Microbial iron management mechanisms in extremely acidic environments: comparative genomics evidence for diversity and versatility

<p>Abstract</p> <p>Background</p> <p>Iron is an essential nutrient but can be toxic at high intracellular concentrations and organisms have evolved tightly regulated mechanisms for iron uptake and homeostasis. Information on iron management mechanisms is available for organisms living at circumneutral pH. However, very little is known about how acidophilic bacteria, especially those used for industrial copper bioleaching, cope with environmental iron loads that can be 10¹⁸times the concentration found in pH neutral environments. This study was motivated by the need to fill this lacuna in knowledge. An understanding of how microorganisms thrive in acidic ecosystems with high iron loads requires a comprehensive investigation of the strategies to acquire iron and to coordinate this acquisition with utilization, storage and oxidation of iron through metal responsive regulation. <it>In silico </it>prediction of iron management genes and Fur regulation was carried out for three Acidithiobacilli: <it>Acidithiobacillus ferrooxidans </it>(iron and sulfur oxidizer) <it>A. thiooxidans </it>and <it>A. caldus </it>(sulfur oxidizers) that can live between pH 1 and pH 5 and for three strict iron oxidizers of the <it>Leptospirillum </it>genus that live at pH 1 or below.</p> <p>Results</p> <p>Acidithiobacilli have predicted FeoB-like Fe(II) and Nramp-like Fe(II)-Mn(II) transporters. They also have 14 different TonB dependent ferri-siderophore transporters of diverse siderophore affinity, although they do not produce classical siderophores. Instead they have predicted novel mechanisms for dicitrate synthesis and possibly also for phosphate-chelation mediated iron uptake. It is hypothesized that the unexpectedly large number and diversity of Fe(III)-uptake systems confers versatility to this group of acidophiles, especially in higher pH environments (pH 4–5) where soluble iron may not be abundant. In contrast, Leptospirilla have only a FtrI-Fet3P-like permease and three TonB dependent ferri-dicitrate siderophore systems. This paucity of iron uptake systems could reflect their obligatory occupation of extremely low pH environments where high concentrations of soluble iron may always be available and were oxidized sulfur species might not compromise iron speciation dynamics. Presence of bacterioferritin in the Acidithiobacilli, polyphosphate accumulation functions and variants of FieF-like diffusion facilitators in both Acidithiobacilli and Leptospirilla, indicate that they may remove or store iron under conditions of variable availability. In addition, the Fe(II)-oxidizing capacity of both <it>A. ferrooxidans </it>and Leptospirilla could itself be a way to evade iron stress imposed by readily available Fe(II) ions at low pH. Fur regulatory sites have been predicted for a number of gene clusters including iron related and non-iron related functions in both the Acidithiobacilli and Leptospirilla, laying the foundation for the future discovery of iron regulated and iron-phosphate coordinated regulatory control circuits.</p> <p>Conclusion</p> <p><it>In silico </it>analyses of the genomes of acidophilic bacteria are beginning to tease apart the mechanisms that mediate iron uptake and homeostasis in low pH environments. Initial models pinpoint significant differences in abundance and diversity of iron management mechanisms between Leptospirilla and Acidithiobacilli, and begin to reveal how these two groups respond to iron cycling and iron fluctuations in naturally acidic environments and in industrial operations. Niche partitions and ecological successions between acidophilic microorganisms may be partially explained by these observed differences. Models derived from these analyses pave the way for improved hypothesis testing and well directed experimental investigation. In addition, aspects of these models should challenge investigators to evaluate alternative iron management strategies in non-acidophilic model organisms.</p

Extending the models for iron and sulfur oxidation in the extreme Acidophile Acidithiobacillus ferrooxidans

<p>Abstract</p> <p>Background</p> <p><it>Acidithiobacillus ferrooxidans </it>gains energy from the oxidation of ferrous iron and various reduced inorganic sulfur compounds at very acidic pH. Although an initial model for the electron pathways involved in iron oxidation has been developed, much less is known about the sulfur oxidation in this microorganism. In addition, what has been reported for both iron and sulfur oxidation has been derived from different <it>A. ferrooxidans </it>strains, some of which have not been phylogenetically characterized and some have been shown to be mixed cultures. It is necessary to provide models of iron and sulfur oxidation pathways within one strain of <it>A. ferrooxidans </it>in order to comprehend the full metabolic potential of the pangenome of the genus.</p> <p>Results</p> <p>Bioinformatic-based metabolic reconstruction supported by microarray transcript profiling and quantitative RT-PCR analysis predicts the involvement of a number of novel genes involved in iron and sulfur oxidation in <it>A. ferrooxidans </it>ATCC23270. These include for iron oxidation: <it>cup </it>(copper oxidase-like), <it>ctaABT </it>(heme biogenesis and insertion), <it>nuoI </it>and <it>nuoK </it>(NADH complex subunits), <it>sdrA1 </it>(a NADH complex accessory protein) and <it>atpB </it>and <it>atpE </it>(ATP synthetase F0 subunits). The following new genes are predicted to be involved in reduced inorganic sulfur compounds oxidation: a gene cluster (<it>rhd, tusA, dsrE, hdrC, hdrB, hdrA, orf2, hdrC, hdrB</it>) encoding three sulfurtransferases and a heterodisulfide reductase complex, <it>sat </it>potentially encoding an ATP sulfurylase and <it>sdrA2 </it>(an accessory NADH complex subunit). Two different regulatory components are predicted to be involved in the regulation of alternate electron transfer pathways: 1) a gene cluster (<it>ctaRUS</it>) that contains a predicted iron responsive regulator of the Rrf2 family that is hypothesized to regulate cytochrome <it>aa</it>₃oxidase biogenesis and 2) a two component sensor-regulator of the RegB-RegA family that may respond to the redox state of the quinone pool.</p> <p>Conclusion</p> <p>Bioinformatic analysis coupled with gene transcript profiling extends our understanding of the iron and reduced inorganic sulfur compounds oxidation pathways in <it>A. ferrooxidans </it>and suggests mechanisms for their regulation. The models provide unified and coherent descriptions of these processes within the type strain, eliminating previous ambiguity caused by models built from analyses of multiple and divergent strains of this microorganism.</p

Acidithiobacillus ferrianus sp. nov.:an ancestral extremely acidophilic and facultatively anaerobic chemolithoautotroph

Strain MG, isolated from an acidic pond sediment on the island of Milos (Greece), is proposed as a novel species of ferrous iron- and sulfur-oxidizing Acidithiobacillus. Currently, four of the eight validated species of this genus oxidize ferrous iron, and strain MG shares many key characteristics with these four, including the capacities for catalyzing the oxidative dissolution of pyrite and for anaerobic growth via ferric iron respiration. Strain MG also grows aerobically on hydrogen and anaerobically on hydrogen coupled to ferric iron reduction. While the 16S rRNA genes of the iron-oxidizing Acidithiobacillus species (and strain MG) are located in a distinct phylogenetic clade and are closely related (98–99% 16S rRNA gene identity), genomic relatedness indexes (ANI/dDDH) revealed strong genomic divergence between strain MG and all sequenced type strains of the taxon, and placed MG as the first cultured representative of an ancestral phylotype of iron oxidizing acidithiobacilli. Strain MG is proposed as a novel species, Acidithiobacillus ferrianus sp. nov. The type strain is MGT(= DSM 107098T= JCM 33084T).Similar strains have been found as isolates or indicated by cloned 16S rRNA genes from several mineral sulfide mine sites.Indexación: Scopu

Domestication of Local Microbial Consortia for Efficient Recovery of Gold Through Top-Down Selection in Airlift Bioreactors

Extreme acidophiles play central roles in the geochemical cycling of diverse elements in low pH environments. This has been harnessed in biotechnologies such as biomining, where microorganisms facilitate the recovery of economically important metals such as gold. By generating both extreme acidity and a chemical oxidant (ferric iron) many species of prokaryotes that thrive in low pH environments not only catalyze mineral dissolution but also trigger both community and individual level adaptive changes. These changes vary in extent and direction depending on the ore mineralogy, water availability and local climate. The use of indigenous versus introduced microbial consortia in biomining practices is still a matter of debate. Yet, indigenous microbial consortia colonizing sulfidic ores that have been domesticated, i.e., selected for their ability to survive under specific polyextreme conditions, are claimed to outperform un-adapted foreign consortia. Despite this, little is known on the domestication of acidic microbial communities and the changes elicited in their members. In this study, high resolution targeted metagenomic techniques were used to analyze the changes occurring in the community structure of local microbial consortia acclimated to growing under extreme acidic conditions and adapted to endure the conditions imposed by the target mineral during biooxidation of a gold concentrate in an airlift reactor over a period of 2 years. The results indicated that operative conditions evolving through biooxidation of the mineral concentrate exerted strong selective pressures that, early on, purge biodiversity in favor of a few Acidithiobacillus spp. over other iron oxidizing acidophiles. Metagenomic analysis of the domesticated consortium present at the end of the adaptation experiment enabled reconstruction of the RVS1-MAG, a novel representative of Acidithiobacillus ferrooxidans from the Andacollo gold mineral district. Comparative genomic analysis performed with this genome draft revealed a net enrichment of gene functions related to heavy metal transport and stress management that are likely to play a significant role in adaptation and survival to adverse conditions experienced by these acidophiles during growth in presence of gold concentrates

The Type IV Secretion System of ICEAfe1: Formation of a Conjugative Pilus in Acidithiobacillus ferrooxidans

The dispersal of mobile genetic elements and their gene cargo relies on type IV secretion systems (T4SS). In this work the ICEAfe1 Tra-type T4SS nanomachine, encoded in the publicly available genome of Acidithiobacillus ferrooxidans ATCC 23270TY, was characterized in terms of its organization, conservation, expression and mating bridge formation. Twenty-one conjugative genes grouped in four genetic clusters encode the ICEAfe1 T4SS, containing all the indispensable functions for the formation and stabilization of the pili and for DNA processing. The clusters’ organization resembles that of other mobile genetic elements (such as plasmids and integrative and conjugative elements–ICEs). Sequence conservation, genetic organization and distribution of the tra system in the genomes of other sequenced Acidithiobacillus spp. suggests that the ICEAfe1 T4SS could mediate the lateral gene transfer between related bacteria. All ICEAfe1 T4SS genes are transcriptionally active and expressed from four independent operons. The transcriptional levels of selected marker genes increase in response to Mitomycin C treatment, a DNA damage elicitor that has acknowledged stimulatory effects on excision rates and gene expression of other ICEs, including ICEAfe1. Using a tailor-made pilin-antiserum against ICEAfe1 T4SS TraA pilin and epifluorescence microscopy, the presence of the conjugative pili on the cell surface of A. ferrooxidans could be demonstrated. Additionally, immunodetection assays, by immunogold, allowed the identification of pili-like extracellular structures. Together, the results obtained in this work demonstrate that the ICEAfe1 T4SS is phylogenetically conserved within the taxon, is expressed at mRNA and protein levels in vivo in the A. ferrooxidans type strain, and produces a pili-like structure of extracellular and intercellular localization in this model acidophile, supporting its functionality. Additional efforts will be required to prove conjugation of the ICEAfe1 or parts of this element through the cognate T4SS

Nucleotide Second Messenger-Based Signaling in Extreme Acidophiles of the Acidithiobacillus Species Complex: Partition Between the Core and Variable Gene Complements

Cyclic and linear nucleotides are key elements of the signal transduction networks linking perception of the environment to specific cellular behavior of prokaryotes. These molecular mechanisms are particularly important in bacteria exposed to different, and frequently simultaneous, types of extreme conditions. This is the case in acidithiobacilli, a group of extremophilic bacteria thriving in highly acidic biotopes, that must also cope with significant variations in temperature, osmotic potentials and concentrations of various transition metals and metalloids. Environmental cues sensed by bacteria are transduced into differential levels of nucleotides acting as intracellular second messengers, promoting the activation or inhibition of target components and eliciting different output phenotypes. Cyclic (c) di-GMP, one of the most common bacterial second messengers, plays a key role in lifestyle changes in many bacteria, including acidithiobacilli. The presence of functional c-di-GMP-dependent signal transduction pathways in representative strains of the best-known linages of this species complex has been reported. However, a comprehensive panorama of the c-di-GMP modulated networks, the cognate input signals and output responses, are still missing for this group of extremophiles. Moreover, little fundamental understanding has been gathered for other nucleotides acting as second messengers. Taking advantage of the increasing number of sequenced genomes of the taxon, here we address the challenge of disentangling the nucleotide-driven signal transduction pathways in this group of polyextremophiles using comparative genomic tools and strategies. Results indicate that the acidithiobacilli possess all the genetic elements required to establish functional transduction pathways based in three different nucleotide-second messengers: (p)ppGpp, cyclic AMP (cAMP), and c-di-GMP. The elements related with the metabolism and transduction of (p)ppGpp and cAMP appear highly conserved, integrating signals related with nutrient starvation and polyphosphate metabolism, respectively. In contrast, c-di-GMP networks appear diverse and complex, differing both at the species and strain levels. Molecular elements of c-di-GMP metabolism and transduction were mostly found scattered along the flexible genome of the acidithiobacilli, allowing the identification of probable control modules that could be critical for substrate colonization, biofilm development and intercellular interactions. These may ultimately convey increased endurance to environmental stress and increased potential for gene sharing and adaptation to changing conditions

Global phylogenomic novelty of the Cas1 gene from hot spring microbial communities

The Cas1 protein is essential for the functioning of CRISPR-Cas adaptive systems. However, despite the high prevalence of CRISPR-Cas systems in thermophilic microorganisms, few studies have investigated the occurrence and diversity of Cas1 across hot spring microbial communities. Phylogenomic analysis of 2,150 Cas1 sequences recovered from 48 metagenomes representing hot springs (42–80°C, pH 6–9) from three continents, revealed similar ecological diversity of Cas1 and 16S rRNA associated with geographic location. Furthermore, phylogenetic analysis of the Cas1 sequences exposed a broad taxonomic distribution in thermophilic bacteria, with new clades of Cas1 homologs branching at the root of the tree or at the root of known clades harboring reference Cas1 types. Additionally, a new family of casposases was identified from hot springs, which further completes the evolutionary landscape of the Cas1 superfamily. This ecological study contributes new Cas1 sequences from known and novel locations worldwide, mainly focusing on under-sampled hot spring microbial mat taxa. Results herein show that circumneutral hot springs are environments harboring high diversity and novelty related to adaptive immunity systems.This work was financed in part by FONDECYT regular N° 1190998 (ANID) and Iniciativa de Investigación UnACh 2021-157-Unach. OS and JT-L were supported in part by ANID National Doctoral Scholarship (Beca de Doctorado Nacional ANID) N° 21172022 and 21171048, respectively. SG-L was supported by ANID FONDECYT Postdoctoral N° 3210547. AM-B and RQ were supported by Centro Ciencia and Vida, FB210008, Financiamiento Basal para Centros Científicos y Tecnológicos de Excelencia de ANID, and FONDECYT regular N° 1221035 (ANID). FJMM acknowledged research support by the Conselleria d’Innovació, Universitats, Ciència i Societat Digital from Generalitat Valenciana, research project PROMETEO/2021/057. BD acknowledged the Millennium Institute Center for Genome Regulation, Project ICN2021-044 supported by the ANID Millennium Scientific Initiative (Chile)

Benchmarking de métodos de binning a partir de un metagenoma conceptual

106 p.La metagenómica implica el estudio de comunidades microbianas enteras mediante el muestreo directo y la secuenciación de los genomas presentes en un entorno determinado. En los últimos años, la disponibilidad de secuencias metagenomicas ha crecido de manera significativa, debido principalmente al enorme impacto de las tecnologías de secuenciación de nueva generación (NGS). A diferencia del análisis clásico de un genoma, que implica la manipulación y ensamble de un número moderado de lecturas (20,000 – 30,000) largas (650-800 pb), la metagenómica tiene el reto de analizar millones de lecturas cortas (35-250 pb) de múltiples microorganismos, muchos de los cuales se desconocen. Uno de los problemas subyacentes del análisis metagenómico es la recuperación de los genomas individuales presentes en la muestra en estudio. La clasificación y el agrupamiento de lecturas o contigs de NGS en unidades taxonómicas operacionales putativos se conoce como binning. Varios métodos de bioinformática para binning se han descrito en los últimos años. Estos métodos se basan en principios diferentes (dependientes de la taxonomía e independientes de la taxonomía) y los softwares se ejecutan de forma local o a través de un servicio web. A pesar de la diversidad de métodos, no hay consenso sobre cuál es el mejor. Con esto en mente, 11 de los métodos de agrupación disponibles han sido evaluados utilizando un metagenoma conceptual construido con el software Grinder (*). Este metagenoma incluye 9 genomas procariontes completamente secuenciados y 28 genomas virales de diversa filiación taxonómica, consta de 2,5 millones de lecturas. El rendimiento de los softwares de binning se evaluó a través de cuatro criterios (el porcentaje de bases clasificadas, el porcentaje de bases correctamente clasificadas, el puntaje taxonómico y la distancia de la variación de la información). Los métodos de clasificación se evaluaron para todo el conjunto de datos, y las fracciones microbianas y virales. * //sourceforge.net/projects/biogrinder/ Se evaluaron cuarenta y siete programas de binning diferentes. De estos: 18 resultaron ser obsoletos, 16 fueron limitados en su aplicación y 13 estaban funcionales para realizar binning. De este último grupo, 2 no fueron evaluados por razones técnicas, dejando a 11 programas de binning que fueron evaluados en profundidad. Estos programas fueron clasificados de acuerdo a los criterios antes mencionados. Los tres programas de binning mejor evaluados fueron: DiScRIBinATE, MEGAN y Sort-ITEMS. El ranking completo se muestra en la Tabla 24./ABSTRACT: Metagenomics entails the study of entire microbial communities via direct sampling and sequencing of genomes present in a given environment. In recent years, the availability of metagenomic sequences has grown significantly, mainly due to the enormous impact of Next Generation Sequencing (NGS) technologies. Unlike classical genome analysis involving the handling and assembly of a moderate number (20,000 – 30,000) of long reads (650-800 bp), metagenomics is challenged to analyze millions of short reads (35-250 bp) of multiple microorganisms, many of which are unknown.One of the underlying problems of metagenomic analysis is the retrieval of the individual genomes present in the sample under study. Classification and grouping of NGS reads or contigs into putative operational taxonomic units is known as binning. Several bioinformatics methods for binning have been described in recent years. These methods are based on different principles (taxonomy dependent and taxonomy independent) and executable programs are run either locally or through a web service. Despite the diversity of methods there is no consensus on which is best.With this in mind, 11 of the available binning methods have been benchmarked using a conceptual metagenome built with Grinder (*). This metagenome includes 9 completely sequenced prokaryotic and 28 viral genomes of diverse taxonomic affiliation and consists of 2.5 million reads. The binning performance of each softwares was evaluated and ranked through four criteria (percent of classified bases, percent of correctly classified bases, taxonomic score and distance of variation of information). Best scoring methods for the whole data set, and the microbial and viral fractions are sourceforge.net/projects/biogrinder/ Fourty-seven different softwares programs for binning were evaluated. Of these: 18 were found to be obsolete, 16 were limited in their application and 13 were determined to be suitable for metagenomic binning. Of this latter group, 2 were not evaluated further for technical reasons, leaving 11 softwares programs that were evaluated in depth. These programs were ranked according to the aforementioned criteria. The top three programs according to correct binning were: DiScRIBinATE, MEGAN and SOrt-ITEMS. The complete ranking is shown in “Tabla 24”