José Eduardo González-Pastor

Novel Nickel Resistance Genes from the Rhizosphere Metagenome of Plants Adapted to Acid Mine Drainage

ABSTRACT Metal resistance determinants have traditionally been found in cultivated bacteria. To search for genes involved in nickel resistance, we analyzed the bacterial community of the rhizosphere of Erica andevalensis, an endemic heather which grows at the banks of the Tinto River, a naturally metal-enriched and extremely acidic environment in southwestern Spain. 16S rRNA gene sequence analysis of rhizosphere DNA revealed the presence of members of five phylogenetic groups of Bacteria and the two main groups of Archaea mostly associated with sites impacted by acid mine drainage (AMD). The diversity observed and the presence of heavy metals in the rhizosphere led us to construct and screen five different metagenomic libraries hosted in Escherichia coli for searching novel nickel resistance determinants. A total of 13 positive clones were detected and analyzed. Insights about their possible mechanisms of resistance were obtained from cellular nickel content and sequence similarities. Two clones encoded putative ABC transporter components, and a novel mechanism of metal efflux is suggested. In addition, a nickel hyperaccumulation mechanism is proposed for a clone encoding a serine O-acetyltransferase. Five clones encoded proteins similar to well-characterized proteins but not previously reported to be related to nickel resistance, and the remaining six clones encoded hypothetical or conserved hypothetical proteins of uncertain functions. This is the first report documenting nickel resistance genes recovered from the metagenome of an AMD environment.

SpoIIIE and a novel type of DNA translocase, SftA, couple chromosome segregation with cell division in Bacillus subtilis

Cell division must only occur once daughter chromosomes have been fully separated. However, the initiating event of bacterial cell division, assembly of the FtsZ ring, occurs while chromosome segregation is still ongoing. We show that a two‐step DNA translocase system exists in Bacillus subtilis that couples chromosome segregation and cell division. The membrane‐bound DNA translocase SpoIIIE assembled very late at the division septum, and only upon entrapment of DNA, while its orthologue, SftA (YtpST), assembled at each septum in B. subtilis soon after FtsZ. Lack of SftA resulted in a moderate segregation defect at a late stage in the cell cycle. Like the loss of SpoIIIE, the absence of SftA was deleterious for the cells during conditions of defective chromosome segregation, or after induction of DNA damage. Lack of both proteins exacerbated all phenotypes. SftA forms soluble hexamers in solution, binds to DNA and has DNA‐dependent ATPase activity, which is essential for its function in vivo. Our data suggest that SftA aids in moving DNA away from the closing septum, while SpoIIIE translocates septum‐entrapped DNA only when septum closure precedes complete segregation of chromosomes.

Novel acid resistance genes from the metagenome of the Tinto River, an extremely acidic environment

Microorganisms that thrive in acidic environments are endowed with specialized molecular mechanisms to survive under this extremely harsh condition. In this work, we performed functional screening of six metagenomic libraries from planktonic and rhizosphere microbial communities of the Tinto River, an extremely acidic environment, to identify genes involved in acid resistance. This approach has revealed 15 different genes conferring acid resistance to Escherichia coli, most of which encoding putative proteins of unknown function or previously described proteins not known to be related to acid resistance. Moreover, we were able to assign function to one unknown and three hypothetical proteins. Among the recovered genes were the ClpXP protease, the transcriptional repressor LexA and nucleic acid-binding proteins such as an RNA-binding protein, HU and Dps. Furthermore, nine of the retrieved genes were cloned and expressed in Pseudomonas putida and Bacillus subtilis and, remarkably, most of them were able to expand the capability of these bacteria to survive under severe acid stress. From this set of genes, four presented a broad-host range as they enhance the acid resistance of the three different organisms tested. These results expand our knowledge about the different strategies used by microorganisms to survive under extremely acid conditions.

Extracellular DNA Release by Undomesticated Bacillus subtilis Is Regulated by Early Competence

Extracellular DNA (eDNA) release is a widespread capacity described in many microorganisms. We identified and characterized lysis-independent eDNA production in an undomesticated strain of Bacillus subtilis. DNA fragments are released during a short time in late-exponential phase. The released eDNA corresponds to whole genome DNA, and does not harbour mutations suggesting that is not the result of error prone DNA synthesis. The absence of eDNA was linked to a spread colony morphology, which allowed a visual screening of a transposon library to search for genes involved in its production. Transposon insertions in genes related to quorum sensing and competence (oppA, oppF and comXP) and to DNA metabolism (mfd and topA) were impaired in eDNA release. Mutants in early competence genes such as comA and srfAA were also defective in eDNA while in contrast mutations in late competence genes as those for the DNA uptake machinery had no effect. A subpopulation of cells containing more DNA is present in the eDNA producing strains but absent from the eDNA defective strain. Finally, competent B. subtilis cells can be transformed by eDNA suggesting it could be used in horizontal gene transfer and providing a rationale for the molecular link between eDNA release and early-competence in B. subtilis that we report.

The role of nitric-oxide-synthase-derived nitric oxide in multicellular traits of Bacillus subtilis 3610: biofilm formation, swarming, and dispersal

BackgroundBacillus subtilis 3610 displays multicellular traits as it forms structurally complex biofilms and swarms on solid surfaces. In addition, B. subtilis encodes and expresses nitric oxide synthase (NOS), an enzyme that is known to enable NO-mediated intercellular signalling in multicellular eukaryotes. In this study, we tested the hypothesis that NOS-derived NO is involved in the coordination of multicellularity in B. subtilis 3610.ResultsWe show that B. subtilis 3610 produces intracellular NO via NOS activity by combining Confocal Laser Scanning Microscopy with the NO sensitive dye copper fluorescein (CuFL). We further investigated the influence of NOS-derived NO and exogenously supplied NO on the formation of biofilms, swarming motility and biofilm dispersal. These experiments showed that neither the suppression of NO formation with specific NOS inhibitors, NO scavengers or deletion of the nos gene, nor the exogenous addition of NO with NO donors affected (i) biofilm development, (ii) mature biofilm structure, and (iii) swarming motility in a qualitative and quantitative manner. In contrast, the nos knock-out and wild-type cells with inhibited NOS displayed strongly enhanced biofilm dispersal.ConclusionThe results suggest that biofilm formation and swarming motility in B. subtilis represent complex multicellular processes that do not employ NO signalling and are remarkably robust against interference of NO. Rather, the function of NOS-derived NO in B. subtilis might be specific for cytoprotection against oxidative stress as has been proposed earlier. The influence of NOS-derived NO on dispersal of B. subtilis from biofilms might be associated to its well-known function in coordinating the transition from oxic to anoxic conditions. Here, NOS-derived NO might be involved in fine-tuning the cellular decision-making between adaptation of the metabolism to (anoxic) conditions in the biofilm or dispersal from the biofilm.

Functional metagenomics of extreme environments.

The bioprospecting of enzymes that operate under extreme conditions is of particular interest for many biotechnological and industrial processes. Nevertheless, there is a considerable limitation to retrieve novel enzymes as only a small fraction of microorganisms derived from extreme environments can be cultured under standard laboratory conditions. Functional metagenomics has the advantage of not requiring the cultivation of microorganisms or previous sequence information to known genes, thus representing a valuable approach for mining enzymes with new features. In this review, we summarize studies showing how functional metagenomics was employed to retrieve genes encoding for proteins involved not only in molecular adaptation and resistance to extreme environmental conditions but also in other enzymatic activities of biotechnological interest.

The ζ Toxin Induces a Set of Protective Responses and Dormancy

The ζε module consists of a labile antitoxin protein, ε, which in dimer form (ε2) interferes with the action of the long-living monomeric ζ phosphotransferase toxin through protein complex formation. Toxin ζ, which inhibits cell wall biosynthesis and may be bactericide in nature, at or near physiological concentrations induces reversible cessation of Bacillus subtilis proliferation (protective dormancy) by targeting essential metabolic functions followed by propidium iodide (PI) staining in a fraction (20–30%) of the population and selects a subpopulation of cells that exhibit non-inheritable tolerance (1–5×10−5). Early after induction ζ toxin alters the expression of ∼78 genes, with the up-regulation of relA among them. RelA contributes to enforce toxin-induced dormancy. At later times, free active ζ decreases synthesis of macromolecules and releases intracellular K+. We propose that ζ toxin induces reversible protective dormancy and permeation to PI, and expression of ε2 antitoxin reverses these effects. At later times, toxin expression is followed by death of a small fraction (∼10%) of PI stained cells that exited earlier or did not enter into the dormant state. Recovery from stress leads to de novo synthesis of ε2 antitoxin, which blocks ATP binding by ζ toxin, thereby inhibiting its phosphotransferase activity.

Exploring the diversity of arsenic resistance genes from acid mine drainage microorganisms

The microbial communities from the Tinto River, a natural acid mine drainage environment, were explored to search for novel genes involved in arsenic resistance using a functional metagenomic approach. Seven pentavalent arsenate resistance clones were selected and analysed to find the genes responsible for this phenotype. Insights about their possible mechanisms of resistance were obtained from sequence similarities and cellular arsenic concentration. A total of 19 individual open reading frames were analysed, and each one was individually cloned and assayed for its ability to confer arsenic resistance in Escherichia coli cells. A total of 13 functionally active genes involved in arsenic resistance were identified, and they could be classified into different global processes: transport, stress response, DNA damage repair, phospholipids biosynthesis, amino acid biosynthesis and RNA-modifying enzymes. Most genes (11) encode proteins not previously related to heavy metal resistance or hypothetical or unknown proteins. On the other hand, two genes were previously related to heavy metal resistance in microorganisms. In addition, the ClpB chaperone and the RNA-modifying enzymes retrieved in this work were shown to increase the cell survival under different stress conditions (heat shock, acid pH and UV radiation). Thus, these results reveal novel insights about unidentified mechanisms of arsenic resistance.

The Presence of Conjugative Plasmid pLS20 Affects Global Transcription of Its Bacillus subtilis Host and Confers Beneficial Stress Resistance to Cells

ABSTRACT Conjugation activity of plasmid pLS20 from Bacillus subtilis subsp. natto is induced when cells are diluted into fresh medium and diminishes as cells enter into stationary-phase growth. Transcriptional profiling shows that during mid-exponential growth, more than 5% of the host genes are affected in the presence of the plasmid, in contrast to the minor changes seen in freshly diluted and stationary-phase cells. Changes occurred in many metabolic pathways, although pLS20 does not confer any detectable burden on its host cell, as well as in membrane and cell wall-associated processes, in the large motility operon, and in several other cellular processes. In agreement with these changes, we found considerable alterations in motility and enzyme activity and increased resistance against several different forms of stress in cells containing the plasmid, revealing that the presence of pLS20 has a broad impact on the physiology of its host cell and increases its stress resistance in multiple aspects. Additionally, we found that the lack of chromosomal gene yueB, known to encode a phage receptor protein, which is upregulated in cells containing pLS20, strongly reduced conjugation efficiency, revealing that pLS20 not only increases fitness of its host but also employs host proteins for efficient transfer into a new cell.

Novel Metal Resistance Genes from Microorganisms: A Functional Metagenomic Approach

Most of the known metal resistance mechanisms are based on studies of cultured microorganisms, and the abundant uncultured fraction could be an important source of genes responsible for uncharacterized resistance mechanisms. A functional metagenomic approach was selected to recover metal resistance genes from the rhizosphere microbial community of an acid-mine drainage (AMD)-adapted plant, Erica andevalensis, from Rio Tinto, Spain. A total of 13 nickel resistant clones were isolated and analyzed, encoding hypothetical or conserved hypothetical proteins of uncertain functions, or well-characterized proteins, but not previously reported to be related to nickel resistance. The resistance clones were classified into two groups according to their nickel accumulation properties: those preventing or those favoring metal accumulation. Two clones encoding putative ABC transporter components and a serine O-acetyltransferase were found as representatives of each group, respectively.