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.

Pseudomonas pseudoalcaligenes CECT5344 grows in minimal medium containing cyanide as the sole nitrogen source. Under these conditions, an O 2 -dependent respiration highly resistant to cyanide was detected in cell extracts. The structural genes for the cyanide-resistant terminal oxidase, cioA and cioB, are clustered and encode the integral membrane proteins that correspond to subunits I and II of classical cytochrome bd, although the presence of heme d in the membrane could not be detected by difference spectra. The cio operon from P. pseudoalcaligenes presents a singular organization, starting upstream of cioAB by the coding sequence of a putative ferredoxin-dependent sulfite or nitrite reductase and spanning downstream two additional open reading frames that encode uncharacterized gene products. PCR amplifications of RNA (reverse transcription-PCR) indicated the cyanide-dependent up-regulation and cotranscription along the operon. The targeted disruption of cioA eliminates both the expression of the cyanide-stimulated respiratory activity and the growth with cyanide as the nitrogen source, which suggests a critical role of this cytochrome bd-related oxidase in the metabolism of cyanide by P. pseudoalcaligenes CECT5344.

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The effect of initial pH on bacterial cell-growth and its change over time was studied under aerobic heterotrophic conditions by using three bacterial strains: Escherichia coli ATCC 25922, Pseudomonas putida KT2440, and Pseudomonas pseudoalcaligenes CECT 5344. In Luria-Bertani (LB) media, pH evolved by converging to a certain value that is specific for each bacterium. By contrast, in the buffered Minimal Medium (MM), pH was generally more stable along the growth curve. In MM with glucose as carbon source, a slight acidification of the medium was observed for all strains. In the case of E. coli, a sudden drop in pH was observed during exponential cell growth that was later recovered at initial pH 7 or 8, but was irreversible below pH 6, thus arresting further cell-growth. When using other carbon sources in MM at a fixed initial pH, pH changes depended mainly on the carbon source itself. While glucose, glycerol, or octanoate slightly decreased extracellular pH, more oxidized carbon sources, such as citrate, 2-furoate, 2-oxoglutarate, and fumarate, ended up with the alkalinization of the medium. These observations are in accordance with pH change predictions using genome-scale metabolic models for the three strains, thus revealing the metabolic reasons behind pH change. Therefore, we conclude that the composition of the medium, specifically the carbon source, determines pH change during bacterial growth to a great extent and unravel the main molecular mechanism behind this phenotype. These findings pave the way for predicting pH changes in a given bacterial culture and may anticipate the interspecies interactions and fitness of bacteria in their environment.

Malate synthase (EC 2.3.3.9, formerly EC 4.1.2.2) has been investigated in the unicellular green algae Chlamydomonas reinhardtii. The molecular characteristics and the regulation of gene expression have been investigated for the enzyme. A full-length malate synthase cDNA has been isolated, containing an open reading frame of 1,641 bp encoding a polypeptide of 546 amino acids. This protein shares the conserved signature of the malate synthase family, along with the catalytic residues essential for enzymatic activity and a C-terminal motif that matches the consensus for glyoxysome import. Functionality studies have been facilitated by heterologous expression of the malate synthase cDNA in Escherichia coli. The remarkable metabolic versatility of the alga has been used to analyse the metabolic control of malate synthase gene expression. The data strongly support the role of acetate and light as the main regulatory effectors, and the existence of cross-talk between the two signalling pathways.