Redox linked flavin sites in extracellular decaheme proteins involved in microbe-mineral electron transfer

Extracellular microbe-mineral electron transfer is a major driving force for the oxidation of organic carbon in many subsurface environments. Extracellular multi-heme cytochromes of the Shewenella genus play a major role in this process but the mechanism of electron exchange at the interface between cytochrome and acceptor is widely debated. The 1.8 Å x-ray crystal structure of the decaheme MtrC revealed a highly conserved CX8C disulfide that, when substituted for AX8A, severely compromised the ability of S. oneidensis to grow under aerobic conditions. Reductive cleavage of the disulfide in the presence of flavin mononucleotide (FMN) resulted in the reversible formation of a stable flavocytochrome. Similar results were also observed with other decaheme cytochromes, OmcA, MtrF and UndA. The data suggest that these decaheme cytochromes can transition between highly reactive flavocytochromes or less reactive cytochromes, and that this transition is controlled by a redox active disulfide that responds to the presence of oxygen

The Pseudomonas aeruginosa Vfr Regulator Controls Global Virulence Factor Expression through Cyclic AMP-Dependent and -Independent Mechanisms

Vfr is a global regulator of virulence factor expression in the human pathogen Pseudomonas aeruginosa. Although indirect evidence suggests that Vfr activity is controlled by cyclic AMP (cAMP), it has been hypothesized that the putative cAMP binding pocket of Vfr may accommodate additional cyclic nucleotides. In this study, we used two different approaches to generate apo-Vfr and examined its ability to bind a representative set of virulence gene promoters in the absence and presence of different allosteric effectors. Of the cyclic nucleotides tested, only cAMP was able to restore DNA binding activity to apo-Vfr. In contrast, cGMP was capable of inhibiting cAMP-Vfr DNA binding. Further, we demonstrate that vfr expression is autoregulated and cAMP dependent and involves Vfr binding to a previously unidentified site within the vfr promoter region. Using a combination of in vitro and in vivo approaches, we show that cAMP is required for Vfr-dependent regulation of a specific subset of virulence genes. In contrast, we discovered that Vfr controls expression of the lasR promoter in a cAMP-independent manner. In summary, our data support a model in which Vfr controls virulence gene expression by distinct (cAMP-dependent and -independent) mechanisms, which may allow P. aeruginosa to fine-tune its virulence program in response to specific host cues or environments

A Bioelectrochemical Approach to Characterize Extracellular Electron Transfer by Synechocystis sp. PCC6803

Biophotovoltaic devices employ photosynthetic organisms at the anode of a microbial fuel cell to generate electrical power. Although a range of cyanobacteria and algae have been shown to generate photocurrent in devices of a multitude of architectures, mechanistic understanding of extracellular electron transfer by phototrophs remains minimal. Here we describe a mediatorless bioelectrochemical device to measure the electrogenic output of a planktonically grown cyanobacterium, Synechocystis sp. PCC6803. Light dependent production of current is measured, and its magnitude is shown to scale with microbial cell concentration and light intensity. Bioelectrochemical characterization of a Synechocystis mutant lacking Photosystem II demonstrates conclusively that production of the majority of photocurrent requires a functional water splitting aparatus and electrons are likely ultimately derived from water. This shows the potential of the device to rapidly and quantitatively characterize photocurrent production by genetically modified strains, an approach that can be used in future studies to delineate the mechanisms of cyanobacterial extracellular electron transport

Characterization of the Promoter, MxiE Box and 5′ UTR of Genes Controlled by the Activity of the Type III Secretion Apparatus in Shigella flexneri

Activation of the type III secretion apparatus (T3SA) of Shigella flexneri, upon contact of the bacteria with host cells, and its deregulation, as in ipaB mutants, specifically increases transcription of a set of effector-encoding genes controlled by MxiE, an activator of the AraC family, and IpgC, the chaperone of the IpaB and IpaC translocators. Thirteen genes carried by the virulence plasmid (ospB, ospC1, ospD2, ospD3, ospE1, ospE2, ospF, ospG, virA, ipaH1.4, ipaH4.5, ipaH7.8 and ipaH9.8) and five genes carried by the chromosome (ipaHa-e) are regulated by the T3SA activity. A conserved 17-bp MxiE box is present 5′ of most of these genes. To characterize the promoter activity of these MxiE box-containing regions, similar ∼67-bp DNA fragments encompassing the MxiE box of 14 MxiE-regulated genes were cloned 5′ of lacZ in a promoter probe plasmid; β-galactosidase activity detected in wild-type and ipaB strains harboring these plasmids indicated that most MxiE box-carrying regions contain a promoter regulated by the T3SA activity and that the relative strengths of these promoters cover an eight-fold range. The various MxiE boxes exhibiting up to three differences as compared to the MxiE box consensus sequence were introduced into the ipaH9.8 promoter without affecting its activity, suggesting that they are equally efficient in promoter activation. In contrast, all nucleotides conserved among MxiE boxes were found to be involved in MxiE-dependent promoter activity. In addition, we present evidence that the 5′ UTRs of four MxiE-regulated genes enhance expression of the downstream gene, presumably by preventing degradation of the mRNA, and the 5′ UTRs of two other genes carry an ancillary promoter

Sequence-Based Prediction of Type III Secreted Proteins

The type III secretion system (TTSS) is a key mechanism for host cell interaction used by a variety of bacterial pathogens and symbionts of plants and animals including humans. The TTSS represents a molecular syringe with which the bacteria deliver effector proteins directly into the host cell cytosol. Despite the importance of the TTSS for bacterial pathogenesis, recognition and targeting of type III secreted proteins has up until now been poorly understood. Several hypotheses are discussed, including an mRNA-based signal, a chaperon-mediated process, or an N-terminal signal peptide. In this study, we systematically analyzed the amino acid composition and secondary structure of N-termini of 100 experimentally verified effector proteins. Based on this, we developed a machine-learning approach for the prediction of TTSS effector proteins, taking into account N-terminal sequence features such as frequencies of amino acids, short peptides, or residues with certain physico-chemical properties. The resulting computational model revealed a strong type III secretion signal in the N-terminus that can be used to detect effectors with sensitivity of ∼71% and selectivity of ∼85%. This signal seems to be taxonomically universal and conserved among animal pathogens and plant symbionts, since we could successfully detect effector proteins if the respective group was excluded from training. The application of our prediction approach to 739 complete bacterial and archaeal genome sequences resulted in the identification of between 0% and 12% putative TTSS effector proteins. Comparison of effector proteins with orthologs that are not secreted by the TTSS showed no clear pattern of signal acquisition by fusion, suggesting convergent evolutionary processes shaping the type III secretion signal. The newly developed program EffectiveT3 (http://www.chlamydiaedb.org) is the first universal in silico prediction program for the identification of novel TTSS effectors. Our findings will facilitate further studies on and improve our understanding of type III secretion and its role in pathogen–host interactions

Pseudomonas aeruginosa Eliminates Natural Killer Cells via Phagocytosis-Induced Apoptosis

Pseudomonas aeruginosa (PA) is an opportunistic pathogen that causes the relapse of illness in immunocompromised patients, leading to prolonged hospitalization, increased medical expense, and death. In this report, we show that PA invades natural killer (NK) cells and induces phagocytosis-induced cell death (PICD) of lymphocytes. In vivo tumor metastasis was augmented by PA infection, with a significant reduction in NK cell number. Adoptive transfer of NK cells mitigated PA-induced metastasis. Internalization of PA into NK cells was observed by transmission electron microscopy. In addition, PA invaded NK cells via phosphoinositide 3-kinase (PI3K) activation, and the phagocytic event led to caspase 9-dependent apoptosis of NK cells. PA-mediated NK cell apoptosis was dependent on activation of mitogen-activated protein (MAP) kinase and the generation of reactive oxygen species (ROS). These data suggest that the phagocytosis of PA by NK cells is a critical event that affects the relapse of diseases in immunocompromised patients, such as those with cancer, and provides important insights into the interactions between PA and NK cells

Timing is everything: the regulation of type III secretion

Type Three Secretion Systems (T3SSs) are essential virulence determinants of many Gram-negative bacteria. The T3SS is an injection device that can transfer bacterial virulence proteins directly into host cells. The apparatus is made up of a basal body that spans both bacterial membranes and an extracellular needle that possesses a channel that is thought to act as a conduit for protein secretion. Contact with a host-cell membrane triggers the insertion of a pore into the target membrane, and effectors are translocated through this pore into the host cell. To assemble a functional T3SS, specific substrates must be targeted to the apparatus in the correct order. Recently, there have been many developments in our structural and functional understanding of the proteins involved in the regulation of secretion. Here we review the current understanding of protein components of the system thought to be involved in switching between different stages of secretion