A fluorescent, supramolecular chemosensor to follow steroid depletion in bacterial cultures.

Steroids have been identified as endocrine-disrupting agents, which are thought to impact the fertility of aquatic organisms and may even have direct effects on humans. The removal of steroids from wastewater is therefore essential, and this is most efficiently achieved by microbial treatment. We report herein a simple fluorescent method to identify microorganisms that are capable of steroid degradation and to optimize the conditions for steroid removal. The method is based on the supramolecular macrocycle cucurbit[8]uril (CB8), which can bind either the fluorescent dye berberine or a steroid in their inner cavity. In absence of steroid, the cavity is free to bind the dye, leading to a strong increase in fluorescence. In contrast, in the presence of steroid, the dye is displaced into the bulk solution. This principle affords a stable (no thermal or photodegradation was noted), fluorescent chemosensor (excitation ca. 450 nm, maximum emission at 525 nm), which can detect testosterone at concentrations > 0.7 µM. We show that this displacement principle can be applied to follow the removal of micromolar concentrations of the steroid testosterone from a bacterial culture of Buttiauxella sp. S19-1. The reliability of the chemosensor in screening applications is demonstrated by an excellent Z-factor, which was in the range of 0.52 to 0.74 for all experiments carried out with this assay. Graphical abstract Steroid depletion by bacterial cultures can be followed by fluorescence spectroscopy using a supramolecular chemosensor based on berberine and cucurbit[8]uril.

Development of a MALDI-TOF MS-Based Protein Fingerprint Database of Common Food Fish Allowing Fast and Reliable Identification of Fraud and Substitution.

Fish substitution and fish fraud are widely observed in the global food market. To detect and prevent substitution, DNA-based methods do not always meet the demand of being time- and cost-efficient; therefore, methodology improvements are needed. The use of species-specific protein patterns, as determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, has recently improved species identification of prokaryotes both time- and cost-wise. We used the method to establish a database containing protein patterns of common food fish prone to substitution. The database currently comprises 54 fish species. Aspects such as the sensitivity of identification on the species level and the impact of bacterial contamination of fish filets are assessed. Most database entries are characterized by low intraspecies but high interspecies variability. Hitherto, 118 validation samples were successfully determined. The results presented herein underline the potential and reliability of eukaryotic species identification via MALDI-TOF mass spectrometry.

Marinobacter adhaerens HP15 harbors two CzcCBA efflux pumps involved in zinc detoxification.

Several members of the ubiquitously found γ-proteobacterial genus Marinobacter were described or assumed to inhabit marine environments naturally enriched in heavy metals. However, direct studies that describe the ability of this genus to occupy such environments have not been conducted. To cope with heavy metal stress, bacteria possess specific efflux pumps as tools for detoxification, among which the CzcCBA type efflux system is one representative. Previous studies showed that this system plays an important role in resistance towards cadmium, zinc, and cobalt. Up to now, no study had focused on characterization of Czc pumps in Marinobacter sp. or other marine prokaryotes. Herein, we elucidated the function of two CzcCBA pumps encoded by Marinobacter adhaerens HP15's genome during exposure to cadmium, zinc, and cobalt. Single and double knock-out mutants lacking the corresponding two czcCBA operons were generated and analyzed in terms of their resistance profiles. Both operons appeared to be important for zinc resistance but had no role in tolerance towards cadmium or cobalt. One of the mutations was genetically complemented thereby restoring the wild type phenotype. In accordance with the resistance pattern, expression of the genes coding for both CzcCBA pumps was induced by zinc but neither by cadmium nor cobalt.

Role of the cell envelope stress regulators BaeR and CpxR in control of RND-type multidrug efflux pumps and transcriptional cross talk with exopolysaccharide synthesis in Erwinia amylovora.

The purpose of this study was to identify the role of the cell envelope stress-sensing systems BaeSR and CpxARP in regulation of multidrug efflux and exopolysaccharide synthesis in Erwinia amylovora. We have previously reported that BaeR activates transcription of the RND-type efflux pumps AcrD and MdtABC. In this study, we found that a cpxR-deficient mutant was highly susceptible to ß-lactams, aminoglycosides and lincomycin, whereas a baeR mutant showed no change in antimicrobial sensitivity. However, overexpression of BaeR in a mutant lacking the major RND pump AcrB increased resistance of E. amylovora to several compounds that are not substrates of AcrD or MdtABC. Furthermore, we observed that overexpression of BaeR significantly increased amylovoran production. Moreover, the expression of RND-type efflux pumps was changed in regulatory mutants of exopolysaccharide production. Our data suggest that BaeSR and CpxARP regulate additional mechanisms, beside efflux, which are responsible for antimicrobial resistance of E. amylovora.

The conserved upstream region of lscB/C determines expression of different levansucrase genes in plant pathogen Pseudomonas syringae.

BACKGROUND: Pseudomonas syringae pv. glycinea PG4180 is an opportunistic plant pathogen which causes bacterial blight of soybean plants. It produces the exopolysaccharide levan by the enzyme levansucrase. Levansucrase has three gene copies in PG4180, two of which, lscB and lscC, are expressed while the third, lscA, is cryptic. Previously, nucleotide sequence alignments of lscB/C variants in various P. syringae showed that a ~450-bp phage-associated promoter element (PAPE) including the first 48 nucleotides of the ORF is absent in lscA. RESULTS: Herein, we tested whether this upstream region is responsible for the expression of lscB/C and lscA. Initially, the transcriptional start site for lscB/C was determined. A fusion of the PAPE with the ORF of lscA (lscB(UpN)A) was generated and introduced to a levan-negative mutant of PG4180. Additionally, fusions comprising of the non-coding part of the upstream region of lscB with lscA (lscBUpA) or the upstream region of lscA with lscB (lscA(Up)B) were generated. Transformants harboring the lscB(UpN)A or the lscBUpA fusion, respectively, showed levan formation while the transformant carrying lscA(Up)B did not. qRT-PCR and Western blot analyses showed that lscB(UpN)A had an expression similar to lscB while lscB(Up)A had a lower expression. Accuracy of protein fusions was confirmed by MALDI-TOF peptide fingerprinting. CONCLUSIONS: Our data suggested that the upstream sequence of lscB is essential for expression of levansucrase while the N-terminus of LscB mediates an enhanced expression. In contrast, the upstream region of lscA does not lead to expression of lscB. We propose that lscA might be an ancestral levansucrase variant upstream of which the PAPE got inserted by potentially phage-mediated transposition events leading to expression of levansucrase in P. syringae.