\u3ci\u3ePseudomonas syringae\u3c/i\u3e Type III Secretion System Targeting Signals and Novel Effectors Studied with a Cya Translocation Reporter

Pseudomonas syringae pv. tomato strain DC3000 is a pathogen of tomato and Arabidopsis. The hrp-hrcencoded type III secretion system (TTSS), which injects bacterial effector proteins (primarily called Hop or Avr proteins) into plant cells, is required for pathogenicity. In addition to being regulated by the HrpL alternative sigma factor, most avr or hop genes encode proteins with N termini that have several characteristic features, including (i) a high percentage of Ser residues, (ii) an aliphatic amino acid (Ile, Leu, or Val) or Pro at the third or fourth position, and (iii) a lack of negatively charged amino acids within the first 12 residues. Here, the well-studied effector AvrPto was used to optimize a calmodulin-dependent adenylate cyclase (Cya) reporter system for Hrp-mediated translocation of P. syringae TTSS effectors into plant cells. This system includes a cloned P. syringae hrp gene cluster and the model plant Nicotiana benthamiana. Analyses of truncated AvrPto proteins fused to Cya revealed that the N-terminal 16 amino acids and/or codons of AvrPto are sufficient to direct weak translocation into plant cells and that longer N-terminal fragments direct progressively stronger translocation. AvrB, tested because it is poorly secreted in cultures by the P. syringae Hrp system, was translocated into plant cells as effectively as AvrPto. The translocation of several DC3000 candidate Hop proteins was also examined by using Cya as a reporter, which led to identification of three new intact Hop proteins, designated HopPtoQ, HopPtoT1, and HopPtoV, as well as two truncated Hop proteins encoded by the naturally disrupted genes hopPtoS4::tnpA and hopPtoAG::tnpA. We also confirmed that HopPtoK, HopPtoC, and AvrPphEPto are translocated into plant cells. These results increased the number of Hrp system-secreted proteins in DC3000 to 40. Although most of the newly identified Hop proteins possess N termini that have the same features as the N termini of previously described Hop proteins, HopPtoV has none of these characteristics. Our results indicate that Cya should be a useful reporter for exploring multiple aspects of the Hrp system in P. syringae

Murine Model for Measuring Effects of Humanized-Dosing of Antibiotics on the Gut Microbiome.

There is a current need for enhancing our insight in the effects of antimicrobial treatment on the composition of human microbiota. Also, the spontaneous restoration of the microbiota after antimicrobial treatment requires better understanding. This is best addressed in well-defined animal models. We here present a model in which immune-competent or neutropenic mice were administered piperacillin-tazobactam (TZP) according to human treatment schedules. Before, during and after the TZP treatment, fecal specimens were longitudinally collected at established intervals over several weeks. Gut microbial taxonomic distribution and abundance were assessed through culture and molecular means during all periods. Non-targeted metabolomics analyses of stool samples using Quadrupole Time of Flight mass spectrometry (QTOF MS) were also applied to determine if a metabolic fingerprint correlated with antibiotic use, immune status, and microbial abundance. TZP treatment led to a 5-10-fold decrease in bacterial fecal viability counts which were not fully restored during post-antibiotic follow up. Two distinct, relatively uniform and reproducible restoration scenarios of microbiota changes were seen in post TZP-treatment mice. Post-antibiotic flora could consist of predominantly Firmicutes or, alternatively, a more diverse mix of taxa. In general, the pre-treatment microbial communities were not fully restored within the screening periods applied. A new species, closely related t

Extensive Gene Amplification as a Mechanism for Piperacillin-Tazobactam Resistance in Escherichia coli.

Although the TEM-1 β-lactamase (BlaTEM-1) hydrolyzes penicillins and narrow-spectrum cephalosporins, organisms expressing this enzyme are typically susceptible to β-lactam/β-lactamase inhibitor combinations such as piperacillin-tazobactam (TZP). However, our previous work led to the discovery of 28 clinical isolates of Escherichia coli resistant to TZP that contained only blaTEM-1 One of these isolates, E. coli 907355, was investigated further in this study. E. coli 907355 exhibited significantly higher β-lactamase activity and BlaTEM-1 protein levels when grown in the presence of subinhibitory concentrations of TZP. A corresponding TZP-dependent increase in blaTEM-1 copy number was also observed, with as many as 113 copies of the gene detected per cell. These results suggest that TZP treatment promotes an increase in blaTEM-1 gene dosage, allowing BlaTEM-1 to reach high enough levels to overcome inactivation by the available tazobactam in the culture. To better understand the nature of the blaTEM-1 copy number proliferation, whole-genome sequence (WGS) analysis was performed on E. coli 907355 in the absence and presence of TZP. The WGS data revealed that the blaTEM-1 gene is located in a 10-kb genomic resistance module (GRM) that contains multiple resistance genes and mobile genetic elements. The GRM was found to be tandemly repeated at least 5 times within a p1ESCUM/p1ECUMN-like plasmid when bacteria were grown in the presence of TZP.IMPORTANCE Understanding how bacteria acquire resistance to antibiotics is essential for treating infected patients effectively, as well as preventing the spread of resistant organisms. In this study, a clinical isolate of E. coli was identified that dedicated more than 15% of its genome toward tandem amplification of a ~10-kb resistance module, allowing it to escape antibiotic-mediated killing. Our research is significant in that it provides one possible explanation for clinical isolates that exhibit discordant behavior when tested for antibiotic resistance by different phenotypic methods. Our research also shows that GRM amplification is difficult to detect by short-read WGS technologies. Analysis of raw long-read sequence data was required to confirm GRM amplification as a mechanism of antibiotic resistance. MBio 2018 Apr 24; 9(2):e00583-18

Functional and Computational Analysis of Amino Acid Patterns Predictive of Type III Secretion System Substrates in Pseudomonas syringae

Bacterial type III secretion systems (T3SSs) deliver proteins called effectors into eukaryotic cells. Although N-terminal amino acid sequences are required for translocation, the mechanism of substrate recognition by the T3SS is unknown. Almost all actively deployed T3SS substrates in the plant pathogen Pseudomonas syringae pathovar tomato strain DC3000 possess characteristic patterns, including (i) greater than 10% serine within the first 50 amino acids, (ii) an aliphatic residue or proline at position 3 or 4, and (iii) a lack of acidic amino acids within the first 12 residues. Here, the functional significance of the P. syringae T3SS substrate compositional patterns was tested. A mutant AvrPto effector protein lacking all three patterns was secreted into culture and translocated into plant cells, suggesting that the compositional characteristics are not absolutely required for T3SS targeting and that other recognition mechanisms exist. To further analyze the unique properties of T3SS targeting signals, we developed a computational algorithm called TEREE (Type III Effector Relative Entropy Evaluation) that distinguishes DC3000 T3SS substrates from other proteins with a high sensitivity and specificity. Although TEREE did not efficiently identify T3SS substrates in Salmonella enterica, it was effective in another P. syringae strain and Ralstonia solanacearum. Thus, the TEREE algorithm may be a useful tool for identifying new effector genes in plant pathogens. The nature of T3SS targeting signals was additionally investigated by analyzing the N-terminus of FtsX, a putative membrane protein that was classified as a T3SS substrate by TEREE. Although the first 50 amino acids of FtsX were unable to target a reporter protein to the T3SS, an AvrPto protein substituted with the first 12 amino acids of FtsX was translocated into plant cells. These results show that the T3SS targeting signals are highly mutable and that secretion may be directed by multiple features of substrates

The Atlas3D project -- XIII. Mass and morphology of HI in early-type galaxies as a function of environment

We present the Atlas3D HI survey of 166 nearby early-type galaxies (ETGs) down to M(HI)~10^7 M_sun. We detect HI in ~40% of all ETGs outside the Virgo cluster and in ~10% of all ETGs inside it. This demonstrates that it is common for non-cluster ETGs to host HI. The HI morphology varies from regular discs/rings (the majority of the detections) to unsettled gas distributions. The former are either small discs (M(HI)<10^8 M_sun) confined within the stellar body and sharing the same kinematics of the stars, or large discs/rings (M(HI) up to 5x10^9 M_sun) extending to tens of kpc from the host galaxy and frequently kinematically decoupled from the stars. Neutral hydrogen provides material for star formation in ETGs. Galaxies with central HI exhibit signatures of star formation in ~70% of the cases, ~5 times more frequently than galaxies without central HI. The central ISM is dominated by molecular gas. In ETGs with a small gas disc the conversion of HI into H_2 is as efficient as in spirals. The ETG HI mass function has M*~2x10^9 M_sun and slope=-0.7. ETGs host much less HI than spirals as a family. However, a significant fraction of them is as HI-rich as spirals. The main difference between ETGs and spirals is that the former lack the high-column-density HI typical of the bright stellar disc of the latter. We find an envelope of decreasing M(HI) with increasing environment density. The gas-richest ETGs live in the poorest environments (where star-formation is more common), galaxies in the centre of Virgo have the lowest HI content, and the cluster outskirts are a transition region. We find an HI morphology-density relation. At low environment density HI is mostly distributed on large discs/rings. More disturbed HI morphologies dominate environment densities typical of rich groups, confirming the importance of processes occurring on a galaxy-group scale for the evolution of ETGs.Comment: Accepted for publication on MNRA

\u3ci\u3ePseudomonas syringae\u3c/i\u3e Type III Secretion System Targeting Signals and Novel Effectors Studied with a Cya Translocation Reporter

Pseudomonas syringae pv. tomato strain DC3000 is a pathogen of tomato and Arabidopsis. The hrp-hrcencoded type III secretion system (TTSS), which injects bacterial effector proteins (primarily called Hop or Avr proteins) into plant cells, is required for pathogenicity. In addition to being regulated by the HrpL alternative sigma factor, most avr or hop genes encode proteins with N termini that have several characteristic features, including (i) a high percentage of Ser residues, (ii) an aliphatic amino acid (Ile, Leu, or Val) or Pro at the third or fourth position, and (iii) a lack of negatively charged amino acids within the first 12 residues. Here, the well-studied effector AvrPto was used to optimize a calmodulin-dependent adenylate cyclase (Cya) reporter system for Hrp-mediated translocation of P. syringae TTSS effectors into plant cells. This system includes a cloned P. syringae hrp gene cluster and the model plant Nicotiana benthamiana. Analyses of truncated AvrPto proteins fused to Cya revealed that the N-terminal 16 amino acids and/or codons of AvrPto are sufficient to direct weak translocation into plant cells and that longer N-terminal fragments direct progressively stronger translocation. AvrB, tested because it is poorly secreted in cultures by the P. syringae Hrp system, was translocated into plant cells as effectively as AvrPto. The translocation of several DC3000 candidate Hop proteins was also examined by using Cya as a reporter, which led to identification of three new intact Hop proteins, designated HopPtoQ, HopPtoT1, and HopPtoV, as well as two truncated Hop proteins encoded by the naturally disrupted genes hopPtoS4::tnpA and hopPtoAG::tnpA. We also confirmed that HopPtoK, HopPtoC, and AvrPphEPto are translocated into plant cells. These results increased the number of Hrp system-secreted proteins in DC3000 to 40. Although most of the newly identified Hop proteins possess N termini that have the same features as the N termini of previously described Hop proteins, HopPtoV has none of these characteristics. Our results indicate that Cya should be a useful reporter for exploring multiple aspects of the Hrp system in P. syringae

The Small Nucleoid-Binding Proteins H-NS, HU, and Fis Affect hilA Expression in Salmonella enterica Serovar Typhimurium

hilA encodes an activator of Salmonella enterica serovar Typhimurium virulence genes and is transcriptionally modulated by environmental conditions. We show that H-NS represses hilA under low-osmolarity conditions. H-NS, HU, and Fis also appear to affect the derepression of hilA by HilD. Modulation of hilA by counteracting repressing and derepressing mechanisms may allow Salmonella serovar Typhimurium to regulate its virulence genes in response to different situations in vivo

Pseudomonas syringae Type III Secretion System Targeting Signals and Novel Effectors Studied with a Cya Translocation Reporter

Pseudomonas syringae pv. tomato strain DC3000 is a pathogen of tomato and Arabidopsis. The hrp-hrc-encoded type III secretion system (TTSS), which injects bacterial effector proteins (primarily called Hop or Avr proteins) into plant cells, is required for pathogenicity. In addition to being regulated by the HrpL alternative sigma factor, most avr or hop genes encode proteins with N termini that have several characteristic features, including (i) a high percentage of Ser residues, (ii) an aliphatic amino acid (Ile, Leu, or Val) or Pro at the third or fourth position, and (iii) a lack of negatively charged amino acids within the first 12 residues. Here, the well-studied effector AvrPto was used to optimize a calmodulin-dependent adenylate cyclase (Cya) reporter system for Hrp-mediated translocation of P. syringae TTSS effectors into plant cells. This system includes a cloned P. syringae hrp gene cluster and the model plant Nicotiana benthamiana. Analyses of truncated AvrPto proteins fused to Cya revealed that the N-terminal 16 amino acids and/or codons of AvrPto are sufficient to direct weak translocation into plant cells and that longer N-terminal fragments direct progressively stronger translocation. AvrB, tested because it is poorly secreted in cultures by the P. syringae Hrp system, was translocated into plant cells as effectively as AvrPto. The translocation of several DC3000 candidate Hop proteins was also examined by using Cya as a reporter, which led to identification of three new intact Hop proteins, designated HopPtoQ, HopPtoT1, and HopPtoV, as well as two truncated Hop proteins encoded by the naturally disrupted genes hopPtoS4::tnpA and hopPtoAG::tnpA. We also confirmed that HopPtoK, HopPtoC, and AvrPphE(Pto) are translocated into plant cells. These results increased the number of Hrp system-secreted proteins in DC3000 to 40. Although most of the newly identified Hop proteins possess N termini that have the same features as the N termini of previously described Hop proteins, HopPtoV has none of these characteristics. Our results indicate that Cya should be a useful reporter for exploring multiple aspects of the Hrp system in P. syringae

Translocation of NopP by Sinorhizobium fredii USDA257 into Vigna unguiculata Root Nodules▿

Sinorhizobium fredii is a nitrogen-fixing legume symbiont that stimulates the formation of root nodules. S. fredii nodulation of roots is influenced by Nop proteins, which are secreted through a type III secretion system (T3SS). We demonstrate that S. fredii injects NopP into Vigna unguiculata nodules in a T3SS-dependent manner