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Archives of Microbiology (v.193, #9)

Use of qPCR for the study of hepatotoxic cyanobacteria population dynamics by António Martins; Vitor Vasconcelos (pp. 615-627).

Toxic cyanobacteria blooms are increasingly frequent and object of greater concern due to its ecological and health impacts. One important lack in the toxic cyanobacteria research field is to understand which parameters influence most and how they operate to regulate the overall levels of cyanotoxins in a body of water. MC concentration is believed to be influenced by changes in several seasonal environmental factors that influence the succession of toxic cyanobacteria. In the last years, qPCR (quantitative polymerase chain reaction) has been applied to determine the seasonal and temporal shifts in the proportions of MC-producing and non-MC-producing subpopulations by quantifying both mcy genotypes and total population numbers. We discuss the most prominent and recent studies using qPCR to address hepatotoxic cyanobacteria population dynamics and evaluate how they helped understanding the factors promoting the growth of toxic strains in situ and the succession of hepatotoxin-producing genera in natural populations.

Keywords: qPCR; Cyanobacteria; Cyanotoxins; Monitoring; Dynamics

Evidences of autoregulation of hfq expression in Sinorhizobium meliloti strain 2011 by Patricio Sobrero; Claudio Valverde (pp. 629-639).

Riboregulation comprises gene expression regulatory mechanisms that rely upon the activity of small non-coding RNAs (sRNAs) and in most cases RNA binding proteins. In γ-proteobacteria, the Sm-like protein Hfq is a key player in riboregulatory processes, because it promotes sRNA–mRNA interactions and influences mRNA polyadenylation or translation. In the α-proteobacterium Sinorhizobium meliloti, the large number of detected small RNA transcripts and the pleiotropic effects of hfq mutations lead to the hypothesis that riboregulatory mechanisms are important in this soil microorganism to adjust gene expression both in free-living conditions and as a nitrogen-fixing endosymbiont within legume root nodules. In this study, homology modeling of S. meliloti Hfq protein and cross-complementation experiments of S. meliloti and Escherichia coli mutants indicates that hfq Sm encodes an RNA chaperone that can be functionally exchanged by its homolog from E. coli. A transcriptional and translational analysis of S. meliloti hfq expression by means of lacZ reporter fusions strongly suggests that the S. meliloti Hfq protein autocontrols its expression at the translational level, a phenomenon that was evident in the natural host S. meliloti as well as in the heterologous host E. coli.

Keywords: Sinorhizobium meliloti ; Hfq ; Autoregulation; Riboregulation

Genomic analysis of the phenylacetyl-CoA pathway in Burkholderia xenovorans LB400 by Marianna A. Patrauchan; J. Jacob Parnell; Michael P. McLeod; Christine Florizone; James M. Tiedje; Lindsay D. Eltis (pp. 641-650).

The phenylacetyl-CoA (Paa) catabolic pathway and genome-wide gene expression responses to phenylacetate catabolism were studied in the polychlorinated biphenyl (PCB)-degrading strain Burkholderia xenovorans LB400. Microarray and RT-qPCR analyses identified three non-contiguous chromosomal clusters of genes that are predicted to encode a complete Paa pathway that were induced up to 40-fold during growth of LB400 on phenylacetate: paaGHIJKR, paaANEBDF, and paaC. Comparison of the available genome sequences revealed that this organization is unique to Burkholderiaceae. Parallel proteomic studies identified 7 of the 14 predicted Paa proteins, most of which were detected only in phenylacetate-grown cells, but not in benzoate- or succinate-grown cells. Finally, the transcriptomic and proteomic analyses revealed the induction of at least 7 predicted catabolic pathways of aromatic compounds and some aromatic plant products (phenols, mandelate, biphenyl, C₁ compounds, mevalonate, opine, and isoquinoline), as well as an oxidative stress response and a large group of transporters. Most of these genes were not induced during growth on benzoate or biphenyl, suggesting that phenylacetate or a metabolite may act as a signal that triggers multiple physiological processes. Identifying the components of the Paa pathway is important since the pathway appears to contribute to virulence of Burkholderia pathogens.

Keywords: Phenylacetyl-CoA pathway; Burkholderia xenovorans ; 2DGE proteomics; Microarray; Quantitative PCR

Interacting domains of P14-3-3 and actin involved in protein–protein interactions of living cells by Daliang Luo; Yi Yang; Jing Guo; Jianhua Zhang; Zhongzhen Guo; Shide Liu; Shengli Tian (pp. 651-663).

14-3-3 proteins are conserved regulatory proteins present in all eukaryotic cells that control numerous cellular activities via targeted protein interactions. To elucidate the interaction between P14-3-3 from Physarum polycephalum and actin in living cells, PCR and DNA recombination were used to generate various P14-3-3 and actin constructs. Yeast two-hybrid assay and FRET were employed to characterize the interaction between P14-3-3 and actin. The two-hybrid assay indicated that P14-3-3 N-terminal 76–108 amino acids and the C-terminal 207–216 amino acids played an important role in mediating interactions with actin, and the actin N-terminal 1–54 amino acids and the C-terminal 326–376 amino acids are also crucial in the interactions with the mPa, a P14-3-3 with mutations at Ser62 (Ser62 → Gly62). Mutations to potential phosphorylation sites did not affect interactions between P14-3-3 and actin. FRET results demonstrated that P14-3-3 co-localized with actin with a FRET efficiency of 22.2% and a distance of 7.4 nm and that P14-3-3 N-terminal 76–108 and C-terminal 207–216 amino acids were important in mediating this interaction, the truncated actin peptides without either the N-terminal 1–54 or C-terminal 326–376 amino acids interacted with P14-3-3, consistent with the results obtained from the yeast two-hybrid assay. Based on data obtained, we identified critical actin and P14-3-3 contact regions.

Keywords: Physarum polycephalum ; 14-3-3 proteins; Actin; Protein–protein interactions; Yeast two-hybrid assay; FRET

Metagenomic analysis of the 1-aminocyclopropane-1-carboxylate deaminase gene (acdS) operon of an uncultured bacterial endophyte colonizing Solanum tuberosum L by Branislav Nikolic; Helmut Schwab; Angela Sessitsch (pp. 665-676).

Deamination of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) is a key plant-beneficial trait found in many plant growth-promoting bacteria. In this study, we analysed ACC deaminase genes (acdS) of bacterial endophytes colonizing field-grown potato plants. PCR analysis revealed the presence of two types of acdS genes, the dominant one showing high homology to an acdS gene derived from Pseudomonas fluorescens. Construction, functional screening and sequence analysis of metagenomic libraries revealed clones containing the acdS gene identified in the PCR library. Sequence analysis of one metagenomic clone identified the entire acdS operon of an uncultivated endophyte and revealed that the acdS gene is coupled upstream with an acdR transcriptional regulator gene as previously found in P. putida strain UW4 (Grichko and Glick 2000). However, in-silico analysis of 195 fully sequenced, acdS-containing bacterial genomes revealed that the majority of strains, including numerous strains belonging to the genus Pseudomonas, do not contain an acdR regulatory gene in the vicinity of the acdS gene or elsewhere in the genome. The acdR ⁺–acdS ⁺ operon was exclusively found in several Alpha- and Betaproteobacteria most prominently in the genus Burkholderia.

Keywords: Endophytes; Metagenomics; 1-aminocyclopropane-1-carboxylate deaminase; acdS ; acdR ; Pseudomonas ; Burkholderia

Genetic evidence for involvement of the alternative sigma factor SigI in controlling expression of the cell wall hydrolase gene lytE and contribution of LytE to heat survival of Bacillus subtilis by Chi-Ling Tseng; Jung-Tze Chen; Ju-Hui Lin; Wan-Zhen Huang; Gwo-Chyuan Shaw (pp. 677-685).

The Bacillus subtilis cell wall hydrolase LytE is involved in cell wall turnover and cell separation during vegetative growth. lytE transcription is known to be driven by a YycF-activated SigA-dependent promoter. The cell wall regulator SigI is an alternative sigma factor that has been shown to be heat stress-inducible and to be essential for survival of B. subtilis at high temperature. However, none of the previously identified target genes of SigI contribute to heat resistance. We now demonstrate that lytE expression is heat-inducible and that heat induction of lytE expression is strongly dependent on SigI. We have also found that the lytE mutant shows the same growth defect at high temperature as the sigI mutant. Introducing an extra copy of lytE into the sigI mutant could rescue its growth defect. Our data strongly suggest that SigI-dependent lytE expression under heat stress is important for heat survival of B. subtilis.

Keywords: Bacillus subtilis ; SigI; Cell wall hydrolase; LytE; Heat stress

Reverse-transcriptase quantitative PCR method to detect uptake of hydrogen produced from cyanobacteria by Alcaligenes hydrogenophilus, an aerobic hydrogen-oxidising bacterium by Sarah Schroeder; Anthony Ranchou-Peyruse; Magali Ranchou-Peyruse; Jim C. Spain (pp. 687-692).

Hydrogen-oxidising bacteria play a key ecological role in a variety of habitats including the rhizosphere and hot springs. To investigate the possibly of interspecies hydrogen exchange between cyanobacteria and hydrogen-oxidising bacteria, we developed a sensitive and reliable reverse-transcriptase qPCR assay for up-regulation of the hupS gene in the knallgas bacterium Alcaligenes hydrogenophilus DSM2625. The assay detected up-regulation of the gene at initial hydrogen concentrations as low as 0.12 μM. Expression of hupS also increased in the presence of hydrogen-producing cyanobacteria, both when Ah DSM2625 was directly added to a hydrogen-producing culture of the cyanobacteria, and when cultures were physically separated in a vessel that allowed gas exchange. Additional refinements and development of the sensitive assay will lead to a better understanding of hydrogen exchange in aerobic ecosystems and development of reporter strains to detect hydrogen-producing organisms.

Keywords: Hydrogen exchange; Reverse-transcriptase real time PCR; Cyanobacteria; Hydrogen-oxidising

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Archives of Microbiology (v.193, #9)