Ammonia concentration determines differential growth of ammonia-oxidising archaea and bacteria in soil microcosms.

The first step of nitrification, oxidation of ammonia to nitrite, is performed by both ammonia-oxidising archaea (AOA) and ammonia-oxidising bacteria (AOB) in soil, but their relative contributions to ammonia oxidation and existence in distinct ecological niches remain to be determined. To determine whether available ammonia concentration has a differential effect on AOA and AOB growth, soil microcosms were incubated for 28 days with ammonium at three concentrations: native (control), intermediate (20 µg NH(4)(+)-N per gram of soil) and high (200 µg NH(4)(+)-N per gram of soil). Quantitative PCR demonstrated growth of AOA at all concentrations, whereas AOB growth was prominent only at the highest concentration. Similarly, denaturing gradient gel electrophoresis (DGGE) analysis revealed changes in AOA communities at all ammonium concentrations, whereas AOB communities changed significantly only at the highest ammonium concentration. These results provide evidence that ammonia concentration contributes to the definition of distinct ecological niches of AOA and AOB in soil.

Autotrophic ammonia oxidation by soil thaumarchaea.

Nitrification plays a central role in the global nitrogen cycle and is responsible for significant losses of nitrogen fertilizer, atmospheric pollution by the greenhouse gas nitrous oxide, and nitrate pollution of groundwaters. Ammonia oxidation, the first step in nitrification, was thought to be performed by autotrophic bacteria until the recent discovery of archaeal ammonia oxidizers. Autotrophic archaeal ammonia oxidizers have been cultivated from marine and thermal spring environments, but the relative importance of bacteria and archaea in soil nitrification is unclear and it is believed that soil archaeal ammonia oxidizers may use organic carbon, rather than growing autotrophically. In this soil microcosm study, stable isotope probing was used to demonstrate incorporation of (13)C-enriched carbon dioxide into the genomes of thaumarchaea possessing two functional genes: amoA, encoding a subunit of ammonia monooxygenase that catalyses the first step in ammonia oxidation; and hcd, a key gene in the autotrophic 3-hydroxypropionate/4-hydroxybutyrate cycle, which has been found so far only in archaea. Nitrification was accompanied by increases in archaeal amoA gene abundance and changes in amoA gene diversity, but no change was observed in bacterial amoA genes. Archaeal, but not bacterial, amoA genes were also detected in (13)C-labeled DNA, demonstrating inorganic CO(2) fixation by archaeal, but not bacterial, ammonia oxidizers. Autotrophic archaeal ammonia oxidation was further supported by coordinate increases in amoA and hcd gene abundance in (13)C-labeled DNA. The results therefore provide direct evidence for a role for archaea in soil ammonia oxidation and demonstrate autotrophic growth of ammonia oxidizing archaea in soil.

DegU and Spo0A jointly control transcription of two loci required for complex colony development by Bacillus subtilis.

Biofilm formation is an example of a multicellular process which depends on cooperative behavior and differentiation within a bacterial population. Our findings indicate that there is a complex feedback loop that maintains the stoichiometry of the extracellular matrix and other proteins required for complex colony development by Bacillus subtilis. Analysis of the transcriptional regulation of two DegU-activated genes that are required for complex colony development by B. subtilis revealed additional involvement of global regulators that are central to controlling biofilm formation. Activation of transcription from both the yvcA and yuaB promoters requires DegU approximately phosphate, but transcription is inhibited by direct AbrB binding to the promoter regions. Inhibition of transcription by AbrB is relieved when Spo0A approximately phosphate is generated due to its known role in inhibiting abrB expression. Deletion of SinR, a key coordinator of motility and biofilm formation, enhanced transcription from both loci; however, no evidence of a direct interaction with SinR for either the yvcA or yuaB promoter regions was observed. The enhanced transcription in the sinR mutant background was subsequently demonstrated to be dependent on biosynthesis of the polysaccharide component that forms the major constituent of the B. subtilis biofilm matrix. Together, these findings indicate that a genetic network dependent on activation of both DegU and Spo0A controls complex colony development by B. subtilis.

DegU co-ordinates multicellular behaviour exhibited by Bacillus subtilis.

Unicellular organisms use a variety of mechanisms to co-ordinate activity within a community and accomplish complex multicellular processes. Because some of the processes that are exhibited by one species can be physiologically incompatible, it raises the question of how entry into these different pathways is regulated. In the Gram-positive bacterium Bacillus subtilis, genetic competence, swarming motility, biofilm formation, complex colony architecture and protease production are all regulated by the response regulator DegU. DegU appears to integrate environmental signals and co-ordinate multicellular behaviours that are subsequently manifested at different levels of DegU phosphorylation. Data are presented which indicate that: (i) swarming motility is activated by very low levels of DegU approximately P that can be generated independently from its cognate sensor kinase DegS; (ii) complex colony architecture is activated by low levels of DegU approximately P that are produced in a DegS-dependent manner to activate transcription of yvcA, a novel gene required for complex colony architecture; and (iii) high levels of DegU approximately P inhibit complex colony architecture and swarming motility but are required prior to the activation of exoprotease production. A model is proposed to explain why such a system may have evolved within B. subtilis to control these multicellular processes through a single regulator.

Cooperativity in signal transfer through the Uhp system of Escherichia coli.

The UhpABC regulatory system in enterobacteria controls the expression of the hexose phosphate transporter UhpT. Signaling is initiated through sensing of extracellular glucose 6-phosphate by membrane-bound UhpC, which in turn modulates the histidine-protein kinase UhpB. Together with the cytoplasmic response regulator UhpA, they constitute a typical two-component regulatory system based on His-to-Asp phosphoryl transfer. Activated (i.e., phosphorylated) UhpA binds to the promoter region of uhpT, resulting in initiation of transcription. We have investigated the contribution of transmembrane signaling (through UhpBC) and intracellular activation (through UhpA) to the overall Uhp response (UhpT expression) in vivo. UhpA activation could be made independent of transmembrane signaling when (Delta)uhpBC cells were grown on pyruvate. Inorganic phosphate interfered with glucose 6-phosphate-dependent, UhpBC-mediated, as well as pyruvate-mediated activation of UhpA. The relationship between the concentration of inducer (glucose 6-phosphate) and the Uhp induction rate was nonhyperbolic, indicating positive cooperativity. The degree of cooperativity was affected by the carbon or energy source available to the cells for growth. As pyruvate-mediated activation of UhpA in (Delta)uhpBC cells could result in considerably stronger UhpT expression than glucose 6-phosphate-dependent activation through UhpBC, the observed positive cooperativity for the overall pathway in wild-type cells may reflect the previously described cooperative binding of UhpA to the uhpT promoter (J. L. Dahl et al., J. Biol. Chem. 272:1910-1919, 1997).

Investigation of in vivo cross-talk between key two-component systems of Escherichia coli.

Intracellular signal transfer in bacteria is dominated by phosphoryl transfer between conserved transmitter and receiver domains in regulatory proteins of so-called two-component systems. Escherichia coli contains 30 such systems, which allow it to modulate gene expression, enzyme activity and the direction of flagellar rotation. The authors have investigated whether, and to what extent, these separate systems form (an) interacting network(s) in vivo, focussing on interactions between four major systems, involved in the responses to the availability of phosphorylated sugars (Uhp), phosphate (Pho), nitrogen (Ntr) and oxygen (Arc). Significant cross-talk was not detectable in wild-type cells. Decreasing expression levels of succinate dehydrogenase (reporting Arc activation), upon activation of the Pho system, appeared to be independent of signalling through PhoR. Cross-talk towards NtrC did occur, however, in a ntrB deletion strain, upon joint activation of Pho, Ntr and Uhp. UhpT expression was demonstrated when cells were grown on pyruvate, through non-cognate phosphorylation of UhpA by acetyl phosphate.