Transcriptional and translational adaptation to aerobic nitrate anabolism in the denitrifier Paracoccus denitrificans.

Transcriptional adaptation to nitrate-dependent anabolism by Paracoccus denitrificans PD1222 was studied. A total of 74 genes were induced in cells grown with nitrate as N-source compared with ammonium, including nasTSABGHC and ntrBC genes. The nasT and nasS genes were cotranscribed, although nasT was more strongly induced by nitrate than nasS The nasABGHC genes constituted a transcriptional unit, which is preceded by a non-coding region containing hairpin structures involved in transcription termination. The nasTS and nasABGHC transcripts were detected at similar levels with nitrate or glutamate as N-source, but nasABGHC transcript was undetectable in ammonium-grown cells. The nitrite reductase NasG subunit was detected by two-dimensional polyacrylamide gel electrophoresis in cytoplasmic fractions from nitrate-grown cells, but it was not observed when either ammonium or glutamate was used as the N-source. The nasT mutant lacked both nasABGHC transcript and nicotinamide adenine dinucleotide (NADH)-dependent nitrate reductase activity. On the contrary, the nasS mutant showed similar levels of the nasABGHC transcript to the wild-type strain and displayed NasG protein and NADH-nitrate reductase activity with all N-sources tested, except with ammonium. Ammonium repression of nasABGHC was dependent on the Ntr system. The ntrBC and ntrYX genes were expressed at low levels regardless of the nitrogen source supporting growth. Mutational analysis of the ntrBCYX genes indicated that while ntrBC genes are required for nitrate assimilation, ntrYX genes can only partially restore growth on nitrate in the absence of ntrBC genes. The existence of a regulation mechanism for nitrate assimilation in P. denitrificans, by which nitrate induction operates at both transcriptional and translational levels, is proposed.

Draft whole genome sequence of the cyanide-degrading bacterium Pseudomonas pseudoalcaligenes CECT5344.

Pseudomonas pseudoalcaligenes CECT5344 is a Gram-negative bacterium able to tolerate cyanide and to use it as the sole nitrogen source. We report here the first draft of the whole genome sequence of a P. pseudoalcaligenes strain that assimilates cyanide. Three aspects are specially emphasized in this manuscript. First, some generalities of the genome are shown and discussed in the context of other Pseudomonadaceae genomes, including genome size, G + C content, core genome and singletons among other features. Second, the genome is analysed in the context of cyanide metabolism, describing genes probably involved in cyanide assimilation, like those encoding nitrilases, and genes related to cyanide resistance, like the cio genes encoding the cyanide insensitive oxidases. Finally, the presence of genes probably involved in other processes with a great biotechnological potential like production of bioplastics and biodegradation of pollutants also is discussed.

Poly(3-hydroxybutyrate) hyperproduction by a global nitrogen regulator NtrB mutant strain of Paracoccus denitrificans PD1222.

Paracoccus denitrificans PD1222 accumulates short-length polyhydroxyalkanoates, poly(3-hydroxybutyrate), under nitrogen-deficient conditions. Polyhydroxybutyrate metabolism requires the 3-ketoacyl-CoA thiolase PhaA, the acetoacetyl-CoA dehydrogenase/reductase PhaB and the synthase PhaC for polymerization. Additionally, P. denitrificans PD1222 grows aerobically with nitrate as sole nitrogen source. Nitrate assimilation is controlled negatively by ammonium through the two-component NtrBC system. NtrB is a sensor kinase that autophosphorylates a histidine residue under low-nitrogen concentrations and, in turn, transfers a phosphoryl group to an aspartate residue of the response regulator NtrC protein, which acts as a transcriptional activator of the P. denitrificans PD1222 nasABGHC genes. The P. denitrificans PD1222 NtrB mutant was unable to use nitrate efficiently as nitrogen source when compared to the wild-type strain, and it also overproduced poly(3-hydroxybutyrate). Acetyl-CoA concentration in the P. denitrificans PD1222 NtrB mutant strain was higher than in the wild-type strain. The expression of the phaC gene was also increased in the NtrB mutant when compared to the wild-type strain. These results suggest that accumulation of poly(3-hydroxybutyrate) in the NtrB mutant strain of PD1222 responds to the high levels of acetyl-CoA that accumulate in the cytoplasm as consequence of its inability to efficiently use nitrate as nitrogen source.

A way to start transanal total mesorectal excision for rectal cancer

Introduction: The optimal rectal cancer care is achieved by a multidisciplinary approach, with a high-quality surgical resection, with complete mesorectal excision and adequate margins. New approaches like the transanal total mesorectal excision (TaTME) aim to achieve these goals, maximizing the sphincter preservation ratio, with good oncologic and functional results. This report describes a way to implement TaTME without a proctor, presents the first case series of this approach in a center experienced in rectal cancer, and compares the results with those of the international literature. Methods: We performed a retrospective study of the first 10 consecutive patients submitted to TaTME for rectal cancer at our institution. The primary outcomes were postoperative complications, pathological specimen quality and local recurrence rate. The results and performance were compared with the outcomes of a known structured program with proctorship and with the largest meta-analysis on this topic. Results: All patients had locally advanced cancer; therefore, all underwent neoadjuvant therapy. A total of 30% had postoperative complications, without mortality or re-admissions. In comparison with the structured training program referred, no differences were found in postoperative complications and reintervention rates, resulting in a similar quality of resection. Comparing these results with those of the largest meta-analysis on the subject, no differences in the postoperative complication rates were found, and very similar outcomes regarding anastomotic leaks and oncological quality of resection were registered. Conclusion: The results of this study validate the safety and effectiveness of our pathway regarding the implementation of the TaTME approach, highlighting the fact that it should be done in a center with proficiency in minimally invasive rectal surgery. (AU)

The nit1C gene cluster of Pseudomonas pseudoalcaligenes CECT5344 involved in assimilation of nitriles is essential for growth on cyanide.

A proteomic approach was used to identify several proteins induced by cyanide in the alkaliphilic bacterium Pseudomonas pseudoalcaligenes CECT5344, two of them, NitB and NitG, encoded by genes that belong to the nit1C gene cluster. The predicted products of the nit1C gene cluster are a Fis-like σ(54) -dependent transcriptional activator (NitA), a nitrilase (NitC), an S-adenosylmethionine superfamily member (NitD), an N-acyltransferase superfamily member (NitE), a trifunctional polypeptide of the AIRS/GARS family (NitF), an NADH-dependent oxidoreductase (NitH) and two hypothetical proteins of unknown function (NitB and NitG). RT-PCR analysis suggested that nitBCDEFGH genes were co-transcribed, whereas the regulatory nitA gene was divergently transcribed. Real-time RT-PCR revealed that expression of the nitBCDEFGH genes was induced by cyanide and repressed by ammonium. The P. pseudoalcaligenes CECT5344 nit1C gene cluster was found to be involved in assimilation of free and organic cyanides (nitriles) as deduced for the inability to grow with cyanides showed by the NitA, NitB and NitC mutant strains. The wild-type strain CECT5344 showed a nitrilase activity that allows growth on cyanide or hydroxynitriles. The NitB and NitC mutants only presented low basal levels of nitrilase activity that were not enough to support growth on either free cyanide or aliphatic nitriles, suggesting that nitrilase NitC is specific and essential for cyanide and aliphatic nitriles assimilation.

3-Hydroxyphenylpropionate and phenylpropionate are synergistic activators of the MhpR transcriptional regulator from Escherichia coli.

The degradation of the aromatic compound phenylpropionate (PP) in Escherichia coli K-12 requires the activation of two different catabolic pathways coded by the hca and the mhp gene clusters involved in the mineralization of PP and 3-hydroxyphenylpropionate (3HPP), respectively. The compound 3-(2,3-dihydroxyphenyl)propionate (DHPP) is a common intermediate of both pathways which must be cleaved by the MhpB dioxygenase before entering into the primary cell metabolism. Therefore, the degradation of PP has to be controlled by both its specific regulator (HcaR) but also by the MhpR regulator of the mhp cluster. We have demonstrated that 3HPP and DHPP are the true and best activators of MhpR, whereas PP only induces no response. However, in vivo and in vitro transcription experiments have demonstrated that PP activates the MhpR regulator synergistically with the true inducers, representing the first case of such a peculiar synergistic effect described for a bacterial regulator. The three compounds enhanced the interaction of MhpR with its DNA operator in electrophoretic mobility shift assays. Inducer binding to MhpR is detected by circular dichroism and fluorescence spectroscopies. Fluorescence quenching measurements have revealed that the true inducers (3HPP and DHPP) and PP bind with similar affinities and independently to MhpR. This type of dual-metabolite synergy provides great potential for a rapid modulation of gene expression and represents an important feature of transcriptional control. The mhp regulatory system is an example of the high complexity achievable in prokaryotes.

The role of FIS protein in the physiological control of the expression of the Escherichia coli meta-hpa operon.

Expression from the Escherichia coli W meta-hpa operon promoter (Pg) is under a strict catabolic repression control mediated by the cAMP-catabolite repression protein (CRP) complex in a glucose-containing medium. The Pg promoter is also activated by the integration host factor (IHF) and repressed by the specific transcriptional regulator HpaR when 4-hydroxyphenylacetate (4HPA) is not present in the medium. Expression from the hpa promoter is also repressed in undefined rich medium such as LB, but the molecular basis of this mechanism is not understood. We present in vitro and in vivo studies to demonstrate the involvement of FIS protein in this catabolic repression. DNase I footprinting experiments show that FIS binds to multiple sites within the Pg promoter. FIS-site I overlaps the CRP-binding site. By using an electromobility shift assay, we demonstrated that FIS efficiently competes with CRP for binding to the Pg promoter, suggesting an antagonist/competitive mechanism. RT-PCR showed that the Pg repression effect is relieved in a FIS deleted strain. The repression role of FIS at Pg was further demonstrated by in vitro transcription assays. These results suggest that FIS contributes to silencing the Pg promoter in the exponential phase of growth in an undefined rich medium when FIS is predominantly expressed.

Genetic modification of the penicillin G acylase surface to improve its reversible immobilization on ionic exchangers.

A new mutant of the industrial enzyme penicillin G acylase (PGA) from Escherichia coli has been designed to improve its reversible immobilization on anionic exchangers (DEAE- or polyethyleneimine [PEI]-coated agarose) by assembling eight new glutamic residues distributed homogeneously through the enzyme surface via site-directed mutagenesis. The mutant PGA is produced and processed in vivo as is the native enzyme. Moreover, it has a similar specific activity to and shows the same pH activity profile as native PGA; however, its isoelectric point decreased from 6.4 to 4.3. Although the new enzyme is adsorbed on both supports, the adsorption was even stronger when supports were coated with PEI, allowing us to improve the enzyme stability in organic cosolvents. The use of restrictive conditions during the enzyme adsorption on anionic exchangers (pH 5 and high ionic strength) permitted us to still further increase the strength of adsorption and the enzyme stability in the presence of organic solvents, suggesting that these conditions allow the penetration of the enzyme inside the polymeric beds, thus becoming fully covered with the polymer. After the enzyme inactivation, it can be desorbed to reuse the support. The possibility to improve the immobilization properties on an enzyme by site-directed mutagenesis of its surface opens a promising new scenario for enzyme engineering.