Catalytic Properties of
 Staphylococcus aureus
 and
 Bacillus
 Members of the Secondary Cation/Proton Antiporter-3 (Mrp) Family Are Revealed by an Optimized Assay in an
 Escherichia coli
 Host

Swartz, Talia H.; Ito, Masahiro; Ohira, Takayuki; Natsui, Shinsuke; Hicks, David; Krulwich, Terry A.

doi:10.1128/jb.00021-07

Cited by 47 publications

(60 citation statements)

References 71 publications

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“…Time-dependent DpH changes were monitored by measuring the quenching of acridine orange fluorescence using a Shimadzu-5300PC instrument (Shimadzu) with the excitation wavelength set at 492±1.5 nm and the emission wavelength set at 525±3.0 nm. Antiport activities were expressed as percentage fluorescence recovery of the total quenching values, as suggested by Swartz et al (2007) (Kiriyama et al, 2012).…”

Section: Methodsmentioning

confidence: 99%

“…In bacteria, monovalent cation/H + antiporters are often considered to be major determinant factors for alkaline and Na + tolerance of cells (Padan et al, 1989(Padan et al, , 2005Putnoky et al, 1998). It has been shown that most of the cation/H + antiporters involved in alkaline tolerance are fully active in alkaline but inactive in neutral to acidic conditions, and such pH dependency is considered to be a critical feature for the maintenance of adequate intracellular pH (Taglicht et al, 1991;Swartz et al, 2007;Yamaguchi et al, 2009). Therefore, the molecular mechanism of the pH-dependent regulation of antiport activity has drawn particular attention for more than two decades (Padan, 2008).…”

Section: Introductionmentioning

confidence: 99%

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Diversities and similarities in pH dependency among bacterial NhaB-like Na+/H+ antiporters

et al. 2013

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NhaB-like antiporters were the second described class of Na + /H + antiporters, identified in bacteria more than 20 years ago. While nhaB-like gene sequences have been found in a number of bacterial genomes, only a few of the NhaB-like antiporters have been functionally characterized to date. Although earlier studies have identified a few pH-sensitive and -insensitive NhaB-like antiporters, the mechanisms that determine their pH responses still remain elusive. In this study, we sought to investigate the diversities and similarities among bacterial NhaB-like antiporters, with particular emphasis on their pH responsiveness. Our phylogenetic analysis of NhaB-like antiporters, combined with pH profile analyses of activities for representative members of several phylogenetic groups, demonstrated that NhaB-like antiporters could be classified into three distinct types according to the degree of their pH dependencies. Interestingly, pH-insensitive NhaB-like antiporters were only found in a limited proportion of enterobacterial species, which constitute a subcluster that appears to have diverged relatively recently among enterobacterial NhaB-like antiporters. Furthermore, kinetic property analyses of NhaB-like antiporters at different pH values revealed that the degree of pH sensitivity of antiport activities was strongly correlated with the magnitude of pH-dependent change in apparent K m values, suggesting that the dramatic pH sensitivities observed for several NhaB-like antiporters might be mainly due to the significant increases of apparent K m at lower pH. These results strongly suggested the possibility that the loss of pH sensitivity of NhaB-like antiporters had occurred relatively recently, probably via accumulation of the mutations that impair pH-dependent change of K m in the course of molecular evolution.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Diversities and similarities in pH dependency among bacterial NhaB-like Na+/H+ antiporters

et al. 2013

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“…After the dequenching reached saturation, 25 mM ammonium chloride was added to dissipate remaining DpH in order to calculate the total quenching value. Antiport activities (dequenching) were expressed as a percentage of the total quenching value, as suggested by Swartz et al (2007). Activities of the Pha1 system were calculated by subtracting the values of TO114/pTrcHis2C vesicles from those of TO114/pTrcHis2C-pha1 under each condition.…”

Section: Methodsmentioning

confidence: 99%

“…Most of the Mrp antiporters studied have been shown to be Na + /H + antiporters that play an important role in cytosolic pH homeostasis under alkaline conditions (Dzioba-Winogrodzki et al, 2009; Ito et al, 1999; Kosono et al, 1999;Swartz et al, 2007;Yang et al, 2006). The TO114/pTrcHis2C control strain showed almost no growth at pH 7.0, but the growth of the TO114/pTrcHis2C-pha1 strain in LBNa100 medium was significantly enhanced at pH values up to 8.5, but not at pH 9.0 (Fig.…”

Section: Functional Expression Of the Pha1 Complex In E Colimentioning

confidence: 99%

“…Recently, two groups have experimentally shown that gene products of the mrp operon from Bacillus species actually form complexes in B. subtilis cells (Kajiyama et al, 2007) and in heterologous Escherichia coli cells (Morino et al, 2008). Detailed functional analysis of group 1 Mrp antiporters from Staphylococcus aureus and several Bacillus species (Swartz et al, 2007) determined that these antiporters are electrogenic Na + (Li + )/H + antiporters with no detectable K + transport activity. In comparison, a group 2 Mrp antiporter, VcMrp, was shown to be a Na + (Li + )/H + antiporter with low-affinity K + /H + antiport activity (Dzioba-Winogrodzki et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

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pH-dependent regulation of the multi-subunit cation/proton antiporter Pha1 system from Sinorhizobium meliloti

et al. 2009

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The pha1 gene cluster (pha1A′-G) of Sinorhizobium meliloti has previously been characterized as a necessary component for proper invasion into plant root tissue. It has been suggested to encode a multi-subunit K+/H+ antiporter, since mutations in the pha1 region rendered S. meliloti cells sensitive to K+ and alkali, and because there is high amino acid sequence similarity to previously characterized multi-subunit cation/H+ antiporters (Mrp antiporters). However, the detailed transport properties of the Pha1 system are yet to be determined. Interestingly, most of the Mrp antiporters are highly selective for Na+, unlike the Pha1 system. Here, we report the functional expression of the Pha1 system in Escherichia coli and the measurement of cation/H+ antiport activity. We showed that the Pha1 system is indeed a K+/H+ antiporter with a pH optimum under mildly alkaline conditions. Moreover, we found that the Pha1 system can transport Na+; this was unexpected based on previous phenotypic analyses of pha1 mutants. Furthermore, we demonstrated that the cation selectivity of the Pha1 system was altered when the pH was lowered from the optimum. The downregulation of Na+/H+ and K+/H+ antiport activities upon acidic shift appeared to occur via different processes, which might indicate the presence of distinct mechanisms for the regulation of the K+/H+ and Na+/H+ antiport activities of the Pha1 system.

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A cytochrome c fusion protein domain for convenient detection, quantification, and enhanced production of membrane proteins in Escherichia coli—Expression and characterization of cytochrome‐tagged Complex I subunits

et al. 2010

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Overproduction of membrane proteins can be a cumbersome task, particularly if high yields are desirable. NADH:quinone oxidoreductase (Complex I) contains several very large membrane-spanning protein subunits that hitherto have been impossible to express individually in any appreciable amounts in Escherichia coli. The polypeptides contain no prosthetic groups and are poorly antigenic, making optimization of protein production a challenging task. In this work, the Cterminal ends of the Complex I subunits NuoH, NuoL, NuoM, and NuoN from E. coli Complex I and the bona fide antiporters MrpA and MrpD were genetically fused to the cytochrome c domain of Bacillus subtilis cytochrome c 550 . Compared with other available fusion-protein tagging systems, the cytochrome c has several advantages. The heme is covalently bound, renders the proteins visible by optical spectroscopy, and can be used to monitor, quantify, and determine the orientation of the polypeptides in a plethora of experiments. For the antiporter-like subunits NuoL, NuoM, and NuoN and the real antiporters MrpA and MrpD, unprecedented amounts of holo-cytochrome fusion proteins could be obtained in E. coli. The NuoHcyt polypeptide was also efficiently produced, but heme insertion was less effective in this construct. The cytochrome c 550 domain in all the fusion proteins exhibited normal spectra and redox properties, with an E m of about 1170 mV. The MrpA and MrpD antiporters remained functional after being fused to the cytochrome c-tag. Finally, a his-tag could be added to the cytochrome domain, without any perturbations to the cytochrome properties, allowing efficient purification of the overexpressed fusion proteins.

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Catalytic Properties of Staphylococcus aureus and Bacillus Members of the Secondary Cation/Proton Antiporter-3 (Mrp) Family Are Revealed by an Optimized Assay in an Escherichia coli Host

Cited by 47 publications

References 71 publications

Diversities and similarities in pH dependency among bacterial NhaB-like Na+/H+ antiporters

Diversities and similarities in pH dependency among bacterial NhaB-like Na+/H+ antiporters

pH-dependent regulation of the multi-subunit cation/proton antiporter Pha1 system from Sinorhizobium meliloti

A cytochrome c fusion protein domain for convenient detection, quantification, and enhanced production of membrane proteins in Escherichia coli—Expression and characterization of cytochrome‐tagged Complex I subunits

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