Validation of a Spectroscopic Screening Method for Secondary Metabolites from New Zealand Extremophilic Microbes

<p>The existing protocol for the screening of natural products from marine invertebrates and macroalgae has been demonstrated successfully at VUW on multiple occasions. This study describes the evaluation of an adapted version of the protocol to screen liquid microbial cultures. The adapted protocol was carried out using an NMR-guided approach and was used to screen 10 New Zealand extremophilic microbes. Investigation of the bacteria Anoxybacillus flavithermus led to the isolation of the known compound 1-acetyl-β-carboline (25), which was briefly evaluated for its biological activity. A detailed investigation on the marine bacteria Shewanella sp. resulted in the isolation of indole-3-carboxaldehyde (93) and 14 known 2,5-diketopiperazines (36, 38, 41–44, 53, 54, 94–99). Three of the isolated 2,5-DKPs were tested against Saccharomyces cerevisiae to evaluate their antifungal activity, the results of which are also described. Overall, the use of the adapted screening method proved successful to guide the isolation of secondary metabolites from microbial cultures.</p>

Validation of a Spectroscopic Screening Method for Secondary Metabolites from New Zealand Extremophilic Microbes

<p>The existing protocol for the screening of natural products from marine invertebrates and macroalgae has been demonstrated successfully at VUW on multiple occasions. This study describes the evaluation of an adapted version of the protocol to screen liquid microbial cultures. The adapted protocol was carried out using an NMR-guided approach and was used to screen 10 New Zealand extremophilic microbes. Investigation of the bacteria Anoxybacillus flavithermus led to the isolation of the known compound 1-acetyl-β-carboline (25), which was briefly evaluated for its biological activity. A detailed investigation on the marine bacteria Shewanella sp. resulted in the isolation of indole-3-carboxaldehyde (93) and 14 known 2,5-diketopiperazines (36, 38, 41–44, 53, 54, 94–99). Three of the isolated 2,5-DKPs were tested against Saccharomyces cerevisiae to evaluate their antifungal activity, the results of which are also described. Overall, the use of the adapted screening method proved successful to guide the isolation of secondary metabolites from microbial cultures.</p>

Molecular Characterization and Biofilm Formation Study of Contaminant Bacteria Isolated from Domiaty and Hungarian Cheeses in Jeddah City

The aim was to study the microbiological quality of Domiaty and Hungarian cheeses, molecular identification and biofilm formation of some selected contaminant bacteria. Samples were collected from two M and P big markets in Jeddah City through the period from February to October 2018, nine visits for two types of natural cheese. Results showed that the total bacterial counts (CFU/ml) from Domiaty cheese from two markets (M and P) were 0.1 x 105, 8 x 105 and 1 x 10 5 CFU/ml respectively (3 visits of M market) and 4 x 106, 0.4 x 106, 6.5 x 103, 1 x 103, 0.1 x 103 and 0.1 x 103 CFU/ml respectively (six samples from 6 visits from P market). Results showed that the total bacterial counts (CFU/ml) from Hungarian cheese were 1.5 x 10 5, 1x 10 4, 11 x 10 4 and 4 x10 6 CFU/ml respectively from (4 visits of M market) and 0.18 x 104, 3 x 106, 22 x 106, 6 x 106 and 5 x 104 CFU/ml respectively (5 visits from P market).Different bacterial isolates from cheese were identified by morphology and biochemical test. Bacterial isolates from cheeses were identified by VITEK MS as follow: Serratia liquefaciens (D6-1, D6-2, D14-1, D13-1 and D13-2), and Pseudomonas fluorescens (D14-2) were isolated from Domiaty cheese while Enterococcus faecium (H11-2), Serratia liquefaciens (H15-1) and Streptococcus thermophilus (H14-1) were isolated from Hungarian cheese. Some selected bacterial isolates were identified by 16S rRNA. Isolates were belong to MK757978 (Raoultilla terrigena (D15-1)), MK757979 (Bacillus cereus (D16-1)), MK757980 (Enterococcus faecalis (H10-2)), MK757982 (Enterococcus fiscalism (H11-1)), MK757981 (Serratia liquefactions (H13-1)), MK757984 (Anoxybacillus flavithermus (H17-1). All bacterial isolates have been tested for the formation of biofilm using a Tissue Culture Plate (TCP). Results revealed 12.5% and 46.15% of high biofilm formation respectively for bacterial isolates of Domiaty and Hungarian cheeses.

Structure and mechanism of the Mrp complex, an ancient cation/proton antiporter

Multiple resistance and pH adaptation (Mrp) antiporters are multi-subunit Na+ (or K+)/H+ exchangers representing an ancestor of many essential redox-driven proton pumps, such as respiratory complex I. The mechanism of coupling between ion or electron transfer and proton translocation in this large protein family is unknown. Here, we present the structure of the Mrp complex from Anoxybacillus flavithermus solved by cryo-EM at 3.0 Å resolution. It is a dimer of seven-subunit protomers with 50 trans-membrane helices each. Surface charge distribution within each monomer is remarkably asymmetric, revealing probable proton and sodium translocation pathways. On the basis of the structure we propose a mechanism where the coupling between sodium and proton translocation is facilitated by a series of electrostatic interactions between a cation and key charged residues. This mechanism is likely to be applicable to the entire family of redox proton pumps, where electron transfer to substrates replaces cation movements.

Spore-Forming Bacteria Associated with Dairy Powders Can Be Found in Bacteriological Grade Agar–Agar Supply

ABSTRACT Thermophilic spore-forming bacteria are found ubiquitously in natural environments and, therefore, are present in a number of agricultural food products. Spores produced by these bacteria can survive harsh environmental conditions encountered during food processing and have been implicated in food spoilage. During research efforts to develop a standardized method for enumerating spores in dairy powders, the dairy powder–associated thermophilic sporeformer Anoxybacillus flavithermus was discovered growing in uninoculated control plates of tryptic soy agar (TSA) supplemented with 1% (w/v) starch, after incubation at thermophilic (55°C) growth temperatures. This article reports the investigation into the source of this thermophilic sporeformer in TSA medium components and characterization of the bacterial isolates collected. Aqueous solutions of tryptic soy broth powder from four suppliers and four agar–agar powders (two manufacturing lots from one supplier [agar A_1 and agar A_2] and two from separate suppliers [agar B and agar C]) were subjected to two different autoclave cycle times (121°C for 15 min or 121°C for 30 min) and then prepared as TSA. After incubation at 55°C for 48 h, bacterial growth was observed only in media prepared from both lots of agar A agar–agar powder, and only when they were subjected to a 15-min autoclave cycle, implicating these powders as a source of the sporeformer contamination. Genetic characterization of 49 isolates obtained indicated the presence of five unique rpoB allelic types of the thermophilic sporeformer Geobacillus spp. in agar–agar powder from agar A. These results not only highlight the importance of microbiological controls but also alert researchers to the potential for survival of thermophilic sporeformers such as Anoxybacillus and Geobacillus in microbiological media used for detection and enumeration of these same thermophilic sporeformers in products such as dairy powders. HIGHLIGHTS