Proteomics and lipidomics integrated analysis of outer membrane vesicles indicates that glycerophospholipid metabolism contributes to the early symbiosis between Sinorhizobium fredii HH103 and soybean

Gram-negative bacteria can produce outer membrane vesicles (OMVs), and most functional studies of OMVs have been focused on mammalian-bacterial interactions. However, research on the OMVs of rhizobia is still limited so far. In this work, we isolated and purified OMVs from Sinorhizobium fredii HH103 under free-living conditions that was set as control (C-OMVs) and symbiosis-mimicking conditions that was induced by genistein (G-OMVs). The soybean roots treated with G-OMVs displayed significant deformation of root hairs. G-OMVs significantly induced the expression of nodulation genes related to early symbiosis, while inhibited that of the defense genes of soybean. Proteomics analysis identified a total of 93 differential proteins between C-OMVs and G-OMVs, which are mainly associated with ribosome synthesis, flagellar assembly, two-component system, ABC transporters, oxidative phosphorylation, nitrogen metabolism, quorum sensing, glycerophospholipid metabolism and peptidoglycan biosynthesis. A total of 45 differential lipids were identified in lipidomics analysis. Correlation analysis of OMV proteome and lipidome data revealed that glycerophospholipid metabolism is the enriched KEGG metabolic pathway, and the expression of phosphatidylserine decarboxylase was significantly up-regulated in G-OMVs. The changes in three lipids related to symbiosis in the glycerophospholipid metabolism pathway were verified by ELISA. Our results indicate that glycerophospholipid metabolism contributes to rhizobia-soybean symbiosis via OMVs.

Gene Analysis of Listeria monocytogenes Suspended Aggregates Induced by Ralstonia insidiosa Cell-Free Supernatants under Nutrient-Poor Environments

Listeria monocytogenes is a zoonotic food-borne pathogen. The production of food-borne pathogenic bacteria aggregates is considered to be a way to improve their resistance and persistence in the food chain. Ralstonia insidiosa has been shown to induce L. monocytogenes to form suspended aggregates, but induction mechanisms remain unclear. In the study, the effect of R. insidiosa cell-free supernatants cultured in 10% TSB medium (10% RIS) on the formation of L. monocytogenes suspended aggregates was evaluated. Next, the Illumina RNA sequencing was used to compare the transcriptional profiles of L. monocytogenes in 10% TSB medium with and without 10% RIS to identify differentially expressed genes (DEGs). The result of functional annotation analysis of DEGs indicated that these genes mainly participate in two component system, bacterial chemotaxis and flagellar assembly. Then the reaction network of L. monocytogenes suspended aggregates with the presence of 10% RIS was summarized. The gene-deletion strain of L. monocytogenes was constructed by homologous recombination. The result showed that cheA and cheY are key genes in the formation of suspended aggregates. This research is the preliminary verification of suspended aggregates’ RNA sequencing and is helpful to analyze the aggregation mechanisms of food-borne pathogenic bacteria from a new perspective.

The short flagella 1 (SHF1) gene in Chlamydomonas encodes a Crescerin TOG-domain protein required for late stages of flagellar growth

Length control of flagella represents a simple and tractable system to investigate the dynamics of organelle size. Models for flagellar length control in the model organism, Chlamydomonas reinhardtii have focused on the length-dependence of the intraflagellar transport (IFT) system which manages the delivery and removal of axonemal subunits at the tip of the flagella. One of these cargoes, tubulin, is the major axonemal subunit, and its frequency of arrival at the tip plays a central role in size control models. However, the mechanisms determining tubulin dynamics at the tip are still poorly understood. We discovered a loss-of-function mutation that leads to shortened flagella, and found that this was an allele of a previously described gene, SHF1, whose molecular identity had not previously been determined. We found that SHF1 encodes a Chlamydomonas ortholog of Crescerin, previously identified as a cilia-specific TOG-domain array protein that can bind tubulin via its TOG domains and increase tubulin polymerization rates. In this mutant, flagellar regeneration occurs with the same initial kinetics as wild-type cells, but plateaus at a shorter length. Using a computational model in which the flagellar microtubules are represented by a differential equation for flagellar length combined with a stochastic model for cytoplasmic microtubule dynamics, we found that our experimental results are best described by a model in which Crescerin/SHF1 binds tubulin dimers in the cytoplasm and transports them into the flagellum. We suggest that this TOG-domain protein is necessary to efficiently and preemptively increase intra-flagella tubulin levels to offset decreasing IFT cargo at the tip as flagellar assembly progresses.

Involvement of the Heat Shock Protein HtpG of Salmonella Typhimurium in Infection and Proliferation in Hosts

Salmonella Typhimurium is a common pathogen infecting the gastrointestinal tract of humans and animals, causing host gastroenteritis and typhoid fever. Heat shock protein (HtpG) as a molecular chaperone is involved in the various cellular processes of bacteria, especially under environmental stress. However, the potential association of HtpG with S. Typhimurium infection remains unknown. In this study, we clarified that HtpG could also play a role as an effector in S. Typhimurium infection. RNA-seq indicated that the flagellar assembly pathway, infection pathway, and chemotaxis pathway genes of S. Typhimurium were downregulated after the mutation of HtpG, which resulted in compromises of S. Typhimurium motility, biofilm formation, adhesion, invasion, and inflammation-inducing ability. In addition, HtpG recombinant protein was capable of promoting the proliferation of S. Typhimurium in host cells and the resultant inflammation. Collectively, our results illustrated an important role of HtpG in S. Typhimurium infection.

A Combination of Genomics, Transcriptomics, and Genetics Provides Insights into the Mineral Weathering Phenotype of Pseudomonas azotoformans F77

Silicate mineral weathering (dissolution) plays important roles in soil formation and global biogeochemical cycling. In this study, a combination of genomics, transcriptomics, and genetics was used to identify the molecular basis of mineral weathering activity and acid tolerance in Pseudomonas azotoformans F77. Biotite was chosen as a silicate mineral to investigate mineral weathering. The genome of strain F77 was sequenced, and the genes significantly upregulated when grown in the presence of biotite included mineral weathering-related genes associated with gluconic acid metabolism, flagellar assembly, and pilus biosynthesis and acid tolerance-related genes associated with neutralizing component production, reducing power, and proton efflux. Then, the biotite-weathering behaviors of strain F77 and its mutants that were created by deleting the tkt , tal , gntP , potF , nuoF , and gdtO genes, which are involved in gluconic acid metabolism and acid tolerance, respectively, were determined. The Fe and Al concentrations in the strain F77-inoculated medium increased 2.2- to 13.7-fold compared to the controls. The cell numbers of strain F77 increased over time, while the pH values in the medium ranged from 3.75 to 3.90 between 20 and 36 h of incubation. The release of Al and Fe was significantly reduced in the mutants F77Δ tal , F77Δ gntP , F77Δ potF , and F77Δ nuoF . Bacterial growth was significantly reduced in the presence of biotite in the mutants F77Δ potF and F77Δ nuoF . Our results demonstrated the acid tolerance of strain F77 and suggested that multiple genes and metabolic pathways in strain F77 are involved in biotite weathering and acid tolerance during the mineral weathering process. IMPORTANCE Acid production and tolerance play important roles in effective and persistent mineral weathering in bacteria, although the molecular mechanisms governing acid production and acid tolerance in bacteria have not been fully elucidated. In this study, the molecular mechanisms underlying biotite (as a silicate mineral) weathering (dissolution) and acid tolerance of P. azotoformans F77 were characterized using genomics, transcriptomics, and genetics analyses. Our results showed that the genes and metabolic pathways for gluconic acid metabolism, flagellar assembly, and pilus biosynthesis may play important roles in mineral weathering by strain F77. Notably, the genes associated with neutralizing component production, reducing power, and proton efflux may be related to acid tolerance in strain F77. The expression of these acid production- and acid tolerance-related genes was observed to be increased by biotite in strain F77. Our findings may help to elucidate the molecular mechanisms governing mineral weathering and, especially, acid tolerance in mineral-weathering bacteria.

The short flagella 1 (SHF1) gene in Chlamydomonas encodes a Crescerin TOG-domain protein required for late stages of flagellar growth.

Length control of flagella represents a simple and tractable system to investigate the dynamics of organelle size. Models for flagellar length control in the model organism, Chlamydomonas reinhardtii have focused on the length-dependence of the intraflagellar transport (IFT) system which manages the delivery and removal of axonemal subunits at the tip of the flagella. One of these cargoes, tubulin, is the major axonemal subunit, and its frequency of arrival at the tip plays a central role in size control models. However, the mechanisms determining tubulin dynamics at the tip are still poorly understood. We discovered a loss-of-function mutation that leads to shortened flagella, and found that this was an allele of a previously described gene, SHF1, whose molecular identity had not previously been determined. We found that SHF1 encodes a Chlamydomonas ortholog of Crescerin, previously identified as a cilia specific TOG-domain array protein that can bind tubulin via its TOG domains and increase tubulin polymerization rates. In this mutant, flagellar regeneration occurs with the same initial kinetics as wild-type cells, but plateaus at a shorter length. Using a computational model in which the flagellar microtubules are represented by a differential equation for flagellar length combined with a stochastic model for cytoplasmic microtubule dynamics, we found that our experimental results are best described by a model in which Crescerin/SHF1 binds tubulin dimers in the cytoplasm and transports them into the flagellum. We suggest that this TOG-domain protein is necessary to efficiently and preemptively increase intra-flagella tubulin levels to offset decreasing IFT cargo at the tip as flagellar assembly progresses.

In vivo imaging shows continued association of several IFT A, B and dynein complexes while IFT trains U-turn at the tip

Flagellar assembly depends on intraflagellar transport (IFT), a bidirectional motility of protein carriers, the IFT trains. The trains are periodic assemblies of IFT-A and IFT-B subcomplexes and the motors kinesin-2 and IFT dynein. At the tip, anterograde trains are remodeled for retrograde IFT, a process that in Chlamydomonas involves kinesin-2 release and train fragmentation. However, the degree of train disassembly at the tip remains unknown. Two-color imaging of fluorescent protein-tagged IFT components indicates that IFT-A and IFT-B proteins from a given anterograde train usually return in the same set of retrograde trains. Similarly, concurrent turnaround was typical for IFT-B proteins and the IFT dynein subunit D1bLIC-GFP but severance was observed as well. Our data support a simple model of IFT turnaround, in which IFT-A, IFT-B, and IFT dynein typically remain associated at the tip and anterograde trains convert directly into retrograde trains without disassembly but for possible splitting into strings of IFT complexes. Continuous association of IFT-A, IFT-B and IFT dynein during tip remodeling could balance protein entry and exit preventing the build-up of IFT material in flagella.

Characterization of Bacterial Communities From The Surface And Adjacent Bottom Layers Water of Billings Reservoir

Here, we describe the microbial diversity and physicochemical properties in freshwater samples from the surface and bottom layer of Billings reservoir in São Paulo state, Brazil. Twenty-two matched samples were characterized using the 16S rRNA gene Illumina MiSeq platform. Taxonomical composition revealed an abundance of Cyanobacteria phyla, followed by Proteobacteria, with 1903 and 2689 known bacterial genera in the surface and deep-water layers, respectively. Chroobacteria, Actinobacteria, Betaproteobacteria, Alphaproteobacteria, Sphingobacteriia, and Acidimicrobiia were the most dominant classes. Shannon diversity index ranging from 2.3 - 5.39 and 4.04 - 6.86 in the surface and bottom layer, respectively. Among the diverse pathogenic genera identified, Flavobacterium was the most predominant genus. Temperature and phosphorus concentration were among the most influential factors in shaping the microbial communities of both layers. Predictive functional analysis suggests that the reservoir is enriched in motility genes involved in the flagellar assembly. The overall results present new information on the significantly altered diversity composition of the bacterial community detected in Billings freshwater reservoir.