A Tar aspartate receptor and Rubisco-like protein substitute biotin in the growth of rhizobial strains.

Biotin is a key cofactor of metabolic carboxylases, although many rhizobial strains are biotin auxotrophs. When some of these strains were serially subcultured in minimal medium, they showed diminished growth and increased excretion of metabolites. The addition of biotin, or genetic complementation with biotin synthesis genes resulted in full growth of Rhizobium etli CFN42 and Rhizobium phaseoli CIAT652 strains. Half of rhizobial genomes did not show genes for biotin biosynthesis, but three-quarters had genes for biotin transport. Some strains had genes for an avidin homologue (rhizavidin), a protein with high affinity for biotin but an unknown role in bacteria. A CFN42-derived rhizavidin mutant showed a sharper growth decrease in subcultures, revealing a role in biotin storage. In the search of biotin-independent growth of subcultures, CFN42 and CIAT652 strains with excess aeration showed optimal growth, as they also did, unexpectedly, with the addition of aspartic acid analogues α- and N-methyl aspartate. Aspartate analogues can be sensed by the chemotaxis aspartate receptor Tar. A tar homologue was identified and its mutants showed no growth recovery with aspartate analogues, indicating requirement of the Tar receptor in such a phenotype. Additionally, tar mutants did not recover full growth with excess aeration. A Rubisco-like protein was found to be necessary for growth as the corresponding mutants showed no recovery either with high aeration or aspartate analogues; also, diminished carboxylation was observed. Taken together, our results indicate a route of biotin-independent growth in rhizobial strains that included oxygen, a Tar receptor and a previously uncharacterized Rubisco-like protein.

Nitrogen Fixation in Pozol, a Traditional Fermented Beverage.

Traditional fermentations have been widely studied from the microbiological point of view, but little is known from the functional perspective. In this work, nitrogen fixation by free-living nitrogen-fixing bacteria was conclusively demonstrated in pozol, a traditional Mayan beverage prepared with nixtamalized and fermented maize dough. Three aspects of nitrogen fixation were investigated to ensure that fixation actually happens in the dough: (i) the detection of acetylene reduction activity directly in the substrate, (ii) the presence of potential diazotrophs, and (iii) an in situ increase in acetylene reduction by inoculation with one of the microorganisms isolated from the dough. Three genera were identified by sequencing the 16S rRNA and nifH genes as Kosakonia, Klebsiella, and Enterobacter, and their ability to fix nitrogen was confirmed.IMPORTANCE Nitrogen-fixing bacteria are found in different niches, as symbionts in plants, in the intestinal microbiome of several insects, and as free-living microorganisms. Their use in agriculture for plant growth promotion via biological nitrogen fixation has been extensively reported. This work demonstrates the ecological and functional importance that these bacteria can have in food fermentations, reevaluating the presence of these genera as an element that enriches the nutritional value of the dough.

Proteins in the periplasmic space and outer membrane vesicles of Rhizobium etli CE3 grown in minimal medium are largely distinct and change with growth phase.

Rhizobium etli CE3 grown in succinate-ammonium minimal medium (MM) excreted outer membrane vesicles (OMVs) with diameters of 40 to 100 nm. Proteins from the OMVs and the periplasmic space were isolated from 6 and 24 h cultures and identified by proteome analysis. A total of 770 proteins were identified: 73.8 and 21.3 % of these occurred only in the periplasm and OMVs, respectively, and only 4.9 % were found in both locations. The majority of proteins found in either location were present only at 6 or 24 h: in the periplasm and OMVs, only 24 and 9 % of proteins, respectively, were present at both sampling times, indicating a time-dependent differential sorting of proteins into the two compartments. The OMVs contained proteins with physiologically varied roles, including Rhizobium adhering proteins (Rap), polysaccharidases, polysaccharide export proteins, auto-aggregation and adherence proteins, glycosyl transferases, peptidoglycan binding and cross-linking enzymes, potential cell wall-modifying enzymes, porins, multidrug efflux RND family proteins, ABC transporter proteins and heat shock proteins. As expected, proteins with known periplasmic localizations (phosphatases, phosphodiesterases, pyrophosphatases) were found only in the periplasm, along with numerous proteins involved in amino acid and carbohydrate metabolism and transport. Nearly one-quarter of the proteins present in the OMVs were also found in our previous analysis of the R. etli total exproteome of MM-grown cells, indicating that these nanoparticles are an important mechanism for protein excretion in this species.

Qualitative changes in proteins contained in outer membrane vesicles produced by Rhizobium etli grown in the presence of the nod gene inducer naringenin.

In this work, we compared the proteomic profiles of outer membrane vesicles (OMVs) isolated from Rhizobium etli CE3 grown in minimal medium (MM) with and without exogenous naringenin. One-hundred and seven proteins were present only in OMVs from naringenin-containing cultures (N-OMVs), 57 proteins were unique to OMVs from control cultures lacking naringenin (C-OMVs) and 303 proteins were present in OMVs from both culture conditions (S-OMVs). Although we found no absolute predominance of specific types of proteins in the N-, C- or S-OMV classes, there were categories of proteins that were significantly less or more common in the different OMV categories. Proteins for energy production, translation and membrane and cell wall biogenesis were overrepresented in C-OMVs relative to N-OMVs. Proteins for carbohydrate metabolism and transport and those classified as either general function prediction only, function unknown, or without functional prediction were more common in N-OMVs than C-OMVs. This indicates that naringenin increased the proportion of these proteins in the OMVs, although NodD binding sites were only slightly more common in the promoters of genes for proteins found in the N-OMVs. In addition, OMVs from naringenin-containing cultures contained nodulation factor.

Analysis of differentially upregulated proteins in ptsHIcrr- and rppH- mutants in Escherichia coli during an adaptive laboratory evolution experiment.

The previous deletion of the cytoplasmic components of the phosphotransferase system (PTS) in Escherichia coli JM101 resulted in the PTS- derivative strain PB11 with severely impaired growth capability in glucose as the sole carbon source. Previous adaptive laboratory evolution (ALE) experiment led to select a fast-growing strain named PB12 from PB11. Comparative genome analysis of PB12 showed a chromosomal deletion, which result in the loss of several genes including rppH which codes for the RNA pyrophosphohydrolase RppH, involved in the preparation of hundreds of mRNAs for further degradation by RNase E. Previous inactivation of rppH in PB11 (PB11rppH-) improved significantly its growing capabilities and increased several mRNAs respect its parental strain PB11. These previous results led to propose to the PB11rppH- mutant as an intermediate between PB11 and PB12 strains merged during the early ALE experiment. In this contribution, we report the metabolic response to the PTS- and rppH- mutations in the deep of a proteomic approach to understanding the relevance of rppH- phenotype during an ALE experiment. Differentially upregulated proteins between the wild-type JM101/PB11, PB11/PB11rppH-, and PB11/PB12 comparisons led to identifying 45 proteins between strain comparisons. Downregulated or upregulated proteins in PB11rppH- were found expressed at an intermediate level with respect to PB11 and PB12. Many of these proteins were found involved in non-previously metabolic traits reported in the study of the PTS- strains, including glucose, amino acids, ribose transport; amino acid biosynthesis; NAD biosynthesis/salvage pathway, biosynthesis of Ac-CoA precursors; detoxification and degradation pathways; stress response; protein synthesis; and possible mutator activities between comparisons. No changes were found in the expression of galactose permease GalP, previously proposed as the primary glucose transporter in the absence of PTS selected by the PTS- derivatives during the ALE experiment. This result suggests that the evolving PTS- population selected other transporters such as LamB, MglB, and ManX instead of GalP for glucose uptake during the early ALE experiment. Analysis of the biological relevance of the metabolic traits developed by the studied strains provided valuable information to understand the relevance of the rppH- mutation in the PTS- background during an ALE experiment as a strategy for the selection of valuable phenotypes for metabolic engineering purposes.

The naringenin-induced exoproteome of Rhizobium etli CE3.

Flavonoids excreted by legume roots induce the expression of symbiotically essential nodulation (nod) genes in rhizobia, as well as that of specific protein export systems. In the bean microsymbiont Rhizobium etli CE3, nod genes are induced by the flavonoid naringenin. In this study, we identified 693 proteins in the exoproteome of strain CE3 grown in minimal medium with or without naringenin, with 101 and 100 exoproteins being exclusive to these conditions, respectively. Four hundred ninety-two (71%) of the extracellular proteins were found in both cultures. Of the total exoproteins identified, nearly 35% were also present in the intracellular proteome of R. etli bacteroids, 27% had N-terminal signal sequences and a significant number had previously demonstrated or possible novel roles in symbiosis, including bacterial cell surface modification, adhesins, proteins classified as MAMPs (microbe-associated molecular patterns), such as flagellin and EF-Tu, and several normally cytoplasmic proteins as Ndk and glycolytic enzymes, which are known to have extracellular "moonlighting" roles in bacteria that interact with eukaryotic cells. It is noteworthy that the transmembrane ß (1,2) glucan biosynthesis protein NdvB, an essential symbiotic protein in rhizobia, was found in the R. etli naringenin-induced exoproteome. In addition, potential binding sites for two nod-gene transcriptional regulators (NodD) occurred somewhat more frequently in the promoters of genes encoding naringenin-induced exoproteins in comparison to those ofexoproteins found in the control condition.

Genomic studies of nitrogen-fixing rhizobial strains from Phaseolus vulgaris seeds and nodules.

BACKGROUND: Rhizobia are soil bacteria that establish symbiotic relationships with legumes and fix nitrogen in root nodules. We recently reported that several nitrogen-fixing rhizobial strains, belonging to Rhizobium phaseoli, R. trifolii, R. grahamii and Sinorhizobium americanum, were able to colonize Phaseolus vulgaris (common bean) seeds. To gain further insight into the traits that support this ability, we analyzed the genomic sequences and proteomes of R. phaseoli (CCGM1) and S. americanum (CCGM7) strains from seeds and compared them with those of the closely related strains CIAT652 and CFNEI73, respectively, isolated only from nodules. RESULTS: In a fine structural study of the S. americanum genomes, the chromosomes, megaplasmids and symbiotic plasmids were highly conserved and syntenic, with the exception of the smaller plasmid, which appeared unrelated. The symbiotic tract of CCGM7 appeared more disperse, possibly due to the action of transposases. The chromosomes of seed strains had less transposases and strain-specific genes. The seed strains CCGM1 and CCGM7 shared about half of their genomes with their closest strains (3353 and 3472 orthologs respectively), but a large fraction of the rest also had homology with other rhizobia. They contained 315 and 204 strain-specific genes, respectively, particularly abundant in the functions of transcription, motility, energy generation and cofactor biosynthesis. The proteomes of seed and nodule strains were obtained and showed a particular profile for each of the strains. About 82 % of the proteins in the comparisons appeared similar. Forty of the most abundant proteins in each strain were identified; these proteins in seed strains were involved in stress responses and coenzyme and cofactor biosynthesis and in the nodule strains mainly in central processes. Only 3 % of the abundant proteins had hypothetical functions. CONCLUSIONS: Functions that were enriched in the genomes and proteomes of seed strains possibly participate in the successful occupancy of the new niche. The genome of the strains had features possibly related to their presence in the seeds. This study helps to understand traits of rhizobia involved in seed adaptation.

Transcriptomic analysis of the process of biofilm formation in Rhizobium etli CFN42.

Organisms belonging to the genus Rhizobium colonize leguminous plant roots and establish a mutually beneficial symbiosis. Biofilms are structured ecosystems in which microbes are embedded in a matrix of extracellular polymeric substances, and their development is a multistep process. The biofilm formation processes of R. etli CFN42 were analyzed at an early (24-h incubation) and mature stage (72 h), comparing cells in the biofilm with cells remaining in the planktonic stage. A genome-wide microarray analysis identified 498 differentially regulated genes, implying that expression of ~8.3 % of the total R. etli gene content was altered during biofilm formation. In biofilms-attached cells, genes encoding proteins with diverse functions were overexpressed including genes involved in membrane synthesis, transport and chemotaxis, repression of flagellin synthesis, as well as surface components (particularly exopolysaccharides and lipopolysaccharides), in combination with the presence of activators or stimulators of N-acyl-homoserine lactone synthesis This suggests that R. etli is able to sense surrounding environmental conditions and accordingly regulate the transition from planktonic and biofilm growth. In contrast, planktonic cells differentially expressed genes associated with transport, motility (flagellar and twitching) and inhibition of exopolysaccharide synthesis. To our knowledge, this is the first report of nodulation and nitrogen assimilation-related genes being involved in biofilm formation in R. etli. These results contribute to the understanding of the physiological changes involved in biofilm formation by bacteria.

Proteomic and morphometric study of the in vitro interaction between Oncidium sphacelatum Lindl. (Orchidaceae) and Thanatephorus sp. RG26 (Ceratobasidiaceae).

Orchidaceae establish symbiotic relationships with fungi in the Rhizoctonia group, resulting in interactions beneficial to both organisms or in cell destruction in one of them (pathogenicity). Previous studies have focused mostly on terrestrial species with a few, preliminary studies, on epiphytes. To further our understanding of the molecular mechanisms involved in these symbioses, we evaluated the interaction between Oncidium sphacelatum Lindl. and the mycorrhizal fungus Thanatephorus sp. strain RG26 (isolated from a different orchid species) in vitro using morphometric and proteomic analyses. Evidence from the morphometric and microscopic analysis showed that the fungus promoted linear growth and differentiation of orchid protocorms during 98 days interaction. On day 63, protocorm development was evident, so we analyzed the physiological response of both organisms at that moment. Proteome results suggest that orchid development stimulated by the fungus apparently involves cell cycle proteins, purine recycling, ribosome biogenesis, energy metabolism, and secretion that were up-regulated in the orchid; whereas in the fungus, a high expression of proteins implicated in stress response, protein-protein interaction, and saccharides and protein biosynthesis were found in the symbiotic interaction. This is the first work reporting proteins differentially expressed in the epiphytic orchid-fungus interaction and will contribute to the search for molecular markers that will facilitate the study of this symbiosis in both wild orchids and those in danger of extinction.

A small heat-shock protein (Hsp20) regulated by RpoS is essential for cyst desiccation resistance in Azotobacter vinelandii.

In Azotobacter vinelandii, a cyst-forming bacterium, the alternative sigma factor RpoS is essential to the formation of cysts resistant to desiccation and to synthesis of the cyst-specific lipids, alkylresorcinols. In this study, we carried out a proteome analysis of vegetative cells and cysts of A. vinelandii strain AEIV and its rpoS mutant derivative AErpoS. This analysis allowed us to identify a small heat-shock protein, Hsp20, as one of the most abundant proteins of cysts regulated by RpoS. Inactivation of hsp20 did not affect the synthesis of alkylresorcinols or the formation of cysts with WT morphology; however, the cysts formed by the hsp20 mutant strain were unable to resist desiccation. We also demonstrated that expression of hsp20 from an RpoS-independent promoter in the AErpoS mutant strain is not enough to restore the phenotype of resistance to desiccation. These results indicate that Hsp20 is essential for the resistance to desiccation of A. vinelandii cysts, probably by preventing the aggregation of proteins caused by the lack of water. To our knowledge, this is the first report of a small heat-shock protein that is essential for desiccation resistance in bacteria.

Nitrogen-fixing rhizobial strains isolated from common bean seeds: phylogeny, physiology, and genome analysis.

Rhizobial bacteria are commonly found in soil but also establish symbiotic relationships with legumes, inhabiting the root nodules, where they fix nitrogen. Endophytic rhizobia have also been reported in the roots and stems of legumes and other plants. We isolated several rhizobial strains from the nodules of noninoculated bean plants and looked for their provenance in the interiors of the seeds. Nine isolates were obtained, covering most known bean symbiont species, which belong to the Rhizobium and Sinorhizobium groups. The strains showed several large plasmids, except for a Sinorhizobium americanum isolate. Two strains, one Rhizobium phaseoli and one S. americanum strain, were thoroughly characterized. Optimal symbiotic performance was observed for both of these strains. The S. americanum strain showed biotin prototrophy when subcultured, as well as high pyruvate dehydrogenase (PDH) activity, both of which are key factors in maintaining optimal growth. The R. phaseoli strain was a biotin auxotroph, did not grow when subcultured, accumulated a large amount of poly-ß-hydroxybutyrate, and exhibited low PDH activity. The physiology and genomes of these strains showed features that may have resulted from their lifestyle inside the seeds: stress sensitivity, a ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) complex, a homocitrate synthase (usually present only in free-living diazotrophs), a hydrogenase uptake cluster, and the presence of prophages. We propose that colonization by rhizobia and their presence in Phaseolus seeds may be part of a persistence mechanism that helps to retain and disperse rhizobial strains.

Functional modules, structural topology, and optimal activity in metabolic networks.

Modular organization in biological networks has been suggested as a natural mechanism by which a cell coordinates its metabolic strategies for evolving and responding to environmental perturbations. To understand how this occurs, there is a need for developing computational schemes that contribute to integration of genomic-scale information and assist investigators in formulating biological hypotheses in a quantitative and systematic fashion. In this work, we combined metabolome data and constraint-based modeling to elucidate the relationships among structural modules, functional organization, and the optimal metabolic phenotype of Rhizobium etli, a bacterium that fixes nitrogen in symbiosis with Phaseolus vulgaris. To experimentally characterize the metabolic phenotype of this microorganism, we obtained the metabolic profile of 220 metabolites at two physiological stages: under free-living conditions, and during nitrogen fixation with P. vulgaris. By integrating these data into a constraint-based model, we built a refined computational platform with the capability to survey the metabolic activity underlying nitrogen fixation in R. etli. Topological analysis of the metabolic reconstruction led us to identify modular structures with functional activities. Consistent with modular activity in metabolism, we found that most of the metabolites experimentally detected in each module simultaneously increased their relative abundances during nitrogen fixation. In this work, we explore the relationships among topology, biological function, and optimal activity in the metabolism of R. etli through an integrative analysis based on modeling and metabolome data. Our findings suggest that the metabolic activity during nitrogen fixation is supported by interacting structural modules that correlate with three functional classifications: nucleic acids, peptides, and lipids. More fundamentally, we supply evidence that such modular organization during functional nitrogen fixation is a robust property under different environmental conditions.

A proteomic overview of the major venom components from Tityus championi from Panama.

In recent years, morbidity caused by scorpion sting of the species Tityus championi has increased in Panama. Therefore, the LD50 was determined by intravenous injection in 2.9 mg/kg and the venom of T. championi was separated using a HPLC system and their fractions were tested for biological activities in mice to identify the most toxic fractions to mammals. In addition, the venom fractions were also tested against invertebrates to look for insect-specific toxin peptides. The most toxic fractions were analyzed by MS/MS spectrometry. The primary structures of T. championi venom peptides with the most relevant activity were obtained, and the primary structure of one of most neurotoxic peptides was found at least in other four species of Tityus from Panama. This neurotoxin is quite important to be used as a protein target to be neutralized if developing antivenoms against the sting of this Panamanian scorpion or other relevant species of genera Tityus in the country.

New insights into Escherichia coli metabolism: carbon scavenging, acetate metabolism and carbon recycling responses during growth on glycerol.

BACKGROUND: Glycerol has enhanced its biotechnological importance since it is a byproduct of biodiesel synthesis. A study of Escherichia coli physiology during growth on glycerol was performed combining transcriptional-proteomic analysis as well as kinetic and stoichiometric evaluations in the strain JM101 and certain derivatives with important inactivated genes. RESULTS: Transcriptional and proteomic analysis of metabolic central genes of strain JM101 growing on glycerol, revealed important changes not only in the synthesis of MglB, LamB and MalE proteins, but also in the overexpression of carbon scavenging genes: lamB, malE, mglB, mglC, galP and glk and some members of the RpoS regulon (pfkA, pfkB, fbaA, fbaB, pgi, poxB, acs, actP and acnA). Inactivation of rpoS had an important effect on stoichiometric parameters and growth adaptation on glycerol. The observed overexpression of poxB, pta, acs genes, glyoxylate shunt genes (aceA, aceB, glcB and glcC) and actP, suggested a possible carbon flux deviation into the PoxB, Acs and glyoxylate shunt. In this scenario acetate synthesized from pyruvate with PoxB was apparently reutilized via Acs and the glyoxylate shunt enzymes. In agreement, no acetate was detected when growing on glycerol, this strain was also capable of glycerol and acetate coutilization when growing in mineral media and derivatives carrying inactivated poxB or pckA genes, accumulated acetate. Tryptophanase A (TnaA) was synthesized at high levels and indole was produced by this enzyme, in strain JM101 growing on glycerol. Additionally, in the isogenic derivative with the inactivated tnaA gene, no indole was detected and acetate and lactate were accumulated. A high efficiency aromatic compounds production capability was detected in JM101 carrying pJLBaroG(fbr)tktA, when growing on glycerol, as compared to glucose. CONCLUSIONS: The overexpression of several carbon scavenging, acetate metabolism genes and the absence of acetate accumulation occurred in JM101 cultures growing on glycerol. To explain these results it is proposed that in addition to the glycolytic metabolism, a gluconeogenic carbon recycling process that involves acetate is occurring simultaneously in this strain when growing on glycerol. Carbon flux from glycerol can be efficiently redirected in JM101 strain into the aromatic pathway using appropriate tools.

Impact of c-di-GMP on the Extracellular Proteome of Rhizobium etli.

Extracellular matrix components of bacterial biofilms include biopolymers such as polysaccharides, nucleic acids and proteins. Similar to polysaccharides, the secretion of adhesins and other matrix proteins can be regulated by the second messenger cyclic diguanylate (cdG). We have performed quantitative proteomics to determine the extracellular protein contents of a Rhizobium etli strain expressing high cdG intracellular levels. cdG promoted the exportation of proteins that likely participate in adhesion and biofilm formation: the rhizobial adhesion protein RapA and two previously undescribed likely adhesins, along with flagellins. Unexpectedly, cdG also promoted the selective exportation of cytoplasmic proteins. Nearly 50% of these cytoplasmic proteins have been previously described as moonlighting or candidate moonlighting proteins in other organisms, often found extracellularly. Western blot assays confirmed cdG-promoted export of two of these cytoplasmic proteins, the translation elongation factor (EF-Tu) and glyceraldehyde 3-phosphate dehydrogenase (Gap). Transmission Electron Microscopy immunolabeling located the Gap protein in the cytoplasm but was also associated with cell membranes and extracellularly, indicative of an active process of exportation that would be enhanced by cdG. We also obtained evidence that cdG increases the number of extracellular Gap proteoforms, suggesting a link between cdG, the post-translational modification and the export of cytoplasmic proteins.

The human bile salt sodium deoxycholate induces metabolic and cell envelope changes in Salmonella Typhi leading to bile resistance.

Introduction. Salmonella enterica serovar Typhi (S. Typhi) is the etiological agent of typhoid fever. To establish an infection in the human host, this pathogen must survive the presence of bile salts in the gut and gallbladder.Hypothesis. S. Typhi uses multiple genetic elements to resist the presence of human bile.Aims. To determine the genetic elements that S. Typhi utilizes to tolerate the human bile salt sodium deoxycholate.Methodology. A collection of S. Typhi mutant strains was evaluated for their ability to growth in the presence of sodium deoxycholate and ox-bile. Additionally, transcriptomic and proteomic responses elicited by sodium deoxycholate on S. Typhi cultures were also analysed.Results. Multiple transcriptional factors and some of their dependent genes involved in central metabolism, as well as in cell envelope, are required for deoxycholate resistance.Conclusion. These findings suggest that metabolic adaptation to bile is focused on enhancing energy production to sustain synthesis of cell envelope components exposed to damage by bile salts.

argC Orthologs from Rhizobiales show diverse profiles of transcriptional efficiency and functionality in Sinorhizobium meliloti.

Several factors can influence ortholog replacement between closely related species. We evaluated the transcriptional expression and metabolic performance of ortholog substitution complementing a Sinorhizobium meliloti argC mutant with argC from Rhizobiales (Agrobacterium tumefaciens, Rhizobium etli, and Mesorhizobium loti). The argC gene is necessary for the synthesis of arginine, an amino acid that is central to protein and cellular metabolism. Strains were obtained carrying plasmids with argC orthologs expressed under the speB and argC (S. meliloti) and lac (Escherichia coli) promoters. Complementation analysis was assessed by growth, transcriptional activity, enzymatic activity, mRNA levels, specific detection of ArgC proteomic protein, and translational efficiency. The argC orthologs performed differently in each complementation, reflecting the diverse factors influencing gene expression and the ability of the ortholog product to function in a foreign metabolic background. Optimal complementation was directly related to sequence similarity with S. meliloti, and was inversely related to species signature, with M. loti argC showing the poorest performance, followed by R. etli and A. tumefaciens. Different copy numbers of genes and amounts of mRNA and protein were produced, even with genes transcribed from the same promoter, indicating that coding sequences play a role in the transcription and translation processes. These results provide relevant information for further genomic analyses and suggest that orthologous gene substitutions between closely related species are not completely functionally equivalent.

Regulation by transcription factors in bacteria: beyond description.

Transcription is an essential step in gene expression and its understanding has been one of the major interests in molecular and cellular biology. By precisely tuning gene expression, transcriptional regulation determines the molecular machinery for developmental plasticity, homeostasis and adaptation. In this review, we transmit the main ideas or concepts behind regulation by transcription factors and give just enough examples to sustain these main ideas, thus avoiding a classical ennumeration of facts. We review recent concepts and developments: cis elements and trans regulatory factors, chromosome organization and structure, transcriptional regulatory networks (TRNs) and transcriptomics. We also summarize new important discoveries that will probably affect the direction of research in gene regulation: epigenetics and stochasticity in transcriptional regulation, synthetic circuits and plasticity and evolution of TRNs. Many of the new discoveries in gene regulation are not extensively tested with wetlab approaches. Consequently, we review this broad area in Inference of TRNs and Dynamical Models of TRNs. Finally, we have stepped backwards to trace the origins of these modern concepts, synthesizing their history in a timeline schema.

Metaproteomic Insights Into the Microbial Community in Pozol.

Pozol is an acidic, refreshing, and non-alcoholic traditional Mayan beverage made with nixtamalized corn dough that is fermented spontaneously. The extensive analysis of the microbiology, biochemistry and metaproteomics of pozol allowed the construction of a comprehensive image of the fermentation system. The main changes in both the substrate and the microbiota occurred in the first 9 h of fermentation. The increase in microorganisms correlated with the drop in pH and with the decrease in the contents of carbohydrates, lipids, and nitrogen, which shows that this stage has the highest metabolic activity. Bacterial proteins were mainly represented by those of lactic acid bacteria, and among them, the proteins from genus Streptococcus was overwhelmingly the most abundant. Yeast proteins were present in all the analyzed samples, while proteins from filamentous fungi increased up to 48 h. The metaproteomic approach allowed us to identify several previously unknown enzyme complexes in the system. Additionally, enzymes for hydrolysis of starch, hemicellulose and cellulose were found, indicating that all these substrates can be used as a carbon source by the microbiota. Finally, enzymes related to the production of essential intermediates involved in the synthesis of organic acids, acetoin, butanediol, fatty acids and amino acids important for the generation of compounds that contribute to the sensorial quality of pozol, were found.

The CRISPR-Cas System Is Involved in OmpR Genetic Regulation for Outer Membrane Protein Synthesis in Salmonella Typhi.

The CRISPR-Cas cluster is found in many prokaryotic genomes including those of the Enterobacteriaceae family. Salmonella enterica serovar Typhi (S. Typhi) harbors a Type I-E CRISPR-Cas locus composed of cas3, cse1, cse2, cas7, cas5, cas6e, cas1, cas2, and a CRISPR1 array. In this work, it was determined that, in the absence of cas5 or cas2, the amount of the OmpC porin decreased substantially, whereas in individual cse2, cas6e, cas1, or cas3 null mutants, the OmpF porin was not observed in an electrophoretic profile of outer membrane proteins. Furthermore, the LysR-type transcriptional regulator LeuO was unable to positively regulate the expression of the quiescent OmpS2 porin, in individual S. Typhi cse2, cas5, cas6e, cas1, cas2, and cas3 mutants. Remarkably, the expression of the master porin regulator OmpR was dependent on the Cse2, Cas5, Cas6e, Cas1, Cas2, and Cas3 proteins. Therefore, the data suggest that the CRISPR-Cas system acts hierarchically on OmpR to control the synthesis of outer membrane proteins in S. Typhi.