Identification of Listeria monocytogenes Determinants Required for Biofilm Formation

Listeria monocytogenes is a Gram-positive, food-borne pathogen of humans and animals. L. monocytogenes is considered to be a potential public health risk by the U.S. Food and Drug Administration (FDA), as this bacterium can easily contaminate ready-to-eat (RTE) foods and cause an invasive, life-threatening disease (listeriosis). Bacteria can adhere and grow on multiple surfaces and persist within biofilms in food processing plants, providing resistance to sanitizers and other antimicrobial agents. While whole genome sequencing has led to the identification of biofilm synthesis gene clusters in many bacterial species, bioinformatics has not identified the biofilm synthesis genes within the L. monocytogenes genome. To identify genes necessary for L. monocytogenes biofilm formation, we performed a transposon mutagenesis library screen using a recently constructed Himar1 mariner transposon. Approximately 10,000 transposon mutants within L. monocytogenes strain 10403S were screened for biofilm formation in 96-well polyvinyl chloride (PVC) microtiter plates with 70 Himar1 insertion mutants identified that produced significantly less biofilms. DNA sequencing of the transposon insertion sites within the isolated mutants revealed transposon insertions within 38 distinct genetic loci. The identification of mutants bearing insertions within several flagellar motility genes previously known to be required for the initial stages of biofilm formation validated the ability of the mutagenesis screen to identify L. monocytogenes biofilm-defective mutants. Two newly identified genetic loci, dltABCD and phoPR, were selected for deletion analysis and both ΔdltABCD and ΔphoPR bacterial strains displayed biofilm formation defects in the PVC microtiter plate assay, confirming these loci contribute to biofilm formation by L. monocytogenes.     

Pectin Biosynthesis Is Critical for Cell Wall Integrity and Immunity in Arabidopsis thaliana

Plant cell walls are important barriers against microbial pathogens. Cell walls of Arabidopsis thaliana leaves contain three major types of polysaccharides: cellulose, various hemicelluloses, and pectins. UDP-d-galacturonic acid, the key building block of pectins, is produced from the precursor UDP-d-glucuronic acid by the action of glucuronate 4-epimerases (GAEs). Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) repressed expression of GAE1 and GAE6 in Arabidopsis, and immunity to Pma ES4326 was compromised in gae6 and gae1 gae6 mutant plants. These plants had brittle leaves and cell walls of leaves had less galacturonic acid. Resistance to specific Botrytis cinerea isolates was also compromised in gae1 gae6 double mutant plants. Although oligogalacturonide (OG)-induced immune signaling was unaltered in gae1 gae6 mutant plants, immune signaling induced by a commercial pectinase, macerozyme, was reduced. Macerozyme treatment or infection with B. cinerea released less soluble uronic acid, likely reflecting fewer OGs, from gae1 gae6 cell walls than from wild-type Col-0. Although both OGs and macerozyme-induced immunity to B. cinerea in Col-0, only OGs also induced immunity in gae1 gae6. Pectin is thus an important contributor to plant immunity, and this is due at least in part to the induction of immune responses by soluble pectin, likely OGs, that are released during plant-pathogen interactions.     

The Biosynthesis of Flavin Cofactors in Listeria monocytogenes

Listeria monocytogenes is riboflavin auxotrophic, but it has two genes envisaged to transform riboflavin into FMN and FAD after its uptaked by specialized transporters. One encodes a bifunctional type I FAD synthase (FADS, herein LmFADS-1), while the other produces a protein similar to type I at the FMN:ATP adenylyltransferase (FMNAT) site but with a shorter C-terminal that lacks any riboflavin kinase (RFK) motif. This second protein is rare among bacteria and has been named FADS type II (LmFADS-2). Here we present a biochemical and biophysical study of LmFADS-1 and LmFADS-2 by integrating kinetic and thermodynamic data together with sequence and structural prediction methods to evaluate their occurrence in Listeria, as well as their function and molecular properties. Despite LmFADS-1 similarities to other type I FADSs, (i) its RFK activity has not riboflavin substrate inhibition and occurs under reducing and oxidizing conditions, (ii) its FMNAT activity requires strong reducing environment, and (iii) binding of reaction products, but not substrates, favors binding of the second ligand. LmFADS-2 produces FAD under oxidizing and reducing environments, but its C-terminus module function remains unknown. Listeria species conserve both FADSs, being sequence identity high within L. monocytogenes strains. Our data exemplify alternative strategies for FMN and FAD biosynthesis and homeostasis, envisaging that in Listeria two FADSs might be required to fulfill the supply of flavin cofactors under niches that can go from saprophytism to virulence. As FADSs are attractive antimicrobial targets, understanding of FADSs traits in different species is essential to help in the discovery of specific antimicrobials.     

Syntaxin Is Required for Cell Division

We recently identified a single family member homologue of syntaxin in the sea urchin. Syntaxin is present throughout development, and in rapidly dividing cleavage stage embryos it is present on numerous vesicles at the cell cortex. We hypothesized that syntaxin mediates essential membrane fusion events during early embryogenesis, reasoning that the vesicles and/or their contents are important for development. Here we show that functional inactivation of syntaxin with either Botulinum neurotoxin C1, which specifically proteolyzes syntaxin, or antibodies against syntaxin results in an inhibition of cell division. These observations suggest that syntaxin is essential for membrane fusion events critical for cell division.     

Teichoic Acid Glycosylation Mediated by gtcA Is Required for Phage Adsorption and Susceptibility of Listeria monocytogenes Serotype 4b

An insertion mutant of gtcA, responsible for serotype-specific glycosylation of the cell wall teichoic acid in serotype 4b strains of Listeria monocytogenes, was also resistant to both Listeria genus- and serotype 4b-specific phages. The sugar substituents on teichoic acid appeared essential for the adsorption of phages A500 (serotype 4b specific) and A511 (Listeria genus specific) to serotype 4b L. monocytogenes.     

Tolerance of Listeria monocytogenes to Cell Envelope-Acting Antimicrobial Agents Is Dependent on SigB

Mutation of sigB impairs the ability of Listeria monocytogenes to grow in sublethal levels, and to survive in lethal concentrations, of the bacteriocins nisin and lacticin 3147 and the antibiotics ampicillin and penicillin G. SigB may therefore represent an attractive target for the development of new control and treatment strategies for this important pathogen.     

Caenorhabditis elegans Is a Model Host for Listeria monocytogenes

Here we report that Caenorhabditis elegans nematodes fed Listeria monocytogenes die over the course of several days, as a consequence of an accumulation of bacteria in the worm intestine. Mutant strains previously shown to be important for virulence in mammalian models were also found to be attenuated in their virulence in C. elegans. However, ActA, which is required for actin-based intracellular motility, appears to be dispensable during infection of C. elegans, indicating that L. monocytogenes remains extracellular in C. elegans.     

The Listeria monocytogenes Hibernation-Promoting Factor Is Required for the Formation of 100S Ribosomes,Optimal Fitness,and Pathogenesis

During exposure to certain stresses, bacteria dimerize pairs of 70S ribosomes into translationally silent 100S particles in a process called ribosome hibernation. Although the biological roles of ribosome hibernation are not completely understood, this process appears to represent a conserved and adaptive response that contributes to optimal survival during stress and post-exponential-phase growth. Hibernating ribosomes are formed by the activity of one or more highly conserved proteins; gammaproteobacteria produce two relevant proteins, ribosome modulation factor (RMF) and hibernation promoting factor (HPF), while most Gram-positive bacteria produce a single, longer HPF protein. Here, we report the formation of 100S ribosomes by an HPF homolog in Listeria monocytogenes. L. monocytogenes 100S ribosomes were observed by sucrose density gradient centrifugation of bacterial extracts during mid-logarithmic phase, peaked at the transition to stationary phase, and persisted at lower levels during post-exponential-phase growth. 100S ribosomes were undetectable in bacteria carrying an hpf::Himar1 transposon insertion, indicating that HPF is required for ribosome hibernation in L. monocytogenes. Additionally, epitope-tagged HPF cosedimented with 100S ribosomes, supporting its previously described direct role in 100S formation. We examined hpf mRNA by quantitative PCR (qPCR) and identified several conditions that upregulated its expression, including carbon starvation, heat shock, and exposure to high concentrations of salt or ethanol. Survival of HPF-deficient bacteria was impaired under certain conditions both in vitro and during animal infection, providing evidence for the biological relevance of 100S ribosome formation.     

A Differential Fluorescence-Based Genetic Screen Identifies Listeria monocytogenes Determinants Required for Intracellular Replication

Listeria monocytogenes is a Gram-positive, facultative intracellular pathogen capable of causing severe invasive disease with high mortality rates in humans. While previous studies have largely elucidated the bacterial and host cell mechanisms necessary for invasion, vacuolar escape, and subsequent cell-to-cell spread, the L. monocytogenes factors required for rapid replication within the restrictive environment of the host cell cytosol are poorly understood. In this report, we describe a differential fluorescence-based genetic screen utilizing fluorescence-activated cell sorting (FACS) and high-throughput microscopy to identify L. monocytogenes mutants defective in optimal intracellular replication. Bacteria harboring deletions within the identified gene menD or pepP were defective for growth in primary murine macrophages and plaque formation in monolayers of L2 fibroblasts, thus validating the ability of the screening method to identify intracellular replication-defective mutants. Genetic complementation of the menD and pepP deletion strains rescued the in vitro intracellular infection defects. Furthermore, the menD deletion strain displayed a general extracellular replication defect that could be complemented by growth under anaerobic conditions, while the intracellular growth defect of this strain could be complemented by the addition of exogenous menaquinone. As prior studies have indicated the importance of aerobic metabolism for L. monocytogenes infection, these findings provide further evidence for the importance of menaquinone and aerobic metabolism for L. monocytogenes pathogenesis. Lastly, both the menD and pepP deletion strains were attenuated during in vivo infection of mice. These findings demonstrate that the differential fluorescence-based screening approach provides a powerful tool for the identification of intracellular replication determinants in multiple bacterial systems.     

Tam41 Is a CDP-Diacylglycerol Synthase Required for Cardiolipin Biosynthesis in Mitochondria

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Proline Biosynthesis Is Required for Endoplasmic Reticulum Stress Tolerance in Saccharomyces cerevisiae

The amino acid proline is uniquely involved in cellular processes that underlie stress response in a variety of organisms. Proline is known to minimize protein aggregation, but a detailed study of how proline impacts cell survival during accumulation of misfolded proteins in the endoplasmic reticulum (ER) has not been performed. To address this we examined in Saccharomyces cerevisiae the effect of knocking out the PRO1, PRO2, and PRO3 genes responsible for proline biosynthesis. The null mutants pro1, pro2, and pro3 were shown to have increased sensitivity to ER stress relative to wild-type cells, which could be restored by proline or the corresponding genetic complementation. Of these mutants, pro3 was the most sensitive to tunicamycin and was rescued by anaerobic growth conditions or reduced thiol reagents. The pro3 mutant cells have higher intracellular reactive oxygen species, total glutathione, and a NADP⁺/NADPH ratio than wild-type cells under limiting proline conditions. Depletion of proline biosynthesis also inhibits the unfolded protein response (UPR) indicating proline protection involves the UPR. To more broadly test the role of proline in ER stress, increased proline biosynthesis was shown to partially rescue the ER stress sensitivity of a hog1 null mutant in which the high osmolality pathway is disrupted.     

Plant Cell Wall Matrix Polysaccharide Biosynthesis

The wall of an expanding plant cell consists primarily of cellulose microfibrils embedded in a matrix of hemicellulosic and pectic polysaccharides along with small amounts of structural and enzymatic proteins. Matrix polysaccharides are synthesized in the Golgi and exported to the cell wall by exocytosis, where they intercalate among cellulose microfibrils, which are made at the plasma membrane and directly deposited into the cell wall. Involvement of Golgi glucan synthesis in auxin-induced cell expansion has long been recognized; however, only recently have the genes corresponding to glucan synthases been identified. Biochemical purification was unsuccessful because of the labile nature and very low abundance of these enzymes. Mutational genetics also proved fruitless. Expression of candidate genes identified through gene expression profiling or comparative genomics in heterologous systems followed by functional characterization has been relatively successful. Several genes from the cellulose synthase-like （Csl） family have been found to be involved in the synthesis of various hemicellulosic glycans. The usefulness of this approach, however, is limited to those enzymes that probably do not form complexes consisting of unrelated proteins. Nonconventional approaches will continue to incrementally unravel the mechanisms of Golgi polysaccharide biosynthesis.     

GUN4 Is Required for Posttranslational Control of Plant Tetrapyrrole Biosynthesis

In aerobic photosynthetic organisms, GUN4 binds the chlorophyll intermediates protoporphyrin and Mg protoporphyrin, stimulates Mg chelatase activity, and is implicated in plastidic retrograde signaling. GUN4 expression is most abundant in young and greening tissues and parallels the activity of 5-aminolevulinic acid （ALA） ALA and Mg porphyrin biosynthesis during photoperiodic growth. We explored function and mode of action of GUN4 using GUN4- deficient and overexpressing plants. GUN4 overexpression leads to a general activation of the enzymes of chlorophyll biosynthesis. During photoperiodic growth GUN4 deficiency prevents ALA synthesis and chlorophyll accumulation. All these metabolic changes do not correlate with altered gene expression or changes of protein abundance in tetrapyrrole biosynthesis. While ALA feeding fails to compensate GUN4 deficiency during light-dark growth, this approach results in chlorophyll accumulation under continuous dim light. A new model defines the involvement of GUN4 in posttranslational regulation of ALA and Mg porphyrin synthesis, to sustain chlorophyll synthesis, namely under varying environmental conditions.     

Aflatoxin Biosynthesis Cluster Gene cypA Is Required for G Aflatoxin Formation

Aspergillus flavus isolates produce only aflatoxins B1 and B2, while Aspergillus parasiticus and Aspergillus nomius produce aflatoxins B1, B2, G1, and G2. Sequence comparison of the aflatoxin biosynthesis pathway gene cluster upstream from the polyketide synthase gene, pksA, revealed that A. flavus isolates are missing portions of genes (cypA and norB) predicted to encode, respectively, a cytochrome P450 monooxygenase and an aryl alcohol dehydrogenase. Insertional disruption of cypA in A. parasiticus yielded transformants that lack the ability to produce G aflatoxins but not B aflatoxins. The enzyme encoded by cypA has highest amino acid identity to Gibberella zeae Tri4 (38%), a P450 monooxygenase previously shown to be involved in trichodiene epoxidation. The substrate for CypA may be an intermediate formed by oxidative cleavage of the A ring of O-methylsterigmatocystin by OrdA, the P450 monooxygenase required for formation of aflatoxins B1 and B2.     

Immunochemistry of the Cell Walls of Listeria monocytogenes

The antigenic specificity of Listeria monocytogenes types I, II, III, IVa, and IVb was studied by immunochemical techniques. Immunologically active carbohydrates of the various types were extracted from cell walls and were chemically analyzed. Types I and II contained predominantly glucosamine and rhamnose; type III, galactose, rhamnose, and glucosamine; and types IVa and IVb, glucose and galactose. Quantitative precipitin inhibition tests with purified monosaccharides indicated that the major antigenic determinant of types I and II is rhamnose. Precipitin reactions could not be detected with type III carbohydrate and homologous or heterologous antisera. The major determinants of types IVa and IVb were found to be galactose and glucose, respectively. As much as 87% inhibition of the quantitative precipitin test for types I and II was obtained with rhamnose, 72% for type IVa with galactose, and 72% for type IVb with glucose. The immunochemical basis for the antigenic specificity of L. monocytogenes types I, II, IVa, and IVb was further confirmed by using agar gel diffusion. Cross-reactions among the various type-specific carbohydrates and heterologous antisera were also studied. Type II carbohydrate was found to contain galactose and react with type IVa antisera. This reaction could be blocked by galactose. Type I carbohydrate did not contain galactose nor did it react with antiserum prepared from type IVa cells. Therefore, the somatic antigens of type I and type II L. monocytogenes, previously thought to be identical, appeared to differ. The dominant immuno-specific group in the cross-reaction between type IVb carbohydrate and type IVa antisera was found to be galactose. Type IVa absorbed antisera did not produce a significant cross-reaction with type IVb carbohydrate. The results obtained from this investigation indicate a lesser degree of antigenic relationship between type IVa and type IVb L. monocytogenes than was previously believed to exist.