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A fundamental question in the assembly of microbial communities is how complex systems arise from a few components. Synthetic communities allow addressing the dynamics and mechanisms of complex microbial interactions. Most studies on microbial interactions are done in lapses of hours and even days, but microbes must be able to sense danger in seconds. We assembled a three-strain synthetic community from the phylum Bacillota that, based on previously evaluated paired interactions, appear to have different ecological roles: resistant (R), antagonists (A), and sensitive (S). The BARS synthetic community (Bacillota A + S + R) reproduces features of complex communities and exhibits higher-order interaction dynamics. The majority of the S strain population, Sutclifiella horikoshii 20a, dies within 5 min in a paired interaction with A strain, Bacillus pumilus 145. An emergent property appears upon adding the third interactor, as antagonism of strain A over S is not observed in the presence of the R strain, Bacillus cereus 111. After the first five min a change of state of the cells is observed, as the surviving population of the S strain seemed to have acquired tolerance to A. In summary, our model allows the study of the assembly dynamics of a three-species community and to evaluate the immediate outcome within a 30 min frame. The BARS has features of a complex system where the paired interactions do not predict the community dynamics. The model is amenable to mechanistic dissection and to modeling how the parts integrate to achieve collective properties.1IMPORTANCEMicrobial communities are of utmost importance, given their roles in health, agriculture, and all biogeochemical cycles on Earth. Synthetic ecology studies communities by reducing the number of variables. Microbial interactions are usually evaluated in hours or days, however, upon a first encounter, bacteria must respond in minutes, particularly when competition involves killing of neighboring cells. We generated a synthetic community of three species that allows the study of community dynamics in a 30 min frame. We denominated our model BARS as it comprises Bacillota strains that in paired interactions are Antagonist, Resistant, or Sensitive. Even though in paired interaction the antagonist kills the sensitive strain, in a triple interaction, the resistant strain provides stability to the community by neutralizing the antagonism. Therefore, BARS is a rapid response model with features of a complex system where the paired interactions do not predict the community dynamics and exhibit emergent properties.

Phenotypic plasticity allows individuals to respond to the selective forces of a new environment, followed by adaptive evolution. We do not know to what extent phenotypic plasticity allows thermal tolerance evolution in bacteria at the border of their physiological limits. We analyzed growth and reaction norms to temperature of strains of two bacterial lineages, Bacillus cereus sensu lato and Bacillus subtilis sensu lato, that evolved in two contrasting environments, a temperate lagoon (T) and a hot spring (H). Our results showed that despite the co-occurrence of members of both lineages in the two contrasting environments, norms of reactions to temperature exhibited a similar pattern only in strains within the lineages, suggesting fixed phenotypic plasticity. Additionally, strains from the H environment showed only two to three degrees centigrade more heat tolerance than strains from the T environment. Their viability decreased at temperatures above their optimal for growth, particularly for the B. cereus lineage. However, sporulation occurred at all temperatures, consistent with the known cell population heterogeneity that allows the Bacillus to anticipate adversity. We suggest that these mesophilic strains survive in the hot-spring as spores and complete their life cycle of germination and growth during intermittent opportunities of moderate temperatures. The limited evolutionary changes towards an increase in heat tolerance in bacteria should alert us of the negative impact of climate change on all biological cycles in the planet, which at its most basic level depends on microorganisms.

Despite the crucial role of microorganisms to sustain life on Earth, there is little research on the evolution of thermal tolerance of bacteria in the face of the challenge that global warming poses. Phenotypic adaptation to a new environment requires plasticity to allow individuals to respond to selective forces, followed by adaptive evolution. We do not know to what extent phenotypic plasticity allows thermal tolerance evolution in bacteria at the border of their physiological limits. We analyzed growth and thermal reaction norms to temperature of strains of two bacterial lineages, Bacillus cereus sensu lato and Bacillus subtilis sensu lato, that evolved in two contrasting environments, a temperate lagoon (T) and a hot spring (H). Our results showed that despite co-occurrence of members of both lineages in the two contrasting environments, norms of reactions to temperature exhibited a similar pattern only within the lineages, suggesting fixed phenotypic plasticity. Additionally, within the B. cereus lineage, strains from the H environment showed only two to three °C more heat tolerance than strains from the T environment. The limited evolutionary changes towards an increase in heat tolerance in bacteria should alert us of the negative impact that climate change can have on all biological cycles in the planet.

Coffee rust is a fungal disease that has affected every coffee-producing region in the world. Given that the effectivity of the protectant and systemic fungicides applied routinely to control the spread of the causative agent of the disease (Hemileia vastatrix) has gradually diminished, besides are harmful to mammals and ecosystems, the objective of this work was to search for a mixture of harmless natural compounds with the potential to be applied in the field. So, a yeast strain producing a battery of long-chain carboxylic acids (CA) with fungicide properties was isolated from soil of coffee crop and identified as Pichia membranifaciens by ITS sequencing. Culture conditions of the yeast were optimized and the CA in the solution were characterized by Gas Chromatography-Mass Spectrometry (GC-MS) as ethyl formate (55.5 g L-1), octadecenoic acid (3.5 g L-1), propionic acid (7.2 g L-1), 3-(octadecanoyl)-propionic acid (7.2 g L-1) and methyl acetate (8.4 g L-1). Randomized field studies were conducted in three different locations in Chiapas, México. Five treatments were tested including three concentrations of the CA solution (389, 584 and 778 ppm) and copper oxychloride (5 000 ppm) as conventional control. The initial coffee rust incidence averages varied between sites: Maravillas (3-9%), Santo Domingo (10-16%) and Búcaro (16-22%). The treatments of CA solution proved to be effective at slowing down the progress of the rust disease even for the sites where initial incidence was high. Likewise, the CA solution reduced the viability of H. vastatrix spores, as assessed by fluorescence microscopy. RESUMO A ferrugem do cafeeiro é uma doença que tem afetado as regiões produtoras de café no mundo. Considerando que os funguicidas protetores e sistemáticos que são aplicados rotinariamente para controlar a difusão do agente causador da doença (Hemileia vastatrix) são nocivos para os humanos, animais e ecossistemas, o objetivo deste trabalho é determinar um componente natural com potencial de uso. Uma cepa de levedura produzindo uma bateria de ácidos carboxílicos com ação funguicida foi isolada do solo e identificada como Pichia membranifaciens por sequenciamento ITS. Foram optimizadas as condições de cultura e os ácidos carboxílicos presentes na semi purificada sobrenadante (solução CA) foram caracterizados por cromatografia de gás espectometia de massa (GC-MS) como formato de etilo (55.5 g L-1), ácido octadecenóico (3.5 g L-1), ácido propiônico (7.2 g L-1), 3-(octadecanoil)-ácido propiônico 7.2 g L-1) e acetato de metilio (8.4 g L-1). Estudos do campo foram conduzidos em três locais na região de Chiapas, Mexico. Cinco tratamentos foram testados incluindo três concentrações da solução CA (389, 584 e 778 ppm) e o funguicida comercial contendo oxicloreto de cobre (500 ppm). As médias iniciais de incidência da ferrugem do cafeeiro variou em função do local: Maravilla (3-9%), Santo domingo (10-16%) e Búcaro (16-22%). A solução CA provou ser efetiva retardando o progresso da doença, mesmo nos locais onde a incidência inicial foi elevada. A solução CA reduziu a viabilidade dos esporos de H. Vastatrix, observado por meio de fluorescência.

Herein we describe the Archaea diversity in a shallow pond in the Cuatro Ciénegas Basin (CCB), Northeast Mexico, with fluctuating hypersaline conditions containing elastic microbial mats that can form small domes where their anoxic inside reminds us of the characteristics of the Archaean Eon, rich in methane and sulfur gases; thus, we named this site the Archaean Domes (AD). These domes only form after heavy rains that are rare in the Chihuahuan desert. CCB is a unique oasis with hundreds of ponds, containing endemic species of animals, plants and highly diverse and unique microbial communities, despite its very biased stoichiometry, due mostly to extreme low phosphorus content (soils, water columns and sediments). This extreme oligotrophy has favored survival of ancestral microorganisms. Whole metagenome sequencing approach was performed for this unusual site in three different seasons to assess the extent of the Archaea biodiversity, with a focus on extremophiles, since members of the Archaea had been underrepresented in different study sites within the oasis. We found a highly diverse Archaea community compassing ∼5% of the metagenomes. The archaeal portion in all three metagenomes maintained its abundance and most of the strains showed to form a resilient core during three seasonal samplings (2016-2017), despite environmental fluctuations. However, relative abundances of all 230 archaeal OTUs (defined using a 97% cutoff) were low enough (

Phosphorus (P) plays a fundamental role in the physiology and biochemistry of all living things. Recent evidence indicates that organisms in the oceans can break down and use P forms in different oxidation states (e.g., +5, +3, +1, and −3); however, information is lacking for organisms from soil and sediment. The Cuatro Ciénegas Basin (CCB), Mexico, is an oligotrophic ecosystem with acute P limitation, providing a great opportunity to assess the various strategies that bacteria from soil and sediment use to obtain P. We measured the activities in sediment and soil of different exoenzymes involved in P recycling and evaluated 1,163 bacterial isolates (mainly Bacillus spp.) for their ability to use six different P substrates. DNA turned out to be a preferred substrate, comparable to a more bioavailable P source, potassium phosphate. Phosphodiesterase activity, required for DNA degradation, was observed consistently in the sampled-soil and sediment communities. A capability to use phosphite (PO 3 3− ) and calcium phosphate was observed mainly in sediment isolates. Phosphonates were used at a lower frequency by both soil and sediment isolates, and phosphonatase activity was detected only in soil communities. Our results revealed that soil and sediment bacteria are able to break down and use P forms in different oxidation states and contribute to ecosystem P cycling. Different strategies for P utilization were distributed between and within the different taxonomic lineages analyzed, suggesting a dynamic movement of P utilization traits among bacteria in microbial communities. IMPORTANCE Phosphorus (P) is an essential element for life found in molecules, such as DNA, cell walls, and in molecules for energy transfer, such as ATP. The Valley of Cuatro Ciénegas, Coahuila (Mexico), is a unique desert characterized by an extreme limitation of P and a great diversity of microbial life. How do bacteria in this valley manage to obtain P? We measured the availability of P and the enzymatic activity associated with P release in soil and sediment. Our results revealed that soil and sediment bacteria can break down and use P forms in different oxidation states and contribute to ecosystem P cycling. Even genetically related bacterial isolates exhibited different preferences for molecules, such as DNA, calcium phosphate, phosphite, and phosphonates, as substrates to obtain P, evidencing a distribution of roles for P utilization and suggesting a dynamic movement of P utilization traits among bacteria in microbial communities.

The associated article was published under the title Genome Announcements (ISSN: 2169-8287). In July 2018 , Genome Announcements was renamed Microbiology Resource Announcements. Volume 6, no. 17, e00364-18, 2018, [https://doi.org/10.1128/genomeA.00364-18][1]. Page 1: The author affiliations should

We assembled the complete genome sequences of Bacillus pumilus strains 145 and 150a from Cuatrociénegas, Mexico. We detected genes codifying for proteins potentially involved in antagonism (bacteriocins) and defense mechanisms (abortive infection bacteriophage proteins and 4-azaleucine resistance). Both strains harbored prophage sequences. Our results provide insights into understanding the establishment of microbial interactions.

To understand microbial life and get the full picture, we ought to zoom out in order to grasp its magnitud and diversity and zoom in to dissect their genes and functions. We have obtained a significant amount of data on the great diversity of bacteria in the different environments of the Cuatro Cienegas Basin (CCB), equivalent to obtaining the census of a city. However, what are these bacteria doing and what is their role? How do they get along? We have chosen the genus Bacillus to answer these questions. Bacillus can form highly resistant spores that disperse readily and can remain dormant for years, which complicates their study. On the other hand, bacteria of the Bacillus genus are abundant, diverse, and easily cultivated, so they are a good model, both to understand their biology and to study their interactions in communities. With 2500 isolates from CCB and data obtained from microbiological and genomic approaches, we can now write Bacillus tale in Cuatrocienegas.

In this chapter, we describe some genomic studies that have been conducted in the CCB to describe the microdiversity of different microbial taxa. Firstly, we describe the genomic variation in the endemic species B. coahuilensis, for which we analyzed three strains, and the evolutionary mechanism that have shaped their genomes, in particular for their adaptations to a low phosphorus environment. Secondly, we describe population genomics of Proteobacteria such as Pseudomonas, Vibrio, Photobacterium, and Aeromonas, finding many genes related to bacterial interactions, such as type III and VI secretion systems, which led us to propose that these systems play an important role in interactions among bacterial communities. Many of these interaction genes have been classified previously as virulence factors. However, at CCB these genes were not found in a cassette configuration—such as an integron or a pathogenicity island—in any strain suggesting that they are very ancestral adaptive responses to keep both the cohesion within the community and the cheaters out of the community. Interestingly, even if B. coahuilensis presents many mobile elements, these elements are rare in the surveyed Proteobacteria, whose genomes present many CRISPS elements. The presence of CRISPR regions may also explain why there are few ISs or plasmids since it has been reported that CRISPR spacers can act as barriers against HGT.

Given the fragility of the Cuatro Cienegas Basin and the danger of its loss, we started to study the variables affecting the resilience of the microbial community using different experimental approaches. How do microbial communities react to different kinds of perturbations and global change scenarios? We analyzed a series of experimental models that represent different ecosystem compartments: bulk soil, soil crusts, water, and sediment. The experiments were performed in mesocosm or microcosm model systems, which we call in general “cosm” experiments. Different questions were addressed. How does water availability affect the recovery of microbial communities in disturbed soil patches? How do changes in temperature affect microbial crusts? How do bacterioplankton and bacterial mat communities respond to changes in temperature and UV radiation? What would happen to an oligotrophic environment if there was a high nutrient input? Our results suggested perturbations that influenced community structure and community cohesion were stronger in less fluctuating environments. Although it had been suggested that there was a functional (ecological) equivalence between microbial communities, our results on N2-fixing microorganisms of two arid ecosystems showed functional differences, even though similar species occur in both systems. Currently, new experiments are being carried out in “cosms” with replicates of the sediment and water interphase. Sadly, in one of the sites we have studied better, the Churince ecosystem, its conditions were dire the last time that sediment and water were sampled there. This ecosystem has now nearly disappeared. Nevertheless, the sediment in these fish tanks seems to be recovering its original structure. While seeming like a white rhino in a zoo, it might be the only ecosystem of this kind left to learn about what we lost.

Wendell Minckley had just graduated from college in 1957, and his love was aquatic environments and wildlife management. After his first fortuitous trip to Cuatro Cienegas Basin (CCB) in 1958, his life changed and devoted his career to understand this amazing oasis. In 1997 Minckley, by then a well-established professor at Arizona State University, brought along a new colleague, Jim Elser, a limnologist interested in the basic stoichiometric composition of life. Jim was fascinated by the abundance of stromatolites and microbial mats as the base of the aquatic food web, just as in the early Cambrian sea. What made CCB so special was its extremely low phosphorus and the unbalanced stoichiometry; those were the ancient conditions of the sea in the Precambrian. Jim got the Astrobiology Institute of NASA interested, but they needed Mexican scientists involved as well as experts in microbiology and evolution. Valeria Souza and Luis Eguiarte visited the CCB in 1999, and this chapter is about the fascination that they felt at the mysteries behind the enormous microbial biodiversity in the blue-eye ponds (locally called pozas). However, this lost world that had survived eons did not give its secrets lightly, and the hardships to study it are many, so they needed more and more colleagues to join into the task. In the last decade, the now large scientific team has been trying to understand why there are so many species in a place without food, and trying to describe as much as possible and as fast as possible. They all feel this sense of urgency since they are working against the implacable clock of ecosystem degradation. Paradise was disappearing fast given the unsustainable use of water from the deep aquifer to irrigate thirsty alfalfa cultivars in the desert. The decision to save CCB and its amazing biota required to transform a sleepy rural society by translating science to the public and more importantly to give scientific tools to the kids of the owners of the land. Bright-eyed children became conscious about the ecosystem through art, and teenagers are taking actions by discovering that they are the stewards of the biodiversity they are describing in the lab of their high school using molecular tools. There is a revolution occurring in this oasis: science is the tool; kids are the drivers.

We sequenced the Bacillus horikoshii 20a genome, isolated from sediment collected in Cuatro Cienegas, Mexico. We identified genes involved in establishing antagonistic interactions in microbial communities (antibiotic resistance and bacteriocins) and genes related to the metabolism of cyanophycin, a reserve compound and spore matrix material potentially relevant for survival in an oligotrophic environment.

The draft genome sequence of Avibacterium paragallinarum strain CL serovar C is reported here. The genome comprises 154 contigs corresponding to 2.4 Mb with 41% G+C content and many insertion sequence (IS) elements, a characteristic not previously reported in A. paragallinarum .

Understanding the relationship between phylogeny and predicted traits is important to uncover the dimension of the predictive power of a microbial composition approach. Numerous works have addressed the taxonomic composition of bacteria in communities, but little is known about trait heterogeneity in closely related bacteria that co-occur in communities. We evaluated a sample of 467 isolates from the Churince water system of the Cuatro Cienegas Basin (CCB), enriched for Bacillus spp. The 16S rRNA gene revealed a random distribution of taxonomic groups within this genus among 11 sampling sites. A subsample of 141 Bacillus spp. isolates from sediment, with seven well-represented species was chosen to evaluate the heterogeneity and the phylogenetic signal of phenotypic traits that are known to diverge within small clades, such as substrate utilization, and traits that are conserved deep in the lineage, such as prototrophy, swarming and biofilm formation. We were especially interested in evaluating social traits, such as swarming and biofilm formation, for which cooperation is needed to accomplish a multicellular behavior and for which there is little information from natural communities. The phylogenetic distribution of traits, evaluated by the Purvis and Fritz's D statistics approached a Brownian model of evolution. Analysis of the phylogenetic relatedness of the clusters of members sharing the trait using consenTRAIT algorithm, revealed more clustering and deeper phylogenetic signal for prototrophy, biofilm and swimming compared to the data obtained for substrate utilization. The explanation to the observed Brownian evolution of social traits could be either loss due to complete dispensability or to compensated trait loss due to the availability of public goods. Since many of the evaluated traits can be considered to be collective action traits, such as swarming, motility and biofilm formation, the observed microdiversity within taxonomic groups might be explained by distributed functions in structured communities.

Members of the Bacillus genus have been extensively studied because of their ability to produce enzymes with high biotechnological value. Here, we report the draft of the whole-genome sequence of the type strain Bacillus horikoshii DSM 8719, an alkali-tolerant strain.

Bacillus aquimaris TF12 is a Gram-positive bacteria isolated from a tidal flat of the Yellow Sea in South Korea. We report the draft whole-genome sequence of Bacillus aquimaris TF12, the type strain of a set of bacteria typically associated with marine habitats and with a potentially high biotechnology value.

Understanding the relationship between phylogeny and predicted traits is important to uncover the dimension of the predictive power of a microbial composition approach. Numerous works have addressed the taxonomic composition of bacteria in communities, but little is known about trait heterogeneity in closely related bacteria that co-occur in communities. We evaluated a sample of 467 isolates from the Churince water system of the Cuatro Cienegas Basin (CCB), enriched for Bacillus spp. The 16S rRNA gene revealed a random distribution of taxonomic groups within this genus among 11 sampling sites. A subsample of 141 Bacillus spp. isolates from sediment, with seven well-represented species was chosen to evaluate the heterogeneity and the phylogenetic signal of phenotypic traits that are known to diverge within small clades, such as substrate utilization, and traits that are conserved deep in the lineage, such as prototrophy, swarming and biofilm formation. We were especially interested in evaluating social traits, such as swarming and biofilm formation, for which cooperation is needed to accomplish a multicellular behavior and for which there is little information from natural communities. The phylogenetic distribution of traits, evaluated by the Purvis and Fritz’s D statistics approached a Brownian model of evolution. Analysis of the phylogenetic relatedness of the clusters of members sharing the trait using consenTRAIT algorithm, revealed more clustering and deeper phylogenetic signal for prototrophy, biofilm and swimming compared to the data obtained for substrate utilization. The explanation to the observed Brownian evolution of social traits could be either loss due to complete dispensability or to compensated trait loss due to the availability of public goods. Since many of the evaluated traits can be considered to be collective action traits, such as swarming, motility and biofilm formation, the observed microdiversity within taxonomic groups might be explained by distributed functions in structured communities.

Bacterial genomes undergo numerous events of gene losses and gains that generate genome variability among strains of the same species (microevolution). Our aim was to compare the genomes and relevant phenotypes of three Bacillus coahuilensis strains from two oligotrophic hydrological systems in the Cuatro Cienegas Basin (Mexico), to unveil the environmental challenges that this species cope with, and the microevolutionary differences in these genotypes. Since the strains were isolated from a low P environment, we placed emphasis on the search of different phosphorus acquisition strategies. The three B. coahuilensis strains exhibited similar numbers of coding DNA sequences, of which 82% (2, 893) constituted the core genome, and 18% corresponded to accessory genes. Most of the genes in this last group were associated with mobile genetic elements or were annotated as hypothetical proteins. Ten percent of the pangenome consisted of strain-specific genes. Alignment of the three B. coahuilensis genomes indicated a high level of synteny and revealed the presence of several genomic islands. Unexpectedly, one of these islands contained genes that encode the 2-keto-3-deoxymannooctulosonic acid (Kdo) biosynthesis enzymes, a feature associated to cell walls of Gram-negative bacteria. Some microevolutionary changes were clearly associated with mobile genetic elements. Our analysis revealed inconsistencies between phenotype and genotype, which we suggest result from the impossibility to map regulatory features to genome analysis. Experimental results revealed variability in the types and numbers of auxotrophies between the strains that could not consistently be explained by in silico metabolic models. Several intraspecific differences in preferences for carbohydrate and phosphorus utilization were observed. Regarding phosphorus recycling, scavenging, and storage, variations were found between the three genomes. The three strains exhibited differences regarding alkaline phosphatase suggesting that in addition to gene gain and loss; regulation adjustment of gene expression also has contributed to the intraspecific diversity of B. coahuilensis.

Understanding the principles that govern community assemblages is a central goal of ecology. There is limited experimental evidence in natural settings showing that microbial assembly in communities are influenced by antagonistic interactions. We, therefore, analyzed antagonism among bacterial isolates from a taxonomically related bacterial guild obtained from five sites in sediments from a fresh water system. We hypothesized that if antagonistic interactions acted as a shaping force of the community assembly, then the frequency of resistance to antagonism among bacterial isolates originating from a given site would be higher than the resistance to conspecifics originating from a different assemblage. Antagonism assays were conducted between 78 thermoresistant isolates, of which 72 were Bacillus spp. Sensitive, resistant and antagonistic isolates co-occurred at each site, but the within-site frequency of resistance observed was higher than that observed when assessed across-sites. We found that antagonism results from bacteriocin-like substances aimed at the exclusion of conspecifics. More than 6000 interactions were scored and described by a directed network with hierarchical structure that exhibited properties that resembled a food chain, where the different Bacillus taxonomic groups occupied specific positions. For some tested interacting pairs, the unidirectional interaction could be explained by competition that inhibited growth or completely excluded one of the pair members. This is the first report on the prevalence and specificity of Bacillus interactions in a natural setting and provides evidence for the influence of bacterial antagonist interactions in the assemblage of a taxonomically related guild in local communities.

The resiliency of prokaryotic life has provided colonization across the globe and in the recesses of Earth's most extreme environments. Horizontal gene transfer provides access to a global bank of genetic resources that creates diversity and allows real-time adaptive potential to the clonal prokaryotic world. We assess the likelihood that this Earth-based strategy could provide survival and adaptive potential, in the case of microbial stowaways off Earth. Key Words: Bacillus—Horizontal gene transfer—Bacteria—Earth Mars—Evolution. Astrobiology 12, 710–715.

DEAD-box proteins are found in all domains of life and participate in almost all cellular processes that involve RNA. The presence of DEAD and Helicase_C conserved domains distinguish these proteins. DEAD-box proteins exhibit RNA-dependent ATPase activity in vitro, and several also show RNA helicase activity. In this study, we analyzed the distribution and architecture of DEAD-box proteins among bacterial genomes to gain insight into the evolutionary pathways that have shaped their history. We identified 1,848 unique DEAD-box proteins from 563 bacterial genomes. Bacterial genomes can possess a single copy DEAD-box gene, or up to 12 copies of the gene, such as in Shewanella. The alignment of 1,208 sequences allowed us to perform a robust analysis of the hallmark motifs of DEAD-box proteins and determine the residues that occur at high frequency, some of which were previously overlooked. Bacterial DEAD-box proteins do not generally contain a conserved C-terminal domain, with the exception of some members that possess a DbpA RNA-binding domain (RBD). Phylogenetic analysis showed a separation of DbpA-RBD-containing and DbpA-RBD-lacking sequences and revealed a group of DEAD-box protein genes that expanded mainly in the Proteobacteria. Analysis of DEAD-box proteins from Firmicutes and γ-Proteobacteria, was used to deduce orthologous relationships of the well-studied DEAD-box proteins from Escherichia coli and Bacillus subtilis. These analyses suggest that DbpA-RBD is an ancestral domain that most likely emerged as a specialized domain of the RNA-dependent ATPases. Moreover, these data revealed numerous events of gene family expansion and reduction following speciation. Electronic supplementary material The online version of this article (doi:10.1007/s00239-011-9441-8) contains supplementary material, which is available to authorized users.

We generated a conditional CCase mutant ofBacillus subtilisto explore the participationin vivoof the tRNA nucleotidyltransferase (CCA transferase or CCase) in the maturation of the single-copy tRNACys, which lacks an encoded CCA 3′ end. We observed that shorter tRNACysspecies, presumably lacking CCA, only accumulated when the inducible Pspac : ccawas introduced into anrnrmutant strain, but not in combination withpnp. We sequenced the tRNA 3′ ends produced in the various mutant tRNACysspecies to detect maturation and decay intermediates and observed that decay of the tRNACysoccurs through the addition of poly(A) or heteropolymeric tails. A few clones corresponding to full-size tRNAs contained either CCA or other C and/or A sequences, suggesting that these are substrates for repair and/or decay. We also observed editing of tRNACysat position 21, which seems to occur preferentially in mature tRNAs. Altogether, our results providein vivoevidence for the participation of theB. subtilis ccagene product in the maturation of tRNAs lacking CCA. We also suggest that RNase R exoRNase inB. subtilisparticipates in the quality control of tRNA.

The ribosomal RNA (rrn) operon is a key suite of genes related to the production of protein synthesis machinery and thus to bacterial growth physiology. Experimental evidence has suggested an intrinsic relationship between the number of copies of this operon and environmental resource availability, especially the availability of phosphorus (P), because bacteria that live in oligotrophic ecosystems usually have few rrn operons and a slow growth rate. The Cuatro Ciénegas Basin (CCB) is a complex aquatic ecosystem that contains an unusually high microbial diversity that is able to persist under highly oligotrophic conditions. These environmental conditions impose a variety of strong selective pressures that shape the genome dynamics of their inhabitants. The genus Bacillus is one of the most abundant cultivable bacterial groups in the CCB and usually possesses a relatively large number of rrn operon copies (6-15 copies). The main goal of this study was to analyze the variation in the number of rrn operon copies of Bacillus in the CCB and to assess their growth-related properties as well as their stoichiometric balance (N and P content). We defined 18 phylogenetic groups within the Bacilli clade and documented a range of from six to 14 copies of the rrn operon. The growth dynamic of these Bacilli was heterogeneous and did not show a direct relation to the number of operon copies. Physiologically, our results were not consistent with the Growth Rate Hypothesis, since the copies of the rrn operon were decoupled from growth rate. However, we speculate that the diversity of the growth properties of these Bacilli as well as the low P content of their cells in an ample range of rrn copy number is an adaptive response to oligotrophy of the CCB and could represent an ecological mechanism that allows these taxa to coexist. These findings increase the knowledge of the variability in the number of copies of the rrn operon in the genus Bacillus and give insights about the physiology of this bacterial group under extreme oligotrophic conditions.

Most of the studies in Ecology have been devoted to analyzing the effects the environment has on individuals, populations, and communities, thus neglecting the effects of biotic interactions on the system dynamics. In the present work we study the structure of bacterial communities in the oligotrophic shallow water system of Churince, Cuatro Cienegas, Mexico. Since the physicochemical conditions of this water system are homogeneous and quite stable in time, it is an excellent candidate to study how biotic factors influence the structure of bacterial communities. In a previous study, the binary antagonistic interactions of 78 bacterial strains, isolated from Churince, were experimentally determined. We employ these data to develop a computer algorithm to simulate growth experiments in a cellular grid representing the pond. Remarkably, in our model, the dynamics of all the simulated bacterial populations is determined solely by antagonistic interactions. Our results indicate that all bacterial strains (even those that are antagonized by many other bacteria) survive in the long term, and that the underlying mechanism is the formation of bacterial community patches. Patches corresponding to less antagonistic and highly susceptible strains are consistently isolated from the highly-antagonistic bacterial colonies by patches of neutral strains. These results concur with the observed features of the bacterial community structure previously reported. Finally, we study how our findings depend on factors like initial population size, differential population growth rates, homogeneous population death rates, and enhanced bacterial diffusion.

Although there are numerous bacteria of the genus Bacillus of great importance for biological control, little is known about their ecology in the soil. We wanted to test illegitimate recombination as a tagging system that would allow us to study selected or genetically engineered Bacillus soil isolates. Strains carrying the plasmid integrated into the chromosome were obtained by growing at a non-permissive temperature after transformation with a plasmid carrying a thermo-sensitive replication origin with selection for erythromycin. A laboratory strain, a commercial strain (Kodiak), and four other soil isolates were generated through this procedure and analysed. In all of these strains the integrated plasmid was maintained in multicopy. The erythromycin resistance gene (ermB) placed on the plasmid was used as a target for polymerase chain reaction (PCR). The tagged strains could be then detected when inoculated into microcosms prepared with non-sterile soil.

DNA extraction techniques for endospore-forming bacteria in soil are often labour-intensive and unreliable. Our objective in this work was to investigate whether good quality DNA could be obtained from spores germinated in soil. To this end, endospores from Bacillus subtilis, B. megaterium and B. thuringiensis were inoculated into soil microcosms and germination was induced by addition of LB medium supplemented with l-alanine, glucose, fructose and KCl. Heat resistance count was reduced to 80% for B. subtilis and more than 90% for B. thuringiensis and B. megaterium after a few minutes. Isolation of DNA from soil with a procedure which did not work on spores was shown to be as efficient for in situ-germinated spores as for inoculated vegetative cells. Furthermore, we developed a simple procedure that allowed us to use the recovered DNA in PCR amplifications. The present methodology is simple and efficient; it avoids the use of special equipment and harsh spore rupturing methods and can be carried out with multiple samples.

The stability of integration and amplification of an integrational plasmid in Bacillus subtilis was analyzed. A cat-containing plasmid was constructed that could be integrated into the amy locus to facilitate measurement of excision events. Pulse-field gel electrophoresis was used to measure the copy number in strains that were resistant to different levels of chloramphenicol. The stability of the amplified unit in strains containing from 2 to 18 tandem copies of the amplicon in the presence and absence of chloramphenicol and through different generation times was then determined. Our results demonstrate that, for any given strain, the copy number of the amplicon remains stable. Furthermore, this stability is maintained when a clone containing an amplicon of defined size is cultured through as many as 100 generations in the absence of selective pressure.

The Cuatro Ciénegas Basin (CCB) has been identified as a center of endemism for many life-forms. Nearly half the bacterial species found in the spring systems have their closest relatives in the ocean. This raises the question of whether the high diversity observed today is the product of an adaptive radiation similar to that of the Galapagos Islands or whether the bacterial groups are "survivors" of an ancient sea, which would be of interest for astrobiology. To help answer this question, we focused on Firmicutes from Cuatro Ciénegas (mainly Bacillus and Exiguobacterium). We reconstructed the phylogenetic relationships of Firmicutes with 28 housekeeping genes and dated the resulting tree using geological events as calibration points. Our results show that marine Bacillus diverged from other Bacillus strains 838 Ma, while Bacillus from Cuatro Ciénegas have divergence dates that range from 770 to 202 Ma. The members of Exiguobacterium from the CCB conform to a much younger group that diverged from the Andes strain 60 Ma and from the one in Yellowstone 183 Ma. Therefore, the diversity of Firmicutes in Cuatro Ciénegas is not the product of a recent radiation but the product of the isolation of lineages from an ancient ocean. Hence, Cuatro Ciénegas is not a Galapagos Archipelago for bacteria but is more like an astrobiological "time machine" in which bacterial lineages survived in an oligotrophic environment that may be very similar to that of the Precambrian. Key Words: Firmicutes-Cuatro Ciénegas-Precambrian-Molecular dating-Western Interior Seaway.

Microbial mats are self-sustained, functionally complex ecosystems that make good models for the understanding of past and present microbial ecosystems as well as putative extraterrestrial ecosystems. Ecological theory suggests that the composition of these communities might be affected by nutrient availability and disturbance frequency. We characterized two microbial mats from two contrasting environments in the oligotrophic Cuatro Ciénegas Basin: a permanent green pool and a red desiccation pond. We analyzed their taxonomic structure and composition by means of 16S rRNA clone libraries and metagenomics and inferred their metabolic role by the analysis of functional traits in the most abundant organisms. Both mats showed a high diversity with metabolically diverse members and strongly differed in structure and composition. The green mat had a higher species richness and evenness than the red mat, which was dominated by a lineage of Pseudomonas. Autotrophs were abundant in the green mat, and heterotrophs were abundant in the red mat. When comparing with other mats and stromatolites, we found that taxonomic composition was not shared at species level but at order level, which suggests environmental filtering for phylogenetically conserved functional traits with random selection of particular organisms. The highest diversity and composition similarity was observed among systems from stable environments, which suggests that disturbance regimes might affect diversity more strongly than nutrient availability, since oligotrophy does not appear to prevent the establishment of complex and diverse microbial mat communities. These results are discussed in light of the search for extraterrestrial life. Key Words: Cuatro Ciénegas—Metagenomics—Microbial mats—Oligotrophic—Phosphorus limitation—Stromatolites. Astrobiology 12, 659–673.

The Cuatro Ciénegas Basin (CCB) is an oasis in the desert of Mexico characterized by low phosphorus availability and by its great diversity of microbial mats. We compared the metagenomes of two aquatic microbial mats from the CCB with different nutrient limitations. We observed that the red mat was P-limited and dominated by Pseudomonas, while the green mat was N-limited and had higher species richness, with Proteobacteria and Cyanobacteria as the most abundant phyla. From their gene content, we deduced that both mats were very metabolically diverse despite their use of different strategies to cope with their respective environments. The red mat was found to be mostly heterotrophic, while the green mat was more autotrophic. The red mat had a higher number of transporters in general, including transporters of cellobiose and osmoprotectants. We suggest that generalists with plastic genomes dominate the red mat, while specialists with minimal genomes dominate the green mat. Nutrient limitation was a common scenario on the early planet; despite this, biogeochemical cycles were performed, and as a result the planet changed. The metagenomes of microbial mats from the CCB show the different strategies a community can use to cope with oligotrophy and persist. Key Words: Microbial mats—Metagenomics—Metabolism. Astrobiology 12, 648–658.

Luis David Alcaraz1, Varinia Lopez-Ramirez2, Alejandra Moreno-Letelier3, Luis Herrera-Estrella4, Valeria Souza5 and Gabriela Olmedo-Alvarez2 1Department of Genomics and Health, Center for Advanced Research in Public Health, Valencia, 2Departamento de Ingenieria Genetica, Cinvestav Unidad Irapuato, 3Division of Biology, Imperial College London, Silwood Park Campus, Ascot, 4Langebio, Cinvestav, Mexico 5Departamento de Ecologia Evolutiva, Instituto de Ecologia, Universidad Nacional Autonoma de Mexico 1Spain 2,4.5Mexico 3UK

Polyadenylation plays a role in decay of some bacterial mRNAs, as well as in the quality control of stable RNA. In Escherichia coli , poly(A) polymerase I (PAP I) is the main polyadenylating enzyme, but the addition of 3′ tails also occurs in the absence of PAP I via the synthetic activity of polynucleotide phosphorylase (PNPase). The nature of 3′-tail addition in Bacillus subtilis , which lacks an identifiable PAP I homologue, was studied. Sizing of poly(A) sequences revealed a similar pattern in wild-type and PNPase-deficient strains. Sequencing of 152 cloned cDNAs, representing 3′-end sequences of nontranslated and translated RNAs, revealed modified ends mostly on incomplete transcripts, which are likely to be decay intermediates. The 3′-end additions consisted of either short poly(A) sequences or longer heteropolymeric ends with a mean size of about 40 nucleotides. Interestingly, multiple independent clones exhibited complex heteropolymeric ends of very similar but not identical nucleotide sequences. Similar polyadenylated and heteropolymeric ends were observed at 3′ ends of RNA isolated from wild-type and pnpA mutant strains. These data demonstrated that, unlike the case of some other bacterial species and chloroplasts, PNPase of Bacillus subtilis is not the major enzyme responsible for the addition of nucleotides to RNA 3′ ends.

We analyzed the influence of the 5' and 3' untranslated regions of the Bacillus thuringiensis cry1Aa on its mRNA stability. Although the cry1Aa gene has a stable transcript (8 min), its 5' UTR did not provide stability to the reporter gene uidA. Stability of cry1Aa could be increased to 40 min by addition of an SP82 stability element at the 5' UTR, suggesting that once the 5' and 3' ends were protected initiation of decay could be effectively blocked. We generated mutations in the transcription terminator and found that changes that reduced the stability of the stem, a larger loop, or elimination of the U-trail sharply decreased the half-life of the transcript. Therefore, unlike some stable bacterial transcripts, cry1Aa lacks special features at the end 5' to prevent decay, but its terminator is the main determinant of its stability.

We undertook the study of the decay process of the cry1Aa mRNA of Bacillus thuringiensis expressed in B. subtilis. The cry1Aa transcript is a 3.7-kb mRNA expressed during sporulation whose transcriptional control has previously been studied in both B. subtilis and B. thuringiensis. We found that the cry1Aa mRNA has a half-life of around 9 min and that its decay occurs through endoribonucleolytic cleavages which result in three groups of high-molecular-weight mRNA intermediates ranging in size from 2.7 to 0.5 kb. A comparative study carried out with Escherichia coli showed a similar pattern of degradation intermediates. Primer extension analysis carried out on RNA from B. subtilis revealed that most cleavages occur within two regions located toward the 5' and 3' ends of the mRNA. The most prominent processing site observed for the cry1Aa mRNA isolated from B. subtilis is only two bases away from that occurring on RNA isolated from E. coli. Most cleavage sites occur at seemingly single-stranded RNA segments rich in A and U nucleotides, suggesting that a common and conserved mechanism may process the cry1Aa mRNA.