Involved in Intracellular Replication and It Is Necessary for Bacterial Cell Wall Integrity.

D-alanyl-D-alanine carboxypeptidase, product of dacD gene in Francisella, belongs to penicillin binding proteins (PBPs) and is involved in remodeling of newly synthetized peptidoglycan. In E. coli, PBPs are synthetized in various growth phases and they are able to substitute each other to a certain extent. The DacD protein was found to be accumulated in fraction enriched in membrane proteins from severely attenuated dsbA deletion mutant strain. It has been presumed that the DsbA is not a virulence factor by itself but that its substrates, whose correct folding and topology are dependent on the DsbA oxidoreductase and/or isomerase activities, are the primary virulence factors. Here we demonstrate that Francisella DacD is required for intracellular replication and virulence in mice. The dacD insertion mutant strain showed higher sensitivity to acidic pH, high temperature and high osmolarity when compared to the wild-type. Eventually, transmission electron microscopy revealed differences in mutant bacteria in both the size and defects in outer membrane underlying its SDS and serum sensitivity. Taken together these results suggest DacD plays an important role in Francisella pathogenicity.

Mutation and Suppressor Analysis of the Essential Lipopolysaccharide Transport Protein LptA Reveals Strategies To Overcome Severe Outer Membrane Permeability Defects in Escherichia coli.

In Gram-negative bacteria, lipopolysaccharide (LPS) contributes to the robust permeability barrier of the outer membrane (OM), preventing the entry of toxic molecules, such as detergents and antibiotics. LPS is transported from the inner membrane (IM) to the OM by the Lpt multiprotein machinery. Defects in LPS transport compromise LPS assembly at the OM and result in increased antibiotic sensitivity. LptA is a key component of the Lpt machine that interacts with the IM protein LptC and chaperones LPS through the periplasm. We report here the construction of lptA41, a quadruple mutant in four conserved amino acids potentially involved in LPS or LptC binding. Although viable, the mutant displays increased sensitivity to several antibiotics (bacitracin, rifampin, and novobiocin) and the detergent SDS, suggesting that lptA41 affects LPS transport. Indeed, lptA41 is defective in Lpt complex assembly, and its lipid A carries modifications diagnostic of LPS transport defects. We also selected and characterized two phenotypic bacitracin-resistant suppressors of lptA41 One mutant, in which only bacitracin sensitivity is suppressed, harbors a small in-frame deletion in mlaA, which codes for an OM lipoprotein involved in maintaining OM asymmetry by reducing accumulation of phospholipids in the outer leaflet. The other mutant, in which bacitracin, rifampin, and SDS sensitivity is suppressed, harbors an additional amino acid substitution in LptA41 and a nonsense mutation in opgH, encoding a glycosyltransferase involved in periplasmic membrane-derived oligosaccharide synthesis. Characterization of the suppressor mutants highlights different strategies adopted by the cell to overcome OM defects caused by impaired LPS transport.IMPORTANCE Lipopolysaccharide (LPS) is the major constituent of the outer membrane (OM) of most Gram-negative bacteria, forming a barrier against antibiotics. LPS is synthesized at the inner membrane (IM), transported across the periplasm, and assembled at the OM by the multiprotein Lpt complex. LptA is the periplasmic component of the Lpt complex, which bridges IM and OM and ferries LPS across the periplasm. How the cell coordinates the processes involved in OM biogenesis is not completely understood. We generated a mutant partially defective in lptA that exhibited increased sensitivity to antibiotics and selected for suppressors of the mutant. The analysis of two independent suppressors revealed different strategies adopted by the cell to overcome defects in LPS biogenesis.

Outer membrane defect and stronger biofilm formation caused by inactivation of a gene encoding for heptosyltransferase I in Cronobacter sakazakii ATCC BAA-894

To investigate the role of lipopolysaccharide (LPS) structure in the stability of outer membrane and the ability of biofilm formation in Cronobacter sakazakii. A C. sakazakii mutant strain LWW02 was constructed by inactivating the gene ESA_04107 encoding for heptosyltransferase I. LPS were purified from LWW02, and changes in their structure were confirmed by thin-layer chromatography and electrospray ionization mass spectrometry. Comparing with the wild-type strain BAA-894, slower growth, higher membrane permeability, higher surface hydrophobicity, stronger ability of autoaggregation and biofilm formation were observed for the mutant strain LWW02. The gene ESA_04107 encodes heptosyltransferase I in C. sakazakii ATCC BAA-894. The cleavage of LPS in C. sakazakii could cause its outer membrane defects and increase its ability to form biofilms. The study is important for understanding the pathogenic mechanism and efficient control of C. sakazakii.

Selective Ribosome Profiling Reveals the Cotranslational Chaperone Action of Trigger Factor In Vivo

As nascent polypeptides exit ribosomes, they are engaged by a series of processing, targeting, and folding factors. Here, we present a selective ribosome profiling strategy that enables global monitoring of when these factors engage polypeptides in the complex cellular environment. Studies of the Escherichia coli chaperone trigger factor (TF) reveal that, though TF can interact with many polypeptides, β-barrel outer-membrane proteins are the most prominent substrates. Loss of TF leads to broad outer-membrane defects and premature, cotranslational protein translocation. Whereas in vitro studies suggested that TF is prebound to ribosomes waiting for polypeptides to emerge from the exit channel, we find that in vivo TF engages ribosomes only after ~100 amino acids are translated. Moreover, excess TF interferes with cotranslational removal of the N-terminal formyl methionine. Our studies support a triaging model in which proper protein biogenesis relies on the fine-tuned, sequential engagement of processing, targeting, and folding factors.

Escherichia coli tat mutant strains are able to transport maltose in the absence of an active malE gene

The twin-arginine transport (Tat) system is a prokaryotic protein transport system. Escherichia coli mutants in this pathway show a defect in cell separation during cell division, resulting in destabilization and permeability of the outer membrane. Maltose uptake is catalysed by a membrane-bound transporter of the ATP binding cassette (ABC) superfamily, where MalE is the essential periplasmic binding protein component. Here, we report that tat mutants are unexpectedly able to transport maltose in the absence of malE. This observation is specific to the MalE component since co-inactivation of malF, which encodes one of the channel components of the transporter, completely abolishes maltose transport even when the Tat system is inactivated. Genetic repair of the outer membrane leaky phenotype of the tat mutant strain re-established the absolute requirement for MalE in maltose uptake. In addition, we demonstrate that phenotypic repair of the outer membrane defect of the tat strain can also be achieved chemically by the inclusion of high concentrations of calcium or magnesium in the growth medium.

Role of the Omp25/Omp31 Family in Outer Membrane Properties and Virulence ofBrucella ovis

The genes coding for the five outer membrane proteins (OMPs) of the Omp25/Omp31 family expected to be located in the outer membrane (OM) of rough virulent Brucella ovis PA were inactivated to evaluate their role in virulence and OM properties. The OM properties of the mutant strains and of the mutants complemented with the corresponding wild-type genes were analyzed, in comparison with the parental strain and rough B. abortus RB51, in several tests: (i) binding of anti-Omp25 and anti-Omp31 monoclonal antibodies, (ii) autoagglutination of bacterial suspensions, and (iii) assessment of susceptibility to polymyxin B, sodium deoxycholate, hydrogen peroxide, and nonimmune ram serum. A tight balance of the members of the Omp25/Omp31 family was seen to be essential for the stability of the B. ovis OM, and important differences between the OMs of B. ovis PA and B. abortus RB51 rough strains were observed. Regarding virulence, the absence of Omp25d and Omp22 from the OM of B. ovis PA led to a drastic reduction in spleen colonization in mice. While the greater susceptibility of the Deltaomp22 mutant to nonimmune serum and its difficulty in surviving in the stationary phase might be on the basis of its dramatic attenuation, no defects in the OM able to explain the attenuation of the Deltaomp25d mutant were found, especially considering that the fully virulent Deltaomp25c mutant displayed more important OM defects. Accordingly, Omp25d, and perhaps Omp22, could be directly involved in the penetration and/or survival of B. ovis inside host cells. This aspect, together with the role of Omp25d and Omp22 in the virulence both of B. ovis in rams and of other Brucella species, should be thoroughly evaluated in future studies.

Role of the Escherichia coli Tat pathway in outer membrane integrity.

The Escherichia coli Tat system serves to export folded proteins harbouring an N-terminal twin-arginine signal peptide across the cytoplasmic membrane. Previous work has demonstrated that strains mutated in genes encoding essential Tat pathway components are highly defective in the integrity of their cell envelope. Here, we report the isolation, by transposon mutagenesis, of tat mutant strains that have their outer membrane integrity restored. This outer membrane repair of the tat mutant arises as a result of upregulation of the amiB gene, which encodes a cell wall amidase. Overexpression of the genes encoding the two additional amidases, amiA and amiC, does not compensate for the outer membrane defect of the tatC strain. Analysis of the amiA and amiC coding sequences indicates that the proteins may be synthesized with plausible twin-arginine signal sequences, and we demonstrate that they are translocated to the periplasm by the Tat pathway. A Tat+ strain that has mislocalized AmiA and AmiC proteins because of deletion of their signal peptides displays an identical defective cell envelope phenotype. The presence of genes encoding amidases with twin-arginine signal sequences in the genomes of other Gram-negative bacteria suggests that a similar cell envelope defect may be a common feature of tat mutant strains.

Escherichia coli strains blocked in Tat-dependent protein export exhibit pleiotropic defects in the cell envelope.

The Tat system is a recently discovered protein export pathway that serves to translocate folded proteins, often containing redox cofactors, across the bacterial cytoplasmic membrane. Here we report that tat strains are associated with a mutant cell septation phenotype, where chains of up to 10 cells are evident. Mutant strains are also hypersensitive to hydrophobic drugs and to lysis by lysozyme in the absence of EDTA, and they leak periplasmic enzymes, characteristics that are consistent with an outer membrane defect. Both phenotypes are similar to those displayed by strains carrying point mutations in the lpxC (envA) gene. The phenotype was not replicated by mutations affecting synthesis and/or activity of all known or predicted Tat substrates.

Outer membrane translocation arrest of the TcpA pilin subunit in rfb mutants of Vibrio cholerae O1 strain 569B.

The toxin-coregulated pilus (TCP) of Vibrio cholerae is a type 4-related fimbrial adhesin and a useful model for the study of type 4 pilus biogenesis and related bacterial macromolecular transport pathways. Transposon mutagenesis of the putative perosamine biosynthesis genes in the rfb operon of V. cholerae 569B eliminates lipopolysaccharide (LPS) O-antigen biosynthesis but also leads to a specific defect in TCP export. Localization of TcpA is made difficult by the hydrophobic nature of this bundle-forming pilin, which floats anomalously in sucrose density gradients, but the processed form of TcpA can be found in membrane and periplasmic fractions prepared from these strains. While TcpA cannot be detected by surface immunogold labelling in transmission electron microscope preparations, EDTA pretreatment facilitates immunofluorescent antibody labelling of whole cells, and ultrathin cryosectioning techniques confirm membrane and periplasmic accumulation of TcpA. Salt and detergent extraction, protease accessibility, and chemical cross-linking experiments suggest that although TcpA has not been assembled on the cell surface, subunit interactions are otherwise identical to those within TCP. In addition, TcpA-mediated fucose-resistant hemagglutination of murine erythrocytes is preserved in whole-cell lysates, suggesting that TcpA has obtained its mature conformation. These data localize a stage of type 4 pilin translocation to the outer membrane, at which stage export failure leads to the accumulation of pilin subunits in a configuration similar to that within the mature fiber. Possible candidates for the outer membrane defect are discussed.

Effect of Ssc protein mutations on the outer membrane permeability barrier function in Salmonella typhimurium: a study using ssc mutant alleles made by site-directed mutagenesis

We have previously discovered and characterized a novel essential enterobacterial protein, the Ssc protein of Salmonella typhimurium and found that the mutation rmVal 291 → Met in this protein inhibits bacterial growth at 42°C and the function of its outer membrane permeability barrier at 37°C [7]. In the present paper we prepared, by site-directed mutagenesis, a series of novel plasmid-encoded Ssc mutant proteins and tested their ability to compensate the loss of wild-type Ssc. The mutant proteins Met 288 → Lys and Gly 289 → Asp completely lacked this ability, and accordingly, were very defective. Ssc mutants Met 288 → LeuMet 290 → Lys, and Met 292 → Lys were partially defective. Mutants Met 290 → Leu and Met 292 → Leu were non-defective as were also four randomly made mutant proteins with mutations outside the 288–292 region. The S. typhimurium derivative which contained both the chromosomally encoded Ssc Val 291 → Met and the plasmid-encoded Ssc Gly 289 → Asp had an outer membrane defect more severe than that caused by Ssc Met291 only. The mutant Ssc proteins had very little, if any, effect on the outer membrane function in the presence of wild-type Ssc. Even though the function of Ssc is not yet known, our results indicate that region 288–292 is important and that Ssc Asp289 is thus far the most defective mutant Ssc.

Tetracyclines of various hydrophobicities as a probe for permeability of Escherichia coli outer membranes.

The outer membrane of gram-negative cells excludes hydrophobic molecules and is responsible for the resistance of these cells to a number of dyes, detergents, and antibiotics. We describe a test for hydrophobic permeability in which a series of tetracyclines with various hydrophobicities are used. Normal Escherichia coli cells became more resistant as hydrophobicity was increased in this series, but mutants altered in outer membrane permeability remained susceptible. A mutant lacking all polysaccharide except 2-keto-3- deoxyoctonic acid in its lipopolysaccharide is virtually as susceptible to the hydrophobic drug 13- phenylmercapto -alpha-6- deoxytetracycline as to oxytetracycline (MIC 100 times lower than that of the wild type), and a mutant with another, as yet undefined outer membrane defect, acrA , also shows increased, although somewhat lesser, susceptibility (MIC 20 times lower than that of the wild type). Increased susceptibility to this tetracycline derivative is associated with greater fluorescence of the derivative when added to the cells, which we interpret as increased interaction of the derivative with hydrophobic domains, such as membranes, in the mutants. This series of tetracyclines may provide an assay for measuring the permeability of gram-negative organisms and their mutants to hydrophobic molecules.

Biosynthesis and structure of lipopolysaccharide in an outer membrane-defective mutant of Escherichia coli J5

Membrane-defective mutants of Escherichia coli J5 were isolated on the basis of supersensitivity to the antibiotic novobiocin. These mutants display an increased sensitivity to a wide range of antibiotics and to several dyes and detergents. In addition, several mutants leak the periplasmic enzymes, alkaline phosphatase and ribonuclease. This evidence indicates an outer membrane defect in these mutants. The inner and outer membranes of one mutant were separated and subjected to compositional analysis. A deficiency in galactose-containing lipopolysaccharide in the outer membrane of the mutant was observed. Two possible causes of this deficiency were examined and discounted: defective galactose uptake into the cell, and defective translocation of lipopolysaccharide from the inner membrane. Extraction and chemical analysis of mutant and wild type lipopolysaccharides suggests that the mutant is defective in the enzyme which transfers glucose to the growing lipopolysaccharide core, UDPglucose transferase. Thus, the mutant's deficiency in galactose-containing lipopolysaccharide can be ascribed to the fact that addition of glucose to the lipopolysaccharide core is a prerequisite for galactose addition. The physiological implications of this alteration are discussed.