Protein Export by the Mycobacterial SecA2 System Is Determined by the Preprotein Mature Domain

At the core of the bacterial general secretion (Sec) pathway is the SecA ATPase, which powers translocation of unfolded preproteins containing Sec signal sequences through the SecYEG membrane channel. Mycobacteria have two nonredundant SecA homologs: SecA1 and SecA2. While the essential SecA1 handles “housekeeping” export, the nonessential SecA2 exports a subset of proteins and is required for Mycobacterium tuberculosis virulence. Currently, it is not understood how SecA2 contributes to Sec export in mycobacteria. In this study, we focused on identifying the features of two SecA2 substrates that target them to SecA2 for export, the Ms1704 and Ms1712 lipoproteins of the model organism Mycobacterium smegmatis. We found that the mature domains of Ms1704 and Ms1712, not the N-terminal signal sequences, confer SecA2-dependent export. We also demonstrated that the lipid modification and the extreme N terminus of the mature protein do not impart the requirement for SecA2 in export. We further showed that the Ms1704 mature domain can be efficiently exported by the twin-arginine translocation (Tat) pathway. Because the Tat system exports only folded proteins, this result implies that SecA2 substrates can fold in the cytoplasm and suggests a putative role of SecA2 in enabling export of such proteins. Thus, the mycobacterial SecA2 system may represent another way that bacteria solve the problem of exporting proteins that can fold in the cytoplasm

Design of synthetic bacterial communities for predictable plant phenotypes

Specific members of complex microbiota can influence host phenotypes, depending on both the abiotic environment and the presence of other microorganisms. Therefore, it is challenging to define bacterial combinations that have predictable host phenotypic outputs. We demonstrate that plant–bacterium binary-association assays inform the design of small synthetic communities with predictable phenotypes in the host. Specifically, we constructed synthetic communities that modified phosphate accumulation in the shoot and induced phosphate starvation–responsive genes in a predictable fashion. We found that bacterial colonization of the plant is not a predictor of the plant phenotypes we analyzed. Finally, we demonstrated that characterizing a subset of all possible bacterial synthetic communities is sufficient to predict the outcome of untested bacterial consortia. Our results demonstrate that it is possible to infer causal relationships between microbiota membership and host phenotypes and to use these inferences to rationally design novel communities

Genomic features of bacterial adaptation to plants

Author(s): Levy, A; Salas Gonzalez, I; Mittelviefhaus, M; Clingenpeel, S; Herrera Paredes, S; Miao, J; Wang, K; Devescovi, G; Stillman, K; Monteiro, F; Rangel Alvarez, B; Lundberg, DS; Lu, TY; Lebeis, S; Jin, Z; McDonald, M; Klein, AP; Feltcher, ME; Rio, TG; Grant, SR; Doty, SL; Ley, RE; Zhao, B; Venturi, V; Pelletier, DA; Vorholt, JA; Tringe, SG; Woyke, T; Dangl, JL | Abstract: © 2017 The Author(s). Plants intimately associate with diverse bacteria. Plant-associated bacteria have ostensibly evolved genes that enable them to adapt to plant environments. However, the identities of such genes are mostly unknown, and their functions are poorly characterized. We sequenced 484 genomes of bacterial isolates from roots of Brassicaceae, poplar, and maize. We then compared 3,837 bacterial genomes to identify thousands of plant-associated gene clusters. Genomes of plant-associated bacteria encode more carbohydrate metabolism functions and fewer mobile elements than related non-plant-associated genomes do. We experimentally validated candidates from two sets of plant-associated genes: one involved in plant colonization, and the other serving in microbe-microbe competition between plant-associated bacteria. We also identified 64 plant-associated protein domains that potentially mimic plant domains; some are shared with plant-associated fungi and oomycetes. This work expands the genome-based understanding of plant-microbe interactions and provides potential leads for efficient and sustainable agriculture through microbiome engineering

An integrated workflow for phenazine-modifying enzyme characterization

Increasing availability of new genomes and putative biosynthetic gene clusters (BGCs) has extended the opportunity to access novel chemical diversity for agriculture, medicine, environmental and industrial purposes. However, functional characterization of BGCs through heterologous expression is limited because expression may require complex regulatory mechanisms, specific folding or activation. We developed an integrated workflow for BGC characterization that integrates pathway identification, modular design, DNA synthesis, assembly and characterization. This workflow was applied to characterize multiple phenazine-modifying enzymes. Phenazine pathways are useful for this workflow because all phenazines are derived from a core scaffold for modification by diverse modifying enzymes (PhzM, PhzS, PhzH, and PhzO) that produce characterized compounds. We expressed refactored synthetic modules of previously uncharacterized phenazine BGCs heterologously in Escherichia coli and were able to identify metabolic intermediates they produced, including a previously unidentified metabolite. These results demonstrate how this approach can accelerate functional characterization of BGCs

Biogenesis and functions of bacterial S-layers.

The outer surface of many archaea and bacteria is coated with a proteinaceous surface layer (known as an S-layer), which is formed by the self-assembly of monomeric proteins into a regularly spaced, two-dimensional array. Bacteria possess dedicated pathways for the secretion and anchoring of the S-layer to the cell wall, and some Gram-positive species have large S-layer-associated gene families. S-layers have important roles in growth and survival, and their many functions include the maintenance of cell integrity, enzyme display and, in pathogens and commensals, interaction with the host and its immune system. In this Review, we discuss our current knowledge of S-layer and related proteins, including their structures, mechanisms of secretion and anchoring and their diverse functions

Cutaneous recording of electroencephalograms in electrically stunned broiler chickens

Methodology was developed to record electroencephalograms (EEGs) from chickens using skin surface contact electrodes and telemetry transmitter and receiving units prior to and immediately after electrical stunning. Optimal location of the three electrodes was determined using scaleless ”featherless” chickens. Broilers required plucking of feathers on the neck caudal to the comb ( 2 x 3 cm) under mild anesthesia the day prior to recording EEGs. The telemetry transmitter was protected from the stunning voltage with a custom-built circuit designed to reduce high amplitude AC and DC voltages to less than 0.8 V. This configuration permitted recording of EEG signals prior to and within 3.5 s after termination of the applied stunning current. EEGs were recorded during two different electrical stunning protocols with the current applied to a standing chicken (wattle + and vent -). The first stun protocol was at 8 mA, 12 V (500 Hz) pulse DC for 11 s immediately followed by 12 V (60 Hz) AC for 4 s. The broilers were given several minutes to recover and then stunned again using the second stun protocol set at 103 mA (60 Hz AC) for 4 s, which was sufficient to induce cardiac arrest. The EEG recordings of the second stun protocol were evaluated to determine wave characteristics and the duration of poststun brain activity. The poststun EEG recordings depicted a brief period of high amplitude spikes, which progressively diminished in amplitude with time. This high amplitude polyspike wave form has been assumed to be analogous to the insensibility period that occurs during epileptic seizures in humans. This poststun data, in both wave form and duration of brain activity (39 s), appears similar to that described in the literature for chickens (32 s). Use of the cutaneous-telemetry system to record brain EEG activity in chickens following electrical stunning may provide the opportunity to quantitatively optimize stunning voltage, current, and frequency. Optimal stun parameters should minimize the time to death, and diminish skeletal muscle contraction and the carcass defects associated with electrical stunning

Cutaneous recording of electroencephalograms in electrically stunned broiler chickens

Methodology was developed to record electroencephalograms (EEGs) from chickens using skin surface contact electrodes and telemetry transmitter and receiving units prior to and immediately after electrical stunning. Optimal location of the three electrodes was determined using scaleless ”featherless” chickens. Broilers required plucking of feathers on the neck caudal to the comb ( 2 x 3 cm) under mild anesthesia the day prior to recording EEGs. The telemetry transmitter was protected from the stunning voltage with a custom-built circuit designed to reduce high amplitude AC and DC voltages to less than 0.8 V. This configuration permitted recording of EEG signals prior to and within 3.5 s after termination of the applied stunning current. EEGs were recorded during two different electrical stunning protocols with the current applied to a standing chicken (wattle + and vent -). The first stun protocol was at 8 mA, 12 V (500 Hz) pulse DC for 11 s immediately followed by 12 V (60 Hz) AC for 4 s. The broilers were given several minutes to recover and then stunned again using the second stun protocol set at 103 mA (60 Hz AC) for 4 s, which was sufficient to induce cardiac arrest. The EEG recordings of the second stun protocol were evaluated to determine wave characteristics and the duration of poststun brain activity. The poststun EEG recordings depicted a brief period of high amplitude spikes, which progressively diminished in amplitude with time. This high amplitude polyspike wave form has been assumed to be analogous to the insensibility period that occurs during epileptic seizures in humans. This poststun data, in both wave form and duration of brain activity (39 s), appears similar to that described in the literature for chickens (32 s). Use of the cutaneous-telemetry system to record brain EEG activity in chickens following electrical stunning may provide the opportunity to quantitatively optimize stunning voltage, current, and frequency. Optimal stun parameters should minimize the time to death, and diminish skeletal muscle contraction and the carcass defects associated with electrical stunning

The twin arginine translocation system is essential for aerobic growth and full virulence of Burkholderia thailandensis.

The twin arginine translocation (Tat) system in bacteria is responsible for transporting folded proteins across the cytoplasmic membrane, and in some bacteria, Tat-exported substrates have been linked to virulence. We report here that the Tat machinery is present in Burkholderia pseudomallei, B. mallei, and B. thailandensis, and we show that the system is essential for aerobic but not anaerobic growth. Switching off of the Tat system in B. thailandensis grown anaerobically resulted in filamentous bacteria, and bacteria showed increased sensitivity to some β-lactam antibiotics. In Galleria mellonella and zebrafish infection models, the Tat conditional mutant was attenuated. The aerobic growth-restricted phenotype indicates that Tat substrates may play a functional role in oxygen-dependent energy conservation. In other bacteria, aerobic growth restriction in Tat mutants has been attributed to the inability to translocate PetA, the Rieske iron-sulfur protein which forms part of the quinol-cytochrome c oxidoreductase complex. Here, we show that PetA is not responsible for aerobic growth restriction in B. thailandensis. However, we have identified an operon encoding 2 proteins of unknown function (BTH_I2176 and BTH_I2175) that play a role in aerobic growth restriction, and we present evidence that BTH_I2176 is Tat translocated