Single cell-level detection and quantitation of leaky protein expression from any strongly regulated bacterial system

Arora, Kanika; Mangale, Sachin S.; Guptasarma, Purnananda

doi:10.1016/j.ab.2015.06.011

Cited by 9 publications

(7 citation statements)

References 16 publications

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“…In a previous article, we have described engineered forms of HU-A and HU-B, in which each protein has been genetically fused with a fluorescent protein at its N-terminus (19). In that article, we had created chimeric forms of HU in which a monomeric red fluorescent protein (tag-RFP) was fused with HU-A (to make RFP-HU-A).…”

Section: Edited By Patrick Sungmentioning

confidence: 99%

The DNA-binding protein HU is a molecular glue that attaches bacteria to extracellular DNA in biofilms

Thakur

Arora

Gupta

et al. 2021

Journal of Biological Chemistry

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In biofilms, bacteria that possess a negatively charged surface are embedded within a matrix of polymers consisting mainly of negatively charged extracellular DNA (e-DNA). In all likelihood, a multivalent positively charged substance, for example, a basic protein, exists within biofilms to neutralize charge–charge repulsions and act as a ‘glue’ attaching negatively charged bacteria to negatively charged e-DNA; however, no protein capable of doing so has yet been identified. We decided to investigate whether a highly abundant nucleoid-associated histone-like protein (HU) happens to be the glue in question. In recent years, HU has been shown to possess qualities that could be considered desirable in the proposed glue, for example, (a) availability in association with e-DNA; (b) multivalent DNA binding; (c) non–sequence-specific DNA-binding; (d) enhancement of biofilm formation upon exogenous addition, and (e) disruption of biofilms, upon removal by HU–cognate antibodies. Geometric considerations suggest that basic residues in HU's canonical and noncanonical DNA-binding sites can interact with sugar-linked terminal phosphates in lipopolysaccharide (LPS) molecules in bacterial outer membranes. Here, using genetic, spectroscopic, biophysical–chemical, microscopy-based, and cytometry-based experiments, we demonstrate that HU's DNA-binding sites also bind to LPS, that this facilitates DNA–DNA, DNA–LPS, and LPS–LPS interactions, and that this facilitates bacterial clumping and attachment of bacteria to DNA. Exogenous addition of HU to bacteria in (nonshaken) cultures is shown to cause cells to become engulfed in a matrix of DNA, potentially arising from the lysis of bacteria with vulnerable cell walls (as they strain to grow, divide, and move away from each other, in opposition to the accreting influence of HUs).

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Section: Edited By Patrick Sungmentioning

confidence: 99%

The DNA-binding protein HU is a molecular glue that attaches bacteria to extracellular DNA in biofilms

Thakur

Arora

Gupta

et al. 2021

Journal of Biological Chemistry

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“…The sequences of primers for cloning Tag-RFP and fusing it to HU-A, based on previous work, are given below: RFP-NdeI-Forward, 5′-ATATATCATATGGCTAGCATGACTGGTGGACAGCAAATG-3′. All primers other than the one described below may be found in ref .…”

Section: Methodsmentioning

confidence: 99%

N-Terminal Extensions Appear to Frustrate HU Heterodimer Formation by Strengthening Intersubunit Contacts and Blocking the Formation of a Heterotetrameric Intermediate

et al. 2021

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HU is a bacterial nucleoid-associated protein. Two homologues, known as HU-A, and HU-B, are found in Escherichia coli within which the early, late, and stationary phases of growth are dominated by HU-AA, HU-BB, and HU-AB dimers, respectively. Here, using genetic manipulation, mass spectrometry, spectroscopy, chromatography, and electrophoretic examination of glutaraldehyde-mediated cross-linking of subunits, in combination with experiments involving mixing, co-expression, unfolding, and refolding of HU chains, we show that the spontaneous formation of HU-AB heterodimers that is reported to occur upon mixing of wild-type HU-AA and HU-BB homodimers does not occur if chains possess N-terminal extensions. We show that N-terminal extensions interfere with the conversion of homodimers into heterodimers. We also show that heterodimers are readily formed at anticipated levels by chains possessing N-terminal extensions in vivo, when direct chain–chain interactions are facilitated through production of HU-A and HU-B chains from proximal genes located upon the same plasmid. From the data, two explanations emerge regarding the mechanism by which N-terminal extensions happen to adversely affect the conversion of homodimers into heterodimers. (1) The disappearance of the α-amino group at HU’s N-terminus impacts the intersubunit stacking of β-sheets at HU’s dimeric interface, reducing the ease with which subunits dissociate from each other. Simultaneously, (2) the presence of an N-terminal extension appears to sterically prevent the association of HU-AA and HU-BB homodimers into a critically required, heterotetrameric intermediate (within which homodimers could otherwise exchange subunits without releasing monomers into solution, by remaining physically associated with each other).

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“…Four oligonucleotides designed to associate to create the four strands of a holiday junction intermediate were mixed together in equimolar proportions according to known protocols, 22 heated to 90 °C and cooled from 90 °C to 25 °C over an extended period of time to allow strands to anneal into 4WJ-DNA. The sequences of the oligonucleotides used for creating 4WJ-DNA are detailed below:…”

Section: Methodsmentioning

confidence: 99%

N-terminal extensions strengthen hydrophobic inter-subunit interactions between HU’s C-terminal domains to frustrate heterodimer formation

Arora

Thakur

Mrigwani

et al. 2021

Preprint

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HU is a nucleoid-associated protein (NAP) that helps bacterial chromosomal DNA to remain compact. Escherichia coli contains two homologs of HU that are ~ 70 % identical: HU-A and HU-B. The early log phase, late log phase, and stationary phase of E. coli growth are reported to be dominated, respectively, by HU-AA homodimers, HU-BB homo-dimers, and HU-AB heterodimers. Here, we show that the formation of HU-AB heterodimers occurs to a much lower degree in HU chains that have a displaced N-terminus, whether through addition of an N-terminal affinity (polyhistidine) tag, or fusion of a fluorescent protein. A combination of mass spectrometry, spectroscopy, chromatography, and electrophoresis (exploring glutaraldehyde crosslinking of subunits) was used to study the mixing, co-expression, unfolding and refolding of HU-AA and HU-BB homodimers. The data suggests that, in HU polypeptides with N-terminal extension, whereas inter-subunit contacts between the alpha helical N-terminal domains (NTDs) undergo facile unfolding and dissociation, inter-subunit contacts between the beta sheet- and IDR-dominated C-terminal domains (CTDs) fail to do so, due to persistence of hydrophobic inter-subunit interactions between two beta sheets. This persistence causes HU to remain nominally dimeric even after substantive unfolding, and frustrates subunit exchange and heterodimer formation.

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Single cell-level detection and quantitation of leaky protein expression from any strongly regulated bacterial system

Cited by 9 publications

References 16 publications

The DNA-binding protein HU is a molecular glue that attaches bacteria to extracellular DNA in biofilms

The DNA-binding protein HU is a molecular glue that attaches bacteria to extracellular DNA in biofilms

N-Terminal Extensions Appear to Frustrate HU Heterodimer Formation by Strengthening Intersubunit Contacts and Blocking the Formation of a Heterotetrameric Intermediate

N-terminal extensions strengthen hydrophobic inter-subunit interactions between HU’s C-terminal domains to frustrate heterodimer formation

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