Biology subjects

Lima-Mendez, G.

Publications and source records attributed to Lima-Mendez, G..

5 recordsLinked to original sources

bioRxiv2024

TamL is a key player of the outer membrane homeostasis in Bacteroidetes.

In Proteobacteria, the outer membrane protein TamA and the inner membrane-anchored protein TamB form the Translocation and Assembly Module (TAM) complex, which facilitates the transport of autotransporters, virulence factors, and likely lipids across the two membranes. In Bacteroidetes TamA is replaced by TamL, a TamA-like lipoprotein with a lipid modification at its N-terminus that likely anchors it to the outer membrane. This structural difference suggests that TamL may have a distinct function compared to TamA. However, the role of TAM in bacterial phyla other than Proteobacteria remains unexplored. Our study aimed to elucidate the functional importance of TamL in Flavobacterium johnsoniae, an environmental Bacteroidetes. Unlike its homologues in Proteobacteria, we found that TamL and TamB are essential in F. johnsoniae. Through genetic, phenotypic, proteomic, and lipidomic analyses, we discovered that TamL depletion severely compromises outer membrane integrity, as evidenced by reduced cell viability, altered cell shape, increased susceptibility to membrane-disrupting agents, and elevated levels of outer membrane lipoproteins. Notably, we did not observe any impact on outer membrane lipid composition. Via pull-down protein assays, we confirmed that TamL interacts with TamB in F. johnsoniae, likely forming the TAM complex. Furthermore, our in silico analysis revealed that the presence of TamL and TamB monocistronic genes is a shared genetic feature among Bacteroidetes members, including the human pathogen Capnocytophaga canimorsus where we also confirmed the essentiality of the TamL and TamB homologs. To our knowledge, this study is the first to provide functional insights into a TAM subunit beyond Proteobacteria. SignificanceIn Proteobacteria, the outer membrane (OM) protein TamA forms with the inner membrane (IM)-anchored protein TamB the Translocation and Assembly Module Complex (TAM). which contributes to efficient biogenesis of the OM. In Bacteroidetes TamA is replaced by TamL, a TamA-like lipoprotein of unknown role. In this work, we studied TamL in the Bacteroidetes Flavobacterium johnsoniae. We found that TamL and TamB are essential for cell viability, and that TamL depletion disrupts outer membrane stability, increases outer membrane vesicle size, and lead to higher sensitivity to OM stressors. These findings highlight TamL critical role in maintaining OM structure in Bacteroidetes. To our surprise, we also identified multiple TamL, TamB and TamA homologs in Bacteroidetes. Altogether, our findings extend the current knowledge on TAM and provide novel insights into a field of research barely investigated outside Proteobacteria.

microbiology↗

bioRxiv2024

LolA and LolB are conserved in Bacteroidetes and are crucial for gliding motility and Type IX secretion.

In Gram-negative bacteria, lipoproteins are major components of the outer membrane (OM) where they play a variety of roles, from the involvement in membrane biogenesis to virulence. Bacteroidetes, a widespread phylum of Gram-negative bacteria, including free-living organisms, commensals and pathogens, encode an exceptionally high number of outer membrane lipoproteins. These proteins are crucial in this phylum mainly because they are key components of SUS-like nutrient acquisition systems as well as of the Type 9 secretion (T9SS) and gliding motility machineries. The transport of lipoproteins to the OM has mainly been studied in E. coli and relies on the Lol system, composed of the inner membrane extraction machinery LolCDE, the periplasmic carrier LolA and the OM lipoprotein LolB. While most Lol proteins are essential and conserved across Gram-negative bacteria, to date, no LolB homologs have been identified outside of {gamma}- and {beta}-proteobacteria. How lipoproteins reach and are inserted in the OM of Bacteroidetes is not known. Here we identified LolB homologs in Bacteroidetes and disclosed the co-existence of several LolA and LolB in several species. We provide evidence that one LolA (LolA1) and one LolB (LolB1) of F. johnsoniae are devoted to targeting gliding and T9SS lipoproteins to the OM. A proteomic analysis of the OM composition of the lolA1 and lolB1 mutants supports this evidence. Furthermore, we show that, while LolB1 and LolA1 have conserved functions in Bacteroidetes, they are functionally different from their E. coli counterparts. We also show that surface lipoprotein transport is LolA and LolB independent. Finally, the finding that, in the absence of LolA and LolB homologs, lipoproteins still localize to the OM, suggests the presence in Bacteroidetes of yet unidentified LolAB-alternative lipoprotein transport pathways. In conclusion, Bacteroidetes have evolved different and more complex lipoprotein transport pathways than other Gram-negative bacteria and further research is required to uncover their complexity. SignificanceIn Gram-negative bacteria, lipoproteins are key components of the outer membrane (OM), essential for functions like membrane biogenesis and virulence. Bacteroidetes, a widespread phylum, encode a high number of OM lipoproteins crucial for nutrient acquisition, Type IX secretion, and gliding motility. While lipoprotein transport in E. coli depends on the Lol system, LolB homologs were previously unidentified outside {gamma}- and {beta}-proteobacteria. Here we identify LolB homologs in Bacteroidetes, revealing the co-existence of multiple LolA and LolB proteins in various species. In F. johnsoniae, LolA1 and LolB1 specifically target gliding and Type 9 secretion system lipoproteins to the OM. Despite this, lipoproteins still localize to the OM without LolA and LolB, suggesting alternative transport pathways. These findings indicate that Bacteroidetes have evolved more complex lipoprotein transport mechanisms than other Gram-negative bacteria, requiring further research to fully understand them.

microbiology↗

bioRxiv2021

Disentangling environmental effects in microbial association networks

BackgroundEcological interactions among microorganisms are fundamental for ecosystem function, yet they are mostly unknown or poorly understood. High-throughput-omics can indicate microbial interactions through associations across time and space, which can be represented as association networks. Associations could result from either ecological interactions between microorganisms, or from environmental selection, where the associations are environmentally-driven. Therefore, before downstream analysis and interpretation, we need to distinguish the nature of the association, particularly if it is due to environmental selection or not. ResultsWe present EnDED (Environmentally-Driven Edge Detection), an implementation of four approaches as well as their combination to predict which links between microorganisms in an association network are environmentally-driven. The four approaches are Sign Pattern, Overlap, Interaction Information, and Data Processing Inequality. We tested EnDED on networks from simulated data of 50 microorganisms. The networks contained on average 50 nodes and 1087 edges, of which 60 were true interactions but 1026 false associations (i.e. environmentally-driven or due to chance). Applying each method individually, we detected a moderate to high number of environmentally-driven edges--87% Sign Pattern and Overlap, 67% Interaction Information, and 44% Data Processing Inequality. Combining these methods in an intersection approach resulted in retaining more interactions, both true and false (32% of environmentally-driven associations). After validation with the simulated datasets, we applied EnDED on a marine microbial network inferred from 10 years of monthly observations of microbial-plankton abundance. The intersection combination predicted that 8.3% of the associations were environmentally-driven, while individual methods predicted 24.8% (Data Processing Inequality), 25.7% (Interaction Information), and up to 84.6% (Sign Pattern as well as Overlap). The fraction of environmentally-driven edges among negative microbial associations in the real network increased rapidly with the number of environmental factors. ConclusionsTo reach accurate hypotheses about ecological interactions, it is important to determine, quantify, and remove environmentally-driven associations in marine microbial association networks. For that, EnDED offers up to four individual methods as well as their combination. However, especially for the intersection combination, we suggest using EnDED with other strategies to reduce the number of false associations and consequently the number of potential interaction hypotheses.

microbiology↗

bioRxiv2019

Tn3-associated toxin-antitoxin gene pairs appear to be an integral part of the transposition module

Much of the diversity of prokaryotic genomes is contributed by the tightly controlled recombination activity of transposons (Tn). The Tn3 family is arguably one of the most widespread transposon families. Members carry a large range of passenger genes incorporated into their structures. Family members undergo replicative transposition using a DDE transposase to generate a cointegrate structure which is then resolved by site-specific recombination between specific DNA sequences (res) on each of the two Tn copies in the cointegrate. These sites also carry promoters controlling expression of the recombinase and transposase. We report here that a number of Tn3 members encode a type II toxin-antitoxin (TA) system, typically composed of a stable toxin and a labile antitoxin that binds the toxin and inhibits its lethal activity. This system serves to improve plasmid maintenance in a bacterial population and, until recently, was believed to be associated with bacterial persistence. At least six different TA gene pairs are associated with various Tn3 members. Our data suggest that several independent acquisition events have occurred. In contrast to most Tn3 family passenger genes which are generally located away from the transposition module, the TA gene pairs abut the res site upstream of the resolvase genes. Although their role when part of Tn3 family transposons is unclear, this finding suggests a potential role for the embedded TA in stabilizing the associated transposon with the possibility that TA expression is coupled to expression of transposase and resolvase during the transposition process itself. ImportanceTransposable Elements (TEs) are important in genetic diversification due to their recombination properties and their ability to promote horizontal gene transfer. Over the last decades, much effort has been made to understand TE transposition mechanisms and their impact on prokaryotic genomes. For example, the Tn3 family is ubiquitous in bacteria, moulding their host genomes by the paste-and-copy mechanism. In addition to the transposition module, Tn3 members often carry additional passenger genes (e.g., conferring antibiotic or heavy metal resistance and virulence) and three were previously known to carry a toxin-antitoxin (TA) system often associated with plasmid maintenance; however, the role of TA systems within the Tn3 family is unknown. The genetic context of TA systems in Tn3 members suggests that they may play a regulatory role in ensuring stable invasion of these Tn during transposition.

microbiology↗

bioRxiv2019

Metabolic Architecture of the Deep Ocean Microbiome

The deep sea, the largest compartment of the ocean, is an essential component of the Earth system, but the functional exploration of its microbial communities lags far behind that of other marine realms. Here we analyze 58 bathypelagic microbial metagenomes from the Atlantic, Indian, and Pacific Oceans in an unprecedented sampling effort from the Malaspina Global Expedition, to resolve the metabolic architecture of the deep ocean microbiome. The Malaspina Deep-Sea Gene Collection, 71% of which consists of novel genes, reveals a strong dichotomy between the functional traits of free-living and particle-attached microorganisms, and shows relatively patchy composition challenging the paradigm of a uniform dark ocean ecosystem. Metagenome Assembled Genomes uncovered 11 potential new phyla, establishing references for deep ocean microbial taxa, and revealed mixotrophy to be a widespread trophic strategy in the deep ocean. These results expand our understanding of the functional diversity, metabolic versatility, and carbon cycling in the largest ecosystem on Earth.\n\nOne Sentence SummaryA whole community genomic survey of the deep microbiome sheds light on the microbial and functional diversity of the dark ocean.

microbiology↗