methods-to-inhibit-bacterial-pyomelanin-production-determine

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism ...

University of Wisconsin Milwaukee

Archaeal flagellins are initially synthesized as preflagellins with a short, positively charged leader peptide, which is cleaved prior to the incorporation of the mature flagellins into the filament. While preflagellin peptidase activity had previously been detected in methanogen membranes, the enzyme responsible for this activity had not been identified. We show here that FlaK of Methanococcus maripaludis has preflagellin peptidase activity. In an in vitro preflagellin peptidase assay, Escherichia coli membranes overexpressing Methanococcus voltae preflagellin FlaB2 (as substrate) were combined with E. coli membranes overexpressing M. maripaludis FlaK (as enzyme). Cleavage of the preflagellin was demonstrated by immunoblotting using antibody to FlaB2 and detection of a faster migrating cross-reactive species. This activity required detergent in the assay, and was not detected in membranes previously heated to 95 degrees C. This is the first reported identification of the preflagellin peptidase, and aside from the flagellins, this is the first assignment of function to a gene involved in archaeal flagellation.

FlaK of the archaeon Methanococcus maripaludis possesses preflagellin peptidase activity.

Methanococcus voltae possesses four flagellin genes, two of which (flaB1 and flaB2) have previously been reported to encode major components of the flagellar filament. The remaining two flagellin genes, flaA and flaB3, are transcribed at lower levels, and the corresponding proteins remained undetected prior to this work. Electron microscopy examination of flagella isolated by detergent extraction of whole cells revealed a curved, hook-like region of varying length at the end of a long filament. Enrichment of the curved region of the flagella resulted in the identification of FlaB3 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and N-terminal sequencing, and the localization of this flagellin to the cell-proximal portion of the flagellum was confirmed through immunoblotting and immunoelectron microscopy with FlaB3-specific antibodies, indicating that FlaB3 likely composes the curved portion of the flagella. This could represent a unique case of a flagellin performing the role of the bacterial hook protein. FlaA-specific antibodies were used in immunoblotting to determine that FlaA is found throughout the flagellar filament. M. voltae cells were transformed with a modified flaA gene containing a hemagglutinin (HA) tag introduced into the variable region. Transformants that had replaced the wild-type copy of the flaA gene with the HA-tagged version incorporated the HA-tagged version of FlaA into flagella which appeared normal by electron microscopy.

Identification and localization of flagellins FlaA and FlaB3 within flagella of Methanococcus voltae.

Prokaryotes use a wide variety of structures to facilitate motility. The majority of research to date has focused on swimming motility and the molecular architecture of the bacterial flagellum. While intriguing questions remain, especially concerning the specialized export system involved in flagellum assembly, for the most part the structural components and their location within the flagellum and function are now known. The same cannot be said of the other apparati including archaeal flagella, type IV pili, the junctional pore, ratchet structure and the contractile cytoskeleton used by a variety of organisms for motility. In these cases, many of the structural components have yet to be identified and the mechanism of action that results in motility is often still poorly understood. Research on the bacterial flagellum has greatly aided our understanding of not only motility but also protein secretion and genetic regulation systems. Continued study and understanding of all prokaryotic motility structures will provide a wealth of knowledge that is sure to extend beyond the bounds of prokaryotic movement.

Prokaryotic motility structures.

The correct delivery of noncytoplasmic proteins to locations both within and outside the cell depends on the appropriate targeting signals. Protein translocation across the bacterial plasma membrane and the eukaryal endoplasmic reticulum membrane relies on cleavable N-terminal signal peptides. Although the signal peptides of secreted proteins in Bacteria and Eukarya have been extensively studied at the sequence, structure, and functional levels, little is known of the nature of archaeal signal peptides. In this report, genome-based analysis was performed in an attempt to define the amino acid composition, length, and cleavage sites of various signal peptide classes in a wide range of archaeal species. The results serve to present a picture of the archaeal signal peptide, revealing the incorporation of bacterial, eukaryal, and archaeal traits.

Archaeal signal peptides--a comparative survey at the genome level.

The differences between archaeal and bacterial flagella are becoming more apparent as research on the archaeal structure progresses. One crucial difference is the presence of a leader peptide on archaeal preflagellins, which is removed from the flagellin prior to its incorporation into the flagellar filament. The enzyme responsible for the removal of the flagellin leader peptide was identified as FlaK. FlaK of Methanococcus voltae retains its preflagellin peptidase activity when expressed in Escherichia coli and used in an in vitro assay. Homologous recombination of an integration vector into the chromosomal copy of flaK resulted in a non-motile, non-flagellated phenotype. The flagellins of the mutant had larger molecular weights than their wild-type counterparts, as expected if they retained their 11- to 12-amino-acid leader peptide. Membranes of the flaK mutant were unable to process preflagellin in the in vitro assay. Site-directed mutagenesis demonstrated that two aspartic acid residues conserved with ones in type IV prepilin peptidases were necessary for proper recognition or processing of the preflagellin. As bacterial flagellins lack a leader peptide and a peptidase is not required for export and assembly, the requirement for FlaK further emphasizes the similarity archaeal flagella have with type IV pili, rather than with bacterial flagella.

Cleavage of preflagellins by an aspartic acid signal peptidase is essential for flagellation in the archaeon Methanococcus voltae.

Archaeal motility occurs through the rotation of flagella that are distinct from the flagella found on bacteria. The differences between the two structures include the multi-flagellin nature of the archaeal filament, the widespread posttranslational modification of the flagellins and the presence of a short signal peptide on each flagellin that is cleaved by a specific signal peptidase prior to the incorporation of the mature flagellin into the flagellar filament. Research has revealed similarities between the archaeal flagellum and the type IV pilus, including the presence of similar unusual signal peptides on the flagellins and pilins, similarities in the amino acid sequences of the major structural proteins themselves, as well as similarities between potential assembly and processing components. The recent suggestion that type IV pili are part of a family of cell surface complexes, coupled with the similarities between type IV pili and archaeal flagella, raise questions about the evolution of these systems and possible inclusion of archaeal flagella into this surface complex family.

Recent advances in the structure and assembly of the archaeal flagellum.

Site-directed mutagenesis studies of the signal peptidase of the methanogenic archaeon Methanococcus voltae identified three conserved residues (Ser52, His122, and Asp148) critical for activity. The requirement for one conserved aspartic acid residue distinguishes the archaeal enzyme from both the Escherichia coli and yeast Sec11 enzymes.

Site-directed mutagenesis analysis of amino acids critical for activity of the type I signal peptidase of the archaeon Methanococcus voltae.

The flagellum of Methanococcus voltae is composed of four structural flagellin proteins FlaA, FlaB1, FlaB2, and FlaB3. These proteins possess a total of 15 potential N-linked sequons (NX(S/T)) and show a mass shift on an SDS-polyacrylamide gel indicating significant post-translational modification. We describe here the structural characterization of the flagellin glycan from M. voltae using mass spectrometry to examine the proteolytic digests of the flagellin proteins in combination with NMR analysis of the purified glycan using a sensitive, cryogenically cooled probe. Nano-liquid chromatography-tandem mass spectrometry analysis of the proteolytic digests of the flagellin proteins revealed that they are post-translationally modified with a novel N-linked trisaccharide of mass 779 Da that is composed of three sugar residues with masses of 318, 258, and 203 Da, respectively. In every instance the glycan is attached to the peptide through the asparagine residue of a typical N-linked sequon. The glycan modification has been observed on 14 of the 15 sequon sites present on the four flagellin structural proteins. The novel glycan structure elucidated by NMR analysis was shown to be a trisaccharide composed of beta-ManpNAcA6Thr-(1-4)-beta-Glc-pNAc3NAcA-(1-3)-beta-GlcpNAc linked to Asn. In addition, the same trisaccharide was identified on a tryptic peptide of the S-layer protein from this organism implicating a common N-linked glycosylation pathway.

Identification and characterization of the unique N-linked glycan common to the flagellins and S-layer glycoprotein of Methanococcus voltae.

A soluble methyl-accepting chemotaxis protein (MCP) of Pseudomonas aeruginosa, McpS, showed polar localization by immunofluorescence microscopy. Overexpression of McpS resulted in a dominant-negative effect on chemotaxis and caused a loss of polar clustering of the general MCP population. The polar localization of a soluble MCP defines a third, and unexpected, paradigm for cellular MCP localization.

Polar localization of a soluble methyl-accepting protein of Pseudomonas aeruginosa.

It is now well established in the microbiology community that the spatial organization of bacterial cells is quite complex with proteins and protein complexes localized to specific subcellular regions. Unresolved for the most part, however, are the mechanisms by which asymmetric proteins are localized. A variety of mechanisms are utilized to achieve polarity in bacteria. In this article, we focus on recent findings that support specific mechanisms for the establishment of polarity in rod shaped bacteria.

Polar explorations Recent insights into the polarity of bacterial proteins.

In this research, tetracycline resistant (tet(R)) and tetracycline susceptible (tet(S)) Escherichia coli isolates were retrieved from dairy manure and the influence of tetracycline resistance on the transport of E. coli in saturated porous media was investigated through laboratory column transport experiments. Experimental results showed that tet(R)E. coli strains had higher mobility than the tet(S) strains in saturated porous media. Measurements of cell surface properties suggested that tet(R)E. coli strains exhibited lower zeta potentials than the tet(S) strains. Because the surface of clean quartz sands is negatively charged, the repulsive electrostatic double layer (EDL) interaction between the tet(R) cells and the surface of sands was stronger and thus facilitated the transport of the tet(R) cells. Although no difference was observed in surface acidity, cell size, lipopolysaccharides (LPS) sugar content and cell-bound protein levels between the tet(R) and tet(S) strains, they displayed distinct outer membrane protein (OMP) profiles. It was likely that the difference in OMPs, some potentially related to drug efflux pumps, between the tet(R) and tet(S) strains led to alteration in cell surface properties which in turn affected cell transport in saturated porous media. Findings from this research suggested that manure-derived tet(R)E. coli could spread more widely in the groundwater system and pose serious public health risks.

Influence of tetracycline resistance on the transport of manure-derived Escherichia coli in saturated porous media.

In this research, we investigate the effects of starvation on the transport of Escherichia coli K12 in saturated porous media. Particularly, we examine the relationship between the starvation effects and the pH and ionic strength of the electrolyte solutions used for cell starvation. E. coli K12 was cultured using either Luria-Bertani Miller (LB-Miller) broth, which contained 10 g/L of NaCl, or LB-Luria broth, which contained 0.5 g/L of NaCl. As both types of broths had similar pH (~7.2) they differed in ionic strengths. The bacterial cells were harvested at late-exponential phase and resuspended in buffered (pH=7.2) and non-buffered (pH=5.7) electrolyte solutions that had ionic strengths of 8.4mM or 168 mM, respectively. Column transport experiments were performed following 4, 25 and 52 h of cell starvation to evaluate the temporal changes in cell mobility. Our results showed that starvation led to a significant increase in the mobility of E. coli K12, particularly between 4 and 25 h, when both pH and ionic strength of the electrolyte solution were different from those of the growth media. The size, viability and surface properties (e.g., zeta potential, hydrophobicity, LPS sugar content, outer membrane protein profiles) of the bacterial cells were determined and related to the observed temporal variation patterns of cell mobility. We found that starvation in electrolyte solutions that had different pH and ionic strength from the growth media significantly lowered cell viability, which may be related to the temporal change in cell mobility under these specific conditions.

The effects of starvation on the transport of Escherichia coli in saturated porous media are dependent on pH and ionic strength.

It was recently reported that tetracycline could enhance the mobility of manure-derived Escherichia coli within saturated porous media (Walczak et al. (Water Research 45:1681-1690, 2011)). It was also shown, however, that E. coli from various sources could display marked variation in their mobility (Bolster et al. (Journal of Environmental Quality 35:1018-1025, 2009)). The focus of this research was to examine if the observed difference in the mobility of manure-derived tetracycline-resistant (tet(R)) and tetracycline-susceptible (tet(S)) E. coli strains was source-dependent. Specifically, E. coli were isolated from Lake Michigan, and the influence of tetracycline resistance on Lake Michigan-derived E. coli was investigated through column transport experiments. Additionally, a variety of cell morphology and surface properties were determined and related to the observed bacterial transport behavior. Our experimental results showed that, consistent with previous observations, the deposition rate coefficients of the tet(R)E. coli strain was ~20-100% higher than those of the tet(S)E. coli strain. The zeta potential of the tet(R)E. coli cells was ~25 mV more negative than the tet(S)E. coli cells. Because the surfaces of the E. coli cells and the quartz sands were negatively charged, the repulsive electrostatic double-layer interaction between the tet(R)E. coli cells and the quartz sands was stronger, and the mobility of the tet(R)E. coli cells in the sand packs was thus higher. The tet(R)E. coli cells were also more hydrophilic than the tet(S)E. coli cells. Results from migration to hydrocarbon phase (MATH) tests showed that about ~35% more tet(S)E. coli cells partitioned to the hydrocarbon phase. As it was previously shown that cell hydrophobicity could enhance the attachment of bacterial cells to quartz sand, the difference in cell hydrophobicity could also have contributed to the observed higher mobility of the tet(R)E. coli cells. The size of the tet(R) and tet(S)E. coli cells were similar, suggesting that the observed difference in their mobility was not size-related. Characterization of cell surface properties also showed that tet(R) and tetS E. coli cells differed slightly in cell-bound lipopolysaccharide contents and had distinct outer membrane protein profiles. Such difference could alter cell surface properties which in turn led to changes in cell mobility.

Comparison of the Transport of Tetracycline-Resistant and Tetracycline-Susceptible Escherichia coli Isolated from Lake Michigan.

The outer membrane protein (OMP) TolC is the cell surface component of several drug efflux pumps that are responsible for bacterial resistance against a variety of antibiotics. In this research, we investigated the effects of OMP TolC on E. coli transport within saturated sands through column experiments using a wild-type E. coli K12 strain (with OMP TolC), as well as the corresponding transposon mutant (tolC::kan) and the markerless deletion mutant (ΔtolC). Our results showed OMP TolC could significantly enhance the transport of E. coli when the ionic strength was 20 mM NaCl or higher. The deposition rate coefficients for the wild-type E. coli strain (with OMP TolC) was usually >50% lower than those of the tolC-negative mutants. The measurements of contact angles using three probe liquids suggested that TolC altered the surface tension components of E. coli cells and lead to lower Hamaker constants for the cell-water-sand system. The interaction energy calculations using the extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory suggested that the deposition of the E. coli cell primarily occurred at the secondary energy minimum. The depth of the secondary energy minimum increased with ionic strength, and was greater for the TolC-deletion strains under high ionic strength conditions. Overall, the transport behavior of three E. coli strains within saturated sands could be explained by the XDLVO calculations. Results from this research suggested that antibiotic resistant bacteria expressing OMP TolC could spread more widely within sandy aquifers.

Effects of outer membrane protein TolC on the transport of Escherichia coli within saturated quartz sands.

Pyomelanin is a brown/black extracellular pigment with antioxidant and iron acquisition properties that is produced by a number of different bacteria. Production of pyomelanin in Pseudomonas aeruginosa contributes to increased resistance to oxidative stress and persistence in chronic infections. We demonstrate that pyomelanin production can be inhibited by 2-[2-nitro-4-(trifluoromethyl) benzoyl]-1,3-cyclohexanedione (NTBC). This treatment increases sensitivity of pyomelanogenic P. aeruginosa strains to oxidative stress, without altering the growth rate or resistance to aminoglycosides. As such, NTBC has potential to function as an anti-virulence factor in treating pyomelanogenic bacterial infections.

NTBC treatment of the pyomelanogenic Pseudomonas aeruginosa clinical isolate PA1111 inhibits pigment production and increases sensitivity to oxidative stress.

Erratum to: NTBC treatment of the pyomelanogenic Pseudomonas aeruginosa clinical isolate PA1111 inhibits pigment production and increases sensitivity to oxidative stress.

Sonia L. Bardy

Department of Biological Sciences

University of Wisconsin Milwaukee

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Sonia L. Bardy

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biological Sciences

University of Wisconsin Milwaukee

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Department of Biological Sciences