Kurt W. Miller

Cyclic di-GMP-dependent Signaling Pathways in the Pathogenic Firmicute Listeria monocytogenes

We characterized key components and major targets of the c-di-GMP signaling pathways in the foodborne pathogen Listeria monocytogenes, identified a new c-di-GMP-inducible exopolysaccharide responsible for motility inhibition, cell aggregation, and enhanced tolerance to disinfectants and desiccation, and provided first insights into the role of c-di-GMP signaling in listerial virulence. Genome-wide genetic and biochemical analyses of c-di-GMP signaling pathways revealed that L. monocytogenes has three GGDEF domain proteins, DgcA (Lmo1911), DgcB (Lmo1912) and DgcC (Lmo2174), that possess diguanylate cyclase activity, and three EAL domain proteins, PdeB (Lmo0131), PdeC (Lmo1914) and PdeD (Lmo0111), that possess c-di-GMP phosphodiesterase activity. Deletion of all phosphodiesterase genes (ΔpdeB/C/D) or expression of a heterologous diguanylate cyclase stimulated production of a previously unknown exopolysaccharide. The synthesis of this exopolysaccharide was attributed to the pssA-E (lmo0527-0531) gene cluster. The last gene of the cluster encodes the fourth listerial GGDEF domain protein, PssE, that functions as an I-site c-di-GMP receptor essential for exopolysaccharide synthesis. The c-di-GMP-inducible exopolysaccharide causes cell aggregation in minimal medium and impairs bacterial migration in semi-solid agar, however, it does not promote biofilm formation on abiotic surfaces. The exopolysaccharide also greatly enhances bacterial tolerance to commonly used disinfectants as well as desiccation, which may contribute to survival of L. monocytogenes on contaminated food products and in food-processing facilities. The exopolysaccharide and another, as yet unknown c-di-GMP-dependent target, drastically decrease listerial invasiveness in enterocytes in vitro, and lower pathogen load in the liver and gallbladder of mice infected via an oral route, which suggests that elevated c-di-GMP levels play an overall negative role in listerial virulence.

Listeria monocytogenes exopolysaccharide: origin, structure, biosynthetic machinery and c-di-GMP-dependent regulation

Elevated levels of the second messenger c‐di‐GMP activate biosynthesis of an unknown exopolysaccharide (EPS) in the food‐borne pathogen Listeria monocytogenes. This EPS strongly protects cells against disinfectants and desiccation, indicating its potential significance for listerial persistence in the environment and for food safety. We analyzed the potential phylogenetic origin of this EPS, determined its complete structure, characterized genes involved in its biosynthesis and hydrolysis and identified diguanylate cyclases activating its synthesis. Phylogenetic analysis of EPS biosynthesis proteins suggests that they have evolved within monoderms. Scanning electron microscopy revealed that L. monocytogenes EPS is cell surface‐bound. Secreted carbohydrates represent exclusively cell‐wall debris. Based on carbohydrate composition, linkage and NMR analysis, the structure of the purified EPS is identified as a β‐1,4‐linked N‐acetylmannosamine chain decorated with terminal α‐1,6‐linked galactose. All genes of the pssA‐E operon are required for EPS production and so is a separately located pssZ gene. We show that PssZ has an EPS‐specific glycosylhydrolase activity. Exogenously added PssZ prevents EPS‐mediated cell aggregation and disperses preformed aggregates, whereas an E72Q mutant in the presumed catalytic residue is much less active. The diguanylate cyclases DgcA and DgcB, whose genes are located next to pssZ, are primarily responsible for c‐di‐GMP‐dependent EPS production.

Novel Activator of Mannose-Specific Phosphotransferase System Permease Expression in Listeria innocua, Identified by Screening for Pediocin AcH Resistance

ABSTRACT To identify genes that are important for class IIa bacteriocin interaction and resistance in Listeria species, transposon Tn917 knockout libraries were constructed for Listeria innocua strain Lin11 and screened for mutants that are resistant to pediocin AcH. A highly resistant mutant (G7) (MIC > 20 μg/ml; 1,000-fold less susceptible than the wild type), in which the transposon integrated into the putative promoter of the lin0142 gene, was isolated. lin0142 is located immediately upstream of the mpt operon (mptA/mptC/mptD) that encodes the mannose-specific phosphoenolpyruvate-dependent phosphotransferase system permease EIItMan, which serves as a docking protein for class IIa bacteriocins. The transcription of the mpt operon is known to be positively controlled by σ54 factor and ManR (a σ54-associated activator). Transcripts for lin0142 and mpt were undetectable in the G7 mutant, based on quantitative real-time reverse transcriptase PCR analysis. When the wild-type lin0142 gene was expressed at a 7.9-fold-elevated level in the mutant via a multicopy-number plasmid, the level of mpt mRNA became 70% higher than that in the wild-type strain. In addition, the complementation strain reverted back to the pediocin AcH-susceptible phenotype. The levels of manR and rpoN (σ54) mRNAs were not directly influenced by the level of lin0142 transcription. lin0142 is the only one of the three mpt regulatory genes whose transcription is induced, albeit slightly (1.2-fold), by glucose. The combined results show that the lin0142 gene encodes a novel activator of the mpt operon. The Lin0142 protein contains a winged-helix DNA-binding motif and is distantly related to the Crp-Fnr family of transcription regulators.

Regulation of Mannose Phosphotransferase System Permease and Virulence Gene Expression in Listeria monocytogenes by the EIItMan Transporter

ABSTRACT The EIItMan phosphotransferase system (PTS) permease encoded by the mpt operon is the principal glucose transporter in Listeria monocytogenes. EIItMan participates in glucose-mediated carbon catabolite repression (CCR) and downregulation of virulence gene expression, and it is the receptor for class IIa bacteriocins. The regulation of this important protein and its roles in gene control were examined using derivatives of strain EGD-e in which the mpt operon or its regulatory genes, manR and lmo0095, were deleted. Real-time reverse transcription-PCR analysis showed that the mpt mRNA level was 10- and 100-fold lower in the lmo0095 and manR deletion strains, respectively. The manR mRNA level was higher in the mpt deletion mutant in medium lacking glucose, possibly due to disruption of a regulatory process that normally downregulates manR transcription in the absence of this sugar. Analysis of the mpt deletion mutant also showed that EIItMan participates to various degrees in glucose-mediated CCR of PTS operons. CCR of the lmo0027 gene, which encodes a β-glucoside PTS transporter, required expression of EIItMan. In contrast, genes in two mannose PTS operons (lmo0024, lmo1997, and lmo2002) were repressed by glucose even when EIItMan was not synthesized. A third mannose PTS operon, mpo, was not regulated by glucose or by the level of EIItMan. Finally, the mRNA levels for five genes in the prfA virulence gene cluster were two- to fourfold higher in the mpt deletion mutant. The results show that EIItMan participates to various extents in glucose-mediated CCR of PTS operons and makes a small, albeit significant, contribution to downregulation of virulence gene transcription by glucose in strain EGD-e.

Regulation of the mpt Operon in Listeria innocua by the ManR Protein

ABSTRACT The phosphotransferase system regulation domain (PRD)-containing activator, ManR, is required for glucose-controlled transcription of the mannose permease two (mpt) operon in Listeria innocua. His-871 in ManR PRD-II is needed for mpt repression in glucose-free media. His-506 in PRD-I is needed for mpt induction by glucose.

Exogenous or l-Rhamnose-Derived 1,2-Propanediol Is Metabolized via a pduD-Dependent Pathway in Listeria innocua

ABSTRACT 1,2-Propanediol (1,2-PD) added exogenously to cultures or produced endogenously from l-rhamnose is metabolized to n-propanol and propionate in Listeria innocua Lin11. The pduD gene, which encodes a diol dehydratase ß subunit homolog, is required for 1,2-PD catabolism. pduD and 16 other genes within the pduA-to-pduF region of a large gene cluster are induced in medium containing 1,2-PD.

Efficient Insertion of Odd-numbered Transmembrane Segments of the Tetracycline Resistance Protein Requires Even-numbered Segments

Functional membrane insertion elements in the pBR322 tetracycline resistance protein were identified by comparing the ability of odd-numbered transmembrane segments and their attached periplasmic loops to insert into the membrane individually or when combined with the next even-numbered segment in the tetracycline resistance protein sequence. The efficiency with which individual odd-numbered segments and periplasmic loops inserted was probed by treating proteins truncated at the distal ends of periplasmic loops P2-P6 with carboxypeptidases and endoproteases in inside-out membrane vesicles. Insertion of odd-numbered segments and attached loops is inefficient when they occupy a C-terminal position in the protein. The C-terminal odd-numbered segment and loop sequences of 34-54% of the molecules of periplasmic loop truncation mutants could be removed by carboxypeptidase Y. In contrast, odd-numbered segments and loops insert efficiently if the next even-numbered segment in the sequence is present. In such cytoplasmic loop truncation mutants, only the cytoplasmic tail sequences of the proteins could be removed by carboxypeptidases. Remarkably, insertion of individual odd-numbered segments and loops is inefficient even though free energies for insertion of these sequences are highly favorable. The results indicate that pairs of adjacent segments, possibly “helical hairpins,” are necessary for efficient membrane insertion of the tetracycline resistance protein.

Expression of recombinant pro‐neuropeptide Y, proopiomelanocortin, and proenkephalin: relative processing by ‘prohormone thiol protease’ (PTP)

The preference of the ‘prohormone thiol protease’ (PTP), a candidate prohormone processing enzyme, for different peptide precursors was assessed in vitro with recombinant prohormones near estimated in vivo levels. Pro‐neuropeptide Y (pro‐NPY), prooplomelanocortin (POMC), and proenkephalin (PE) were expressed at high levels in E. coli. Purification of prohormones utilized a combination of DEAE‐Sepharose, Mono Q, and preparative electrophoresis. PTP cleaved PE most readily, and also cleaved pro‐NPY. The processing of POMC by PTP was minimal. These results demonstrate PTPs preference for certain prohormone substrates.

Mutational analysis of tetracycline resistance protein transmembrane segment insertion.

The tetracycline resistance proteins (TetA) of gram-negative bacteria are secondary active transport proteins that contain buried charged amino acids that are important for tetracycline transport. Earlier studies have shown that insertion of TetA proteins into the cytoplasmic membrane is mediated by helical hairpin pairs of transmembrane (TM) segments. However, whether helical hairpins direct spontaneous insertion of TetA or are required instead for its interaction with the cellular secretion (Sec) machinery is unknown. To gain insight into how TetA proteins are inserted into the membrane, we have investigated how tolerant the class C TetA protein encoded by plasmid pBR322 is to placement of charged residues in TM segments. The results show that the great majority of charge substitutions do not interfere with insertion even when placed at locations that cannot be shielded internally within helical hairpins. The only mutations that frequently block insertion are proline substitutions, which may interfere with helical hairpin folding. The ability of TetA to broadly tolerate charge substitutions indicates that the Sec machinery assists in its insertion into the membrane. The results also demonstrate that it is feasible to engineer charged residues into the interior of TetA proteins for the purpose of structure-function analysis.

Mutational and sequence analysis of transmembrane segment 6 orientation in TetA proteins

The packing orientations of the 8 transmembrane (TM) segments that line the central, aqueous transport channel within tetracycline resistance proteins (TetA) have been established. However, the orientations of the remaining 4 segments, TMs 3, 6, 9, and 12, located at the periphery, and away from the transport channel, have not yet been determined. In this study, the packing orientation of TM6 within the class C TetA protein encoded by plasmid pBR322 was evaluated by substitution mutagenesis and analysis of sequence conservation and amphipathicity. The combined data support a model in which the conserved and polar face of the TM6 alpha-helix containing Asn170 and Asn173 orients towards channel-lining TM segments, and the relatively non-conserved and hydrophobic face of TM6 points towards membrane lipids.