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LyrA (SpdC), a homologue of eukaryotic CAAX proteases that act on prenylated substrates, has been implicated in the assembly of several pathways of the envelope of Staphylococcus aureus. We described earlier the lysostaphin resistance (Lyr) and staphylococcal protein A display (Spd) phenotypes associated with loss of the lyrA (spdC) gene. However, a direct contribution to the assembly of pentaglycine cross bridges, the target of lysostaphin cleavage in S. aureus peptidoglycan or of staphylococcal protein A attachment to peptidoglycan, could not be attributed directly to LyrA (SpdC). These two processes are catalyzed by the Fem factors and Sortase A, respectively. To gain insight into the function of LyrA (SpdC), here we use affinity chromatography and LC-MS/MS analysis and report that LyrA interacts with SagB. SagB cleaves glycan strands of peptidoglycan to achieve physiological length. Similar to sagB peptidoglycan, lyrA peptidoglycan contains extended glycan strands. Purified lyrA peptidoglycan can still be cleaved to physiological length by SagB in vitro. LyrA does not modify or cleave peptidoglycan, and it also does not modify or stabilize SagB. The membrane-bound domain of LyrA is sufficient to support SagB activity, but predicted “CAAX enzyme” catalytic residues in this domain are dispensable. We speculate that LyrA exerts its effect on bacterial prenyl substrates, specifically undecaprenol-bound peptidoglycan substrates of SagB, to help control glycan length. Such an activity also explains the Lyr and Spd phenotypes observed earlier. IMPORTANCE Peptidoglycan is assembled on the trans side of the plasma membrane from lipid II precursors into glycan chains that are cross-linked at stem peptides. In S. aureus, SagB, a membrane-associated N-acetylglucosaminidase, cleaves polymerized glycan chains to their physiological length. Deletion of sagB is associated with longer glycan strands in peptidoglycan, altered protein trafficking and secretion in the envelope, and aberrant excretion of cytosolic proteins. It is not clear whether SagB, with its single transmembrane segment, serves as the molecular ruler of glycan chains or whether other factors modulate its activity. Here, we show that LyrA (SpdC), a protein of the CAAX type II prenyl endopeptidase family, modulates SagB activity via interaction though its transmembrane domain.

Gram-negative bacteria such as Escherichia coli build a peptidoglycan (PG) cell wall in their periplasm using the precursor known as lipid II. Lipid II is a large amphipathic molecule composed of undecaprenyl diphosphate and a disaccharide-pentapeptide that PG-synthesizing enzymes use to build the PG sacculus. During PG biosynthesis, lipid II is synthesized at the cytoplasmic face of the inner membrane and then flipped across the membrane. This translocation of lipid II must be assisted by flippases thought to shield the disaccharide-pentapeptide as it crosses the hydrophobic core of the membrane. The inner membrane protein MurJ is essential for PG biogenesis and homologous to known and putative flippases of the MOP ( m ultidrug/ o ligo-saccharidyl-lipid/ p olysaccharide) exporter superfamily, which includes flippases that translocate undecaprenyl diphosphate-linked oligosaccharides across the cytoplasmic membranes of bacteria. Consequently, MurJ has been proposed to function as the lipid II flippase in E. coli . Here, we present a three-dimensional structural model of MurJ generated by the I-TASSER server that suggests that MurJ contains a solvent-exposed cavity within the plane of the membrane. Using in vivo topological studies, we demonstrate that MurJ has 14 transmembrane domains and validate features of the MurJ structural model, including the presence of a solvent-exposed cavity within its transmembrane region. Furthermore, we present functional studies demonstrating that specific charged residues localized in the central cavity are essential for function. Together, our studies support the structural homology of MurJ to MOP exporter proteins, suggesting that MurJ might function as an essential transporter in PG biosynthesis.

The integrity of the bacterial cell envelope is essential to sustain life by countering the high turgor pressure of the cell and providing a barrier against chemical insults. In Bacillus subtilis , synthesis of both peptidoglycan and wall teichoic acids requires a common C 55 lipid carrier, undecaprenyl-pyrophosphate (UPP), to ferry precursors across the cytoplasmic membrane. The synthesis and recycling of UPP requires a phosphatase to generate the monophosphate form Und-P, which is the substrate for peptidoglycan and wall teichoic acid synthases. Using an optimized c lustered r egularly i nterspaced s hort p alindromic r epeat (CRISPR) system with catalytically inactive (“ d ead”) C RISPR- as sociated protein 9 (dCas9)-based transcriptional repression system (CRISPR interference [CRISPRi]), we demonstrate that B. subtilis requires either of two UPP phosphatases, UppP or BcrC, for viability. We show that a third predicted lipid phosphatase (YodM), with homology to diacylglycerol pyrophosphatases, can also support growth when overexpressed. Depletion of UPP phosphatase activity leads to morphological defects consistent with a failure of cell envelope synthesis and strongly activates the σ M -dependent cell envelope stress response, including bcrC , which encodes one of the two UPP phosphatases. These results highlight the utility of an optimized CRISPRi system for the investigation of synthetic lethal gene pairs, clarify the nature of the B. subtilis UPP-Pase enzymes, and provide further evidence linking the σ M regulon to cell envelope homeostasis pathways. IMPORTANCE The emergence of antibiotic resistance among bacterial pathogens is of critical concern and motivates efforts to develop new therapeutics and increase the utility of those already in use. The lipid II cycle is one of the most frequently targeted processes for antibiotics and has been intensively studied. Despite these efforts, some steps have remained poorly defined, partly due to genetic redundancy. CRISPRi provides a powerful tool to investigate the functions of essential genes and sets of genes. Here, we used an optimized CRISPRi system to demonstrate functional redundancy of two UPP phosphatases that are required for the conversion of the initially synthesized UPP lipid carrier to Und-P, the substrate for the synthesis of the initial lipid-linked precursors in peptidoglycan and wall teichoic acid synthesis.

Although peptidoglycan synthesis is one of the best-studied metabolic pathways in bacteria, the mechanism underlying the membrane translocation of lipid II, the undecaprenyl-disaccharide pentapeptide peptidoglycan precursor, remains mysterious. Recently, it was proposed that the essential Escherichia coli mviN gene encodes the lipid II flippase. Bacillus subtilis contains four proteins that are putatively homologous to MviN, including SpoVB, previously reported to be necessary for spore cortex peptidoglycan synthesis during sporulation. MviN complemented the sporulation defect of a Δ spoVB mutation, and SpoVB and another of the B. subtilis homologs, YtgP, complemented the growth defect of an E. coli strain depleted for MviN. Thus, these B. subtilis proteins are likely to be MviN homologs. However, B. subtilis strains lacking these four proteins have no defects in growth, indicating that they likely do not serve as lipid II flippases in this organism.

Multimodular penicillin-binding proteins (PBPs) are essential enzymes responsible for bacterial cell wall peptidoglycan (PG) assembly. Their glycosyltransferase activity catalyzes glycan chain elongation from lipid II substrate (undecaprenyl-pyrophosphoryl- N -acetylglucosamine- N -acetylmuramic acid-pentapeptide), and their transpeptidase activity catalyzes cross-linking between peptides carried by two adjacent glycan chains. Listeria monocytogenes is a food-borne pathogen which exerts its virulence through secreted and cell wall PG-associated virulence factors. This bacterium has five PBPs, including two bifunctional glycosyltransferase/transpeptidase class A PBPs, namely, PBP1 and PBP4. We have expressed and purified the latter and have shown that it binds penicillin and catalyzes in vitro glycan chain polymerization with an efficiency of 1,400 M −1 s −1 from Escherichia coli lipid II substrate. PBP4 also catalyzes the aminolysis ( d -Ala as acceptor) and hydrolysis of the thiolester donor substrate benzoyl-Gly-thioglycolate, indicating that PBP4 possesses both transpeptidase and carboxypeptidase activities. Disruption of the gene lmo2229 encoding PBP4 in L. monocytogenes EGD did not have any significant effect on growth rate, peptidoglycan composition, cell morphology, or sensitivity to β-lactam antibiotics but did increase the resistance of the mutant to moenomycin.

Structural analysis of compounds identified as lipid I and II from Mycobacterium smegmatis demonstrated that the lipid moiety is decaprenyl phosphate; thus, M. smegmatis is the first bacterium reported to utilize a prenyl phosphate other than undecaprenyl phosphate as the lipid carrier involved in peptidoglycan synthesis. In addition, mass spectrometry showed that the muropeptides from lipid I are predominantly N- acetylmuramyl- l -alanine- d -glutamate- meso -diaminopimelic acid- d -alanyl- d -alanine, whereas those isolated from lipid II form an unexpectedly complex mixture in which the muramyl residue and the pentapeptide are modified singly and in combination. The muramyl residue is present as N -acetylmuramic acid, N- glycolylmuramic acid, and muramic acid. The carboxylic functions of the peptide side-chains of lipid II showed three types of modification, with the dominant one being amidation. The preferred site for amidation is the free carboxyl group of the meso -diaminopimelic acid residue. Diamidated species were also observed. The carboxylic function of the terminal d -alanine of some molecules is methylated, as are all three carboxylic acid functions of other molecules. This study represents the first structural analysis of mycobacterial lipid I and II and the first report of extensive modifications of these molecules. The observation that lipid I was unmodified strongly suggests that the lipid II intermediates of M. smegmatis are substrates for a variety of enzymes that introduce modifications to the sugar and amino acid residues prior to the synthesis of peptidoglycan.

A soluble derivative of the Enterococcus faecalis JH2-2 class A PBP1 ( * PBP1) was overproduced and purified. It exhibited a glycosyltransferase activity on the Escherichia coli 14 C-labeled lipid II precursor. As a dd- peptidase, it could hydrolyze thiolester substrates with efficiencies similar to those of other class A penicillin-binding proteins (PBPs) and bind β-lactams, but with k 2 / K (a parameter accounting for the acylation step efficiency) values characteristic of penicillin-resistant PBPs.

The bacterial peptidoglycan consists of glycan chains of repeating β-1,4-linked N -acetylglucosaminyl- N -acetylmuramyl units cross-linked through short peptide chains. The polymerization of the glycans, or glycosyltransfer (GT), and transpeptidation (TP) are catalyzed by bifunctional penicillin-binding proteins (PBPs). The β-lactam antibiotics inhibit the TP reaction, but their widespread use led to the development of drug resistance in pathogenic bacteria. In this context, the GT catalytic domain represents a potential target in the antibacterial fight. In this work, the in vitro polymerization of glycan chains by the extracellular region of recombinant Streptococcus pneumoniae PBP2a, namely, PBP2a* (the asterisk indicates the soluble form of the protein) is presented. Dansylated lipid II was used as the substrate, and the kinetic parameters K m and k cat / K m were measured at 40.6 μM (± 15.5) and 1 × 10 −3 M −1 s −1 , respectively. The GT reaction catalyzed by PBP2a* was inhibited by moenomycin and vancomycin. Furthermore, the sequence between Lys 78 and Ser 156 is required for enzymatic activity, whereas it is dispensable for lipid II binding. In addition, we confirmed that this region of the protein is also involved in membrane interaction, independently of the transmembrane anchor. The characterization of the catalytically active GT domain of S . pneumoniae PBP2a may contribute to the development of new inhibitors, which are urgently needed to renew the antibiotic arsenal.

Previous studies suggested that a Gly-containing branch of cell wall precursor [C 55 -MurNAc-(peptide)-GlcNAc], which is often referred to as lipid II, might serve as a nucleophilic acceptor in sortase-catalyzed anchoring of surface proteins in Staphylococcus aureus. To test this hypothesis, we first simplified the procedure for in vitro biosynthesis of Gly-containing lipid II by using branched UDP-MurNAc-hexapeptide isolated from the cytoplasm of Streptomyces spp. Second, we designed a thin-layer chromatography-based assay in which the mobility of branched but not linear lipid II is shifted in the presence of both sortase and LPSTG-containing peptide. These results and those of additional experiments presented in this study further suggest that lipid II indeed serves as a natural substrate in a sorting reaction.

In gram-positive bacteria, the ɛ-amino group of l-lysine in the pentapeptide stem of peptidoglycan precursors is often replaced by amino acid chains of various lengths and compositions (19) (Fig. ​(Fig.1).1). These amino acids form cross bridges between l-Lys3 and d-Ala4 following cross-linking of the peptide stems from nascent chains of peptidoglycan by the d,d-transpeptidases. The ligases for the addition of glycine and l-amino acids to the ɛ-amino group of l-lysine use aminoacyl-tRNAs as substrates, whereas d-amino acids are added in a tRNA-independent reaction (15, 20). FIG. 1 Structure of cross-linked peptidoglycan. (A) Fragment of the primary structure of peptidoglycan. The position of the peptide bond formed by the d,d-transpeptidases (penicillin-binding proteins) between the acceptor and donor peptide stems is indicated ... In Staphylococcus aureus, the femA, femB, and fmhB genes were shown to be essential for incorporation of glycine into the side chain of peptidoglycan precursors (18, 21). The femAB locus was initially identified as a factor essential for methicillin resistance (fem) based on random insertional inactivation of chromosomal genes and a screen for reduced expression of resistance mediated by the penicillin binding protein 2A (PBP2A) (2, 5). Inactivation of femA or femB was subsequently reported to prevent incorporation of glycine residues at positions 2 to 5 or positions 4 to 5 of the penta-glycine cross bridge since muropeptides cross-linked by one or three glycine residues were detected in the corresponding mutants (4, 21). Inactivation of fmhB, formerly femX, is lethal, but the construction of a mutant conditionally expressing fmhB under the control of a xylose-inducible promoter showed that the gene was essential for synthesis of branched peptidoglycan precursors (18). These results indicated that the fem gene products were required for incorporation of glycine at positions 1 (FmhB), 2 and 3 (FemA), and 4 and 5 (FemB) of the cross bridge, although the catalytic activity of the proteins was not directly assessed (18). Similarly, inactivation of two fmhB homologues in Streptococcus pneumoniae, designated murM (fibA) and murN (fibB), reduced addition of l-Ala or l-Ser to the ɛ-amino group of l-Lys and subsequent addition of a second l-Ala residue, respectively (6, 7, 23). Overall, disruption of the murMN operon reduced the proportion of branched peptide stems in the peptidoglycan from 89 to 33% (6). In contrast to what occurs in S. aureus (18), direct cross-linking of l-Lys to d-Ala occurs in S. pneumoniae, and the murMN operon was accordingly reported to be unessential (6). However, production of branched peptidoglycan precursors was necessary for expression of penicillin resistance (6) and appeared to affect mainly the activity of mosaic PBP2x (23). In enterococci, streptococci, and staphylococci, the ligation reactions are catalyzed by membrane-associated enzymes acting exclusively or preferentially on late peptidoglycan precursors linked to the undecaprenyl-phosphate lipid carrier, namely undecaprenyl-PP-MurNAc-pentapeptide (lipid I) and undecaprenyl-PP-MurNAc(pentapeptide)-GlcNAc (lipid II) (14). This conclusion is supported by the detection of only small amounts (

The photosynthetic organelles (cyanelles) of the protist Cyanophora paradoxa are surrounded by a peptidoglycan wall, modified through amidation with N -acetylputrescine. Cyanelle envelope membrane preparations were shown to catalyze the lipid-linked steps of peptidoglycan biosynthesis as well as the putrescinylation and subsequent acetylation, occurring at the stage of lipid I and/or lipid II.

The LytR-CpsA-Psr (LCP) proteins are thought to transfer bactoprenol-linked biosynthetic intermediates of wall teichoic acid (WTA) to the peptidoglycan of Gram-positive bacteria. In Bacillus subtilis , mutants lacking all three LCP enzymes do not deposit WTA in the envelope, while Staphylococcus aureus Δ lcp mutants display impaired growth and reduced levels of envelope phosphate. We show here that the S. aureus Δ lcp mutant synthesized WTA yet released ribitol phosphate polymers into the extracellular medium. Further, Δ lcp mutant staphylococci no longer restricted the deposition of LysM-type murein hydrolases to cell division sites, which was associated with defects in cell shape and increased autolysis. Mutations in S. aureus WTA synthesis genes ( tagB , tarF , or tarJ2 ) inhibit growth, which is attributed to the depletion of bactoprenol, an essential component of peptidoglycan synthesis (lipid II). The growth defect of S. aureus tagB and tarFJ mutants was alleviated by inhibition of WTA synthesis with tunicamycin, whereas the growth defect of the Δ lcp mutant was not relieved by tunicamycin treatment or by mutation of tagO , whose product catalyzes the first committed step of WTA synthesis. Further, sortase A-mediated anchoring of proteins to peptidoglycan, which also involves bactoprenol and lipid II, was not impaired in the Δ lcp mutant. We propose a model whereby the S. aureus Δ lcp mutant, defective in tethering WTA to the cell wall, cleaves WTA synthesis intermediates, releasing ribitol phosphate into the medium and recycling bactoprenol for peptidoglycan synthesis.

The transglycosylation step of cell wall synthesis is a prime antibiotic target because it is essential and specific to bacteria. Two antibiotics, ramoplanin and moenomycin, target this step by binding to the substrate lipid II and the transglycosylase enzyme, respectively. Here, we compare the ramoplanin and moenomycin stimulons in the Gram-positive model organism Bacillus subtilis . Ramoplanin strongly induces the LiaRS two-component regulatory system, while moenomycin almost exclusively induces genes that are part of the regulon of the extracytoplasmic function (ECF) σ factor σ M . Ramoplanin additionally induces the ytrABCDEF and ywoBCD operons, which are not part of a previously characterized antibiotic-responsive regulon. Cluster analysis reveals that these two operons are selectively induced by a subset of cell wall antibiotics that inhibit lipid II function or recycling. Repression of both operons requires YtrA, which recognizes an inverted repeat in front of its own operon and in front of ywoB . These results suggest that YtrA is an additional regulator of cell envelope stress responses.

Colicin M (ColM) is a bactericidal protein that kills sensitive cells by hydrolyzing lipid II, involved in the biosynthesis of cell wall peptidoglycan. It recognizes FhuA on the outer leaflet, and its translocation through the outer membrane depends on the energized Ton complex in the inner membrane. To be active in the periplasm, ColM must be translocated through the outer membrane and then interact with FkpA, a periplasmic protein that exhibits both cis - and trans -peptidylprolyl isomerase (PPiase) and chaperon activities. In an attempt to directly target ColM to the periplasm of the producing bacteria, we fused the presequence of OmpA to ColM (sp-ColM). We found that expression of this hybrid protein in an Escherichia coli strain devoid of ColM immunity protein (Cmi) was bactericidal. We showed that sp-ColM was correctly expressed, processed, and associated with the inner membrane. sp-ColM toxicity was related to its enzymatic activity and did not rely on the TonB import proteins or the FhuA receptor. The presence of both activity domains of FkpA was still required for sp-ColM activity. Analyses of deletion mutants of sp-ColM show that the domain required for toxicity corresponds to the C-terminal last 153 amino acids of ColM. Like the full-length protein, this domain is not active in the presence of the immunity protein Cmi. On the other hand, it does not require FkpA for toxic activity.

In the Firmicutes , two-component regulatory systems of the LiaSR type sense and orchestrate the response to various agents that perturb cell envelope functions, in particular lipid II cycle inhibitors. In the current study, we found that the corresponding system in Streptococcus pneumoniae displays similar properties but, in addition, responds to cell envelope stress elicited by murein hydrolases. During competence for genetic transformation, pneumococci attack and lyse noncompetent siblings present in the same environment. This phenomenon, termed fratricide, increases the efficiency of horizontal gene transfer in vitro and is believed to stimulate gene exchange also under natural conditions. Lysis of noncompetent target cells is mediated by the putative murein hydrolase CbpD, the key effector of the fratricide mechanism, and the autolysins LytA and LytC. To avoid succumbing to their own lysins, competent attacker cells must possess a protective mechanism rendering them immune. The most important component of this mechanism is ComM, an integral membrane protein of unknown function that is expressed only in competent cells. Here, we show that a second layer of self-protection is provided by the pneumococcal LiaFSR system, which senses the damage inflicted to the cell wall by CbpD, LytA, and LytC. Two members of the LiaFSR regulon, spr 0810 and PcpC ( spr 0351), were shown to contribute to the LiaFSR-coordinated protection against fratricide-induced self-lysis.

Members of the COG2244 protein family are integral membrane proteins involved in synthesis of a variety of extracellular polymers. In several cases, these proteins have been suggested to move lipid-linked oligomers across the membrane or, in the case of Escherichia coli MviN, to flip the lipid II peptidoglycan precursor. Bacillus subtilis SpoVB was the first member of this family implicated in peptidoglycan synthesis and is required for spore cortex polymerization. Three other COG2244 members with high similarity to SpoVB are encoded within the B. subtilis genome. Mutant strains lacking any or all of these genes ( yabM , ykvU , and ytgP ) in addition to spoVB are viable and produce apparently normal peptidoglycan, indicating that their function is not essential in B. subtilis . Phenotypic changes associated with loss of two of these genes suggest that they function in peptidoglycan synthesis. Mutants lacking YtgP produce long cells and chains of cells, suggesting a role in cell division. Mutants lacking YabM exhibit sensitivity to moenomycin, an antibiotic that blocks peptidoglycan polymerization by class A penicillin-binding proteins. This result suggests that YabM may function in a previously observed alternate pathway for peptidoglycan strand synthesis.

The glycosyltransferase (GT) module of class A penicillin-binding proteins (PBPs) and monofunctional GTs (MGTs) belong to the GT51 family in the sequence-based classification of GTs. They both possess five conserved motifs and use lipid II precursor (undecaprenyl-pyrophosphate- N -acetylglucosaminyl- N -acetylmuramoyl- pentapeptide) to synthesize the glycan chain of the bacterial wall peptidoglycan. MGTs appear to be dispensable for growth of some bacteria in vitro. However, new evidence shows that they may be essential for the infection process and development of pathogenic bacteria in their hosts. Only a small number of class A PBPs have been characterized so far, and no kinetic data are available on MGTs. In this study, we present the principal enzymatic properties of the Staphylococcus aureus MGT. The enzyme catalyzes glycan chain polymerization with an efficiency of ∼5,800 M −1 s −1 and has a pH optimum of 7.5, and its activity requires metal ions with a maximum observed in the presence of Mn 2+ . The properties of S. aureus MGT are distinct from those of S. aureus PBP2 and Escherichia coli MGT, but they are similar to those of E. coli PBP1b. We examined the role of the conserved Glu100 of S. aureus MGT (equivalent to the proposed catalytic Glu233 of E. coli PBP1b) by site-directed mutagenesis. The Glu100Gln mutation results in a drastic loss of GT activity. This shows that Glu100 is also critical for catalysis in S. aureus MGT and confirms that the conserved glutamate of the first motif EDXXFXX(H/N)X(G/A) is likely the key catalytic residue in the GT51 active site.

The antibiotic peptide nisin is the first known lantibiotic that uses a docking molecule within the bacterial cytoplasmic membrane for pore formation. Through specific interaction with the cell wall precursor lipid II, nisin forms defined pores which are stable for seconds and have pore diameters of 2 to 2.5 nm.

The lantibiotic mersacidin inhibits peptidoglycan biosynthesis by binding to the peptidoglycan precursor lipid II. Mersacidin contains an unsaturated thioether bridge, which is proposed to be synthesized by posttranslational modifications of threonine residue +15 and the COOH-terminal cysteine residue of the mersacidin precursor peptide MrsA. We show that the flavoprotein MrsD catalyzes the oxidative decarboxylation of the COOH-terminal cysteine residue of MrsA to an aminoenethiol residue. MrsD belongs to the recently described family of homo-oligomeric flavin-containing Cys decarboxylases (i.e., the HFCD protein family). Members of this protein family include the bacterial Dfp proteins (which are involved in coenzyme A biosynthesis), eukaryotic salt tolerance proteins, and further oxidative decarboxylases such as EpiD. In contrast to EpiD and Dfp, MrsD is a FAD and not an FMN-dependent flavoprotein. HFCD enzymes are characterized by a conserved His residue which is part of the active site. Exchange of this His residue for Asn led to inactivation of MrsD. The lantibiotic-synthesizing enzymes EpiD and MrsD have different substrate specificities.

The polysaccharide chains of enterobacterial common antigen (ECA) are comprised of the trisaccharide repeat unit Fuc4NAc-ManNAcA-GlcNAc, where Fuc4NAc is 4-acetamido-4,6-dideoxy -d- galactose, ManNAcA is N -acetyl -d- mannosaminuronic acid, and GlcNAc is N -acetyl -d- glucosamine. Individual trisaccharide repeat units are assembled as undecaprenyl-linked intermediates in a sequence of reactions that culminate in the transfer of Fuc4NAc from TDP-Fuc4NAc to ManNAcA-GlcNAc-pyrophosphorylundecaprenol (lipid II) to yield Fuc4NAc-ManNAcA-GlcNAc-pyrophosphorylundecaprenol (lipid III), the donor of trisaccharide repeat units for ECA polysaccharide chain elongation. Most of the genes known to be involved in ECA assembly are located in the wec gene cluster located at ca. 85.4 min on the Escherichia coli chromosome. The available data suggest that the structural gene for the TDP-Fuc4NAc:lipid II Fuc4NAc transferase also resides in the wec gene cluster; however, the location of this gene has not been unequivocally defined. Previous characterization of the nucleotide sequence of the wec gene cluster in the region between o416 and wecG revealed that it contained three open reading frames: o74 , o204 , and o450 . In contrast, the results of experiments described in the current investigation revealed that it contains only two open reading frames, o359 and o450 . Mutants of E. coli possessing null mutations in o359 were unable to synthesize ECA, and they accumulated lipid II. In addition, the in vitro incorporation of [ 3 H]FucNAc from TDP-[ 3 H]Fuc4NAc into lipid II was not observed in reaction mixtures using cell extracts obtained from these mutants as a source of enzyme. The ECA-negative phenotype of these mutants was complemented by plasmid constructs containing the wild-type o359 allele, and Fuc4NAc transferase activity was demonstrated by using cell extracts obtained from the complemented mutants. Furthermore, partially purified o359 gene product, expressed as recombinant C-terminal His-tagged protein, was able to catalyze the in vitro transfer of [ 3 H]Fuc4NAc from TDP-[ 3 H]Fuc4NAc to lipid II. Our data support the conclusion that o359 of the wec gene cluster of E. coli is the structural gene for the TDP-Fuc4NAc:lipid II Fuc4NAc transferase involved in the synthesis ECA trisaccharide repeat units.

In Escherichia coli , transcription of the degP locus, which encodes a heat-shock-inducible periplasmic protease, is controlled by two parallel signal transduction systems that each monitor extracytoplasmic protein physiology. For example, the heat-shock-inducible sigma factor, ς E , controls degP transcription in response to the overproduction and folded state of various extracytoplasmic proteins. Similarly, the CpxA/R two-component signal transduction system increases degP transcription in response to the overproduction of a variety of extracytoplasmic proteins. Since degP transcription is attuned to the physiology of extracytoplasmic proteins, we were interested in identifying negative transcriptional regulators of degP . To this end, we screened for null mutations that increased transcription from a strain containing a degP-lacZ reporter fusion. Through this approach, we identified null mutations in the wecE , rmlA ECA , and wecF loci that increase degP transcription. Interestingly, each of these loci is responsible for synthesis of the enterobacterial common antigen (ECA), a glycolipid situated on the outer leaflet of the outer membrane of members of the family Enterobacteriaceae . However, these null mutations do not stimulate degP transcription by eliminating ECA biosynthesis. Rather, the wecE , rmlA ECA , and wecF null mutations each impede the same step in ECA biosynthesis, and it is the accumulation of the ECA biosynthetic intermediate, lipid II, that causes the observed perturbations. For example, the lipid II-accumulating mutant strains each (i) confer upon E. coli a sensitivity to bile salts, (ii) confer a sensitivity to the synthesis of the outer membrane protein LamB, and (iii) stimulate both the Cpx pathway and ς E activity. These phenotypes suggest that the accumulation of lipid II perturbs the structure of the bacterial outer membrane. Furthermore, these results underscore the notion that although the Cpx and ς E systems function in parallel to regulate degP transcription, they can be simultaneously activated by the same perturbation.

The rff genes of Salmonella typhimurium include structural genes for enzymes involved in the conversion of UDP N-acetyl-D-glucosamine (UDP-GlcNAc) to UDP N-acetyl-D-mannosaminuronic acid (UDP-ManNAcA), the donor of ManNAcA residues in enterobacterial common antigen (ECA) synthesis. An rff mutation (rff-726) of Escherichia coli has been described (U. Meier and H. Mayer, J. Bacteriol. 163:756-762, 1985) that abolished ECA synthesis but which did not affect the synthesis of UDP-ManNAcA or any other components of ECA. The nature of the enzymatic defect resulting from the rff-726 lesion was investigated in the present study. The in vitro synthesis of GlcNAc-pyrophosphorylundecaprenol (lipid I), an early intermediate in ECA synthesis, was demonstrated by using membranes prepared from a mutant of E. coli possessing the rff-726 lesion. However, in vitro synthesis of the next lipid-linked intermediate in the biosynthetic sequence, ManNAcA-GlcNAc-pyrophosphorylundecaprenol (lipid II), was severely impaired. Transduction of wild-type rff genes into the mutant restored the ability to synthesize both lipid II and ECA as determined by in vitro assay and Western blot (immunoblot) analyses done with anti-ECA monoclonal antibody, respectively. Our results are consistent with the conclusion that the rff-726 mutation is located in the structural gene for the transferase that catalyzes the transfer of ManNAcA from UDP-ManNAcA to lipid I.

The heteropolysaccharide chains of enterobacterial common antigen (ECA) are composed of linear trisaccharide repeat units having the structure----3)-alpha-Fuc4NAc-(1----4)-beta-D-ManNAcA-(1---- 4)-alpha-D-GlcNAc- (1----. Mutants of Salmonella typhimurium lacking the structural gene for dTDP-glucose pyrophosphorylase (rfbA) are severely impaired in their ability to synthesize dTDP-glucose, which is a precursor of dTDP-4-acetamido-4,6-dideoxy-D-galactose (Fuc4NAc), the donor of Fuc4NAc residues for ECA synthesis. These mutants synthesize only trace amounts of ECA, and they are hypersensitive to sodium dodecyl sulfate (SDS). Incubation of delta rfbA mutants with [3H]N-acetylglucosamine ([3H]GlcNAc) resulted in the accumulation of radioactivity in N-acetyl-D-mannosaminuronic acid (ManNAcA)-GlcNAc-pyrophosphorylundecaprenol (lipid II), the putative acceptor of Fuc4NAc residues in ECA synthesis. Lipid II did not accumulate in either wild-type cells or in rff mutants unable to synthesize ManNAcA. Both the accumulation of lipid II and the synthesis of trace amounts of ECA were abolished when delta rfbA mutants were grown in the presence of the antibiotic tunicamycin. Tunicamycin also prevented the SDS-mediated lysis of the mutants. SDS-resistant derivatives of delta rfbA mutants were isolated that were no longer able to synthesize trace amounts of ECA. Characterization of these derivatives revealed that they were defective in various steps of ECA synthesis leading to the synthesis of lipid II. The data support the conclusion that accumulation of lipid II is responsible in some way for the hypersensitivity of delta rfbA mutants to SDS.