Mycobacterium smegmatis is a fast-growing, saprophytic, mycobacterial species that contains two cAMP-receptor protein (CRP) homologues designated herein as Crp1 and Crp2. Phylogenetic analysis suggests that Crp1 (Msmeg_0539) is uniquely present in fast-growing environmental mycobacteria, whereas Crp2 (Msmeg_6189) occurs in both fast- and slow-growing species. A crp1 mutant of M. smegmatis was readily obtained, but crp2 could not be deleted, suggesting it was essential for growth. A total of 239 genes were differentially regulated in response to crp1 deletion (loss of function), including genes coding for mycobacterial energy generation, solute transport and catabolism of carbon sources. To assess the role of Crp2 in M. smegmatis, the crp2 gene was overexpressed (gain of function) and transcriptional profiling studies revealed that 58 genes were differentially regulated. Identification of the CRP promoter consensus in M. smegmatis showed that both Crp1 and Crp2 recognized the same consensus sequence (TGTGN8CACA). Comparison of the Crp1- and Crp2-regulated genes revealed distinct but overlapping regulons with 11 genes in common, including those of the succinate dehydrogenase operon (MSMEG_0417-0420, sdh1). Expression of the sdh1 operon was negatively regulated by Crp1 and positively regulated by Crp2. Electrophoretic mobility shift assays with purified Crp1 and Crp2 demonstrated that Crp1 binding to the sdh1 promoter was cAMP-independent whereas Crp2 binding was cAMP-dependent. These data suggest that Crp1 and Crp2 respond to distinct signalling pathways in M. smegmatis to coordinate gene expression in response to carbon and energy supply.
The present study was conducted to determine the potential of five cyanobacteria strains isolated from aquatic zones to induce lipid production. The phylogenetic affiliation of the isolates was determined by 16S rRNA gene sequencing. Amongst the isolates, an efficient cyanobacterium, Synechococcus sp. HS01 showing maximal biomass and lipid productivity, was selected for further studies. In order to compare lipid productivity, the HS01 strain was grown in different media to screen potential significant culture ingredients and to evaluate mixotrophic cultivation. Mixotrophic cultivation of the strain using ostrich oil as a carbon source resulted in the best lipid productivity. GC analysis of fatty acid methyl esters of the selected cyanobacterial strain grown in media supplemented with ostrich oil showed a high content of C16 (palmitoleic acid and palmitic acid) and C18 (linoleic acid, oleic acid and linolenic acid) fatty acids of 42.7 and 42.8 %, respectively. Transmission electron micrographs showed that the HS01 cells exhibited an elongated rod-shaped appearance, either isolated, paired, linearly connected or in small clusters. According to initial experiments, ostrich oil, NaNO3 and NaCl were recognized as potential essential nutrients and selected for optimization of media with the goal of maximizing lipid productivity. A culture optimization technique using the response surface method demonstrated a maximum lipid productivity of 56.5 mg l−1 day−1. This value was 2.82-fold higher than that for the control, and was achieved in medium containing 1.12 g l−1 NaNO3, 1 % (v/v) ostrich oil and 0.09 % (w/v) NaCl.
S-Adenosyl-l-methionine (AdoMet) is an essential metabolite, serving in a very wide variety of metabolic reactions. The enzyme that produces AdoMet from l-methionine and ATP (methionine adenosyltransferase, MAT) is thus an attractive target for antimicrobial agents. We previously showed that a variety of methionine analogues are MAT substrates, yielding AdoMet analogues that function in specific methyltransfer reactions. However, this left open the question of whether the modified AdoMet molecules could support bacterial growth, meaning that they functioned in the full range of essential AdoMet-dependent reactions. The answer matters both for insight into the functional flexibility of key metabolic enzymes, and for drug design strategies for both MAT inhibitors and selectively toxic MAT substrates. In this study, methionine analogues were converted in vitro into AdoMet analogues, and tested with an Escherichia coli strain lacking MAT (ΔmetK) but that produces a heterologous AdoMet transporter. Growth that yields viable, morphologically normal cells provides exceptionally robust evidence that the analogue functions in every essential reaction in which AdoMet participates. Overall, the S-adenosylated derivatives of all tested l-methionine analogues modified at the carboxyl moiety, and some others as well, showed in vivo functionality sufficient to allow good growth in both rich and minimal media, with high viability and morphological normality. As the analogues were chosen based on incompatibility with the reactions via which AdoMet is used to produce acylhomoserine lactones (AHLs) for quorum sensing, these results support the possibility of using this route to selectively interfere with AHL biosynthesis without inhibiting bacterial growth.