Ines Mandic-Mulec

Thaumarchaeal Ammonia Oxidation in an Acidic Forest Peat Soil Is Not Influenced by Ammonium Amendment

ABSTRACT Both bacteria and thaumarchaea contribute to ammonia oxidation, the first step in nitrification. The abundance of putative ammonia oxidizers is estimated by quantification of the functional gene amoA, which encodes ammonia monooxygenase subunit A. In soil, thaumarchaeal amoA genes often outnumber the equivalent bacterial genes. Ecophysiological studies indicate that thaumarchaeal ammonia oxidizers may have a selective advantage at low ammonia concentrations, with potential adaptation to soils in which mineralization is the major source of ammonia. To test this hypothesis, thaumarchaeal and bacterial ammonia oxidizers were investigated during nitrification in microcosms containing an organic, acidic forest peat soil (pH 4.1) with a low ammonium concentration but high potential for ammonia release during mineralization. Net nitrification rates were high but were not influenced by addition of ammonium. Bacterial amoA genes could not be detected, presumably because of low abundance of bacterial ammonia oxidizers. Phylogenetic analysis of thaumarchaeal 16S rRNA gene sequences indicated that dominant populations belonged to group 1.1c, 1.3, and “deep peat” lineages, while known amo-containing lineages (groups 1.1a and 1.1b) comprised only a small proportion of the total community. Growth of thaumarchaeal ammonia oxidizers was indicated by increased abundance of amoA genes during nitrification but was unaffected by addition of ammonium. Similarly, denaturing gradient gel electrophoresis analysis of amoA gene transcripts demonstrated small temporal changes in thaumarchaeal ammonia oxidizer communities but no effect of ammonium amendment. Thaumarchaea therefore appeared to dominate ammonia oxidation in this soil and oxidized ammonia arising from mineralization of organic matter rather than added inorganic nitrogen.

Specific activation of the Bacillus quorum‐sensing systems by isoprenylated pheromone variants

Natural genetic competence in Bacillus subtilis is controlled by quorum‐sensing (QS). The ComP– ComA two‐component system detects the signalling molecule ComX, and this signal is transduced by a conserved phosphotransfer mechanism. ComX is synthesized as an inactive precursor and is then cleaved and modified by ComQ before export to the extracellular environment. The comQXP′ loci of a set of natural Bacillus isolates have been sequenced and shown to possess a striking polymorphism that determines specific patterns of both activation and inhibition of the quorum‐sensing response. We have developed a simple purification method for the modified peptide signalling pheromones allowing the characterization of four distinct ComX molecules representing different pherotypes. Genetic and biochemical evidence demonstrate that all the ComX variants are isoprenylated by the post‐translational modification of a conserved tryptophan residue and that the modifications on the ComX peptide backbones vary in mass among the various pherotypes. These results give new insights into peptidemediated quorum‐sensing signalling in Gram‐positive bacteria and emphasize the role of isoprenylation in bacterial signal transduction.

Influence of temperature and soil water content on bacterial, archaeal and denitrifying microbial communities in drained fen grassland soil microcosms

In this study, microcosms were used to investigate the influence of temperature (4 and 28 degrees C) and water content (45% and 90% WHC) on microbial communities and activities in carbon-rich fen soil. Bacterial, archaeal and denitrifier community composition was assessed during incubation of microcosms for 12 weeks using terminal restriction fragment length polymorphism (T-RFLP) profiling of 16S rRNA and nitrous oxide reductase (nosZ) genes. In addition, microbial and denitrifier abundance, potential denitrification activity and production of greenhouse gases were measured. No detectable changes were observed in prokaryote or denitrifier abundance. In general, cumulatively after 12 weeks more carbon was respired at the higher temperature (3.7 mg CO(2) g(-1) soil), irrespective of the water content, whereas nitrous oxide production was greater under wet conditions (98-336 microg N(2)O g(-1) soil). After an initial lag phase, methane emissions (963 microg CH(4) g(-1) soil) were observed only under warm and wet conditions. T-RFLP analyses of bacterial 16S rRNA and nosZ genes revealed small or undetectable community changes in response to temperature and water content, suggesting that bacterial and denitrifying microbial communities are stable and do not respond significantly to seasonal changes in soil conditions. In contrast, archaeal microbial community structure was more dynamic and was strongly influenced by temperature.

Influence of pharmaceutical residues on the structure of activated sludge bacterial communities in wastewater treatment bioreactors

Concern is growing over contamination of the environment with pharmaceuticals because of their widespread use and incomplete removal during wastewater treatment, where microorganisms drive the key processes. The influence of pharmaceuticals on bacterial community structure in activated sludge was assessed in small-scale wastewater treatment bioreactors containing different concentrations (5, 50, 200 and 500microgL(-1)) of several commonly used pharmaceuticals (ibuprofen, naproxen, ketoprofen, diclofenac and clofibric acid). T-RFLP analyses of the bacterial 16S rRNA genes indicated a minor but consistent shift in the bacterial community structure in the bioreactor R50 supplied with pharmaceuticals at a concentration of 50microgL(-1), compared to the control reactor R0, which was operated without addition of pharmaceuticals. In the reactors operated with higher concentrations of pharmaceuticals, a greater structural divergence was observed. Bacterial community composition was further investigated by preparation of two clone libraries of bacterial 16S rRNA genes from reactors R0 and R50. Most clones in both libraries belonged to the Betaproteobacteria, among which Thauera, Sphaerotilus, Ideonella and Acidovorax-related spp. dominated. Nitrite-oxidizing bacteria of the genus Nitrospira sp., which are key organisms for the second stage of nitrification in wastewater treatment plants, were found only in the clone library of the reactor without pharmaceuticals. In addition, diversity indices were calculated for the two clone libraries, indicating a reduced diversity of activated sludge bacterial community in the reactor supplied with 50microgL(-1) of each of selected pharmaceuticals.

Stimulation of thaumarchaeal ammonia oxidation by ammonia derived from organic nitrogen but not added inorganic nitrogen

Ammonia oxidation, the first step in nitrification, is performed by autotrophic bacteria and thaumarchaea, whose relative contributions vary in different soils. Distinctive environmental niches for the two groups have not been identified, but evidence from previous studies suggests that activity of thaumarchaea, unlike that of bacterial ammonia oxidizers, is unaffected by addition of inorganic N fertilizer and that they preferentially utilize ammonia generated from the mineralization of organic N. This hypothesis was tested by determining the influence of both inorganic and organic N sources on nitrification rate and ammonia oxidizer growth and community structure in microcosms containing acidic, forest soil in which ammonia oxidation was dominated by thaumarchaea. Nitrification rate was unaffected by the incubation of soil with inorganic ammonium but was significantly stimulated by the addition of organic N. Oxidation of ammonia generated from native soil organic matter or added organic N, but not added inorganic N, was accompanied by increases in abundance of the thaumarchaeal amoA gene, a functional gene for ammonia oxidation, but changes in community structure were not observed. Bacterial amoA genes could not be detected. Ammonia oxidation was completely inhibited by 0.01% acetylene in all treatments, indicating ammonia monooxygenase-dependent activity. The findings have implications for current models of soil nitrification and for nitrification control strategies to minimize fertilizer loss and nitrous oxide production.

Bioinformatic Analysis Reveals High Diversity of Bacterial Genes for Laccase-Like Enzymes

Fungal laccases have been used in various fields ranging from processes in wood and paper industries to environmental applications. Although a few bacterial laccases have been characterized in recent years, prokaryotes have largely been neglected as a source of novel enzymes, in part due to the lack of knowledge about the diversity and distribution of laccases within Bacteria. In this work genes for laccase-like enzymes were searched for in over 2,200 complete and draft bacterial genomes and four metagenomic datasets, using the custom profile Hidden Markov Models for two- and three- domain laccases. More than 1,200 putative genes for laccase-like enzymes were retrieved from chromosomes and plasmids of diverse bacteria. In 76% of the genes, signal peptides were predicted, indicating that these bacterial laccases may be exported from the cytoplasm, which contrasts with the current belief. Moreover, several examples of putatively horizontally transferred bacterial laccase genes were described. Many metagenomic sequences encoding fragments of laccase-like enzymes could not be phylogenetically assigned, indicating considerable novelty. Laccase-like genes were also found in anaerobic bacteria, autotrophs and alkaliphiles, thus opening new hypotheses regarding their ecological functions. Bacteria identified as carrying laccase genes represent potential sources for future biotechnological applications.

The N‐ and C‐terminal domains of MecA recognize different partners in the competence molecular switch

ComK is a transcription factor required for the expression of competence genes in Bacillus subtilis. Binding to MecA targets ComK for degradation by the ClpCP protease. MecA therefore acts as an adapter protein recruiting a regulatory protein for proteolysis. However, when ComS is synthesized, ComK is released from binding by MecA and thereby protected from degradation. MecA binds to three protein partners during these processes: ComK, ClpC and ComS. Using limited proteolysis, we have defined N‐ and C‐terminal structural domains of MecA and evaluated the interactions of these domains with the protein partners of MecA. Using surface plasmon resonance, we have determined that the N‐terminal domain of MecA interacts with ComK and ComS and the C‐terminal domain with ClpC. MecA is shown to exist as a dimer with dimerization sites on both the N‐ and C‐terminal domains. The C‐terminal domain stimulates the ATPase activity of ClpC and is degraded by the ClpCP protease, while the N‐terminal domain is inactive in both of these assays. In vivo data were consistent with these findings, as comG–lacZ expression was decreased in a strain overproducing the N‐terminal domain, indicating reduced ComK activity. We propose a model in which binding of ClpC to the C‐terminal domain of MecA induces a conformational change enabling the N‐terminal domain to bind ComK with enhanced affinity. MecA is widespread among Gram‐positive organisms and may act generally as an adapter protein, targeting proteins for regulated degradation.

Ammonium supply rate influences archaeal and bacterial ammonia oxidizers in a wetland soil vertical profile

Oxidation of ammonia, the first step in nitrification, is carried out in soil by bacterial and archaeal ammonia oxidizers and recent studies suggest possible selection for the latter in low-ammonium environments. In this study, we investigated the selection of ammonia-oxidizing archaea and bacteria in wetland soil vertical profiles at two sites differing in terms of the ammonium supply rate, but not significantly in terms of the groundwater level. One site received ammonium through decomposition of organic matter, while the second, polluted site received a greater supply, through constant leakage of an underground septic tank. Soil nitrification potential was significantly greater at the polluted site. Quantification of amoA genes demonstrated greater abundance of bacterial than archaeal amoA genes throughout the soil profile at the polluted site, whereas bacterial amoA genes at the unpolluted site were below the detection limit. At both sites, archaeal, but not the bacterial community structure was clearly stratified with depth, with regard to the soil redox potential imposed by groundwater level. However, depth-related changes in the archaeal community structure may also be associated with physiological functions other than ammonia oxidation.

Effects of low-density static magnetic fields on the growth and activities of wastewater bacteria Escherichia coli and Pseudomonas putida

The aim of this study was to explore the influence of a moderate static magnetic field (SMF) of different densities on Escherichia coli and Pseudomonas putida that are commonly found in wastewater treatment plants. In line with literature reports that SMF increases the efficiency of wastewater treatment the findings of this study indicated that SMF negatively influenced the growth but positively influenced the enzymatic activities and ATP levels of the two model bacteria. The inhibitory effect of SMF on growth of E. coli and P. putida was most pronounced at their optimal growth temperature (37°C and 28°C respectively) and was reversible shortly after the SMF had been terminated. Finally, the results suggested that the induced energy metabolism reflected in higher dehydrogenase activities and ATP levels may be more important for survival, and adaptation to SMF induced stress than the increase in the expression of the rpoS gene.

The quorum sensing diversity within and between ecotypes of Bacillus subtilis

Ecological sociobiology is an emerging field that aims to frame social evolution in terms of ecological adaptation. Here we explore the ecological context for evolution of quorum sensing diversity in bacteria, where social communication is limited to members of the same quorum sensing type (pherotype). We sampled isolates of Bacillus subtilis from soil on a microgeographical scale and identified three ecologically distinct phylogenetic groups (ecotypes) and three pherotypes. Each pherotype was strongly associated with a different ecotype, suggesting that it is usually not adaptive for one ecotype to listen to the signalling of another. Each ecotype, however, contained one or more minority pherotypes shared with the other B.u2003subtilis ecotypes and with more distantly related species taxa. The pherotype diversity within ecotypes is consistent with two models: first, a pherotype cycling model, whereby minority pherotypes enter a population through horizontal genetic transfer and increase in frequency through cheating the social interaction; and second, an occasional advantage model, such that when two ecotypes are each below their quorum densities, they may benefit from listening to one another. This is the first survey of pherotype diversity in relation to ecotypes and it will be interesting to further test the hypotheses raised and supported here, and to explore other bacterial systems for the role of ecological divergence in fostering pherotype diversity.