Flow cytometry as a rapid analytical tool to determine physiological responses to changing O2 and iron concentration by Magnetospirillum gryphiswaldense strain MSR-1

Abstract

Magnetotactic bacteria (MTB) are a diverse group of bacteria that synthesise magnetosomes, magnetic membrane-bound nanoparticles that have a variety of diagnostic, clinical and biotechnological applications. We present the development of rapid methods using flow cytometry to characterize several aspects of the physiology of the commonly-used MTB Magnetospirillum gryphiswaldense MSR-1. Flow cytometry is an optical technique that rapidly measures characteristics of individual bacteria within a culture, thereby allowing determination of population heterogeneity and also permitting direct analysis of bacteria. Scatter measurements were used to measure and compare bacterial size, shape and morphology. Membrane permeability and polarization were measured using the dyes propidium iodide and bis-(1,3-dibutylbarbituric acid) trimethine oxonol to determine the viability and ‘health’ of bacteria. Dyes were also used to determine changes in concentration of intracellular free iron and polyhydroxylakanoate (PHA), a bacterial energy storage polymer. These tools were then used to characterize the responses of MTB to different O2 concentrations and iron-sufficient or iron-limited growth. Rapid analysis of MTB physiology will allow development of bioprocesses for the production of magnetosomes, and will increase understanding of this fascinating and useful group of bacteria.

Development of a simple intensified fermentation strategy for growth of Magnetospirillum gryphiswaldense MSR-1: Physiological responses to changing environmental conditions

“…Nitrite concentration was also very low throughout, except for the last sampling point (15.6 μM NO 2 -). This suggests concurrent denitrification and aerobic respiration; this has previously been observed by Li et al [ 24 ]. For oxygen-limited growth, nitrate was rapidly utilized when the pO 2 decreased to zero ( Fig.…”

“…Flow cytometry (FCM) was recently used by our group as a rapid, single-cell technique to study the physiology of MTB during growth in flasks [ 24 ]. In the present study, we have applied FCM to analyze and optimize fermentations.…”

“…In this study, we describe the development of a simple pH-stat based fermentation strategy that affords growth of M. gryphiswaldense MSR-1 to high cell densities. We have employed flow cytometry methods recently developed in our laboratory [ 24 ] to evaluate a range of physiological and stress parameters (cell morphology, aspects of metabolism, and the accumulation of intracellular polyhydroxyalkanoate (PHA)), and investigated the impact of different concentrations of lactate and nitrate on cell growth and magnetosome formation. We aim to better understand the bacterial physiology and metabolism of MTB, so that they might be grown industrially at large scale to high cell densities with elevated magnetosome content, allowing development of magnetosome-related applications [ 2 , 14 , 25 ].…”

Metabolic characterisation ofMagnetospirillum gryphiswaldenseMSR-1 using LC-MS-based metabolite profiling

“…These results are in agreement with ndings from our previous studies: 29 using a 20% headspace volume allows relatively high growth under free air exchange conditions, whereas it maintains the magnetic properties of Mgryph in shake ask cultures. 29 The presence of PHAs has been previously reported and might be due to the redirection of nutrients and energy competition between PHAs and magnetosome formation. In contrast, magnetosome formation is inhibited under aerobic conditions.…”

Nanoparticle Tracking Analysis: A powerful tool for characterizing magnetosome preparations

“…Despite numerous efforts to improve the yields of magnetosomes [4,12,13], their large-scale biomanufacture remains significant barrier to future widespread industrial application. Fundamental to this are: (i) appropriate means for analysing MTB growth and physiology [14,15]; (ii) investigating the biomineralization of magnetic iron-containing minerals, which is still not fully understood [16 -18]; (iii) the ability to both produce batches of nanoparticles that are consistent in structure, chain length and functionality -this is key to unlocking the potential of magnetosomes as therapeutic agents; and (iv) to be able to rapidly analyse these characteristics.…”