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Abstract: Pharmaceuticals represent a group of the new emerging contaminants, which might influence microbial communities in the activated sludge. Nitrification activity and Nitrospira community structure in the small-scale reactors supplied with different concentrations (0, 50, 200, 500μgL−1) of the selected pharmaceuticals (ibuprofen, naproxen, ketoprofen, diclofenac and clofibric acid) were evaluated. Ammonia removal was not influenced by selected pharmaceuticals. However, in the two reactors operated with 50μgL−1 of pharmaceuticals (R50 and R50P), the effluent concentration of N-(NO2 − +NO3 −) was significantly higher than in the other reactors. Nitrospira community structure was assessed by terminal restriction fragment length polymorphism (T-RFLP) and by cloning and sequencing of the partial genes for 16S rRNA. Nitrospira spp. were detected in all reactors. The two dominant T-RFs represented the sublineages I and II of the genus Nitrospira. Main shifts were observed in the reactors R50 and R50P, where the T-RF representing sublineage II was much higher as compared to the other reactors. Consistent with this, the Nitrospira sublineage II was detected only in the clone libraries from the reactors R50 and R50P. Our results suggest that the relative abundance of Nitrospira sublineage II could be related to the effluent N-(NO2 − +NO3 −) concentration. [Copyright &y& Elsevier]

Abstract: Peatlands play an important role in emissions of the greenhouse gases CO2, CH4 and N2O, which are produced during mineralization of the peat organic matter. To examine the influence of soil type (fen, bog soil) and environmental factors (temperature, groundwater level), emission of CO2, CH4 and N2O and soil temperature and groundwater level were measured weekly or biweekly in loco over a one-year period at four sites located in Ljubljana Marsh, Slovenia using the static chamber technique. The study involved two fen and two bog soils differing in organic carbon and nitrogen content, pH, bulk density, water holding capacity and groundwater level. The lowest CO2 fluxes occurred during the winter, fluxes of N2O were highest during summer and early spring (February, March) and fluxes of CH4 were highest during autumn. The temporal variation in CO2 fluxes could be explained by seasonal temperature variations, whereas CH4 and N2O fluxes could be correlated to groundwater level and soil carbon content. The experimental sites were net sources of measured greenhouse gases except for the drained bog site, which was a net sink of CH4. The mean fluxes of CO2 ranged between 139 mg m−2 h−1 in the undrained bog and 206 mg m−2 h−1 in the drained fen; mean fluxes of CH4 were between −0.04 mg m−2 h−1 in the drained bog and 0.05 mg m−2 h−1 in the drained fen; and mean fluxes of N2O were between 0.43 mg m−2 h−1 in the drained fen and 1.03 mg m−2 h−1 in the drained bog. These results indicate that the examined peatlands emit similar amounts of CO2 and CH4 to peatlands in Central and Northern Europe and significantly higher amounts of N2O. [Copyright &y& Elsevier]

Bacillus subtilis strains communicate through the comQXPA quorum sensing (QS) system, which regulates genes expressed during early stationary phase. A high polymorphism of comQXP' loci was found in closely related strains isolated from desert soil samples separated by distances ranging from meters to kilometers. The observed polymorphism comprised four communication groups (pherotypes), such that strains belonging to the same pherotype exchanged information efficiently but strains from different pherotypes failed to communicate. To determine whether the same level of polymorphism in the comQXP' QS system could be detected at microscale, B. subtilis isolates were obtained from two separate 1-cm³ soil samples, which were progressively divided into smaller sections. Cross-activation studies using pherotype-responsive reporter strains indicated the same number of communication pherotypes at microscale as previously determined at macroscale. Sequencing of the housekeeping gene gyrA and the QS comQ gene confirmed different evolutionary rates of these genes. Furthermore, an asymmetric communication response was detected inside the two pherotype clusters, suggesting continuous evolution of the QS system and possible development of new languages. To our knowledge, this is the first microscale study demonstrating the presence of different QS languages among isolates of one species, and the implications of this microscale diversity for microbial interactions are discussed. [ABSTRACT FROM AUTHOR]

Examines the genome of Bacillus subtilis. Detection of DNA binding in the bacteria; Effects of adapter protein on sporulation; Control of transcription factor.

Examines the regulation of sinIR operon protein expression in Bacillus subtilis. Transcription of sinIR from the P1 promoter; Repression of P1 induction by the transition-state regulatory protein Hpr; Synthesis of beta-galactosidase.

Explores the regulatory role of Bacillus subtilis SinR in sporulation gene spo0A expression. Identification of guanine residues important for SinR recognition of the DNA sequence; Role of SinR in spo0A expression; SinR binding to the spo0A promoter; Localization of the SinR binding site on spo0A; Mutagenesis of the SinR-spo0A binding site.

Summary: Here, we review the multiple mechanisms that the Gram‐positive bacterium Bacillus subtilis uses to allow it to communicate between cells and establish community structures. The modes of action that are used are highly varied and include routes that sense pheromone levels during quorum sensing and control gene regulation, the intimate coupling of cells via nanotubes to share cytoplasmic contents, and long‐range electrical signalling to couple metabolic processes both within and between biofilms. We explore the ability of B. subtilis to detect 'kin' (and 'cheater cells') by looking at the mechanisms used to potentially ensure beneficial sharing (or limit exploitation) of extracellular 'public goods'. Finally, reflecting on the array of methods that a single bacterium has at its disposal to ensure maximal benefit for its progeny, we highlight that a large future challenge will be integrating how these systems interact in mixed‐species communities. 'Bacillus subtilis is a Gram‐positive bacterium that has evolved multiple mechanisms for communication and cooperation. Here we review processes which include division of labour, nanotube formation, metabolic signalling, and quorum sensing'. [ABSTRACT FROM AUTHOR]

The production pathway and carbon source of CH4 in the surface sediment of a eutrophic alpine lake (Lake Bled, northwest Slovenia), in which the hypolimnion is anoxic for most of the year, were determined from molecular and biogeochemical studies. The average δ13CCH4 value of -69.5‰ ± 1.2‰, associated with low acetate concentrations, suggested that CH4 should be formed, predominantly, hydrogenotrophically. The proportion of "fresh" autochthonous lipids in total extractable lipids in sediment decreased from 62% at the surface to 41% at a depth of 20 cm. The contribution of lipids of bacterial origin was more pronounced at the surface, comprising 13%. Terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S ribosomal ribonucleic acid (rRNA) sequences of bacterial and archaeal community members suggested that larger sediment-depth-dependent changes occurred in the latter. The majority of archaeal sequences belonged to Euryarchaeota. The methanogenic population accounted for 73% and 38% of the archaeal community at depths of 0-2 cm and 10-12 cm, respectively. In the upper 2 cm, hydrogenotrophs, mostly Methanomicrobiaceae, were dominant. In the deeper sediment, archaeal sequences were mostly those of unknown affiliation with Euryarchaeota, Thermoplasmatales, and related linkages, and only 21% of the hydrogenotrophic methanogenic archaea were detected. Somewhat lower percentages (< 18%) of sequences representing acetotrophic archaea (Methanosaetaceae) were present in the two layers. The biogeochemical processes and structure of the archaeal community support the hypothesis that hydrogenotrophic methanogenesis is the dominant pathway in the sediment of alpine Lake Bled, despite low temperature and the prevalence of "fresh" autochthonous-derived organic matter. [ABSTRACT FROM AUTHOR]

We investigated microbial growth in increasing concentrations of hexavalent chromium (Cr(VI)) and its reduction by a microbial community enriched from tannery effluent and by the bacterial strains isolated from the enriched community. The bacterial growth was monitored by measuring the optical cell density (OD650), while the Cr(VI) concentration in the samples was determined using spectrophotometry and liquid chromatography hyphenated to inductively coupled plasma mass spectrometry (HPLC–ICP–MS). At a Cr(VI) concentration of 100 mg/L, the isolates affiliated with Pseudomonas aeruginosa (P. aeruginosa) reached higher optical cell densities, but were in general less effective for Cr(VI) reduction than the isolates affiliated with Mammaliicoccus sciuri (M. sciuri). All three M. sciuri isolates and only one of the seven P. aeruginosa isolates were able to reduce 50% of the Cr(VI) with an initial concentration of 100 mg/L within 24 h (pH 7.1), while the six isolates affiliated with P. aeruginosa were less effective. Compared to the isolated, individual bacterial strains, the enriched microbial community was better adapted to the elevated Cr(VI) concentrations, but needed a longer time (48 h) to reduce the Cr(VI) with the same efficacy as the most efficient individual isolates. The ability of the enriched microbial community and the isolated bacterial strains to reduce the Cr(VI) highlights their potential for use in the rapid bioremediation of wastewaters contaminated with Cr(VI). [ABSTRACT FROM AUTHOR]

Bacillus is a genus of microorganisms (bacteria) and contains many important commercial species used in industry, agriculture and healthcare. Many different Bacilli are relatively well understood at the single-cell level; however, molecular tools that determine the diversity and ecology of Bacillus community are limited, which limits our understanding of how the Bacillus community works. In the present study, we investigated the potential of the housekeeping gene gyrA as a molecular marker for determining the diversity of Bacillus species. The amplification efficiency for Bacillus species diversity could be greatly improved by primer design. Therefore, we designed a novel primer pair gyrA3 that can detect at least 92 Bacillus species and related species. For B. amyloliquefaciens, B. pumilus, and B. megaterium, we observed that the high variability of the gyrA gene allows for more detailed clustering at the subspecies level that cannot be achieved by the 16S rRNA gene. Since gyrA provides better phylogenetic resolution than 16S rRNA and informs on the diversity of the Bacillus community, we propose that the gyrA gene may have broad application prospects in the study of Bacillus ecology. [ABSTRACT FROM AUTHOR]

Microorganisms in nature form multicellular groups called biofilms. In biofilms, bacteria embedded in the extracellular matrix (ECM) interact intensely due to their proximity. Most studies have investigated genetically homogeneous biofilms, leaving a gap in knowledge on genetically heterogeneous biofilms. Recent insights show that a Gram-positive model bacterium, Bacillus subtilis, discriminates between strains of high (kin) and low (nonkin) genetic similarity, reflected in merging (kin) and boundaries (nonkin) between swarms. However, it is unclear how kinship between interacting strains affects their fitness, the genotype assortment, and incorporation of the mutant lacking the main structural ECM polysaccharide (EpsA-O) into floating biofilms (pellicles). We cultivated Bacillus subtilis strains as mixtures of isogenic, kin, and nonkin strain combinations in the biofilm-promoting minimal medium under static conditions, allowing them to form pellicles. We show that in nonkin pellicles, the dominant strain strongly reduced the frequency of the other strain. Segregation of nonkin mixtures in pellicles increased and invasion of nonkin EpsA-O-deficient mutants into pellicles decreased compared to kin and isogenic floating biofilms. Kin and isogenic strains had comparable relative frequencies in pellicles and showed more homogenous cell mixing. Overall, our results emphasize kin discrimination as a social behavior that shapes strain distribution, spatial segregation, and ECM mutant ability to incorporate into genetically heterogenous biofilms of B. subtilis. [ABSTRACT FROM AUTHOR]

Bacillus sp. strains that are beneficial to plants are widely used in commercial biofertilizers and biocontrol agents for sustainable agriculture. Generally, functional Bacillus strains are applied as single-strain communities since the principles of synthetic microbial consortia constructed with Bacillus strains remain largely unclear. Here, we demonstrated that the mutual compatibility directly affects the survival and function of two-member consortia composed of Bacillus velezensis SQR9 and FZB42 in the rhizosphere. A mutation in the global regulator Spo0A of SQR9 markedly reduced the boundary phenotype (appearance of a visible boundary line at the meeting point of two swarms) with wild-type FZB42, and the combined use of the SQR9(Î"spo0A) mutant and FZB42 improved biofilm formation, root colonization, and the production of secondary metabolites that are beneficial to plants. Furthermore, alleviation of antagonistic interactions of two-member Bacillus consortia improved its beneficial effects to cucumber in a greenhouse experiment. Our results provide evidence that social interactions among bacteria could be an influencing factor for achieving a desired community-level function. [ABSTRACT FROM AUTHOR]

Abstract: Laccases of fungal origin have been intensively studied due to their importance in various biotechnological applications. There is a constant demand for new laccases with improved properties such as stability at higher temperatures or at an alkaline pH. Growing molecular evidence suggests that laccases may also be widespread in bacteria. While only a handful of bacterial laccases have been purified and characterized, several novel traits have already been discovered (e.g. pH-stability and 2-domain organization of the enzyme as opposed to the usual 3-domain structure of fungal laccases). The aim of this study was to examine the diversity of bacterial laccase-like genes in two types of high-organic peat soil using a cloning and sequencing approach. Gene libraries prepared of small fragments (150 base pairs) revealed an amazing diversity of bacterial laccases. The fragments clustered in 11 major lineages, and one third of the 241 sequences resembled laccase-like genes of Acidobacteria. Additionally, a new primer was used to retrieve several larger fragments of the putative bacterial laccase genes that spanned all four copper-binding sites. Both “conventional” 3-domain laccases and the recently described 2-domain small laccases have been obtained using this approach, demonstrating the potential of the primer. The present study thus contributes to the understanding of the diversity of bacterial laccases and provides a new tool for finding laccase-like sequences in bacterial strains and soil samples. [Copyright &y& Elsevier]

Abstract: The microbial activity and bacterial community structure were investigated in two types of peat soil in a temperate marsh. The first, a drained grassland fen soil, has a neutral pH with partially degraded peat in the upper oxic soil horizons (16% soil organic carbon). The second, a bog soil, was sampled in a swampy forest and has a very high soil organic carbon content (45%), a low pH (4.5), and has occasional anoxic conditions in the upper soil horizons due to the high water table level. The microbial activity in the two soils was measured as the basal and substrate-induced respiration (SIR). Unexpectedly, the SIR (μlCO2 g−1 dry soil) was higher in the bog than in the fen soil, but lower when CO2 production was expressed per volume of soil. This may be explained by the notable difference in the bulk densities of the two soils. The bacterial communities were assessed by terminal restriction fragment length polymorphism (T-RFLP) profiling of 16S rRNA genes and indicated differences between the two soils. The differences were determined by the soil characteristics rather than the season in which the soil was sampled. The 16S rRNA gene libraries, constructed from the two soils, revealed high proportions of sequences assigned to the Acidobacteria phylum. Each library contained a distinct set of phylogenetic subgroups of this important group of bacteria. [Copyright &y& Elsevier]

Bacterial quorum sensing (QS) is based on signal molecules (SM), which increase in concentration with cell density. At critical SM concentration, a variety of adaptive genes sharply change their expression from basic level to maximum level. In general, this sharp transition, a hallmark of true QS, requires an SM dependent positive feedback loop, where SM enhances its own production. Some communication systems, like the peptide SM-based ComQXPA communication system of Bacillus subtilis, do not have this feedback loop and we do not understand how and if the sharp transition in gene expression is achieved. Based on experiments and mathematical modeling, we observed that the SM peptide ComX encodes the information about cell density, specific cell growth rate, and even oxygen concentration, which ensure power-law increase in SM production. This enables together with the cooperative response to SM (ComX) a sharp transition in gene expression level and this without the SM dependent feedback loop. Due to its ultra-sensitive nature, the ComQXPA can operate at SM concentrations that are 100–1000 times lower than typically found in other QS systems, thereby substantially reducing the total metabolic cost of otherwise expensive ComX peptide. Iztok Dogsa et al. use mathematical modeling and experimental approaches to investigate how the ComQXPA communication system in Bacillus subtilis initiate a quorum-sensing response without the positive feedback loop dependent on signal molecules seen in many systems. They find that the peptide ComX encodes information about cell density, specific cell growth rate and oxygen concentration, which ensure power-law increase in signal molecule production and enables a sharp transition in gene expression level without the typical feedback loop. [ABSTRACT FROM AUTHOR]

Metagenomics is a powerful tool that allows identifying enzymes with novel properties from the unculturable component of microbiomes. However, thus far only a limited number of laccase or laccase -like enzymes identified through metagenomics has been subsequently biochemically characterized. This work describes the successful bio-mining of bacterial laccase-like enzymes in an acidic bog soil metagenome and the characterization of the first acidobacterial laccase-like multicopper oxidase (LMCO). LMCOs have hitherto been mostly studied in fungi and some have already found applications in diverse industries. However, improved LMCOs are in high demand. Using molecular screening of a small metagenomic library (13,500 clones), a gene encoding a three-domain LMCO (LacM) was detected, showing the highest similarity to putative copper oxidases of Candidatus Solibacter (Acidobacteria). The encoded protein was expressed in Escherichia coli, purified by affinity chromatography and biochemically characterized. LacM oxidized a variety of phenolic substrates, including two standard laccase substrates (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), k / k = 8.45 s mM; 2,6-dimethoxyphenol (2,6-DMP), k / k = 6.42 s mM), next to L-3,4-dihydroxyphenylalanine (L-DOPA), vanillic acid, syringaldazine, pyrogallol, and pyrocatechol. With respect to the latter two lignin building blocks, LacM showed the highest catalytic activity ( k / k = 173.6 s mM) for pyrogallol, with ca. 20% activity preserved even at pH 8.0. The enzyme was thermostable and heat-activated in the interval 40-60 °C, with an optimal activity on ABTS at 50 °C. It was rather stable at high salt concentration (e.g., 34% activity preserved at 500 mM NaCl) and in the presence of organic solvents. Remarkably, LacM decolored azo and triphenylmethane dyes, also in the absence of redox mediators. [ABSTRACT FROM AUTHOR]

Summary Multicellularity inherently involves a number of cooperative behaviors that are potentially susceptible to exploitation but can be protected by mechanisms such as kin discrimination. Discrimination of kin from non-kin has been observed in swarms of the bacterium Bacillus subtilis , but the underlying molecular mechanism has been unknown. We used genetic, transcriptomic, and bioinformatic analyses to uncover kin recognition factors in this organism. Our results identified many molecules involved in cell-surface modification and antimicrobial production and response. These genes varied significantly in expression level and mutation phenotype among B. subtilis strains, suggesting interstrain variation in the exact kin discrimination mechanism used. Genome analyses revealed a substantial diversity of antimicrobial genes present in unique combinations in different strains, with many likely acquired by horizontal gene transfer. The dynamic combinatorial effect derived from this plethora of kin discrimination genes creates a tight relatedness cutoff for cooperation that has likely led to rapid diversification within the species. Our data suggest that genes likely originally selected for competitive purposes also generate preferential interactions among kin, thus stabilizing multicellular lifestyles. [ABSTRACT FROM AUTHOR]

Laccases are oxidoreductases mostly studied in fungi, while bacterial laccases remain poorly studied despite their high genetic diversity and potential for biotechnological application. Our previous bioinformatic analysis identified alkaliphilic bacterial strains Thioalkalivibrio sp. as potential sources of robust bacterial laccases that would be stable at high pH. In the present work, a gene for a laccase-like enzyme from Thioalkalivibrio sp. ALRh was cloned and expressed as a 6× His-tagged protein in Escherichia coli. The purified enzyme was a pH-tolerant laccase stable in the pH range between 2.1 and 9.9 at 20 °C as shown by intrinsic fluorescence emission spectrometry. It had optimal activities at pH 5.0 and pH 9.5 with the laccase substrates 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,6-dimethoxyphenol, respectively. In addition, it could oxidize several other monophenolic compounds and potassium hexacyanoferrate(II) but not tyrosine. It showed highest activity at 50 °C, making it suitable for prolonged incubations at this temperature. The present study shows that Thioalkalivibrio sp. encodes an active, alkaliphilic, and thermo-tolerant laccase and contributes to our understanding of the versatility of bacterial laccase-like multicopper oxidases in general. [ABSTRACT FROM AUTHOR]

Kin discrimination, broadly defined as differential treatment of conspecifics according to their relatedness, could help biological systems direct cooperative behavior toward their relatives. Here we investigated the ability of the soil bacterium Bacillus subtilis to discriminate kin from nonkin in the context of swarming, a cooperative multicellular behavior. We tested a collection of sympatric conspecifics from soil in pairwise combinations and found that despite their history of coexistence, the vast majority formed distinct boundaries when the swarms met. Some swarms did merge, and most interestingly, this behavior was only seen in the most highly related strain pairs. Overall the swarm interaction phenotype strongly correlated with phylogenetic relatedness, indicative of kin discrimination. Using a subset of strains, we examined cocolonization patterns on plant roots. Pairs of kin strains were able to cocolonize roots and formed a mixed-strain biofilm. In contrast, inoculating roots with pairs of nonkin strains resulted in biofilms consisting primarily of one strain, suggestive of an antagonistic interaction among nonkin strains. This study firmly establishes kin discrimination in a bacterial multicellular setting and suggests its potential effect on ecological interactions. [ABSTRACT FROM AUTHOR]

A mix of oligonucleotide probes was used to hybridize soil metagenomic DNA from a fosmid clone library spotted on high density membranes. The pooled radio-labeled probes were designed to target genes encoding glycoside hydrolases GH18, dehalogenases, bacterial laccases and mobile genetic elements (integrases from integrons and insertion sequences). Positive hybridizing spots were affiliated to the corresponding clones in the library and the metagenomic inserts were sequenced. After assembly and annotation, new coding DNA sequences related to genes of interest were identified with low protein similarity against the closest hits in databases. This work highlights the sensitivity of DNA/DNA hybridization techniques as an effective and complementary way to recover novel genes from large metagenomic clone libraries. This study also supports that some of the identified catabolic genes might be associated with horizontal transfer events. [ABSTRACT FROM AUTHOR]

The comQXPA locus of Bacillus subtilis encodes a quorum sensing (QS) system typical of Gram positive bacteria. It encodes four proteins, the ComQ isoprenyl transferase, the ComX pre-peptide signal, the ComP histidine kinase, and the ComA response regulator. These are encoded by four adjacent genes all situated on the same chromosome strand. Here we present results of a comprehensive census of comQXPA-like gene arrangements in 2620 complete and 6970 draft prokaryotic genomes (sequenced by the end of 2013). After manually checking the data for false-positive and false-negative hits, we found 39 novel com-like predictions. The census data show that in addition to B. subtilis and close relatives, 20 comQXPA-like loci are predicted to occur outside the B. subtilis clade. These include some species of Clostridiales order, but none outside the phylum Firmicutes. Characteristic gene-overlap patterns were observed in comQXPA loci, which were different for the B. subtilis-like and non-B. subtilis-like clades. Pronounced sequence variability associated with the ComX peptide in B. subtilis clade is evident also in the non-B. subtilis clade suggesting grossly similar evolutionary constraints in the underlying quorum sensing systems. [ABSTRACT FROM AUTHOR]

Bacteria coordinate their behavior using quorum sensing (QS), whereby cells secrete diffusible signals that generate phenotypic responses associated with group living. The canonical model of QS is one of extracellular signaling, where signal molecules bind to cognate receptors and cause a coordinated response across many cells. Here we study the link between QS input (signaling) and QS output (response) in the ComQXPA QS system of Bacillus subtilis by characterizing the phenotype and fitness of comQ null mutants. These lack the enzyme to produce the ComX signal and do not activate the ComQXPA QS system in other cells. In addition to the activation effect of the signal, however, we find evidence of a second, repressive effect of signal production on the QS system. Unlike activation, which can affect other cells, repression acts privately: the de-repression of QS in comQ cells is intracellular and only affects mutant cells lacking ComQ. As a result, the QS signal mutants have an overly responsive QS system and overproduce the secondary metabolite surfactin in the presence of the signal. This surfactin overproduction is associated with a strong fitness cost, as resources are diverted away from primary metabolism. Therefore, by acting as a private QS repressor, ComQ may be protected against evolutionary competition from loss-of-function mutations. Additionally, we find that surfactin participates in a social selection mechanism that targets signal null mutants in coculture with signal producers. Our study shows that by pleiotropically combining intracellular and extracellular signaling, bacteria may generate evolutionarily stable QS systems. [ABSTRACT FROM AUTHOR]

Exopolymeric substances (EPS) are important for biofilm formation and their chemical composition may influence biofilm properties. To explore these relationships the chemical composition of EPS from Bacillus subtilis NCIB 3610 biofilms grown in sucrose-rich (SYM) and sucrose-poor (MSgg and Czapek) media was studied. We observed marked differences in composition of EPS polymers isolated from all three biofilms or from spent media below the biofilms. The polysaccharide levan dominated the EPS of SYM grown biofilms, while EPS from biofilms grown in sucrose-poor media contained significant amounts of proteins and DNA in addition to polysaccharides. The EPS polymers differed also in size with very large polymers (Mw>2000 kDa) found only in biofilms, while small polymers (Mw<200 kD) dominated in the EPS isolated from spent media. Biofilms of the eps knockout were significantly thinner than those of the tasA knockout in all media. The biofilm defective phenotypes of tasA and eps mutants were, however, partially compensated in the sucrose-rich SYM medium. Sucrose supplementation of Czapek and MSgg media increased the thickness and stability of biofilms compared to non-supplemented controls. Since sucrose is essential for synthesis of levan and the presence of levan was confirmed in all biofilms grown in media containing sucrose, this study for the first time shows that levan, although not essential for biofilm formation, can be a structural and possibly stabilizing component of B. subtilis floating biofilms. In addition, we propose that this polysaccharide, when incorporated into the biofilm EPS, may also serve as a nutritional reserve. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

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. subtilis 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. [ABSTRACT FROM AUTHOR]

Industrial-scale bioreactors treat wastewater of temporally variable composition under different weather conditions, while the microbial populations of wastewater treatment plants are often studied in controlled laboratory-scale systems with defined influent at a constant temperature. 16S rRNA- and ammonia oxidising amo A-gene-defined bacterial community structure was investigated in industrial and laboratory-scale moving bed biofilm bioreactors (MBBRs) treating municipal wastewater (WW) or synthetic ammonium solution (AS). Nitrification activity, 16S rRNA and amo A gene T-RFLP profiles were comparable between industrial and laboratory scale WW bioreactors. AS bioreactors exhibited higher nitrification and higher relative abundances of Nitrosomonadaceae and Nitrospiraceae families but only small changes in the general bacterial community structure was detected compared to WW MBBRs. Nitrosomonas europaea lineage dominated WW, while uncultivated Nitrosomonas -like sequences prevailed in AS bioreactors. These results suggest that influent type has a stronger influence on community structure than operational conditions, such as temperature or bioreactor size. [ABSTRACT FROM AUTHOR]

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 threedomain 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Shifts in oxic and anoxic conditions in soil are most frequently caused by water table fluctuations, heavy rain, snowmelt or flooding, with potentially significant impacts on microbial processes and the ability of soils to convert mineral nitrogen to nitrogen gases efficiently. The impact of oxic/anoxic cycles on nitrogen transformation rates was therefore explored in the upper layer (0–30 cm) of partially degraded peat soil. We hypothesized that high denitrification potential would be conserved due to the high organic matter content of this soil. Mineral nitrogen was applied to approximately 1-cm deep layers of homogenized soil in microcosms, with no external source of readily degradable carbon. Microcosms were subjected to three cycles, each consisting of an oxic phase of 8–11 days and an anoxic phase of 21–28 days. Approximately 2% of the ammonium load was lost through ammonia volatilization during oxic phases and the remainder was nitrified. The accumulated nitrate decreased soil pH from 8.0 to 6.8 before its transformation through denitrification. Nitrification and denitrification rates during the three oxic/anoxic cycles (approximately three months) were 2.9–3.2 kg N ha−1 d−1 and 1.0–2.3 kg N ha−1 d−1, respectively. Extrapolation of these values to 30-cm deep soil layers gave rates that were sufficient for complete transformation of at least 1700 kg N ha−1 of ammonium to nitrogen gases, which is ten-fold greater than the annual nitrogen application of 170 kg N ha−1 permitted by the European directive. Denitrification rates decreased linearly during the three cycles (from 36 ± 2 to 16 ± 1 μg N g−1 d−1 dry soil), projecting cessation of denitrification activity and CO2 production during the fifth cycle. Storage of peat soil at 4 °C most probably allowed slow degradation of organic matter that was completely oxidized to CO2 after the soil was exposed to higher temperature (28 °C). Storage of soil for one year did not affect nitrification rate, but reduced denitrification rate, unless soil was amended with a readily degradable carbon source. The data suggest that, despite the high carbon content of this soil, it cannot sustain transformations of high N loads to nitrogen gases for prolonged periods without amendment with readily available carbon. [Copyright &y& Elsevier]

Abstract: 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 500μgL−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 50μgL−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 50μgL−1 of each of selected pharmaceuticals. [Copyright &y& Elsevier]

In this study, microcosms were used to investigate the influence of temperature (4 and 28 °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 CO2 g−1 soil), irrespective of the water content, whereas nitrous oxide production was greater under wet conditions (98–336 μg N2O g−1 soil). After an initial lag phase, methane emissions (963 μg CH4 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. [ABSTRACT FROM AUTHOR]

Abstract: Fen peatlands are specific wetland ecosystems containing high soil organic carbon (SOC). There is a general lack of knowledge about the microbial communities that abound in these systems. We examined the microbial activity and community structure in two fen soils differing in SOC content sampled from the Ljubljana Marsh under different seasonal conditions. Substrate-induced respiration and dehydrogenase activity were used as indicators of total microbial activity. Both methods indicated higher microbial activities in the fen soil with the higher SOC content on all dates of sampling. To determine whether the differences in microbial activity were associated with differences in the microbial community structures, terminal restriction fragment length polymorphism (T-RFLP) of bacterial 16S rRNA genes was performed. Comparison of the T-RFLP profiles revealed very similar community structures in both fens and in the two seasonal extremes investigated. This suggested a stable community structure in the two fens, which is not affected by the SOC content or seasonal variation. In addition, a bacterial 16S ribosomal RNA gene based clone library was prepared from the fen soil with the higher SOC content. Out of 114 clones analysed, approximately 53% belonged to the Proteobacteria, 23% to the Acidobacteria, 21% to a variety of other taxa, and less than 3% were affiliated with the Firmicutes. [Copyright &y& Elsevier]

Rok is a repressor of the transcriptional activator ComK and is therefore an important regulator of competence in Bacillus subtilis (T. T. Hoa, P. Tortosa, M. Albano, and D. Dubnau, Mol. Microbiol. 43:15–26, 2002). To address the wider role of Rok in the physiology of B. subtilis, we have used a combination of transcriptional profiling, gel shift experiments, and the analysis of lacZ fusions. We demonstrate that Rok is a repressor of a family of genes that specify membrane-localized and secreted proteins, including a number of genes that encode products with antibiotic activity. We present evidence for the recent introduction of rok into the B. subtilis-Bacillus licheniformis-Bacililus amyloliquefaciens group by horizontal transmission. [ABSTRACT FROM AUTHOR]

Summary 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. [ABSTRACT FROM AUTHOR]

Summary Bactericidal/permeability-increasing protein (BPI) of neutrophils is a lipopolysaccharide (LPS)-binding antibacterial protein with specificity for Gramnegative bacteria. BPI binding to the bacterial surface rapidly triggers potentially reversible bacterial growth inhibition and alterations of the outer membrane and, later, disruption of the inner membrane and lethal injury. Initial effects include selective OmpR-dependent changes in the synthesis of outer membrane porins (OmpF and OmpC). Because OmpR is a global transcriptional regulator, we have examined its possible role in responses of E. coli to sublethal injury caused by BPI. Early (< 15 min) reversible effects of BPI on bacterial colony-forming ability and outer membrane permeability were virtually identical in isogenic wild-type (wt) and ompR - E. coli. Both strains could repair the outer membrane permeability barrier after Mg2+ -induced displacement of bound BPI. However, OmpR was essential for the ability of E. coli to tolerate low doses of BPI and escape the progression of sublethal to lethal damage. Scanning electron microscopy revealed that BPI treatment produced greater membrane perturbations in the ompR - strain, apparent even before lethal injury. These findings suggest that the fate of E. coli exposed to BPI depends on both OmpR-independent mechanisms engaged in outer membrane repair and OmpR- dependent processes that modulate porin synthesis and retard progression of injury from the outer to the inner membrane. [ABSTRACT FROM AUTHOR]