NAD+ biosynthesis in bacteria is controlled by global carbon/nitrogen levels via PII signaling.

NAD+ is a central metabolite participating in core metabolic redox reactions. The prokaryotic NAD synthetase enzyme NadE catalyzes the last step of NAD+ biosynthesis, converting nicotinic acid adenine dinucleotide (NaAD) to NAD+ Some members of the NadE family use l-glutamine as a nitrogen donor and are named NadEGln Previous gene neighborhood analysis has indicated that the bacterial nadE gene is frequently clustered with the gene encoding the regulatory signal transduction protein PII, suggesting a functional relationship between these proteins in response to the nutritional status and the carbon/nitrogen ratio of the bacterial cell. Here, using affinity chromatography, bioinformatics analyses, NAD synthetase activity, and biolayer interferometry assays, we show that PII and NadEGln physically interact in vitro, that this complex relieves NadEGln negative feedback inhibition by NAD+ This mechanism is conserved in distantly related bacteria. Of note, the PII protein allosteric effector and cellular nitrogen level indicator 2-oxoglutarate (2-OG) inhibited the formation of the PII-NadEGln complex within a physiological range. These results indicate an interplay between the levels of ATP, ADP, 2-OG, PII-sensed glutamine, and NAD+, representing a metabolic hub that may balance the levels of core nitrogen and carbon metabolites. Our findings support the notion that PII proteins act as a dissociable regulatory subunit of NadEGln, thereby enabling the control of NAD+ biosynthesis according to the nutritional status of the bacterial cell.

In silico prediction and expression profile analysis of small non-coding RNAs in Herbaspirillum seropedicae SmR1.

BACKGROUND: Herbaspirillum seropedicae is a diazotrophic bacterium from the ß-proteobacteria class that colonizes endophytically important gramineous species, promotes their growth through phytohormone-dependent stimulation and can express nif genes and fix nitrogen inside plant tissues. Due to these properties this bacterium has great potential as a commercial inoculant for agriculture. The H. seropedicae SmR1 genome is completely sequenced and annotated but despite the availability of diverse structural and functional analysis of this genome, studies involving small non-coding RNAs (sRNAs) has not yet been done. We have conducted computational prediction and RNA-seq analysis to select and confirm the expression of sRNA genes in the H. seropedicae SmR1 genome, in the presence of two nitrogen independent sources and in presence of naringenin, a flavonoid secreted by some plants. RESULTS: This approach resulted in a set of 117 sRNAs distributed in riboswitch, cis-encoded and trans-encoded categories and among them 20 have Rfam homologs. The housekeeping sRNAs tmRNA, ssrS and 4.5S were found and we observed that a large number of sRNAs are more expressed in the nitrate condition rather than the control condition and in the presence of naringenin. Some sRNAs expression were confirmed in vitro and this work contributes to better understand the post transcriptional regulation in this bacterium. CONCLUSIONS: H. seropedicae SmR1 express sRNAs in the presence of two nitrogen sources and/or in the presence of naringenin. The functions of most of these sRNAs remains unknown but their existence in this bacterium confirms the evidence that sRNAs are involved in many different cellular activities to adapt to nutritional and environmental changes.

Defining the impact of exoribonucleases in the shift between exponential and stationary phases.

The transition between exponential and stationary phase is a natural phenomenon for all bacteria and requires a massive readjustment of the bacterial transcriptome. Exoribonucleases are key enzymes in the transition between the two growth phases. PNPase, RNase R and RNase II are the major degradative exoribonucleases in Escherichia coli. We analysed the whole transcriptome of exponential and stationary phases from the WT and mutants lacking these exoribonucleases (Δpnp, Δrnr, Δrnb, and ΔrnbΔrnr). When comparing the cells from exponential phase with the cells from stationary phase more than 1000 transcripts were differentially expressed, but only 491 core transcripts were common to all strains. There were some differences in the number and transcripts affected depending on the strain, suggesting that exoribonucleases influence the transition between these two growth phases differently. Interestingly, we found that the double mutant RNase II/RNase R is similar to the RNase R single mutant in exponential phase while in stationary phase it seems to be closer to the RNase II single mutant. This is the first global transcriptomic work comparing the roles of exoribonucleases in the transition between exponential and stationary phase.

Labeled Azospirillum brasilense wild type and excretion-ammonium strains in association with barley roots.

Soil bacteria colonization in plants is a complex process, which involves interaction between many bacterial characters and plant responses. In this work, we labeled Azospirillum brasilense FP2 (wild type) and HM053 (excretion-ammonium) strains by insertion of the reporter gene gusA-kanamycin into the dinitrogenase reductase coding gene, nifH, and evaluated bacteria colonization in barley (Hordeum vulgare). In addition, we determined inoculation effect based on growth promotion parameters. We report an uncommon endophytic behavior of A. brasilense Sp7 derivative inside the root hair cells of barley and highlight the promising use of A. brasilense HM053 as plant growth-promoting bacterium.

Quinolone resistance and ornithine decarboxylation activity in lactose-negative Escherichia coli.

Quinolones and fluoroquinolones are widely used to treat uropathogenic Escherichia coli infections. Bacterial resistance to these antimicrobials primarily involves mutations in gyrA and parC genes. To date, no studies have examined the potential relationship between biochemical characteristics and quinolone resistance in uropathogenic E. coli strains. The present work analyzed the quinolone sensitivity and biochemical activities of fifty-eight lactose-negative uropathogenic E. coli strains. A high percentage of the isolates (48.3%) was found to be resistant to at least one of the tested quinolones, and DNA sequencing revealed quinolone resistant determining region gyrA and parC mutations in the multi-resistant isolates. Statistical analyses suggested that the lack of ornithine decarboxylase (ODC) activity is correlated with quinolone resistance. Despite the low number of isolates examined, this is the first study correlating these characteristics in lactose-negative E. coli isolates.

Quinolone resistance and ornithine decarboxylation activity in lactose-negative Escherichia coli

Quinolones and fluoroquinolones are widely used to treat uropathogenic Escherichia coli infections. Bacterial resistance to these antimicrobials primarily involves mutations in gyrA and parC genes. To date, no studies have examined the potential relationship between biochemical characteristics and quinolone resistance in uropathogenic E. coli strains. The present work analyzed the quinolone sensitivity and biochemical activities of fifty-eight lactose-negative uropathogenic E. coli strains. A high percentage of the isolates (48.3%) was found to be resistant to at least one of the tested quinolones, and DNA sequencing revealed quinolone resistant determining region gyrA and parC mutations in the multi-resistant isolates. Statistical analyses suggested that the lack of ornithine decarboxylase (ODC) activity is correlated with quinolone resistance. Despite the low number of isolates examined, this is the first study correlating these characteristics in lactose-negative E. coli isolates.

Rapid identification of bacterial isolates from wheat roots by high resolution whole cell MALDI-TOF MS analysis.

Whole-cell mass spectrometry analysis is a powerful tool to rapidly identify microorganisms. Several studies reported the successful application of this technique to identify a variety of bacterial species with a discriminatory power at the strain level, mainly for bacteria of clinical importance. In this study we used matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) to assess the diversity of wheat-associated bacterial isolates. Wheat plants cultivated in non-sterile vermiculite, under greenhouse conditions were used for bacterial isolation. Total cellular extracts of 138 isolates were analyzed by MALDI-TOF MS and the mass spectra were used to cluster the isolates. Taxonomic identification and phylogenetic reconstruction based on 16S rRNA gene sequences showed the presence of Pseudomonas, Pantoea, Acinetobacter, Enterobacter and Curtobacterium. The 16S rRNA gene sequence analyses were congruent with the clusterization from mass spectra profile. Moreover, MALDI-TOF whole cell mass profiling allowed a finer discrimination of the isolates, suggesting that this technique has the potential of differentiating bacterial isolates at the strain level.

Ardra profiles of bacteria and archaea in mangrove sediments with different levels of contamination in the estuarine complex of Paranaguá, Brazil

The mangrove's sediments from the coastal areas under human activities may contain significant contaminations by hydrocarbons, even when there are no visual evidences of it. The microorganisms are essential to these ecosystems, especially in the control of their chemical environment. Sediment samples were collected in two regions under different environment conditions (pristine and contaminated) of the Paranaguá Estuarine Complex (Paranaguá Bay and Laranjeiras Bay), Brazil. Aliphatic hydrocarbons were determined by the GC-FID to assess the status of contamination of the studied areas. The total DNA was extracted from these samples. The 16S rRNA gene was amplified by the PCR reactions with the pair of primers 21F and 958R for the archaeal domain, and 27F and 1492R for the bacterial domain. Comparisons of communities were made by the ARDRA technique, using the HinfI restriction enzyme. The phosphate concentration showed significant differences between the two regions. The aliphatic hydrocarbons analysis showed the presence of unresolved complex mixture (UCM), an indicator of oil contamination, in the samples from the Paranaguá Bay, which was corroborated by the concentration of total aliphatic hydrocarbons. The ARDRA profile indicated that the structure of archaeal and bacterial communities of the sampled areas was very similar. Therefore, the anthropogenic influences in the Paranaguá Bay showed to be not sufficient to produce disturbances in the prokaryotic dominant groups.

Morphological and genetic characterization of endophytic bacteria isolated from roots of different maize genotypes.

Maize is one of the most important crops worldwide, and in Brazil, the state of Paraná stands as its largest producer. The crop demands high inputs of N fertilizers, therefore all strategies aiming to optimize the grain production with lower inputs are very relevant. Endophytic bacteria have a high potential to increment maize grain yield by means of input via biological nitrogen fixation and/or plant growth promotion, in this last case increasing the absorption of water and nutrients by the plants. In this study, we established a collection of 217 endophytic bacteria, isolated from roots of four lineages and three hybrid genotypes of maize, and isolated in four different N-free culture media. Biochemical-comprising growth in different carbon sources, intrinsic tolerance to antibiotics, and biochemical tests for catalase, nitrate reductase, urease, and growth in N-free media in vitro-and genetic characterization by BOX-PCR revealed great variability among the isolates. Both commercial hybrids and homozygous lineages were broadly colonized by endophytes, and sequencing of the 16S rRNA gene revealed the presence of bacteria belonging to the genera Pantoea, Bacillus, Burkholderia, and Klebsiella. Qualitative differences in endophytic colonization were detected between lineages and hybrid genotypes.

Draft genome sequence of Herbaspirillum lusitanum P6-12, an endophyte isolated from root nodules of Phaseolus vulgaris.

Herbaspirillum lusitanum strain P6-12 (DSM 17154) is, so far, the only species of Herbaspirillum isolated from plant root nodules. Here we report a draft genome sequence of this organism.

Structural characterization of the RNA chaperone Hfq from the nitrogen-fixing bacterium Herbaspirillum seropedicae SmR1.

The RNA chaperone Hfq is a homohexamer protein identified as an E. coli host factor involved in phage Qß replication and it is an important posttranscriptional regulator of several types of RNA, affecting a plethora of bacterial functions. Although twenty Hfq crystal structures have already been reported in the Protein Data Bank (PDB), new insights into these protein structures can still be discussed. In this work, the structure of Hfq from the ß-proteobacterium Herbaspirillum seropedicae, a diazotroph associated with economically important agricultural crops, was determined by X-ray crystallography and small-angle X-ray scattering (SAXS). Biochemical assays such as exclusion chromatography and RNA-binding by the electrophoretic shift assay (EMSA) confirmed that the purified protein is homogeneous and active. The crystal structure revealed a conserved Sm topology, composed of one N-terminal α-helix followed by five twisted ß-strands, and a novel π-π stacking intra-subunit interaction of two histidine residues, absent in other Hfq proteins. Moreover, the calculated ab initio envelope based on small-angle X-ray scattering (SAXS) data agreed with the Hfq crystal structure, suggesting that the protein has the same folding structure in solution.