Carbon metabolism of intracellular bacteria

Bacterial metabolism has been studied intensively since the first observations of these 'animalcules' by Leeuwenhoek and their isolation in pure cultures by Pasteur. Metabolic studies have traditionally focused on a small number of model organisms, primarily the Gram negative bacillus Escherichia coli, adapted to artificial culture conditions in the laboratory. Comparatively little is known about the physiology and metabolism of wild microorganisms living in their natural habitats. For approximately 500-1000 species of commensals and symbionts, and a smaller number of pathogenic bacteria, that habitat is the human body. Emerging evidence suggests that the metabolism of bacteria grown in vivo differs profoundly from their metabolism in axenic cultures.

Rapid detection of bacterial resistance to antibiotics using AFM cantilevers as nanomechanical sensors

Livan Bladimir Alonso Sarduy, Giovanni Dietler, Sandor Kasas, Giovanni Longo, Justin Terence Notz Pekkanen, Laura Rio Marques

The widespread misuse of drugs has increased the number of multiresistant bacteria1, and this means that tools that can rapidly detect and characterize bacterial response to antibiotics are much needed in the management of infections. Various techniques, such as the resazurin-reduction assays2, the myco- bacterial growth indicator tube3 or polymerase chain reaction- based methods4, have been used to investigate bacterial metabolism and its response to drugs. However, many are rela- tively expensive or unable to distinguish between living and dead bacteria. Here we show that the fluctuations of highly sen- sitive atomic force microscope cantilevers can be used to detect low concentrations of bacteria, characterize their metabolism and quantitatively screen (within minutes) their response to antibiotics. We applied this methodology to Escherichia coli and Staphylococcus aureus, showing that live bacteria produced larger cantilever fluctuations than bacteria exposed to anti- biotics. Our preliminary experiments suggest that the fluctu- ation is associated with bacterial metabolism.

Nature Publishing Group2013

Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase

John McKinney

Mycobacterium tuberculosis claims more human lives each year than any other bacterial pathogen. Infection is maintained in spite of acquired immunity and resists eradication by antimicrobials. Despite an urgent need for new therapies targeting persistent bacteria, our knowledge of bacterial metabolism throughout the course of infection remains rudimentary. Here we report that persistence of M. tuberculosis in mice is facilitated by isocitrate lyase (ICL), an enzyme essential for the metabolism of fatty acids. Disruption of the icl gene attenuated bacterial persistence and virulence in immune-competent mice without affecting bacterial growth during the acute phase of infection. A link between the requirement for ICL and the immune status of the host was established by the restored virulence of delta icl bacteria in interferon-gamma knockout mice. This link was apparent at the level of the infected macrophage: Activation of infected macrophages increased expression of ICL, and the delta icl mutant was markedly attenuated for survival in activated but not resting macrophages. These data suggest that the metabolism of M. tuberculosis in vivo is profoundly influenced by the host response to infection, an observation with important implications for the treatment of chronic tuberculosis.

2000

Mechanism(s) of electron transfer and metabolic changes during iron reduction by Clostridium acetobutylicum

Cornelia List

Microorganisms catalyze many of the redox transformations driving the iron cycle in the geosphere. For instance, iron cycling includes the reduction of solid-phase and soluble ferric iron (Fe(III)) to ferrous iron (Fe(II)). However, in order to reduce solid-phase Fe(III), the reduction requires the delivery of electrons extracellularly, requiring a specialized electron transfer system. The mechanism of iron reduction by Gram-negative bacteria has been de-scribed in some detail, but very little is known for Gram-positive bacteria. These microorgan-isms are likely to contribute to iron reduction significantly, particularly in environments where Gram-negative bacteria fare poorly, such as metal contaminated sites, or hydrother-mal environments. Some Gram-positive bacteria use metal reduction as a form of enhanced fermentation, where the metal is a sink for excess reducing equivalents. In these cases, metal reduction is facultative and is not a mechanism for direct energy conservation. Here, we use Clostridium acetobutylicum, a Gram-positive fermenter, as a model organism for its ability to reduce iron and contaminant heavy metals. A major advantage of this model organism is that genetic tools are available to study genes involved in the metal reduction process. We investigated the reduction of solid-phase and soluble Fe(III) by C. acetobutyli-cum and found that it reduces both forms of iron. The bacteria do not require direct contact with solid-phase Fe(III) (as hydrous ferric oxide, HFO) for the reduction to occur. In fact, flavins were identified as likely to be involved in the extracellular electron transfer (EET) in this organism. We found that HFO reduction is increased by utilizing exogenous (resazurin, resorufin, anthraquinone-2,6-disulfonate) or endogenous (riboflavin, RF) electron shuttles. Inactivation of a putative flavin transporter (CAC2841) decreased HFO and Fe(III)-citrate reduction. We proposed that a complex CAC2841/ CAC3100/ CAC3101/ CAC3102 imports RF and CAC2841/ CAC3100 exports flavin adenine dinucleotide (FAD). FAD might be hy-drolyzed by CAC2761 and CAC2766, which interact with membrane-anchored flavoproteins potentially involved in EET (CAC2762 and CAC2767). Additionally, we performed a transcriptomic analysis of Fe(III)-citrate- and HFO-reducing cultures and compared them to fermentation alone. We found two Fe-S oxidoreductases (CA1254 and CAC1021) upregulated with Fe(III)-citrate, which have an unknown function and could be putative Fe(III)-citrate reductases. Additional upregulated redox active pro-teins with potential involvement in solid-phase Fe(III) reduction were identified as CA1044 and CAC3371. These proteins should be considered for further studies. Furthermore, iron reduction impacts bacterial metabolism by buffering the pH and shifting the carbon and electron flow to less reduced metabolites as compared to fermentation. In addition to catabolic pathways, metabolic modeling showed that iron reduction influences anabolic pathways. Finally, metabolic modeling suggests that NADH is the physiological elec-tron donor for iron reduction in this system. This study contributes to a better understanding of the mechanism of iron reduction by Gram-positive bacteria and provides implications for further studies to understand the EET by fermenters in natural environments.

EPFL2019

Mechanism(s) of electron transfer and metabolic changes during iron reduction by Clostridium acetobutylicum

Cornelia List

EPFL2019