Characterization of 2-(2-nitro-4-trifluoromethylbenzoyl)- 1,3-cyclohexanedione resistance in pyomelanogenic Pseudomonas aeruginosa DKN343

Pyomelanin is a reddish-brown pigment that provides bacteria and fungi protection from oxidative stress, and is reported to contribute to infection persistence. Production of this pigment can be inhibited by the anti-virulence agent 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3- cyclohexanedione (NTBC). The Pseudomonas aeruginosa clinical isolate DKN343 exhibited high levels of resistance to NTBC, and the mechanism of pyomelanin production in this strain was uncharacterized. We determined that pyomelanin production in the clinical Pseudomonas aeruginosa isolate DKN343 was due to a loss of function in homogentisate 1,2- dioxygenase (HmgA). Several potential resistance mechanisms were investigated, and the MexAB-OprM efflux pump is required for resistance to NTBC. DKN343 has a frameshift mutation in NalC, which is a known indirect repressor of the mexAB-oprM operon. This frameshift mutation may contribute to the increased resistance of DKN343 to NTBC. Additional studies investigating the prevalence of resistance in pyomelanogenic microbes are necessary to determine the future applications of NTBC as an anti-virulence therapy

Identification and Characterization of the Unique N-Linked Glycan Common to the Flagellins and S-layer Glycoprotein of 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

Polar Localization of a Soluble Methyl-Accepting Protein of Pseudomonas aeruginosa

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

Resistance to NTBC is mediated through the MexAB-OprM multidrug efflux pump.

<p>(A) Deletion of <i>mexA</i> in PAO1 <i>hmgA</i>::<i>tn</i> and DKN343 increased sensitivity to NTBC as indicated by reduced levels of pyomelanin. Strains complemented with His-MexA showed restoration of pyomelanin production in the presence of NTBC. PAO1 <i>hpd</i>::<i>tn</i> (V) was the pyomelanin non-producer control. Cultures were grown in LB with gentamicin and 0.05% arabinose with the indicated concentrations of NTBC. (B) The relative levels of His-MexA was determined through western blotting of whole cell lysates from PAO1 <i>hmgA</i>::<i>tn</i>Δ<i>mexA</i> and DKN343Δ<i>mexA</i>. MexA was expressed at higher levels in in DKN343Δ<i>mexA</i> compared to PAO1 <i>hmgA</i>::<i>tn</i>Δ<i>mexA</i>. Cultures were grown for 24 hours in LB with gentamicin and 0.05% arabinose.</p

NTBC treatment reduced pigment production in pyomelanogenic <i>P</i>. <i>aeruginosa</i>.

<p>Strains were grown in LB with the indicated concentrations of NTBC. The non-pyomelanogenic control PAO1 <i>hpd</i>::<i>tn</i> showed no pigment change in response to NTBC treatment. The pyomelanin producers PAO1 <i>hmgA</i>::<i>tn</i> and DKN343 showed dose dependent reductions in pigmentation with increasing concentrations of NTBC. PAO1 <i>hmgA</i>::<i>tn</i> was the positive control for pyomelanin production. The clinical isolate DKN343 was less affected by NTBC than the laboratory strain PAO1 <i>hmgA</i>::<i>tn</i>, as indicated by the higher concentrations of NTBC required to reduce pyomelanin production.</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

Recent advances and future prospects in bacterial and archaeal locomotion and signal transduction

The structure and function of two-component and chemotactic signaling and different aspects related to the motility of bacteria and archaea are key research areas in modern microbiology. Escherichia coli is the traditional model organism used to study chemotaxis signaling and motility. However, the recent study of a wide range of bacteria and even some archaea with different lifestyles has provided new insight into the ecophysiology of chemotaxis, which is essential for the establishment of different pathogens or beneficial bacteria in a host. The expanded range of model organisms has also permitted the study of chemosensory pathways unrelated to chemotaxis, multiple chemotaxis pathways within an organism, and new types of chemoreceptors. This research has greatly benefitted from technical advances in the field of cryomicroscopy, which continues to reveal with increasing resolution the complexity and diversity of large protein complexes like the flagellar motor or chemoreceptor arrays. In addition, sensitive instruments now allow an increasing number of experiments to be conducted at the single-cell level, thereby revealing information that is beginning to bridge the gap between individual cells and population behavior. Evidence has also accumulated showing that bacteria have evolved different mechanisms for surface sensing, which appears to be mediated by flagella and possibly type IV pili, and that the downstream signaling involves chemosensory pathways and two-component-system-based processes. Herein, we summarize the recent advances and research tendencies in this field as presented at the latest Bacterial Locomotion and Signal Transduction (BLAST XIV) conference.Research in our laboratories is supported by grants from the Spanish Ministry for Economy and Competitiveness (grants BIO2013-42297 and 585 BIO2016-76779-P to T.K.) and the Canada Foundation for Innovation (SR).Peer Reviewe