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

Abstract

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). 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). Bacteria in biofilms are embedded within a matrix of extracellular polymeric substances, consisting mainly of extracellular DNA (e-DNA) released through cell lysis (1Thomas V.C. Thurlow L.R. Boyle D. Hancock L.E. Regulation of autolysis-dependent extracellular DNA release by Enterococcus faecalis extracellular proteases influences biofilm development.J. Bacteriol. 2008; 190: 5690-5698Crossref PubMed Scopus (230) Google Scholar), or cellular secretions (2Whitchurch C.B. Tolker-Nielsen T. Ragas P.C. Mattick J.S. Extracellular DNA required for bacterial biofilm formation.Science. 2002; 295: 1487Crossref PubMed Scopus (1532) Google Scholar). A conceptual with the of bacteria in a matrix of DNA is that the of bacteria and DNA to be highly negatively charged of the of in and the A of lipopolysaccharide (LPS) molecules the outer of In in of negatively charged molecules are upon bacterial of the of DNA and bacteria are to within biofilms for the of extracellular DNA in biofilm formation by Bacteriol. PubMed Scopus Google Scholar). that they the of of a glue that to bacteria and DNA. In glue capable of to DNA and cells could be a protein that is with surface extracellular matrix of biofilms that with the cell surface in to Scopus Google Scholar). in a protein could be protein of to DNA and glue be that is capable of to cells and DNA with and We this by that the glue in a biofilm to the of to be a protein capable of to DNA and to that a positively charged the protein to be and in the extracellular by or in association with to to cells and through sites of of could a protein to bind to cells and in the protein to act as a of cells and the of from all the DNA-binding in the of the is the highly abundant nucleoid-associated histone-like protein for the HU is a highly nucleoid-associated protein of the HU has been to be in bacterial biofilms a in biofilm PubMed Scopus Google Scholar). HU has also been to be for biofilm that been to biofilms DNA-binding of biofilms in and bacterial in PubMed Scopus Google Scholar). HU is to be upon DNA, of to DNA in a non–sequence-specific D. by HU and PubMed Scopus Google Scholar). HU is to be of the abundant in of to cell of the nucleoid-associated protein from and DNA PubMed Scopus Google Scholar). HU is to be released the extracellular through lysis of cells (1Thomas V.C. Thurlow L.R. Boyle D. Hancock L.E. Regulation of autolysis-dependent extracellular DNA release by Enterococcus faecalis extracellular proteases influences biofilm development.J. Bacteriol. 2008; 190: 5690-5698Crossref PubMed Scopus (230) Google Scholar), cell lysis cell lysis as a for the of bacterial and PubMed Scopus Google Scholar), and (2Whitchurch C.B. Tolker-Nielsen T. Ragas P.C. Mattick J.S. Extracellular DNA required for bacterial biofilm formation.Science. 2002; 295: 1487Crossref PubMed Scopus (1532) Google by by in DNA and Bacteriol. PubMed Scopus Google DNA and a and of the PubMed Scopus Google DNA a PubMed Scopus Google Scholar). HU is to be multivalent in to bind to DNA HU protein is a PubMed Scopus Google DNA in PubMed Scopus Google D. HU PubMed Scopus Google Scholar), HU and each HU within HU for DNA and the protein, for the glue that cells to DNA and to HU is a DNA-binding protein, to be (a) whether HU is capable of to and to and (b) whether HU is capable of of and of In HU exists as and are of DNA-binding of HU and from PubMed Scopus Google and is mainly in the and of and exists as a or is mainly in the and exists as a or HU protein is a PubMed Scopus Google of DNA-binding of HU and from PubMed Scopus Google and in a PubMed Scopus Google Scholar). In the and is that of and in with DNA in HU of is required for Scopus Google Scholar). In a we of and in each protein has been with a protein and of protein from bacterial PubMed Scopus Google Scholar). In that we of HU in a protein with a protein with In the the of HU to and DNA, we and of and protein in and also with a protein in and as as of in addition to HU in residues been by the In this using a of spectroscopic, and we that and as as of of bind to and the outer of bacterial cells We that the of HU to or or of HU's or DNA-binding We that addition of to HU of We also that addition of to cells cellular bacterial charged in the A of terminal of and potentially the of the and the of We that bind to the residues of DNA-binding through charge–charge and and We demonstrate that and DNA are to with each for to the we that HU to bind to DNA and the that DNA-binding also to act as in biofilms, in in the of the canonical DNA in HU that are each of the of HU through DNA in PubMed Scopus Google Scholar), are shown upon the of the HU also the noncanonical DNA-binding that using of the HU D. HU PubMed Scopus Google Scholar). that HU of for the is also of for DNA PubMed Scopus Google Scholar), is that are for the canonical DNA-binding of HU in the of DNA, and for HU to of in the of the HU in with a of DNA the canonical DNA-binding A and HU from the and are from a in of HU each DNA is also to a HU within the through the terminal of the DNA In the of we to HU can also bind to DNA through HU's noncanonical DNA-binding in we the HU of from a by in of a the of the HU of and of HU with DNA the noncanonical in a of the DNA in the to the canonical of a can be to be with the noncanonical of the shown in in a of in the is with each with of the DNA canonical and with a of the DNA noncanonical In this the HU is shown from a is from and could be to be that that and and of HU with DNA to the HU could also potentially interact with DNA canonical and noncanonical the we decided to whether HU's DNA-binding sites could also potentially bind to negatively charged through HU's to in the of that has been the extracellular or to that is in association with bacterial outer membranes. We negatively charged in and positively charged residues the canonical and noncanonical DNA-binding of the A of are and the in DNA. the in are or in the A of are the in the DNA of the of the in the formation of in in the of phosphates are terminal phosphates could potentially possess negatively charged upon the of the of the in a terminal are and a of of with a of be to a of molecules could potentially that the A could as with a or as with in in the is and example, is that the of is and also that in the of of to the of the be that a of phosphates in the A of or a the with a of in with a of the of the of terminal phosphates the the terminal phosphates of the A of LPS, and the phosphates of the DNA could be the phosphates the A of are to bind to through T. of of PubMed Scopus Google Scholar). interactions, and phosphates could also potentially bind to the DNA-binding sites of using the of the for of the canonical and noncanonical DNA-binding sites of In the of the and residues been shown in of the HU been shown the of the of from the of to the T. sites of A a for negatively charged PubMed Scopus Google Scholar). the in the A of each the of the can potentially bind to or positively by and in HU's canonical DNA-binding and are and and also in the HU from bacteria of bacterial histone-like HU Google Scholar). the terminal of the of A could potentially bind to or by and the of a in HU's noncanonical DNA-binding and suggest that a exists for to bind to through potentially through and considered also of we of experiments, demonstrate that the HU bind to A and a of of and from to the of is to whether the of of is of of to and also whether is in the and of of of molecules away from or a of within a of HU and the of the of of as as the of of of the of the that the of away from a of of molecules and the to be for of that to also no the and of that the A in for of HU in and of the is a for from to be of HU with of for of all molecules the the are in the and the HU of and HU the of be to be of the of the of HU to and as for of a and for of to this to in surface using a and association of the with the is and so is a association of with the is to cause the association to the association of by of could cause the to away from the or cause this association is in with the that from of and of from the that to of and for in of and In this and are in of a with a to of the to through in the and also of the of the to be through of the of the for the the of the of of potentially by of through of the in the of of the in each and the of the of through of and the of the of the through of the A in of through is and are the in the as a from the of of the of to to the are no interactions, no in is of the as to a is a of and a of and bind to each and neutralize each PubMed Scopus Google and in and Scopus Google Scholar). In the shown in is shown to as to in the of HU of in of HU's residues HU no or and also of in of the in the and that bind to that of upon addition to a of of a of of in the of that are of in of HU of is of to HU and are to upon of negatively charged to the protein exists as a of and in each could sites of with negatively charged HU to be that in experiments, in all the in this the through a we the of in as that we to be in the of in the has a of to consisting of or that also to in the and to the of LPS, as shown in that upon of with and the HU in and is by is so that is to the in the the a of is In has no HU that and of the HU are through in the of with to of and with the no that all no of with that in is in the that of with through of to in that no is with the addition of to this is the bind to in that to a the of and are by the upon addition of and is to is to be of the of is by that and that become by that no the of A and the upon in of and and DNA-binding upon addition of the the canonical DNA-binding in in to the the noncanonical DNA-binding that is of by addition of to by addition of to for this are from the to the residues the DNA-binding sites in A and in the that the of to the HU's noncanonical DNA-binding to of a to the canonical to that is a with that to of of HU to upon of of with In to in D. A to by using PubMed Scopus Google Scholar), we that the in in the and to a a as in the are the a in the and a from to as in is upon the of the from to a as shown in in no upon as in the is charged the the and with the is no by the of to the can a that the to from to in is to the as a in for the of protein to as D. A to by using PubMed Scopus Google Scholar). In the of experiments, a from to is upon addition of to as in that to a for the by protein to that that in the of as as cells as as that are of in through cell clumping is to that is to cells to cause clumping clumping as a the of the the for or are to the is a for from and to be the to bacterial all are to cause clumping in the of and is also in the cell and that can be that with addition of are with to the with addition of and a is for that for in the in the the of to the of bacteria in of is as the of no are shown for this addition of to cells of strain happens to be with the bacteria in the and in the In addition of to cells no the to bacterial cells in the no of and is in the that is the HU of the the to with bacterial is shown that HU to the of bacterial cells and to the cells cells for this is that HU to the outer with the in the the of the HU upon bacterial cell be that the cells that In that the of a with of with with the of a of within bacteria and we also that cells that and and to each and a of the cell in addition to the with the bacterial in the cell in In experiments, to to or the the cell surface all and that (as can be in clumping and the is with as the of to the of the HU to bind to bacterial cell through to In addition, they that HU can cause clumping of bacteria by using to bind to is that is in with HU HU's this the formation of and by HU the of the as a in the with the of to as in We to the of through cell lysis (1Thomas V.C. Thurlow L.R. Boyle D. Hancock L.E. Regulation of autolysis-dependent extracellular DNA release by Enterococcus faecalis extracellular proteases influences biofilm development.J. Bacteriol. 2008; 190: 5690-5698Crossref PubMed Scopus (230) Google Scholar), in through cell lysis bacteria and become to release and DNA, is in a of bacterial cells cell lysis as a for the of bacterial and PubMed Scopus Google Scholar). is that the of released by bacteria through the of the to bacterial cell with the of multivalent and abundant as that addition of HU to a of cells could the of cells to each and to of cell lysis and of of We that bacteria are in the and of bacterial are in the to is that a DNA-binding is to of and are for the bacteria to are to be embedded in a matrix of to In bacteria are to be embedded in a matrix of DNA is also that HU a could a in cell through of cells and and cells and a for lysis of cells through upon could in the cell the sites of cell in addition to in and cause that the availability of HU in the extracellular can the of and the of a through to of and lysis of to shown in a of cell is upon of with DNA. that of DNA for to HU sites and the for of sites for of the HU to upon cell In of of with of with DNA, that is a in cell as the of is from to to that of for to HU HU's sites and the for the of sites for of the HU to In molecules that are to DNA or are cell clumping through multivalent to molecules are to DNA or be in of that are of and DNA, for and the of upon the of of be to influence of the cells cells in the of and cells in the of with of and that is to interactions, to the of the of DNA and with we HU are in are and and for and canonical DNA-binding HU of from each HU as shown in A and and the noncanonical DNA-binding sites of a each consisting of residues and as shown in A and We of and HU in canonical and noncanonical DNA-binding or (as shown in in the from to in and and by to the canonical DNA-binding or (as shown in in and by the to the noncanonical DNA-binding and and or (as shown in in canonical and noncanonical DNA-binding sites through In addition, we also in and HU and and with each in of to that and all of the to DNA-binding that and with in the of to and and arising from the of of that the and all in the HU to a of from of HU each of a and a HU with in the DNA-binding to the In the to the and of and DNA-binding also using by the of to in DNA, that or the canonical or noncanonical DNA is to bind to DNA. In or sites is to bind to DNA. In DNA is shown in and and and are all to bind DNA as is of the or or residues and to bind to DNA with and and is that or residues or is to bind to DNA, as be of the of the canonical DNA-binding as with the canonical DNA-binding is and or all of the noncanonical is also that the or or residues or no of DNA is all of is that the of of HU's DNA-binding sites canonical or the noncanonical is required by HU for of DNA. of is and the HU to DNA, of sites through or removal of or is and is no DNA by In the we that of the sites also of the sites the In we demonstrate that the DNA-binding and in the canonical and noncanonical We the of by and and using the to the shown in to whether LPS, the and and the that to the the of the and In and the of protein of the of the and the of and the protein and the by of and all of the protein or the protein in also the that and that the and that no is is no with are in and and with in and with the with and are of and to interactions, the that are to the the of the of and We shown that HU are capable of to lipopolysaccharide as as to the outer of HU of DNA-binding of sites canonical with the of the of the HU in with DNA. noncanonical we that of positively charged residues each of sites residues and the canonical DNA-binding and residues and the noncanonical DNA-binding to be for of the in the of and of we that HU and In using we in the of a of however, we that this is and by this mainly to of in the of of the LPS, and of the of the in the of of experiments, the experiments, using in the of and experiments, with protein in in with we that to the of We also that HU the of cells a of experiments, in we with bacterial cells with or or DNA, we that can cause the clumping of bacterial and that DNA, and the clumping by HU's of the canonical and noncanonical DNA-binding sites of and in we that each is capable of to DNA, and LPS, that HU is to bind to DNA or of sites are in that is a of the DNA-binding and sites of the In this is that the of of in with is to the and phosphates in the of DNA. In DNA, the are of in LPS, they are terminal each in DNA a each of the in can or the of the can to in each in DNA is a each in the A of is a the of A in is or DNA, and DNA. is to capable of to the DNA-binding of a DNA-binding protein, in with of and to is that can bind to the DNA-binding of a DNA-binding protein by to the the in the of the and the with cells that HU is capable of as a glue that negatively charged cell could be to each to bind to positively charged of the We shown that cells HU to to each the of the cell surface could be We also shown that of and HU the in association with the cell In this to the of HU the cell and also association with the cell surface and in is that HU can the cell in the extracellular that biofilms a in biofilm PubMed Scopus Google Scholar). is also that HU can be by DNA and a and of the PubMed Scopus Google Scholar), or by cells through cell with DNA cell lysis as a for the of bacterial and PubMed Scopus Google Scholar). We that HU in the extracellular in a biofilm to the of cells that are also in and that this in upon cells that become the cause for cell In of lysis that the DNA the matrix of a is by cells and by cells cell lysis as a for the of bacterial and PubMed Scopus Google Scholar). We that this is the DNA is with molecules of HU and that all of the DNA-binding sites HU molecules are in the of DNA, with and for with the of bacterial is that DNA-binding sites bind to cell cells to bind to through with the In this has the for a that each of cell lysis the of the cause of the of cell by for cells to bind to as cells grow, divide, and move a bacterial we shown that exogenous addition of HU to bacterial cells in cultures to the of of in bacterial cells are to be that addition of exogenous HU can the of biofilm with cell lysis by and bacteria sites of in outer cell and cell is in the to suggest that addition of the HU the formation of biofilms a in biofilm PubMed Scopus Google Scholar), to of addition of the HU of cells in of DNA. we that HU could be a in biofilms, (a) by in (b) by upon the matrix within biofilms, and (c) also by to and negatively charged of bacterial as as as shown in this we that the and of from could be in the of association of cells to biofilms, of of the of a cells the DNA through upon of DNA of cells in and of cells in to the by HU could cause cells to to each other, as we as as cause cells to to of through to of to the of is to that be and LPS, for to HU's DNA-binding We shown that HU to cause HU also to cause and with the of HU with cause is that the DNA-binding the canonical of HU cause of bacteria from biofilms DNA-binding of biofilms in and bacterial in PubMed Scopus Google Scholar). of the of HU in the formation and of be that all bacteria negatively charged and that they possess they possess negatively charged a also has all bacteria possess this protein could be a glue for biofilms by all and strain of for all using the of all of the using for cells by of in the using and from the HU through of all HU protein and with of to and removal of all DNA and and from DNA and the sites of the the and that the all of or and protein as could be with that with in HU's that with in HU's that in residues to HU's canonical DNA-binding are and by that in and HU's noncanonical DNA-binding by and that the and and through DNA for and by or protein from DNA or from DNA in the of from DNA or through and DNA through addition of the to each in for DNA from DNA and from and from or or from of for with for protein a using a with a of for a of of with of LPS, and of of the through of the of the as and bind to each and neutralize each PubMed Scopus Google and in and Scopus Google Scholar). for of and upon to of using and and a using a and or protein of and from to to the protein using a using a of and protein in the of to of protein using a and using a protein of and of using a and from to the HU and LPS, using of and of and of a with with through a to protein as a of to protein for and the in the and through of the by of bacterial and the with of to bacteria a of the protein and for and using to cells embedded in a DNA matrix using a using a to association with of the and and using a with and a using a for of with using and protein to and using a for DNA and using or and DNA and from as D. A to by using PubMed Scopus Google Scholar). of protein are in the and the of the as in the also the for of protein to of the and and of the disruption of of upon the by of the HU been in to the and to this are in the and the that they no of with the of this We and of the of of and for for for to whether highly abundant nucleoid-associated histone-like protein to lipopolysaccharide using We for and the of and the and the of of for the of of for the of a of in of and in the of and and the of and of experiments, as as the of the by T. and and HU by also the of bacteria HU and the that HU to LPS, with of the of HU by also of with and the the addition of HU to with to

Topics & Concepts