Oxygen-Dependent Globin Coupled Sensor Signaling Modulates Motility and Virulence of the Plant Pathogen Pectobacterium carotovorum.

Bacterial pathogens utilize numerous signals to identify the presence of their host and coordinate changes in gene expression that allow for infection. Within plant pathogens, these signals typically include small molecules and/or proteins from their plant hosts and bacterial quorum sensing molecules to ensure sufficient bacterial cell density for successful infection. In addition, bacteria use environmental signals to identify conditions when the host defenses are weakened and potentially to signal entry into an appropriate host/niche for infection. A globin coupled sensor protein (GCS), termed PccGCS, within the soft rot bacterium Pectobacterium carotovorum ssp. carotovorum WPP14 has been identified as an O2 sensor and demonstrated to alter virulence factor excretion and control motility, with deletion of PccGCS resulting in decreased rotting of a potato host. Using small molecules that modulate bacterial growth and quorum sensing, PccGCS signaling also has been shown to modulate quorum sensing pathways, resulting in the PccGCS deletion strain being more sensitive to plant-derived phenolic acids, which can function as quorum sensing inhibitors, and exhibiting increased N-acylhomoserine lactone (AHL) production. These findings highlight a role for GCS proteins in controlling key O2-dependent phenotypes of pathogenic bacteria and suggest that modulating GCS signaling to limit P. carotovorum motility may provide a means to decrease rotting of plant hosts.

Oxygen and Bis(3',5')-cyclic Dimeric Guanosine Monophosphate Binding Control Oligomerization State Equilibria of Diguanylate Cyclase-Containing Globin Coupled Sensors.

Bacteria sense their environment to alter phenotypes, including biofilm formation, to survive changing conditions. Heme proteins play important roles in sensing the bacterial gaseous environment and controlling the switch between motile and sessile (biofilm) states. Globin coupled sensors (GCS), a family of heme proteins consisting of a globin domain linked by a central domain to an output domain, are often found with diguanylate cyclase output domains that synthesize c-di-GMP, a major regulator of biofilm formation. Characterization of diguanylate cyclase-containing GCS proteins from Bordetella pertussis and Pectobacterium carotovorum demonstrated that cyclase activity is controlled by ligand binding to the heme within the globin domain. Both O2 binding to the heme within the globin domain and c-di-GMP binding to a product-binding inhibitory site (I-site) within the cyclase domain control oligomerization states of the enzymes. Changes in oligomerization state caused by c-di-GMP binding to the I-site also affect O2 kinetics within the globin domain, suggesting that shifting the oligomer equilibrium leads to broad rearrangements throughout the protein. In addition, mutations within the I-site that eliminate product inhibition result in changes to the accessible oligomerization states and decreased catalytic activity. These studies provide insight into the mechanism by which ligand binding to the heme and I-site controls activity of GCS proteins and suggests a role for oligomerization-dependent activity in vivo.

Globin domain interactions control heme pocket conformation and oligomerization of globin coupled sensors.

Globin coupled sensors (GCS) are O2-sensing proteins used by bacteria to monitor the surrounding gaseous environment. To investigate the biphasic O2 dissociation kinetics observed for full-length GCS proteins, isolated globin domains from Pectobacterium carotovorum ssp. carotovorum (PccGlobin), and Bordetella pertussis (BpeGlobin), have been characterized. PccGlobin is found to be dimeric, while BpeGlobin is monomeric, indicating key differences in the globin domain dimer interface. Through characterization of wild type globin domains and globin variants with mutations at the dimer interface and within the distal pocket, dimerization of the globin domain is demonstrated to correlate with biphasic dissociation kinetics. Furthermore, a distal pocket tyrosine is identified as the primary hydrogen bond donor, while a secondary hydrogen bond donor within the distal heme pocket is involved in conformation(s) that lead to the second O2 dissociation rate. These findings highlight the role of the globin dimer interface in controlling properties of both the heme pocket and full-length GCS proteins.

Oligomeric state affects oxygen dissociation and diguanylate cyclase activity of globin coupled sensors.

Bacterial biofilm formation is regulated by enzymes, such as diguanylate cyclases, that respond to environmental signals and alter c-di-GMP levels. Diguanylate cyclase activity of two globin coupled sensors is shown to be regulated by gaseous ligands, with cyclase activity and O2 dissociation affected by protein oligomeric state.

Identification of a molecular signature unique to metal-reducing Gammaproteobacteria.

Functional genes required for microbial (dissimilatory) metal reduction display high sequence divergence, which limits their utility as molecular biomarkers for tracking the presence and activity of metal-reducing bacteria in natural and engineered systems. In the present study, homologs of the outer membrane beta-barrel protein MtrB of metal-reducing Gammaproteobacteria were found to contain a unique N-terminal CXXC motif that was missing from MtrB homologs of nonmetal-reducing Gammaproteobacteria and metal- and nonmetal-reducing bacteria outside the Gammaproteobacteria. To determine whether the N-terminal CXXC motif of MtrB was required for dissimilatory metal reduction, each cysteine in the CXXC motif of the representative metal-reducing gammaproteobacterium Shewanella oneidensis was replaced with alanine, and the resulting site-directed mutants were tested for metal reduction activity. Anaerobic growth experiments demonstrated that the first, but not the second, conserved cysteine was required for metal reduction by S. oneidensis. The ability to predict metal reduction by Gammaproteobacteria with unknown metal reduction capability was confirmed with Vibrio parahaemolyticus, a pathogen whose genome encodes an MtrB homolog with an N-terminal CXXC motif. MtrB homologs with an N-terminal CXXC motif may thus represent a molecular signature unique to metal-reducing members of the Gammaproteobacteria.

Electron transport and protein secretion pathways involved in Mn(III) reduction by Shewanella oneidensis.

Soluble Mn(III) represents an important yet overlooked oxidant in marine and freshwater systems. The molecular mechanism of microbial Mn(III) reduction, however, has yet to be elucidated. Extracellular reduction of insoluble Mn(IV) and Fe(III) oxides by the metal-reducing γ-proteobacterium Shewanella oneidensis involves inner (CymA) and outer (OmcA) membrane-associated c-type cytochromes, the extracellular electron conduit MtrCAB, and GspD, the secretin of type II protein secretion. CymA, MtrCAB and GspD mutants were unable to reduce Mn(III) and Mn(IV) with lactate, H2, or formate as electron donor. The OmcA mutant reduced Mn(III) and Mn(IV) at near wild-type rates with lactate and formate as electron donor. With H2 as electron donor, however, the OmcA mutant was unable to reduce Mn(III) but reduced Mn(IV) at wild-type rates. Analogous Fe(III) reduction rate analyses indicated that other electron carriers compensated for the absence of OmcA, CymA, MtrCAB and GspD during Fe(III) reduction in an electron donor-dependent fashion. Results of the present study demonstrate that the S. oneidensis electron transport and protein secretion components involved in extracellular electron transfer to external Mn(IV) and Fe(III) oxides are also required for electron transfer to Mn(III) and that OmcA may function as a dedicated component of an H2 oxidation-linked Mn(III) reduction system.

Outer membrane-associated serine protease involved in adhesion of Shewanella oneidensis to Fe(III) oxides.

The facultative anaerobe Shewanella oneidensis MR-1 respires a variety of anaerobic electron acceptors, including insoluble Fe(III) oxides. S. oneidensis employs a number of novel strategies for respiration of insoluble Fe(III) oxides, including localization of respiratory proteins to the cell outer membrane (OM). The molecular mechanism by which S. oneidensis adheres to and respires Fe(III) oxides, however, remains poorly understood. In the present study, whole cell fractionation and MALDI-TOF-MS/MS techniques were combined to identify a serine protease (SO3800) associated with the S. oneidensis OM. SO3800 contained predicted structural motifs similar to cell surface-associated serine proteases that function as bacterial adhesins in other gram-negative bacteria. The gene encoding SO3800 was deleted from the S. oneidensis genome, and the resulting mutant strain (DeltaSO3800) was tested for its ability to adhere to and respire Fe(III) oxides. DeltaSO3800 was severely impaired in its ability to adhere to Fe(III) oxides, yet retained wild-type Fe(III) respiratory capability. Laser Doppler velocimetry and cryoetch high-resolution SEM experiments indicated that DeltaSO3800 displayed a lower cell surface charge and higher amount of surface-associated exopolysaccharides. Results of this study indicate that S. oneidensis may respire insoluble Fe(III) oxides at a distance, negating the requirement for attachment prior to electron transfer.

Anaerobic respiration of elemental sulfur and thiosulfate by Shewanella oneidensis MR-1 requires psrA, a homolog of the phsA gene of Salmonella enterica serovar typhimurium LT2.

Shewanella oneidensis MR-1, a facultatively anaerobic gammaproteobacterium, respires a variety of anaerobic terminal electron acceptors, including the inorganic sulfur compounds sulfite (SO3(2-)), thiosulfate (S2O3(2-)), tetrathionate (S4O6(2-)), and elemental sulfur (S(0)). The molecular mechanism of anaerobic respiration of inorganic sulfur compounds by S. oneidensis, however, is poorly understood. In the present study, we identified a three-gene cluster in the S. oneidensis genome whose translated products displayed 59 to 73% amino acid similarity to the products of phsABC, a gene cluster required for S(0) and S2O3(2-) respiration by Salmonella enterica serovar Typhimurium LT2. Homologs of phsA (annotated as psrA) were identified in the genomes of Shewanella strains that reduce S(0) and S2O3(2-) yet were missing from the genomes of Shewanella strains unable to reduce these electron acceptors. A new suicide vector was constructed and used to generate a markerless, in-frame deletion of psrA, the gene encoding the putative thiosulfate reductase. The psrA deletion mutant (PSRA1) retained expression of downstream genes psrB and psrC but was unable to respire S(0) or S2O3(2-) as the terminal electron acceptor. Based on these results, we postulate that PsrA functions as the main subunit of the S. oneidensis S2O3(2-) terminal reductase whose end products (sulfide [HS-] or SO3(2-)) participate in an intraspecies sulfur cycle that drives S(0) respiration.

Shewanella putrefaciens produces an Fe(III)-solubilizing organic ligand during anaerobic respiration on insoluble Fe(III) oxides.

The mechanism of Fe(III) reduction was investigated using voltammetric techniques in anaerobic incubations of Shewanella putrefaciens strain 200 supplemented with Fe(III) citrate or a suite of Fe(III) oxides as terminal electron acceptor. Results indicate that organic complexes of Fe(III) are produced during the reduction of Fe(III) at rates that correlate with the reactivity of the Fe(III) phase and bacterial cell density. Anaerobic Fe(III) solubilization activity is detected with either Fe(III) oxides or Fe(III) citrate, suggesting that the organic ligand produced is strong enough to destabilize Fe(III) from soluble or solid Fe(III) substrates. Results also demonstrate that Fe(III) oxide dissolution is not controlled by the intrinsic chemical reactivity of the Fe(III) oxides. Instead, the chemical reaction between the endogenous organic ligand is only affected by the number of reactive surface sites available to S. putrefaciens. This report describes the first application of voltammetric techniques to demonstrate production of soluble organic-Fe(III) complexes by any Fe(III)-reducing microorganism and is the first report of a Fe(III)-solubilizing ligand generated by a metal-reducing member of the genus Shewanella.

Terminal electron acceptors influence the quantity and chemical composition of capsular exopolymers produced by anaerobically growing Shewanella spp.

Bacterial exopolymers perform various roles, including acting as a carbon sink, a protective layer against desiccation or antimicrobial agents, or a structural matrix in biofilms. Despite such varied roles, little is known about the heterogeneity of bacterial exopolymer production under varying growth conditions. Here we describe experiments designed to characterize the quantity and quality of exopolymers produced by two commonly studied members of the widely distributed genus Shewanella. Electrokinetic, spectroscopic, and electron microscopic techniques were employed to demonstrate that cell surfaces of Shewanella oneidensis MR-1 (electrophoretic softness, lambda(-1), range from 0.4 to 2.6 nm) are associated with less extracellular polymeric material than surfaces of Shewanella putrefaciens 200R (lambda(-1) range from 1.6 to 3.0 nm). Both species exhibit similar responses to changes in electron acceptor with nitrate- and fumarate-grown cells producing relatively little exopolymer compared to trimethylamine N-oxide (TMAO)-grown cells. In S. oneidensis, the increase in exopolymers has no apparent effect upon cell-surface fixed charge density (-7.7 to -8.7 mM), but for S. putrefaciens a significant drop in fixed charge density is observed between fumarate/nitrate-grown cells (-43 mM) and TMAO-grown cells (-20.8 mM). For both species, exopolymers produced during growth on TMAO have significant amide functionality, increasing from approximately 20-25% of C-containing moieties in nitrate-grown cells to over 30% for TMAO-grown cells (determined from X-ray photoelectron spectroscopy). The increased exopolymer layer associated with TMAO-grown cells appears as a continuous, convoluted layer covering the entire cell surface when viewed by low-temperature, high-resolution scanning electron microscopy. Such significant changes in cell-surface architecture, dependent upon the electron acceptor used for growth, are likely to influence a variety of cell interactions, including aggregation and attachment to surfaces, and the binding of aqueous metal species.