The isolation and characterization of a Rhizobium etli glutamate auxotroph, TAD12, harbouring a single Tn5 insertion, is reported. This mutant produced no detectable glutamate synthase (GOGAT) activity. The cloning and physical characterization of a 72 kb fragment of R. etli DNA harbouring the structural genes gltB and gltD encoding the two GOGAT subunits GltB and GltD is also reported. In comparison with the wild-type strain (CFN42), the GOGAT mutant strain utilized less succinate and glutamate and grew less with this and other amino acids as nitrogen source. R. etli assimilates ammonium by the glutamine synthetase (GS)-GOGAT pathway and a GOGAT mutant prevents the cycling of glutamine by this pathway, something that impairs nitrogen and carbon metabolism and explains the decrease in the amino-nitrogen during exponential growth, with glutamate as nitrogen source. GOGAT activity also has a role in ammonium turnover and in the synthesis of amino acids and proteins, processes that are necessary to sustain cell viability in non-growing conditions. The assimilation of ammonium is important during symbiosis and glutamate constitutes 20-40 % of the total amino-nitrogen. In symbiosis, the blockage of ammonium assimilation by a GOGAT mutation significantly decreases the amino-nitrogen pool of the bacteroids and may explain why more N 2 is fixed in ammonium, excreted to the plant cell, transported to the leaves and stored in the seeds.

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The Naica Underground System (NUS) in Northern Mexico comprises a lead, zinc, and silver producing mine and displays the largest gypsum crystals ever found in natural caves. The caves are now closed to the public and mining activities have been suspended for an undefined period since October 2015. Besides its geological, economical, and tourist importance, the bacterial diversity in the NUS has not been fully explored yet. This study surveyed for bacteria present on different mineral substrates (gypsum crystals, iron oxide crusts) and hot spring samples collected before the NUS was inaccessible, using culture-dependent and culture-independent (PCR-DGGE) methods. This study is the first reporting the isolation of microorganisms from Naica. Cluster analysis of DGGE fingerprints revealed slight differences between communities from caves and tunnels and according to their mineral substrate type while communities from solid substrates and water samples appeared to be more distant. Both approaches, culture-dependent and independent, revealed the presence of bacteria from the Firmicutes, Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria in gypsum crystals, iron oxide crusts, and hot springs, respectively. Deinococcus-Thermus and Actinobacteria were only detected by DGGE in hot spring samples. According to 16S rRNA sequencing, heterotrophic bacteria isolated under aerobic conditions were affiliated with Bacillus, Brevibacillus, Paenibacillus, Schlegelella, Cupriavidus, Pseudoxanthomonas, and Lysobacter. Most of the isolates and sequences retrieved by DGGE were related to organisms previously detected in other extreme subsurface environments. Some of the isolates were able to precipitate calcium carbonate and precipitate Fe(III) in solid media but their possible participation in biomineralization processes in situ has still to be investigated. Microbial communities found in the NUS are likely autochthonous with some allochtonous components due to human intervention. Their role in geobiological processes requires further investigation. gypsum crystals, iron oxide crusts, hot springs, 16S rRNA, bacterial isolates, Naica

Abstract:Las Karmidas Cave (Puebla State, Mexico) is an unusual type of pseudokarstic cavity generated by piping and erosive processes within the contact of a diamicton and an overlying Quaternary ignimbrite. Morphological evidence suggests that the cave was developed in two stages: a phreatic stage and a vadose stage. The latter was characterized by the formation of carbonate speleothems. The absolute upper-age limit for the cave (168 +7.1/-7.5 ka) was established by U-Th dating of zircons grains extracted from the overlying ignimbrite, whilst a minimum age for the transition from a phreatic to vadose regime (95.6 ± 2.1 ka) was constrained by U-Th dating of carbonate speleothems within the cave. The geochronological results indicate a very rapid evolution of this pseudokarstic system, and suggest that similar systems might evolve and degrade at a very fast pace; consequently, making them hard to be preserved. Despite this, and considering the rather common geological context in which this system was developed, it is likely that similar pseudokarstic systems are yet to be detected worldwide.pseudokarst, cave, speleogenesis, Xaltipan ignimbrite, Las Karmidas Cave