- Volume 162, Issue 11, 2016

In Saccharomyces cerevisiae, Adr1 is a zinc-finger transcription factor involved in the transcriptional activation of ADH2. Deletion of KlADR1, its putative ortholog in Kluyveromyces lactis, led to reduced growth in glycerol, oleate and yeast extract-peptone medium suggesting, as in S. cerevisiae, its requirement for glycerol, fatty acid and nitrogen utilization. Moreover, growth comparison on yeast extract and peptone plates showed in K. lactis a KlAdr1-dependent growth trait not present in S. cerevisiae, indicating different metabolic roles of the two factors in their environmental niches. KlADR1 is required for growth under respiratory and fermentative conditions like Kl ADH, alcohol dehydrogenase genes necessary for metabolic adaptation during the growth transition. Using in-gel native alcohol dehydrogenase assay, we showed that this factor affected the Adh pattern by altering the balance between these activities. Since the activity most affected by KlAdr1 is KlAdh3, a deletion analysis of the KlADH3 promoter allowed the isolation of a DNA fragment through which KlAdr1 modulated its expression. The expression of the KlADR1-GFP gene allowed the intracellular localization of the factor in K. lactis and S. cerevisiae, suggesting in the two yeasts a common mechanism of KlAdr1 translocation under fermentative and respiratory conditions. Finally, the chimeric Kl/ScADR1 gene encoding the zinc-finger domains of KlAdr1 fused to the transactivating domains of the S. cerevisiae factor activated in Sc adr1 Δ the transcription of ADH2 in a ScAdr1-dependent fashion.

Pseudomonas alkylphenolica is an important strain in the biodegradation of toxic alkylphenols and mass production of bioactive polymannuronate polymers. This strain forms a diverse, 3D biofilm architecture, including mushroom-like aerial structures, circular pellicles and surface spreading, depending on culture conditions. A mutagenesis and complementation study showed that a predicted transmembrane kinase, PSAKL28_21690 (1164 aa), harbouring a periplasmic CHASE3 domain flanked by two transmembrane helices in addition to its cytoplasmic GAF, histidine kinase and three CheY-like response regulator domains, plays a positive role in the formation of the special biofilm architecture and a negative role in swimming activity. In addition, the gene, named here as bmsA, is co-transcribed with three genes encoding proteins with CheR (PSAKL28_21700) and CheB (PSAKL28_21710) domains and response regulator and histidine kinase domains (PSAKL28_21720). This gene cluster is thus named bmsABCD and is found widely distributed in pseudomonads and other bacteria. Deletion of the genes in the cluster, except forbmsA, did not result in changes in biofilm-related phenotypes. The RNA-seq analysis showed that the expression of genes coding for flagellar synthesis was increased when bmsA was mutated. In addition, the expression of rsmZ, which is one of final targets of the Gac regulon, was not significantly altered in the bmsA mutant, and overexpression of bmsA in the gacA mutant did not produce the WT phenotype. These results indicate that the sensory Bms regulon does not affect the upper cascade of the Gac signal transduction pathway for the biofilm-related phenotypes in P. alkylphenolica.

All cells are subjected to oxidative stress, a condition under which reactive oxygen species (ROS) production exceeds elimination. Bacterial defences against ROS include synthesis of antioxidant enzymes like peroxidases and catalases. Vibrio cholerae can produce two distinct catalases, KatB and KatG, which contribute to ROS homeostasis. In this study, we analysed the mechanism behind katG and katB expression in two V. cholerae O1 pandemic strains, O395 and N16961, of classical and El Tor biotypes, respectively. Both strains express these genes, especially at stationary phase. However, El Tor N16961 produces higher KatB and KatG levels and is much more resistant to peroxide challenge than the classical strain, confirming a direct relationship between catalase activity and oxidative stress resistance. Moreover, we showed that katG and katB expression levels depend on inorganic phosphate (Pi) availability, in contrast to other bacterial species. In N16961, katB and katG expression is reduced under Pi limitation relative to Pi abundance. Total catalase activity in N16961 and its phoB mutant cells was similar, independently of growth conditions, indicating that the PhoB/PhoR system is not required for katB and katG expression. However, N16961 cells from Pi-limited cultures were 50–100-fold more resistant to H2O2 challenge and accumulated less ROS than phoB mutant cells. Together, these findings suggest that, besides KatB and KatG, the PhoB/PhoR system is an important protective factor against ROS in V. cholerae N16961. They also corroborate previous results from our and other groups, suggesting that the PhoB/PhoR system is fundamental for V. cholerae biology.

The filamentous fungus Phycomyces blakesleeanus provides a renewable biosource of industrial high-value compounds such as carotenes, other isoprenoids (ubiquinone and sterols), organic acids and fatty acids. Several Phycomyces mutants involved in the formation of β-carotene are available. For example, the carA mutants have a leaky mutation in the phytoene synthase and produce significantly lower amounts of carotenes, while the carB and carR mutants produce phytoene and lycopene, respectively, due to a null mutation in the genes encoding the phytoene dehydrogenase and lycopene cyclase, respectively. The carS mutants are mutated in the gene encoding the oxygenase responsible for the conversion of β-carotene into apocarotenoids and, as a result, β-carotene accumulates. In order to ascertain further the biochemical changes arising in these potential industrial strains, a metabolite profiling workflow was implemented for Phycomyces. GC-MS and ultra-performance liquid chromatography–photodiode array platforms enabled the identification of over 100 metabolites in 11 carA, carB, carR and carS mutant strains and their wild-type comparator. All mutant strains possessed decreased TCA cycle intermediates, galactose, alanine and ribitol, while dodecanol and valine showed a general increase. As predicted, other terpenoid levels were affected in the carB, carR and carS mutants but not in the carA mutants. The global changes across intermediary metabolism of the mutants suggest that complex metabolic networks exist between intermediary and secondary metabolism or that other mutations beyond the carotene pathway may exist in these mutants. These data show the utility of the methodology in metabolically phenotyping Phycomyces strains with potential industrial exploitation.

Clostridium perfringens type A can cause both food poisoning (FP) and non-food-borne (NFB) gastrointestinal diseases. Our previous study reported that a mixture of l-asparagine and KCl (AK)-germinated spores of FP and NFB isolates well, but KCl and, to a lesser extent, l-asparagine induced spore germination only in FP isolates. We now report that the germination response of FP and NFB spores differsignificantly in several defined germinants and rich media. Spores of NFB strain F4969 gerAA, gerKA-KC or gerKC mutants lacking specific germinant receptor proteins germinated more slowly than wild-type spores with rich media, did not germinate with AK and germinated poorly compared to wild-type spores with l-cysteine. The germination defects in the gerKA-KC spores were largely due to loss of GerKC as (i) gerKA spores germinated significantly with all tested germinants, while gerKC spores exhibited poor or no germination; and (ii) germination defects in gerKC spores were largely restored by expressing the wild-type gerKA-KC operon in trans. We also found that gerKA-KC, gerAA and gerKC spores, but not gerKA spores, released dipicolinic acid at a slower rate than wild-type spores with AK. The colony-forming efficiency of F4969 gerKC spores was also ~35-fold lower than that of wild-type spores, while gerAA and wild-type spores had similar viability. Collectively, these results suggest that the GerAA and GerKC proteins play roles in normal germination of C. perfringens NFB isolates and that GerKC, but not GerAA, is important in these spores' apparent viability.

The spirochaete bacterium Borrelia burgdorferi sensu lato is the aetiologic agent of Lyme disease. Borrelia is transmitted to mammals through tick bite and is adapted to survive at tick and mammalian physiological temperatures. We have previously shown that B. burgdorferi can exist in different morphological forms, including the antibiotic-resistant biofilm form, in vitro and in vivo. B. burgdorferi forms aggregates in ticks as well as in humans, indicating potential of biofilm formation at both 23 and 37 °C. However, the role of various environmental factors that influence Borrelia biofilm formation remains unknown. In this study, we investigated the effect of tick (23 °C), mammalian physiological (37 °C) and standard in vitro culture (33 °C) temperatures with the objective of elucidating the effect of temperature on Borrelia biofilm phenotypes in vitro using two B. burgdorferi sensu stricto strains (B31 and 297). Our findings show increased biofilm quantity, biofilm size, exopolysaccharide content and enhanced adherence as well as reduced free spirochaetes at 37 °C for both strains, when compared to growth at 23 and 33 °C. There were no significant variations in the biofilm nano-topography and the type of extracellular polymeric substance in Borrelia biofilms formed at all three temperatures. Significant variations in extracellular DNA content were observed in the biofilms of both strains cultured at the three temperatures. Our results indicate that temperature is an important regulator of Borrelia biofilm development, and that the mammalian physiological temperature favours increased biofilm formation in vitro compared to tick physiological temperature and in vitro culture temperature.

Regulation of the Neisseria gonorrhoeae pilE gene is ill-defined. In this study, post-transcriptional effects on expression were assessed. In silico analysis predicts the formation of three putative stable stem–loop structures with favourable free energies within the 5′ untranslated region of the pilE message. Using quantitative reverse transcriptase PCR analyses, we show that each loop structure forms, with introduced destabilizing stem–loop mutations diminishing loop stability. Utilizing a series of pilE translational fusions, deletion of either loop 1 or loop 2 caused a significant reduction of pilE mRNA resulting in reduced expression of the reporter gene. Consequently, the formation of the loops apparently protects the pilE transcript from degradation. Putative loop 3 contains the pilE ribosomal binding site. Consequently, its formation may influence translation. Analysis of a small RNA transcriptome revealed an antisense RNA being produced upstream of the pilE promoter that is predicted to hybridize across the 5′ untranslated region loops. Insertional mutants were created where the antisense RNA is not transcribed. In these mutants, pilE transcript levels are greatly diminished, with any residual message apparently not being translated. Complementation of these insertion mutants in trans with the antisense RNA gene facilitates pilE translation yielding a pilus + phenotype. Overall, this study demonstrates a complex relationship between loop-dependent transcript protection and antisense RNA in modulating pilE expression levels.