BackgroundThe presence of Bacillus in very diverse environments reflects the versatile metabolic capabilities of a widely distributed genus. Traditional phylogenetic analysis based on limited gene sampling is not adequate for resolving the genus evolutionary relationships. By distinguishing between core and pan-genome, we determined the evolutionary and functional relationships of known Bacillus.ResultsOur analysis is based upon twenty complete and draft Bacillus genomes, including a newly sequenced Bacillus isolate from an aquatic environment that we report for the first time here. Using a core genome, we were able to determine the phylogeny of known Bacilli, including aquatic strains whose position in the phylogenetic tree could not be unambiguously determined in the past. Using the pan-genome from the sequenced Bacillus, we identified functional differences, such as carbohydrate utilization and genes involved in signal transduction, which distinguished the taxonomic groups. We also assessed the genetic architecture of the defining traits of Bacillus, such as sporulation and competence, and showed that less than one third of the B. subtilis genes are conserved across other Bacilli. Most variation was shown to occur in genes that are needed to respond to environmental cues, suggesting that Bacilli have genetically specialized to allow for the occupation of diverse habitats and niches.ConclusionsThe aquatic Bacilli are defined here for the first time as a group through the phylogenetic analysis of 814 genes that comprise the core genome. Our data distinguished between genomic components, especially core vs. pan-genome to provide insight into phylogeny and function that would otherwise be difficult to achieve. A phylogeny may mask the diversity of functions, which we tried to uncover in our approach. The diversity of sporulation and competence genes across the Bacilli was unexpected based on previous studies of the B. subtilis model alone. The challenge of uncovering the novelties and variations among genes of the non-subtilis groups still remains. This task will be best accomplished by directing efforts toward understanding phylogenetic groups with similar ecological niches.

Ethylene is a gaseous plant growth regulator that controls a multitude of developmental and stress responses. Recently, the levels of Arabidopsis EIN3 protein, a key transcription factor mediating ethylene-regulated gene expression, have been demonstrated to increase in response to the presence of ethylene gas. Furthermore, in the absence of ethylene, EIN3 is quickly degraded through a ubiquitin͞proteasome pathway mediated by two F-box proteins, EBF1 and EBF2. Here we report the identification of ETHYLENE-INSENSITIVE5 as the 533 exoribonuclease XRN4. Specifically, we demonstrate that EIN5 is a component of the ethylene signal transduction cascade acting downstream of CTR1 that is required for ethylene-mediated gene expression changes. Furthermore, we find that the ethylene insensitivity of ein5 mutant plants is a consequence of the over-accumulation of EBF1 and EBF2 mRNAs resulting in the under-accumulation of EIN3 even in the presence of ethylene gas. Together, our results suggest that the role of EIN5 in ethylene perception is to antagonize the negative feedback regulation on EIN3 by promoting EBF1 and EBF2 mRNA decay, which consequently allows the accumulation of EIN3 protein to trigger the ethylene response.Arabidopsis ͉ growth regulation ͉ signal transduction

Functioning of the spoIIE locus of Bacillus subtilis is required for formation of a normal polar septum during sporulation and for activation of the transcription factor sigma F, which directs early forespore-specific gene expression. We have determined the DNA sequence of the wild type and several mutant alleles of the spoIIE gene of B. subtilis and sequenced a substantial portion of its presumptive homologue in Bacillus megaterium. We show that the spoIIE locus encodes a single large protein with a predicted molecular mass of 92 kDa. Each of five point-mutation alleles, which have traditionally defined the locus, and two transposon-generated mutations were shown to fall within the coding sequence for the 92 kDa gene product or within sequences expected to be required for its expression. The amino-terminal portion of the predicted SpoIIE gene product, comprising approximately 40% of the protein, is extremely hydrophobic and is expected to contain up to 12 membrane-spanning segments. The remainder of the protein contains no hydrophobic segments long enough to span a lipid bilayer and is therefore presumed to comprise one or more globular, aqueous-phase exposed domains. An in-frame fusion joining the 3' end of the B. megaterium spoIIE coding sequence to the 5' end of gfp, a gene encoding the green fluorescent protein (GFP) of Aquorea victoria, resulted in a strong, sporulation-specific fluorescent signal localized to the sites of sporulation septum assembly. We speculate that SpoIIE plays a role in assembling the sporulation septum, perhaps determining the special properties of the structure that permit intercompartment signalling during development.

The Cuatro Cié negas Basin (CCB) in the central part of the Chihuahan desert (Coahuila, Mexico) hosts a wide diversity of microorganisms contained within springs thought to be geomorphological relics of an ancient sea. A major question remaining to be answered is whether bacteria from CCB are ancient marine bacteria that adapted to an oligotrophic system poor in NaCl, rich in sulfates, and with extremely low phosphorus levels (<0.3 M). Here, we report the complete genome sequence of Bacillus coahuilensis, a sporulating bacterium isolated from the water column of a desiccation lagoon in CCB. At 3.35 Megabases this is the smallest genome sequenced to date of a Bacillus species and provides insights into the origin, evolution, and adaptation of B. coahuilensis to the CCB environment. We propose that the size and complexity of the B. coahuilensis genome reflects the adaptation of an ancient marine bacterium to a novel environment, providing support to a ''marine isolation origin hypothesis'' that is consistent with the geology of CCB. This genomic adaptation includes the acquisition through horizontal gene transfer of genes involved in phosphorous utilization efficiency and adaptation to high-light environments. The B. coahuilensis genome sequence also revealed important ecological features of the bacterial community in CCB and offers opportunities for a unique glimpse of a microbe-dominated world last seen in the Precambrian.evolution ͉ genomic adaptation ͉ horizontal gene transfer ͉ phosphorus stress ͉ sulfolipids T he Cuatro Ciénegas Basin (CCB) is located in a valley Ϸ740 m above sea level in the state of Coahuila, Mexico, that measures Ϸ30 km by 40 km and is surrounded by high mountains (Ͼ3,000 m) (Fig. 1). CCB is an enclosed evaporitic basin that receives Ϸ150 mm of annual precipitation. Despite the dry climate of the valley, the CCB harbors an extensive system of springs, streams, and pools (1). The CCB ecosystem is not only characterized by a high endemism of macrooganisms and biodiversity of microorganisms (1, 2), but also by extremely oligotrophic waters that are unable to sustain algal growth, making microbial mats the base of the food web (3). In particular, phosphorus (P) levels in CCB appear to be rather low, because they were below the level of detection of several methods used (0.3 M) and the extremely high biomass C:P and N:P ratios (Ͼ100 by moles) previously reported for CCB stromatolites (3, 4). Unlike the present sea, the Churince spring water is poor in NaCl and carbonates, but it is rich in sulfates, magnesium, and calcium (4). Characterization of the microbiological diversity by sequencing 16S rRNA genes revealed that nearly half of the phylotypes from the CCB were closely related to bacteria from marine environments (2). Bacillus coahuilensis is a free-living, spore-forming bacteria isolated from the water column of a shallow desiccation lagoon in the Churince system at CCB (4) (Fig. 1 A and B). A molecular phylogenetic analysis of 16S rRNA sequences indicates that B. coahuilensis is closely ...

Barriers to microbial migrations can lead adaptive radiations and increased endemism. We propose that extreme unbalanced nutrient stoichiometry of essential nutrients can be a barrier to microbial immigration over geological timescales. At the oasis in the Cuatro Ciénegas Basin in Mexico, nutrient stoichiometric proportions are skewed given the low phosphorus availability in the ecosystem. We show that this endangered oasis can be a model for a lost world. The ancient niche of extreme unbalanced nutrient stoichiometry favoured survival of ancestral microorganisms. This extreme nutrient imbalance persisted due to environmental stability and low extinction rates, generating a diverse and unique bacterial community. Several endemic clades of Bacillus invaded the Cuatro Cienegas region in two geological times, the late Precambrian and the Jurassic. Other lineages of Bacillus, Clostridium and Bacteroidetes migrated into the basin in isolated events. Cuatro Ciénegas Basin conservation is vital to the understanding of early evolutionary and ecological processes.

NprR belongs to the RNPP family of quorum-sensing receptors, a group of intracellular regulators activated directly by signaling oligopeptides in Gram-positive bacteria. In Bacillus thuringiensis (Bt), nprR is located in a transcriptional cassette with nprRB that codes for the precursor of the signaling peptide NprRB. NprR is a transcriptional regulator activated by binding of reimported NprRB; however, several reports suggest that NprR also participates in sporulation but the mechanism is unknown. Our in silico results, based on the structural similarity between NprR from Bt and Spo0F-binding Rap proteins from Bacillus subtilis, suggested that NprR could bind Spo0F to modulate the sporulation phosphorelay in Bt. Deletion of nprR-nprRB cassette from Bt caused a delay in sporulation and defective trigger of the Spo0A∼P-activated genes spoIIA and spoIIIG. The DNA-binding domain of NprR was not necessary for this second function, since truncated NprRΔHTH together with nprRB gene was able to restore the sporulation wild type phenotype in the ΔnprR-nprRB mutant. Fluorescence assays showed direct binding between NprR and Spo0F, supporting that NprR is a bifunctional protein. To understand how the NprR activation by NprRB could result in two different functions, we studied the molecular recognition mechanism between the signaling peptide and the receptor. Using synthetic variants of NprRB, we found that SSKPDIVG displayed the highest affinity (Kd = 7.19 nM) toward the recombinant NprR and demonstrated that recognition involves conformational selection. We propose that the peptide concentration in the cell controls the oligomerization state of the NprR-NprRB complex for switching between its two functions.

The high affinity phosphate transport system (pst) is crucial for phosphate uptake in oligotrophic environments. Cuatro Cienegas Basin (CCB) has extremely low P levels and its endemic Bacillus are closely related to oligotrophic marine Firmicutes. Thus, we expected the pst operon of CCB to share the same evolutionary history and protein similarity to marine Firmicutes. Orthologs of the pst operon were searched in 55 genomes of Firmicutes and 13 outgroups. Phylogenetic reconstructions were performed for the pst operon and 14 concatenated housekeeping genes using maximum likelihood methods. Conserved domains and 3D structures of the phosphate-binding protein (PstS) were also analyzed. The pst operon of Firmicutes shows two highly divergent clades with no correlation to the type of habitat nor a phylogenetic congruence, suggesting horizontal gene transfer. Despite sequence divergence, the PstS protein had a similar 3D structure, which could be due to parallel evolution after horizontal gene transfer events.

Poly(A)-binding proteins (PABPs) play an important role in the regulation of translation and the control of mRNA stability in eukaryotes, and their functions are known to be essential in many organisms. PABPs contain a highly conserved C-terminal segment termed the PABC domain. The PABC domain from human PABP interacts with the proteins PAIP1, PAIP2 and RF3 via its PAM2 motifs. These interactions are important for modulating translation. Arabidopsis has eight PABPs, an unexpectedly large number in comparison to other eukaryotes whose genomes have been sequenced. Six of the Arabidopsis PABPs contain the conserved PABC domain. In this work, we have identified PABC-interacting proteins in Arabidopsis. Two proteins, which we named CID1 and CID7, were initially isolated in a two-hybrid screen, and eleven more were predicted to be present in the Arabidopsis proteome and eleven in the rice proteome. Among the 24 PAM2-containing proteins in this set, we observed a diversity of modules of intriguing function, ranging from acidic regions similar to the PAM1 motif found in human PAIP1 and PAIP2, to domains such as the small MutS-related domain, the Lsm domains of Ataxin-2, and RNA recognition motifs (RRMs). We suggest that the large number of PABPs and PAM2-containing proteins may have evolved to provide plants with greater flexibility in modulating the metabolism of specific transcripts. We also found that two PABP genes, PAB2 (ubiquitously expressed) and PAB5 (expressed in reproductive tissues), are essential for viability, suggesting that each has a vital and specific function.